Download Abstracts of the 12 European Biological Inorganic Chemistry

J Biol Inorg Chem (2014) 19 (Suppl 2):S697–S698
DOI 10.1007/s00775-014-1150-5
Abstracts of the 12th European Biological
Inorganic Chemistry Conference
August 24–28, 2014
University of Zurich, Zurich, Switzerland
123
Organizing Committee of EuroBIC 12
International Organizing Committee
Eva Freisinger, co-Chair
Roland Sigel, co-Chair
Roger Alberto
Gilles Gasser
Bernhard Spingler
Felix Zelder
Daniela Donghi
Silke Johannsen, Head of Conference Secretariat
Sofia Gallo, Conference Secretary
Ramona Erni, Conference Secretary
Beatrice Spichtig, Sponsoring
Bernhard Lippert, Technical University of Dortmund, Secretary
Josefa Marı́a González-Pérez, University of Granada, co-Chair
EuroBIC 11
Juan Niclós-Gutierrez, University of Granada, co-Chair EuroBIC 11
Eva Freisinger, University of Zurich, co-Chair EuroBIC 12
Roland Sigel, University of Zurich, co-Chair EuroBIC 12
Tamás Kiss, University of Szeged, co-Chair EuroBIC 13
Imre Sóvágó, University of Debrecen, co-Chair EuroBIC 13
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J Biol Inorg Chem (2014) 19 (Suppl 2):S699
DOI 10.1007/s00775-014-1152-3
EDITORIAL
Preface
Eva Freisinger • Roland K. O. Sigel
Ó SBIC 2014
Dear colleagues,
We cordially welcome you to the 12th European Biological
Inorganic Chemistry Conference, EuroBIC 12, to be held
August 24–28, 2014. 22 years after EuroBIC 1 took place
in Newcastle, UK, this conference series is now coming to
Switzerland for the first time.
The biannual EuroBIC conferences have become one of
the major international events in the field of Biological
Inorganic Chemistry. Its interdisciplinarity is well reflected
in the broad wealth of topics covered in this year’s event.
The presentations at EuroBIC 12 will include the latest
developments and results from the interface between
Coordination Chemistry and the Life Sciences: All aspects
of Metalloproteins—Structure and Function, Bioinspired
and Biomimetic Systems, Artificial Photosystems—From
Light to Chemical Energy, Metal-Nucleic Acid Interactions, Bioorganometallic Chemistry, Environmental and
Geological Bioinorganic Chemistry, New Trends in Bioinorganic Chemistry, Metal Homeostasis and Detoxification, Metal-related Diseases, and Metals in Medicine—
Diagnosis and Therapy will be covered. More than 180 oral
contributions, delivered by Plenary, Keynote, Session,
Invited, and Selected Speakers will provide the basis for
vivid and stimulating discussions. These discussions will
be intensified in a relaxed atmosphere during the two
Poster Sessions with more than 260 contributions. Special
sessions of the Scientific Program encompass the Alfred
Werner Lecturer series, the Young Researcher Presentations, Poster Flash Presentations as well as the concluding
EuroBIC Medal 2014 Award Lecture by Xile Hu.
EuroBIC 12 would not be possible without the financial
support of numerous companies and societies, many of
which will be part of the exhibition during the conference
and to all of whom we are very grateful for their support.
We are also expressing our sincere gratitude to the staff,
our coworkers, and students of the Department of Chemistry, University of Zurich, who are and will be involved
during the organization and the conference itself. Only
their help and efforts will ensure a memorable EuroBIC 12.
We are hence looking very much forward to an exciting
scientific program and a stimulating environment at the
Irchel Natural Science Campus of the University of Zurich,
and are very excited to seeing you all at EuroBIC 12.
With our best wishes,
Sincerely,
Eva Freisinger and Roland Sigel
On behalf of the Organizing Committee
E. Freisinger R. K. O. Sigel (&)
Zurich, Switzerland
e-mail: [email protected]
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J Biol Inorg Chem (2014) 19 (Suppl 2):S701–S702
DOI 10.1007/s00775-014-1151-4
LAUDATIO
EuroBIC Medal 2014
Kay Severin
Xile Hu
Award lecture PL 6
Born in 1978 in Fujian, China, Xile Hu received a Bachelor’s
degree from Peking University in 2000 and a Ph.D. degree from the
University of California, San Diego in 2004. Following a postdoctoral study in the California Institute of Technology from 2005 to
2007, he was appointed tenure-track assistant professor in inorganic
chemistry at the Ecole Polytechnique Fédérale de Lausanne (EPFL)
in Switzerland. He was promoted to associate professor with tenure
at EPFL in 2013. Among his many research interests is the biomimetic and bio-inspired chemistry of redox-active metalloenzymes.
During the last several years, he has developed an exciting and
highly successful research program in the bio-mimetic chemistry of
[Fe]-hydrogenase that forms the basis for this year’s EuroBIC
Medal.
Hydrogenases are enzymes that catalyze the production and utilization of hydrogen in the biosphere. Given the essential role of
hydrogen in energy technology (fuel cell) and chemical synthesis
(hydrogenation), the study of hydrogenases is of significant contemporary interest. One exciting recent story in bioinorganic chemistry is
the discovery and structural elucidation of [Fe]-hydrogenase. R. Thauer, S. Shima, and co-workers show that [Fe]-hydrogenase is a
mononuclear Fe-containing enzyme with a unique pyridinylacyl ligand.
The proposed active site is unprecedented in both biology and synthetic
chemistry that it warrants confirmation by model complexes. This task
is largely accomplished by Xile Hu’s research in the area.
In 2010, the Hu group synthesized the first Fe acyl complexes that
reproduced the donor set of the active site of [Fe]-hydrogenase,
namely, one acyl carbon, one sulfur, two cis-CO, and one pyridinyl
nitrogen. This work, published in J. Am. Chem. Soc. 2010, 132,
928–929, demonstrated that an iron complex with the donor set proposed
for the active site of [Fe]-hydrogenase could exist outside a protein
environment. The work was a strong encouragement for the biomimetic
community to explore the model chemistry of [Fe]-hydrogenase.
Later in 2010, the Hu group used model chemistry to confirm
another major uncertainty in the proposed active site of [Fe]hydrogenase, the pyridinyl acyl ligand. They elegantly applied
organometallic synthetic chemistry to install an acylmethylpyridinyl
ligand on Fe. Subsequent transformation led to the first model complex to mimic this unique prosthetic group (Angew. Chem. Int. Ed.
2010, 49, 7512–7515). This achievement was not only a synthetic
triumph, it also cleared the remaining doubts in the proposed structure
of [Fe]-hydrogenase.
In 2011, the Hu group reported an unexpected finding: they found
that a model complex reproducing the structural features of [Fe]hydrogenase existed as a five-coordinate species (Angew. Chem. Int.
Ed. 2011, 50, 5670–5672). This was a highly relevant finding because
X-ray crystallography study of [Fe]-hydrogenase was not able to
reveal whether the active site was five or six-coordinate. Furthermore,
the catalytically active state of iron has to be five-coordinate to enable
the chemistry. This work set the stage for reactivity study of model
complexes.
In 2012, using the five-coordinate model mentioned above, the Hu
group demonstrated that the thiolate ligand could be reversibly protonated (Angew. Chem. Int. Ed. 2012, 51, 1919–1921). This reactivity
provided insight into the proton transport process during H2 activation
in [Fe]-hydrogenase. A proton is yielded during H2 activation; the
proton acceptor is however not clear. Xile Hu’s chemistry showed
that the thiolate ligand in the active site of [Fe]-hydrogenase was a
viable proton acceptor.
The attention of the Hu group then turned to H2 activation using
model complexes. They found that the model complexes had similar
spectroscopic properties to the [Fe]-hydrogenase, and they also
exhibited similar ligand binding properties towards CO, CN-, and
isocyanide. However, the models do not react with H2 while the
enzyme does. His group applied DFT calculations to probe the origin
of this difference. They found that the binding of H2 was the main
problem in synthetic compounds as it is unfavorable by about 10 kcal/
mol (Eur. J. Inorg. Chem. 2013, 3993–3999). Once H2 is bound,
splitting of H2 is facile. Xile Hu proposed that the enzyme was
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S702
efficient because it used the protein scaffold and the substrate to
achieve favorable H2 binding.
This leads to the latest and a very impressive result of his research
in the area. In a collaboration with Dr. S. Shima, they successfully
incorporated some of his model complexes into the apoenzyme of
[Fe]-hydrogenase. Spectroscopic data indicate that the artificial
enzymes have a similar electronic property to the native enzyme;
reactivity essays confirm the hydrogenase activity of the artificial
enzymes. These results provide unprecedented, molecular-level
insights into the mechanism of [Fe]-hydrogenase activity. The work is
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J Biol Inorg Chem (2014) 19 (Suppl 2):S701–S702
not only a convincing validation of Xile Hu’s model chemistry, but
also a great example of a new trend in biomimetic chemistry—the
marriage between coordination chemistry and enzymology.
In summary, Xile Hu’s work in the biomimetic chemistry of [Fe]hydrogenase is a truly outstanding example of bioinorganic chemistry
in Europe and is now duly recognized with this year’s EuroBIC
Medal.
Kay Severin, EPFL
Ecole Polytechnique Fédérale de Lausanne (EPFL), Switzerland
J Biol Inorg Chem (2014) 19 (Suppl 2):S703–S705
DOI 10.1007/s00775-014-1153-2
ORAL PRESENTATION
Plenary lectures
PL 1
Metal-templated design: from enzyme inhibition
to asymmetric catalysis
Eric Meggers1,2
Department of Chemistry, Philipps-University Marburg, HansMeerwein-Strasse, 35043 Marburg.
2
College of Chemistry and Chemical Engineering, Xiamen
University, Xiamen 361005, People’s Republic of China
Octahedral coordination geometries permit the straightforward generation of structures with high shape and stereochemical complexity
and furthermore simplify the design of defined globular and rigid
structures because molecular geometries are basically constructed
from a single center with chelating ligands limiting the degree of
conformational flexibility [1]. This presentation will demonstrate how
this sophisticated octahedral stereochemistry can be exploited for the
design of enzyme inhibitors [2] and chiral-at-metal asymmetric catalysts [3]. Molecular recognition and catalysis by the here disclosed
inert, rigid metal complexes does not involve any direct metal coordination but operates through cooperative weak interactions with
functional groups properly arranged in the ligand sphere of the metal
complexes.
Financial support was provided by the German Research Foundation, by the US National Institutes of Health and the National
Natural Science Foundation of P. R. China. The author would like to
acknowledge the contribution of the COST Action CM1105.
1
PL 2
Utilization of phosphorescent iridium(III)
and rhenium(I) complexes as functional cellular
reagents
Kenneth Kam-Wing Lo
Department of Biology and Chemistry, City University of Hong
Kong, Tat Chee Avenue, Kowloon, Hong Kong, People’s Republic
of China, [email protected]
We believe that the interesting phosphorescence properties of iridium(III) and rhenium(I) complexes can be exploited in biological
sensing, labeling, and imaging. Using a range of biologically relevant
substrates, chemically reactive functional groups, and spacer-arms of
different lipophilicity, we have designed new phosphorescent complexes and applied them as [1] sugar uptake indicators for cancer
cells, [2] organelle-selective photocytotoxic agents [3], bioorthogonal
probes for glycans on cell membrane, and [4] intracellular sensors for
ions and nitric oxide. We have focused on the photophysical properties, cytotoxicity, cellular-uptake behavior, intracellular
localization, and protein targets of these reagents.
+
+
S
CO
OH
OC
N
O
HO
HO
C
HO O
NHC 6H12NH
NH
N
C
N
N
CH3
S
NH
glucose uptake indicator
photocytotoxic agent
+
+
N
HOOC
HOOC
C
O
N
NH
O
CO
3
OC
O
OC
N
N
N
Re
Ir
C
O
n
CH3
Ir
N
S
S
NH
O
N
Re
OC
O
N
N
N
CH3
CH3O
NH
NH2
bioorthogonal probe
References
1. Dörr M, Meggers E (2004) Curr Opin Chem Biol 19:76–81
2. Feng L, Geisselbrecht Y, Blanck S, Wilbuer A, Atilla-Gokcumen
GE, Filippakopoulos P, Kräling K, Celik MA, Harms K, Maksimoska
J, Marmorstein R, Frenking G, Knapp S, Essen L-O, Meggers E
(2011) J Am Chem Soc 133:5976–5986
3. Chen L-A, Xu W, Huang B, Ma J, Wang L, Xi J, Harms K, Gong L,
Meggers E (2013) J Am Chem Soc 135:10598–10601
nitric oxide sensor
References
1. Li SPY, Lau CTS, Louie MW, Lam YW, Cheng SH, Lo KKW
(2013) Biomaterials 34:7519–7532
2. Lo KKW, Chan BTN, Liu HW, Zhang KY, Li SPY, Tang TSM
(2013) Chem Commun 49:4271–4273
3. Li SPY, Tang TSM, Yiu KSM, Lo KKW (2012) Chem Eur J
18:13342–13354
4. Louie MW, Liu HW, Lam MHC, Lam YW, Lo KKW (2011) Chem
Eur J 17:8304–8308
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PL 3
Copper-oxygen intermediates relevant
to metalloenzymes
William B. Tolman
Department of Chemistry, University of Minnesota, 207 Pleasant St.
SE, Minneapolis, MN 55410, USA. [email protected]
Inspired by the unusual active site structures and reactivities exhibited
by copper enzymes, we seek to prepare and characterize synthetic
complexes in order to test hypotheses developed to explain the novel
functions of the biological sites. Mono- and dicopper oxo and/or
hydroxo complexes at high oxidation states are proposed intermediates in oxidation catalysis by copper enzymes and other catalysts, and
thus are key targets for synthesis and characterization. Recently, we
identified several Cu(III)-OH complexes supported by a pyridine(dicarboxamide) ligand, and found that they performed hydrogen
atom abstraction reactions at high rates [1,2]. In this lecture, progress
toward understanding the basis for these high rates will be presented.
In addition, motivated by lessons learned from these studies, new
research has focused on novel oxidized dicopper species supported by
binucleating ligands with pyridine(dicarboxamide) donors. Preliminary data in support of the formulations of these species will be
discussed.
Financial support by the National Institutes of Health and DARPA
is acknowledged.
References
1. Donoghue PJ, Tehranchi J, Cramer CJ, Sarangi R, Solomon EI,
Tolman WB (2011) J Am Chem Soc 133:17602–17605
2. Tehranchi J, Donoghue PJ, Cramer CJ, Tolman WB (2013) Eur J
Inorg Chem 2013:4077–4084
PL 4
Drawing iron pathways in the ferritin nanocage
Paola Turano
CERM & Department of Chemistry, University of Florence, via L.
Sacconi 6, 50019 Sesto Fiorentino, Italy. [email protected]
Ferritins, the main iron storage proteins in eukaryotes and prokaryotes, are formed by 24 4-helical bundle subunits that self-assemble in
a nanocage architecture. The highly symmetric structure, with three
fourfold, four threefold and six twofold axes, makes the system
accessible to NMR studies, despite the high molecular mass
(480 kDa) of the protein [1, 2]. We have developed a combined
solution-solid state NMR approach to monitor the transient interactions between the protein cage and the ferric-products that form from
the catalytic reaction at the ferroxidase site [1]. The role in iron
management of specific amino acids located in different protein
structural domains has been extensively studied by a combination of
X-ray crystallography and solution spectroscopy [3, 4]. New protein
variants have been produced that enabled the redirection of iron
trafficking within the protein cage.
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J Biol Inorg Chem (2014) 19 (Suppl 2):S703–S705
References
1. Turano P, Lalli D, Felli IC, Theil EC, Bertini I (2010) Proc Natl
Acad Sci USA 107:545–550
2. Lalli D, Turano P (2013) Acc Chem Res 46:2676–2685
3. Bertini I, Lalli D, Mangani S, Pozzi C, Rosa C, Theil EC, Turano P
(2012) J Am Chem Soc 134:6169–6176
4. Theil EC, Turano P, Ghini V, Allegrozzi M, Bernacchioni C (2014)
J Biol Inorg Chem (epub ahead of print)
PL 5
Redox tuning in biological electron transfer: sacrificing
efficiency to survive life in O2
A. William Rutherford
Molecular Biosciences, Imperial College, London SW7 2AZ, UK.
[email protected]
The energy-converting redox enzymes perform productive reactions
efficiently despite the involvement of high energy intermediates in
their catalytic cycles. This is achieved by kinetic control: with forward reactions being faster than competing, energy-wasteful
reactions. This requires appropriate cofactor spacing, driving forces
and reorganizational energies. These features evolved in ancestral
enzymes in a low O2 environment. When O2 appeared, energy-converting enzymes had to deal with its troublesome chemistry. Various
protective mechanisms duly evolved that are not directly related to
the enzymes’ principal redox roles. These protective mechanisms
involve fine-tuning of reduction potentials, switching of pathways and
the use of short circuits, back-reactions and side-paths, all of which
compromise efficiency. This energetic loss is worth it since it minimises damage from reactive derivatives of O2 and thus gives the
organism a better chance of survival. Here I will discuss photosynthetic reaction centres from this viewpoint. This allows several
mysteries and anomalies to be explained and provides a new perspective on reaction centre bioenergetics. This ‘‘sacrifice-ofefficiency-for-protection’’ concept should be generally applicable to
bioenergetic enzymes in aerobic environments. It also has repercussions on plans to improve efficiency of photosynthesis and other
biological electron transfer systems and on aspects of artificial
photosynthesis.
Reference
1. Rutherford AW, Osycska A, Rappaport F (2012) FEBS Lett
586:603–616
PL 6
Biomimetic chemistry of [Fe]-hydrogenase—from
synthetic models to modified enzymes
Xile Hu
Institute of Chemical Sciences and Engineering, Ecole Polytechnique
Fédérale de Lausanne (EPFL), EPFL-ISIC-LSCI, BCH 3305,
Lausanne, CH 1015, Switzerland. [email protected]
[Fe]-hydrogenase is a newly characterized hydrogenase. It catalyzes the
reversible reduction of methenyl-H4MPT? with H2 to form methyleneH4MPT and H? (Figure) [1, 2]. Current data suggest a unique and
intriguing active site: a single Fe ion is coordinated to a cysteine sulfur
atom, two cis-CO ligands, and a bidentate pyridone cofactor with pyridinyl nitrogen and acyl carbon donors [2]. We are developing mimics
of the active site of [Fe]-hydrogenase as references to clarify the
structural, spectroscopic, and mechanistic puzzles of the enzyme. In
early 2010, we reported the first model complexes that reproduced the
primary coordination sphere of the proposed active site (M-1) [3]. And
later that year, we developed models that mimicked the acylmethylpyridinyl ligand of the enzyme (M-2) [4]. In 2011, we were able
J Biol Inorg Chem (2014) 19 (Suppl 2):S703–S705
S705
to prepare a model complex with an open coordination site (M-3) [5].
And just this year, we succeeded in obtaining the first model complexes
with an acylmethylpyridinol ligand (M-4) [6]. Our model complexes
have provided credence to the proposed structure of the active site, and
have served as useful spectroscopic and reactivity references [7].
Recently, in collaboration with the Shima group, we have incorporated several model complexes into the apoenzyme of [Fe]hydrogenase to produce artificial enzymes. The modified enzymes not
only have similar electronic property to the native enzyme, but also
exhibit typical hydrogenase activity. The study gives significant
mechanistic insights into the activity of [Fe]-hydrogenase.
Financial support by the Swiss National Science Foundation is
gratefully acknowledged.
References
1. Shima S, Pilak O, Vogt S, Schick M, Stagni MS, Meyer-Klaucke
W, Warkentin E, Thauer RK, Ermler U (2008) Science 321:572–575
2. Shima S, Ermler U (2011) Eur J Inorg Chem 2011:963–972
3. Chen DF, Scopelliti R, Hu, XL (2010) J Am Chem Soc
132:928–929
4. Chen DF, Scopelliti R, Hu XL (2010) Angew Chem Int Ed
49:7512–7515
5. Chen DF, Scopelliti R, Hu XL (2011) Angew Chem Int Ed
50:5671–5673
6. Hu BW, Chen DF, Hu XL (2014) Chem Eur J 20:1677–1682
7. Chen DF, Scopelliti R, Hu XL (2012) Angew Chem Int Ed
51:1919–1921
guanyl nucleotide
pro-R
H
N
O
O
HN
H2N N
Fe
(?)
(Cys)S
H
O
CO
C
CO
N
N
H
N
CH3+ H2
H
CH3
Methenyl-H4MPT+
[Fe]-hydrogenase
O
HN
H2N N
H
O
H
N
N
N
H
+ +
CH3 H
H
CH3
Methylene-H4MPT
C
N
N
CO
O
Fe
L
S
CO
L = CN-, RNC, PPh3, CO
M-1
C
Fe
S
Me
CO
N
CO
M-2
N
O
C
Fe
S
OC
M-3
O
CO
CH3
N
O
C
OH
CO
Fe
S
R OC
M-4
Active site of [Fe]-hydrogenase
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J Biol Inorg Chem (2014) 19 (Suppl 2):S707–S708
DOI 10.1007/s00775-014-1154-1
ORAL PRESENTATION
Keynote Lectures
KL 1
The chemical biology of acylated enterobactin-like
siderophores from vbrio species
Alison Butler1, Hannah K. Zane1, Michelle P. Kem1, Hiroaki
Naka2, Margo G. Haygood2
1
Department of Chemistry and Biochemistry, University
of California, Santa Barbara, CA 93106-9510, USA.
2
Institute of Environmental Health, Oregon Health and Science
University, Portland, Oregon 97239-3098, USA
The bioluminescent marine bacterium Vibrio harveyi produces a
series of acylated enterobactin-like siderophores, called the amphienterobactins, comprised of the cyclic lactone of tris-2,3-dihydroxybenzoyl-L-serine and acyl-L-serine. Although the genome
of Vibrio harveyi reveals a nonribosomal peptide synthetase
(NRPS) gene cluster (aebA-F) resembling that for enterobactin
(entA-F), enterobactin is not produced. A gene encoding a long
chain fatty acid CoA ligase (aebG), which resides nearby the aebAF cluster, is critical for initiating biosynthesis of the amphi-enterobactins (Figure 1). The condensation domain of the AebF NPRS
is shown to catalyze two distinct condensation reactions, i.e.,
acylation of L-Ser and amidation of L-Ser by 2,3-dihydroxybenzoic
acid. The amphi-enterobactins are quite hydrophobic, yet subsequent esterase activity is predicted to release hydrophilic
fragments. We are investigating the structures and importance of
the hydrophilic fragments in the iron uptake process, as well as the
mechanism of their formation.
KL 2
Matching metals to proteins by a metallomic approach
Hongzhe Sun, Ligang Hu, Yuchuan Wang, Yau-Tsz Lai
Department of Chemistry, The University of Hong Kong, Pokfulam
Road, Hong Kong, P. R. China. [email protected]
Metal ions operate, on one hand, as cofactors for around 40 % enzymes,
on the other hand, they also exhibit toxic effects. Some metal ions,
although being not essential, have been widely used in human healthcare
as either therapeutic agents or diagnosis agents. To understand the
molecular mechanism of a metallodrug, it is crucial to match metals to
proteins at a proteome-wide scale [1]. We used an integrated approach
consisting of gel electrophoresis and inductively coupled plasma mass
spectrometry, LA-ICP-MS, IMAC and bioinformatic approach to identify metal-associated proteins using bismuth antiulcer drug as an example
[2,3]. Using continuous-flow gel electrophoresis in combination with
ICP-MS, we developed a comprehensive and robust strategy to readily
identify metal-associated proteins as well as to quantify the metals for
fast metallome/proteome-wide profiling of metal-binding proteins.
To match metals to proteins, we also established a bioinformatic
method which allows potential metal-binding proteins both sequentially and spaciously to be searched [4–6]. Surprisingly, histidine-rich
proteins and motifs (HRMs) are commonly found in proteins. We
systematically analyzed the proteomes of 675 prokaryotes, and show
that HRMs are extensively distributed in prokaryotic proteomes, with
the majority (62 %) of histidine-rich proteins (HRPs) being involved
in metal homeostasis. Importantly, the occurrence of histidine-rich
proteins (motifs) in the proteomes of prokaryotes is related to their
habitats. Our goal is to metal ions and proteins in vivo by an integrative approach for biomedical study.
This work was supported by the RGC of Hong Kong (7046/12P
and 7039/13P), Livzon Pharmaceutical Ltd and the University of
Hong Kong (for an e-SRT on Integrative Biology).
References
1. Li H, Sun H (2012) Curr Opin Chem Biol 16:74–83
2. Tsang CN, Bianga J, Sun H, Szpunar J, Lobinski R (2012) Metallomics 4277–283
3. Hu LG, Cheng TF, He B, Li L, Wang YC, Lai YT, Jiang GB, Sun
H (2013) Angew Chem Int Ed 52:4916–4920
4. Cun S, Lai YT, Chang YY, Sun H (2013) Metallomics 5:904–912
5. Cun S, Sun H (2010) Proc Natl Acad Sci USA 107:4943–4948
6. Cheng T, Xia W, Wang P, Huang F, Wang J, Sun H (2013) Metallomics 5:1423–1429
Financial support by the US NSF CHE1059067 is gratefully
acknowledged.
Figure 1: A, B: Gene Clusters for enterobactin and amphi-enterobactin biosynthesis, respectively; C, D: enterobactin and amphienterobactin-C12:0.
Reference
1. Zane HK, Naka H, Rosconi F, Sandy M, Haygood MG, Butler A
(2014) J Am Chem Soc 134. doi:10.1021/ja5019942
KL 3
Interaction of amyloid-beta with metal ions: structure,
reactivity and biological relevance
Peter Faller1,2, Christelle Hureau1,2, Fabrice Collin3,4, Giovanni
La Penna5
1
CNRS; Laboratoire de Chimie de Coordination, Toulouse, France.
[email protected].
2
Université de Toulouse, UPS, INPT; LCC; Toulouse, France.
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3
Université de Toulouse; UPS; UMR 152 PHARMA-DEV;
Université Toulouse 3, F-31062 Toulouse Cedex 09, France.
4
Institut de Recherche pour le développement (IRD); UMR 152
PHARMA-DEV, F-31062 Toulouse Cedex 09, France.
5
CNR - National Research Council of Italy, ICCOM - Institute
for chemistry of organo-metallic compounds, via Madonna del Piano
10, I-50019 Sesto Fiorentino, Firenze, Italy
According to the amyloid cascade hypothesis, amyloid-b peptides
(Ab) play a causative role in Alzheimer’s disease (AD), of which
oligomeric forms are proposed to be the most neurotoxic by provoking oxidative stress. Copper ions seem to be an important factor as
they are bound to Ab in amyloid plaques, a hallmark of AD. Moreover, in vitro Cu-Ab complexes are able to catalyze the production of
hydrogen peroxide and hydroxyl radicals, and oligomeric Cu-Ab was
reported to be more reactive. The flexibility of the disordered Ab
peptide leads to the formation of a multitude of different states of both
Cu(I) and Cu(II) complexes. This contrast to the classical metalloproteins with a well defined 3D structure (Figure). This raised the
question of the structure–function relationship, in particular in terms
of redox reactions and the connected production of reactive oxygen
species [1–3].
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J Biol Inorg Chem (2014) 19 (Suppl 2):S707–S708
Structured metalloprotein
Metal-binding to IDP
Energy
Energy
minor metalbinding sites
main metalbinding site
metal bound to
one site in structured
protein
We report on our recent advancements in the coordination
chemistry and in the mechanistic understanding of the redox-chemistry Cu-Ab.
References
1. Faller P, Hureau C, La Penna G (2014) Acc Chem Res (in press)
2. Hureau C (2012) Coord Chem Rev 256:2164–2174
3. Cassagnes L-E, Hervé V, Nepveu P, Hureau C, Faller P, Collin F
(2013) Angew Chem Int Ed 52:11110–11113
J Biol Inorg Chem (2014) 19 (Suppl 2):S709–S712
DOI 10.1007/s00775-014-1155-0
ORAL PRESENTATION
Alfred Werner Lectures
AW 1
Vanadium: its role in life and environmental issues
Dieter Rehder
AW 2
Properties of the indole ring in metal complexes
Osamu Yamauchi
Department of Chemistry, University of Hamburg, Martin-LutherKing-Platz 6, 20146 Hamburg, Germany. [email protected]
Vanadium is the second-to-most abundant transition metal in sea
water. Marine macroalgae employ vanadate, built into the active
centre of vanadate-dependent haloperoxidases VHPOs, in the peroxidation of halides (Hal) to Hal+ species. Hal+ can generate
halomethanes that are released into the atmosphere [1] where they are
involved in ozone depletion; see Figure. VHPOs are also present in
some terrestrial fungi, in lichen and in Streptomyces bacteria. The
biocidal (antifouling) and oxidative power of VHPOs have initiated
research into applications, including active centre models, in medicinal (disinfection) and industrial (oxidation catalysis) fields [2]. In the
context of possible medicinal applications of vanadium compounds it
is worth noting that vanadate HVO42- is an antagonist of phosphate:
Vanadate appears to have a cardio- and neuro-protective potential,
and oxidovanadium(IV) and –(V) chelates have been show to exert
in vitro and in vivo anti-cancer, anti-viral and anti-bacterial activity
[3]. The most prominent issue in a medicinal context is the insulinenhancing action of vanadyl chelates such as VO(acac)2 and
VO(maltol)2. Vanadium can enter the cytosol directly as vanadate, or
in the form of VO2+ coordinated to transferrin and serum albumin. Its
possible primary mode of action is the regulation of the tyrosine
phosphorylation level of the insulin receptor (and thus the glucose
signalling path) by interacting with protein tyrosine phosphatase 1B
[2, 3].
Department of Chemistry, School of Science, Nagoya University,
Chikusa-ku, Nagoya 464-8602, Japan.
[email protected]
Indole is an important constituent of natural products. Tryptophan (Trp),
an essential amino acid, has an indole ring and is known to give a cation
radical in cytochrome c peroxidase [1]. Indole exists as tautomers, 1Hand 3H-indoles, and when incorporated into the side chain of
Cu(I) complexes, it can form a Cu(I)-indole g2-bond through the C(2)–
C(3) moiety and influence the Cu(I) complex-O2 reactivity [2]. It also
forms a r-bond with metal ions such as Pd(II) and Pt(II) through the
nitrogen or the carbon atom, and the indole ring bound to Pd(II) through
C(2) gives a cation radical upon one-electron oxidation [3,4]. Recently the
indole ring of Trp has been reported to interact weakly with Cu(I) in a Cu
chaperone CusF [5]. Pd(II) complexes with ligands having a pendent
indole exhibit a unique exchange of donors from the phenolate oxygen or
the chloride ion to the indole carbon and vice versa [6]. Due to the high
electron density the side chain indole ring of metal complexes undergoes
stacking interactions with the coordinated aromatic ligand such as 1,10phenanthroline. Similar interactions of the Trp residue are seen at the
metal sites of proteins, suggesting various functions of the indole ring.
These and some other properties of the indole ring in metal-containing systems will be reviewed and discussed.
Br + NO2
NO
H2SO4
Ox.
(CH3)2SO
(CH3)2S
O2
Br2
Br in HOBr
sea spray
HO2
BrO
h
HOBr, O2
O3
Hg
Br
h
CH2Br2, CHBr3
{CH}
HgBr2
BrCN
[HPO]
HOBr, Br2, Br3
Br
+ H2O2
Br + HCO3
[VBrPO]
References
1. Wever R, van der Horst MA (2013) Dalton Trans 42:11778–11786
2. Rehder D (2013) Dalton Trans 42:11749–11761
3. Rehder D (2013) Met Ions Life Sci 13:139–169
References
1. Sivaraja M, Goodin DB, Smith M, Hoffman BM (1989) Science
245:738–740
2. Shimazaki Y, Nogami T, Tani F, Odani A, Yamauchi O (2001)
Angew Chem Int Ed 40:3859–3862
3. Shimazaki Y, Yajima T, Takani M, Yamauchi O (2009) Coord
Chem Rev 253:479–492
4. Shimazaki Y, Yamauchi O (2012) Chem Biodiv 9:1635–1658
5. Xue Y, Davis AV, Balakrishnan G, Stasser J P, Staehlin BM, Focia
P, Spiro TG, Penner-Hahn JE, O’Halloran TV (2008) Nat Chem Biol
4:107–109
6. Iwatsuki S, Suzuki T, Tanooka S, Yajima T, Shimazaki Y (2014)
Pure Appl Chem 86:151–162
AW 3
‘Fine-tuning’ of biomimetic siderophore analogs
to optimize biological activity
Abraham Shanzer1, Jenny Besserglick1, Evgenia
Olshvang1, Joseph Englander1, Elzbieta GumiennaKontecka2, Henryk Kozłowski2, Yitzhak Hadar3
1
Department of Organic Chemistry, The Weizmann Institute
of Science, Rehovot, Israel.
123
S710
2
Faculty of Chemistry, University of Wroclaw, Wroclaw, Poland.
Department of Plant Pathology and Microbiology, Faculty
of Agriculture Food and Environment, The Hebrew University
of Jerusalem, Rehovot, Israel
A misfit in receptor-substrate recognition is easily identified by
observing alteration in the microbial growth and proliferation, compared to growth characteristics of the natural counterpart. Since the
sensitivity of these interactions is generally very high, minor structural changes in the substrate, induce dramatic effects ranging from
low activity to the lack of it all together.
In this lecture, I’ll present a methodology capable in modifying
some of these minor structural changes to improve biological activity
even in cases where original activity was very low.
The methodology is based on a ‘reductive’ process, at the refining
stage, and includes several changes among them: (i) removal of side
arms (ii) templates are replaced by complementary templates and the
(iii) spacing between the ‘essential’ building-blocks are systematically shortened. The newly obtained ‘reduced’ library is than
synthesized and subjected for microbial screening.
Several examples demonstrating the feasibility of this approach
will be described and discussed.
3
AW 4
On the structural variability of exocyclic amino groups
in free and metalated cytosine nucleobases
Bernhard Lippert1, Célia Fonseca Guerra2, Pablo J. Sanz
Miguel3, Andrea Cebollada3, F. Matthias Bickelhaupt2,4
1
Fakultät Chemie und Chemische Biologie, TU Dortmund, 44221
Dortmund, Germany.
2
Department of Theoretical Chemistry, Vrije Universiteit, 1081 HV
Amsterdam, The Netherlands.
3
Departamento de Quı́mica Inorgánica, Universidad de ZaragozaCSIC, 50009 Zaragoza, Spain.
4
Institute for Molecules and Materials, Radboud University
Nijmegen, 6525 AJ Nijmegen, The Netherlands
There exists a long-standing and seemingly paradoxical situation
regarding the behaviour of the exocyclic amino groups of nucleobases
(cytosine, adenine, guanine) in that on one hand they behave as
nucleophiles in numerous organic reactions, yet that they do not coordinate metal ions through their lone electron pairs unless deprotonated.
In fact, numerous structural studies have revealed that the lone electron
pair at the N atom is delocalized into the heterocyclic ring, and that
consequently the exocyclic N atoms are only very slightly pyramidal.
By applying a combination of synthetic chemistry [1] and DFT calculations [2], an attempt has been made to reconcile these two features. As
a result, it is demonstrated that the hybridization state of the exocyclic
amino group of cytosine nucleobases can switch from (essentially) sp2 to
(largely) sp3 in dependence of its environment (involvement in hydrogen
bond formation) and metalation state. Thus, involvement in H-donor
properties or displacement of a proton by a monofunctionally bonded
metal ion cause a marked pyramidalization of the exocyclic N atom.
References
1. Yin L, Sanz Miguel P J, Shen W-Z, Lippert B (2009) Chem Eur J
15:10723–10726
2. Fonseca Guerra C, Sanz Miguel PJ, Cebollada A, Bickelhaupt FM,
Lippert B (2014) submitted for publication
AW 5
Brain metallothionein-3: structural and functional
insights
Milan Vašák
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J Biol Inorg Chem (2014) 19 (Suppl 2):S709–S712
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland. [email protected]
Zinc and copper homeostasis plays a crucial role in brain physiology
and in neurodegenerative diseases. The homeostasis of both metals is
regulated by a small intra- and extracellular metalloprotein metallothionein-3 (Zn7MT-3). The seven metal ions in Zn7MT-3 are
organized in a Zn4(CysS)11 and a Zn3(CysS)9 cluster. The protein is
mainly expressed in the brain and was found downregulated in Alzheimer’s (AD), prion and Parkinson’s (PD) diseases. Aberrant
interactions of Cu(II) with amyloid-b (Ab) in Alzheimer’s, prion
protein (PrP) in Creutzfeldt-Jakob and a-synuclein (a-Syn) in Parkinson’s diseases potentiate their progression by participating in the
aggregation process and the production of reactive oxygen species
(ROS) [1]. In AD, Cu(II) and Zn(II) are involved in the disease
progression by modulating the formation and toxicity of soluble and
insoluble oligomers and aggregates of the Ab peptide. Whereas the
copper-induced Ab aggregation is related to the ROS production and
neurotoxicity, the zinc-induced Ab aggregation is considered to be
neuroprotective. The protective effect of extracellular Zn7MT-3 from
Ab toxicity in neuronal cell cultures has been demonstrated, but its
origin remained unexplained. By using several complementary
spectroscopic, biochemical, and cell biological techniques we showed
that Zn7MT-3 not only scavenges free Cu(II), but also Cu(II) bound to
Ab. We found that a metal swap between Zn7MT-3 and soluble and
aggregated Ab-Cu(II) is the underlying molecular mechanism by
which the ROS production and related cellular toxicity is abolished
[2]. In this process, Cu(II) is reduced by the protein thiolates forming
Cu(I)4Zn4MT-3, in which an air stable Cu(I)4-thiolate cluster and two
disulfide bonds are present. To examine a potential protective effect
of the protein in other metal-linked neurodegenerative pathologies,
similar studies using a-Syn and the prion protein/peptides in complex
with Cu(II) were conducted. Our demonstration that Zn7MT-3 in a
similar reaction also removes Cu(II) from the binding sites in a-Syn
[3] and PrP [4] signifies a general protective role of Zn7MT-3 from
Cu(II) toxicity in the brain.
References
1 Barnham KJ, Masters CL, Bush AI (2004) Nat Rev Drug Discovery
3:205–214
2 Meloni G, Sonois V, Delaine T, Guilloreau L, Gillet A, Teissié J,
Faller P, Vašák M (2008) Nat Chem Biol 4:366–372
3. Meloni G, Vašák M (2011) Free Rad Biol Med 50:1471–1479
4. Meloni G, Crameri A, Fritz G, Davies P, Brown DR, Kroneck
PMH, Vašák M (2012) ChemBioChem 13:1261–1265
AW 6
Dysfunction of metal homeostasis
and neurodegeneration
Robert R. Crichton, David T. Dexter, Roberta J. Ward
Institute of Life Sciences, University of Louvain, Place Croix du Sud,
1348 Louvain-la-Neuve, Belgium. [email protected]
Life expectancy in Switzerland has increased from 71.3 years in 1960
to 82.7 years in 2010, (typical for most of Europe), yet as we live
longer, the incidence of neurological diseases increases tremendously.
In the last decade, an increasing number of these debilitating diseases,
including Alzheimer’s and Parkinson’s disease have been associated
with local increases in the concentration of transition metal ions in the
specific brain regions associated with disease symptoms [1].
Under normal physiological conditions, metals play essential roles
in brain function––Na+ and K+ in transmission of nervous impulses,
Ca+ in mediating physiological events within neuronal cells including
neurotransmitter exocytosis/secretion, Zn2+, localized in synaptic
vesicles and co-released with glutamate when hippocampal fibres are
stimulated and Fe2/3+ and Cu1/2+ in the synthesis of neurotransmitters,
neurononal myelination and neuroprotection.
J Biol Inorg Chem (2014) 19 (Suppl 2):S709–S712
S711
However dysfunction of homoestasis of these three transition
metals is associated with both neuroinflammation and neurodegeneration, as we will demonstrate for a number of cognitive and motor
neurological conditions.
Current ideas on transition metal homeostasis in brain, and dysfunction of metal homeostasis associated with oxidative stress and
neuroinflammation will be presented [2]. The potential for developing
chatation-based therapeutic approaches evidence in appropriate animal models of neurodegenerative diseases as well as results from
preliminary clinical studies using chelation therapy in both Friedreich’s ataxia and Parkinson’s disease patients will be discussed.
Financial support from the Distinguished Scientists Fellowship
Program (DSFP), King Saud University, is gratefully acknowledged.
References
1. Crichton RR, Ward RJ (2014) Metal-based neurodegeneration:
from molecular mechanisms to therapeutic strategies, 2nd ed. John
Wiley & Sons, Chichester pp. 423
2. Ward RH, Dexter DT, Crichton RR (2012) Curr Med Chem
19:2760–2772
Department of Biology, University of Konstanz, Universitaetsstrasse
10, 78457 Konstanz, Germany. [email protected]
Many essential life processes on Earth, such as photosynthesis, respiration, or nitrogen fixation, depend on transition metals and their ability to
catalyze multi-electron redox and hydrolytic transformations. The focus
of this contribution will be on structural and functional aspects of two
complex multi-site enzymes with unique heme iron centers. These are
involved in energy conserving processes of important branches within
the biogeochemical cycles of nitrogen and sulfur:
(1) six-electron reduction of NO2- to NH3 catalyzed by pentaheme cytochrome c nitrite reductase (ccNiR), and (2) the six-electron
reduction of SO32- to H2S by sulfite reductase (SIR). The figure
shows the active site of Archaeoglobus fulgidus SIR, with sulfite
coordinated to the siroheme iron. The structural and mechanistic
information has been obtained by X-ray crystallography and spectroscopic techniques spectroscopy [1,2].
Financial support by the University of Konstanz and Deutsche
Forschungsgemeinschaft is gratefully acknowledged.
AW 7
Hydrogen bonding in bio-coordination chemistry:
Synergy between metal binding and hydrogen bonding
in nucleic-acid metal coordination compounds
Jan Reedijk1,2
1
Leiden Institute of Chemistry, Leiden University, P.O. Box 9502,
2300 RA Leiden, The Netherlands.
2
Department of Chemistry, College of Science, King Saud University,
P.O. Box 2455 Riyadh 11451, Kingdom of Saudi Arabia
Reference
1. Reedijk, J (2009) Eur J Inorg Chem 2009:1303–1312
AW 8
Multi-electron, multi-proton transfer reactions
performed by unique heme iron centers
Peter M. H. Kroneck
Coordination to metals and hydrogen bonds often operate in concert;
3–5 hydrogen bonds comprise about the same energy as a coordination bond. When Alfred Werner realized that the number of
neighbours to a metal could be larger than the known valence of the
metal, hydrogen bonding was not known to exist. The non-existence
lasted till Linus Pauling first hinted towards hydrogen bonds. Nowadays, with accurate X-ray structure determinations routinely
available, it is clear that hydrogen bonding does play a key role in
building up structures, even of Werner-type metal coordination
compounds, both intramolecularly and intermolecularly.
In this short lecture I will focus on the influence that
(intramolecular) hydrogen bonding may have on the coordination of
ligands and anions to metals. How the molecular and lattice structures are stabilised by a combination of both effects will be
illustrated for some simply cases, and for binding of metal compounds to nucleic acid fragments, like cisplatin binding at G-N7 of
the C:G base pair in nucleic acids (see Figure). The ultimate
applications of the synergism between H bonding and coordination
are found in structure of metalloproteins, metal-DNA binding and in
crystal engineering.
References
1. Parey K, Fritz G, Ermler U, Kroneck PMH (2013) Metallomics
5:302–317
2. Simon J, Kroneck PMH (2013) Adv Microbial Physiol 62:45–117
H 3N
H
H
C
H NH
Pt
N
H
O
X
N
7
3
N
1
HN1
N
Sugar
O
HN H
3
N
G
N
AW 9
Warns against unapproved ‘chelation therapy’
Guido Crisponi1, Valeria M. Nurchi1, Joanna I.
Lachowicz1, Miriam Crespo-Alonso1, M. Antonietta
Zoroddu2, Massimiliano Peana2
1
Sugar
Dipartimento di Scienze Chimiche e Geologiche, University
of Cagliari, Cittadella Universitaria, 09042 Monserrato-Cagliari,
Italy.
123
S712
2
Dipartimento di Chimica e Farmacia, University of Sassari, Via
Vienna 2, 07100, Sassari, Italy
At Eurobic11 in Granada we presented a Keynote Lecture on chelation therapy, a consolidated medical procedure used primarily to
hinder the effects of toxic metal ions on human tissues [1–2]. Its
application spans a broad spectrum of serious disorders, ranging from
acute metal intoxication to genetic metal-overload. The use of chelating agents is compromised by a number of serious side effects,
mainly attributable to perturbed equilibrium of essential metal ion
homeostasis and dislocation of complexed metal ions to dangerous
body sites. For this reason, chelation therapy has been limited to
specific critical and otherwise untreatable conditions and it needs to
be monitored within an appropriate clinical context [3–4].
In this meeting we want warn against the widespread fraudulent
use of the term ‘‘chelation therapy’’ to take advantage of and make
profit from people with tragic health problems. We believe that scientists working in this field have the corollary obligation to deter
these frauds and to inform the scientific community of the possible
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J Biol Inorg Chem (2014) 19 (Suppl 2):S709–S712
side effects and complications of chelation therapy. This duty is all
the more important if we consider the detrimental and even life
threatening consequences that can occur in subjects with no clear
clinical and laboratory evidence of metal intoxication. The aim of this
communication is to present how this ‘‘false chelation therapy’’
developed and in which diseases it is currently applied [5].
This research was supported by the Regione Autonoma Sardegna
[CRP-27564].
References
1. Crisponi G., Nurchi VM (2012) Eurobic 11 Granada
2. Crisponi G, Nurchi VM, Crespo-Alonso M, Toso L (2012) Curr
Med Chem 19:2794–2815
3. Crisponi G, Nurchi VM, Fanni D, Gerosa C, Nemolato S, Faa G
(2010) Coord Chem Rev 254:876–889
4. Crisponi G, Remelli M (2008) Coord Chem Rev 252:1225–1240
5. Crisponi G, Nurchi VM, Lachowicz IJ, Crespo-Alonso M, Zoroddu
MA, Peana, M (2014) Coord Chem Rev (accepted for publication)
J Biol Inorg Chem (2014) 19 (Suppl 2):S713–S724
DOI 10.1007/s00775-014-1156-z
ORAL PRESENTATION
Session lectures
SL 1
Recent advances in bioinorganic anticancer drug
development
SL 2
Light activation of anticancer metallodrugs with blue,
yellow, and red photons
Bernhard K. Keppler1,2
1
Institute of Inorganic Chemistry, University of Vienna, Währinger
Straße 42, 1090 Vienna, Austria. [email protected]
2
Research Platform ‘‘Translational Cancer Therapy Research’’,
University of Vienna, Währinger Straße 42, 1090 Vienna, Austria
Current anticancer drug development aims at finding new therapies
with increased clinical efficacy and tolerability by improved targeting
of the specificities of malignant tumours. Promising compounds have
been identified in various classes of metal complexes which are now
in different stages of preclinical or clinical development.
Most notably, the ruthenium complex NKP-1339 has recently
been evaluated in a clinical phase I/IIa study led by D. Von Hoff
(Virginia G. Piper Cancer Center, Scottsdale, AZ, USA) and H. Burris
(Sarah Cannon Research Institute, Nashville, TN, USA). Remarkable
activity in patients with gastro-intestinal neuroendocrine tumours and
disease stabilizations in various other solid tumours have been
reported [1]. The mild clinical side effects and preclinical synergies
with other anticancer drugs make NKP-1339 an attractive candidate
for combination therapies [2]. E.g., the combination with the multikinase inhibitor sorafenib synergizes in vitro and in vivo, as reflected
in mutually enhanced cellular accumulation and attenuated cellular
stress response to NKP-1339, and a substantially prolonged survival
compared to single-drug treatment in a Hep3B xenograft model [3].
New developments in the platinum arena will be exemplified by
methyl-substituted oxaliplatin derivatives with improved preclinical
efficacy and therapeutic window, lower systemic toxicity and
neurotoxicity in particular, as well as reduced dependence on
immunogenic cell death induction as compared to the parent drug
[4,5]. Together with examples of gallium and lanthanum compounds,
these advances emphasize the vital role that metallopharmaceuticals
continue to have in anticancer drug development.
Sylvestre Bonnet, Sven H. C. Askes, Azadeh Bahreman, JordiAmat Cuello-Garibo
Leiden University, Leiden Institute of Chemistry, Einsteinweg 55,
2300RA Leiden, The Netherlands. [email protected]
Ruthenium-based polypyridyl compounds have recently been proposed as light-activated anticancer prodrugs. Upon blue light
irradiation, they typically photosubstitute a ligand from their
coordination sphere [1], which unleashes their ability to bind to
biomolecules such as DNA or proteins and provoke cell death.
Two strategies will be presented that allow for shifting such bluelight photoreactivity towards the photodynamic window
(600–900 nm), where light penetrates better human tissues. First,
an organic dye can be covalently attached to the complex to
increase the light absorption properties of the metallodrug at higher
wavelengths. By doing so, the photosubstitution quantum yield is
not necessarily diminished, leading to fast photosubstitution reactions with, for example, yellow light [2]. The second strategy
consists in using upconverting liposomes, which are able to
transform in situ the red photons of a commercial PDT laser
(630 nm) into blue light. These blue photons are then absorbed by
the ruthenium complex, thus obtaining efficient activation and
release of the metal prodrug from a PEGylated liposome support
using red light irradiation [3].
Financial support of this research by NWO-CW and ERC is
gratefully acknowledged.
References
1. Thompson DS, Weiss GJ, Fields Jones S, Burris HA, Ramanathan
RK, Infante JR, Bendell JC, Ogden A, Von Hoff DD (2012) J Clin
Oncol 30, Suppl: abstract 3033
2. Baerga R, Cobb J, Ogden A, Sheshbaradaran H (2011) Mol Cancer
Ther 10, Suppl 1: abstract B223
3. Heffeter P, Atil B, Kryeziu K, Groza D, Koellensperger G, Körner
W, Jungwirth U, Mohr T, Keppler BK, Berger W (2013) Eur J Cancer
49:3366–3375
4. Abramkin SA, Jungwirth U, Valiahdi SM, Dvorak C, Habala L,
Meelich K, Berger W, Jakupec MA, Hartinger CG, Nazarov AA,
Galanski M, Keppler BK (2010) J Med Chem 53:7356–7364
5. Jungwirth U, Xanthos DN, Gojo J, Bytzek AK, Körner W, Heffeter
P, Abramkin SA, Jakupec MA, Hartinger CG, Windberger U, Galanski M, Keppler BK, Berger W (2012) Mol Pharmacol 81:719–728
References
1. Goldbach, RE; Rodriguez-Garcia, I; Lenthe, JHV; Siegler, MA;
Bonnet, S (2011) Chem Eur J 17:9924
2. Bahreman, A; Cuello-Garibo, J-A; Bonnet, S (2014) Dalton Trans
43:4494
3. Askes, SHC; Bahreman, A; Bonnet, S (2014) Angew Chem Int Ed
53:1029
123
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SL 3
Determining and adjusting the set-point for metal
homeostasis: structure–function of transcriptional
regulator InrS
Nigel Robinson
Department of Chemistry and School of Biological and Biomedical
Sciences, Durham University, Durham, DH1 3LE, UK
As multiple metalloregulators from different families are characterized in a single organism it becomes possible to identify aspects of
metal sensing that are a function of their combined actions. Furthermore, as multiple metal sensors from a single family are
characterized in different organisms, it becomes possible to confirm
(or refute) correlations between structure and function that were
inferred from early work. Recently we have adopted both approaches
to explore the factors that determine the metal-selectivity of metalloregulators such as Ni(II)-responsive InrS (Foster et al., 2014,
Molecular Microbiology 92, 797–812). InrS KZn(II) (5.6 9 10-13 M)
is comparable to the sensory sites of zinc sensors ZiaR (and Zur), but
the coupling free energy connecting zinc binding to altered DNA
is less for InrS than DGZn(II)-ZiaRDNA
for
binding, DGZn(II)-InrSDNA
C
C
ZiaR. These data imply that relative to other sensors, DGZn(II)-SenC
sorDNA
rather than KZn(II) determines the final detection threshold for
Zn(II).
The discernment of Zn(II) between InrS and ZiaR in Zn(II)adapted cells (at 48 h) is also consistent with an associative regime in
which a pool of readily exchangeable Zn(II), bound to an excess of
relatively weak ligands (amino acids, organic acids, lipids, polypeptides and adventitious ligands on the surfaces of macromolecules
including proteins) constituting a polydisperse buffer, partitions to
and from metal sensors and indeed other Zn(II) proteins via nonspecific, ligand-exchange reactions.
Finally, experiments will be described in which the cellular set
point for nickel homeostasis has been adjusted by changing the nickel
affinity of InrS.
SL 4
Illuminating biological trace metals with high- and lowenergy photons
Christoph J. Fahrni1, M. Thomas Morgan1, Pritha Bagchi1, Daisy
Bourassa1, Irina Issaeva1, Adam McCallum1, Sophie-Charlotte
Gleber2, Stefan Vogt2
1
School of Chemistry and Biochemistry, Petit Institute
for Bioengineering and Bioscience, Georgia Institute of Technology.
901 Atlantic Drive, Atlanta, GA 30332, USA.
[email protected]
2
Advanced Photon Source, X-ray Science Division, Argonne National
Laboratory, 9700 S. Cass Avenue, Argonne, IL 60439, USA.
The identification and quantification of transition metals, ideally in
the context of their native physiological environment [1], is of critical
importance for a comprehensive understanding of metal homeostasis
in cells, tissues, and whole organisms. To this end, we designed and
systematically optimized a new water-soluble Cu(I)-selective fluorescent probe that offers a 180-fold fluorescence contrast with a limit
of detection in the parts-per-trillion concentration range. Furthermore,
we developed water-soluble affinity standards for the reliable determination of the Cu(I) stability constants of proteins and small
molecule ligands [2]. As a complementary approach we employed
synchrotron X-ray fluorescence (SXRF) microscopy and microtomography to visualize the redistribution of copper, zinc, and iron in
proliferating cells and in developing zebrafish embryos. These studies
revealed a sharp increase in mitotic zinc compared to interphase cells
and an intriguing redistribution dynamics during mitosis [3]. While in
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J Biol Inorg Chem (2014) 19 (Suppl 2):S713–S724
the zebrafish embryo (24 hpf) more than 80 % of the total zinc is
localized in maternally derived yolk stores, we also observed a distinct accumulation at the posterior end, which coincides with areas of
progenitor cell differentiation and cellular proliferation. Together,
these data imply a new physiological role for zinc in development that
differs from its established function as a cofactor in metalloproteins or
as a messenger in signaling pathways.
Financial support by NIH (GM067169), NSF (CHE-1306943), and
DOE (DE-AC02-06CH11357) is gratefully acknowledged.
References
1. McRae R, Bagchi P, Sumalekshmy S, Fahrni CJ (2009) Chem Rev
109:4780–4827
2. Bagchi P, Morgan MT, Fahrni CJ (2013) J Am Chem Soc
135:18549–18559
3. McRae R, Lai B, Fahrni CJ (2013) Metallomics 5:52–61
SL 5
High-valent metal-oxo and imido cores in chemistry
and biology
Kallol Ray
Humboldt-Universität zu Berlin, Brook-Taylor Strasse 2, Berlin,
Germany. [email protected]
Although terminal CoIV–O, NiIII–O and CuIII–O intermediates have
been implicated as active intermediates in a number of important
chemical transformations, no spectroscopic evidences for the species
are available, leaving the pathway uncertain [1,2]. Evidences of the
presence of terminal M–O units [M = Cu(III), Ni(III) or Co(IV)] are
to date limited to mass spectrometric studies in the gas phase [2].
Theory suggests that they should be powerful oxidants [2], perhaps
even more reactive than the related [FeIV=O]2+ units that have been
extensively studied [3]. In this presentation, we will summarize some
of our recent efforts to stabilize the elusive metal-oxo and isoelectronic metal-imido units of Cu(III), Co(IV) [4] and Ni(III) [5]) in
solution phase at low temperatures. The high-valent metal-oxo or
metal-imido assignments are made on the basis of a variety of
spectroscopic methods. The reactivity of the intermediates in hydrogen atom abstraction and oxo transfer reactions are also discussed.
References
1. a) Nam W, Kim I, Kim Y, Kim C (2001) Chem Commun 14:1262;
b) Chang CJ, 2. Loh Z-H, Shi HC, Anson FC, Nocera DG (2004) J
Am Chem Soc 126:10013
2. Schröder D, Schwarz H (1995) Angew Chem Int Ed 34:1973
3. Hohenberger J, Ray K, Meyer K (2012) Nat Commun 3:720
4. Pfaff FF, Kundu S, Risch M, Heims F, Ray IP, Haack P, Metzinger
R, Bill E, Dau H, Comba P, Ray K (2011) Angew Chem Int Ed
50:1711
5. Pfaff FF, Heims F, Kundu S, Mebs S, Ray K (2012) Chem Commun 48:3730
SL 6
Iron-bispidine-catalyzed oxidation reactions-ligand
control of structure, electronics and reactivity
Peter Comba
Universität Heidelberg, Anorganisch-Chemisches Institut, Im
Neuenheimer Feld 270, D-69120 Heidelberg, Germany.
[email protected]
Bispidine ligands are extremely rigid, easy to synthesize and available
in a large variety. They enforce coordination geometries derived from
cis-octahedral, and the two vacant coordination sites with the
J Biol Inorg Chem (2014) 19 (Suppl 2):S713–S724
tetradentate ligand systems are sterically and electronically distinct.
Substrates coordinated trans to N3 have strong and short bonds, those
trans to N7 are more labile. Implications with respect to the mechanism of formation and the structure, spin state, redox properties and
reactivity of high-valent iron oxidants are analyzed based on experimental and computational studies. Possibilities to tune the spin state,
structure and reactivity of high-valent iron complexes, specifically
with bispidine systems, are discussed. Novel reactions of these systems will also be presented.
S715
4. Zhao Y, Farrer NJ, Li H, Butler JS, McQuitty RJ, Habtemariam A,
Wang F, Sadler PJ (2013) Angew Chem Int Ed 52:13633–13637
5. Fu Y, Habtemariam A, Pizarro AM, van Rijt SH, Healey DJ, Cooper
PA, Shnyder SD, Clarkson GJ, Sadler PJ (2010) J Med Chem
53:8192–8196; Shnyder SD, Fu Y, Habtemariam A, van Rijt SH, Cooper
PA, Loadman PM, Sadler PJ (2011) MedChemComm 2:666–668
6. Romero-Canelón I, Sadler PJ (2013) Inorg Chem 52:12276–12291
7. Betanzos-Lara S, Liu Z, Pizarro AM, Qamar B, Habtemariam A,
Sadler PJ (2012) Angew Chem Int Ed 51:3897–3900; Z. Liu, et al.
(2014) Angew Chem Int Ed 53:3941–3946; Liu Z, Deeth RJ, Butler
JS, Habtemariam A, Newton ME, Sadler PJ (2013) Angew Chem Int
Ed 52:4194–4197
8. Liu Z, Sadler PJ (2014) Acc Chem Res 47:1174–1185
9 Noffke AL, Habtemariam A, Pizarro AM, Sadler PJ (2012) Chem
Commun 48:5219–5246
SL 8
De novo metalloprotein and metalloenzyme design
Figure 1. Bispidine Ligand Structure and Geometry of Iron Bispidine
Complexes
SL 7
Precious metal anticancer complexes with new
mechanisms of action
Peter J. Sadler
Department of Chemistry, University of Warwick, Coventry CV4
7AL, UK. [email protected]
Inorganic compounds, and metal complexes in particular, offer much
potential for greatly widening the scope of structural and electronic
diversity in drug design [1]. I will describe our recent research on
complexes of low-spin d6 precious metal ions RuII, OsII, IrIII and PtIV.
complexes
such
as
trans,trans-[Pt(pyriDiazido
PtIV
dine)2(N3)2(OH)2] are stable in the dark, but have potent antiproliferative
activity towards cancer cells when irradiated with low doses of light [2].
They produce reactive PtII species which form unusual cross-links on
DNA, and also azidyl radicals and singlet oxygen [3, 4].
Organometallic phenylazopyridine OsII arene complexes are
highly potent against a wide range of cancer cells and active in vivo
[5]. They can target mitochondria and perturb the redox balance in
cancer cells, giving selectivity over normal cells [6]. IrIII cyclopentadienyl complexes can also have potent antiproliferative redox
activity and can catalyse the transfer of hydride from coenzyme
NADH to give e.g. H2 from H+, semiquinones from quinones, or
H2O2 from O2 [7]. These complexes can lower the NADH/NAD+
ratio in cells, raising the possibility of catalytic anticancer drug
activity [8, 9].
Acknowledgements. I thank the ERC, EPSRC, BBSRC and Science City (ERDF/AWM) for funding, all my co-workers and
collaborators, and COST Action CM1105 for stimulating discussions.
References
1. Barry NPE, Sadler PJ (2013) Chem Commun 49:5106–5131
2. Farrer NJ, Woods JA, Salassa L, Zhao Y, Robinson KS, Clarkson
G, Mackay FS, Sadler PJ (2010) Angew Chem Int Ed 49:8905–8908
3. Butler JS, Woods JA, Farrer NJ, Newton ME, Sadler PJ (2012) J
Am Chem Soc 134:16508–16511
Vincent L. Pecoraro1, Cathy Mocny1, Jefferson Plegaria1, Alison
Tebo1, Fangting Yu1, Anniruddha Deb2, James E. Penner-Hahn1,2
1
Department of Chemistry, University of Michigan, Ann Arbor, MI,
USA. [email protected] 2Biophysics Research Division, University
of Michigan, Ann Arbor, MI, USA
de Novo protein design has been investigated for over three decades,
but only in the past several years have functional de Novo designed
metalloenzymes been realized [1–5]. In this presentation, we will
describe examples of such chemistry using the newest constructs for
preparing asymmetric site and redox active sites. Systems that could
be presented are Rubredoxins and Cupredoxins using either three
stranded coiled coils or 3-stranded bundles.
References
1. Yu F, Cangelosi VM, Zastrow ML, Tegoni M, Plegaria JS, Tebo
AG, Mocny CS, Ruckthong L, Qayyum H, Pecoraro VL (2014) Chem
Rev 114:3495–3578
2. Tegoni M, Yu F, Berseloni M, Pecoraro VL (2012) Proc Nat Acad
Sci USA 109:21234–21239
3. Zastrow ML, Peacock AFA, Stuckey JA, Pecoraro VL (2012)
Nature Chem 4:118–123
4. Yu F, Penner-Hahn JE, Pecoraro VL (2013) J Am Chem Soc
135:18096
5. Zastrow M, Pecoraro VL (2013) J Am Chem Soc 135:5895–5903
SL 9
Siderophore iron uptake pathways in gram-negative
bacteria
Isabelle Schalk
University of Strasbourg-CNRS, ESBS, 300 Boulevard Sébastien
Brant, 67 400 Illkirch, France
Siderophores, are organic chelators produced by bacteria in order to
get access to iron. Pseudomonas aeruginosa, a human opportunist
pathogen, produces two major siderophores pyoverdine (PVD) and
pyochelin (PCH), two molecules with very different chemical structures. These chelators are released in the environment of the bacteria,
where they chelate iron with a very high affinity. Bacteria capture
back the ferric forms of PVD and PCH from their environment and
the uptake across the bacterial membranes involves highly siderophore specific transport proteins and molecular mechanisms. New
insights in these molecular mechanisms involved in the ferri-siderophore capture and transport will be presented. Besides, we used
chromosomal replacement to generate P. aeruginosa strains
123
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J Biol Inorg Chem (2014) 19 (Suppl 2):S713–S724
producing fluorescent fusions with proteins involved in PVD and
PCH iron uptake pathways. These strains were used to investigate the
expression of PVD and PCH pathways in planktonic and biofilm
growth conditions, in order to understand why bacteria need and use
several siderophore iron uptake pathways.
SL 10
Syntheses of DNA duplexes containing metal ion
mediated base pairs
Akira Ono1, Itaru Okamoto1, Hideo Urata2, Hidetake Torigoe3,
Hisao Saneyoshi1, Jiro Kondo4, Yoshiyuki Tanaka5
1
Department of Material & Life Chemistry, Faculty of Engineering,
Kanagawa University, 3-27-1 Rokkakubashi, Kanagawa-ku,
Yokohama, Kanagawa-ken 221-8686 Japan 2Osaka University
of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka
569-1094 Japan
3
Department of Applied Chemistry, Faculty of Science, Tokyo
University of Science, 1-3 Kagurazaka, Shinjuku-ku, Tokyo
162-8601, Japan
4
Department of Materials and Life Sciences, Faculty of Science
and Technology, Sophia University, 7-1 Kioi-cho, Chiyoda-ku,
102-8554 Tokyo, Japan
5
Laboratory of Molecular Transformation, Graduate School
of Pharmaceutical Sciences, Tohoku University, 6-3 Aza-Aoba,
Aramaki, Aoba-ku, Sendai, Miyagi 980-8578, Japan
Pyrimidine base pairs in DNA duplexes selectively capture metal ions
to form metal ion-mediated base pairs, which can be evaluated by
thermal denaturation, isothermal titration calorimetry, nuclear magnetic resonance spectroscopy, and crystallography [1, 2]. In this
paper, we discuss the metal ion binding of pyrimidine bases (thymine,
cytosine, 4-thiothymine, 2-thiothymine, 5-fluorouracil) in DNA
duplexes. Thymine–thymine (T–T) and cytosine–cytosine (C–C) base
pairs selectively capture Hg(II) and Ag(I) ions, respectively, and the
metallo-base pairs, T–Hg(II)–T and C–Ag(I)–C, are formed in DNA
duplexes. The metal ion binding properties of the pyrimidine–
pyrimidine pairs can be changed by small chemical modifications.
The binding selectivity of a metal ion to a 5-fluorouracil–5-fluorouracil pair in a DNA duplex can be switched by changing the pH of
the solution. Two silver ions bind to each thiopyrimidine–thiopyrimidine pair in the duplexes, and the duplexes are largely stabilized.
Oligonucleotides containing these bases are commercially available
and can readily be applied in many scientific fields.
H
O
N H
N
N
N
N
O
O
O
O
N
C 1'
N
O
O
N
N
N
C 1'
C 1'
C 1'
F
O
5fU-Hg(II)-5fU
F
O
O
N
N
N
C 1'
C 1'
C 1'
O
O
N
N
C 1'
S
S
F
N
S
S
N
N
N
C 1'
5fU-Ag(I):Ag(I)-5fU
C 1'
C 1'
O
4sT-Hg(II)-4sT
O
N
C 1'
N
S
S
N
C1'
2sT-Ag(I):Ag(I)-2sT
N
O
O
N
2sT-Hg(II)-2sT
N
O
O
N
N
C-Ag(I)-C
N
O
H N
N
T-Hg(II)-T
F
H
O
S
S
N
N
O
O
N
C1'
C 1'
N
C 1'
4sT-Ag(I):Ag(I)-4sT
Metal ion mediated base pairs
This work was supported by a Grant-in-Aid for Scientific Research
(A) (No. 24245037).
References
1. Ono A, Torigoe H, Tanaka Y, Okamoto I (2011) Chem Soc Rev
40:5855–5866
123
2. Kondo J, et al. (2014) Angew Chem Int Ed 53:2385–2388
SL 11
Modular building of high capacity copper-thioneins
in a pathogenic fungus: cryptococcus neoformans MTs
Òscar Palacios1, Selene Gil1, Anna Espart2, Dennis J. Thiele3,
Sı́lvia Atrian2, Mercè Capdevila1
1
Departament de Quimica, Universitat Autònoma de Barcelona,
08193 Cerdanyola del Valles, Barcelona, Spain.
[email protected]
2
Departament de Genetica, Universitat de Barcelona, 08028
Barcelona, Spain
3
Department of Pharmacology and Cancer Biology, Duke University,
Durham, NC 27710, USA
Pathogenic microorganisms often rely on host Cu for growth because
it is an essential metal. However, host cells also hyperaccumulate Cu
to exert antimicrobial effects, e.g. the macrophages of the mammalian
immune system, because this metal is toxic at high concentrations.
The human fungal pathogen C. neoformans causes respiratory
infections that may end up in lethal meningitis if disseminated to the
brain. In lung alveoli, C. neoformans cells sense high copper concentrations, which result in the induction of many Cu-responsive
genes, among which those encoding two metallothioneins (CnMT1
and CnMT2) clearly stand out. CnMT1 and CnMT2 are not only
essential for the fungus survival but also for its virulence and pathogenicity, by performing a Cu-detoxification role [1]. The analysis of
the Cu+ binding properties of these recombinantly expressed and
purified MTs has shown that they are extraordinary in their overall
length and presence of repeated Cu coordinating domains, which
confer them a large capacity for Cu+ binding in relation to other
fungal MTs. The study of the recombinant Cu-CnMT1 and CuCnMT2 complexes, as well as those in vitro formed by Zn2+/Cu+
displacement on the Zn-CnMTs species, allowed us to observe the
formation of unusual Cu5-building blocks in both isoforms. In the
peptide, each building module encompasses seven Cys and some
variable intercalating residues. This ‘‘Cys-box’’ appears triplicated in
CnMT1 and five times in CnMT2. An also conserved ‘‘spacer region’’
separates adjacent Cys-boxes [2]. It is feasible that under selection
pressure in pathogenic fungi, MT evolved by tandem amplification of
a basic Cu-binding block, which surprisingly appears very similar to
the well-known N. crassa or A. bisporus Cu-thioneins. To lay
experimental grounds on this model, we constructed and recombinantly-expressed a series of CnMT1-derived peptides consisting of a
different number of repetitions of the basic Cys-box, with and without
flanking spacers, N- or C-terminally located. Our results fully corroborate the aforementioned hypothesis and yield additional
information on the coordination features of these unusual Cu5clusters.
Financial support by the Spanish Ministerio de Economı́a y
Competitividad, grants BIO2012-39682-C02-01 (to SA) and 02 (to
MC) is gratefully acknowledged.
References
1. Ding C, Festa, RA, Chen YL, Espart A, Palacios O, Espin J,
Capdevila M, Atrian S, Heitman J, Thiele D (2013) Cell Host &
Microbe 13:265–276
2. Palacios O, Espart A, Espı́n J, Ding C, Thiele DJ, Atrian S, Capdevila M (2014) Metallomics 6:279–291
J Biol Inorg Chem (2014) 19 (Suppl 2):S713–S724
S717
SL 12
Paradigm shift in heme degradation; heme oxygenase
and IsdG
Shusuke Nambu1, Toshitaka Matsui1, Celia W. Goulding2,
Kouhei Tsumoto3, Satoshi Takahasi1, Masao Ikeda-Saito1
1
Institute of Multidisciplinary Research for Advanced Materials,
Tohoku University, Katahira, Aoba, Sendai 980-8577, Japan.
[email protected]
2
Departments of Molecular Biology & Biochemistry
and Pharmaceutical Sciences, University of California, Irvine,
California 96297, United States
3
Department of Bioengineering, School of Engineering, and Institute
of Medical Science, University of Tokyo, Tokyo 108-8639, Japan
Heme oxygenase (HO), the central enzyme in iron recycling, bacterial
iron acquisition, and heme degradation, catalyses conversion of heme
to biliverdin, free iron, and CO by three successive mono-oxygenation
reactions with a-meso-hydroxyheme and verdoheme intermediates
[1]. The recent discovery of the structurally distinct bacterial IsdGlike heme degrading proteins of S. aureus and M. tuberculosis has
expanded the reaction manifold of heme degradation. The S. aureus
IsdG reaction products are novel chromophores termed staphylobilins,
where the meso-carbon is released as formaldehyde [2]. The structurally related MhuD cleaves heme to a product mycobilin that retains
the meso-carbon as an aldehyde group [3]. A highly ruffled heme
group, a unique feature of the IsdG-like proteins, appears to be
responsible for the novel heme degradation reaction.
Supported by JSPS, MEXT Japan, and NIH.
CO, Fe2+
O O
HN
NH
HN
N
cylinder arrays [1–3] will be discussed together with work on new
agents that recognise tetraplex DNAs found in gene promoter regions.
We have shown that our cylinders bind strongly and preferentially
to DNA fork structures and prevent DNA transactions in vivo, that
they are taken up readily into cells and rapidly localise in cell nuclei,
where they interfere with the processing of DNA leading to cell cycle
arrest followed by apoptosis, without inducing genotoxicity or
mutagenicity.
The key challenge of how to build from in vitro biophysical
observations to demonstrate what chemistry is happening in the cell,
where and how quickly it is occurring and how that induces the
biological response will be addressed.
References
1. Phongtongpasuk S, Paulus S, Schnabl J, Sigel RKO, Spingler B,
Hannon MJ, Freisinger E (2013) Angew Chem Int Ed
52:11513–11516
2. Ducani C, Leczkowska A, Hodges NJ, Hannon MJ (2010) Angew
Chem Int Ed 49:8942–8945
3. Boer D, Kerckhoffs J, Parajo Y, Pascu M, Usón I, Lincoln P,
Hannon MJ, Coll M (2010) Angew Chem Int Ed 49:2336–2339
CHOO
HN
Fe2+
NH
HN
O
O O
HN
N
CH2O +Fe2+
NH
HN
NH
O
References
1. Matsui T, Unno M, Ikeda-Saito M (2010) Acc Chem Res
43:240–247
2. Matsui T, Nambu S, Ono Y, Goulding CW, Tsumoto K, IkedaSaito M (2013) Biochemistry 52:3025–3027
3. Nambu S, Matsui T, Goulding C W, Takahashi S, Ikeda-Saito M
(2013) J Biol Chem 288:10101–10109
SL 13
Non-covalent recognition of ‘unusual’ but active DNA
and RNA structures
Michael J. Hannon
PSIBS Biomedical Imaging Centre and School of Chemistry,
University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.
[email protected]
DNA occupies its familiar duplex form when it is ‘asleep’ and not
being processed. When it is in action its structures are very different
indeed.
The recognition of DNA replication forks and other Y-shaped
DNA and RNA junctions using nanosized metallo-supramolecular
SL 14
Nanoparticles coated with luminescent metal complexes
for imaging in cells
Zoe Pikramenou1, A. Davies2, D. J. Lewis1, S. Claire2, N.
J. Rogers1, R. M. Harris3, S. Farabi1, I. B. Styles4, S. P. Watson5,
S. G. Thomas5, N. J. Hodges3
1
School of Chemistry,
2
PSIBS Doctoral Training Centre,
3
School of Biosciences,
4
School of Computer Sciences,
5
Institute for Biomedical Research, University of Birmingham,
Edgbaston, Birmingham B15 2TT, UK. [email protected]
Nanoscale probes are ideal for monitoring cellular events based on the
spatial resolution offered by imaging techniques. We have used
labeling approaches to prepare gold nanoparticles coated with luminescent probes, so that the nanoprobes bear the distinct optical
signature of the luminescent agent, independent of the particle
properties. Such designed EuL-pHLIP-Au probes offer multimodal
detection taking advantage of gold’s high electron density, and also
lack the blinking effect observed in quantum dots. We demonstrated
13 nm AuNP coated with neutral (uncharged) lanthanide lumophores
and a pH sensitive peptide, pHLIP, were taken up by platelets within
10 min in a pH selective manner [1, 2]. Selective cell uptake was
demonstrated with different imaging modalities. We have also prepared ruthenium- [3] and iridium-coated [4] nanoparticles using a
surfactant to increase the metal complex coating. The 100 nm gold
nanoparticles have shown uptake in cancer cell lines and can be
123
S718
visualized as single particles with conventional confocal luminescence microscopy. Their localisation indicating interaction with
nuclear chromatin was visualized by both confocal and electron
microscopy techniques.
References
1. Davies A, Lewis, DJ, Watson SP, Thomas SG, Pikramenou Z
(2012) Proc Natl Acad Sci USA 109:1862
2. Lewis DJ, Bruce C, Bohic S, Hammond SP, Arbon D, Pikramenou
Z, Kysela B (2010) Nanomedicine 5:1547
3. Rogers NJ, Claire S, Harris RM, Farabi S, Zikeli G, Styles IB,
Hodges NJ, Pikramenou Z (2014) Chem. Commun 50:617
4. Lewis DJ, Dore V, Rogers NJ, Mole TK, Nash GB, Angeli P,
Pikramenou Z (2013) Langmuir 29:14701
SL 15
Role of poly-his-tags in synthetic and natural proteins
in metal ion binding
Henryk Kozlowski1, Joanna Watly1, Eyal Simonovsky2,3, Yifat
Miller2,3, Robert Wieczorek1, Nuno Barbosa1
1
Faculty of Chemistry, University of Wroclaw, F. Joliot-Curie 14,
50383 Wroclaw, Poland. [email protected]
2
Department of Chemistry, Ben-Gurion University of the Negev,
Beer-Sheva 84105, Israel.
3
Ilse Katz Institute for Nanoscale Science and Technology, BenGurion University of the Negev, Beer-Sheva 84105, Israel
His-Tags are specific sequences containing six histydyl residues and
they are used for purification of recombinant proteins by use of IMAC
chromatography [0]. Such polyhistydyl Tags, often used in molecular
biology, can be also found in nature [0]. More than 650 proteins
containing of His-Tag motifs have been found in archaeal and bacterial
organisms. Proteins containing histidine-rich domains play a crucial
role in metal regulation and homeostasis [3]. Also other functions of
histidine-rich proteins in eukaryotes have been reported [4].
Binding mode and the thermodynamic properties of the system
depends on the specific metal ion and the histidine-rich sequence.
Despite the wide application of the His-Tag for purification of proteins,
little is known about the properties of metal binding to such domains.
The experimental studies have shown that the Cu2+ ion binds most
likely to two imidazoles and one/two or three amide nitrogen
depending on the pH. The MD simulations and DFT calculations have
shown that Cu2+-HHHHHH-NH2 demonstrates polymorphic states
with different sets of two bound imidazoles. This could suggest that
His-Tag motifs in proteins may serve as the dynamic site able to move
metal ions along the ‘‘tag’’ sequence. The Cu2+ binding by AcHHHHHH-NH2 is much more efficient compared to other histidinerich protein domains with separated histydyl residues [5].
The comparison of the potentiometric measurements for Cu2+-AcHHHHHH-NH2 and Cu2+-Ac-EDDHHHHHHHHHG-NH2 (sequence
occurs in nature-from venom glands of the snake) show that at
physiological pH, Cu2+ complexes are thermodynamically very stable
123
J Biol Inorg Chem (2014) 19 (Suppl 2):S713–S724
and contrary to IMAC, in which the most effective in metal ion
binding is His6 sequence, when the metal ion is coordinated to chelator immobilized in the column the sequence with 9 residues is
distinctly more stable than that of 6 His [6].
References
1. Gaberc-Porekar V, Menartr V (2001) J Biochem Biophys Methods
49:335–360
2. Rowinska-Zyrek M, Witkowska D, Potocki S, Remelli M, Kozlowski H (2013) New J Chem 37:58–70
3. Cheng T, Xia W, Wang P, Huang F, Wang J, Sun H (2013) Metallomics 5:1423–1429
4. Stanczak P, Valensin D, Porciatti E, Jankowska E, Grzonka Z,
Molteni E, Gaggelli E, Valensin G, Kozlowski H (2006) Biochem
45:12227–12239
5. Watly J, Simonovsky E, Wieczorek R, Barbosa N, Miller Y,
Kozlowski H (2014) Inorg Chem
6. Knecht S, Ricklin D, Eberle A.N, Ernst B (2009) J Mol Recognit
22:270–279
SL 16
New specific copper chelators as drug-candidates
for Alzheimer’s disease
Bernard Meunier1,2
1
Laboratoire de Chimie de Coordination du CNRS, 205 route de
Narbonne, 31077 Toulouse cedex 4, France.
2
University of Technology of Guangdong, Department of Chemical
Engineering, 100 Waihuan Xi road, Higher Education Mega Center,
Guangzhou, P. R. China. [email protected]
The design of efficient drugs able to inhibit the evolution of Alzheimer disease (AD) in the early stages of the disease is currently one
of the most important challenges in medicinal chemistry. The progressive cognitive decline of patients associated to complete dependence for
basic functions of daily life place a considerable burden on patients,
families and public health costs. The currently approved drugs (acetylcholine inhibitors like tacrine, donepezil, rivastigmine and
galantamine, or a non-competitive antagonist of NMDA receptor like
memantine) are weakly effective and their efficiency/cost ratios are
questionable. There is an urgent need for new disease-modifying therapies able to slow or to stop the neurodegenerative process. However,
Alzheimer’s disease is not a simple monogenic disease and all classical
approaches based on the concept of a single identified target failed.
Considering the deregulation of copper ions in AD brain should
contribute to pathological situations in aging brain, because of the easy
reduction of non-controlled copper(II) ions in physiological conditions
by endogenous reductants, we designed new copper chelating agents
having four coordination sites within the same ligand.[1, 2] One of
these tetradentate copper ligands, PA1637, was a full inhibitor of the
loss of episodic memory in a non-transgenic mouse model created by
one intra-cerebroventricular (icv) injection of Ab1–42 oligomer (a fast
and predictive animal model for the evaluation of episodic memory
deficits). After a three-week treatment of mice by oral route, PA1637
was able to fully reverse the cognitive deficits at 25 mg/kg per dose as
published in reference 1 and also at 12.5 mg/kg (unpublished data). No
toxicity was observed with PA1637 after a single oral dose of 400 mg/
kg on mice. These specific chelators should be considered as copper
homeostasis regulators for neurodegenerative diseases.
References
1. Ceccom J, Cosledan F, Halley H, Frances B, Lassalle JM, Meunier
B (2012) PLoS One 7:e43105
2. Nguyen M, Robert A, Sournia-Saquet A, Vendier L, Meunier B
(2014) Chem Eur J DOI: 10.1002/chem.201402143
J Biol Inorg Chem (2014) 19 (Suppl 2):S713–S724
SL 17
Enzyme-triggered CO-releasing molecules (ETCORMs)
Hans-Günther Schmalz
Department of Chemistry, University of Cologne, Greinstrasse 4,
50939 Köln, Germany. [email protected]
In recent years, carbon monoxide has been recognized as an important
signaling molecule in mammals. It is endogenously produced in the
course of oxidative heme degradation and induces several beneficial
biological effects, such as cytoprotection, vasodilation, or inhibition
of inflammation [1, 2]. In the search of new therapeutically useful
CO-releasing molecules, which deliver CO to a target tissue only after
activation of a specific release mechanism, we have recently introduced acyloxybutadiene-Fe(CO)3 complexes as enzyme-triggered
CO-releasing molecules (ET-CORMs) [3,4]. While the esters (1)
represent stable (and storable) compounds, their intracellular enzymatic cleavage leads to highly oxidation-sensitive dienol complexes
of type 2, which rapidly decompose under physiological condition to
liberate up to three molecules of CO together with the enone ligand
(3) and Fe ions.
The lecture will summarize the conceptual, synthetic and mechanistic aspects, and the potential of the ET-CORMs as potential
therapeutic agents will be discussed.
Financial support by the DFG and the University of Cologne is
gratefully acknowledged.
References
1. Motterlini R, Otterbein LE (2010) Nature Rev Drug Discov 9:728
2. Romão CC et al. (2012) Chem Soc Rev 41:3571
3. Romanski S, Kraus B, Schatzschneider U, Neudörfl J-M, Amslinger S, Schmalz H-G (2011) Angew Chem Int Ed 50:2392–2396
4. Romanski S, Kraus B, Guttentag M, Schlundt W, Rücker H, Adler
A, Neudörfl J-M, Alberto R, Amslinger S, Schmalz H-G (2012)
Dalton Trans 41:13862–13875
5. Romanski S, Rücker H, Stamellou E, Guttentag M, Neudörfl J-M,
Alberto R, Amslinger S, Yard B, Schmalz H-G (2012), Organometallics 31:5800–5809.
6. Botov S, Stamellou E, Romanski S, Guttentag M, Alberto R,
Neudörfl J-M, Yard B, Schmalz H-G (2013) Organometallics 32:
3587–3594
7. Romanski S, Stamellou E, Jaraba JT, Storz D, Kramer BK, Hafner
M, Amslinger S, Schmalz H-G, Yard BA (2013) Free Radical Bio
Med 65:78–88
SL 18
Organometallic B12-Chemistry
Bernhard Kräutler
Institute of Organic Chemistry and Centre for Molecular Biosciences
(CMBI), University of Innsbruck, A-6020 Innsbruck, Austria
Vitamin B12-derivatives have fascinating structural properties [1–3]
and chemical reactivities [4]. Due to the scarcity of the corrinoids,
most living organisms have developed a complex system for B12uptake and intercellular transport [5, 6]. The proteins involved here,
may also help shuttling-in unnatural B12-derivatives, such as those
that eventually act as ‘Trojan Horses’, by carrying a toxic load [7].
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The biological roles of the natural B12-derivatives are largely
associated with their organometallic reactivity and their redox-properties [8]. Formation and cleavage of the organometallic bond of B12cofactors are essential for catalysis by B12-dependent enzymes [4], as
well as for ‘tailoring’ of B12-derivatives for their biological tasks by
some B12-biosynthetic enzymes [5, 9].
In this lecture novel organometallic B12-derivatives will be presented (among them aryl-cobalamins [10] and alkynyl-cobalamins
[11]) that have been designed to explore B12-biology in humans and
animals.
References
1. Eschenmoser A (2011) Angew Chem Int Ed 50:12412–12472
2. Kratky C, Kräutler B (1999) in Chemistry and Biochemistry of B12
(Banerjee R, ed.) pp. 9–41
3. Randaccio L, Geremia S, Demitri N, Wuerges J (2010) Molecules
15:3228–3259
4. Kräutler B, Puffer B (2012) in Handbook of Porphyrin Science,
Vol. 25 (Kadish KM, Smith KM, Guilard R, eds.), World Scientific
pp. 133–265
5. Banerjee R, Gherasim C, Padovani D (2009) Curr Opin Chem Biol
13:484–491
6. Nielsen MJ, Rasmussen MR, Andersen CBF, Nexo E, Moestrup SK
(2012) Nat Rev 9:345–354
7. Zelder Z, Alberto R (2012) in Handbook of Porphyrin Science, Vol.
25 (Kadish KM, Smith KM, Guilard R, eds.) World Scientific
pp. 84–132
8. Gruber K, Puffer B, Kräutler B (2011) Chem Soc Rev
40:4346–4363
9. Warren MJ, Raux E, Schubert HL, Escalante-Semerena JC (2002)
Nat Prod Rep 19:390–412
10. Ruetz M, Gherasim C, Fedosov SN, Gruber K, Banerjee R,
Kräutler B (2013) Angew Chem Int Ed 52:2606–2610
11. Ruetz M, Salchner R, Wurst K, Fedosov S, Kräutler B (2013)
Angew Chem Int Ed 52:11406–11409
SL 19
Metal-mediated quadruplexes with and without DNA
Guido H. Clever, David M. Engelhard, Marcel Krick, Fernanda
Pereira
Institute for Inorganic Chemistry, Georg-August-University
Göttingen, Tammannstraße 4, 37077 Göttingen, Germany.
[email protected]
The square-planar metal(pyridine)4-motif has proven its value in
many self-assembled structures such as helicates, spherical cages and
coordination networks. We have prepared a number of discrete monoand oligonuclear (n = 2–5) architectures based on various organic
and DNA-derived ligands containing this motif with PdII, PtII, CuII or
NiII as the metal centers. The talk will highlight a couple of functional
assemblies including reversibly stabilized DNA-quadruplexes, anionbinding coordination cages and knots [1].
We have developed a series of interpenetrated double-cages [template@Pd4Ligand8] comprising a variety of organic ligands and
different templates in their central pockets [2]. Recently, we found that
the redoxactive compounds phenothiazine and anthraquinone can also
serve as suitable backbones. This opens up a very simple approach for
the study of densely packed redox centers in a spontaneously formed
self-assembly. Such structures might help to understand multi-electron
accumulation and transfer processes found in nature and may foster
future developments in molecular electronics and photovoltaics [3].
By transferring the metal(pyridine)4 coordination motif onto
G-quadruplex DNA, we were able to stabilize these structures
against thermal denaturation by the addition of transition metal
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cations such as CuII and NiII [4]. The stabilizing effect is reversed
by removal of the metal with a competing chelator. Currently, we
study the effect of varying the type, number and position of the
DNA-incorporated ligands. This approach promises to lead to
metal-switchable DNA nanostructures with applications as aptamers, sensors and catalysts. Furthermore, we envision that the
research on DNA origami will benefit from incorporating metalbased functions such as redox activity, magnetism, color and
enhanced stability.
References
1. Han M, Engelhard DM, Clever GH (2014) Chem Soc Rev
43:1848–1860
2. Freye S, Michel R, Stalke D, Pawliczek M, Frauendorf H, Clever
GH (2013) J Am Chem Soc 135:8476–8479
3. Frank M, Hey J, Balcioglu I, Chen YS, Stalke D, Suenobu T,
Fukuzumi S, Frauendorf H, Clever GH (2013) Angew Chem Int Ed
52:10102–10106
4. Engelhard DM, Pievo R, Clever GH (2013) Angew Chem Int Ed
52:12843–12847
SL 20
Towards artificial photosynthesis: catalytic reduction
of CO2
Marc Fontecave1,2
1
Laboratoire de Chimie des Processus Biologiques, UMR 8229 CDF/
CNRS/UPMC, Collège de France, 11 Place Marcelin Berthelot,
75231 Paris Cedex 05, France. [email protected]
2
Laboratoire de Chimie et Biologie des Métaux, Université Joseph
Fourier, CNRS, CEA/DSV/iRTSV, CEA-Grenoble 17 rue des martyrs
38054 Grenoble cedex 9, France. [email protected]
The development of renewable energies, such as solar energy in
particular, requests new efficient technologies for storing them in the
form of energy-dense chemicals. One example is hydrogen, derived
from water, which can be used as an energy vector. Another example
is carbon-based compounds, such as carbon monoxide, formic acid,
methanol and hydrocarbons, derived from reduction of CO2. To
achieve this goal one needs to optimize cheap and stable metal-based
catalysts for electro-reduction of CO2 and also to combine them with
photosensitizers/semiconductors to achieve photoreduction of CO2.
This field has recently been revivified with the objective to generate
artificial photosynthetic devices. Here we discuss various strategies to
achieve this goal based on: (i) homogeneous Co- and Ni-complexes;
(ii) bioinspired molecular W- and Mo-based complexes (iii) heterogenous Cu-based materials; (iv) supramolecular Metal–OrganicFrameworks.
Financial support by French National Research Agency (ANR,
Carbiored ANR-12-BS07-0024-03; Labex program ARCANE, ANR11-LABX-0003-01 and DYNAMO, ANR-11-LABX-0011) and from
Fondation de l’Orangerie for individual Philanthropy and its donors is
gratefully acknowledged.
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J Biol Inorg Chem (2014) 19 (Suppl 2):S713–S724
References
1. Elgrishi N, Artero V, Fontecave M (2013) L’Actualité Chimique
371–372:95–100
2 Elgrishi N, Chambers MB, Artero V, Fontecave M (2014) Phys
Chem Chem Phys (in press)
SL 21
Bio-inspired interfacial materials with super-wettability
Lei Jiang
Institute of Chemistry, Chinese Academy of Sciences, Beijing
100190, China. [email protected]
Learning from nature and based on lotus leaves and fish scale, we
developed super-wettability system: superhydrophobic, superoleophobic, superhydrophilic, superoleophilic surfaces in air and
superoleophobic, superareophobic, superoleophilic, superareophilic
surfaces under water [1]. Further, we revealed smart switchable superwettability [2]. The smart super-wettability system has great applications in various fields, such as self-cleaning glasses, water/oil
separation, anti-biofouling interfaces, and water collection system [3].
The smart property was further extended into 1D system. Energy
conversion systems that based on artificial ion channels have been
fabricated [4]. Also, we discovered the spider silk’s and cactus’s
amazing water collection and transportation capability [5], and based
on these nature systems, artificial water collection fibers and oil/water
separation system have been designed successfully [6].
Learning from nature, the constructed smart multiscale interfacial
materials system not only has new applications, but also presents new
knowledge: Super wettability based chemistry including basic
chemical reactions, crystallization, nanofabrication arrays such as
small molecule, polymer, nanoparticles, and so on [7].
References
1. Adv Mater 2006 18:3063–3078
2. Adv Mater 2008 20:2842–2858
3. Adv Mater 2011 23:719–734
4. a) Chem Soc Rev 2011 40:2385–2401; b) Acc Chem Res 2013
46:2834–2846; c) Adv Mater 2010 22:1021–1024; d) ACS Nano 2009
3:3339–3342; e) Angew Chem Int Ed 2012 51:5296–5307
5. a) Nature 2010 463:640-643; b) Nat Commun 2012 3:1247
6. a) Nat Commun 2013 4:2276; b) Adv Mater 2010 22:5521–5525
7. a) Chem Soc Rev2012 41:7832–7856; b) Adv Funct Mater 2011
21:3297–3307; c) Adv Mater 2012 24:559–564; d) Nano Research
2011 4:266–273; e) Soft Matter 2011 7:5144–5149; f) Soft Matter
2012 8:631–635; g) Adv Mater 2012 24:2780–2785; h) Adv Mater
2013 25:3968–3972; i) J Mater Chem A 2013 1:8581–8586; j) Adv
Mater 2013 25:6526–6533; k) Adv Funct Mater 2012 22:4569–4576;
l) ACS Nano 2012 6:9005–9012
SL 22
Cisplatin-induced duplex dissociation of RNA-influence
of structure on kinetics
Sofi. K. C Elmroth
Biochemistry and Structural Biology, Department of Chemistry, Lund
University, POBox 124, SE-221 00 Lund, Sweden.
[email protected]
The adduct profile of nucleophiles such as cisplatin is often referred to
as being under kinetic control. In duplex DNA, where the structural
variations are small, adduct formation with consecutive guanines
predominates. In the structurally more diverse RNA world, no
J Biol Inorg Chem (2014) 19 (Suppl 2):S713–S724
consensus has so far been reached. Thus, the relative contributions
from e.g. variations of local melting behaviour and overall dynamics
to binding rates remain to be determined. In an attempt to resolve
these issues, our laboratory is currently exploring metal-induced RNA
duplex melting as a tool for investigating the interaction between
cisplatin and small duplex RNAs as models for interactions with
endogenous microRNAs [1]. The approach takes advantage of the
hyperchromicity associated with nucleic acid duplex melting [2, 3],
and follows concentration-dependent first-order kinetics under in vivo
relevant concentrations of both metal reagent and RNAs, see Figure
below. Further, the sensitivity of the method is good enough to allow
for kinetic discrimination between closely related structures caused
by e.g. single base variations. Implications for biological processing
of RNAs will be discussed.
Financial support from Cancerfonden, FLÄK, Kgl. Fyiografiska
Sällskapet, Crafoordstiftelsen, and COST CM1105 is gratefully
acknowledged.
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Financial support by the National Science Foundation NSF CHE1153147 is gratefully acknowledged.
References
1. Hostetter AA, Osborn MF, DeRose VJ (2012) ACS Chem Biol
7:218–225
2. Osborn MF, White JN, DeRose VJ (2014) submitted
3. White JN, Osborn MF, Moghaddam AN, Guzman LE, Haley MM,
DeRose VJ (2013) J Am Chem Soc 135:11680–11683
SL 24
Fuels from solar energy and water-from natural
to artificial photosynthesis
References
1. Polonyi C, Elmroth SKC (2013) Dalton Trans 42:14959–14962
2. Horacek P, Drobnik J (1971) Biochim Biophys Acta 254:341–347
3. Polonyi C, Albertsson I, Damian MS Elmroth SKC (2013) Z Anorg
Allg Chem 639:1655–1660
SL 23
Platinum therapeutic target analysis using click
chemistry
Victoria J. DeRose, Jonathan D. White, Maire F. Osborn, Alan D.
Moghaddam, Kory Plakos, Lindsay E. Guzman, Rachael
Cunningham, Michael M. Haley
Department of Chemistry and Biochemistry, University of Oregon,
Eugene, OR, 97403-1253, USA. [email protected]
Platinum anticancer therapeutics, broadly used in frontline treatments
of solid tumors, are known to crosslink cellular DNA and trigger
apoptosis. Despite prevalent use, broad molecular identification of
cellular targets that may lead to resistance, side effects, and alternative apoptotic pathways is not available. Of particular interest may be
targeting of cellular RNA and downstream effects on ensuing regulatory pathways. We have previously shown that cisplatin treatment
results in significant platinum accumulation on yeast ribosomal RNA
[1], with specificity towards high-impact sites [2]. To further identify,
isolate, and visualize such targets we have developed platinum
compounds modified for post-treatment ‘click’ azide-alkyne cycloaddition reactions. Picazoplatin, an azide-modified picoplatin, readily
undergoes binding and subsequent fluorescent labelling with DNA
and RNA [3] as well as proteins. Post-treatment fluorescent labelling
of RNA extracted from S. cerevisiae treated with picazoplatin demonstrates utility of this compound for in vivo work. The properties of
additional Pt(II) compounds with azide and alkyne modifications, and
their efficacies in identifying and visualizing Pt targets, will be
presented.
Stenbjörn Styring
Molecular Biomimetics, Department of Chemistry-Ångström,
Uppsala University, Box 523, SE-751 20 Uppsala, Sweden
The lecture will discuss the need for Solar Fuels and overview the
different scientific paths to achieve this goal. Visions and strategies in
research in the Swedish Consortium for Artificial Photosynthesis will
be covered [1, 2]. Our research aims for the production of hydrogen
from solar energy and water. Water shall be oxidized in a catalytic
process using solar energy. The electrons from water shall be used in a
second process to reduce protons to hydrogen. We apply a biomimetic
approach where we copy key principles from natural enzymes that
accomplish partial reactions. Water oxidation using solar energy is
carried out by Photosystem II using a catalytic Mn4 complex. In our
chemistry we use a photoactive Ru-center (instead of chlorophyll) that
is coupled to synthetic multinuclear manganese-complexes. I will
describe some of our research on light driven, multi-electron transfer in
these Ru–Mn systems [1]. The lecture will also cover a water oxidizing
catalyst based on a cobalt nano-particle [3]. This nano-particle has been
linked to a photosensitizer to form a water splitting photosensitizercatalyst complex [4]. To accomplish reduction of protons to hydrogen
we mimic the di-iron center in hydrogenase enzymes. Some recent
results on these biomimetic Fe–Fe complexes will be described [5].
Financial support by the Swedish Energy Agency and the Knut
and Alice Wallenberg Foundation is gratefully acknowledged
References
1. Magnuson, A, Anderlund M, Johansson O, Lindblad P, Lomoth R,
Polivka T, Ott S, Stensjö K, Styring S, Sundström V, Hammarström L
(2009) Acc Chem Res 42:1899–1909
2. Styring S (2012) Faraday Discuss 155:357–376
3. Shevchenko D, Anderlund MF, Thapper A, Styring S (2011)
Energy Environ Sci 4:1284–1287
4. Wang H-Y, LiuJ, Zhu J, Styring S, Ott S, Thapper A (2014) Phys
Chem Chem Phys 16:3661–3669
5. Pullen S, Fei H, Orthaber A, Cohen SM, Ott S (2013) J Am Chem
Soc 135:16997–17003
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SL 25
Hydrogen evolution: bioinspired catalysts and artificial
hydrogenases
Vincent Artero
Laboratory of Chemistry and Biology of Metals, Univ. Grenoble
Alpes, CNRS, CEA Grenoble, 17 rue des martyrs, 38000 Grenoble,
France. [email protected]
Hydrogen production, through the reduction of water in electrolysers,
is currently one of the most convenient ways to store energy durably,
if the electrical energy is initially obtained from renewable resources.
However, while electrolysis is a mature and robust technology, the
most promising devices, based on proton exchange membranes, relay
on the use of platinum as electrocatalyst to accelerate both hydrogen
evolution and water oxidation reactions. However, this rare and
expensive metal is not itself a renewable resource, so the viability of a
hydrogen economy depends on the design of new efficient and robust
electrocatalytic materials based on earth-abundant elements [1]. A
competitive alternative to platinum could be found in living microorganisms metabolizing hydrogen thanks to hydrogenases. Catalysis
in hydrogenases only requires base-metal centers (nickel and iron)
and we will show how their active sites can be used as an inspiration
to design new synthetic catalysts. We found that cobalt diimine–
dioxime complexes are efficient and stable electro-catalysts for
hydrogen evolution form acidic non-aqueous solutions with slightly
lower overvoltages [2, 3] and much larger stabilities towards hydrolysis as compared to previously reported cobaloxime catalysts [4, 5].
We will report on our approach for the covalent functionalization of
electrode materials with such catalysts and their activity under fully
aqueous conditions [6]. Integration of cobalt-based catalysts into
protein frameworks to prepare artificial hydrogenases and their
combination with photosensitizers to design photocatalytic systems
able to achieve the photochemical production of hydrogen will also be
discussed [7–9].
References
1. Artero V, Chavarot-Kerlidou M, Fontecave M (2011) Angew Chem
Int Ed 50:7238–7266
2. Jacques P-A, Artero V, Pécaut J, Fontecave M (2009) Proc Natl
Acad Sci USA 106:20627–20632
3. Bhattacharjee A, Andreiadis ES, Chavarot-Kerlidou M, Fontecave
M, Field MJ, Artero V (2013) Chem Eur J 19:15166–15174
4. Razavet M, Artero V, Fontecave M (2005) Inorg Chem
44:4786–4795
5. Baffert C, Artero V, Fontecave M (2007) Inorg Chem
46:1817–1824
6. Andreiadis ES, Jacques P-A, Tran PD, Leyris A, Chavarot-Kerlidou M, Jousselme B, Matheron M, Pécaut J, Palacin S, Fontecave M,
Artero V (2013) Nat Chem 5:48–53
7. Fihri A, Artero V, Pereira A, Fontecave M (2008) Dalton Trans
(2008) 5567–5569
8. Fihri A, Artero V, Razavet M, Baffert C, Leibl W, Fontecave M
(2008) Angew Chem Int Ed 47:564–567
9. Zhang P, Jacques P-A, Chavarot-Kerlidou M, Wang M, Sun L,
Fontecave M, Artero V (2012) Inorg Chem 51:2115–2120
SL 26
Towards molecular systems biology of metal trafficking
in cells
Lucia Banci
CERM, University of Florence, Via L. Sacconi 6, Sesto Fiorentino,
Italy. [email protected]
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Metal transfer processes, through which a metal ion is transferred
from metal transporters to the final recipient proteins, occur
through weak, transient protein–protein interactions [1]. The
transfer is determined by metal affinity gradients among the various proteins, with kinetic factors contributing to the selectivity of
the processes [2]. The characterization of these functional processes
requires their description both at system (e.g. a cell) and at
molecular level, (e.g. atomic-resolution characterization of biomolecules) for which NMR is a quite powerful technique. NMR is
indeed suitable not only for characterizing the structure and
dynamics of biomolecules but, even more importantly, can describe
functional events. The presence of paramagnetic centers, such as
iron-sulfur clusters, dramatically affects the NMR spectra, requiring
tailored experiments also integrated with EPR spectra. In-cell NMR
can provide the description of these processes within living cells.
The power of NMR in describing cellular pathways at atomic
resolution in a cellular environment will be presented for a few
pathways responsible for copper trafficking in the cell and for the
biogenesis of iron-sulfur proteins. New major advancements in incell NMR [3] and in the characterization of highly paramagnetic
systems [4] will be also discussed within an integrated approach
where, from single structures to protein complexes, the processes
are described in their cellular context within a molecular
perspective.
References
1. Banci L, Bertini I, McGreevy KS, Rosato A (2010) Nat Prod Rep
27:695–710
2. Banci L, et al. (2010) Nature 465:645–648
3. Banci L, Barbieri L, Bertini I, Luchinat E, Secci E, Zhao Y, Aricescu AR (2013) Nat Chem Biol 9:297–299
4. Banci L, Bertini I, Calderone V, Ciofi-Baffoni S, Giachetti A,
Jaiswal D, Mikolajczyk M, Piccioli M, Winkelmann J (2013) Proc
Natl Acad Sci USA 110:7136–7141
SL 27
Artificial metalloenzymes: challenges and opportunities
Thomas R. Ward
Department of Chemistry, University of Basel, Spitalstrasse 51, 4056
Basel, Switzerland
Artificial metalloenzymes result from incorporation of a catalytically
competent organometallic moiety within a host protein. We and
others have been exploiting the potential of the biotin-(strept)avidin
technology for the creation of artificial metalloenzymes, Figure.
Thanks to the remarkable affinity of biotin for either avidin or
streptavidin (KD [ 10–13 M), linking of a biotin anchor to a catalyst
precursor ensures that, upon stoichiometric addition of (strept)avidin,
the metal moiety is quantitatively incorporated within the host
protein.
Such artificial metalloenzymes are optimized either by chemical
(variation of the biotin-spacer-ligand moiety) or genetic- (mutation of
(strept)avidin) means. These chemogenetic schemes were applied to
optimize the performance for eight different catalyzed transformations as well reaction cascades in the presence of natural enzymes
[1–3].
More recently, we have been exploiting the tetrameric nature of
streptavidin to fine-tune the performance of such supramolecular
catalysts. For this purpose, we rely on two adjacent biotin binding
sites to localize both the substrate and the catalyst within a well
defined second coordination sphere environment. As a proof of
principle, we have been investigating the olefin metathesis as a model
reaction.
J Biol Inorg Chem (2014) 19 (Suppl 2):S713–S724
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Reactions implemented thus far:
Hydrogenation (up to 96 % ee)
Transfer Hydrogenation of
ketones (up to 98 % ee)
imines (up to 96 % ee)
enones (up to 1000 TONs)
Allylic Alkylation (up to 95% ee)
C-H Activation (up to 86 % ee)
Olefin Metathesis (up to 40 TONs)
Alcohol Oxidation (up to 250 TONs)
Sulfoxidation (up to 93 % ee)
Dihydroxylation (up to 98 % ee)
Figure Artificial metalloenzymes obtained upon supramolecular incorporation of a
biotinylated organometallic catalyst within streptavidin
1. Ward TR (2011) Acc Chem Res 44:47–57
2. Köhler V, et al. (2012) Nat Chem 5:93–99
3. Hyster, TK, Knorr L, Ward, TR, Rovis T (2012) Science
338:500–503
SL 28
Discrimination of cytosine and thymine via metalmediated base pairing with an artificial nucleoside
analogue
Jens Müller1, Philipp Scharf1, Dominik A. Megger2, Célia
Fonseca Guerra3
1
Institut für Anorganische und Analytische Chemie, Westfälische
Wilhelms-Universität Münster, Corrensstr. 28/30, 48149 Münster,
Germany. [email protected] 2Medizinisches Proteom-Center,
Ruhr-Universität Bochum, Universitätsstr. 150, 44801 Bochum,
Germany. 3Department of Theoretical Chemistry and Amsterdam
Center for Multiscale Modeling, Vrije Universiteit, De Boelelaan
1083, 1081 HV Amsterdam, The Netherlands
Nucleic acids and particularly DNA are nowadays regularly being
applied as scaffolds for the attachment of various functional entities.
One possibility to introduce functionality is the use of metal-mediated
base pairs [1, 2]. In these artificial base pairs, transition metal ions are
located inside the double helix between complementary nucleobases,
formally replacing hydrogen bonds [3,4]. To expand the scope of
metal-mediated base pairs, new silver(I)-mediated base pairs
involving the ligand 1H-imidazo[4,5-f][1,10]phenanthroline (imphen)
have been developed. Imphen can form stable hydrogen-bond-mediated base pairs with both natural pyrimidine nucleobases. In the
presence of silver(I), a highly stable silver(I)-mediated base pair is
formed with cytosine, whereas under acidic and neutral conditions the
imphen: thymine base pair is strongly destabilized, as shown by UV
and CD spectroscopy and confirmed by computational methods.
Moreover, imphen can form silver(I)-mediated hetero base pairs with
imidazole nucleoside and homo base pairs with itself. Interestingly,
the thermal stabilization of the imphen–Ag(I)–imidazole base pair
depends strongly on the sequence context.
Support by the DFG (SFB 858), COST CM1105, and the NWO is
gratefully acknowledged.
References
1. Scharf P, Müller J (2013) ChemPlusChem 78:20–34
2. Megger DA, Megger N, Müller J (2012) Met Ions Life Sci
10:295–317
3. Johannsen S, Megger N, Böhme D, Sigel RKO, Müller J (2010) Nat
Chem 2:229–234
4. Kumbhar S, Johannsen S, Sigel RKO, Waller MP, Müller J (2013) J
Inorg Biochem 127:203–210
SL 29
Theoretical spectroscopy of open shell transition metals
in enzymes and model complexes
Frank Neese
Max-Planck Institut für Chemische Energiekonversion, Stiftstr.
34-36, D-45470 Mülheim an der Ruhr, Germany
Open Shell transition metal ions play fundamental roles in the active
sites of enzymes, in catalysis, in materials science and molecular
magnetism to only name a few important research fields. The reactivities and spectroscopic properties of these open-shell transition
metal ions are complex and represent a substantial challenge for
theoretical chemistry. In the past years we have been involved in
developing and applying theoretical methods ranging from density
functional theory to multireference wavefunction approaches to
problems in transition metal chemistry (e.g. [1–10]). The talk will
present selected examples from this work.
References
1. Cox N, Pantazis DA, Neese F, Lubitz W (2013) Acc Chem Res
46:1588–1596
2. Kampa M, Pandelia M-E, Lubitz W, van Gastel M, Neese F (2013)
J Am Chem Soc 135:3915–3925
3. Krahe O, Neese F, Engeser M (2013) ChemPlusChem
78:1053–1057
4. Krewald V, Lassalle-Kaiser B, Boron III TT, Pollock CJ, Kern J,
Beckwith MA, Yachandra VK, Pecoraro VL, Yano J, Neese F,
DeBeer S (2013) Inorg Chem 52:12904–12914.
5. Krewald V, Neese F, Pantazis DA (2013) J Am Chem Soc
135:5726–5739
6. Lassalle-Kaiser B, Boron III TT, Krewald V, Kern J, Beckwith
MA, Delgado-Jaime MU, Schroeder H, Alonso-Mori R, Nordlund D,
Weng T-C, Sokaras D, Neese F, Bergmann U, Yachandra VK,
DeBeer S, Pecoraro VL, Yano J (2013) Inorg Chem 52:12915–12922
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J Biol Inorg Chem (2014) 19 (Suppl 2):S713–S724
7. Navarro MP, Ames WM, Nilsson H, Lohmiller T, Pantazis DA,
Rapatskiy L, Nowaczyk MM, Neese F, Boussac A, Messinger J,
Lubitz W, Cox N (2013) Proc Natl Acad Sci USA 110:15561–15566
8. Pandelia M-E, Bykov D, Izsak R, Infossi P, Giudici-Orticoni M-T,
Bill E, Neese F, Lubitz W (2013) Proc Natl Acad Sci USA
110:483–488
9. Retegan M, Neese F, Pantazis DA (2013) J Chem Theo Comp
9:3832–3842
10. Saracini C, Liakos DG, Rivera JEZ, Neese F, Meyer GJ, Karlin
KD (2014) J Am Chem Soc 136:1260–1263
SL 30
Trilateration of metal centers in biomolecules with EPR
Dinar Abdulin, Andreas Meyer, Gregor Hagelueken, Olav
Schiemann
Institute of Physical and Theoretical Chemistry, University of Bonn,
Wegelerstr. 12, 53115 Bonn, Germany
Metal ions play an important role in the catalysis and folding of
proteins and oligonucleotides. Their localization within the threedimensional fold of a biomacromolecule is therefore an important aim
in understanding structure–function relationships. Here an approach is
presented for the localization of paramagnetic metal ions based on site
directed spin labeling and electron paramagnetic resonance (EPR)
methods, as e.g. pulsed electron–electron double resonance (PELDOR
or DEER) [1] or relaxation Induced dipolar modulation enhancement
(RIDME) [2]. The location of the spin label in the structure of the
biomolecule is determined with the program mtsslWizard [3] and the
actual trilateration of the metal center is done with the program
mtsslTrilaterate [4]. The approach is tested on the Cu2? center of
azurin and the influence of distance errors, number of constraints and
quality of starting structures are discussed
Financial support by the DFG via SFB813 project A6 is gratefully
acknowledged.
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References
1. Reginsson GW, Schiemann O (2011) Biochem J 434:353–363
2. Konov KB, Knyazev AA, Galyametdinov YG, Isaev NP, Kulik LV
(2013) Appl Magn Reson 44:949–966
3. Hagelueken G, Ward R, Naimsith JH, Schiemann O (2012) Appl
Magn Reson 42:377–391
4. Hagelueken G, Abdullin D, Ward R, Schiemann O (2013) Mol
Phys 111:2757-2766
J Biol Inorg Chem (2014) 19 (Suppl 2):S725–S747
DOI 10.1007/s00775-014-1157-y
ORAL PRESENTATION
Invited lectures
IL 1
Nanoparticle-mediated photoactivation of anticancer
metal complexes
IL 2
Selective detection of cancer cells by luminescent
ruthenium probes
Emmanuel Ruggiero1, Abraha Habtemariam1,2,3, Silvia Alonso-de
Castro1, Juan C. Mareque-Rivas1,2, Luca Salassa1
1
CIC biomaGUNE, Paseo Miramón 182, 20009, Donostia, Spain;
2
IKERBASQUE, Basque Foundation for Science, 48011, Bilbao,
Spain;
3
Department of Chemistry, University of Warwick, Coventry, CV4
7AL, UK. [email protected]
Fostered by the success of photodynamic therapy (PDT), the use of
light to activate prodrugs is currently gaining momentum in biomedical research. In principle, photoactivation allows to spatially and
temporally control the biological effects of a prodrug, hence conferring selectivity to its action and reducing unwanted drawbacks.
In the last few years, photoactivatable transition metal complexes
have been studied intensively for application in chemotherapy [1].
The interest in this class of derivatives is motivated by a rich photochemistry which can be tailored to promote novel mechanisms of
action. Nevertheless, the poor absorption properties of metal complexes in the therapeutic windows of the visible spectrum
(k [ 600 nm) pose a key limitation for further advancing their use
towards preclinical and clinical PDT.
Nanoparticles (NPs) and their outstanding optical properties offer
a superior and effective strategy to achieve efficient photoactivation
of metal complexes and overcome their intrinsic flaws [2]. Notably,
NPs can be exploited to access unconventional excited-state chemistry in metal complexes and design innovative hybrid materials for
imaging and therapy. In the present contribution we will discuss our
advances in this emerging field.
Financial support by the Spanish Ministry of Economy and Competitiveness (Grant CTQ2012-39315 and Ramón y Cajal Fellowship
RYC-2011-07787), the EU PF7 program (MC-CIG fellowship
PCIG11-GA-2012-321791) and the Department of Industry of the
Basque Country (Grant ETORTEK) is acknowledged. A.H. is grateful
to IKERBASQUE for a Visiting Professor fellowship.
Dumitru Arian, Roland Krämer
Inorganic Chemistry Institute, University of Heidelberg, Im
Neuenheimer Feld 270, 69120 Heidelberg, Germany
The selective detection of rare circulating cancer cells that have shed
into the vasculature from a primary tumor could be an invaluable tool
for early stage detection of cancer by a simple blood test [1]. Accurate
detection of these rare cells (one among millions) is challenging and
requires highly effective and selective labelling strategies.
Building on our studies of biomolecule detection in human blood
plasma by small-molecule conjugates of luminescent ruthenium probes
[2], we have developed folate derivatives of Ru(II)-tris(bipyridine)
complexes. Unlike normal cells, many cancer cell types overexpress a
folate-receptor at the cell surface so that folate coupled with a signalling
moiety enables the selective labelling of these cells. We will demonstrate the selective detection of cancer cells by folate-ruthenium probes,
including application to cell- spiked human plasma samples.
While the photoluminescence of Ru-bipyridine type complexes is
relatively weak, they are preferred labels for fluorescence lifetime and
electrochemiluminescence based imaging and cytometry techniques.
These emerging methods [3, 4] promise much enhanced labelling
sensitivity what is essential to the reliable quantification of circulating
cancer cells in view of in vitro-diagnostic applications.
References
1. Plaka V, Koopman CD, Werb Z (2013) Science 341:1186–1188
2. Szelke H, Harenberg J, Krämer R (2009) Thromb Haemost
102:859–864
3. Jin D, et al. (2009) J Biomed Optics 14:024023
4. Dolci LS, et al. (2009) Anal Chem 81:6234–6241
IL 3
Solid State NMR as a method to characterize biosilicaentrapped enzymes
References
1. Schatzschneider U (2010) Eur J Inorg Chem 10:1451–1467
2. Ruggiero E, Habtemariam A, Yate L, Mareque-Rivas JC, Salassa L
(2014) Chem Commun 50:1715–1718
Marco Fragai1, Claudio Luchinat1, Tommaso Martelli1, Enrico
Ravera1
1
CERM and Department of Chemistry ‘‘Ugo Schiff’’, Via L. Sacconi
6, 50019, Sesto Fiorentino (FI), Italy
Enzymes immobilized in inorganic matrices find a wide range of
applications for analysis and catalysis in industrial and academic
research. Among the protein immobilization methods, bio-inspired
silicification has attracted great attention, because the formation of the
biosilica matrix around the protein takes place at room temperature
and goes to completion in few minutes. We show that solid-state
123
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NMR is a quick and very efficient way to characterize metalloenzymes trapped in bioinspired silica and immediately assess their
structural integrity. Having a method to assess whether an enzyme
retains its native conformation in atomic detail allows for the
exploration of many candidate processes for industrial application and
selection of the most reliable and robust ones. Further investigations
on the methods to improve SSNMR sensitivity in those systems will
also be presented.
IL 4
Novel insights in zinc biochemistry from quantitative
research
Wojciech Bal, Ilona Marszałek, Wojciech Goch
Institute of Biochemistry and Biophysics, Polish Academy
of Sciences, Pawińskiego 5A, 02-106 Warsaw, Poland
The aim of this research is to provide a consistent perspective on
quantitative description of interactions of biological Zn(II) ions, using
theoretical concepts and experimental approaches. The definitions of
absolute, conditional and apparent binding constants will be discussed
for various types of binary and ternary Zn(II) complexes, e.g. ZnA,
ZnA2, ZnAB. Main methods of their determination will be reviewed,
with particular attention to possible errors resulting from an incomplete
description of chemistry of studied systems. Our studies of fluorescent
zinc sensors, including ZnAF and FluoZin-3 will provide relevant
examples [1]. The relationship between complex stoichiometry and
Zn(II) binding abilities will be described for the example of glutathione.
The availability of Zn(II) for binding sites characterized with various
binding constant values will be discussed using literature data and our
recent results for zinc fingers of DNA repair proteins. Finally, the effects
of volume of cellular structures on Zn(II) distribution among its ligands
will be described. Such critical analysis and rigorously determined
binding constants of Zn(II) complexes with biomolecules leads to novel
testable hypotheses in the field of zinc homeostasis and signaling.
Financial support by the National Science Centre of Poland (NCN)
grant no. 2012/07/B/ST5/02390 is gratefully acknowledged.
Reference
1. Staszewska A, Kurowska E, Bal W (2013) Metallomics
5:1483–1490
IL 5
Biochemical and physiological evidence characterising
chromium as a new essential ultra-micronutrient
in plants
Hendrik Küpper1,2, Jürgen Mattusch3, Edgar Peiter4, Christian
Schmelzer5, Hans-Joachim Stärk3
1
Biology Center of the Czech Academy of Sciences, Institute of Plant
Molecular Biology, Department of Biophysics and Biochemistry
of Plants, and University of South Bohemia, Faculty of Sciences,
Department of Experimental Plant Biology, CZ-370 05 České
Budejovice, Czech Republic;
2
University of Konstanz, Faculty of Sciences, Department of Biology,
D-78457 Konstanz, Germany;
3
UFZ-Helmholtz Centre for Environmental Research, Department
of Analytical Chemistry, Permoserstr. 15, D-04318 Leipzig,
Germany;
4
Martin Luther University Halle-Wittenberg, Institute of Agricultural
and Nutrition Sciences, Wolfgang-Langenbeck-Str. 4, 06120 Halle
(Saale), Germany;
5
Martin Luther University Halle-Wittenberg, Institute of Pharmacy,
Wolfgang-Langenbeck-Str. 4, 06120 Halle (Saale), Germany
Chromium is considered as non-essential for plants, in contrast to
animals (incl. humans) where it has been linked to glucose metabolism
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J Biol Inorg Chem (2014) 19 (Suppl 2):S725–S747
by activation of insulin. These processes do not exist in plants, so that
an essential role of Cr in plants would have to concern other physiological processes. We used conditions that allowed work in the subnanomolar range to characterise Cr effects on plant growth, photosynthesis biophysics, pigment content, Cr compartmentation, Cr
binding to soluble and membrane proteins, as well as uptake of various
metals and other nutrients. Thus we found that the water plant Ceratophyllum demersum stopped growth unless Cr(III) as Cr3+ or Cr(VI)
as CrO2
4 became available, as extrapolated from the growth decrease
towards the lowest achievable Cr (0.17 nM). Chromium deficiency
was furthermore found, although not at severe likely due to lower
demands comparable to the lowest achievable Cr concentration, in the
crop plants Glycine soja (soybean) and Triticum aestivum (wheat). In
all species chromium deficiency led to retarded formation of new
meristems and stunted branches, in soybean sometimes to death of the
primary meristems. Investigating Cr deficiency effects further in C.
demersum as a plant shoot model revealed that non-photochemical
exciton quenching and photosynthetic oxygen release were affected by
deficient Cr. Metalloproteomics applying radioactive 51Cr, native gel
electrophoresis and size exclusion chromatography with elementsensitive detection of extracts from C. demersum, several terrestrial
plants and the cyanobacterium Trichodesmium erythraeum showed
chromium in at least two [30 kDa membrane proteins plus several
\7 kDa soluble proteins. Purification of the two membrane proteins
plus the largest (about 6 kD) soluble protein was successful already, so
that their spectroscopic characterisation as well as their identification
by de mass spectrometry is in progress- so far we preliminarily
identified 4 Cr-binding proteins and compared them with genomic
databases. This proteomic/genomic approach, measurements of metal
binding to the proteins and also element uptake into the plants did not
suggest a replacement of Mo by Cr. All these results strongly suggest
that Cr is an essential ultra-micronutrient in plants, which also means
that Cr has biochemical functions other than insulin activation.
IL 6
Biomimetic metal–oxygen intermediates in dioxygen
activation chemistry
Wonwoo Nam
Department of Chemistry, Ewha Womans University, Seoul 120-750,
Korea. [email protected]
Dioxygen is essential in life processes, and enzymes activate dioxygen
to carry out a variety of biological reactions. One primary goal in
biomimetic research is to elucidate structures of reactive intermediates
and mechanistic details of dioxygen activation and oxygenation
reactions occurring at the active sites of enzymes, by utilizing synthetic metal–oxygen complexes. A growing class of metal–oxygen
complexes, such as metal–superoxo, –peroxo, –hydroperoxo, and –oxo
species, have been isolated, characterized spectroscopically, and
investigated in various oxygenation reactions. During the past decade,
we have been studying the chemical and physical properties of various
reactive intermediates in oxygenation reactions, such as high-valent
iron(IV)- and manganese(V)-oxo complexes of heme and non-heme
ligands in oxo-transfer and C–H activation reactions, non-heme metalperoxo complexes in nucleophilic reactions, and non-heme metalsuperoxo complexes in electrophilic reactions. The effects of supporting and axial ligands on structural and spectroscopic properties
and reactivities of metal–oxygen adducts have been extensively
investigated as well. In this presentation, I will present our recent
results on the reactivities of various metal–oxygen intermediates in
electrophilic and nucleophilic oxidation reactions. The synthesis and
structural and spectroscopic characterization of mononuclear nonheme
metal-dioxygen intermediates will be discussed as well.
J Biol Inorg Chem (2014) 19 (Suppl 2):S725–S747
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IL 7
Role of dioxygen in the oxidative dehydrogenation
at mononuclear hexadentate N6 iron complexes
Martha E. Sosa-Torres, Juan P. Saucedo-Vázquez, Pedro D.
Sarmiento-Pavı́a Ricardo D. Páez-López
Departamento de Quı́mica Inorgánica y Nuclear, Facultad de
Quı́mica, Universidad Nacional Autónoma de México, Ciudad
Universitaria, 04510, México, DF. [email protected]
A mechanism for the oxidative dehydrogenation of a hexamine
coordinated to iron(III), [FeIIIL3]3+ (1), L3 = 1,9-bis(20 -pyridyl)-5[(ethoxy-200 -pyridyl)methyl]-2,5,8-triazanonane in the presence of
molecular oxygen has been derived based on kinetic and structural
investigations. The dehydrogenation reaction, under oxic conditions,
proceeds via successive oxidations of ligand-centered FeII-radical
2
intermediates by O2, and reduced oxygen species ðO
2 ; O2 Þ via
II 4 2+
outer sphere electron transfer to yield the product [Fe L ] (2),
(L4 = 1,9-bis(20 -pyridyl)-5-[(ethoxy-200 -pyridyl)methyl]-2,5,8-triazanon-1-ene). The absence of general base catalysis as well as the
experimental and theoretical rate laws show that the first reductive
step O2 ! O
2 becomes rate determining (in contrast to the reaction
under anoxic conditions [1, 2]). In biological and chemical systems,
the most common mechanism for reduction of O2 to H2O includes
an inner sphere electron transfer process. However, several studies
have reported an outer sphere mechanism for the reduction of O2.
We did not observe any evidence for the formation of Fe–OO
adducts, or high valent iron-oxo (FeIV=O, FeV=O) intermediates as
proof of inner sphere electron transfer mechanism. Moreover, the
detection of free peroxide ðO2
2 Þ in a coupled reaction with heme
catalase demonstrates that the stepwise O2 reduction by FeII-radical
intermediates goes via outer sphere single electron transfer. In
conclusion, we present experimental data that reveal a decisive role
of O2 for the dehydrogenation of a polyamine coordinated to iron.
Notably, the rate constant determined for the oxidative dehydrogenation of [FeIIIL3]3+ to [FeIIL4]2+, in the presence of O2, is almost
one order of magnitude larger than the one obtained under N2,
(kEtO- = 3.09 9 105 M-1 s-1 vs kEtO- = 4.92 9 104 M-1 s-1).
Interestingly, under oxic conditions the product [FeIIL4]2+, is obtained
with an enantiomeric excess.
Financial support by DGAPA_UNAM (Research project
IN231111) and CONACYT (Research Project 128921) is gratefully,
acknowledged. JPSV thanks CONACYT for the Ph.D. scholarship.
References
1. Saucedo-Vázquez JP, Ugalde-Saldı́var, VM, Toscano AR, Kroneck
P, Sosa-Torres ME (2009) Inorg Chem 48:1214–1222
2. Ugalde-Saldı́var, VM, Sosa-Torres ME, Ortiz-Frade L, Bernès S,
Höpfl H (2001) J Chem Soc Dalton Trans 3099–3107
IL 8
New heme analogs with iron in unusual spin states
Martin Bröring, Dimitri Sakow, Jörn Rösner, Thomas
Ostapowicz, Silke Köhler
Institut für Anorganische und Analytische Chemie, Technical
University Braunschweig, Hagenring 30, 38106 Braunschweig,
Germany
One special feature of naturally occurring and catalytically active
metal porphyrins like the hemes, cobalamine, or co-factor F430, is
their ability to change binding affinities and redox potentials by nonplanar ligand distortions within a given protein scaffold [1]. These
distortions result in variations of both, electronic and steric metal–
ligand interactions, and this fine-tuning is often the clue to the
understanding of a specific function.
A possible way to vary porphyrin-metal interactions in the laboratory is the exchange of the natural ligand by ring-contracted
porphyrinoids like the corroles [2], heterocorroles [3, 4], or the like.
In fact, the characteristics of transition metal chelates of such bioinspired ligand systems clearly deviate from the natural archetype, and
the differences observed in transition metal electronic structure relates
to largely changed reaction characteristics. The presentation will
discuss current selected examples of such metal chelates from the
group.
Financial support by the Deutsche Forschungsgemeinschaft (DFG
Grant Br2010/13-1) is gratefully acknowledged.
References
1. MacGowan SA, Senge MO (2011) Chem Commun 11621
2. Aviv I, Gross Z (2007) Chem Commun 1987
3. Sakow D, Böker B, Brandhorst K, Burghaus O, Bröring M (2013)
Angew Chem Int Ed 52:4912
4. Sakow D, Baabe D, Böker B, Burghaus O, Funk M, Kleeberg C,
Menzel D, Pietzonka C, Bröring M (2014) Chem Eur J 20:2913
IL 9
Mono- and dinuclear iron complexes as catalysts/
catalyst precursors for the oxidation of alkanes
and alkenes
Mainak Mitra1, Biswanath Das1, Julio Lloret-Fillol2, Afnan AlHunaiti3, Timo Repo3, Ivan Castillo Pérez4, Miquel Costas2, Ebbe
Nordlander1
1
Chemical Physics, Department of Chemistry, Lund University, Box
124, SE-221 00, Lund, Sweden. [email protected];
2
QBIS Group, Department of Chemistry, University of Girona,
Campus Montilivi, Girona E-17071, Spain;
3
Department of Chemistry, Laboratory of Inorganic Chemistry,
University of Helsinki, FI-00014, Helsinki, Finland;
4
Instituto de Quı́mica, Universidad Nacional Autónoma de México,
Circuito Exterior, CU, México, D.F. 04510, México
The abilities of a number of new mono- or dinuclear iron complexes
to act as catalysts for oxidation of alkanes/alkenes by hydrogen peroxide have been investigated [1, 2]. Good activities have been
observed when Fe(II) or Fe(III)–O–Fe(III) complexes of new tetradentate ligands are used as catalysts/catalyst precursors. The potential
influence of steric and electronic properties of the ligands on the
reactivities of the complexes will be discussed.
123
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J Biol Inorg Chem (2014) 19 (Suppl 2):S725–S747
Financial support by COST Action CM1003 and the EU Erasmus
Mundus program is gratefully acknowledged.
R-H
R-OH, R=O
Fe(II)(L)
(L)Fe(III)-O-Fe(III)(L)
H2O2
OH ,
HO
O
References
1. Mitra M, Lloret-Fillol J, Haukka M, Costas M, Nordlander E
(2014) Chem Commun 50:1408–1410
2. Das B, Al-Hunaiti A, Repo T, Castillo Pérez I, Nordlander E,
unpublished results
IL 10
Tailored gold-dithiocarbamato bioconjugates
for the targeted anticancer chemotherapy
Luca Ronconi
School of Chemistry, National University of Ireland Galway,
University Road, Galway, Ireland. [email protected]
The therapeutic success of the clinically-established platinum drugs
has triggered the development of several metal-based potential chemotherapeutic agents, most of which have failed to enter clinical
trials. In this context, during the last decade, we have been designing
a number of metal-dithiocarbamato complexes that were expected, at
least in principle, to resemble the main features of cisplatin together
with higher activity, improved selectivity and bioavailability, and
lower side-effects [1].
Among all, gold(III) derivatives were reported to exert outstanding
in vitro and in vivo antitumor activity and reduced, or even no, systemic
and renal toxicity, owing to the intrinsic chemoprotective function of
the coordinated dithiocarbamate. In particular, we aimed at designing
‘‘Trojan Horse’’-type complexes characterized by an improved selectivity provided by selected coordinated ligands exploiting specific
receptors up-regulated in cancer cells, so as to achieve biomolecular
recognition and tumor targeting without affecting healthy tissues [2].
Starting from the rationale behind our research work, the main
results achieved to date are here summarized, focusing on the
development of gold-based bioconjugates for the targeted chemotherapy. New prospects opened up by these anticancer agents and
currently under investigation in our group are illustrated and discussed, including the exploitation of either vitamin B12 or glucose
derivatives as tumor site-specific delivery carriers of bioactive
gold(III)-dithiocarbamato species.
Financial support by the National University of Ireland Galway
(CoS Scholarship) and the COST Action CM1105 (STSM Grant) is
gratefully acknowledged.
chemoprotective function (dithiocarbamate)
1. vitamin B12
labile sites
(Cl, Br)
2. glucose derivatives
anticancer core (metal)
References
1. Nagy EM, Ronconi L, Nardon C, Fregona F (2012) Mini-Rev Med
Chem 12:1216–1229
2. Ronconi L, Nardon C, Boscutti G, Fregona D (2013) In: Prudhomme M (ed) Advances in anti-cancer agents in medicinal
chemistry, vol 2. Bentham Science Publishers, Bussum, pp 130–172
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IL 11
Mechanistic studies on cytotoxic gold compounds
Luigi Messori1, Lara Massai1, Federica Scaletti1, Tiziano Marzo1,
Chiara Gabbiani2
1
Department of Chemistry, University of Florence, via della
Lastruccia 3, 50019 Sesto Fiorentino (Firenze), Italy;
2
Department of Chemistry and Industrial Chemistry, University
of Pisa, Italy
Gold compounds form an attractive class of antiproliferative agents of
potential use as anticancer agents. The molecular mechanisms
through which gold compounds produce their biological effects are
still largely unknown and the subject of intense investigations. Recent
studies point out that the mechanism of action of cytotoxic gold
compounds are essentially DNA-independent and cisplatin-unrelated,
most likely relying on gold interactions with a few crucial proteins.
Notably, various cellular proteins playing relevant functional roles
were proposed to represent effective targets for cytotoxic gold compounds but these hypotheses still need validation [1].
Our research group has focused attention on the likely protein
targets for cytotoxic gold compounds and on the elucidation of their
molecular mechanisms. At present this goal is being pursued through
two distinct approaches that will be described in detail.
On one hand, studies are being carried out on isolated proteins;
their interactions with representative gold compounds are studied
through independent biophysical methods. Particularly informative is
the joint use of X-ray diffraction and ESI MS on small model proteins
that allowed us to define the actual molecular mechanisms of protein
metalation [2].
On the other hand, studies are directed to monitor the alterations in
the cell proteome caused by cancer cell exposure to cytotoxic gold
compounds. This goal may be achieved through classical proteomic
strategies. In a recent study we considered the effects of the gold(III)
complex Aubipyc on A2780 ovarian cancer cells. From comparative
analysis of 2D gels of treated versus control cancer cells it was
possible to highlight a few protein spots that showed appreciable
changes in their expression rates following drug exposure [3]. Subsequent bioinformatic analysis of these proteomic alterations provided
valuable insight on the underlying biochemical processes ultimately
leading to cell death.
References
1. Nobili S, Mini E, Landini I, Gabbiani C, Casini A, Messori L
(2010) Med Res Rev 30:550
2. Messori L, Scaletti F, Massai L, Cinellu MA, Gabbiani C, Vergara
A, Merlino A (2013) Chem Commun 49:10100
3. Gamberi T, Massai L, Magherini F, Landini I, Fiaschi T, Scaletti F,
Gabbiani C, Bianchi L, Bini L, Nobili S, Perrone G, Mini E, Messori
L, Modesti A (2014) J Proteomics (in press)
IL 12
The chemistry and mechanistic biology of vanadium
as an anticancer agent
Athanasios Salifoglou
Department of Chemical Engineering, Aristotle University
of Thessaloniki, Thessaloniki 54124, Greece. [email protected]
Vanadium is a transition metal element widely encountered in the
planet in abiotic formations as well as biological systems. Its presence
in biota is linked to essential physiological processes associated with
catalytic transformations vital to plant and marine organism growth.
Beyond the specific functions, vanadium has been found to act as an
exogenous factor influencing insulin mimetic activity and antitumorigenicity [1]. Poised to investigate the impact of well-defined
aqueous forms of vanadium on pathways and target biomolecules
J Biol Inorg Chem (2014) 19 (Suppl 2):S725–S747
(oncogenes-proteins) involved in cancer cell physiology, synthetic
chemistry [2, 3] targeting ternary V(V)-H2O2-betaine systems led to a
well-characterized family of vanadoforms. A select such novel ternary V(V)-peroxido-betaine species (Fig. 1) form that family was
employed in experimental work targeting cell viability, apoptosis,
ROS production, H-ras signaling, and MMP-2 expression in two cell
lines, namely human breast cancer epithelial MCF-7 and lung adenocarcinoma A549 cells. The experimental data reveal that vanadium
has a significant effect on cancer cells, exemplified as a decrease in
cancer cell viability, inducing reduction of the H-ras and MMP-2
expression by increasing ROS-mediated apoptosis. To this end, the
observed chemical reactivity of vanadium distinctly emphasizes the
importance of nature, structure and properties of ternary ligands
formulating vanadium anti-tumor activity [4]. It appears that the
presence of the peroxido as well as betaine ligands bound to
V(V) goes along with the presence of the diperoxo moiety bound to
V(V), inflicting preferential damage to cancer cells, thereby setting
the stage for further perusal into vanadium’s future potential as a
metallodrug.
Fig. 1 Molecular structure of the V(V)-betaine-peroxido species in
aqueous media
References
1. Marzban L, McNeill J (2003) J Trace Elem Exp Med 16:253–267
2. Kaliva M, Raptopoulou CP, Terzis A, Salifoglou A (2003) J Inorg
Biochem 93:161–173
3. Kaliva M, et al. (2004) Inorg Chem 43:2895–2905
4. Petanidis S, Kioseoglou E, Hadzopoulou-Cladaras M, Salifoglou A
(2013) Cancer Lett 335:387–396
IL 13
Biogenesis, function, and catabolism
of the molybdenum cofactor: from biochemistry
to therapy
Guenter Schwarz
Institute of Biochemistry, Department of Chemistry and Center
for Molecular Medicine Cologne, University of Cologne, Germany.
[email protected]
In eukaryotes, the molybdenum cofactor (Moco) is composed of a
reduced metal binding pterin harboring a third pyranoring with an
ene-dithiolate being essential for molybdenum ligation. Amongst five
known eukaryotic molybdenum-dependent enzymes, sulfite oxidase is
most important for animals while the survival of plants is dependent
on nitrate reductase. Moco is synthesized by a complex biosynthetic
pathway, which we have extensively studies in the past [1] and
recently, a fully defined in vitro system of Moco biosynthesis has
been established. Moco deficiency (MoCD) is characterized by severe
and rapidly progressing neurological damage caused by the loss of
sulfite oxidase activity. Without effective therapy, death in early
infancy has been the usual outcome. For MoCD type A patients that
carry a mutation in MOCS1, an experimental substitution therapy
with cyclic pyranopterin monophosphate (PMP), the first intermediate
in the Moco pathway, has been established. In treated patients, Moco
S729
enzyme-dependent biomarkers normalized within days after treatment
was initiated and clinically, significant improvement, in some cases
normal neurological development, has been documented. Substitution
of cPMP represents the first causative therapy available for MoCD
patients and future research focuses on the molecular basis of neurodegeneration in MoCD. Furthermore, catabolism of Moco has
identified novel links between molybdenum and drug metabolism.
Reference
1. Schwarz G, Mendel RR, Ribbe MW (2009) Nature 460:839–847
IL 14
Metal chelating pseudopeptides and their potential
medical applications
Anne-Solène Jullien1, Marie Monestier1, Anaı̈s Pujol1, Colette
Lebrun1, Christelle Gateau1, Peggy Charbonnier2, Martine
Cuillel2, Elisabeth Mintz2, Pascale Delangle1
1
CEA, Univ. Grenoble Alpes, INAC, SCIB, RICC, F-38054
Grenoble, France; 2CEA, CNRS, Univ. Grenoble Alpes, IRTSV,
LCBM, Biomet, F-38054 Grenoble, France. [email protected]
Wilson’s disease (WD) is one of the major genetic disorders of copper
(Cu) metabolism in human. In this rare disease the ATPase ATP7B in
charge of the excretion of excess Cu from the liver cells is defective
and consequently, toxic Cu accumulates in the liver [1]. To treat WD,
we propose innovative Cu chelators, with the following properties:
(i) they show a high affinity for Cu(I), which is the oxidation state of
excess intracellular Cu (ii) they are selective for Cu(I) with respect to
potentially competing endogeneous metals such as Zn(II) and (iii)
they are water soluble. Mimics of binding sites found in proteins
involved in Cu homeostasis are good candidates that meet all these
expectations. Therefore, pseudopeptides were designed to mimic
Cu(I) coordination by metal-sequestering proteins such as metallothioneins. They are built from nitrilotriacetic acid (NTA) as a tripodal
anchor and functionalized with three amino acids cysteine or D-Penicillamine [2, 3]. The C3-symmetric nonbiological scaffold acts as a
platform that orients the three binding arms in the same direction to
coordinate the Cu(I) ion.
To promote intracellular Cu chelation and internalization in
hepatocytes, these sulphur ligands have been functionalized with
sugar units to target liver cells via the asialoglycoprotein receptors
(ASGP-R). The obtained glycoconjugates were demonstrated to
release high affinity Cu chelating agents in the hepatic cells. This
confirms that the use of intracellular Cu(I) chelators is very promising
to treat Cu overload in WD [4].
Financial support by FRM (DCM20111223043), ANR (COPDETOX, ANR-11-EMMA-025) and Labex Arcane (ANR-11-LABX0003-01)
References
1. Delangle P, Mintz E (2012) Dalton Trans 41:6359–6370
2. Jullien AS, et al. (2013) Inorg Chem 52:9954–9961
3. Jullien AS, et al. (2014) Inorg Chem 53 (in press)
4. Pujol AM, et al. (2012) Angew Chem Int Ed 51:7445–7448
123
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IL 15
His-containing peptides: fine-tuning their features
to obtain Cu(II) complexes with interesting properties
Ana Fragoso1, Tiago Carvalho2, Zrinka Aljinović2, Pedro
Lamosa1, Daniela Valensin3, Margarida M. Correia4, Rita
Delgado1, Olga Iranzo1,2
1
Instituto de Tecnologia Quı́mica e Biológica, Universidade Nova de
Lisboa, 2780-157 Oeiras, Portugal;
2
Aix Marseille Université, Centrale Marseille, CNRS, iSm2 UMR
7313, 13397 Marseille, France. [email protected];
3
Department of Biotechnology, Chemistry and Pharmacy, University
of Siena, 53100 Siena, Italy.
4
Centro de Quı́mica Estrutural, Instituto Superior Técnico,
Universidade de Lisboa, 1049-001, Lisboa, Portugal
Designing small peptides capable of binding Cu(II) mainly by the side
chain functionalities and forming single species in the neutral pH
range is a hard task since the amide nitrogens strongly compete for
Cu(II) coordination [1, 2]. However, metalloproteins are proficient on
this and generate the appropriated coordination pocket to avoid amide
coordination. This exquisite control allows copper proteins to attain a
myriad of catalytic activities and thus, a large variety of biological
functions [3, 4]. Achieving this with short peptides will be very
appealing to engineer miniaturized copper proteins with potential
redox and hydrolytic activities.
In this communication we report different peptides containing
several His but backbones with different degree of conformational
constrain. Their metal ion coordination properties have been studied
using electroanalytical (potentiometry and cyclic voltammetry) and
spectroscopic methods (UV–Vis, CD, EPR and NMR) [5, 6]. The
results show that the distinct flexible nature of the scaffolds has
remarkable effects on their metal ion coordination properties as well
as on metal ion exchange rate and redox potentials. Possible consequences of all these findings in catalysis will be discussed.
Financial support by FCT (PTDC/QUI-BIQ/098406/2008, REDE/
1517/RMN/2005, REDE/1504/REM/2005), Marie Curie Actions
(FP7-PEOPLE-IRG) and Leonardo da Vinci and Erasmus Programs
are gratefully acknowledged.
References
1. Sigel H, Martin RB (1982) Chem Rev 82:385–426
2. Kozłowski H, Bal W, Dyba M, Kowalik-Jankowska T (1999)
Coord Chem Rev 184:319–346
3. Holm RH, Kennepohl P, Solomon EI (1996) Chem Rev
96:2239–2314
4. Gaggelli E, Kozłowski H, Valensin D, Valensin G (2006) Chem
Rev 106:1995–2044
5. Fragoso A, Lamosa P, Delgado R, Iranzo O (2013) Chem Eur J
19:2076-2088
6. Fragoso A, Delgado R, Iranzo O (2013) Dalton Trans
42:6182–6192
IL 16
Organometallic anticancer agents
with pyridinecarbothioamide ligands for selective
delivery and enzyme inhibition
Sally Moon1, Stefan Wiezorek1, Kelvin Tong1, Chih-Hsiang Lin1,
Sam Meier2, Muhammad Hanif1, Stephen Jamieson3, Michael A.
Jakupec2, Zenita Adhireksan4, Curt A. Davey4, Bernhard K.
Keppler2, Christian G. Hartinger1
123
J Biol Inorg Chem (2014) 19 (Suppl 2):S725–S747
1
School of Chemical Sciences, University of Auckland, Private Bag
92019, Auckland 1142, New Zealand. [email protected];
2
Institute of Inorganic Chemistry, University of Vienna, Waehringer
Str. 42, 1090 Vienna, Austria;
3
Auckland Cancer Society Research Centre, University of Auckland,
Private Bag 92019, Auckland 1142, New Zealand;
4
Nanyang Technological University, School of Biological Sciences,
Division of Structural Biology and Biochemistry, 60 Nanyang Drive,
Singapore 637551, Singapore
Many widely used chemotherapeutics show low selectivity for tumor
tissue and cells [1]. In order to overcome this issue, we have developed
strategies based on organometallic pharmacophores which feature
bioactive ligand fragments based on the pyridinecarbothioamide
(PCA) scaffold. These ligand systems act as gastric mucosal protectants with low acute toxicity in vivo [2], but upon coordination to RuII–
and OsII–arene moieties they were shown to be highly cytotoxic. Data
on this new class of complexes developed for oral administration will
be presented. Crystallographic studies with the nucleosome core particle (NCP) suggest high affinity to the histone proteins at the NCP and
particularly at histidine residues at an extensive histone dimer–dimer
and dimer–tetramer interface indicating interference with chromatin
dynamics as a possible mode of action [3]. Furthermore, we have
functionalized the PCA ligand with maleimide and salicylic acid
derivatives for selective delivery by human serum albumin as a vector
and as COX inhibitors, respectively.
Financial support by the University of Auckland, the Austrian
Science Fund, COST CM1105, the Royal Society of New Zealand,
Genesis Oncology Trust, and the Higher Education Commission of
Pakistan is gratefully acknowledged.
References
1. Jakupec MA, Galanski M, Arion VB, Hartinger CG, Keppler BK
(2008) Dalton Trans 183
2. Kinney WA, Lee NE, Blank RM, Demerson CA, Sarnella CS,
Scherer NT, Mir GN, Borella LE, Dijoseph JF, Wells C (1990) J Med
Chem 33:327
3. Meier SM, Hanif M, Adhireksan Z, Pichler V, Novak M, Jirkovsky
E, Jakupec MA, Arion VB, Davey CA, Keppler BK, Hartinger CG
(2013) Chem Sci 4:1837
IL 17
CO2 fixation a possible biological function
for the patellamide family of marine cyclic
octapeptides? Insights from model systems
Nina Dovalil1–3, Michael Westphal1–3, Lawrence R. Gahan2,
Marcel Maeder4, Peter Comba3, Graeme R. Hanson1
1
Centre for Advanced Imaging and
2
School of Chemistry and Molecular Biosciences, The University
of Queensland, Brisbane, Queensland, Australia, 4072.
[email protected];
3
Anorganisch-Chemishes Insitut, Universitat Heidelberg, Im
J Biol Inorg Chem (2014) 19 (Suppl 2):S725–S747
Neuenheimer Feld 270, 69120 Heidelberg, Germany.
[email protected]; 4Department of Chemistry, The
University of Newcastle, Callaghan, NSW 2308, Australia
The copper coordination chemistry of six pseudo-octapeptides, synthetic analogues of ascidiacyclamide and the patellamides, found in
ascidians of the Pacific and Indian oceans, has been studied extensively and is sensitive to the choice of heterocyclic rings,
stereochemistry and backbone flexibility [1–3]. The six pseudooctapeptides are also shown to be efficient carbonic anhydrase model
complexes with kcat up to 7.3 9 103 s-1 (uncatalyzed: 3.7 9 10-2 s1
; enzyme-catalyzed: 2 9 105–1.4 9 106 s-1) and a turnover number
(TON) of at least 1700, limited only by the experimental conditions
used [4]. So far, no copper-based natural carbonic anhydrases are
known, no faster model systems have been described and the biological role of the patellamide macrocycles is so far unknown. The
observed CO2 hydration rates depend on the configuration of the
isopropyl side chains of the pseudo-octapeptide scaffold, and the
naturally observed R*,S*,R*,S* geometry is shown to lead to more
efficient catalysts than the S*,S*,S*,S* isomers. The catalytic efficiency also depends on the heterocyclic donor groups of the pseudooctapeptides. Interestingly, the dicopper(II) complex of the ligand
with four imidazole groups is a more efficient catalyst than that of the
close analogue of ascidiacyclamide with two thiazole and two oxazoline rings. The experimental observations indicate that the
nucleophilic attack of a CuII-coordinated hydroxide at the CO2 carbon
center is rate determining, i.e. formation of the catalyst-CO2 adduct
and release of carbonate/bicarbonate are relatively fast processes.
References
1. Comba P, Dovalil N, Gahan LR, Hanson GR, Westphal M (2014)
Dalton Trans 43:1935–1956
2. Comba P, Dovalil N, Gahan LR, Haberhauer G, Hanson GR, Noble
CJ, Seibold B, Vadivelu P (2012) Chem Eur J 18:2578–2590
3. Comba P, Dovalil N, Hanson GR, Linti G (2011) Inorg Chem
50:5165–5174
4. Comba P, Gahan LR, Hanson GR, Maeder M, Westphal M (2014)
Dalton Trans 43:3144–3152
IL 18
Metal substituted rubredoxins: a sulfur rich
coordination site as models for metalloenzymes
José J. G. Moura, Biplab K. Maiti, Cı́ntia Carreira, Luisa B.
Maia, Marta S. P. Carepo, Sofia R. Pauleta, Isabel Moura
REQUIMTE/CQFB, Departamento de Quı́mica, Faculdade de
Ciências e Tecnologia, Universidade Nova de Lisboa, Campus de
Caparica, 2829-516 Caparica, Portugal. [email protected]
The design of metal substituted derivatives in proteins still offers one
of the most emerging fields in the study of metalloproteins. These
‘‘novel’’ compounds can enhance our understanding of the structural/
functional properties and/or design of metalloproteins, whose properties can mimic, enhance, and/or in some cases improve many
features found in the natural ones [1].
Rubredoxin provides an excellent scaffold for the design of other
metal-sulfur substituted derivatives due to its small size. Overall,
S731
metal-substituted rubredoxins containing 57Fe(II), Cu(I), Co(II),
Ni(II), Zn(II), Cd(II), Hg(II), Ga(III) and In(III) have been prepared
and characterized [2]. The Ni-substituted rubredoxin was speculated
as a size scale intermediate model compound of Ni-site of bacterial
hydrogenase [2, 3]. Currently several metal-substituted derivatives of
rubredoxin are available but molybdenum (tungsten)-derivative is
absent from this list. An obvious interest is the possibility of creating
a sulfur rich environment, as the one present in molybdenum enzymes
due to the presence of pyranopterins and in some cases additional
coordinating residues, such as cysteine or selenocysteine [4].
Work was supported by FCT (SFRH/BPD/63066/2009, PTDC/
QUI-BIQ/098071/2008).
References
1. Day MW, Hsu T, Joshua-Tor BL, Park J-B, Zhou ZH, Adams
MWW, Rees DC (1992) Protein Sci 1:1494–1507
2. Saint-Martin P, Lespinat PA, Fauque G, Berlier Y, Legall J, Moura
I, Teixeira M, Xavier AV, Moura JJG (1998) Proc Natl Acad Sci USA
85:9378–9380
3. Thapper A, Rizzi AC, Brondino CD, Wedd AG, Pais RJ, Maiti BK,
Moura I, Pauleta SR, Moura JJG (2013) J Inorg Biochem
127:232–237
4. Hille R (2013) Dalton Trans 42:3029–3042
IL 19
Bio-inspired thiolate metal complexes structural,
spectroscopic and redox properties, reactivity
Marcello Gennari1, Deborah Brazzolotto1, Maylis Orio2, Frank
Neese3, Maurice Van Gastel3, Serena DeBeer3, Jacques Pécaut4,
Carole Duboc1
1
Département de Chimie Moléculaire, Université J. Fourier, CNRS,
Grenoble, France. [email protected];
2
Max Planck Institute for Chemical Energy Conversion, Mülheim,
Germany;
3
Laboratoire de Spectrochimie Infrarouge et Raman, CNRS,
Villeneuve d’Ascq, France;
4
CEA-Grenoble, INAC, Grenoble, France
Metal sulfur bonds are largely found in metalloenzymes, and lead to
specific spectroscopic properties and original reactivity in comparison with complexes containing only N-or/and O-based ligands. On
the other hand, synthetic complexes with metal-thiolate bonds have
revealed some interesting properties that could be involved in biological processes. However, to rationalize the reactivity of such
systems, a deep investigation of their electronic properties is
required. In this context, we have synthesized and characterized
series of aliphatic thiolate complexes with various transition metal
ions (Ni, Co, Zn, Cu, Mn, V) [1–3]. Their electronic properties have
been explored via different spectroscopic techniques combined with
quantum chemistry. More recently, we went a step further by
looking at their reactivity, especially their ability to activate small
molecules, or to form disulfide bridges and how this process can be
controlled.
Financial support by the Labex Arcane (ANR-11-LABX-0003-01)
is acknowledged.
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References
1. Gennari M, Gerey B, Hall N, Pécaut J, Collomb M-N, Rouzières
M, Clérac R, Orio M, Duboc C (2014) Angew Chem Int Ed. doi:
10.1002/anie.201402125
2. Hall N, Orio M, Jorge-Robin A, Gennaro B, Marchi-Delapierre C,
Duboc C (2013) Inorg Chem 52:13424–13431
3. Gennari M, Pécaut J, DeBeer S, Neese F, Collomb M-N, Duboc C
(2011) Angew Chem Int Ed 50:5662–5666
4. Gennari M, Orio M, Pécaut J, Neese F, Collomb M-N, Duboc C
(2010) Inorg Chem 49:6399–6401
IL 20
Periplasmic binding protein can capture several
siderophores
Daniel J. Raines, Axel Müller, Olga V. Moroz, Keith S. Wilson,
Anne-K. Duhme-Klair
Department of Chemistry, University of York, Heslington, York
YO10 5DD, UK. [email protected]
Bacteria produce siderophores to acquire essential Fe(III). Whilst the
most efficient siderophores are hexadentate, others are only tetradentate or even didentate but still bind Fe(III) and mediate its uptake.
The Fe(III) complexes of hexadentate siderophores and mimics, such
as MECAM6-, are coordinatively saturated and interact with their
binding proteins through a combination of electrostatic interactions
and hydrogen bonding (Figure, left) [1].
Tetradentate siderophores, however, can occupy only four of the
six coordination sites of octahedral Fe(III). We determined the crystal
structures of the Fe(III) complexes of a series of tetradentate and
didentate siderophores bound to the periplasmic binding protein CeuE
of Campylobacter jejuni and found that two amino acid side chains
coordinate directly to the Fe(III) centre, thus completing its coordination sphere (Figure, right) [2]. By displaying this switch in binding
mode, CeuE is the first siderophore binding protein to provide
structural insights into the binding of hexadentate, tetradentate and
didentate siderophores. We propose that the two amino acid residues
act as an adaptor that enables the binding protein to capture more than
one type of ferric siderophore.
J Biol Inorg Chem (2014) 19 (Suppl 2):S725–S747
IL 21
Understanding key factors of microbial iron uptake
processes: a physico-chemical study of artificial
siderophores
Elzbieta Gumienna-Kontecka1, Agnieszka Szebesczyk1, Jenny
Besserglick2, Evgenia Olshvang2, Abraham Shanzer2
1
Faculty of Chemistry, University of Wroclaw, F. Joliot-Curie 14,
50-383 Wroclaw, Poland. [email protected];
2
Weizmann Institute of Science, 234 Herzl Street, 76100 Rehovot,
Israel
At the time of increasing number of severe and often lethal infections
caused by multiresistant bacterial strains and fungi, the research in the
field of iron transport in microorganisms seems to be of great
importance. The difficulties in synthesis of structurally complicated
natural siderophores, has prompted research in the direction of artificial siderophores used as structural probes of microbial iron uptake
processes [1].
Our current research is focused on characterization of novel biomimetic compounds, artificial iron-carriers, in terms of iron complex
formation and stability. The interplay between structure and function
has been previously studied on biomimetic examples based on Ferrioxamine B [2], and here we will present a new series of ferrichrome
analogues, based on a tripodal template, where the asymmetric hexapeptide ring of the natural ferrichrome is replaced by a much simpler
C3 symmetric template, anchored to a quaternary carbon. Three
identical arms are comprised of a spacer containing an amino acid and
terminated by hydroxamate metal binding moiety. Various aminoacid
residues are studied, in order to determine the possibility of hydrogen
bonds formation and the role of side groups. Preliminary data show
that Fe(III) binding properties of studied analogs are close to natural
ferrichrome. Moreover, growth promotion studies show that these
biomimetic compounds are able to transfer iron to Pseudomonas
putida as efficiently as natural ferrichrome, and therefore act like
siderophores.
Financial support by the Polish National Science Centre (NCN,
UMO-2011/03/B/ST5/01057) is gratefully acknowledged.
References
1. Shanzer A, Felder CE, Barda Y (2009) In: Rappoport Z, Liebman
JF (eds) The chemistry of hydroxylamines, oximes and hydroxamic
acids. Wiley, Chichester 2:751–815
2. Kornreich Leshem H, Ziv C, Gumienna-Kontecka E, Arad-Yellin
R, Chen Y, Elhabiri M, Albrecht- Gary AM, Hadar Y, Shanzer A
(2005) J Am Chem Soc 127:1137–1145
IL 22
NMR spectroscopic studies on metallo-base-pair
in DNA duplex
References
1.Müller A, Wilkinson AJ, Wilson KS, Duhme-Klair AK (2006)
Angew Chem Int Ed 45:5132–5136
2. Raines DJ, Moroz OV, Wilson KS, Duhme-Klair AK (2013) Angew Chem Int Ed 52:4595–4598
123
Yoshiyuki Tanaka1, Itaru Okamoto2, Kyoko Furuita3, Jakub
Šebera4, Jiro Kondo5, Hidetaka Torigoe6, Hidehito Urata7,
Takenori Dairaku1, Akira Ono2, Chojiro Kojima3, Vladimı́r
Sychrovský4
1
Graduate School of Pharmaceutical Sciences, Tohoku University,
Sendai 980-8578, Japan;
2
Faculty of Engineering, Kangawa University, Yokohama, Kanagawa
221-8686 Japan;
J Biol Inorg Chem (2014) 19 (Suppl 2):S725–S747
3
Institute for Protein Research, Osaka University, Suita, Osaka
565-0871, Japan;
4
Institute of Organic Chemistry and Biochemistry, Academy
of Sciences of the Czech Republic, Flemingovo nám. 2, 16610, Praha
6, Czech Republic;
5
Faculty of Science and Technology, Sophia University, Tokyo
102-8554, Japan;
6
Faculty of Science, Tokyo University of Science; Shinjuku-ku,
Tokyo 162-8601, Japan;
7
Osaka University of Pharmaceutical Sciences, Takatsuki, Osaka
569-1094 Japan
We have studied structures of metal-mediated base-pairs, HgII-mediated T–T (T–HgII–T) and AgI-mediated C–C (C–AgI–C). We have
definitely determined the chemical structure of the T–HgII–T basepair with HgII-mediated 15N–15N J-coupling (2JNN) [1]. Based on this
chemical structure, we further determined 3-dimensional (3D) structure of a DNA duplex with tandem T–HgII–T base-pairs [2]. The T–
HgII–T base-pairs well mimics Watson–Crick base-pairs, which
explains why DNA polymerase elongated a DNA chain using the T–
HgII–T base-pair [3]. Within the duplex, Hg atoms are located along
with the helical axis of the DNA duplex, and are shielded from bulk
water solvent by the thymine bases and the stacked Watson–Crick
base-pairs. This structural feature well explains positive entropy (DS)
for the formation of a T–HgII–T base-pair within a DNA duplex [4],
which is known as a dehydration-entropy. In addition, a recently
determined crystal structure of a DNA duplex with tandem T–HgII–T
base-pairs [5] showed the Hg–Hg distance (3.3 Å) is close enough to
suggest the existence of the metallophillic attraction between heavy
elements [6].
References
1. Tanaka Y, Oda S, Yamaguchi H, Kondo Y, Kojima C, Ono A
(2007) J Am Chem Soc 129:244–245
2. Yamaguchi H, Šebera J, Kondo J, Oda S, Komuro T, Kawamura T,
Daraku T, Kondo Y, Okamoto I, Ono A, Burda JV, Kojima C, et al.
(2014) Nucleic Acids Res 42:4094–4099
3. Urata H, Yamaguchi E, Funai T, Matsumura Y, Wada S (2010)
Angew Chem Int Ed 49:6516–6519
4. Torigoe H, Ono A, Kozasa T (2010) Chem Eur J 16:13218–13225
5. Kondo J, Yamada T, Hirose C, Okamoto I, Tanaka Y, Ono A
(2014) Angew Chem Int Ed 53:2385–2388
6. Benda L, Straka M, Tanaka Y, Sychrovský V (2011) Phys Chem
Chem Phys 13:100–103
IL 23
DNA cleavage ability and cytotoxicity of monoand dinuclear copper complexes
Andrea Erxleben1, Diego Montagner1, Valentina Gandin2
1
School of Chemistry, National University of Ireland, Galway,
Ireland;
Dipartimento di Scienze del Farmaco, Università degli Studi di
Padova, Padova, Italy
Since the discovery of cisplatin, metal-based drugs for cancer therapy
is a rapidly growing field of medicinal chemistry. Cisplatin is one of
the most widely used anticancer drugs, although its high toxicity and
severe side-effects are significant limitations. Consequently, the
attention has shifted to less toxic transition metal ions, such as copper
which is an essential biometal. Cu complexes can interact with DNA
via intercalation or groove binding, cause oxidative DNA damage,
S733
induce apoptosis and a range of Cu complexes were found to exhibit
promising anticancer activity.
This lecture presents our recent work aimed at the development of
Cu-based DNA cleavage agents. As an example, the X-ray structure
of [Cu2{bcmp(-H)}(l-OH)](ClO4)2 (bcmp = 2,6-bis(1,4,7-triazacyclonon-1-ylmethyl)-4-methylphenol is shown below. The Cu complex
cleaves DNA in the presence of reducing agents and exhibits promising in vitro antitumor activity against pancreatic cancer cell lines.
Financial support by the European Commission and Science
Foundation Ireland is gratefully acknowledged.
IL 24
How much ‘‘wrong’’ metal can a metallothionein fold
take?
Claudia A. Blindauer, Maria Tareen
Department of Chemistry, University of Warwick, Gibet Hill Road,
Coventry CV4 7AL, UK. [email protected]
The small, cysteine-rich metallothionein (MT) proteins are thought to
be intrinsically disordered in the absence of metal ions, and to only
adopt an ordered structure upon metal-binding. Generally, MTs can,
in vitro, bind both mono- (Cu(I)) and divalent (Zn(II) and Cd(II))
metal ions. For Cys-only coordination, Cu(I) prefers linear/diagonal
and trigonal planar coordination geometries, and Zn(II) and Cd(II)
adopt tetrahedral geometry. In absolute thermodynamic terms,
Cu(I) always binds more strongly than either Cd(II) or Zn(II), but it is
inevitable that the protein backbone must adopt different conformations to allow for different coordination geometries. Since the
backbones of MTs tend to be highly flexible, it is a common
assumption that either M(I) or M(II) can be accommodated equally
well, but more recently, this idea has been questioned [1]. It is suggested that at least some MTs have evolved to best accommodate
either M(I) or M(II), with others showing no clear preference, leading
to a new classification system based on which metal ion is ‘‘preferred’’ [2]. We hypothesise that the observed preferences are
mediated through protein folding—with an ordered structure observed
only for the ‘‘correct’’ metal ion. We explore this idea using a prototypical Zn-MT, SmtA from the cyanobacterium Synechococcus
PCC7942. Zn4SmtA has a very well-defined 3D structure. Multinuclear NMR spectroscopy and native ESI–MS have been used to
characterise the products of reconstitution of the apo-protein with
Cu(I), as well as mixed-metal species generated during metal
replacement titrations. SmtA formed species with a range of M(I),
M(II) stoichiometries, with some retaining at least partially ordered
structures, whilst complete replacement with Cu(I) led to fully disordered protein. In addition, SmtA expressed recombinantly in the
123
S734
presence of surplus Cu(I) yielded mostly Zn4SmtA. We hypothesise
that structurally disordered metallospecies are either not formed, or
rapidly degraded in vivo, with concomitant Cu(I) remobilisation.
Support by the University of Warwick and Science City is
gratefully acknowledged.
J Biol Inorg Chem (2014) 19 (Suppl 2):S725–S747
4. Popovic-Bijelic A, Voevodskaya N, Domkin V, Thelander L,
Gräslund A (2009) Biochemistry 48:6532–6539
5. Leidel N, Popovic-Bijelic A, Havelius K, Chernev P, Voevodskaya
N, Gräslund A, Haumann M (2012) Biochim Biophys Acta
1817:430–444
6. Andersson CS, Högbom M (2009) Proc Natl Acad Sci USA
106:5633–5638
7. Griese JJ, Roos K, Cox N, Shafaat HS, Branca RMM, Lehtio J,
Gräslund A, Lubitz W, Siegbahn PEM, Högbom M (2013) Proc Nat
Acad Sci USA 110:17189–17194
IL 26
A molecular view of monothiol glutaredoxins in Fe/S
protein biogenesis
References
1. Bofill R, Capdevila M, Atrian S (2009) Metallomics 1:229–234
2. Palacios O, Atrian S, Capdevila M (2011) J Biol Inorg Chem
16:991–1009
IL 25
The dinuclear manganese/iron cluster: a new redoxactive enzyme cofactor
Astrid Gräslund
Department of Biochemistry and Biophysics, Stockholm University,
S-106 91 Stockholm, Sweden. [email protected]
Transition metal ions like iron and manganese are important components of many enzymes. Diiron carboxylate enzymes are wellknown participants in challenging oxidation reactions. Bacterial
multicomponent monooxygenases and protein R2 of class I ribonucleotide reductase (RNR) are typical examples of such proteins.
Some years ago, a new class of RNRs was identified in Chlamydia
trachomatis [1]. The enzyme was named class Ic RNR. EPR spectroscopy studies showed that it lacks the tyrosyl radical found in other
class I RNRs. As a metal cofactor this enzyme has a manganese/iron
cluster instead of the common diiron cluster found in class Ia RNRs
[2–5]. Somewhat later a similar manganese/iron cluster was identified
in a new type of bacterial enzymes, named R2lox proteins, with a
probable oxidase function [6].
In class Ic RNR, the manganese/iron cluster has a similar function
as the tyrosyl radical found in the R2 proteins of other class I RNRs.
This function is to provide a shielded, reversible electron storage site
during the enzymatic reaction, when the reduction of ribonucleotides
takes place at the active site about 35 Å away in protein R1.
The R2lox protein was discovered in Mycobacterium tuberculosis
(6). The protein catalyses 2-electron oxidations. A recently studied
homologue was isolated from the thermophilic bacterium Geobacillus
kaustophilus. The formation and catalytic activities of the manganese/
iron cluster in this enzyme, as well as the EPR spectroscopic properties, have been characterized [7]. The results from the class Ic R2
and the R2lox proteins show that the manganese/iron mixed metal
cofactor is a highly versatile redox-active catalyst.
References
1. Högbom M, Stenmark P, Voevodskaya N, McClarty G, Gräslund
A, Nordlund P (2004) Science 305:245–248
2. Jiang W, Yun D, Saleh L, Barr EW, Xing G, Hoffart LM, Maslak
MA, Krebs C, Bollinger JM Jr (2007) Science 316:1188–1191
3. Voevodskaya N, Lendzian F, Ehrenberg A, Gräslund A (2007)
FEBS Lett 581:3351–3355
123
Simone Ciofi Baffoni1,2, Julia Winkelmann1,2, Maciej
Mikolajczyk1, Riccardo Muzzioli1,2, Mario Piccioli1,2, Lucia
Banci1,2
1
Magnetic Resonance Center CERM, University of Florence, Via
Luigi Sacconi 6, 50019 Sesto Fiorentino, Florence, Italy.
[email protected];
2
Department of Chemistry, University of Florence, Via della
Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy
In eukaryotes, monothiol glutaredoxins have been proposed to play
crucial roles in iron-sulfur (Fe/S) protein biogenesis, intracellular
iron signalling and iron trafficking. Essentially all of them can
coordinate a [2Fe–2S] cluster and the [2Fe–2S]-bound forms of
monothiol glutaredoxins were suggested to be responsible for trafficking [2Fe–2S] clusters within the cell. The investigation at the
molecular level of such process is fundamental to fully define the
functional role of this protein family. Thanks to an integrated
structural biology approach, Fe/S cluster trafficking mechanisms are
described at atomic resolution for mitochondrial and cytoplasmic
pathways implicating human monothiol glutaredoxin-5 and glutaredoxin-3 [1–3]. The presented data show that monothiol
glutaredoxins act as cluster transfer proteins by following a specific
and cluster-mediated protein–protein recognition mechanism [4].
This mechanism guarantees a safe transfer at the cellular level of the
potentially harmful Fe/S clusters from one protein to another, up to
its final target protein.
References
1. Banci L, Bertini I, Calderone V, Ciofi-Baffoni S, Giachetti A,
Jaiswal D, Mikolajczyk M, Piccioli M, Winkelmann J (2013) Proc
Natl Acad Sci USA 110:7136–7141
2. Ciofi-Baffoni S, Gallo A, Muzzioli R, Piccioli M (2014) J Biomol
NMR 58:123–128
3. Banci L, Ciofi-Baffoni S, Mikolajczyk M, Winkelmann J, Bill E,
Pandelia ME (2013) J Biol Inorg Chem. 18:883–893
4. Banci L, Brancaccio D, Ciofi-Baffoni S, Del Conte R, Gadepalli R,
Mikolajczyk M, Neri S, Piccioli M, Winkelmann J (2014) Proc Natl
Acad Sci USA. Epub ahead of print.
IL 27
X-ray spectroscopic insights into the metalloprotein
active sites
Serena DeBeer1,2
1
Max Planck Institute for Chemical Energy Conversion, Stiftstr.
34-36, 45470 Mülheim an der Ruhr, Germany;
2
Department of Chemistry and Chemical Biology, Cornell University,
Ithaca, New York, 14850 USA. [email protected]
J Biol Inorg Chem (2014) 19 (Suppl 2):S725–S747
In recent years, advanced X-ray spectroscopic methods have
revealed key insights into the geometric and electronic structure of
metalloprotein active sites. This has included the identification of a
central carbon atom in the FeMo cluster in nitrogenase by valence to
core X-ray emission spectroscopy (V2C XES), and the presence of a
Mo(III) in nitrogenase by high-resolution fluorescence energy
detected X-ray absorption spectroscopy (HERFD XAS). The
strength of X-ray spectroscopic methods is in large part due the
element selectivity of these methods. This allows the Fe and Mo to
be separately probed in nitrogenase, or the Ni and Fe sites to be
separately probed in hydrogenase active sites. Similarly, the Mn and
Ca sites of the oxygen-evolving complex of photosystem II can be
separately evaluated. The selectivity of X-ray spectra can further
enhanced through resonant measurements. A key component in
understanding the spectra involves the close coupling of experiment
with theory. In this talk, recent work using X-ray absorption and
X-ray emission spectroscopies to understand the processes of dinitrogen reduction, hydrogen production and photosynthetic water
oxidation will be presented. The broad applicability of these methods to understand small molecule activation by transition metals will
be highlighted.
Financial support by the Max Planck Society and the European
Research Council is gratefully acknowledged.
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IL 29
Anion–nucleic acid interactions. Just anecdotal?
Pascal Auffinger, Luigi D’Ascenzo
Architecture et Réactivité de l’ARN, Université de Strasbourg,
Institut de Biologie Moléculaire et Cellulaire du CNRS, 15 rue René
Descartes, 67084 Strasbourg, France
Interactions with nucleic acids involving cationic species (mainly
Na+, K+ and Mg2+) are relatively well characterized. Conversely, less
is known about anionic species interacting with nucleic acids [1, 2].
Although the cytosolic concentration of Cl- ions is much smaller
(&4–20 mmol/L) than the extracellular concentration (&100 mmol/
L), Cl- ions have been recurrently found in nucleic acid crystallographic structures in close contact to electropositive nucleobase
groups. In this respect, Cl- anions present very specific binding
patterns and display strict coordination distances (&3.2 Å). We will
describe associated binding patterns along with binding properties of
other halide ions and put these patterns in relation with those of
3
further inorganic (SO2
4 ; PO4 , …) and organic (CH3 CO2 , …)
anions. Considerations related to the characterization of these anions
in crystallographic structures will also be presented. This is of
importance since some anion (Cl- or SO2
4 ) electron densities were
misattributed to Mg2+ cations, therefore blurring our perception of the
role they play in nucleic acid structure and function.
IL 28
Kinetic regulation of E. coli TPP-riboswitches
Sondes Guedich, Eric Ennifar, Dominique Burnouf, Philippe
Dumas
Biophysics and Structural Biology Team, IBMC, UPR9002 du CNRS
15 rue René Descartes, 67084 Strasbourg, France. [email protected]
Riboswitches are located in the 50 -UTR of mRNAs in bacteria and are
also found in the 30 -UTR of mRNAs in plants. We have studied the
functioning of two thiamine pyrophosphate (TPP) riboswitches, thiC
and thiM, in E. coli that can sense the intra-cellular level of TPP and
regulate either premature transcription arrest, as with thiC, or translation inhibition, as with thiM. We ascertained an ‘induced fit’
mechanism with initial loose binding of TPP subsequently transformed into tight binding after complete RNA folding. Hydroxyl
radical footprinting experiments showed that TPP initially binds
through its pyrimidine ring into the conserved TwC-like loop of the
aptamer. Using our newly developed kinITC technique [1], we
derived all kinetic and thermodynamic parameters for TPP binding
and RNA folding for the two bacterial riboswitches. These results are
clearly in favor of kinetic regulation since the affinity of these riboswithes for their ligand is too high to allow a classical
‘thermodynamic’ regulation. A theoretical analysis allowed us to link
the measured kinetic parameters to the ON- or OFF-state probability
of these riboswitches, which correctly predicts an ON/OFF switch at a
TPP concentration in the micromolar range. This also revealed how
the duration of programmed RNA-polymerase pauses ensuring kinetic
regulation [2] has to be linked to the off-rate of initial TPP interaction.
Remarkably, all such kinetically regulated riboswitches have the same
regulation efficiency, which incites to consider them as ‘standardized
pieces of biological hardware’.
References
1. Guedich S, Puffer-Enders B, Baltzinger M, Hoffmann G, Jossinet J,
Thore S, Ennifar E, Burnouf D, Dumas P (2012) J Am Chem Soc
134:559–565
2. Wickiser JK, Cheah MT, Breaker RR, Crothers DM (2005) Biochemistry, 44:13404–13414
References
1. Auffinger P, Bielecki L, Westhof E (2004) Structure 12:379–388
2. D’Ascenzo L, Auffinger P (2014) In: Ennifar E (ed) Methods in
molecular biology. Nucleic acids crystallography: methods and protocols. Humana Press, in press
IL 30
Cyclometalated DNA-Intercalating ruthenium(II)
complexes that target cancer cell nuclei and exhibit high
anticancer activities
Hui Chao
School of Chemistry and Chemical Engineering, Sun Yat-Sen
University, Guangzhou 510275, People’s Republic of China
Recently, coordinatively saturated and substitutionally inert Ru(II)
complexes have been investigated as anticancer agents. [Ru(bpy)2(dppz)]2+ strongly binds to DNA in aqueous media but does not
exhibit remarkable anticancer activity due to inefficient cellular
uptake. To interact with genomic DNA in living cells, molecules must
not only enter the interior of cells but be able to reach the cell nucleus.
Therefore, a convenient route for substitutionally inert Ru(II) complexes that reach their DNA target in cellulo is still highly sought. In
this paper, we present a structurally similar cyclometalated analogue,
[Ru(bpy)(ppy)(dppz)]+ (ppy = 2-phenylpyridine). Its in vitro
123
S736
anticancer activities were screened against a panel of cancer cell lines
and compared with cisplatin and [Ru(bpy)2(dppz)]2+, and the underlying mechanisms by which the complex caused cancer cell death
were elucidated. Remarkably, [Ru(bpy)(ppy)(dppz)]+ exhibited IC50
values (IC50 = 0.6–4.3 lM) that are lower than those of cisplatin for
the same cell line. Inductively coupled plasma mass spectrometry
(ICP-MS) accompanied by a Hoechst 33258 displacement assay
revealed that [Ru(ppy)(bpy)(dppz)]+ was rapidly incorporated by
cancer cells and accumulated in the nuclei. [Ru(ppy)(bpy)(dppz)]+
intercalated into DNA with high affinity and blocked transcription
factor binding to DNA sequences. Transcription factor hijacking
repressed the expression of backward genes and induced irreversible
cell apoptosis. Our results indicate that cyclometalation is an effective
means of improving the cell nuclei localization and anticancer
activity of DNA-intercalating Ru(II) complexes.
Financial supports by NSFC, the 973 program, and the Research
Fund for the Doctoral Program of Higher Education are gratefully
acknowledged.
References
1. Barry NPE, Sadler PJ (2013) Chem Commun 49:5106–5131
2. Komor CA, Barton JK (2013) Chem Commun 49:3617–3630
3. Kou JF, Qian C, Wang JQ, Chen X, Wang LL, Chao H, Ji LN
(2012) J Biol Inorg Chem 17:81–96
4. Chen X, Wu JH, Lai YW, Zhao R, Chao H, Ji LN (2013) Dalton
Trans 42:4386–4397
5. Zhang PY, Wang JQ, Huang HY, Qiao LP, Ji LN, Chao H (2013)
Dalton Trans 42:8907–8917
6. Wang JQ, Zhang PY, Qian C, Hou XJ, Ji LN, Chao H (2014) J Biol
Inorg Chem 19:335–348
IL 31
Near-infrared emitting lanthanide complexes
for optical imaging applications in biology: polymetallic
dendrimers and metal–organic frameworks
Stéphane Petoud1,2, Alexandra Foucault-Collet1, Svetlana V.
Eliseeva1, Kristy A. Gogick2, Kiley A. White2, Iuliia Nazarenko1,
Virginie Placide1, Sandrine Villette1, Nathaniel L. Rosi2
1
Center for Molecular Biophysics, CNRS UPR4301, Orléans, 45071,
France. [email protected];
2
Department of Chemistry, University of Pittsburgh, Pittsburgh, PA
15260, USA
Fluorescence and luminescence are detection techniques that possess
important advantages for bioanalytical applications and biologic
imaging: high sensitivity, versatility and low costs of instrumentation.
A common characteristic of biologic analytes is their presence in
small quantities among complex matrices such as blood, cells, tissue
and organs. These matrices emit significant background fluorescence
(autofluorescence), limiting detection sensitivity.
The luminescence of lanthanide cations has several complementary advantages over the fluorescence of organic fluorophores and
semiconductor nanocrystals, such as sharp emission bands for spectral
discrimination from background emission, long luminescence lifetimes for temporal discrimination and strong resistance to
photobleaching. In addition, several lanthanides emit near-infrared
(NIR) photons that can cross deeply into tissues for non-invasive
investigations and that result in improved detection sensitivity due to
the absence of native NIR luminescence from tissues and cells
(autofluorescence). The main requirement to generate lanthanide
emission is to sensitize them with an appropriate chromophore
(‘‘antenna effect’’).
An innovative concept for such sensitization of NIR-emitting
lanthanides is proposed herein; the current limitation of low quantum
yields experienced by most mononuclear lanthanide complexes is
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J Biol Inorg Chem (2014) 19 (Suppl 2):S725–S747
compensated for by using a large number of lanthanide cations and by
maximizing the absorption of each discrete molecule, thereby
increasing the number of emitted photons per unit of volume and the
overall sensitivity of the measurement. To apply this concept, we
have created two families of lanthanide compounds: metal–organic
frameworks [1] and dendrimer complexes [2] and succeeded in
generating highly emissive NIR reporters. We will discuss their
designs, structures, photophysical properties and their applications for
biological imaging in NIR microscopy.
This research was supported by a Marie Curie Intra European
Fellowship, La Ligue Contre le Cancer and La Région Centre. S.P.
acknowledges support from the Institut National de la Santé et de la
Recherche Médicale.
References
1. Foucault-Collet A, Gogick KA, White KA, Villette S, Pallier A,
Collet G, Kieda C, Li T, Geibb SJ, Rosi NL, Petoud S (2013) Proc
Natl Acad Sci USA 43:17199–17204
2. Foucault-Collet A, Shade CM, Nazarenko I, Petoud S, Eliseeva SV
(2014) Angew Chem Int Ed 53:2927–2930
IL 32
Effects of membrane and metal ion interactions
with amyloidogenic proteins
Daniela Valensin1, Riccardo De Ricco1, Caterina Migliorini1,
Marek Luczkowski2, Henryk Kozlowski2
1
Department of Biotechnology, Chemistry and Pharmacy, University
of Siena, Via Aldo Moro 2, 53100 Siena, Italy.
[email protected];
2
Faculty of Chemistry, University of Wroclaw, F. Joliot Curie 14,
50383 Wroclaw, Poland
Neurodegenerative disorders such as Alzheimer Disease (AD), Parkinson Disease (PD) and Transmisible Spongform Encelapthaties
(TSEs) are increasingly widespread and highly debilitating diseases,
characterized by the progressive loss of neuron structure and function,
which ultimately leads to neuronal death. The major hallmark of AD,
PD and TSEs pathologies is the presence of inclusion bodies mainly
made of protein aggregates in the brain, consisting of fibers assembled
by misfolded proteins with b-sheet conformation. Amyloid b (Ab), aSynuclein (aS) and human Prion Protein (hPrP) are the amyloidogenic proteins associated to AD, PD and TSEs respectively. They are
native unfolded but, after misfolding aggregate and accumulate as
deposits in the brain. Ab, aS also share the ability to undergo to ahelix structural transition in presence of membrane mimicking environments [see for example 1, 2].
Metals ions, especially copper, zinc and iron play very important
role in neurodegeneration having impact on both protein structure and
oxidative stress. Interestingly Ab, aS and hPrP exist in copper-metallated forms and huge number of evidence has pointed out that
specific binding domain exists for both Cu2+ and Cu+ oxidation states
[see for example 3, 4].
In this study, the effects of either copper binding [5] or membrane
interactions on the structural rearrangements of amyloidogenic proteins is discussed.
Financial support by PRIN (Programmi di Ricerca di Rilevante Interesse Nazionale) (2010M2JARJ_004), CIRMMP (Consorzio
Interuniversitario Risonanze Magnetiche di Metalloproteine Paramagnetiche) and CIRCMSB (Consorzio Interuniversitario di Ricerca in
Chimica dei Metalli nei Sistemi Biologici) is gratefully acknowledged.
References
1. Alderson TR, Markley JL (2013) Intrinsically Disord Proteins
1:18–39
J Biol Inorg Chem (2014) 19 (Suppl 2):S725–S747
2. Abelein A, Kaspersen JD, Nielsen SB, Jensen GV, Christiansen G,
Pedersen JS, Danielsson J, Otzen DE, Gräslund A (2013) J Biol Chem
288:23518–23528
3. Migliorini C, Porciatti E, Luczkowski, Valensin D (2012) Coord
Chem Rev 256:352–368
4. Kozlowski H, Luczkowski M, Remelli M, Valensin D (2012)
Coord Chem Rev 256:2129–2141
5. Migliorini C, Sinicropi A, Luczkowski M, Kozlowski H, Valensin
D (2014) J Biol Inorg Chem. doi:10.1007/s00775-014-1132-7
IL 33
Synergesic effects of dendrimers on metal ion chelators
with potential use in the therapy of Alzheimer’s disease
Tamás Kiss1,2, Éva Sija1, Tamás Jakusch,2, Dietmar Appelhans3
1
MTA-USZ Bioinorganic Chemistry Research Group, Dóm tér 7,
H-6720 Szeged, Hungary;
2
Department of Inorganic and Anylitical Chemistry, University
of Szeged, Dóm tér 7, H-6720 Szeged, Hungary; 3Leibniz Institute
of Polymer Research, Hohe Strasse 6, D-01069 Dresden, Germany
According to the amyloid cascade hypothesis, amyloid peptide
aggregation is related to the onset and development of Alzheimer’s
disease (AD). The first steps of b-amyloid oligomerisation are
assumed to be promoted by metal ions such as Cu2+, Fe3+, and Zn2+.
Various chelator molecules which can bind these metal ions stronger
than amyloids will hinder amyloidogenesis and thus prevent development of AD. Similarly, various organic molecules among others
glycodendrimers can also behave as anti-amyloidogenic agents. In
this work we studied the effects of two different maltose (Mal)
modified poly(propyleneimine)(PPI) type dendrimers [1] on various
efficient metal ion chelators [2, 3].
The dendrimers efficiently in blocked amyloid fibril formation alone
and both in the absence and the presence of chelators if Cu(II) ion was
added to the system in equimolar amount of the chelator. The larger
generation (PPI-G5-Mal) dendrimer was more efficient than the lower
generation (PPI-G4-Mal) dendrimer. It was interesting to observe that
the chelator and the dendrimer together showed more efficient antiamyloidogrenic effect than added separately. The observed synergistic
effect is probably due to some quaternary interactions between the
metal ion, the chelator, the dendrimer and the amyloid.
Financial support by the TAMOP 4.2.2.4A ‘‘Dementia, neurodegenerative diseases: early recognition, new therapies’’ EU project and
the OTKA 77833 is gratefully acknowledged.
References
1. Klementieve O, Benseny-Cases N, Gella A, Appelhans D, Voit B,
Cladera J (2011) Biomacromolecules 12:3903–3909
2. Lakatos A, Zsigó É, Hollender D, Nagy N.V, Fülöp L, Simon D,
Bozsó Zs, Kiss T (2010) 39:1302–1315
3. Lakatos A, Gyurcsik B, Nagy N.V, Csendes Z, Wéber E, Fülöp L,
Kiss T (2012) 41:1713–1726
IL 34
Novel bioorthogonal ‘‘click’’ reactions for flexible access
to Pd and Pt complexes with nanomolar biological
activity
Peter V. Simpson1, Olivier Nguyen1, Florian Geyer1, Luciano
Oehninger2, Ingo Ott2, Ulrich Schatzschneider1
1
Institut für Anorganische Chemie, Julius-Maximilians-Universität
Würzburg, Am Hubland, D-97074 Würzburg, Germany.
S737
[email protected]; 2Institut für Medizinische
und Pharmazeutische Chemie, TU Braunschweig, Beethovenstr. 55,
D-38106 Braunschweig, Germany
Cisplatin is one of the iconic molecules of medicinal inorganic
chemistry. Its anticancer activity is generally thought to be due to
ligand exchange reactions, ultimately leading to DNA adducts,
which, if not repaired, finally result in the triggering of cellular
apoptotic pathways [1]. A plethora of derivatives of this core
structure have been prepared and evaluated over the last few decades [2].
In the present contribution, we will introduce a novel and flexible
[3+1] approach to bioactive palladium(II) and platinum(II) complexes based on a bioorthogonal ‘‘click’’ reaction, leading to square
planar complexes. Surprisingly and against established structure–
activity relationships, the Pd compound was more active than the
isostructural Pt complex by a factor of about 30 and had midnanomolar activity, with IC50 values of 160 (HT-29) and 170 nM
(MDA-MB-231), respectively, thus showing at least one order of
magnitude higher potency than cisplatin used as a reference compound [3]. To probe the mechanism of action, the thioredoxin
reductase (TrxR) assay was carried out and showed significantly
different inhibition of this essential redox enzyme [4]. In line with
the in vitro cytotoxicity data, the Pd complex was about 150 times
more efficient in TrxR inhibition than its Pt analogue (IC50 0.02 vs.
2.44 lM). These results show that even small variations of a
seemingly well-established lead structure can still result in surprising biological activity.
References
1. Todd RC, Lippard SJ (2009) Metallomics 1:280–291
2. Wilson JJ, Lippard SJ (2014) Chem Rev. doi:10.1021/cr4004314
3. Simpson RV, Oehninger L, Ott I, Schatzschneider U, unpublished
results
4. Oehninger L, Küster LN, Schmidt C, Muñoz-Castro A, Prokop A,
Ott I (2013) Chem. Eur J 19:17871–17880
IL 35
Organometallic conjugates of peptide nucleic acids:
applications in biosensors and antibiotics
Nils Metzler-Nolte
Faculty of Chemistry and Biochemistry, Ruhr University Bochum,
Universitätsstrasse 150, 44801 Bochum, Germany. [email protected]
Our group has pioneered the chemical synthesis of metal conjugates
with peptide nucleic acids (PNA), which are mimics of DNA/RNA
with very promising properties. While PNA is very stable towards
chemical and enzymatic degradation, its application in biosensors
requires an added functionality. We have attached metallocenes to
PNA oligomers for electrochemical detection [1]. To this end, the
usual PNA oligomer synthesis scheme by solid phase methods had to
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be modified to accommodate the metal complex at the desired location. Moreover, we have used Cu-catalzed cycloaddition reactions
(‘‘Click chemistry’’) to incorporate different metal complexes with
different electrochemical potentials [2]. This so-called ‘‘four potential’’ detection [3] compares favourably to the well-established ‘‘four
colour’’ detection scheme with fluorescent dyes and allows for the
electrochemical detection of single base mismatches, e.g. in single
nucleobase polymorphism (SNP) [4]. The stability of such metallocene-PNA oligomers as well as their mismatch behaviour has been
studied in detail by UV melting experiments and CD spectroscopy
[5], and electron transfer kinetics through the PNA•DNA oligomers
were studied in detail [6].
Moreover, we have shown that metal complexes can be used to
quantify the cellular uptake of peptides and PNA oligomers.
Applying this strategy to PNA chemistry, we were able to directly
quantify the uptake of PNA oligomers using atomic absorption
spectroscopy [7], and more recently the mechanism of action of
PNA-based antibiotics was elucidated. These experiments underscore the potential of (organo)metal PNA conjugates for biological
applications [8, 9].
Financial support by the German Science Foundation (DFG) is
gratefully acknowledged.
References
1. Maurer A, Kraatz HB, Metzler-Nolte N (2005) Eur J Inorg Chem
3207–3210
2. Gasser G, Hüsken N, Köster SD, Metzler-Nolte N (2008) Chem
Commun 3675–3677
3. Hüsken N, Gasser G, Köster SD, Metzler-Nolte N (2009) Bioconjugate Chem 20:1578
4. Hüsken N, Gebala M, La Mantia F, Schuhmann W, Metzler-Nolte
N (2012) ChemPhysChem 13:131–139
5. Sosniak A, Gasser G, Metzler-Nolte N (2009) Org Biomol Chem
(2009) 7:4992–5000
6. Hüsken N, Gebala M, La Mantia F, Schuhmann W, Metzler-Nolte
N (2011) Chem Eur J 17:9678–9690
7. Kirin SI, Ott I, Gust R, Mier W, Weyhermüller T, Metzler-Nolte N
(2008) Angew Chem Int Ed 47:955
8. Wenzel M, Patra M, Senges CHR, Ott I, Stepanek JJ, Pinto A,
Prochnow P, Vuong C, Langklotz S, Metzler-Nolte N, Bandow JE
(2013) ACS Chem Biol 8:1442–1450
9. Gasser G, Sosniak AM, Metzler-Nolte N (2011) Dalton Trans
40:7061–7076
IL 36
Cyanocobalamin conjugates for live-cell IR imaging
and distribution of active photoCORMs
via synchrotron FTIR spectromicroscopy
Fabio Zobi1, Luca Quaroni1, Giuseppe Santoro1, Theodoa
Zlateva, Martin Obst2, Marcus C. Schaub3, Anna Yu. Bogdanova4
1
Department of Chemistry, University of Fribourg, Chemin de Musée
9, 1700, Fribourg, Switzerland;
2
Institute for Geoscience, Eberhard Karls University Tuebingen,
Hoelderlinstr. 12, 72074 Tuebingen, Germany;
3
Institute of Pharmacology and Toxicology, University of Zurich,
Winterthurerstrasse 190, 8057 Zurich, Switzerland;
4
Institute of Veterinary Physiology, University of Zurich,
Winterthurerstrasse 260, 8057 Zurich, Switzerland
Carbon monoxide releasing molecules (CORMs) are an emerging
class of pharmaceutical compounds currently evaluated in several
preclinical disease models [1–3]. There is general consensus that the
therapeutic effects elicited by the molecules may be directly ascribed
to the biological function of the released CO. It remains unclear,
however, if cellular internalization of CORMs is a critical event in
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J Biol Inorg Chem (2014) 19 (Suppl 2):S725–S747
their therapeutic action. In this contribution we will outlined our
recent efforts in studying the uptake and cellular distribution of
CORM conjugate via synchrotron FTIR spectromicroscopy measurements on living cells [4]. New approaches for the 3D
morphological distribution and localization of CORM species (and
organometallic complexes in general) within a single cell will be
presented. We will show imaging representations capable of correlating specific chemical vibrational fingerprints to subcellular
structures in complex cellular topography.
References
1. Motterlini R, Otterbein L (2010) Nat Drug Discov 9:728–742
2. Mann BE (2010) Top Organomet Chem 32:247–285
3. Zobi F (2013) Fut Med Chem 5:175–188
4. Zobi F, et al. (2013) J Med Chem 56:6719–6731
IL 37
Biomimetic B12-protein cofactor complexes
Felix Zelder, Marjorie Sonnay, Kai Zhou, Christine MännelCroisé
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland. [email protected]
Cobalamins are molecular switches with the nucleotide base either
being coordinated to or dissociated from the cobalt center of the
corrin macrocycle (‘‘base on/base off’’ equilibrium) [1]. In this
manner, the natural product triggers cellular uptake as well as its
chemical properties and biological functions in B12 cofactor dependent reactions.
My group develops modified B12 derivatives for fundamental
studies, analytical purposes and medicinal applications [2]. We are
also interested in biomimetic as well as ‘‘unusual’’ coordination
modes of B12 derivatives. Recently, we developed an immobilisation
strategy for synthesizing the first biomimetic model of a ‘‘base-off/
histidine on’’ B12-protein complex (scheme left) [3].
In another study, we reported the first dimeric cobalamin derivative with an ‘‘intra base off/inter base on’’ coordination mode (scheme
right) [4]. The constitution at the cobalt ion and the introduction of a
J Biol Inorg Chem (2014) 19 (Suppl 2):S725–S747
flexible linker were of utmost importance. Recent developments in the
design of B12 models will be discussed.
Financial support by the University of Zurich, the Forschungskredit of the University of Zurich and the Swiss National Science
Foundation is gratefully acknowledged.
References
1. Kräutler B, Arigoni D, Golding B (1998) Vitamin B12 and B12proteins, Wiley–VCH, Weinheim
2. Zelder F, Zhou K, Sonnay M (2013) Dalton Trans 42:854–862
3. Männel-Croisé C, Zelder F (2011) Chem Commun
47:11249–11251
4. Zhou K, Zelder F (2011) Chem Commun 47:11999–12001.
IL 38
Correlation of trace metal ions and the development
of infectious diseases in acutely Ill hospitalized patients
Peggy L. Carver1, Imad F. Btaiche1, Kathy Welch2
1
University of Michigan College of Pharmacy, Department
of Clinical, Social, and Administrative Sciences, and University
of Michigan Hospitals, 428 Church Street, Ann Arbor, MI, USA.
[email protected];
2
University of Michigan Center for Statistical Consulting
and Research, Ann Arbor, MI, USA
Although patients with : plasma concentrations of Fe have an : risk
of certain bacterial and fungal infections, the effect of : or ; plasma
concentrations of other trace metal ions (TEs) is poorly understood.
TEs are important components of nutrition, are required for cellular
growth and metabolism, and may play a role patients’ susceptibility to
infection [1]. We evaluated hospitalized patients to determine the
relationship between bacterial and fungal infections and plasma
concentrations of select trace metal ions (Zn, Cu, Se, Mn) and Fe.
Methodology: Data collection included all fungal and bacterial
infections, doses and plasma concentrations of TEs, and risk factors
for documented infections (primarily blood stream infections and
pneumonia) in [750 acutely ill, hospitalized patients evaluated from
2000–2013. Descriptive statistics were used for quantitative data, and
a recurrent events survival analysis and a Glimmix procedure to
analyze plasma concentrations of the trace metals, and their correlation with infections.
Results: In hospitalized patients, % Fe saturation and IV dosages of
Fe were predictive of an : risk of infection, particularly for Staphylococcus aureus. Higher plasma levels of Zn and Mn were highly
predictive of an overall : risk of gram negative and Candida infections;
modest correlations were seen for Zn and Mn. : Cu/Zn and ; Mn/Fe
ratios strongly correlated (P = 0.003) with an : risk of infections.
Conclusions: Correlations found between TE doses and plasma [TE]s,
and with [TE]s and infections may help to improve our dosing and
monitoring of trace elements, and minimize the risk of inadequate
nutrition or life-threatening infections in acutely ill hospitalized patients.
Reference
1. Btaiche IF, Carver PL, Welch KB (2011) J Parenter Enter Nutr
35:736–747
IL 39
Regulation of metabolism by heme, redox and carbon
monoxide
Stephen W. Ragsdale1, Eric Carter1, Angela Fleischhacker1,
Andrea M. Spencer1, Ireena Bagai1, Ajay Sharma2, Erik
Zuiderweg1, Donald Becker3, Brian M. Hoffman2, Ritimukta
Sarangi4
S739
1
Department of Biological Chemistry, University of Michigan, Ann
Arbor, Michigan 48109, USA. [email protected];
2
Department of Chemistry, Northwestern University, Evanston, IL
60208, USA; Department of Biochemistry, University of Nebraska,
Lincoln, NE, 68588, USA;
4
Stanford Synchrotron Radiation Lightsource, SLAC National
Accelerator Laboratory, Menlo Park, CA 94025, USA
The lecture describes results supporting a model for the regulation of
various metabolic and systemic processes by linking heme homeostasis to the cellular redox poise and to gas signaling molecules. This
model pertains to proteins that contain a heme-binding domain, which
is regulated by a thiol/disulfide redox switch involving specific cysteine residues that undergo interconversion between dithiol and
disulfide redox states. Because heme is the site of action of gaseous
signaling molecules, these proteins are subject to another layer of
regulation in which binding of CO and NO can reverse, or amplify the
effect of heme. This model is described in relation to recent studies on
heme oxygenase-2 (HO2) [1, 2], the BK potassium ion channel [3]
and the nuclear receptor, Rev-erbb [4]. Furthermore, because HO and
nitric oxide synthase are the sources of endogenous CO and NO in
higher organisms, these enzymes play a crucial role in this model.
Several recent reviews of this work are available [5–7].
Financial support from NIH-NHLBI (HL 102662A) is gratefully
acknowledged.
References
1. Yi L, Jenkins PM, Leichert LI, Jakob U, Martens JR, Ragsdale SW
(2009) J Biol Chem 284:20556–20561
2. Yi L, Ragsdale SW (2007) J Biol Chem 282:20156–21067
3. Yi L, Morgan JT, Ragsdale SW (2010) J Biol Chem
285:20117–20127
4. Gupta N, Ragsdale SW (2011) J Biol Chem 286:4392–4403
5. Ragsdale SW, Yi L (2011) Antioxid Redox Signal 14:1039–1047
6. Ragsdale SW, Yi L, Bender G, Gupta N, Kung Y, Yan L, Stich TA,
Doukov T, Leichert L, Jenkins PM, Bianchetti CM, George SJ,
Cramer SP, Britt RD, Jakob U, Martens JR, Phillips GN, Drennan CL
(2012) Biochem Soc Trans 40:501–507
7. Carter EL, Ragsdale SW (2014) J Inorg Biochem 133:92–103
IL 40
Anti-oxidant Mn-complexes: evaluation in cellular
models of oxidative stress
Clotilde Policar1,2,3, Anne-Sophie Bernard1,2,3, Nicolas Delsuc1,2,3,
Géraldine Gazzah1,2,3, Manon Guille1,2,4, Frédéric Lemaı̂tre1,2,4,
Christian Amatore1,2,4, Maria Bachelet2,5,6, Joelle Masliah2,5,6
1
Ecole Normale Supérieure-PSL Research University, Département
de Chimie, 24 rue Lhomond F-75005 Paris, France;
2
Sorbonne Universités, UPMC Univ Paris 06, F-75005 Paris, France;
3
CNRS, UMR 7203 LBM, F-75005, Paris, France;
5
INSERM-ERL 1157, CHU St Antoine, 27 rue de Chaligny, F-75012
Paris, France;
6
Inflammation-Immunopathology-Biotherapy Department (DHU
i2B), 27 rue de Chaligny, F-75012, Paris, France
Oxidative burst is a relevant biomedical target because it is involved
in a wide range of diseases. The beneficial outcome of superoxide
removal is now well documented [1, 2]. SOD-mimics are small
complexes that reproduce the activity of superoxide dismutases [3],
natural proteins that catalytically dismutate the superoxide anion. A
wide range of SOD-mimics have been studied in the last decade,
showing intrinsic SOD-activity—id est out of any cellular context
[3]—but also anti-oxidant properties in cells or in animals models [4].
Clearly, a SOD-mimic can be active outside any cellular context but
may show inactivity within cells or a whole organism. The
123
S740
implementation into cellular and biological environments is thus not
straightforward.
Activated macrophages, which produce ROS and RNS fluxes,
constitute a relevant model to challenge antioxidant activity in a
cellular context and they were used to test the activity of a SODmimic
developed in our group [5]. Additional cellular model will also be
presented.
Acknowledgements: ENS is gratefully acknowledged for A-S.B.’s
thesis fellowship, and ANR for financial support (ANR-Metabact).
J Biol Inorg Chem (2014) 19 (Suppl 2):S725–S747
Financial support from DGAPA-UNAM IN22713 and Conacyt
CB-2012-178851 are acknowledged
References
1. Alfaro-Fuentes I, López-Sandoval H, Mijangos E, Duarte-Hernández AM, Rodriguez-López G, Bernal-Uruchurtu MI, Contreras R,
Flores-Parra A, Barba-Behrens N (2014) Polyhedron 67:373–380
2. Betanzos-Lara S, Gómez-Ruiz C, Barrón-Sosa LR, Gracia-Mora I,
Flores-Álamo M, Barba-Behrens N (2012) J Inorg Biochem
114:82–93
References
1. McCord JM, Edeas MA (2005) Biomed Pharmacother 59:139–142
2. Batinic-Haberle I, et al. (2010) Antioxid Redox Signal 13:877–918
3. Iranzo O (2011) Bioorg Chem 39:73–87
4. Cisnetti F, et al. (2007) Eur J Inorg Chem 4472–4480
5. Bernard A-S, et al. (2012) Dalton Trans 41:6399–6403
IL 41
Structural and electronic properties of biological active
coordination compounds of imidazole derivatives:
towards understanding the role of the metal ions
Norah Barba-Behrens1, Soledad Betanzos-Lara1, I. AlfaroFuentes1, Rodrigo Castro-Ramı́rez1, I. Gracia-Mora1, R.
Contreras2, A. Flores-Parra2, Julia Brumaghim3, Matthew T.
Zimmerman3
1
Departamento de Quı́mica Inorgánica, Facultad de Quı́mica,
Universidad Nacional Autónoma de México, C.U., Coyoacán,
México, D.F., 04510, México;
2
Departamento de Quı́mica, Centro de Investigación y de Estudios
Avanzados, México D.F., México;
3
Hunter Laboratories, Chemistry Department, Clemson University,
Clemson SC USA
We have been interested to study the coordination ability of biological
active molecules towards transition metal ions. The structural and
electronic properties of these coordination compounds have been
investigated as their biological activity. Along these lines, synergistic
effects on transition metal coordination compounds of imidazole and
azole derivatives have been probed. The complexes hence obtained
display promising therapeutic potential as antibacterial, antineoplastic
or antihelmintic agents [1, 2].
In this work we present a series of cobalt(II) copper(II) and zinc(II) coordination compounds with imidazole and nitroimidazole
derivatives, in an effort to combine the chemotherapeutic properties
of the parental drug with the DNA binding (or other related) capacity
of the central metal atom. The complexes were fully characterised by
analytical and spectrophotometric methods and by X-ray diffraction
in selected cases. The anticancer or antiparasitic activities of these
new complexes were investigated, in order to establish a plausible
mechanism of action.
123
IL 42
Interaction of metal complexes with G-quadruplex
DNA and their effects on gene expression
Verity Stafford1,2, Arun Shivalingam1, Kogularamanan
Suntharalingam1, David Mann2,3, Ramon Vilar1,2
1
Department of Chemistry, Imperial College London, London SW7
2AZ, UK; 2Institute of Chemical Biology, Imperial College London,
London SW7 2AZ, UK; 3Department of Life Sciences, Imperial
College London, London SW7 2AZ, UK
Bio-informatic studies have established that in the human genome
there are ca. 370,000 guanine-rich sequences that can potentially
form quadruplex DNA [1]. Interestingly these putative quadruplex
forming regions, are not randomly distributed in genomes: they are
enriched in regions proximal to the transcription start site of proteincoding genes [2]. Furthermore it has been found that these quadruplex forming regions are most abundantly found at oncogene
promoters but not at onco-suppressor genes. Thus, some of these
quadruplex-forming regions have been proposed as targets for the
development of novel anticancer drugs. Herein we report a series of
biophysical studies to determine the affinity and selectivity of a
series of metal complexes (mono- and bi-metallic) towards a range
of quadruplex DNA structures. We also report on the effect that
these metal complexes have in the cellular levels of the corresponding RNA and protein(s).
Financial support by the Engineering and Physical Sciences
Research Council (EPSRC) of UK is gratefully acknowledged.
References
1. Todd AK, Johnston M, Neidle S (2005) Nucleic Acid Res
33:2901–2907
2. Huppert JL, Balasubramanian S (2007) Nucleic Acid Res
35:406–413
IL 43
Targeting G-quadruplex DNA with metal complexes
Florian Hamon1, Corinne Guetta1, Elodie Morel1, Sophie
Bombard2, Marie-Paule Teulade-Fichou1
Department of Chemistry, CNRS UMR176, Institut Curie University
of Paris-Sud, 91405 Orsay, France Laboratoire de Chimie et
Biochimie Pharmacologiques et Toxicologiques, CNRS UMR8601,
Université Paris Descartes, 45 Rue des Saints-Pères, 75006 Paris,
France. [email protected]
J Biol Inorg Chem (2014) 19 (Suppl 2):S725–S747
DNA has the capacity to adopt secondary structures differing from the
canonical double-stranded B-helix. Over the past 10 years, G-quadruplexes (G4) which result from local fold-over of guanine-rich sequences
have attracted considerable research effort. A wealth of knowledge has
accumulated concerning G-quadruplex motifs and their potential roles
in various processes such as replication, recombination, transcription,
splicing, and telomere maintenance [1]. G-quadruplexes may accommodate small molecules and the search for synthetic or natural G4
interacting compounds has developed dramatically with two major
goals: finding probes to sense quadruplex formation and discovering
domain-targeted DNA-interactive agents of potential therapeutic
interest in anticancer research [2]. We have shown that G-quadruplexes
may be targeted with specifically tailored monovalent Pt(II) or Pd(II)
complexes that induce strong coordination with loop bases as shown on
the figure below. The high efficiency of the reaction is likely due to the
confinement of the reactive metal cation between the loop and the top of
the quadruplex [3, 4]. Results obtained with a panel of cancer cells and
on animal models indicate that this class of metal complexes could
represent new telomere-targeted anti-tumour agents.
S741
Financial support by the Academy of Finland is gratefully
acknowledged.
References
1. Taherpour S, Lönnberg H, Lönnberg T (2013) Org Biomol Chem
11:991–1000
2. Golubev O, Lönnberg T, Lönnberg H (2013) Helv Chim Acta
96:1658–1669
References
1. Maizels N, Gray LT (2013) PLoS Genetics 9:e1003468
2. Monchaud D, Teulade-Fichou M-P (2008) Org Biomol Chem
6:627–636
3. Bertrand H, et al. (2009) Org Biomol Chem 72864
4. Largy E, et al. (2011) Chem Eur J 17:13274
IL 44
Discrimination between natural nucleobases by metal
ion chelates
Tuomas Lönnberg, Oleg Golubev, Sharmin Taherpour
Department of Chemistry, University of Turku, Vatselankatu 2,
FIN-20014 Turku, Finland. [email protected]
Four 3,5-dimethylpyrazol-1-yl-substituted purine nucleosides, serving
as either bi- or tridentate nitrogen ligands, have been prepared and
incorporated in short 20 -O-methyl-RNA oligonucleotides [1]. The
melting temperatures of the duplexes formed by these oligonucleotides with their natural counterparts are markedly increased upon
addition of 1 eq. of a divalent metal ion, in particular Cu2?, consistent
with formation of a Cu2?-mediated base pair. Furthermore, the extent
of stabilization is dependent on the identity of the natural nucleobase
completing the putative Cu2?-mediated base pair, the 2-substituted
purines favouring cytosine and the 6-substituted ones uracil. Similar
trends at the monomeric level were discovered by NMR titrations of
Pd2? chelates of various modified purine and pyrimidine nucleosides
and pyridine derivatives with the natural nucleosides and nucleoside50 -monophosphates [2]. The observed discrimination between natural
nucleobases may be explained in terms of steric and hydrogen
bonding interactions between the two ligands coordinated to the
central metal ion in square planar geometry.
IL 45
Recent findings concerning the mechanism for water
oxidation in photosystem II
Per E.M. Siegbahn
Department of Organic Chemistry, Arrhenius Laboratory, Stockholm
University, SE-106 91, Stockholm, Sweden. [email protected]
A full understanding of most details of the mechanism for water
oxidation by the oxygen evolving complex (OEC) in PSII is very
close [1]. Theoretical model calculations have played a major role in
this context. Based on density functional theory (DFT) calculations, a
mechanism for O–O bond formation was suggested in 2006, which
had not been suggested before [2]. This unique mechanism has
recently (2012) obtained strong support from experiments [3], where
also most other mechanisms suggested could be ruled out. In 2008, a
structure of the OEC was obtained from DFT modelling calculations
[4], quite different from the ones available from X-ray crystallography at the time. This structure was confirmed in 2011 by a highresolution structure [5]. In recent studies the effects from the nearest
surrounding of the OEC have been studied in detail. It has been found
that a critically positioned valine is of importance for the binding of
the substrate water. In the present talk, this mechanism and structure
will be described. There will also be suggestions of how the understanding of this mechanism could be used in designing artificial water
oxidation catalysts.
References
1. Siegbahn PEM (2013) Biochim Biophys Acta 1827:1003–1019
2. Siegbahn PEM (2006) Chem Eur J 12:9217–9227
3. Rapatskiy L, Cox N, Savitsky A, Ames WM, Sander J, Nowacyzk
MM, Rögner M, Boussac A, Neese F, Messinger J, Lubitz W (2013) J
Am Chem Soc 134:16619–16634
4. Siegbahn PEM (2008) Chem Eur J 27:8290–8302
5. Umena Y, Kawakami K, Shen J-R, Kamiya N (2011) Nature
473:55–60
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IL 46
Unmasking the metal-binding frontiers of acyclovir
(acv) through its coordination to copper(II)-polyamine
chelates
Josefa Marı́a González-Pérez1, Inmaculada Pérez-Toro1, Alicia
Domı́nguez-Martı́n1,2, Duane Choquesillo-Lazarte3, Alfonso
Castiñeiras4, Juan Niclós-Gutiérrez1
1
Deparment of Inorganic Chemistry, Faculty of Pharmacy, University
of Granada, 18071 Granada, Spain. [email protected];
2
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland;
3
Laboratorio de Estudios Cristalográficos, IACT, CSIC-UGR, Avda.
de las Palmeras 4, 18100 Armilla, Granada, Spain;
4
Department of Inorganic Chemistry, Faculty of Pharmacy,
University of Santiago de Compostela, 15782 Santiago de
Compostela, Spain
A comprehensive structural study on ternary CuII(polyamine)(acv)
complexes reveals the following insights: (1) The use of tridentate dien
and tripodal-tetradentatetren-Cu(II) complexes evidenced the cooperation of the Cu-N7(acv) bond with one intra-molecular N–HO6(acv)
interaction. This metal binding patter has been indeed recently reported
and requires the presence of a coligand with an appropriate primary
amino-terminal group [1]. (2) Tridentate amines (bis-2-picolylamine
and N,N,N0 ,N00 ,N00 -pentamethyl-dien) yield Cu(II) complexes that bind
N7,O6-acv in the so-called ‘pseudo-chelating mode’ (Cu-N7 * 2.0 Å
and Cu-O6 * 2.7 Å). In these cases, the guanine(acv) moiety falls
perpendicular to the Cu(N4-basal) coordination plane. This bidentate
mode was already reported [2] for [Cu(acv)2(H2O)2](NO3)2. (3) Cu(II)
chelates with the acyclic-tetradentate trien moves the N7-acv donor to
an apical/distal coordination site, thus giving a Cu–N7 bond (2.20(1)–
2.25(1) Å) which also cooperates with an intra-molecular interligand
(trien)N–HO6(acv) interaction. This apical acv-Cu(II)coordination
mode has not previously reported.
Financial support by LEC (IACT-CSIC), UGR and USC Universities and Fundación Ramon Areces are gratefully acknowledged.
References
1. Brandi-Blanco MP, Choquesillo-Lazarte D, Domı́nguez-Marı́n A,
González-Pérez JM, Castiñeiras A, Niclós-Gutiérrez J (2011) J Inorg
Biochem 105:616–623
2. Turel I, Andersen B, Sletten E, White AJP (1998) Polyhedron
17:4195–4201
IL 47
Teaching an old drug new tricks: overcoming
drawbacks associated with classical platinum drugs
as anti-cancer agents
Celine J. Marmion1, Darren Griffith1, Ziga Ude1, James Parker1,
Maria Morgan2, Jana Kasparkova3, Viktor Brabec3
123
J Biol Inorg Chem (2014) 19 (Suppl 2):S725–S747
1
Centre for Synthesis and Chemical Biology, Department
of Pharmaceutical and Medicinal Chemistry;
2
Molecular and Cellular Therapeutics, Royal College of Surgeons
in Ireland, Dublin 2, Ireland;
3
Institute of Biophysics, Academy of Sciences of the Czech Republic,
Brno, Czech Republic
Despite the enormous success of platinum drugs as anti-cancer therapies,
their widespread application and efficacy are hindered by toxic side
effects and drug resistance [1]. To overcome these drawbacks, new
molecular targets beyond DNA are being sought which may present
unique opportunities for therapeutic exploitation. The recent correlation
between the inhibition of enzymes that regulate chromatin structure/
function and tumour growth suppression has, for example, validated
chromatin control as a promising new target in contemporary medical
oncology. Inhibition of histone deacetylases (HDACs), for example,
enzymes which play a key role in maintaining chromatin structure and
function, has been shown to cause cell cycle arrest, differentiation and/or
apoptosis of tumour cells. While several are currently undergoing clinical
trials, already one, suberoylanilide hydroxamic acid, is in clinical use as a
treatment for cutaneous T cell lymphoma. HDAC inhibitors are also
known as ‘sensitizer drugs’ that display synergistic effects with other
anti-cancer agents such as cisplatin. Some have also been shown to be
selective for cancer cells over normal cells [2, 3].
We have designed and developed novel platinum drug candidates
with dual DNA binding and HDAC inhibitory activity. The rationale
behind their development and their syntheses will be described. A
summary of the pharmacological results obtained to date, in which
they have been shown to be highly cytotoxic towards cancer cells as
well as having enhanced selectivity for cancer cells over normal cells,
will also be provided [4, 5].
This material is based upon works supported by the Science
Foundation Ireland under Grants No. [07/RFP/CHEF570] and [11/
RFP.1/CHS/3094]. We also thank colleagues in COST CM1105 for
fruitful collaborations.
References
1. Kelland L (2007) Nat Rev Cancer 7:573–584
2. Bolden JE, Peart MJ, Johnstone RW (2006) Nat Rev Drug Discov
5:769–784 and references therein
3. Marks PA, Xu WS (2009) J Cell Biochem 4:600–608 and references therein
4. Griffith D, Morgan MP, Marmion CJ (2009) Chem Comm
44:6735–6737
5. Brabec V, Griffith D, Kisova A, Kostrhunova H, Lenka Z,
Marmion CJ, Kasparkova J (2012) Mol Pharmaceutics 7:1990–1999
IL 48
A molecular approach for water splitting with sunlight:
the key for the transition from fossil to solar fuels
Antoni Llobet
Institute of Chemical Research of Catalonia (ICIQ), Avinguda Paı̈sos
Catalans 16, E-43007 Tarragona, Spain; Departament de Quı́mica
Universitat Autònoma de Barcelona, Cerdanyola del Vallès, E-08193
Barcelona, Spain. [email protected]
Water oxidation (WO) to molecular dioxygen and its inverse reaction
oxygen reduction (OR) to water, are the two key reactions involved in
water splitting and hydrogen fuel cells respectively. The mastering of
both reactions WO and OR catalyzed by Transition Complexes
(TMC) is an essential requirement for their potential applications in
wide spread energy applications. In both cases the O–O bond formation and breakage step is crucial and needs to be fully understood
as well as all the previous activation pathways. Additionally the
oxidation of water to molecular oxygen is also a reaction of interest
J Biol Inorg Chem (2014) 19 (Suppl 2):S725–S747
from a biological perspective since it is the main reaction occurring at
the Oxygen Evolving Center of Photosystem II (OEC-PSII). These
oxygen related reactions together with proton and CO2 reduction
catalytic reactions constitute the complete set of reactions needed to
be able to come up with efficient models for molecular artificial
photosynthesis [1].
A few Ru and Co complexes will be described that are capable of
catalyzing the water oxidation and the oxygen, proton and CO2
reduction reactions. Additionally their performance and mechanistic
pathways will be disclosed based on electrochemical, kinetics, oxygen labeling experiments and DFT calculations [2–7].
References
1. Berardi S, Drouet S, Francàs L, Gimbert-Suriñach C, Guttentag M,
Richmond C, Stoll T, Llobet A (2014) Chem Soc Rev (in press)
2. Bozoglian F, Romain S, Ertem NZ, Cramer CJ, Gagliardi L, Llobet
A, et al. (2009) J Am Chem Soc 131:15176–15187
3. Sala X, Ertem MZ, Cramer CJ, Gagliardi L, Llobet A, et al. (2010)
Angew Chem Int Ed 49:7745–7747
4. Duan L, Bozoglian F, Privalov T, Llobet A, Sun L, et al. (2012)
Nature Chem 3:2576–2578
5. Rigs S, Mandal S, Nam W, Llobet A, Stahl SS (2012) Chem Sci
3:3058–3062
6. Neudeck S, Maji S, López I, Meyer S, Meyer F, Llobet A (2014) J
Am Chem Soc 136:24–27
7. López I, Ertem MZ, Maji S, Benet-Buchholz J, Keidel A, Kuhlmann U, Hildebrandt P, Cramer CJ, Batista VS, Llobet A (2014)
Angew Chem Int Ed 53:205–210
IL 49
Water oxidation catalysis by manganese:
from bioinorganic model chemistry
towards technological applications
Philipp Kurz
Institut für Anorganische und Analytische Chemie, Albert-LudwigsUniversität Freiburg, Albertstraße 21, 79104 Freiburg, Germany.
[email protected]
In nature, solar energy is converted into chemically stored energy
through the processes of photosynthesis. One of the fundamental
reactions involved is the light-driven oxidation of water to molecular
oxygen, reaction (1). In vivo, this reaction provides the reduction
equivalents for CO2-fixation and takes place in the very large protein
ensemble Photosystem II (PSII), where a Mn4Ca(l-O)5-cluster, the
oxygen-evolving-complex (OEC, see Figure), is the catalytic site for
the following fundamental reaction: 2 H2O ? O2 ? 4 H? ? 4 e-.
In the last 5 years, we have developed bio-inspired solid state catalysts
for water oxidation and demonstrated that layered (calcium) manganese
oxides are very promising heterogeneous catalysts for this reaction with
(for Mn-based materials) unprecedented activity and stability [1].
X-ray absorption spectroscopy (XAS) provided insights into the
structures of the most active oxides and revealed that they belong to
the birnessite mineral family of amorphous, layered manganese oxides. Such materials feature a number of similarities to the OEC, such
as an intermediate oxidation state between MnIII and MnIV, coordination sites for water and a high degree of flexibility. Similar to the
natural paragon, we also found that the incorporation of calcium is
crucial to achieve highest catalytic rates [1, 2].
For an application of these materials in technologically viable setups, it is now necessary to develop immobilization methods to prepare
affordable manganese-based anodes for water-splitting devices like
‘‘artificial leaves’’ [3, 4]. The presentation will outline our approaches
for both the synthesis of ever-better MnOx-catalysts and also their use
in CaMn-oxide electrodes.
S743
References
1. Wiechen M, et al. (2012) Chem Sci 3:2330
2. Frey CE, et al. (2014) Dalton Trans 43:4370
3. Nocera D (2012) Acc Chem Res 45:767
4. Joya KS, et al. (2013) Angew Chem 125:10618
IL 50
Electrochemical studies and in situ characterization
of molecular water oxidation catalysts
Dennis G. H. Hetterscheid
Leiden Institute of Chemistry, Leiden University, Einsteinweg 55,
2333CC Leiden, The Netherlands.
[email protected]
In my group molecular catalysts for the oxidation of water are
studied predominantly by electrochemical techniques rather than
using stoichiometric oxidants in ill defined reaction media [1–3].
Using on line electrochemical mass spectrometry (OLEMS) the
formation of dioxygen is studied as a function of applied potential,
while tracking formation of e.g. CO2 and NO2 illustrates at which
potentials degradation of the organic ligands occurs. Complementary
to these experiments, the deposition of material on the electrode is
studied by electrochemical quartz crystal microbalance (EQCM)
techniques and after the catalytic reaction with surface analysis
techniques such as extended X-ray absorption fine structure (EXAFS) and X-ray photoelectron spectroscopy (XPS). Using these
techniques we have been able to pinpoint the observed catalytic
activity to a molecular species in case Ir(OH)2 (see Figure) was used
as the anticipated catalyst [4], while in case of several other
molecular systems we found that formation of CO2 takes place prior
to evolution of dioxygen and thus that catalysis is most likely
mediated by degradation products of the original molecular systems.
In situ spectro-electrochemical techniques (e.g. Raman, IR, UVvis)
in combination with DFT calculations are used to further unravel the
reaction mechanisms [5]. Currently, the focus of our research is
shifted towards molecular iron and copper catalysts for oxidation of
water and the reduction of dioxygen.
References
1. Hetterscheid DGH, Van der Vlugt JI, de Bruin B, Reek JNH (2009)
Angew Chem Int Ed 48:8178–8181
2. Hetterscheid DGH, Reek JNH (2012) Angew Chem Int Ed
51:9740–9747
3. Hetterscheid DGH, Reek JNH (2014) Eur J Inorg Chem
2014:742–749
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4. Hetterscheid DGH, Reek JNH (2011) Chem Commun
47:2712–2714
5. Venturini A, Barbieri A, Reek JNH, Hetterscheid DGH (2014)
Chem Eur J 18:5358–5368
IL 51
Electronic structure and reactivities of resting
and intermediate forms of the tetranuclear copper
cluster in nitrous oxide reductase
Isabel Moura
Universidade Nova de Lisboa, Departamento de Quı́mica, Caparica
2829-516, Portugal
Denitrification is an important biological pathway with environmental implications. Nitrate accumulation and release of nitrous
oxide in the atmosphere due to use of excess fertilizers are examples
of two environmental problems, where denitrification plays a central
role. The reduction of nitrate to nitrogen gas uses four different
types of metalloenzymes in four simple steps: nitrate is reduced to
nitrite, then to nitric oxide, followed by the reduction to nitrous
oxide and by a final reduction to di-nitrogen: ð2NO
3 ! 2NO2 !
2NO ! N2 O ! N2 Þ: The 3D structures of all these enzymes are
known. We present a concise updated review of the bioinorganic
aspects of denitrification with emphasis on spectroscopic features,
structural and mechanistic aspects of the relevant enzymes involved,
with emphasis on the one involved in the last step of this complex
pathway. The metal diversity detected in this pathway is also
acknowledged. Nitrous oxide reductase the last enzyme of this
pathway contains a new tetranuclear copper center (CuZ). The
structures of two different resting forms of nitrous oxide reductase
have been reported, one with an active site containing a 4CuS tetranuclear copper cluster with a solvent-derived edge ligand, known
as CuZ , [1] and another containing a 4Cu2S cluster, known as CuZ
[2]. We show that these resting sites differ in their redox properties
and reactivity towards N2O, with CuZ accessing the CuII3CuI (1hole) and 4CuI redox states, while CuZ accesses the 2CuII2CuI (2hole) and 1-hole redox states. Additionally, we show that 1-hole
CuZ reacts very slowly with N2O while only fully reduced CuZ
reacts rapidly, at rates that are catalytically competent. Absorption,
MCD, EPR, and resonance Raman spectroscopies were employed to
compare the electronic structures of CuZ and CuZ in the 1-hole
redox state and determine the nature of the edge ligand. We further
employed the correlation between structure, electronic structure and
reactivity in these resting forms to define the nature of the intermediate CuoZ , a 1-hole form observed in single turnover with N2O
that can be rapidly reduced [3]. Absorption, MCD, and resonance
Raman spectroscopies coupled with DFT calculations gave insight
into the electronic structure of CuoZ , leading to a complete catalytic
cycle for N2OR.
We thank E. Pierce, E. Solomon, S.R. Pauleta, S. Dell’Acqua, S.
Ramos, C. Carreira and J. Moura for their contributions. The work
was supported by the project from FCT (PTDC/QUI-BIQ/116481/
2010).
References
1. Brown K, Djinovic-Carugo K, Haltia T, Cabrito I, Saraste M,
Moura JJG, Moura I, Tegoni M, Cambillau C (2000) J Biol Chem
275:41133–41136
2. Pomowski A, Zumft WG, Kroneck PMH, Einsle O (2011) Nature
46:234–237
3. Dell’Acqua S, Pauleta S, Paes de Sousa P, Monzani E, Casella L,
Moura J, Moura I (2010) J Biol Inorg Chem 15:967–976
123
J Biol Inorg Chem (2014) 19 (Suppl 2):S725–S747
IL 52
New artificial metalloenzymes based
on the transcription factor LmrR
Gerard Roelfes, Jeffrey Bos, Ivana Drienovská
Stratingh Institute for Chemistry, University of Groningen,
Nijenborgh 4, 9747 AG Groningen, The Netherlands.
[email protected]
The catalytic efficiency and high selectivities achieved by natural
metalloenzymes are a source of inspiration for the design of novel
bio inspired catalysts. An emerging approach for creating artificial
metalloenzymes involves incorporating synthetic transition metal
catalysts into a biomolecular scaffold such as a protein or DNA
[1].
We have developed a new concept for the design of artificial
metalloenzymes that involves creation of a novel active site at the
dimer interface of the transcription factor LmrR (Lactococcal multidrug resistance Regulator) [2]. LmrR was selected as the protein
scaffold because it contains a large hydrophobic pocket on the dimer
interface [3]. Here, three approaches to the construction of these
LmrR-based artificial metalloenzymes will be presented, involving
covalent or supramolecular anchoring of the metal complex or biosynthetic incorporation of an unnatural metal binding amino acid
using expanded genetic code methodology. Also the application of
these artificial metalloenzymes in catalytic asymmetric C–C bond
forming and hydration reactions will be discussed [3, 4].
Financial support by the Netherlands Research School Combination Catalysis (NRSC-C) and the European Research Council (ERC
Starting grant) is gratefully acknowledged.
References
1. Rosati F, Roelfes G (2010) ChemCatChem 2:916–927
2. Madoori PK, Agustiandari, H, Driessen, AJM, Thunnissen AMWH
(2009) EMBO J 28:156–166
3. Bos J, Fusetti F, Driessen AJM, Roelfes G (2012) Angew Chem Int
Ed 51:7472–7475
4. Bos J, Garcı́a-Herraiz A, Roelfes G (2013) Chem Sci 4:3578–3582
IL 53
Selected aspects of the medicinal chemistry
and chemical biology of metal NHC complexes
Ingo Ott
Institute of Medicinal and Pharmaceutical Chemistry, Technische
Universität Braunschweig, Beethovenstrasse 55, 38106
Braunschweig, Germany. [email protected]
Metal complexes with N-heterocyclic carbene (NHC) ligands have
been playing an important role in catalysis during the last decades.
Their biological and medicinal properties, however, have been
described in more detail only during the last years. The great interest
of medicinal inorganic chemists in this type of metallodrugs is mainly
driven by the high stability of the complexes as well as the structural
versatility of the NHC ligand, which facilitates the preparation of
J Biol Inorg Chem (2014) 19 (Suppl 2):S725–S747
compound libraries and in consequence the development of structure–
activity relationships (SAR) [1, 2].
We have been investigating the medicinal chemistry of NHC metal
complexes with gold(I), ruthenium(II) and rhodium(I) metal centers as
possible antitumor agents (see the below figure for some representative
examples). The triggering of antiproliferative effects in tumor cells, the
inhibition of thioredoxin reductase (TrxR) and other tumor-relevant
enzymes, the induction of apoptosis and the interference with mitochondria have been evaluated among other biochemical pathways [3–5].
High resolution atomic absorption spectroscopy was used to quantify the
cellular uptake and the biodistribution of selected complexes. Both the
biological activity and the cellular trafficking depended on the coordinated NHC moiety, the metal and on the secondary ligands.
In summary our studies showed that development of SAR for metal
NHC complexes is possible and that these organometallics could be
used as partial structures or pharmacophores of novel antitumor drugs.
Financial support by Deutsche Forschungsgemeinschaft (DFG)
and COST CM1105 is gratefully acknowledged.
References
1. Oehninger L, Rubbiani R, Ott I (2013) Dalton Trans 42:3269–3284
2. Liu W, Gust R (2013) Chem Soc Rev 42:755–773
3. Rubbiani R, Can S, Kitanovic I, Alborzinia H, Stefanopoulou M,
Kokoschka M, Mönchgesang S, Sheldrick WS, Wölfl S, Ott I (2011) J
Med Chem 54:8646–8657
4. Oehninger L, Küster N, Schmidt C, Munoz-Castro A, Prokop A,
Ott I (2013) Chem Eur J 19:17871–17880
5. Rubbiani R, Salassa L, De Almeida A, Casini A, Ott I (2014)
ChemMedChem (in press)
IL 54
Substituted Nucleotides: versatile building blocks
in DNA nano-biotechnology
Eugen Stulz, Jonathan R. Burns, Daniel G. Singleton, James W.
Wood, Iwona Mames
School of Chemistry and Institute for Life Sciences, University
of Southampton, Highfield, Southampton SO17 1BJ, UK
DNA has become very attractive as scaffold for functional molecules on
the nanometre scale [1]. The sequence specific insertion of modified
nucleotides using automated DNA synthesis allows for the creation of
new designer molecules with a wide range of potential applications. We
have established a general synthetic route to porphyrin-nucleosides and
their subsequent site specific incorporation into oligonucleotides to
create multiporphyrin arrays. Up to eleven consecutive porphyrins
could be incorporated into DNA giving access to a multiporphyrin array
of approximately 10 nm in length, which corresponds to the highest
amount of DNA modification with a large hydrophobic metal complex
to date [2]. The spectroscopic data and structure calculations indicate
the formation of a stable helical array in the single strand porphyrinDNA. The p-stack of the porphyrins leads to strong electronic interaction between the chromophores. A zipper array with induced stability
and energy transfer properties has recently been realised, providing
access to the first reversible photonic wire based on a DNA scaffold [3].
We will present our latest results in terms of novel modified DNA
structures, including their characterization using CD and EPR
S745
spectroscopy. Applications of modified DNA will be described, such
as genosensors [4], switches [5] and DNA origami nanopores [6, 7].
References
1. Bandy TJ, Brewer A, Burns JR, Marth G, Nguyen T, Stulz E (2011)
Chem Soc Rev 40:138–148
2. Fendt LA, Bouamaied I, Thöni S, Amiot N, Stulz E (2007) J Am
Chem Soc 129:15319–15329
3. Nguyen T, Brewer A, Stulz E (2009) Angew Chem Int Ed
48:1974–1977
4. Grabowska I, Singleton DG, Stachyra A, Gora-Sochacka A, Sirko
A, Zagorski-Ostoja W, Radecka H, Stulz E, Radecki J (2014) Chem
Commun 50:4196–4199
5. Burns JR, Preus S, Singleton DG, Stulz E (2012) Chem Commun
48:11088–11090
6. Burns JR, Stulz E, Howorka S (2013) Nano Lett 13:2351–2356
7. Burns JR, Gopfrich K, Wood JW, Thacker VV, Stulz E, Keyser UF,
Howorka S (2013) Angew Chem Int Ed 52:12069–12072
IL 55
New tasks for multiheme cytochrome c enzymes
Bianca Hermann1, Nienke Kuipers1, Simon Netzer1, Melanie
Kern2, Jörg Simon2, Oliver Einsle1
1
Institute for Biochemistry, University of Freiburg, Albertstrasse 21,
79104 Freiburg im Breisgau, Germany. [email protected];
2
Department of Biology, Microbial Energy Conversion
and Biotechnology, Technische Universität Darmstadt,
Schnittspahnstrasse 10, 64287 Darmstadt, Germany
The heme group, Fe-protoporphyrin IX, is arguably one of nature’s
most versatile cofactors, fulfilling roles in electron transfer, ligand
binding and redox catalysis. Cytochromes of type c have one or
multiple heme groups attached covalently to the protein chain. This
allows for increased protein stability as well as a high heme:protein
ratio. We have studied various multiheme enzymes, in particular
cytochrome c nitrite reductase [1] and have recognized the importance of conserved heme packing motifs that are evolutionarily
conserved between distinct members of this class of proteins [2]. In a
broad screen for multiheme cytochromes that are amenable for
recombinant production in suitable bacterial hosts we have identified
several new enzymes that share the heme arrangement, but that have
proven to possess substantial functional diversity. From novel nitrite
reductases to a unique type of octaheme sulfite reductase [3], the
family of penta-and octaheme cytochromes proves to be rich in redox
enzymes with unexpected functionalities and mechanisms [4].
This work was supported by Deutsche Forschungsgemeinschaft.
123
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References
1. Einsle O, Messerschmidt A, Stach P, Bourenkov GP, Bartunik HD,
Huber R, Kroneck PMH (1999) Nature 400:476–480
2. Einsle O (2011) Methods Enzymol 496:399–422
3. Hartshorne RS, Kern M, Meyer B, Clarke TA, Karas M, Richardson DJ, Simon J (2007) Mol Microbiol 64:1049–1060
4. Kern M, Klotz MG, Simon J (2011) Mol Microbiol 82:1515–1530
IL 56
Generation and reactivity of transient cytochrome P450
oxygen-containing intermediates
John H. Dawson1, Anuja Modi1, Daniel P. Collins1, D. M. Indika
Bandara1, Shengxi Jin1, Thomas A. Bryson1, Eric D. Coulter1,
Zanna Beharry1, David P. Ballou2
1
Dept Chem/Biochem, Univ SC, Columbia, SC 29208 USA.
[email protected];
2
Dept Biol Chem, Univ MI, Ann Arbor, MI 48109 USA
Cytochrome P450 and nitric oxide synthase are both heme-containing
oxygenases. Both ferric enzymes first bind substrate, are reduced and
then bind dioxygen to give the oxyferrous state. Addition of a second
electron yields a peroxo-ferric intermediate, protonation of which
generates a hydroperoxoferric state; loss of water then forms an oxoiron(IV) porphyrin radical (Compound I). Although the latter is
thought to be the ultimate oxidant, the preceding two species have
been proposed as secondary oxidants. T252A P450-CAM, hydroxylates camphor to a very limited extent, indicating it does not form
Compound I. However, it still accepts electrons from NADH to give
H2O2, presumably via the peroxo/hydroperoxoferric intermediates
and is thus the ideal mutant to test whether either of these two species
are capable of O-atom transfer. We have prepared a series of camphor
analogues and related compounds with reactive functional groups for
O-atom transfer to investigate the mechanism of dioxygen activation
by P450 and NOS. We have used this approach to investigate sulfoxidation,
thioether
S-dealkylation,
deformylation
and
N-hydroxyimine denitrosation. Results of these studies, with
emphasis on the involvement of a second active P450 oxidant in
O-atom transfer, will be presented.
We are also investigating the generation of transient intermediates in the reaction cycle of P450-CAM. While monitoring the
reaction of ferric P450-CAM with substituted perbenzoic acids using
rapid scan stopped flow spectroscopy, an intermediate appears en
route to Compound I. We have proposed that this moiety is an
acylperoxo-ferric heme adduct, which then undergoes O–O bond
cleavage to generate Compound I—a rare case where Compound I
formation can be monitored in real time. Singular value decomposition analysis of data for the formation of this species shows that
the energy of its Soret absorption peak is sensitive to the electron
donor properties of the aromatic ring perbenzoic acid substituents. A
linear Hammett correlation plot is seen for the energy of the Soret
peak vs. the Hammett q constant. This correlation requires that the
substituents remain as part of the ligand bound to the heme iron,
providing direct evidence that the intermediate is indeed a ferricacylperoxo derivative. Linear Hammett correlation plots are also
seen for both the rate of intermediate formation as well as for its
conversion to Compound I. Thus, the electron donating/withdrawing
properties of the substituents affect the binding of the peracid to
form the acylperoxo intermediate as well as the propensity of the
substituted benzoic acid to serve as the leaving group during O–O
bond cleavage to yield Compound I.
Funding: NIH GM 26730. We thank Steve Sligar for the T252A
over expression system.
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J Biol Inorg Chem (2014) 19 (Suppl 2):S725–S747
IL 57
Molecular details of indirect extracellular electron
transfer between bacteria and metallic solids
or electrodes
Bruno M Fonseca*, Catarina M. Paquete*, Sónia M. Neto, Davide
Cruz, Isabel Pacheco, Cláudio M. Soares, Ricardo O. Louro
Instituto de Tecnologia Quı́mica e Biológica António Xavier,
Universidade Nova de Lisboa, Av. Da República (EAN) 2780-156
Oeiras, Portugal. [email protected]
*contributed equally
Extracellular electron transfer is the key metabolic trait that
enables dissimilatory metal reducing organisms to play a significant role in the biogeochemical cycling of metals and in the
operation of bioelectrochemical devices such as microbial fuel
cells. In Shewanella oneidensis MR-1, electrons generated in the
cytoplasm by catabolic processes cross the periplasmic space to
reach terminal oxidoreductases found at the cell surface. These
terminal oxidoreductases are outer-membrane multiheme cytochromes responsible for both direct and indirect interaction with
the insoluble electron acceptors. In this type of mechanism, electron shuttles, endogenously produced or scavenged from the
medium are used to facilitate electron transfer.Lack of knowledge
on how the bioenergetic metabolism of these bacteria is linked to
the reduction of insoluble acceptors is one of the unresolved issues
related with extracellular electron transfer. In this work, this subject was explored using a combination of NMR spectroscopy,
stopped-flow kinetics, electrostatic and molecular docking
calculations.
Several periplasmic cytochromes (STC, MtrA, CymA, FccA and
ScyA), as well as all known outer-membrane decaheme cytochromes
from Shewanella oneidensis MR-1 with known metal terminal
reductase activity (OmcA, MtrC and MtrF) were expressed and
purified. The results showed that both STC and FccA interact with
their redox partners, CymA and MtrA, through a single heme
avoiding the establishment of stable redox complexes capable of
spanning the periplasmic space. Among the outer membrane multiheme cytochromes, despite the structural similarities among them, the
detailed characteristics of their interactions with soluble electron
shuttles are distinct. MtrC and OmcA appear to interact with a variety
of different electron shuttles in the close vicinity of some of their
hemes, and with affinities that are biologically relevant for the concentrations typically found in the medium for this type of compounds.
In contrast the data support a view of a distant interaction between the
hemes of MtrF and the electron shuttles. These results provide
guidance for future work of the manipulation of these proteins toward
improving the bioremediation and bioenergy production capabilities
of metal reducing bacteria such as Shewanella oneidensis MR-1.
References
1. Fonseca BM, et al. (2013) Biochem J 449:101–108
2. Paquete CM, et al. (2014) Front Microbiol (under review)
IL 58
Inorganic–organic hybrid drug delivery systems
and their therapeutic efficacy
Zong-Wan Mao
MOE Key Laboratory of Bioinorganic and Synthetic Chemistry,
School of Chemistry and Chemical Engineering, Sun Yat-sen
University, Guangzhou. [email protected]
The therapeutic efficacy of drugs can be improved by the use of drug
delivery vehicles. The unique properties of different materials can be
J Biol Inorg Chem (2014) 19 (Suppl 2):S725–S747
utilized to design multi-component drug delivery systems that can
efficiently overcome the major barriers in the body.
Our research group has developed several inorganic–organic
hybrid particles for the delivery of chemotherapeutics and small
interfering RNA (siRNA). Specifically, quantum dots (QDs), gold,
porous silicon and porous silica have been used as the inorganic
components of the hybrid system. The organic components, in turn,
consist of cyclodextrin (CD) and polyethylenimine (PEI). Quantum
dots permit imaging of the delivery vehicle, while Au NRs can be
utilized for thermal ablation. Porous silicon and silica particles are
biodegradable, thereby enabling sustained release. CDs are watersoluble, can be modified with amino acids, and reduce the toxicity of
cationic compounds, while PEI binds tightly to siRNA, aids in
intracellular uptake and mediates escape from the lysosome. In particular, QDs have been coated with amino acid-modified CDs,
through direct ligand exchange, resulting in particles with different
surface charges (positive, negative and neutral). These platforms have
been used for the delivery of siRNA and chemotherapeutics [1–6].
Furthermore, PEI has been grafted to porous silicon microparticles
[7], porous silica nanoparticles [8], and gold nanorods. The incorporation of CD in the delivery vehicle eliminates the charge-induced
S747
toxicity of PEI [8]. The inorganic–organic hybrid particles have been
evaluated in vivo and in vitro, and display both therapeutic efficacy
and biocompatibility.
References
1. Zhao MX, Ji LN, Mao ZW (2012) Chem Eur J 18:1650–1658
2. Li JM, Wang YY, Zhao MX, Tan CP, Li YQ, Le XY, Ji LN, Mao
ZW (2012) Biomaterials 33:2780–2790
3. Li JM, Zhao MX, Su H, Wang YY, Tan CP, Ji LN, Mao ZW (2011)
Biomaterials 32:7978–7987
4. Zhao MX, Li JM, Du L, Tan CP, Xia Q, Mao ZW, Ji LN (2011)
Chem Eur J 17:5171–5179
5. Zhao MX, Xia Q, Feng XD, Zhu XH, Mao ZW, Ji LN, Wang K
(2010) Biomaterials 31:4401–4408
6. Zhao MX, Huang HF, Xia Q, Ji LN, Mao ZW (2011) J Mater Chem
21:10290–10297
7. Shen J, Xu R, Mai J, Kim HC, Guo X, Qin G, Yang Y, Wolfram J,
Mu C, Xia X, Gu J, Liu X, Mao ZW, Ferrari M, Shen H (2013) ACS
Nano 7:9867–9880
8. Shen J, Kim HC, Su H, Wang F, Wolfram J, Kirui D, Mai J, Mu C,
Ji LN, Mao ZW, Shen H (2014) Theranostics 4:487–497
123
J Biol Inorg Chem (2014) 19 (Suppl 2):S749–S764
DOI 10.1007/s00775-014-1159-9
ORAL PRESENTATION
Oral presentations
OP 1
Development of potential anticancer agents
by coordination of bioactive molecules
to organometallic fragments
Wolfgang Kandioller1,2, Stephan Mokesch1, Carmen
Hackl1, Michael Jakupec1,2, Christian G. Hartinger3,
Bernhard K. Keppler1,2
OP 2
Effect of a hexacationic ruthenium complex as potential
anticancer drug on the cell metabolome studied
by 1H HR-MAS NMR spectroscopy
Martina Vermathen1, Lydia E.H. Paul1, Gaëlle
Diserens2, Peter Vermathen2, Julien Furrer1
1
Institute of Inorganic Chemistry, University of Vienna, Waehringer
Strasse 42, 1090 Vienna, Austria. [email protected]
2
Research Platform ‘‘Translational Cancer Therapy Research’’,
University of Vienna, Waehringer Strasse 42, 1090 Vienna, Austria
3
The University of Auckland, School of Chemical Sciences, Private
Bag 92019, Auckland 1142, New Zealand
Ru(II)-arene complexes are promising alternatives for the clinically
applied platinum-based chemotherapeutics. One approach is the
attachment of bioactive molecules to organometallic moieties,
leading to compounds with potential multi-targeted character which
are able to interact with different biological targets [1,2]. [1,4]Naphthoquinones are known for its broad range of biological
activities such as antibacterial, anti-inflammatory and anticancer
activities and the mode of action is supposed to be related to
reactive oxygen species (ROS) formation. [1,3]-Dioxoindan-2-carboxamides have shown promising topoisomerase inhibiting
properties and this compound class can be easily obtained by
rearrangement of the [1,4]-naphthoquinone backbone. With the aim
to develop novel metallodrugs with potential multi-targeted properties, these bioactive scaffolds were coordinated to organometallic
fragments. The synthesized ligands and the corresponding Ru(II),
Os(II) and Rh(III) complexes were characterized by standard analytical methods and their behaviour under physiological conditions,
binding affinity towards biomolecules, cytotoxicity in human cancer cell lines, ROS generating ability and further mode of action
studies will be discussed.
Financial support by the University of Vienna and the Johanna
Mahlke née Obermann Foundation is gratefully acknowledged.
Department of Chemistry and Biochemistry, University of Bern,
Freiestrasse 3, 3012 Bern, Switzerland
2
Departments of Clinical Research and Radiology, University
of Bern, 3010 Bern, Switzerland
A water soluble hexacationic Ruthenium complex [(p-cymene)6Ru6(tpt)2(dhnq)3](CF3SO3)6 with tri-pyridyl-triazene (tpt) and
dihydroxy-naphthoquinone (dhnq) as bridging ligands was prepared
and tested for its anticancer activity and interaction with potential
biological targets [1]. The complex was found to be highly cytotoxic
against human ovarian carcinoma cells (A2780) with an IC50 value of
0.45 lM. To learn more about the specificity and the mechanism of
action, the effect of the complex on the metabolic profile of three
different human cell lines was studied by high resolution magic angle
spinning (HR-MAS) NMR spectroscopy. HR-MAS NMR allows
obtaining well resolved 1H NMR spectra from living cell suspensions
[2] well suited for chemometric analyses.
Cisplatin-sensitive and -resistant cancer cells (A2780 and
A2780cisR) as well as human embryonic kidney cells (HEK-293) as
healthy model cells were each incubated with the Ru-complex for 24
and 72 h, respectively. The corresponding cell suspensions were submitted to HR-MAS NMR yielding a total of 104 1H NMR spectra of
control and drug treated samples. Multivariate statistical analysis (PCA
and PLS) of the spectra indicated clear metabolic changes between
control and drug-treated cells for all 3 cell lines, as shown in the Figure
for tincub = 24 h. The changes were most pronounced for A2780 cancer
cells mainly due to lipids and choline containing compounds indicating
potential drug-induced membrane breakdown. The single components
responsible for the discrimination between all control and drug treated
groups are discussed in more detail in this presentation.
Financial supports by the University of Bern and SNF are gratefully acknowledged.
References
1. Kilpin KJ, Dyswon PJ (2013) Chem Sci 4:1410–1419
2. Nazarov AA, Hartinger CG, Dyson PJ (2013) J Organomet Chem
751:251–260
References
1. Paul LEH, Therrien B, Furrer J (2012) J Biol Inorg Chem 17:1053
2. Griffin JL, Bollard M, Nicholson JK, Bhakoo K (2002) NMR
Biomed 15:375
1
123
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OP 3
Fluorescent ‘‘crowdoxidation’’ probes
David G. Churchill1
1
Department of Chemistry (Molecular Logic Gate Laboratory), Korea
Advanced Institute of Science and Technology, 373-1 Guseong-dong,
Yuseong-gu, Daejeon, 305-701, Republic of Korea.
[email protected]
The chalcogens, as individual atoms, can be incorporated into the
design of fluorescent (fluorogenic) molecules. Novel classes of
fluorophores can be produced and exploited for, not only sensing,
but also therapeutics and theranostics (e.g., GPx systems). So far,
we have produced four different chalcogen-containing molecular
motifs. A simple electron photoinduced electron transfer (PET)
donor–acceptor design is utilized. The rotation of the electron
donor is critical in fluorescence properties in these systems. Our
laboratory has reported the first well-defined molecular probe
involving chalcogen oxidation (benzothienyl–BODIPY) as the
chemical switch for fluorescence/optical changes. It serves as the
electron donor in a PET donor–acceptor design [1]. Our laboratory
first reported a probe in which there are multiple sites of oxidation
(multi-input) [2]. A total of four oxidation sites are possible here.
Our laboratory first synthesized a BODIPY diselenide probe [3]. It
is the second one, only, to behave as a reversible superoxide probe.
This probe involves BODIPY–Phenyl–Se–Se–Phenyl–BODIPY
attachments. The selenides are connected ortho in the electron
receptor to maximize the impact on sterics. The sensitivity,
selectivity, time response, cell studies, stability are all affirmative
and decent, or excellent. In the process of repairing a diselenide
probe for the non-substituted system, an unexpected reaction
occurred. This is the first annulation product of its class [4];
importantly, it is the first chalcogen annulation product known for
dipyrrins, or aryl-meso porphyrins, etc. The probe is an ‘‘off–on’’
fluorescent probe; it is reversible and \500 Da. The structure
shows large mean planarity and rigidity; the uniquely-placed
chalcogen atom is saddled halfway between the aryl ring and
chromophore. This results in a red-shifted emission band molecule
where, again, the molecule is kept small. Organoselenides also
feature in a chelation site in a BODIPY conjugate in a recent paper
devoted to modelling subsequent Fenton chemistry based on ferrous/ferric ion coordination and detection [5]. Financial support by
the National Research Foundation of Korea and the Center for
Catalytic Hydrocarbon Functionalizations, Institute for Basic
Science.
References
1. Choi SH, Kim K, Jeon J, Meka B, Bucella D, Pang K, Khatua S,
Lee J, Churchill DG (2008) Inorg Chem 47:11071–11083
2. Singh AP, Mun Lee K, Murale DP, Jun T, Liew H, Suh Y-H,
Churchill DG (2012) Chem Commun 48:7298–7300
3. Manjare ST, Kim S, Heo, WD, Churchill DG (2014) Org Lett
16:410–412
4. Manjare ST, Kim J, Lee Y, Churchill DG (2014) Org Lett
16:520–523
5. Murale DP, Manjare ST, Lee Y-S, Churchill DG (2014) Chem
Commun 50:359–361
123
J Biol Inorg Chem (2014) 19 (Suppl 2):S749–S764
OP 4
Sulfite oxidase: A paradigm for the mechanistic
complexities and mysteries of metallo-enzymes
with multiple domains, subunits and cofactors
John H. Enemark, Gordon Tollin, Susan Borowski
Department of Chemistry and Biochemistry, University of Arizona,
Tucson, AZ 85721 USA
Human sulfite oxidase (hSO) is a complex enzyme that is essential for
normal neonatal neurological development. Each of the two identical
subunits of the homodimeric hSO protein possesses two domains that
are linked by a flexible polypeptide tether. One domain contains the
molybdenum cofactor and the other a b-type heme. The generally
accepted catalytic mechanism for the oxidation of sulfite to sulfate by
SO involves five different formal oxidation states for the Fe and Mo
centers and both oxygen atom transfer and electron transfer processes.
However, several recent studies of recombinant variants of hSO have
produced seemingly paradoxical kinetic, structural and spectroscopic
data that are not easily explained by previous mechanistic proposals.
We have developed a comprehensive model for the catalytic mechanism of hSO that involves extensive inter-domain conformational
changes of all five formal oxidation states of the Fe and Mo centers.
This mechanistic model provides a framework for interpreting previous results on hSO and for planning future research. In addition, this
model clearly shows that hSO is not just a medical curiosity related to
a rare inherited disorder. Rather, hSO is an excellent example of a
multi-cofactor, multi-subunit, multi-domain, multi-conformational
state metallo-enzyme whose properties have broad and general
importance for medicine and for bioinorganic chemistry.
OP 5
Molybdenum and tungsten enzyme’s active site models:
some new developments in dithiolene chemistry
Carola Schulzke1, Yulia B. Borozdina1, Christian
Fischer1, Ashta Chandra Ghosh,1 Muhammad Zubair2
1
Institut für Biochemie, Ernst-Moritz-Arndt-Universität Greifswald,
Felix-Hausdorff-Str. 4, 17487 Greifswald, Germany
2
School of Chemistry, Trinity College Dublin, College Green, Dublin
2, Ireland. [email protected]
Some new developments in dithiolene molybdenum model chemistry,
in particular focusing on structural aspects of molybdopterin, will be
presented. These are, for instance, synthesis, characterisation and
reactivity of pyrane dithiolene complexes of molybdenum and tungsten and derivatives thereof. Chemical, electrochemical and catalytic
properties were studied in comparison in order to further understand
the role of the pyrane unit in molybdopterin. The pyrazine ring in
combination with the dithiolene moiety has been addressed separately. Among intended outcomes of the various synthetic approaches,
some unexpected and pleasantly surprising observations were made
[1, 2]. These are for instance unusual binding motifs found crystallographically (e.g. dithiolene-sulfur hydrogen bonds, interactions
involving the M=O moiety and packing motifs; Fig.), the discovery of
a very mild synthetic route to pentathiepins and the unexpectedly
facile oxidation of complexes by air.
J Biol Inorg Chem (2014) 19 (Suppl 2):S749–S764
Generous financial support by the ERC is gratefully acknowledged
(project: MocoModels).
S751
formation of stable metal complexes despite the lack of any common
strongly coordinating donor functions.
In this work we demonstrate through the studies of the above
mentioned peptides those tendencies which finely regulate the equilibrium, structural and electrochemical parameters of metal complexes.
Acknowledgement: The research was supported by the EU and cofinanced by the European Social Fund under the project ENVIKUT
(TÁMOP-4.2.2.A-11/1/KONV-2012-0043)
References
1. Timári S, Cerea R, Várnagy K (2011) J Inorg Biochem
105:1009–1017
2. Kállay C, Dávid Á, Timári S, Nagy EM, Sanna D, Garribba E,
Micera G, De Bona P, Pappalardo G, Rizzarelli E, Sóvágó I (2011)
Dalton Trans 40:9711–9721
References
1. Zubair M, Ghosh AC, Schulzke C (2013) Chem Commun
49:4343–4345
2. Döring A, Fischer C, Schulzke C (2013) Z Anorg Allg Chem
639:1552–1558
OP 6
The role of side chains in the fine tuning of metal
binding ability of peptides
Katalin Várnagy, Gizella Csire, Sarolta Timári,
Ágnes Dávid, Csilla Kállay
Department of Inorganic and Analytical Chemistry, University
of Debrecen, Egyetem tér 1, 4032 Debrecen, Hungary.
[email protected]
It is well known that peptides have high metal binding affinity, but
both the thermodynamic stability and the coordination geometry of
peptide complexes are very much influenced by the amino acid
sequence of the ligands. One field of our present research work is the
synthesis and investigation of polypeptides containing various side
chain donor groups, in which the coordination of side chain donor
atoms comes to the front and their sequences serve as the models of
different metalloproteins. These molecules include peptide fragments
of Cu, Zn-superoxide dismutase enzyme and amylin which is a
37-residue peptide hormone cosecreted with insulin by pancreatic bcells [1, 2].
We synthesized such series of multihistidine peptides in which the
systematic change of the amino acid sequence is carried out and the
equilibrium, structural and electrochemical parameters of their complexes are determined. These molecules include oligopeptides built up
from 4 to 12 amino acid residues containing 2–4 histidines among
them. The thermodynamic, structural and electrochemical properties
of these peptides are primarily determined by the number and location
of histidyl residues. The presence of positively or negatively charged
and polar or bulky side chains of other amino acids in the neighbourhood of the metal binding sites can, however, significantly
contribute to the above mentioned parameters of these complexes. To
understand the specific effects of these side chains aspartic acid,
serine or phenylalanine are inserted into the sequence of the multihistidine peptides [Ac-(HisXaa)n-His, Xaa=Ala, Phe, Asp, Ser etc.].
On the other side, the studies of amylin fragments (-ValArgSerSerAsnAsn-) and their mutants reveal that the presence of more
polar side chains (Ser, Asn etc.) in the peptide could result in the
OP 7
Transition metals alter the biological properties
of dithiocarbamates: formation of metal complexes
and the uses of metal–organic frameworks
Raymond W.-Y. Sun1, Ming Zhang1, Shan Deng2,
Alice S.-T. Wong3, Nikki P.-Y. Lee4
1
Department of Chemistry, Shantou University, No. 243 Daxue Road,
Shantou, Guangdong 515063, People’s Republic of China.
[email protected]
2
Department of Chemistry,
3
School of Biological Sciences and
4
Department of Surgery, The University of Hong Kong, Pokfulam
Road, Hong Kong
Various sulphur-containing molecules including dithiocarbamates and
an anti-alcoholism agent disulfiram have shown promising in vitro and
in vivo anti-cancer activities. Major hurdles in the development of these
molecules include the stability, bioavailability, specificity, drug detoxification and cellular resistance accounting for by the direct binding of
their active sulphurs to the thiol-containing peptides/proteins.
Different metal ions and their coordination compounds present
unique structural features and distinctive physical, chemical and
biological properties, thus rendering them irreplaceable roles over
common organic moieties in biological systems. By appropriate
selection of coordinating metal ions, dithiocarbamates can be tuned
and rationally designed to achieve different biological activities.
Moreover, metal can be used to construct biologically-relevant metal–
organic frameworks (MOFs). These materials can also be used as
carriers to enhance the bioavailability of the encapsulated materials.
In this work, we have demonstrated the alteration in physical properties and the anti-cancer/viral activities of various transition metalbased dithiocarbamato complexes compared to that of their corresponding dithiocarbamates. The encapsulating and sustained-release
properties of MOFs render to these dithiocarbamates and their related
complexes have also been reported.
Financial supports by Shantou University (2013 NTF13005),
General Research Fund (HKU 704812P), University Grants Committee of the Hong Kong Special Administrative Region are
gratefully acknowledged.
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J Biol Inorg Chem (2014) 19 (Suppl 2):S749–S764
References
1. Zhang JJ, Lu W, Sun RWY, Che CM (2012) Angew Chem Int Ed
51:4882
2. Zhang JJ, Lok CN, Ng KM, Sun RWY, Che CM (2013) Chem
Commun 49:5153–5155
OP 8
Ruthenium complexes of redox-active intercalating
ligands as an emerging class of anti-cancer agents
Frederick M. MacDonnell, Nagham Alatrash,
Eugenia S. Narh
Department of Chemistry and Biochemistry, University of Texas
at Arlington, Arlington, TX 76019, USA
The clinical success of cisplatin in cancer therapy has demonstrated
the tremendous potential of metal complexes as therapeutic agents,
however despite decades of subsequent research only a handful of
such metallodrugs exist, and most of these are simple derivatives of
cisplatin. We have reported on a new class of ruthenium complexes
which have low animal toxicity, good cytotoxicity and selectivity for
malignant over normal cells and which show *83 % regression in
H358 tumors implanted in nude mice compared to controls and a
doubling of lifetime [1, 2]. This paper will focus on examining the
mechanism of cytotoxicity which is correlated with their DNA
cleaving properties. The key structural feature common to the most
active complexes is the presence of a redox-active intercalating ligand
(see figure below) which can be bioreduced in situ. One redox product
is a reactive radical intermediate which is held in close juxtaposition
with the DNA backbone, and ultimately responsible for DNA
cleavage reaction. The pO2 is observed to affect the steady-state
concentration of the radical in a manner which results in enhanced
DNA cleavage as the pO2 is lowered. This has therapeutic implications as many tumor cells are often under hypoxic stress.
Financial support by the Robert A. Welch Foundation (Y-1301)
and US NCI-NIH is gratefully acknowledged.
RAIL
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
= {Ru(diimine)22+ fragment
References
1. Yadav A, Janaratne T, Krishnan A, Singhal SS, Yadav S, Dayoub
AS, Hawkins DL, Awasthi S, MacDonnell FM (2013) Mol Cancer
Ther 12:643–653
2. Janaratne TK, Yadav A, Ongeri F, MacDonnell FM (2007) Inorg
Chem 46:3420–3422
OP 9
Ferritin: another ‘dialogue concerning the two chief
world systems’
Kourosh Honarmand-Ebrahimi, Peter-Leon
Hagedoorn, Wilfred R. Hagen
Department of Biotechnology, Delft University of Technology,
Julianalaan 67, 2628BC Delft, The Netherlands. [email protected]
Some four decades of intensive biochemical research on the ubiquitous iron storage protein ferritin has provided a wealth of structural,
123
spectroscopic, kinetic, and thermodynamic data, from whose analysis
in recent years a sub-molecular picture of the mechanism of action is
beginning to emerge. Consensus, however, does not seem to be around
the corner yet, although the spectrum of viewpoints at this time appears
to condense into two well demarcated but mutually exclusive models:
(1) the unifying proposal that all ferritins essentially work according to
a single mode of operation (e.g. [1, 2]), versus (2) the diversifying
proposal that different classes of ferritins exhibit fundamental differences in their respective mechanisms (e.g. [3, 4]).
Drawing from Galileo’s format of a trialogue between two specialists and a layman to juxtapose arguments for/against Copernican
and Ptolemaic systems [5] we will present the main sets of data and
arguments for/against unifying and diversifying systems of ferritin
action with sufficient contrast to allow the informed layman to take
position. The debate centers around the question by what driving
force—after catalytic oxidation—the two iron ions leave the ferroxidase center of ferritin en route to core formation in the protein’s
cavity.
References
1. Honarmand-Ebrahimi K, Bill E, Hagedoorn P-L, Hagen WR (2012)
Nat Chem Biol 8:941–948
2. Watts RK (2013) Chem Bio Chem 14:415–419
3. Turano P, Lalli D, Felli IC, Theil E, Bertini I (2010) Proc Natl
Acad Sci USA 107:545–550
4. Bradley J, Moore GR, Le Brun NE (2014) J Biol Inorg Chem. doi:
10.1007/s00775-014-11363
5. Gallileo G (1632) ‘‘Dialogo sopra I due massimi sistemi del
mondo’’ Landini Firenze
OP 10
Tuning the nuclease activity of macrocyclic copper
complexes
Jan Hormann1, Nora Kulak1
1
Institute of Chemistry and Biochemistry, Freie Universität Berlin,
Fabeckstr. 34/36, 14195 Berlin, Germany. [email protected]
The cleavage of DNA is of high importance for biotechnological and
therapeutic applications [1, 2]. The degradation of DNA in cancer
cells is among the applications that could be carried out by so-called
nucleases. Whereas there are a variety of natural enzymes available,
as synthetic chemists we seek for small metal complexes that do the
same job, but come with some advantages concerning stability, prize
and accessibility to rational design.
Some of the artificial metallonucleases used so far are based on the
macrocyclic ligand cyclen (1,4,7,10-tetraazacyclododecane).
Approaches for increasing the efficiency of such metallonucleases
comprise the design of multinuclear metal complexes and the
attachment of DNA intercalators and positively charged residues to
the ligand moiety in order to increase the affinity to DNA [3].
We show here, that the exchange of one of the nitrogen atoms in
the cyclen ligand by oxygen (oxacyclen) or sulfur (thiacyclen) has an
J Biol Inorg Chem (2014) 19 (Suppl 2):S749–S764
important impact on the oxidative cleavage activity of its copper
complexes (O [ S [ N) [4]. Recent results are also presented to show
that further derivatization in the ligand’s donor set and periphery
leads to an increase in efficiency. The knowledge acquired during
these studies allows us now to tune the nuclease properties of cyclen
copper complexes.
Financial support by the Deutsche Forschungsgemeinschaft (DFG)
is gratefully acknowledged.
References
1. Hormann J, Perera C, Kulak N (2013) Nachr Chem 61:1003–1006
2. Wende C, Lüdtke C, Kulak N (2014) Eur J Inorg Chem (in press).
doi:10.1002/ejic.201400032
3. Mancin F, Scrimin P, Tecilla P (2012) Chem Commun
48:5545–5559
4. Hormann J, Perera C, Deibel N, Lentz D, Sarkar B, Kulak N (2013)
Dalton Trans 42:4357–4360
OP 11
DNA-interacting molecular switches
Andreu Presa1, Guillem Vázquez1, Patrick Gamez1,2
1
Departament de Quı́mica Inorgànica, Facultat de Quı́mica,
Universitat de Barcelona, Martı́ i Franquès 1-11, 08028 Barcelona,
Spain. [email protected]
2
Institució Catalana de Recerca i Estudis Avançats (ICREA), Passeig
Lluı́s Companys 23, 08010 Barcelona, Spain
Photoactivated chemotherapy drugs provide the prospect to achieve
highly controllable activity with reduced side effects [1]. Photoactivation of coordination compounds are commonly based on metalcentred processes [2].
Dithienylcyclopentene (DTE) molecules undergo thermally irreversible cyclization reactions between colourless (open) and coloured
(closed) forms when stimulated with UV and visible light (see figure)
[3]. This closing/opening event gives rise to a contraction or expansion of the molecule, respectively. For instance, the distance between
the methyl groups in 1,2-bis(2,5-dimethyl(3-thienyl))-3,3,4,4,5,5hexafluorocyclopent-1-ene decreases by 1.138 Å upon ring closure
(figure).
In this presentation, a series of photoswitching metal complexes
obtained from DTE-based ligands will be described together with
their properties. Actually, in addition to the expected distinct optical
properties, the open and closed forms of such coordination compounds exhibit different DNA-interacting properties.
Financial support by the Ministerio de Economı́a y Competitividad (MINECO) of Spain (Project CTQ2011-27929-C02-01). COST
Action CM1105 is kindly acknowledged.
S753
OP 12
Reversible transformation between cuboidal Fe4S4
and dinuclear Fe2S2 cores
Kazuki Tanifuji, Kazuyuki Tatsumi, Yasuhiro Ohki
Research Center for Materials Science, and Department of Chemistry,
Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya
464-8602, Japan
Splitting of the [Fe4S4] cluster core into two [Fe2S2] fragments has
not been realized in synthetic inorganic chemistry. On the other hand,
it has been proposed that the [Fe4S4] cubane in the fumarate nitrate
reductase regulatory protein is transformed to dinuclear [Fe2S2] cores
under O2, while the protein dissociates DNA [1]. A similar oxidative
decomposition of [Fe4S4] in Nif-IscA was postulated to occur generating [Fe2S2] cores [2].
In this presentation, we show a series of reactions displaying
interconversion between [Fe4S4] and [Fe2S2] core structures based
on the preformed all-ferric [Fe4S4]4+ cluster, [Fe4S4{N(SiMe3)2}4]
(1). Treatment of 1 with excess pyridine (py) or pyridine derivatives (py-R) resulted in splitting of the cubane core to
[Fe2S2{N(SiMe3)2}2(py-R)2] (2). The 1H NMR spectra of the dinuclear products 2 in C6D5Cl show presence of 1 and py-R,
indicating that 2 and 1 are in equilibrium in solution. Conversely,
fusion of two [Fe2S2] cores of 2 was found to be facilitated by
B(C6F5)3, generating [Fe4S4{N(SiMe3)2}4] (1) and (Py-R)B(C6F5)3.
[Fe4S4] cores with lower oxidation states were formed by chemical
reduction of cluster 2. Treatment of 2 with 1.2 equiv of Na[C10H8]
in THF afforded [Fe4S4{N(SiMe3)2}4]2- (3), while an analogous
reaction of 2 with 0.5 equiv of Na[C10H8] gave rise to [Fe4S4{N(SiMe3)2}4]- (4).
Interestingly, while the reduced forms of [Fe4S4] clusters, 3 and 4,
are intact in the presence of excess pyridine, the addition of excess
oxidant ([Cp2Fe]+) to the reaction systems readily gave [Fe2S2{N(SiMe3)2}2(py-R)2] (2), presumably via formation of all-ferric
[Fe4S4{N(SiMe3)2}4] (1). Thus, the all-ferric [Fe4S4]4+ state is
essential for dissociation of [Fe4S4] into [Fe2S2].
References
1. Popescu C, Bates DM, Beinert H, Münck E, Kiley PJ (1998) Proc
Natl Acad Sci USA 95:13431–13435.
2. D. T. Mapolelo DT, Zhang B, Naik SG, Huynh BH, Johnson MK
(2012) Biochemistry 51:8071–8084
OP 13
Conversion of readout from transcriptional regulator
by electron transfer proteins
Hiroshi Nakaijma1, Souji Miyazaki2, Takaaki Itoh1,
Yoshihito Watanabe2
1
References
1. Farrer NJ, Salassa L, Sadler PJ (2009) Dalton Trans 10690–10701
2. Szaciłowski K, Macyk W, Drzewiecka-Matuszek A, Brindell M,
Stochel G (2005) Chem Rev 105:2647–2694
3. Feringa BL (ed) (2001) Molecular switches. Wiley-VCH,
Weinheim
Department of Chemistry, Nagoya University, Chikusa-ku,
464-8602, Nagoya, Japan
2
Reseach Centre of Materials Science, Nagoya University, Chikusa-ku,
464-8601, Nagoya, Japan. [email protected]
In a biological system there are various transcriptional regulator
proteins which evolved to detect environmental factors, control
appropriate biological events, and retain the homeostasis of living
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cells at the transcriptional level. The high, specific sensitivity of
these proteins is attractive for constructing novel bio-based sensor
modules. However, facile conversion of the biological readout from
the protein to a readily detectable signal is a major issue to be
solved before application. We have recently developed a signaltransducing mechanism consisting of a pair of electron transfer (ET)
proteins [azurin and cytochrome c (Cyt c)] and a stimulus responsive
molecule that was introduced at the hydrophobic surface of azurin so
as to modulate inter-proteins interaction and the subsequent ET step
in the stimulus dependent manner [1]. In this study, we show
application of the signal-transducing mechanism to a transcriptional
regulator (figure). Thereby, the readout from the regulator is converted to a change in the apparent ET rate between the ET proteins
[2]. The hydrophobic surface of azurin was chemically modified
with a double stranded oligo-DNA (DNA-Azu) that contains a recognition sequence of a carbon monoxide (CO) dependent
transcriptional regulator, CooA [3]. CooA showed reversible binding
to DNA-Azu, depending on CO. The apparent ET rate constant (kET)
for Cyt c and DNA-Azu was determined to be 6.2 9 104 M-1 s-1,
which was 16 folds smaller than that for Cyt c and wild type azurin
(1.0 9 106 M-1 s-1) [1], likely due to steric and electrostatic hindrance of DNA. The CooA binding to DNA-Azu partially recovered
the ET rate (5.0 9 105 M-1 s-1). We will discuss this behavior and
possible application to a modified electrode. We gratefully
acknowledge financial support by MEXT, Japan.
References
1. Rosenberger N, Studer A, Takatani N, Nakajima H, Watanabe Y
(2009) Angew Chem Int Ed 48:1946–1949
2. Nakajima H, Miyazaki S, Itoh T, Hayamura M, Watanabe Y (2014)
Chem Lett (in press)
3. Thorsteinsson MV, Kerby RL, Conrad M, Youn H, Staples CR,
Lanzilotta WN, Poulos TJ, Serate J, Roberts GP (2000) J Biol Chem
275:39332–39338
OP 14
Contribution of each Trp residue towards the intrinsic
fluorescence of the Gia1 protein
Duarte Mota de Freitas, Matthew S. Najor, Kenneth W.
Olsen, Daniel J. Graham
Department of Chemistry and Biochemistry, Loyola University
Chicago, USA
Gia1 is the inhibitory G-protein that, upon activation, reduces the
activity of adenylyl cyclase. Comparison of the crystal structures of
Gia1 bound to GDP•AMF or GTPcS with that of the inactive, GPDbound protein indicates that a conformational change occurs in the
activation step centered on three switch regions. The contribution of
each tryptophan residue (W211 in the switch II region, W131 in the ahelical domain, and W258 in the GTPase domain) toward the intrinsic
protein fluorescence was evaluated by using W211F, W131F and
123
J Biol Inorg Chem (2014) 19 (Suppl 2):S749–S764
W258F mutants. Regardless of the conformation, all three tryptophan
residues contributed significantly toward the emission spectra. When
activated by either GDP•AMF or GTPcS, the maximal fluorescence
scaled according to the solvent accessibilities of the tryptophan residues, calculated from molecular dynamics simulations. In the
GDP•AMF and GTPcS, but not in the GDP, conformations, the
residues W211 and R208 are in close proximity and form a p-cation
interaction that results in a red shift in the emission spectra of WT,
and W131F and W258F mutants, but a blue shift for the W211F
mutant. The observed shifts did not correlate with the span of the
W211-R208 bridge but rather with the electrostatic energy of the
interactions in the various proteins. Trypsin digestion of the active
conformations only occurred for the W211F mutant indicating that
the electrostatic p-cation interaction blocks access to R208, which
was consistent with the molecular dynamics simulations. We therefore conclude that solvent accessibility and electrostatic interactions
account for the fluorescence features of Gia1.
OP 15
Label-free DNA-based biosensing using luminescent
metal complexes
Dik-Lung Ma
Department of Chemistry, Hong Kong Baptist University, Kowloon
Tong, Hong Kong. [email protected]
Oligonucleotides represent a versatile sensing platform due to their
ease of synthesis, sensitivity to particular analytes, low cost and
robust stability [1, 2]. Interest in DNA-based detection has exploded
in the scientific literature over the last few years. In particular, the use
of luminescent metal complexes as signal transducers in label-free
DNA sensing holds great promise as they are highly sensitive to
changes in the local environment, making them suitable to monitor
the DNA-switching event. Moreover, the application of luminescent
metal complexes in DNA sensing could further reduce the cost of
assay compared to the use of fluorescently-labelled oligonucleotides.
Transition heavy metal complexes possess salient advantages that
render them suitable for sensing applications: (1) their long emission
life-time allows their phosphorescence to be distinguished in highly
fluorescent media with the use of time-resolved spectroscopy, (2) they
usually display significant Stokes shifts which can prevent selfquenching, and (3) their interaction with biomolecules and their
photophysical properties can be readily tuned without lengthy synthetic procedures [3]. In this poster, I will present continuing progress
in the field of ‘‘label-free’’ luminescent based detection platform for a
variety of biologically and environmentally important analytes based
on oligonucleotides and luminescent metal complexes from our
research group.
J Biol Inorg Chem (2014) 19 (Suppl 2):S749–S764
References
1. Du Y, Li B, Wang E (2012) Acc Chem Res 46:203–213
2. Ma DL, He HZ, Leung KH, Zhong HJ, Chan DSH, Leung CH
(2013) Chem Soc Rev 42:3427–3440
3. Zhao Q, Huang C, Li F (2011) Chem Soc Rev 40:2508–2524
S755
OP 17
pH dependence of amyloid-b–Cu(II) binding
and oligomerization kinetics
Jeppe T. Pedersen1, Christian B. Borg2, Kaare Teilum2,
Lars Hemmingsen3
1
OP 16
Copper(II), nickel(II) and zinc(II) binding ability
of the N-terminal fragments of amyloid-b peptide
Imre Sóvágó, Ágnes Grenács
Department of Inorganic and Analytical Chemistry, University
of Debrecen, 4010 Debrecen, Hungary
Amyloid-b is a 40–43 residue peptide responsible for the development of Alzheimer’s disease. The N-terminus of the peptide is reach
in histidyl residues and contains some other polar side chains which
enhance the metal binding ability of the peptide. Speciation and
characterization of the copper(II), nickel(II) and zinc(II) complexes
of the N-terminal hexadecapeptide fragment, Ab(1–16)-PEG, have
already been reported in our previous publications [1] but the elucidation of the metal binding sites requires further studies. In this
work we report the synthesis of two nonapeptide domains of the
native peptide: Ab(1–9) and Ab(8–16) and their mutants. The
sequences of the six peptides studied are NH2-DAEFRHDSG-NH2,
NH2-DAAAAHAAA-NH2 and NH2-DAAAAAHAA-NH2 for
Ab(1–9) and Ac-SGAEGHHQK-NH2, Ac-SGAEGHAQK-NH2 and
Ac-SGAEGAHQK-NH2 for Ab(8–16). The results obtained from
combined potentiometric and spectroscopic (UV–vis, CD, ESR,
NMR and ESI–MS) studies will be presented here. Both thermodynamic and structural data support the primary role of the amino
termini of peptides in copper(II) and nickel(II) binding. Moreover, it
can be unambiguously stated that the amino acid sequence of the
N-terminal domains of amyloid peptides is especially well suited for
the complexation with copper(II) ions as it is represented by the
figure showing the distribution of copper ions among the native and
two mutant peptides. The enhanced stability of the copper(II) complexes was attributed to the secondary interactions of the polar side
chains of Asp, Glu, Ser and Arg residues present in the native
peptides.
Department of Pharmacy, University of Copenhagen,
Universitetsparken 2, 2100 Copenhagen, Denmark.
[email protected]
2
Department of Biology, University of Copenhagen, Ole Maaløes Vej
5, 2200 Copenhagen, Denmark
3
Department of Chemistry, University of Copenhagen,
Universitetsparken 5, 2100 Copenhagen, Denmark
Extracellular aggregation of amyloid-b peptides (Ab) is implicated in the
pathogenesis of Alzheimer’s disease. Metal ions such as Cu(II) can
promote aggregation of Ab on the millisecond–second time scale upon
binding [1, 2]. Hence, aberrant metal–Ab interaction may play a role in
development of AD. It is well-established that there are multiple coordination states of Cu(II) in soluble Ab and the different states co-exist in a
dynamic equilibrium depending on the pH [3,4]. It is reasonable to think
that distinct Ab–Cu(II) species could have distinct oligomerization propensity. Here, we study the Cu(II) binding mechanism to Ab and the
subsequent oligomerization at different pH using stopped-flow fluorescence and light scattering in combination with NMR relaxation.
Financial support by the Villum Foundation is gratefully acknowledged.
References
1. Noy D, Solomonov I, Sinkevich O, Arad T, Kjaer K, Sagi I (2008) J
Am Chem Soc 130:1376–1383
2. Pedersen JT, Teilum K, Heegaard NHH, Østergaard J, Adolph
H-W, Hemmingsen L (2011) Angew Chem Int Ed 50:2532–2535
3. Drew SC, Noble CJ, Masters CL, Hanson GR, Barnham KJ (2009)
J Am Chem Soc 131:1195–1207
4. Dorlet P, Gambarelli S, Faller P, Hureau C (2009) Angew Chem Int
Ed 48:9273–9276
β
OP 18
Probing the efficacy of novel bismuth (III and V)
complexes as anti-leishmanial agents
Philip C. Andrews,1 Lukasz Kedzierski,2 Yih Ching
Ong1
1
The research was supported by EU and co-financed by the European Social Fund under the project ENVIKUT (TAMOP-4.2.2.A-11/
1/KONV-2012-0043).
Reference
1. Arena G, Pappalardo G, Sóvágó I, Rizzarelli E (2012) Coord Chem
Rev 256:3–12
School of Chemistry, Monash University, Melbourne, VIC 3800,
Australia. [email protected]
2
Walter and Eliza Hall Institute of Medical Research, Parkville, VIC
3052, Melbourne, Australia
Even after 70 years, Leishmaniasis, the deadly parasitic disease
endemic in various forms across the developing world, is treated
primarily with two Sb(V) compounds; sodium stibogluconate and
meglumine antimoniate [1]. While effective, these drugs have significant problems; treatment for visceral leishmania requires
intravascular or intramuscular injections daily for 28 days under strict
medical monitoring, and intracellular reduction processes involving
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trypanothione produce active Sb(III) which is both highly toxic and
biodistributed [2]. Alternative drugs amphotericin B and pentamidine
are expensive and do not overcome the need for parenteral administration or reduce the onset of severe side effects, and miltefosine,
while orally active, is also expensive and teratogenic [3].
Bismuth and its compounds are considered to have low systemic
toxicity in humans, while showing good antimicrobial activities. In this
context, we have been examining and assessing novel organometallic
and metal–organic Bi(III) and highly oxidising Bi(V) compounds for
their anti-leishmanial activity and toxicity towards human fibroblasts. This has involved synthesis using a range of ligand classes
(e.g. carboxylates, thiocarboxylates, thioamides, thioxoketonates,
hydroxamates), full characterisation, including crystal structures, and
an examination of stability in aqueous and biological environments
[4].
In this presentation we will report our most recent results in the
chemistry and biological assays, demonstrating the potential of bismuth compounds in treating leishmaniasis.
References
1. Kedzierski L, Sakthianandeswaren A, Curtis JM, Andrews PC,
Junk PC, Kedzierska K (2009) Curr Med Chem 16:599–614
2. Demicheli C, Frézard F (2005) Drug Design Reviews-Online
2:243–249
3. Richard JV, Werbovetz KA (2010) Curr Opin Chem Biol 14:447
4. Andrews PC, Junk PC, Kedzierski L, Peiris, RM (2013) Aus J
Chem 13:6276–6279.
OP 19
Searching for new aromatic amine N-oxide metal
complexes as prospective agents against infectious
diseases
Esteban Rodrı́guez1, Ignacio Machado1, Leonardo
Biancolino Marino2, Florencia Mosquillo3, Leticia
Pérez3, Clarice Q. F. Leite2, Fernando R. Pavan2,
Lucı́a Otero1, Dinorah Gambino1
1
Cátedra de Quı́mica Inorgánica, Facultad de Quı́mica, Universidad
de la República, Gral. Flores 2124, 11800 Montevideo, Uruguay
2
Facultade de Ciencias Farmaceuticas, Unesp, 14801-902 Araraquara
(SP), Brazil 3Laboratorio de Interacciones Moleculares, Facultad de
Ciencias, Universidad de la República, Iguá 4225, 11400 Montevideo,
Uruguay
Infectious diseases are major causes of human disease worldwide.
Despite the progress in efforts to control the spread of tuberculosis,
this ancient and currently re-emerging infectious disease still remains
a global public health issue. Chagas disease (American Trypanosomiasis) is a chronic infection caused by the protozoan parasite
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Trypanosoma cruzi that affects about 10 million people in Latin
America.
Current chemotherapy for both diseases is inadequate and new
strategies for the discovery of new drugs are needed. Our group is
focused on the development of prospective metal-based drugs mainly
based on bioactive ligands and pharmacologically active metals. As
part of this work, we had previously developed Pd(II), Pt(II) and
Au(I) complexes of pyridine-2-thiol N-oxide (Hmpo). The ligand
blocks T. cruzi’s growth affecting all stages of the life cycle of the
parasite and showing low IC50 values. The complexes showed high
antitrypanosomal activities with adequate selectivity indexes. Results
suggested that the trypanocidal action of the complexes could mainly
rely on the inhibition of the parasite-specific enzyme NADH fumarate
reductase, main known parasite target for the free ligand.
In the search for new metal-based therapeutic tools against
tuberculosis and Chagas disease, and to further address the therapeutic potential of mpo metal complexes, two new octahedral
[MIII(mpo)3] complexes, with M = Ga or Bi, and two new Pd(II) and
Pt(II) heterobimetallic compounds [MII(L)(mpo)](PF6), with
L = ferrocene derivative, were synthesized and characterized in the
solid state and in solution. The compounds showed excellent activity,
both on the standard M. tuberculosis strain H37Rv ATCC 27294 (pansusceptible) and on five clinical isolates that are resistant to the
standard first-line anti-tuberculosis drugs isoniazid and rifampicin. In
addition, the complexes showed an enhancement of the anti-T. cruzi
activity compared with the parent compound.
These new derivatives are highly promising for the development
of prospective agents for the treatment of resistant tuberculosis and/or
Chagas disease.
References
1. Vieites M, Smircich P, Guggeri L, Marchán E, Gómez-Barrio A,
Navarro M, Garat B, Gambino D (2009) J Inorg Biochem
103:1300–1306
2. Vieites M, Smircich P, Parajón-Costa B, Rodrı́guez J, Galaz V,
Olea-Azar C, Otero L, Aguirre G, Cerecetto H, González M, GómezBarrio A, Garat B, Gambino D (2008) J Biol Inorg Chem 13:723–735
OP 20
The role of covalent heme to protein bonds
in the formation and reactivity of redox intermediates
of a bacterial peroxidase with high homology to human
peroxidases
Paul G. Furtmüller1, Markus Auer1, Andrea Nicolussi1,
Georg Schütz1, Marzia Bellei2, Gianantonio
Battistuzzi2, Christian Obinger1
1
Department of Chemistry, Division of Biochemistry, VIBT-Vienna
Institute of BioTechnology, BOKU-University of Natural Resources
and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
[email protected] 2Department of Chemistry and Geology,
University of Modena and Reggio Emilia, 41100 Modena, Italy
Reconstructing the phylogenetic relationships of the evolutionary
lines of the mammalian peroxidases revealed the presence of novel
bacterial heme peroxidase subfamilies [1]. Recently, an ancestral
bacterial heme peroxidase of the peroxidockerin clade was shown to
possess halide oxidation activities similar to human peroxidases.
Moreover, the recombinant protein allowed monitoring of the autocatalytic (i.e. hydrogen peroxide-driven) formation of covalent heme
to protein bonds (which are also found in vertebrate peroxidases [2].
Here, for the first time, the direct impact of the covalent heme to
protein bonds on the formation and reactivity of all relevant redox
intermediates of this peroxidase is demonstrated by transient kinetic
J Biol Inorg Chem (2014) 19 (Suppl 2):S749–S764
S757
measurements. Protein species with covalently bound heme were
compared with those having predominantly unmodified heme b.
We report the kinetics of binding of the low-spin ligand cyanide
and demonstrate the strong influence of this posttranslational modification on the redox reactions including formation of Compound I by
hydrogen peroxide as well as two- and one-electron reduction reactions of Compound I to either the ferric enzyme or Compound II. The
presented data are discussed with respect to the known crystal
structures and kinetic data available from mammalian peroxidases.
This research was supported by the Austrian Funding Fund (FWFstand-alone project P20664 and the doctoral program BioToP- Biomolecular Technology of Proteins W1224).
3. Hofbauer S, Gysel K, Mlynek G, Kostan J, Hagmüller A, Daims H,
Furtmüller PG, Djinovic-Carugo K, Obinger C (2012) Biochim Biophys Acta Proteins Proteomics 1824:1031–1038
4. Hofbauer S, Bellei M, Sündermann A, Pirker KF, Hagmüller A,
Mlynek G, Kostan J, Daims H, Furtmüller PG, Djinović-Carugo K,
Oostenbrink C, Battistuzzi G, Obinger C (2012) Biochemistry
51:9501–9512
5. Hofbauer S, Gysel K, Bellei M, Pirker KF, Hagmüller A, Schaffner
I, Mlynek G, Kostan J, Daims H, Furtmüller PG, Battistuzzi G, Djinovic-Carugo K, Obinger C (2014) Biochemistry 53:77–89
6. Hofbauer S, Schaffner I, Furtmüller PG, Obinger C (2014) Biotechnol J 9:461–473
References
1. Zamocky M, Jakopitsch C, Furtmüller PG, Dunand C, Obinger C
(2008) Proteins 71:589–605
2. Auer M, Gruber C, Bellei M, Pirker KF, Zamocky M, Kroiss D,
Teufer SA, Hofbauer S, Soudi M, Battistuzzi G, Furtmüller PG,
Obinger C (2013) J Biol Chem 288:27181–27199
OP 22
Metal mobilization from waste hydroxide sludge
by sulfur oxidizing bacteria
Helmut Brandl1, Carlotta Fabbri1, Thomas Wüthrich2
1
1
Department of Chemistry, Division of Biochemistry,
Department for Structural and Computational Biology, Max F.
Perutz Laboratories, University of Vienna, 1030 Vienna, Austria
3
Department of Chemistry and Geology, University of Modena
and Reggio Emilia, 41125 Modena, Italy
Chlorite dismutases (Clds) are oligomeric heme b-dependent oxidoreductases capable of catalyzing the conversion of chlorite (ClO2-)
into chloride and dioxygen (designation as ‘‘dismutase’’ is wrong and
should be eliminated in future).
This presentation compares two model Clds [1–3] from the two
main lineages that differ in oligomeric structure and subunit architecture. Here, we compare the available X-ray structures and discuss
the role of conserved heme cavity residues in maintenance of the
active site architecture as well as in catalysis [4, 5]. A reaction
mechanism is presented that underlines the important role of the
highly conserved distal arginine in keeping the transiently formed
intermediate hypochlorous acid in the reaction sphere for recombination with the oxoiron(IV) of Compound I. In this reaction a
covalent oxygen–oxygen bond is formed and O2 is released. Finally,
we discuss the close phylogenetic relationship between Clds and
recently discovered dye-decolorizing peroxidases [6].
Our research was supported by the Austrian Funding Agency
(FWF-doctoral program BioToP-Biomolecular Technology of Proteins, W1224 and the stand alone project P25270).
2
References
1. Kostan J, Sjoeblom B, Maixner F, Mlynek G, Furtmüller PG,
Obinger C, Wagner M, Daims H, Djinovic-Carugo K (2010) J Struct
Biol 172:331–342
2. Mlynek G, Sjöblom B, Kostan J, Füreder S, Maixner F, Gysel K,
Furtmüller PG, Obinger C, Wagner M, Daims H, Djinovic-Carugo K.
(2011) J Bacteriol 193:2408–2417
100
80
mobilization (%)
OP 21
Chlorite to chloride and O2 conversion: new lessons
from structural and mechanistic investigations
of chlorite dismutase
Christian Obinger1, Stefan Hofbauer1, Irene
Schaffner1, Katharina F. Pirker1, Georg Mlynek2,
Kristina Djinovic-Carugo2, Gianantonio Battistuzzi3,
Paul G. Furtmüller1
Institute of Evolutionary Biology and Environmental Studies,
University of Zurich, Winterthurerstrasse 190, 8057 Zurich,
Switzerland 2ALAB AG, In der Luberzen 5, 8902 Urdorf, Switzerland
When applying biological techniques (‘‘biohydrometallurgy’’) in the
mining of valuable metals such as copper and gold (‘‘bioleaching’’,
‘‘biomining’’), sulfur oxidizing microorganisms play a fundamental
role [1]. Sulfur oxidizers belong to the group of acidophilic microbes,
thrive on carbon dioxide, and form sulfuric acid as end product of
their metabolism resulting in the mobilization of elements from solid
materials. However, when treating metal-containing industrial waste,
high salt content along with high alkalinity might inhibit these acidloving microbes. Therefore, we investigated the physiological
potential of Halothiobacillus neapolitanus for the mobilization of
metals from waste hydroxide sludge originating from flue gas purification. H. neapolitanus is salt tolerant and metabolically active over
a pH range of 4–8.5 (with an optimum between 6.5 and 7), which
seems to be ideal for the biological treatment of alkaline waste
materials. Within a growth period of 10 days in a suspension of 10 g
sludge per liter, pH values dropped from 7 to 3.4. It was possible to
solubilize certain metals completely (e.g., Cd, Zn), whereas others
were mobilized to a smaller extent (e.g. Pb 30 %, Cu 50 %). Zn was
the major constituent (*95 %) of the leachate. By gradually
increasing bulk density, H. neapolitanus adapted to suspensions of
30 g sludge per liter. In summary, results showed that H. neapolitanus
can cope with alkaline salt-containing waste materials and mobilize
some metals to a high extent. In perspective, this might be the base for
a biological recovery of metals from hydroxide sludge, all the more
because the selective environment (high salt and metal content, low
pH) might allow a biological treatment of wastes under non-sterile
conditions.
60
40
20
0
Al
Cd
Cu
Fe
Ni
Pb
Zn
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Reference
1. Brandl H (2001) In: Rehm HJ (ed) Biotechnology, vol 10. WileyVCH, Weinheim, pp 191–224
OP 23
Hydride binding to the active-site H-cluster of [FeFe]hydrogenase
Petko Chernev1, Camilla Lambertz2, Nils Leidel1,
Kajsa Sigfridsson1, Ramona Kositzki1, Thomas Happe2,
Michael Haumann1
1
Department of Physics, Free University Berlin, Arnimallee 14,
14195 Berlin, Germany. [email protected]
2
Institute for Biochemistry of Plants, Department
of Photobiotechnology, Universitätsstrasse 150, Ruhr-University
Bochum, 44801 Bochum, Germany
[FeFe]-hydrogenase from green algae (HydA1) is the most efficient
enzyme for hydrogen (H2) production in nature. Its active site is a
unique six-iron center (H-cluster) composed of a [4Fe4S]H cluster
linked to a diiron unit, [2Fe]H. The molecular and electronic configurations of the H-cluster need to be determined to understand the
specific restraints for high-rate H2 production to be implemented in
novel synthetic catalysts. We have probed the electronic configuration of the H-cluster in purified HydA1 protein using site-selective
X-ray absorption and emission spectroscopy experiments for the first
time [1, 2]. This has provided novel and distinct spectroscopic signatures, which were reproduced and interpreted by quantum
chemical calculations (DFT), thereby leading to specific H-cluster
model structures. The electronic configuration of several redox
intermediates thus was determined. We show that iron-hydride bonds
are absent in the oxidized and one-electron reduced states of the
H-cluster. Only in the two-electron (super-)reduced state an ironhydride bond could be directly detected. The hydride binding possibly occurs to the Fe–Fe bridging position at [2Fe]H. These results
suggest a catalytic cycle of [FeFe]-hydrogenases with at least three
main intermediates, involving protonation, hydride binding, and
electron transfer steps prior to the H2 formation chemistry. Our
methods open a new perspective for characterization of metalhydride species in (bio)inorganic chemistry.
MH acknowledges financial support by the DFG (Grants Ha3265/
2-2,/3-1, and/6.1), the BMBF (Grant 05K14KE1 within the RöntgenAngström Cluster), and Unicat (CoE Berlin).
References
1. Chernev P, Lambertz C, Sigfridsson K, Leidel N, Kositzki R, Hsieh
C, Schiwon R, Yao S, Limberg C, Driess M, Happe T, Haumann M
(2014) manuscript submitted
2. Lambertz C, Chernev P, Klingan K, Leidel N, Sigfridsson K,
Happe T, Haumann M (2014) Chem Sci 5:1187–1203
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OP 24
Probing the electron transfer mechanism of diironcarbonyl complexes relevant to the diiron sub-unit
of [FeFe]-hydrogenase
Guifen Qian2, Zhinyin Xiao1, Li Long1, Xiaoming Liu1, 2
1
College of Biological, Chemical Sciences and Engineering, Jiaxing
University, Jiaxing, 314001 Zhejiang, China
2
Department of Chemistry, Nanchang University, Nanchang, 330031
Jiangxi, China. [email protected]
[FeFe]-hydrogenase and its mimicking chemistry have attracted a
great deal of attentions since its structural revelation about 15 years
ago due to its relevance to hydrogen energy, a promising energy
vector in future. Under physiological conditions, this enzyme
catalyses hydrogen evolution with zero-overpotential. It is appealing
to understand the intrinsic chemistry behind this feature. The confirmation of azodithiolate as the bridge of the diiron centre suggests
that PCET contributes certainly to decrease the overpotential [1].
Since either evolution or oxidation of hydrogen, two-electron per
molecule is involved. Therefore, there ought to be other explanation
for the zero-overpotential. Electrochemical investigations into the
mimics of the diiron sub-unit show that the reduction of the diironcarbonyl complexes may involve two-electron process despite a
single reduction wave observed often in their cyclic voltammograms, that is, involving potential inversion caused by isomerisation
upon reduction [2]. By incorporating a ferrocenyl group into the
mimics to calibrate the number of electron [3], the ECE process is
clearly demonstrated and it is concluded that the inversed potential
(E2) can not be more positive than the first potential (E1). In conclusion, PCET and the potential inversion are the main causes for
the zero-overpotential of the enzymatic catalysis in hydrogen
evolution.
Financial support by Natural Science Foundation of China is
gratefully acknowledged.
References
1. Berggren G, Adamska A, Lambertz C, Simmons TR, Esselborn J,
Atta M, Gambarelli S, Mouesca JM, Reijerse E, Lubitz W, Happe T,
Artero V, Fontecave M (2013) Nature 299:66–70
2. Lounissi S, Zampella G, Capon JF, De Gioia L, Matoussi F,
Mahfoudhi S, Petillon FY, Schollhammer P, Talarmin J (2012) Chem
Eur J 18:11123–11138
3. Zeng X, Li Z, Xiao Z, Wang Y, Liu X (2010) Electrochem
Commun 12:342–345
J Biol Inorg Chem (2014) 19 (Suppl 2):S749–S764
S759
OP 25
M–Purine(C8) constructs and their potential
applications in catalysis
Pablo J. Sanz Miguel, Andrea Cebollada, Alba Vellé
OP 26
Geometric and electronic structures of 5d
metallocorroles: Au, Pt, Os
Abhik Ghosh
1
Department of Chemistry, UiT-The Arctic University of Norway,
9037 Tromsø, Norway. [email protected]
Because of the size mismatch between the contracted N4 cores of
corroles and the large ionic radii of the 5d transition metals in their
lower oxidation states, the synthesis of 5d metallocorroles has been a
challenge for synthetic coordination chemists [1]. Against this backdrop, gold corroles were synthesized recently and, in as yet
unpublished work in our laboratory, the first platinum and osmium
corroles have been synthesized. The work provides fascinating
examples of synthetic strategy, heavy-element mediated C–H activation, and ligand noninnocence, the last perhaps best exemplified by
a series of oxidized Pt corroles with the formula Pt(corrole•2-)ArAr0 .
A representative crystal structure is shown below. The potential
anticancer properties of the Pt complexes are currently being examined.
Departamento de Quı́mica Inorgánica, Instituto de Sı́ntesis Quı́mica
y Catálisis Homogénea (ISQCH), Universidad de Zaragoza-CSIC,
50009 Zaragoza (Spain). [email protected]
Coordination of transition metals to the imidazolic positions of purines or their derivatives has been widely studied in the cases of the N7
and N9 sites [1], but only scarcely for C8. There are few examples of
metal coordination to the C8 sites of N7,N9-methylated purines [2].
Houlton et al. prepared several C8-coordinated metal complexes with
purines by cyclometallation [3], with N7/N9 available for metal
coordination. In addition, only three examples of caffeine as C8monodentate ligand for Os, Ru, and Co have been reported [4]. Our
interest on C8-coordination of transition metals at purines is grounded
on their analogy with the N-heterocyclic carbene ligands, commonly
employed in catalysis.
We report on the first examples of twofold metal coordination to
both the C8 and N9 sites of purines, including examples of (1) catalytic active Mn(purine-C8)n cyclic compounds, and (2) a stepwise
formation strategy of a Pt4,Pd4,Ag2 aggregate in which its central
skeleton is supported by dative bonds and strong intermetallic interactions [5].
Financial support by the Spanish Ministerio de Economı́a y
Competitividad (CTQ2011-27593, and Ramón y Cajal Program) is
gratefully acknowledged.
References
1. See e.g.: (a) Lippert B (2000) Coord Chem Rev 200–202:487–516;
(b) Houlton A (2002) Adv Inorg Chem 53:87–158
2. (a) Kascatan-Nebioglu A, Panzner MJ, Garrison JC, Tessier CA,
Youngs WJ (2004) Organometallics 23:1928–1931; (b) Skander M,
Retailleau P, Bourrie B, Schio L, Mailliet P, Marinetti A (2010) J
Med Chem 53:2146–2154; (c) Stefan L, Bertrand B, Richard P, Le
Gendre P, Denat F, Picquet M, Monchaud D (2012) ChemBioChem
13:905–912
3. See e.g.: (a) Price C, Elsegood MRJ, Clegg W, Rees NH, Houlton
A (1997) Angew Chem Int Ed 36:1762–1764; (b) Price C, Shipman
MA, Rees NH, Elsegood MRJ, Edwards AJ, Clegg W, Houlton A
(2001) Chem Eur J 7:1194–1201
4. (a) Krentzien HJ, Clarke MK, Taube H (1975) Bioinorg Chem
4:143–151; (b) Johnson A, O’Connell LA, Clarke MJ (1993) Inorg
Chim Acta 210:151–157; (c) Zhenga T, Suna H, Lua F, Harmsc K, Li
X (2013) Inorg Chem Commun 30:139–142
5. Cebollada A, Velle A, Sanz Miguel PJ (2014) unpublished results.
Reference
1. Thomas KE, Alemayehu A, Conradi, J, Beavers CM, Ghosh A
(2012) Acc Chem Res 45:1203–1214
OP 27
Investigation of metal complexes-RNA interaction
Marianthi Zampakou1, Elena Alberti1, Michael P.
Coogan2, Daniela Donghi1
1
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland. [email protected]
2
Department of Chemistry, Faraday Building, Lancaster University,
Bailrigg, Lancaster, LA1 4YB, UK
The use of metal complexes as therapeutic and diagnostic agents is
well acknowledged [1]. Depending on their chemical nature, these
complexes can interact with their biological target via covalent and
non-covalent binding [1]. The anticancer drug cisplatin and its
derivatives belong to the first class of compounds, and are believed to
mainly target DNA by preferentially binding to N7 atoms of guanine
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J Biol Inorg Chem (2014) 19 (Suppl 2):S749–S764
bases [2]. Conversely, various complexes studied as potential bioimaging agents belong to the second class, and show luminescence
upon DNA intercalation [3]. In addition to DNA, metal complexes
can also target other biomolecules, including RNA [4]. The latter is
involved in several crucial biological processes and its structural
diversity makes it an attractive target for the development of structure-selective RNA targeting molecules [5]. For example, platinum
drugs can inhibit RNA dependent processes [4] and metal complexes
with potential in bio-imaging were shown to accumulate in RNA-rich
regions within the cell [6]. Nevertheless, information on metal containing molecules binding to RNA is still scarce.
We are currently investigating the interaction of different classes
of metal complexes with RNA to rationalize the basis of structureselective recognition. We use as model systems RNA constructs that
contain structural features widespread in RNA, e.g. GU wobbles,
internal and terminal loops. On the one hand, we study RNA interaction with platinum drugs with a special focus on cisplatin and
oxaliplatin. On the other hand, we investigate the RNA binding ability
of mononuclear rhenium(I) metallo-intercalators [6]. The interaction
is studied by several techniques, including gel mobility shift assays,
UV–vis, luminescence and CD spectroscopy and mass spectrometry.
Special attention is given to NMR spectroscopy, which is used to both
localize the interaction site and to evaluate the structural changes
induced by metal complex binding.
Financial support by the Swiss National Science Foundation
(Ambizione fellowship PZ00P2_136726 to DD), by the University of
Zurich (including the Forschungskredit FK-13-107 to DD) and within
the COST Action CM1105 is gratefully acknowledged.
treatments to facilitate thiocyanate formation since it does not affect
oxygen carrying capacity in smoke inhalation victims [2–3]. We
describe our approach to catalytically transfer sulfur from thiosulfate
to cyanide to facilitate thiocyanate formation, using well-tolerated
compounds in small amounts to minimize toxicity without sacrificing
oxygen transport. Data for spontaneous and catalytic sulfur transfer
reactions will be presented along with results from toxicity and efficacy studies. In one instance we show near complete elimination of
cyanide from solution in 20 min, in a reaction of cyanide with thiosulfate solutions containing 10 mol% of a molybdenum sulfur
complex. The molybdenum sulfur complexes were shown non-toxic
in hepatocytes, and safe dose in mice was measured as 0.5 g/kg.
Financial support by the University of Iceland Research Fund, and
NIH NINDS Grant No. 5R21NS067265 is gratefully acknowledged.
References
1. Ma D-L, He H-Z, Leung K-H, Chan DS-H, Leung C-H (2013)
Angew Chem Int Ed 52:7666–7682
2. Alderden RA, Hall MD, Hambley TW (2006) J Chem Educ
83:728–734
3. Zeglis BM, Pierre VC, Barton JK (2007) Chem Commun
4565–4579
4. Chapman EG, Hostetter AA, Osborn MF, Miller AL, DeRose VJ
(2011) Met Ions Life Sci 9:347–377
5. Guan L, Disney MD (2012) ACS Chem Biol 7:73–86
6. Thorp-Greenwood FL, Coogan MP, Mishra L, Kumari N, Rai G,
Saripella S (2012) New J Chem 36:64–72
References
1. Leininger K, Westley J (1968) J Biol Chem 243:1892–1899
2. Ivankovich AD, Braverman B, Kanuru RP Heyman HJ Paulissian R
(1980) Anesthesiology 52:210–216
3. Baud FJ (2007) Hum Exp Toxicol 26:191–201
OP 29
Metal complexes as molecularly-targeted agents against
protein–protein interactions
Hai-Jing Zhong1, Li-Juan Liu1, Daniel Shiu-Hin Chan2,
Dik-Lung Ma2, Chung-Hang Leung1
1
OP 28
Cyanide detoxification by molybdenum sulfur
complexes
Sigridur G. Suman1,2, Johanna M. Gretarsdottir1,
Thorvaldur Snæbjörnsson1, Gerdur R. Runarsdottir1,
Paul E. Penwell2, Shirley Brill3, Carol Green3
1
Science Institute, University of Iceland, Dunhagi 3, 107 Reykjavik,
Iceland
2
Physical Sciences Department, SRI International, 333 Ravenswood
Avenue, Menlo Park, CA 94025, USA
3
Biosciences Department, SRI International, 333 Ravenswood
Avenue, Menlo Park, CA 94025, USA. [email protected]
Thiocyanate is a product of a biocatalytic reaction of cyanide and
sulfur by the rhodanase enzyme in the liver. Mechanistic studies
in vitro of the rhodanase catalyzed reaction of cyanide and thiosulfate
showed the reaction takes place by a conformational change of the
enzyme and metal assisted thiosulfate binding. The rate limiting step
of this reaction is the rupture of the sulfur–sulfur bond in thiosulfate
[1]. Natural sulfur substrates are quickly depleted at toxic levels of
cyanide. Thiosulfate is commonly administered with cyanide
123
Department of Chemistry, Hong Kong Baptist University, Kowloon
Tong, Hong Kong, China
2
State Key Laboratory of Quality Research in Chinese Medicine,
Institute of Chinese Medical Sciences, University of Macau, Macao,
China
Protein–protein interactions (PPIs) are ubiquitous in essential biological processes such as cell proliferation and differentiation, hostpathogen interactions, and signal transduction pathways [1]. Pioneering advances in the field of interactomics have uncovered new
networks of protein interactions within cells, with estimates for the
size of the interactome ranging up to 650,000 PPIs [2]. Hence, PPIs
have emerged as attractive targets in medicinal chemistry and drug
discovery [3]. Meanwhile, transition metals possess variable oxidation states and molecular geometries that enable the design of
intricate coordination sphere architectures. The ability to arrange
organic ligands in a precise three-dimensional arrangement around
the metal centre can be harnessed to generate unique scaffolds for
recognizing the binding sites of proteins. Due to the adverse side
effects associated with ‘‘shotgun’’ cytotoxic metal complexes such as
cisplatin and its analogues, there has been a recent upsurge in interest
in the development of kinetically-inert metal complexes as molecularly-targeted agents against enzymes or PPIs [4–7]. We present
recent examples of biologically active, kinetically-inert metal
J Biol Inorg Chem (2014) 19 (Suppl 2):S749–S764
complexes developed by our group, and highlight possible future
directions for this exciting field. Financial support by the University
of Macau is gratefully acknowledged.
References
1. Lievens S, Eyckerman S, Lemmens I, Tavernier J (2010) Expert
Rev Proteomics 7:679–690
2. Stumpf M, Thorne T, de Silva E, Stewart R, An H, Lappe M, Wiuf
C (2008) Proc Natl Acad Sci USA 105:6959–6964
3. Wells J, McClendon C (2007) Nature 450:1001–1009
4. Meggers E (2011) Angew Chem Int Ed 50:2442–2448
5. Leung CH, Zhong HJ, Yang H, Cheng Z, Chan DS, Ma VP, Abagyan
R, Wong CY, Ma DL (2012) Angew Chem Int Ed 51:9010–9014
6. Zhong HJ, Leung KH, Liu LJ, Lu L, Chan DSH, Leung CH, Ma DL
(2014) ChemPlusChem (in press)
7. Leung CH, He HZ, Liu LJ, Wang M, Chan DSH, Ma DL (2013)
Coord Chem Rev 257:3139–3151
OP 31
Lanthanide complexes as tools for structural biology
Bim Graham1, James D. Swarbrick1, Michael D. Lee1,
Phuc Ung1, Sandeep Chhabra1, Choy Theng Loh2,
Thomas Huber2, Gottfried Otting2
1
Monash Institute of Pharmaceutical Sciences, Monash University,
Parkville, VIC 3052, Australia. [email protected]
2
Research School of Chemistry, Australian National University,
Canberra, ACT 0200, Australia
The tagging of proteins with paramagnetic lanthanide ions produces
large effects that are observable in NMR spectra, including pseudocontact shifts, paramagnetic relaxation enhancements and residual
dipolar couplings [1, 2]. These effects provide valuable structural
restraints to expedite protein structure determination and facilitate
structure analysis of protein–protein and protein–ligand interactions.
In addition, the attachment of pairs of gadolinium complexes to
proteins enables highly accurate distance measurements to be made in
protein assemblies via EPR spectroscopy [3]. Our group has developed a range of new tagging reagents and strategies for attaching
lanthanide ions to proteins in a site-specific manner, which have
greatly facilitated such structural studies. This presentation will
describe the synthesis, testing and utilization of some of our most
successful designs and approaches.
Financial support by the Australian Research Council is gratefully
acknowledged, including a Future Fellowship to B.G.
S761
References
1. Otting G (2010) Annu Rev Biophys 39:387–405
2. Keizers PHJ, Ubbink M (2011) Prog Nucl Magn Reson Spectrosc
58:88–96.
3. Yagi H, Banerjee D, Graham B, Huber T, Goldfarb D, Ottin G
(2011) J Am Chem Soc 133:10418–10421
OP 32
Expanding nature’s toolbox with artificial
metalloenzymes
Jörg Eppinger1, Johannes Fischer1, Anna Zernickel1,
Arwa Makki1
1
Division of Physical Sciences and Engineering and KAUST
Catalysis Centre (KCC), King Abdullah University of Science
and Technology (KAUST), Thuwal 23955-6900, Saudi Arabia.
[email protected]
Artificial metalloenzymes are expected to combine enzymatic selectivity with the broad range of catalytic motifs provided by
homogeneous catalysts. Using specifically designed metal-conjugated
affinity labels to introduce metal centres into the binding pocket of
cysteine proteases, we were able to overcome the lack of structural
definition, which tends to hamper catalytic selectivity. Experimental
results proof, that the protein ligand induces enantioselectivities. The
novel modular platform and the in situ protocol allow fast generation
of diverse libraries of organometallic enzyme hybrid catalysts (see
figure) [1].
Site-selective orthogonal incorporation of metal binding unnatural
amino acids (UAA) into a host protein represents another novel tool
to create catalytically active metalloenzymes in vivo. The incorporated UAA provides stable ligation of late transition metals or serves
as an anchoring point to selectively conjugate metal chelating
motives to the host protein. This presentation details our studies on
the development of an optimized fluorescent host protein (mTFP*)
with minimized metal binding affinity and its conversion into an
artificial metalloenzyme through UAA incorporation and specific
UAA-metal conjugation. X-Ray crystallographic studies, post-translational modification (e.g. CuAAC) and catalytic tests for
asymmetric cyclo-addition and Pd-catalyzed cross-coupling reactions
are presented.
Financial support by the King Abdullah University of Science and
Technology, KAUST (faculty baseline fund and KAUST-GCR project FIC/2010/07) is gratefully acknowledged.
Reference
1. Reiner T, Jantke D, Marziale AM, Raba A, Eppinger J (2013)
ChemistryOpen 2:50–54
123
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J Biol Inorg Chem (2014) 19 (Suppl 2):S749–S764
OP 33
Synthesis of fac-{99(m)TcO3}+ complexes: activation
of [99(m)TcO4]2 by phosphonium cations
Henrik Braband, Michael Benz, Roger Alberto
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland. [email protected]
99m
Tc is a very practical nuclide for nuclear medical applications due
to its availability from generators, its short physical half-life time
(6 h), and the emission of low energy c-rays (140.5 keV). We are
aiming at a general understanding of the reactivity of technetium at its
highest oxidation state +VII. Our research foremost focuses on
compounds containing the fac-{99(m)TcO3}+-core, due to its interesting chemical reactivities ((3+2)-cycloaddition with alkenes) [1].
This reactivity enables an innovative approach for the synthesis of
novel radioconjugates [2]. Recent developments, based on the interaction of phosphonium salts with the robust [99(m)TcO4]- anion in
neutral water, led to a simple procedure for the synthesis of
[99mTcO3(tacnR)]+ type complexes (tacnR = 1,4,7-triazacyclononane
or derivatives) [3]. Due to this new approach fac-{99mTcO3}? complexes are now available in high yields and purity for stereoselective
labeling of biomolecules. The potential of the new bioconjugation
strategy has been demonstrated by labeling of a series of different
vectors (pharmacophores, non-natural amino acids, and carbohydrates) [4]. Furthermore, the labeling via (3?2)-cycloaddition has
been established as a novel procedure for the labeling of silica based
particles, which will help to gain more detailed in vivo data of silica
(nano)particles by non-invasive radioimaging, in the future [5].
H
H
N
N
N
+
Tc
O
O
H
H
O
R
H
N
N
N
H
Tc
O
O
O
R
R = pharmacophores,
amino acids,
carbohydrates
(nano)particles
References
1. Pearlstein RM, Davison A (1988) Polyhedron 7:1981–1989
2. Braband H, Tooyama Y, Fox T, Alberto R (2009) Chem Eur J
15:633–638
3. Braband H, Benz M, Tooyama Y, Alberto R (2014) Chem Commun 50:4126–4129
4. Braband H, Tooyama Y, Fox T, Simms R, Forbes J, Valliant, JF,
Alberto R (2011) Chem Eur J 17:12967–12974
5. Wuillemin, MA, Stuber WT, Fox T, Reber MJ, Brühwiler D,
Alberto R, Braband H (2014) Dalton Trans 43:4260–4263
OP 34
Alkalimetal controlled DNA nanoswitch
Célia Fonseca Guerra1, Jordi Poater1, Marcel Swart1,2,
F. Matthias Bickelhaupt1
1
Department of Theoretical Chemistry, VU University Amsterdam,
1081 HV Amsterdam, The Netherlands
2
Institut de Quı́mica Computacional, Universitat de Girona, 17071
Girona, Spain. [email protected]
The self-assembly capacity of DNA has been an inspiration in the
field of supramolecular chemistry. We show with dispersion-corrected
density functional theory that DNA itself can be used as a nanoswitch,
able to alternate between three states (weak, moderate and strongly
bound). The suitable DNA base pair to act as the switch is the
Watson–Crick GC base pair. Substitution of H8 at the six-membered
123
ring of the purine by OH enables via protonation or deprotonation to
obtain the switching capacity. This capacity is also observed when the
switching aggregate is addressed through coordination of alkali metal
cations to OH instead of protons. The switching behavior is still
preserved when the subsitituent is linked to the DNA base pair via
an acetylene linker (26 Å) turning the substituent into a remote
control. The switching could therefore pass through a membrane
allowing for different experimental conditions of the controller and
the switch. The last step in the computational design of a DNA
switch was to introduce the switch into a DNA helix and ‘‘submerge’’ it into different solvents. This computational investigation
of the artificial DNA nanoswitch showed that the switch conserves
its switching capacities under experimental conditions which in
general involve solvation.
Financial support by the NRSC-C, NWO, MICINN and HPCEuropa2 is gratefully acknowledged.
References
1. Fonseca Guerra C, van der Wijst T, Bickelhaupt FM (2006) Chem
Eur J 12:3032–3042
2. Fonseca Guerra C, Szekeres Z, Bickelhaupt FM (2011) Chem Eur J
17:8816–8818
3. Poater J, Fonseca Guerra C, Swart M, Bickelhaupt FM, submitted
OP 35
The diverse functions of calcium in natural water
oxidation
Dimitrios A. Pantazis, Marius Retegan, Vera Krewald,
Frank Neese, Nicholas Cox
Max Planck Institute for Chemical Energy Conversion, Stiftstr.
34–36, 45470 Mülheim an der Ruhr, Germany
Natural water oxidation, carried out by an inorganic Mn4CaO5 cluster
embedded in the enzyme photosystem II of photosynthetic organisms,
underpins all oxygenic life on earth [1]. Among the many poorly
understood aspects of this process, which serves as the ultimate blueprint
for synthetic efforts towards development of synthetic water splitting
catalysts, is the role of calcium: why does the catalyst depend critically
on calcium for its function, and why is natural water oxidation inhibited
by very similar cations, even though they may be structurally incorporated in the catalytic cluster? We address these questions by combining
recent results from spectroscopy (EPR/ENDOR), information from
kinetics measurements, and extensive theoretical modelling of photosystem II and its oxygen evolving complex [1–4]. Our results suggest
that the calcium ion satisfies not one but several diverse requirements,
J Biol Inorg Chem (2014) 19 (Suppl 2):S749–S764
which are electronic as much as structural in nature. Most importantly,
calcium simultaneously modulates the properties of not only the
Mn4CaO5 cluster itself, but also of the redox-active tyrosine residue that
mediates electron transfer from the water oxidation site to the photodriven charge separation site of the enzyme.
S763
4. Liu H, Li Y, Wang ST et al J Am Chem Soc 135:7603–7609
5. Zhang PC, Chen L, Zhou J, Wang ST et al (2013) Adv Mater
25:3566–3570
6. Wang ST, Liu K, Liu J et al (2011) Angew Chem Int Ed
50:3084–3088
OP 37
A comprehensive platform to investigate protein-metal
ion interactions by affinity capillary electrophoresis
(ACE)
Hassan A. AlHazmi1, Markus Nachbar1, Mona
Mozafari Toshizi1, Sabine Redweik1, Sami El Deeb2,
Deia El Hady3,4, Hassan M. AlBishri3, Hermann
Wätzig1
References
1. Cox N, Pantazis DA, Neese F, Lubitz W (2013) Acc Chem Res
46:1588–1596
2. Pantazis DA, Ames W, Cox N, Lubitz W, Neese F (2012) Angew
Chem Int Ed 51:9935–9940
3. Retegan M, Neese F, Pantazis DA (2013) 9:3832–3842
4. Retegan M, Cox N, Lubitz W, Neese F, Pantazis DA (2014) Phys
Chem Chem Phys. doi:10.1039/C1034CP00696H
OP 36
Engineering biointerface with controlled cell adhesion
towards cancer diagnostics
Gao Yang1, Pengchao Zhang1, Xueli Liu1, Hongliang
Liu1, Shutao Wang1
1
Technical Institute of Physics and Chemistry of the Chinese
Academy of Sciences, Beijing 100190, China. [email protected]
Circulating tumor cells (CTCs) have become an emerging ‘‘biomarker’’ for monitoring cancer metastasis and prognosis. Although
there are existing technologies available for isolating/counting CTCs,
the most common of which using immunomagnetic beads, they are
limited by their low capture efficiencies and low specificities. By
introducing a three-dimensional (3D) nanostructured substrate—specifically, a silicon-nanowire array coated with anti-EpCAM—we can
capture CTCs with much higher efficiency and specificity. The conventional methods of isolating CTCs depend on biomolecular
recognitions, such as antigen–antibody interaction. Unlikely, we here
proposed that nanoscaled local topographic interactions besides biomolecular recognitions inspired by natural immuno-recognizing
system. This cooperative effect of physical and chemical issues
between CTCs and substrate leads to increased binding of CTCs,
which significantly enhance capture efficiency. Recently, we have
also developed a 3D cell capture/release system triggered by aptamer
enzyme, electrical potential and Temperature, which is effective and
of ‘‘free damage’’ to capture and release cancer cells. The bio-inspired
interfaces of cell capture and release open up a light to rare-cell based
diagnostics, such as CTCs, fetal cells, stem cell and so on.
Financial support by the Chinese Academy of Sciences is gratefully acknowledged.
References
1. Liu X, Wang ST(2014) Chem Soc Rev 43:2385–2401
2. Liu H, Liu X, Wang ST et al (2013) Adv Mater 25:922–928
3. Jin J, Wang ST, Liu DS et al (2013) Adv Mater 25:4714–4717
1
Institute of Medicinal and Pharmaceutical Chemistry, University
of Braunschweig, Germany
2
Department of Pharmaceutical Chemistry, Al-Azhar UniversityGaza, Gaza, Palestine
3
Chemistry Department, Faculty of Science-North Jeddah, King
Abdulaziz University, Jeddah, Saudi Arabia
4
Chemistry Department, Faculty of Science, Assiut University,
71516-Assiut, Egypt
Affinity Capillary Electrophoresis (ACE) provides an important
enhancement to characterize molecular interactions. In exciting
recent studies, the influence of various metal ions, including Li?,
Na?, Mg2?, Ca2?, Ba2?, Al3?, Ga3?, La3?, Pd2?, Ir3?, Ru3?, Rh3?,
Pt2?, Pt4?, Os3?, Au3?, Au?, Ag?, Cu2?, Fe2?, Fe3?, Co2?, Ni2?,
Cr3?, V3?, Mn2?, MoO42- and SeO32- was investigated by ACE,
giving deep insight into the functional interactions between these
species and biomolecules. The predominant role of ACE is in the
early screening stage when binding and non-binding compounds are
sorted out. The requirements for sample amount and purity are low,
but high precision of binding information in reasonable short analysis times can be expected [1]. ACE can now be performed in
*5 min including rinsing procedures. An excellent precision, corresponding to RSD % of 0.2–1.0 % was achieved. Long term
stability and appropriate method transfers have also been established. The capillary manufacture batch, the type of temperature
controlling tool, the purity of running buffer constituents and the
quality of the ligands involved, including their stability, have been
identified as main parameters for robustness. Further ACE key
method development parameters include protein concentration,
length of injected plug, applied voltage, and the choice of the
regression method [2]. Now we not only provide a generic concept
and experimental conditions for all relevant metal ions to be
investigated, which could be easily enhanced to each and every
further species, but we also provide reference values for characteristic interactions to a set of reference proteins. These concepts have
already been successfully applied for a number of applications,
namely Extracellular-signal Regulated Kinase (ERK), dehydrins
(metal-ion storing plant proteins), potentially Ca2? binding peptides
and transferrin.
References
1. AlHazmi H, El Deeb S, Nachbar M, Redweik S, AlBishri HM, Abd
El-Hady D, Wätzig H Submitted to electrophoresis, manuscript no.
elps.201400064
2. El Deeb S, Wätzig H, El-Hady D (2013) Trends Anal Chem
48:112–131
123
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OP 38
Specific recognition of DNA depurination
by a luminescent terbium(III) complex
Xiaohui Wang1,3, Xiaoyong Wang2, Zijian Guo1
J Biol Inorg Chem (2014) 19 (Suppl 2):S749–S764
Financial support by National Natural Science Foundation of
China is gratefully acknowledged.
1
State Key Laboratory of Coordination Chemistry, School
of Chemistry and Chemical Engineering, Nanjing University, Nanjing
210093, People’s Republic of China. [email protected]
2
State Key Laboratory of Pharmaceutical Biotechnology, School
of Life Sciences, Nanjing University, Nanjing 210093, People’s
Republic of China. [email protected]
3
College of Sciences, Nanjing Tech University, Nanjing 211816,
People’s Republic of China. [email protected]
Recognition of DNA depurination is of great importance for early
cancer detection [1]. Luminescent lanthanide complexes possess
some fascinating optical properties that have shown potential applications in biomedical researches [2]. In this study, a novel
terbium(III) complex (TbL) has been demonstrated to be capable of
recognizing purine nucleobases in DNA as a selective time-resolved
luminescence probe. The luminescence of TbL is enhanced remarkably upon reaction with oligonucleotides or natural DNA containing
purine bases in aqueous solution, while it is quenched dramatically as
depurination occurs to DNA. Mechanistic studies using the circular
dichroism and fluorescence spectroscopies revealed that the luminescence enhancement results from the preferential intercalations
between nitroimidazole moieties of TbL and purine bases of DNA,
which regulate the electron withdrawing effect of nitro groups via
hydrogen bonds and thereby affect the energy transfer from the ligand
to the metal center of the probe. This mechanism is also supported by
the molecular dynamics simulation results for the reaction. The distinct luminescence responses of TbL in the presence and absence of
purine bases in DNA make it a sensitive probe for DNA depurination
in physiological conditions.
123
References
1. Dahlmann HA, Vaidyanathan VG, Sturla SJ (2009) Biochemistry
48:9347–9359
2. Bünzli JCG, Eliseeva SV (2013) Chem Sci 4:1939–1949
J Biol Inorg Chem (2014) 19 (Suppl 2):S765–S772
DOI 10.1007/s00775-014-1160-3
ORAL PRESENTATION
Young Researcher Presentations
YRP 1
A Fluorescent probe for investigating the activation
of anticancer platinum(IV) prodrugs based on cisplatin
scaffold
Diego Montagner1,2, Siew Qi Yap1, Wee Han Ang1
1
Department of Chemistry, National University of Singapore,
3 Science Drive, Singapore
2
School of Chemistry, NUI Galway, Ireland.
[email protected]
Following the discovery of its potent antitumoral activity in 1965,
the inorganic drug cisplatin has become one of the most important
anticancer agents used in the clinic [1]. After this, others Pt(II)
based complexes are used as anticancer agents (i.e. carboplatin,
oxaliplatin) [2]. In this abstract we present a fluorescent turn-on
probe rationally designed for the study of anticancer Pt complexes
within live cells. This probe (Rho-DDTC, figure) was custombuilt for the detection of Pt(II) drugs such as cisplatin and
demonstrate using confocal microscopy the localization of Pt(IV)
prodrugs in cancer cells in vitro after cell entry and intracellular
reduction [3].
The probe is compatible with biological conditions and designed
to specific towards Pt(II) complexes containing the diam(m)ineplatinum(II) pharmacophore such as cisplatin. The probe is also able
to distinguish between Pt(IV) prodrug complexes and their Pt(II)
products. We showed for several Pt(IV) prodrugs, including satraplatin, that reduction occurred after cell entry and the Pt(II)
products accumulate with the cytoplasm. In summary, Rho-DDTC is
a versatile tool to study localization of clinically important Pt(II)
anticancer drugs and the activation of their Pt(IV) prodrug congeners within live cells.
References
1. (a) Rosenberg B, Van Camp L, Krigas T (1965) Nature
205:698–699; (b) Rosenberg B, Van Camp L, Trosko JE, Mansour
VH (1969) Nature 222:385–386
2. (a) Kelland L (2007) Nat Rev Cancer 7:573–584; (b) Wang D,
Lippard SJ (2005) Nat Rev Drug Discov 4:307–320
3. Montagner D, Yap SQ, Ang WH (2013) Angew Chem Int Ed
125:12001–12005
YRP 2
Ferrocenyl-terpyridine platinum (II) complexes
showing photocyto-toxicity in visible light
Koushambi Mitra1, Uttara Basu1, Imran Khan2,
Basudev Maity1, Paturu Kondaiah2,
Akhil R. Chakravarty1
1
Department of Inorganic and Physical Chemistry, Indian Institute
of Science, Bangalore 560012, India
2
Department of Molecular Reproduction, Development and Genetics,
Indian Institute of Science, Bangalore 560012, India
Photodynamic therapy has emerged as a non-invasive therapeutic
treatment modality of cancer where we can selectively activate a photosensitizer in visible light, and damage only the photo-exposed cancer
cells thus restoring normal organ functioning. The excellent photophysical properties of ferrocenyl terpyridines (Fc-tpy) molecules and the
anticancer properties of Pt(II) terpyridines prompted us to design new
generation of ferrocenyl Pt(II) complexes and explore their photochemotherapeutic properties. Four complexes, viz. [Pt(Fc-tpy)Cl]Cl (1),
[Pt(Fc-tpy)(NPC)]Cl (2, HNPC = N-propargyl carbazole), [Pt(Fcbpa)Cl]Cl (3) and [Pt(Ph-tpy)Cl]Cl (4, as a control complex) were
synthesized and characterized. Crystal structures of 1 and 3 revealed a
distorted square-planar geometry of the Pt(II) centre. Cyclic voltammetry
studies showed reversible Fc?–Fc redox response near 0.6 V vs. SCE in
DMF-0.1 M TBAP for complexes 1–3. Unlike 4, complexes 1–3 lacked
intercalative properties as evident from the DNA binding studies.
Complexes 1 and 2, having an intense charge transfer band at *590 nm,
showed enhanced anti-proliferative effect in HaCaT and MCF-7 cancer
cells (IC50: 9.8–12.2 lM) in visible light of 400–700 nm with low dark
toxicity (IC50 [ 60 lM). Complexes 1 and 4 showed substantial cytoplasmic and nuclear localizations. The Annexin V-FITC/PI assay
indicated that complexes 1–3 are capable of triggering light-induced
apoptosis. The photoredox pathway proceeding via a Fenton type
mechanism involving ferrocenium ion and cytotoxic hydroxyl radicals is
responsible for the pronounced photocytotoxic effect. The theoretical
123
S766
studies revealed that extensive conjugation lowers the HOMO–LUMO
energy gap in 1 and 2 making them better photoinitiators.
Reference
1. Mitra K, Basu U, Khan I, Maity B, Kondaiah P, Chakravarty AR
(2014) Dalton Trans 43:751–763
YRP 3
Exploiting combination therapy for metal-based
anticancer agents
Isolda Romero-Canelón, Magdalena Moss
and Peter J. Sadler
Department of Chemistry, University of Warwick,
Coventry CV4 7AL, UK
Platinum complexes are widely used anticancer drugs [1]. However,
new generations of metal -based compounds offer the prospect of combating Pt resistance and expanding the range of treatable cancers. We are
investigating the possibility that targeting the redox balance in cancer cells
may be a highly effective strategy, especially since it is a multiple site
approach and offers selectivity over normal cells. Metal complexes can
interfere in cellular redox chemistry in several ways: directly though metal
or ligand redox centres, or indirectly by binding to biomolecules involved
in cellular redox pathways [2]. We illustrate that a number of active
organometallic anticancer agents based on RuII, and OsII, have a potential
redox arm in their mechanism of action. For such complexes, the possibility arises of using combination therapy together with redox modulators
to increase their potency– attractive for lowering the doses of metal
complexes that need to be administered. In particular, azopyridine Ru(II)
and Os(II) arene complexes 1 and 2 show promising activity, especially in
the ovarian cancer cell line A2780 in which they are an order of magnitude
more active than cisplatin [3]. Moreover, their potency can be increased by
co-administration of a redox modulator, such as L-buthionine sulfoxime
(L-BSO) an inhibitor of c-glutamylcysteine synthetase. Redox modulation
might provide effective multi-targeting for a new generation of potent
anticancer drugs and allow administration of very low doses of these
metallodrugs. In the present work, we investigate the influence of the
redox modulator on cell cycle, apoptosis and the mitochondrial membrane
potential, as well as, the GSH/GSSG ratio in cells.
Acknowledgements: We thank the ERC (Grant No. 247450),
University of Warwick IAS (fellowship for IRC), Science City
(ERDF/AWM), and EPSRC for support, and members of COST
Action of CM1105 for stimulating discussions.
123
J Biol Inorg Chem (2014) 19 (Suppl 2):S765–S772
References
1. Kelland L (2007) Nat Rev Cancer 7:573–584
2. Romero-Canelón I, Sadler PJ (2013) Inorg Chem 52:12276-12291
3. Fu Y, Habtemariam A, Pizarro AM, et al. (2010) J Med Chem
53:8192–8196
YRP 4
Synthesis and characterization of semisynthetic
[FeFe]-hydrogenases from Chlamydomonas reinhardtii
Judith F. Siebel, Agnieszka Adamska-Venkatesh,
Katharina Weber, Sigrun Rumpel, Edward Reijerse,
Wolfgang Lubitz
Max-Planck-Institut für Chemische Energiekonversion,
Stiftstrasse 34–36, 45470 Mülheim an der Ruhr, Germany.
[email protected]
Hydrogen is considered the ideal fuel of the future, since its combustion
generates only water. Hydrogenases catalyze the reaction of protons and
electrons to hydrogen and are therefore of great biotechnological
interest [1]. The catalytic reaction of [FeFe]-hydrogenases takes place at
a binuclear [2Fe]-subsite containing unusual CO and CN- ligands and
an azadithiolate bridge. The [2Fe]-subsite is connected to a [4Fe–4S]cluster via the bridging sulfur of a cysteine [1]. Recently, it was shown
that apo-hydrogenase containing only the [4Fe–4S]-cluster can be
activated by addition of a binuclear [2Fe]-subsite mimic [2, 3]. Insertion
of the mimic with an azadithiloate bridge yielded fully active hydrogenase, mimics with a propanedithiolate or oxodithiolate bridge could be
inserted, but did not exhibit catalytic activity [2].
We present [2Fe]-model compounds with a thiodithiolate, an
N-methylazadithiolate and a bulky dimethylazadithiolate bridge,
which also bind to Chlamydomonas reinhardtii’s [FeFe]-hydrogenase
apo-HydA1. Furthermore, we show that one CN- ligand instead of
two as in the native binuclear [2Fe]-subsite is sufficient for apoHydA1 binding, but decreases the stability drastically. FTIR spectroscopy analysis give information about the nature of the model
compounds bound to apo-HydA1. Moreover, hydrogen production
and oxidation measurements revealed that apart from the synthetic
mimic with the azadithiolate bridge, only its mono-cyanide version
exhibits good activity. However, also other HydA1-model compound
hybrids showed some remaining activity. Our findings demonstrate
that in the protein pocket of apo-HydA1, several different [2Fe]model compounds can bind, but even very minor changes of the
natural active site strongly affect catalytic activity and stability.
References
1. Lubitz W, Ogata H, Rüdiger O, Reijerse E (2014) Chem Rev, doi:
10.1021/cr4005814
2. Berggren G, Adamska A, Lambertz C, Simmons TR et al. (2013)
Nature 499:66–70
3. Esselborn J, Lambertz L et al. (2013) Nat Chem Biol 9:607–610
J Biol Inorg Chem (2014) 19 (Suppl 2):S765–S772
YRP 5
New insight into the mechanism of NiFe hydrogenases
from IR spectroscopy coupled with protein film
electrochemistry
Philip A. Ash, Ricardo Hidalgo, Min-Wen Chung,
Kylie A. Vincent
University of Oxford, Department of Chemistry, Inorganic Chemistry
Laboratory, South Parks Road, Oxford, UK OX1 3QR.
[email protected]
This talk describes experiments that provide new insight into the
mechanism and reactions of nickel–iron hydrogenases—nature’s
efficient catalysts for oxidation and production of H2. Metalloenzymes are fascinating to chemists because of their ability to carry out
catalysis selectively using cheap, abundant metals. One important
technique used in studying redox catalysis by enzymes is the
approach known as protein film electrochemistry (PFE) in which the
enzyme of interest is immobilised on an electrode and can undergo
rapid and direct exchange of electrons with the electrode surface. The
enzyme is thus handled as a heterogeneous electrocatalyst, directly
comparable to a metal electrode or supported nanoparticle catalyst.
The power of this technique in replacing the biological redox chain of
an enzyme by direct electron transfer to or from an electrode has
spurred the development of electrode configurations designed to allow
spectroscopic sampling to be coupled with PFE [1–3]. By coupling
infrared spectroscopy with direct electrochemical control of hydrogenases adsorbed on a carbon electrode we are able to describe
simultaneous electrocatalytic and in situ infrared measurements on
E. coli hydrogenases. For the first time, these measurements allow
states of the active site to be probed under catalytic as well as noncatalytic conditions, correlating active site states with turnover under
different solution and potential conditions. These data provide new
insight into the catalytic mechanism of H2 activation by nickel–iron
hydrogenases and the approach opens up possibilities for addressing
catalytic states of a range of metalloenzymes.
This research was supported by European Research Council grant
EnergyBioCatalysis-ERC-2010-StG-258600.
References
1. Krzemiński L, et al. (2011) J Am Chem Soc 133:15085
2. Doyle R-MAS, et al. (2013) Electrochim Acta 110:73
3. Ash PA, Vincent KA (2012) Chem Commun 48:1400
YRP 6
Identification of the Hcg enzymes in biosynthesis
of [Fe]-hydrogenase cofactor by a structural
genomics-based approach
Takashi Fujishiro1, Haruka Tamura1, Michael Schick1,
Jörg Kahnt1, Xiulan Xie2, Ulrich Ermler3,
Seigo Shima1,4
1
Max Planck Institute for Terrestrial Microbiology, Karl-von-FrischStrasse 10, 35043 Marburg, Germany
2
Department of Chemistry, Philipps-Universität Marburg, HansMeerwein Strasse, 35032 Marburg, Germany
3
Max Planck Institute for Biophysics, Max-von-Laue-Strasse 3,
60438 Frankfurt am Main, Germany
S767
4
PRESTO, Japan Science and Technology Agency (JST), Honcho,
Kawaguchi, 332-0012 Saitama, Japan. [email protected]
[Fe]-hydrogenase is one of three types of hydrogenases and catalyzes
reversible heterolytic cleavage of molecular hydrogen and stereospecific hydrogenation of methenyl-tetrahydromethanopterin to
methylene-tetrahydromethanopterin [1]. [Fe]-hydrogenase harbours
the mononuclear iron complex, iron-guanylylpyridinol (FeGP)
cofactor, at its active site for hydrogen activation [2]. The FeGP
cofactor shows unique structures such as an organometallic acyl-iron
bond and a guanylyl-pyridionl linkage. Because of its unique structural and functional features, biosynthesis of the FeGP cofactor is of
interest at the viewpoints of chemistry and biology. Previously, we
performed stable isotope-labelling study on the biosynthesis of the
FeGP cofactor, which proposed a possible biosynthetic pathway to the
FeGP cofactor [3]. However, it was unclear what kind of enzymes
could be involved in the biosynthesis of the FeGP cofactor. Here, we
report identification of the Hcg enzymes involved in the FeGP
cofactor biosynthesis by a structural genomics-based approach [4].
References
1. Shima S, Ermler U (2011) Eur J Inorg Chem 963–972
2. Tamura H, Salomone-Stagni M, Fujishiro T, Warkentin E, MeyerKlaucke W, Ermler U, Shima S (2013) Angew Chem Int Ed
52:9656–9659
3. Schick M, Xie X, Ataka K, Kahnt J, Linne U, Shima S (2012) J Am
Chem Soc 134:3271–3280
4. Fujishiro T, Tamura H, Schick M, Kahnt J, Xie X, Ermler U, Shima
S (2013) Angew Chem Int Ed 52:12555–12558
YRP 7
New dihydroxamic acid siderophores from Shewanella
putrefaciens using precursor-directed biosynthesis
Cho Zin Soe, Rachel Codd
Chemical Biology in Drug Discovery Laboratory, Discipline
of Pharmacology and Bosch Institute, The University of Sydney,
NSW 2006, Australia
To acquire iron essential for growth, the marine bacterium Shewanella
putrefaciens produces the macrocyclic dihydroxamate siderophore
putrebactin (PB) [1]. The first step of PB synthesis involves the conversion of L-ornithine to putrescine (1,4-diaminobutane) by the enzyme
ornithine decarboxylase (ODC) [2]. This study aimed to design new
macrocyclic dihydroxamic acids (PB analogues) in S. putrefaciens by
direct media augmentation with a combination of an ODC inhibitor,
1,4-diamino-2-butanone (DBO), and exogenous diamine substrates.
The inhibitor DBO depleted levels of endogenous putrescine in the
cells and attenuated PB biosynthesis. Under such conditions, S. putrefaciens produced a replacement trihydroxamic acid siderophore
desferrioxamine B. The presence of DBO along with alternative
exogenous substrates, cadaverine (1,5-diaminopentane), 15N2-1,4-diaminobutane or 1,4-diamino-2(E)-butene, resulted in the respective
biosynthesis of bisucaberin, 15N-labelled PB and the unsaturated PB
analogue, named E,E-putrebactene. The formation of hybrid macrocyclic dihydroxamic acid complexes comprised of one putrescineand one exogenous substrate-based precursor was also observed [3].
The metal complexation behaviour of these compounds was analysed
using LC–MS/MS in the presence of Fe(III), V(V) or Mo(VI).
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This study opens up new avenues towards the generation of
molecular diversity within the macrocyclic dihydroxamic acid class
of siderophores using a combination of directed-biosynthesis and ex
situ chemical conversion approaches. The scope of this study also
contributes to a better understanding towards bacterial diaminedependent metabolism beyond siderophore biosynthesis.
J Biol Inorg Chem (2014) 19 (Suppl 2):S765–S772
one can state that the substitution of Zn(II) by toxic Cd(II) in
XPAzf can paradoxically increase the persistency of XPA protein
activity in the cell.
Financial support by the National Science Center, Poland
(Grant Preludium 4 no. 2012/07/N/NZ1/03090) is gratefully
acknowledged.
YRP 9
Hydrogen sulfide functions as reversible inhibitor
and electron donor for human myeloperoxidase
Katharina F. Pirker1, Zoltán Pálinkás2,
Paul G. Furtmüller1, Attila Nagy2, Christa Jakopitsch1,
Christian Obinger1, Péter Nagy2
1
References
1. Ledyard KM, Butler A (1997) J Biol Inorg Chem 2:93–97
2. Challis GL (2005) Chem Bio Chem 6:601–611
3. Soe CZ, Codd R (2014) ACS Chem Biol doi:10.1021/cb400901j
YRP 8
The interactions of XPAzf with intracellular carriers
of redox signals
Aleksandra Witkiewicz Kucharczyk1, Anna De˛bska2,
Andrea Hartwig3, Wojciech Bal1
1
Institute of Biochemistry and Biophysics, Polish Academy
of Science, Pawińskiego 5a, 02-106 Warsaw, Poland
2
Institute of Biotechnology, Faculty of Chemistry, Warsaw University
of Technology, ul. Noakowskiego 3, 00-664 Warsaw, Poland
3
Institute of Applied Biosciences, Department of Food Chemistry
and Toxicology, Karlsruhe Institute of Technology, 76131 Karlsruhe,
Germany
Our previous investigations demonstrated that a peptide modelling the
ZF domain of human DNA repair protein XPA undergoes oxidation in
a condition of nitrosative and oxidative stress. Our model peptide, a
synthetic 4-Cys zinc finger domain of 37 aa complexed with a Zn(II)
ion is a target for a representative nitrogen specie (GSNO) and
oxygen specie (H2O2) at a 1:10 molar ratio. The oxidation by H2O2
resulted in a release the metal ion, followed by the formation of
disulfide forms. The reaction of oxidation by GSNO is also preceded
by a reversible S-nitrosylation step. Although H2O2 and GSNO are
both species emerging in cells during nitrosative and oxidative stress,
they are naturally present in the same cells at a metabolic level. The
next step of our research was to investigate the oxidation of the
Zn(II)XPAzf complex by the same species present at a metabolic
level.
Another factor that can affect the activity of zinc finger protein
is the metal ion exchange. Zinc finger proteins are naturally
structurized and activated by Zn(II) complexation, but Zn(II) can
be substituted by Cd(II), a toxic metal ion of similar chemical
properties yet stronger binding to the cysteines in XPAzf binding
core. The reactions of oxidation of both Zn(II)XPAzf and
Cd(II)XPA by GSNO and H2O2 in at low molar ratio were monitored by ESI–MS (detection of the products), HPLC (the formation
of covalent reaction products), CD (the change of a structure) and
UV–VIS (release of metal ion). These reactions have slow kinetics
but still are considered deleterious. Also the slow kinetics of oxidation by GSNO does not allow to consider S-nitrosylation as an
effective cellular switch for XPAzf activity. Finally, Cd(II)XPAzf
was more resistant to oxidation than Zn(II)XPAzf complex. Considering the geometrical similarity of this complex to Zn(II)XPAzf,
123
Division of Biochemistry, Department of Chemistry, Vienna
Institute of BioTechnology, BOKU-University of Natural Resources
and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
[email protected]
2
Department of Molecular Immunology and Toxicology, National
Institute of Oncology, Ráth György utca 7-9, Budapest, Hungary,
1122
Increasing attention is devoted to the actions of hydrogen sulfide in
human physiology. Although it was shown to be an essential mediator of
a variety of biological functions, the underlying molecular mechanisms
of its actions are hardly understood. With the intention to contribute to the
understanding of sulfide’s role in inflammation we investigated its
interactions with human myeloperoxidase (MPO), a major player in
innate immunity and in promoting inflammatory oxidative stress, using
time-resolved UV–vis and electron paramagnetic resonance (EPR)
spectroscopy. The typical high-spin ferric heme of MPO in the EPR
spectrum is drastically reduced in intensity in the presence of sulfide and a
small amount of low-spin ferric species with sulfide as a 6th ligand, was
detected. The subsequent loss of ferric MPO indicates a reduction of
Fe(III) by sulfide. Both reactions (i.e. ligand binding and formation of
ferrous heme) could be confirmed using stopped-flow UV–vis spectroscopy. Interestingly, the presence of hydrogen peroxide promotes the
recovery of the native ferric MPO signal in the EPR spectrum indicating
reversibility of the interaction of MPO with sulfide. Additionally, sulfide
is shown to act as a one-electron donor for MPO Compounds I and II. The
obtained data allow us to propose a mechanism for the reactions of MPO
with sulfide, which includes sulfide’s reversible inhibitor as well as
electron donor functions [1].
Reference
1. Pálinkás Z, Furtmüller PG, Nagy A, Jakopitsch C, Pirker KF,
Magierowski M, Jasnos K, Wallace JL, Obinger C, Nagy P (2014)
Brit J Pharmacol (in press)
YRP 10
Highly resistant anticancer titanium(IV) complexes
and their mechanistic investigation using 19F NMR
Sigalit Meker1, Ori Braitbard2,
Katrin Margulis-Goshen1, Shlomo Magdassi1,
Jacob Hochman2, Edit Y. Tshuva1
1
The Institute of Chemistry, The Hebrew University of Jerusalem,
Jerusalem 91904, Israel
2
Department of Cell and Developmental Biology, Alexander
Silberman Institute of Life Sciences, The Hebrew University
of Jerusalem, 91904 Jerusalem, Israel
J Biol Inorg Chem (2014) 19 (Suppl 2):S765–S772
Titanium(IV) complexes have been studied extensively as anti-cancer
agents due to their high activity toward various cancer cells and low
side effects; however, they have not yet been utilized in the clinic due
to water instability and formation of unidentified aggregates in
aquatic solutions. Therefore, mechanistic aspects remain unresolved,
including the nature of the active species and its identification out of
the multiple hydrolysis products formed.
We recently reported on the synthesis, characterization, and
cytotoxicity of salan titanium(IV) complexes. These compounds are
efficient anticancer agents that exhibit slow and defined hydrolysis to
form stable oxo-bridged salan-bound polynuclear compounds, thus
enabling mechanistic evaluation. The polynuclear hydrolysis products
were inactive when administered directly, due to solubility and cellular penetration limitations. Conversion of these polynuclear
compounds into nanoparticles enabled overcoming these limitations
and thus such nanoformulated complexes demonstrated cytotoxicity
toward human and murine cancer cells [1, 2]. Their activity indicates
that inert hydrolysis products, lacking labile ligands, may act as active
species.
Herein we present the synthesis and characterization of highly
cytotoxic and hydrolytically stable mono- and poly-nuclear Ti(IV)
complexes that lack labile ligands and bare bis- and tetrakis-phenolato
hexadentate ligands. To investigate their mechanism of action, we
have synthesized fluoro-substituted derivatives, which can be detected
in cellular media by 19F NMR. These fluoro-compounds were
administered to cancer cells to examine their bioavailability, cellular
distribution, potential biological targets, and the identity of possible
intracellular active species.
Additionally, we study the pharmacokinetics of these complexes
in murine models. We found that a nanoformulated complex is
present in the urine of mice 24 h post intraperitoneal inoculation.
Preliminary results of drug accumulation in murine tissues, identity of
possible active species, and possible biological targets of these
compounds will be discussed.
This research was funded by the European Research Council under
the European Community’s Seventh Framework Programme (FP7/
2007-2013)/ERC Grant agreement (No. 239603)
References
1. Meker S, Margulis-Goshen K, Weiss E, Magdassi S, Tshuva EY
Angew Chem Int Ed (2012) 51:10515-10517 selected as ‘‘Hot
Paper’’; selected for back cover
2. Meker S, Margulis-Goshen K, Weiss E, Braitbard O, Hochman J,
Magdassi S, Tshuva EY (2014) ChemMedChem doi:10.1002/
cmdc.2014000
YRP 11
Fold-back anchor DNA-templated silver nanoclusters
for miRNA detection
Pratik Shah1, Seok Keun Cho1, Peter W. Thulstrup2,
Suk Won Choi3, YongJoo Bhang3, Jong Cheol Ahn3,
Morten J. Bjerrum2, Seong Wook Yang1
1
UNIK Center for Synthetic Biology, University of Copenhagen,
Thorvaldsensvej 40, 1871 Frederikberg, Denmark. [email protected]
2
Department of Chemistry, University of Copenhagen,
Universitetsparken 5, 2100 Copenhagen, Denmark
3
SeouLin Bioscience Co. Ltd, 4F. #A, KOREA BIO PARK, 700,
Daewangpangyo-ro, Bundang-gu, Seongnam-si, Gyeonggi-do, Korea
Fluorescent properties of DNA encapsulated Silver Nano-Cluster
(DNA/AgNC) has received increasing attention in recent years for the
biosensing purposes. MicroRNAs are small non-protein coding RNA
S769
molecules which can be used as biomarkers for disease diagnostics in
cancer, Alzheimer’s and diabetes. We have developed microRNA
detection method using DNA/AgNC to monitor the presence of target
miRNA simply by drop of the strong fluorescence of a DNA/AgNCs
sensor [1]. Previously we reported that the mis-matched secondary
structure of DNA sensor- composed of cytosine-rich sequence and
miRNA sensing sequence- plays crucial role in forming bright red
emitting AgNC [2]. However, to detect enormously diverse target
miRNA sequences, we have developed more rational sensor design
approach-a fold-back anchor sensor. It consists of a poly-cytosine
loop with partial complementary sequences to the target recognition
sequences by which the loop can be anchored. The anchor allows the
poly-cytosine loop to stably harbour fluorescent AgNC and the thermodynamic strength of the anchor can be modulated for the detection
of miRNA of varying guanine-cytosine content allowing rapid
designing of the DNA/AgNC sensor to detect large number of miRNA. Thus, in the presence of target miRNA, the anchor can be easily
disrupted, leading to the drop in fluorescence. We have employed our
newly designed sensor for the diagnostics of cancer with high
specificity.
Financial support by the ‘‘Centre for Synthetic Biology’’ is
acknowledged
.
References
1. Yang SW, Vosch T (2011) Anal Chem 83:6935
2. Shah P, Rorvig-Lund A, Chaabane SB, Thulstrup PW, Kjaergaard
HG, Fron E, Hofkens J, Yang SW, Vosch T (2012) ACS Nano 6:8803
YRP 12
Coenzyme B12 riboswitch from Klebsiella pneumonia:
an ITC and in-line probing combined study
Miquel Barceló-Oliver, Joana Palou-Mir
Department of Chemistry, University of the Balearic Islands,
Carretera Valldemossa km 7.5, 07122 Palma de Mallorca, Spain.
[email protected]
Proteins lost their exclusivity in the control of the bacterial gene
expression due to the discovery of more and more genes controlled at the non-coding mRNA level (cis- or trans- encoded
targets) [1].
The btuB riboswitch from Klebsiella pneumoniae was found by
bioinformatics comparative analysis thanks to some fragments of
secondary structure conservation in bacterial genomes. It has been
proposed to be a 50 UTR regulatory element responsible for btuB gene
silencing at the translational level in the presence of coenzyme B12
[2].
We report here the detailed elucidation of the thermodynamic
parameters for the riboswitch binding to its metabolite coenzyme B12
by Isothermal Titration Calorimetry (ITC) and the characterization of
switch using in-line probing experiments. Two different RNA
sequences have been used to study the importance of the complete
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untranslated region, including the ribosome-binding site, in contrast
to the minimal length that just includes the pseudoknot interaction
within the riboswitch.
We found that the interaction between coenzyme B12 and the
riboswitch does not follow a single step mechanism (as can be seen
from the ITC diagram shown in the Figure). Instead, a two-step
approximation has to be used to explain experimental data. Finally,
observed dissociation constants are compared to the reported data
from the best-studied B12 riboswitches from E. coli and S.
typhimurium.
Financial support by the ‘‘Govern de les llles Balears’’ (project
numbers AAEE0145/09 and AAEE0019/2012) is gratefully
acknowledged. The authors would like to acknowledge the contribution of the COST Action CM1105. The authors also thank Roland
K.O. Sigel, Sofia Gallo and Anastasia Musiari from University of
Zürich for all the aid and support received.
J Biol Inorg Chem (2014) 19 (Suppl 2):S765–S772
generally present in cells at a lower concentration than iron, while
also having a lower predicted complex stability according to the
Irving-Williams series (MnII \ FeII \ NiII \ CoII \ CuII [ ZnII) [1].
Here we show that a heterodinuclear Mn/Fe cluster with the same
primary protein ligands in both metal sites self-assembles from MnII
and FeII in vitro, thus diverging from the Irving-Williams series
without requiring auxiliary factors such as metallochaperones [2].
Crystallographic, spectroscopic and computational data demonstrate
that one of the two metal sites preferentially binds FeII over MnII as
expected, whereas the other site is non-specific, binding equal
amounts of both metals in the absence of oxygen. Oxygen exposure
results in further accumulation of the Mn/Fe cluster, indicating that
cofactor assembly is at least a two-step process governed by both the
intrinsic metal specificity of the protein scaffold and additional effects
exerted during oxygen binding or activation. Upon oxygen activation,
the mixed-metal cofactor catalyzes a two-electron oxidation of the
protein scaffold, yielding a tyrosine-valine ether crosslink that demonstrates the chemical potential of this novel cofactor.
Financial support was provided by the Swedish Research Council,
the Swedish Foundation for Strategic Research and the Knut and
Alice Wallenberg Foundation, the Max Planck Gesellschaft, the
Alexander von Humboldt Foundation, and BioStruct X.
References
1. Irving H, Williams RJP (1953) J Chem Soc 3192–3210
2. Griese JJ, Roos K, Cox N, Shafaat HS, Branca RM, Lehtio J,
Graslund A, Lubitz W, Siegbahn PE, Hogbom M (2013) Proc Natl
Acad Sci USA 110:17189–17194
References
1. Ferré-D’Amaré AR, Winkler WC (2011) Met Ions Life Sci
9:142–174
2. Vitreschak AG, Rodionov DA, Mironov DA, Gelfand MS (2003)
RNA 9:1084–1097
YRP 13
Structure, function and assembly of an oxygenactivating heterodinuclear Mn/Fe cofactor
Julia J. Griese1, Katarina Roos2, Nicholas Cox3,
Hannah S. Shafaat3, Rui M. M. Branca4, Janne Lehtiö4,
Astrid Gräslund1, Wolfgang Lubitz3,
Per E. M. Siegbahn2, Martin Högbom1
1
Stockholm Center for Biomembrane Research, Department
of Biochemistry and Biophysics, Stockholm University, SE-106 91
Stockholm, Sweden
2
Department of Physics, Stockholm University, SE-106 91
Stockholm, Sweden
3
Max Planck Institute for Chemical Energy Conversion, Stiftstraße
34-36, D-45470 Mülheim an der Ruhr, Germany
4
Clinical Proteomics Mass Spectrometry, Department of OncologyPathology, Science for Life Laboratory, Karolinska Institutet, Box
1031, SE-171 21 Solna, Sweden
Although metallocofactors are ubiquitous in enzyme catalysis, how
metal binding specificity arises remains poorly understood, especially
in the case of metals with similar primary ligand preferences such as
manganese and iron. The biochemical selection of manganese over
iron presents a particularly intricate problem because manganese is
123
YRP 14
Catalytic alkane hydroxylation by myoglobin
containing a manganese porphycene complex
Koji Oohora, Hiroyuki Meichin, Yushi Kihira, Takako
Nishiura, Takashi Hayashi
Department of Applied Chemistry, Graduate School of Engineering,
Osaka University, Yamadaoka 2-1, 565-0871 Suita, Japan.
[email protected]
Myoglobin (Mb), an oxygen storage hemoprotein, has very low peroxidase and monooxygenase activities and does not catalyze alkane
hydroxylation despite including the same cofactor, heme b, as hemedependent enzymes such as horseradish peroxidase and cytochrome
P450. In this decade, our group has demonstrated artificial metalloenzymes by hemoprotein reconstitution with artificial cofactors [1].
Most recently, we focus on Mb reconstituted with a manganese
porphycene complex (MnPc) to catalyze alkane hydroxylation [2],
because the high-valent oxo-species of a manganese complex is
expected to show C–H activation activity (Figure 1).
Mb reconstituted with MnPc (rMb-MnPc) was successfully
obtained by conventional method. The X-ray structure reveals the
incorporation of MnPc to the intrinsic heme binding site and the axial
ligation by His93, which is also the axial ligand of heme in native Mb.
The reaction of rMb-MnPc with mCPBA produced the spectroscopically detectable intermediate, which is EPR silent species, indicating
Mn(V)-oxo species. In addition, we carried out the H2O2-dependent
hydroxylation of ethylbenzene by rMb-MnPc. From the GC analysis,
it is found that rMb-MnPc catalyzes the hydroxylation to provide
1-phenylethanol with the turnover number of 13 at 25 °C and pH 8.5,
whereas native Mb and other reconstituted proteins show no product
under the same condition. Kinetic data, log kobs versus BDE(C(sp3)–
H) for ethylbenzene, toluene, and cyclohexane, indicate a linear
relationship with negative slope, showing that the reaction occurs via
J Biol Inorg Chem (2014) 19 (Suppl 2):S765–S772
a hydrogen-atom abstraction by a Mn(V)-oxo species as a retedetermining step. This is the first example of the hydroxylation catalyzed by myoglobin without any mutations.
Figure 1. Schematic representation of reconstituted myoglobin
References
1. Hayashi T, Hisaeda Y (2002) Acc Chem Res 35:35–43
2. Oohora K, Kihira Y, Mizohata E, Inoue T, Hayashi T (2013) J Am
Chem Soc 135:17282–17285
YRP 15
Spectroscopically consistent Mn oxidation state
assignments of the natural water splitting catalyst
Vera Krewald, Marius Retegan, Frank Neese, Dimitrios
A. Pantazis
Max Planck Institute for Chemical Energy Conversion, Stiftstr. 34-36,
45470 Mülheim an der Ruhr, Germany
Studying nature’s water splitting machinery is not only of fundamental interest, but also driven by the vision that the understanding of its
geometric and electronic construction principles could guide the design
of artificial water oxidizing catalysts, an essential require-ment for a
hydrogen-based energy economy. In nature, sunlight is converted into
chemical energy by oxidizing water and sequentially releasing electrons,
protons and O2 at different steps of the catalytic cycle, known as the Kok
cycle of S0–S4 intermediates (see scheme) [1].
Despite decades of research, a central aspect of the electronic structure
of the natural catalyst is still debated: the oxidation states of the four Mn
ions. Two paradigms exist that differ by two electrons, the ‘‘low’’ oxidation state scheme with Mn(III)3Mn(IV) for the S2 state, and the ‘‘high’’
oxidation state scheme with Mn(III)Mn(IV)3 for the S2 state [2-4].
We use quantum chemical methods5 to evaluate two sets of
models for all S-states of the Kok cycle, conforming to both oxidation
state schemes, analyzing for the first time the two formulations with a
common methodological approach. Comparison of calculated properties with state-specific experimental information available from EXAFS (Mn–Mn distances) and EPR/ENDOR (spin states, 55Mn
hyperfine couplings) reveals that the ‘‘low’’ oxidation state scheme
models are incompatible with experiment. On the other hand the
sequence of models in the ‘‘high’’ oxidation state scheme is fully
consistent with experimental data, and furthermore consistent with the
electron/proton release events of the catalytic cycle.
S771
References
1. Messinger J, Renger G in Primary Processes of Photosynthesis,
Part 2: Principles and Apparatus; The Royal Society of Chemistry:
Cambridge 2008 9:291–349
2. Pace RJ, Jin L, Stranger R (2012) Dalton Trans 41:11145–11160
3. Pantazis DA, Ames W, Cox N, Lubitz W, Neese F (2012) Angew
Chem Int Ed 51:9935–9940
4. Cox N, Pantazis DA, Neese F, Lubitz W (2013) Acc Chem Res
46:1588–1596
5. Krewald V, Neese F, Pantazis DA (2013) J Am Chem Soc
135:5726–5739
YRP 16
MetalS3, a database-mining tool for the identification
of structurally similar metal sites
Yana Valasatava1, Antonio Rosato1, 2,
Claudia Andreini1, 2, Gabriele Cavallaro1
1
Magnetic Resonance Center (CERM), University of Florence, Via L.
Sacconi 6, 50019 Sesto Fiorentino, Italy
2
Department of Chemistry, University of Florence, Via della
Lastruccia 3, 50019 Sesto Fiorentino, Italy
Metals are essential for many physiological processes and widely
used by biological macromolecules to perform their function. The
variability and diversity of metal sites in macromolecules warrants the
development of specific tools for their analysis.
Here we introduce the concept of Minimal Functional Site (MFS)
as a novel viewpoint for the description of metal sites. MFSs extend
the structure of metal site to surrounding residues. Such local environment has a determinant role in tuning the chemical reactivity of
the metal, ultimately contributing to the functional properties of the
whole system.
We have developed various resources to foster adoption and usage
of MFSs: the MetalPDB database [1], which hosts all known MFSs;
the MetalS2 (Metal Sites Superimposition) program and web server,
to quantitatively evaluate the structural similarity of MFS pairs [2];
the MetalS3 (Metal Sites Similarity Search) tool to search the MetalPDB database for MFSs having structural similarity to a query
metal site structure. The latter tool can be accessed through a web
interface at http://metalweb.cerm.unifi.it/tools/metals3/.
This suite of resources and tools will allow researchers in the field
of bioinorganic chemistry to assess the relationships or identify
possible evolutionary links between different groups of metalloproteins as well as help guide experimentalists’ work in understanding
the function of uncharacterized metalloproteins.
Financial support by the Italian Ministry of Research is
acknowledged.
References
1. Andreini C, Cavallaro G, Lorenzini S, Rosato A (2013) Nucleic
Acids Res 41:D312–D319
2. Andreini C, Cavallaro G, Rosato A, Valasatava Y (2013) J Chem
Inf Model 53:3064–3075
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YRP 17
A new dicopper(II) peroxo complex with triplet ground
state
Nicole Kindermann,1 Sebastian Dechert,1 Serhiy
Demeshko,1 Eckhard Bill,2 Franc Meyer1
1
Institut für Anorganische Chemie, Tammannstraße 4, 37077
Göttingen, Germany. [email protected]
2
Max-Planck-Institut für chemische Energiekonversion, Stiftstraße
34-36, 45470 Mülheim an der Ruhr, Germany
Copper containing active centers are responsible for the redoxactivity in a vast array of metalloenzymes resulting in, e.g., their
ability to transport or activate molecular oxygen [1, 2] In order to
understand how reduced binuclear copper sites are able to interact
with dioxygen, many synthetic models have been prepared.
Depending on the design of a particular model, it has been possible
to isolate different intermediates upon the reaction of dicopper(I) complexes with molecular oxygen, namely trans-l-g1:g1
peroxo dicopper(II) (TP), l-g2:g2 peroxo dicopper(II) (P), bis(loxo) dicopper(III) (O), or superoxide species [3,4] The latter three
are relevant in biological systems. In contrast, the cis-l-g1:g1 peroxo dicopper(II) (CP), which is supposed to occur at an early stage of
the binding of 3O2 at dicopper(I) sites [2] has been elusive so far.
Following our recent report of a first structurally characterized CP
model complex [5] we have now developed a preorganized dicopper(I) system, based on a new pyrazole/triazacyclononane ligand,
that binds dioxygen to generate an unprecedented CP complex with
ferromagnetically coupled copper(II) ions and S = 1 spin ground
state. The characterization and the properties of that complex will be
reported.
Financial support from the Fonds der chemischen Industrie, the
Studienstiftung des deutschen Volkes (to N.K.), and the Deutsche
Forschungsgemeinschaft (Metal Sites in Biomolecules: Structures,
Regulation and Mechanisms; IRTG 1422) is gratefully acknowledged.
References
1. Lewis EA, Tolman WB (2004) Chem Rev 104:1047–1076
2. Solomon EI, Ginsbach JW, Heppner DE, et al. (2011) Farad Discuss 148:11–39
3. Rolff M, Schottenheim J, Decker H, Tuczek F (2011) Chem Soc
Rev 40: 4077–4098
4. Hatcher LQ, Karlin KD (2004) J Biol Inorg Chem 9:669–683
5. Dalle KE, Grüne T, Dechert S, Demeshko S, Meyer F (2014) J Am
Chem Soc doi:10.1021/ja5025047
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J Biol Inorg Chem (2014) 19 (Suppl 2):S765–S772
YRP 18
Crystal structure of a crustacean prophenoloxidase
elucidates the functional difference of the type 3 copper
proteins
Taro Masuda1, Kyosuke Momoji2, Takashi Hirata2,
Bunzo Mikami3
1
Division of Agronomy and Horticultural Science, Graduate School
of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611-0011,
Japan
2
Division of Applied Biosciences, Graduate School of Agriculture,
Kyoto University, Kitashirakawa-oiwakecho, Sakyo, Kyoto
606-8502, Japan
3
Division of Applied Life Sciences, Graduate School of Agriculture,
Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan
Phenoloxidase (PO) and hemocyanin (Hc) are commonly classified as a
type 3 copper protein. The former catalyzes the hydroxylation of
monophenol and subsequent oxidation to the corresponding o-quinone,
while the latter has no such enzymatic activity. However, the geometry
and coordination environment of the active site of the arthropod PO and
Hc are closely resembled to each other. Recently, we identified a new
type of proPO from a crustacean and designated it as proPOb. Here, we
determined the crystal structure of proPOb prepared from the hemolymph of kuruma prawns (Marsupenaeus japonicus) at 1.8 Å resolution.
The hexameric structure of M. japonicus proPOb was rather similar to
arthropod Hc. Furthermore, the geometry of the active copper site in
proPOb was nearly identical to that of arthropod Hc. However, the
accessibility to the active site differed in several ways. First, a phenylalanine residue which shields the active site copper A by interacting
with a copper-coordinated histidine in crustacean Hc was substituted by
valine in proPOb structure. Second, two tyrosine residues, Tyr208 and
Tyr209, both of which are absent in Hc, form a pathway accessible to the
reaction center. Thus, the present crystal structure clarified the similarities and differences in the active site of two closely related proteins,
PO and Hc.
References
1. Masuda T, Momoji K, Hirata T, Mikami B (2014) FEBS J in press
2. Masuda T, Otomo R, Kuyama H, Momoji K, Tonomoto M, Sakai
S, Nishimura O, Sugawara T, Hirata T (2012) Fish Shellfish Immunol
32: 61–68
J Biol Inorg Chem (2014) 19 (Suppl 2):S773–S777
DOI 10.1007/s00775-014-1161-2
POSTER PRESENTATION
Metal homeostasis and detoxification
P1
Characterization of two members of the ZIP family
of Zn transporters expressed in Zn accumulating
fungus Russula atropurpurea
Tereza Leonhardt, Pavel Šimek, Pavel Kotrba
Department of Biochemistry and Microbiology, Institute of Chemical
Technology, Technická 3, CZ-16628, Prague,
Czech Republic, [email protected]
Russula atropurpurea is an ectomycorrhizal (EM) species, accumulating even from unpolluted environments up to 1,300 mg Zn kg-1
sporocarp dwt, a concentration that significantly exceeds the levels
found in the sporocarps of most other EM species (median of
98.6 mg kg-1 dwt) [1]. Interestingly, a substantial portion of Zn in R.
atropurpurea sporocarps remains bound with metallothionein-like
peptides [2], which is different from the preferential targeting of
excess Zn ions into subcellular compartments in some other EM
species, including Hebeloma mesophaeum [3]. The purpose of this
study was to identify and characterize the membrane transporters that
may contribute to the Zn overaccumulation phenotype of R. atropurpurea—the Zrt/IRT-like proteins (ZIPs), required in eukaryotes for
the loading of Zn into the cytoplasm. Sequencing of the sporocarp
transcriptome library allowed the identification of two partial transcripts corresponding to ZIPs, facilitating the isolation of the fulllength transcripts designated RaZIP1 and RaZIP2. While the deduced
352-amino acid (AA) RaZIP1 shared 40 % identity with the plasma
membrane ZRT1 from S. cerevisiae, the 422-AA RaZIP2 showed
30 % identity with YKE4 of S. cerevisiae, a member of the LIV-1
subfamily of ZIPs localized to the endoplasmic reticulum. The
transmembrane domain (TMD) prediction identified a model for the
deduced RaZIP proteins consisting of eight TMDs and a histidine rich
region between TM III and IV that is characteristic of Zn-transporting
ZIPs [4]. RaZIP2 further contains a predicted N-terminal targeting
signal and a HEXPHEXGD signature motif of LIV-1 ZIPs [4].
Complementation assays in S. cerevisiae revealed that: (i) expression
of RaZIP1 fully restored the growth of the Dzrt1 mutant under Znlimited conditions, while RaZIP2 restored the growth to some extent;
(ii) expression of RaZIP1, but not RaZIP2, resulted in hypersensitivity
of Zn-sensitive Dzrc1Dcot1 and Cd-sensitive Dyap1 cells to Zn and
Cd respectively. It thus appears reasonable to assume that RaZIP1 is
the Zn (and Cd) uptake transporter of R. atropurpurea and the role of
RaZIP2 may be in the remobilization of Zn ions from the endomembrane compartments. To support this hypothesis, Zn, Cd and
other metal accumulation analyses as well as protein localization
experiments with green fluorescent protein-tagged RaZIPs are currently conducted with model yeasts.
This work is supported by the Czech Science Foundation (P504-110484).
References
1. Borovička J, Řanda Z (2007) Mycol Prog 6:249–259
2. Leonhardt T, Šimek P, Sácký J, Kotrba P (2013) XII Int Symp
Inorg Biochem 72
3. Sácký J, Leonhardt T, Borovička J, Gryndler M, Briksı́ A, Kotrba P
(2014) Fungal Genet Biol doi:10.1016/j.fgb.2014.03.003
4. Dempski RE (2012) Curr Top Membr 69:221–245
P2
Metal ion induced structural diversity of the metal
binding domain from E. Coli Cuer regulatory protein
Dániel Szunyogh1, Attila Jancsó1, Béla Gyurcsik1, Lars
Hemmingsen2, Peter W. Thulstrup2, Flemming H.
Larsen3
1
Department of Inorganic and Analytical Chemistry, University
of Szeged, Dom tér 7, 6720 Szeged, Hungary, szunyogh@chem.
u-szeged.hu
2
Department of Chemistry, University of Copenhagen,
Universitetsparken 5, 2100 Copenhagen, Denmark
3
Department of Food Science, University of Copenhagen,
Rolighedsvej 30, 1958 Copenhagen, Denmark
The metal ion efflux/detoxification systems are activated by metalloregulatory proteins in prokaryotic cells. Members of the large
family of MerR metalloregulators respond to a variety of metal ions.
A common feature of these proteins is the Cys–Xn–Cys (n = 6–10)
metal ion binding loop close to the C-terminus. The sequential/
structural diversity of this domain is a key factor in sensing of the
metal ions. The MerR family member CueR is a CuI efflux regulator
protein exhibiting an outstanding selectivity for monovalent d10 metal
ions (e.g. AgI or CuI) but no activity for HgII or ZnII.
We have synthesized a 12-mer oligopeptide, comprising the metal
binding loop of E. Coli CueR (Ac-ACPGDDSADCPI-NH2, EC). The ZnII
and CdII binding affinity of the ligand has been studied by pH-potentiometric titrations. As previously shown with similar CueR-model
oligopeptides the coordination of the two Cys-thiolates direct the ligand
into a loop structure around ZnII and CdII by pH *7.0 and 6.0, respectively
[1, 2]. Besides, the metal ions are also able to bridge two ligands with a
presumably tetrahedral {4 9 S-} coordination mode at pH[8.
It is an interesting question whether different metal ions force
diverse coordination structures of the metal binding oligopeptide
sequence according to their preferences for donor sets and coordination environment. If metal ions play such a structural role in CueR
protein, this can have an impact on its activity/inactivity.
The peptide coordinated AgI and HgII very tightly by Cys-thiolates
already at pH *2. While a dicoordinate {2S-} binding mode was
observed for HgII in the whole studied pH-range, a monothiolate type
coordination of EC to AgI was suggested below pH *5.5. Neither HgII
nor AgI formed metal-bridged species under the applied conditions.
Acknowledgement: TÁMOP-4.2.2/B-10/1-2010-0012, János Bolyai Research Grant from the Hungarian Academy of Sciences.
References
1. Jancsó A, Szunyogh D, Larsen FH, Thulstrup PW, Christensen NJ,
Gyurcsik B, Hemmingsen L (2011) Metallomics 3:1331–1339
123
S774
2. Jancsó A, Gyurcsik B, Mesterházy E, Berkecz R (2013) J Inorg
Biochem 126:96–103
P3
Toxicity and detoxification of cadmium in the aquatic
macrophyte Ceratophyllum demersum
Elisa Andresen1, Sophie Kroenlein2, Jürgen Mattusch3,
Gerd Wellenreuther4, Uriel Arroyo Abad3, HansJoachim Stärk3, George Thomas2, Hendrik Küpper1,2
1
Department of Plant Biophysics and Biochemistry, Biology Centre
of the Academy of Sciences of the Czech Republic, Branišovska
31/1160, 37005 České Budějovice, Czech Republic, Elisa.
[email protected]
2
Fachbereich Biologie, Mathematisch-Naturwissenschaftliche
Sektion, Universität Konstanz, 78457 Konstanz, Germany
3
Department of Analytical Chemistry, UFZ, Helmholtz Centre
for Environmental Research, Permoserstr. 15, 04318 Leipzig,
Germany.
4
HASYLAB at DESY, Notkestr. 85, 22637 Hamburg, Germany
The heavy metal cadmium (Cd) is an important pollutant and poisonous to many organisms. We studied the effects of Cd on C.
demersum under environmentally relevant conditions. High, moderate
and low concentrations of Cd had different effects. Lethally toxic
concentrations (100–200 nM) led to growth stop and the plants’
ability to perform photosynthesis (measured as Fv/Fm) decreased more
than twofold, consistent with decreased pigment content. Moderately
toxic concentrations (10–50 nM) led to reduced growth, slightly
reduced pigment content, but hardly affected photosynthesis (measured as O2 exchange and as Fv/Fm). Lower concentrations
(0.2–5 nM) even had beneficial effects, like enhanced growth rate.
When applied in low concentrations, Cd was homogeneously distributed in the whole cross-section of the leaves like a nutrient.
Moderate and high Cd concentrations led to sequestration of Cd in the
vascular bundle and the epidermis cells, where Cd does not affect
photosynthetic molecules. At toxic Cd concentrations, Zn was redistributed and mainly found in the vein along with Cd, indicating
inhibition of Zn transporters. Consistently, by metalloproteomics
(HPLC-ICP-MS) we found that during Cd toxicity concentrations of Cd
increased in fractions of the major photosynthetic complexes while Mg
decreased, suggesting the replacement of Mg by Cd in chlorophylls.
Furthermore, the induction of phytochelatins was not proportional
to metal concentration, but had distinct thresholds, specific for each PC
species. PC3 especially was switch-like induced already at 20 nM Cd,
which was previously regarded as non-toxic to most plants. Phytochelatin levels at the lowest Cd concentrations were not detectable or
below 0.1 % of the level at sublethally toxic concentrations, suggesting
that they do not have another function than metal detoxification.
P4
The role of magnesium supplementation and effects
on metal ion homeostasis
Zoltán May1, Klára Szentmihályi1, Mária Rábai1,
Anna Blázovics2
1
Institute of Materials and Environmental Chemistry, Research
Centre for Natural Sciences of the HAS, Magyar tudósok körútja 2,
H-1117 Budapest, Hungary.
2
Department of Pharmacognosy, Semmelweis University, H-1086
Budapest, Hungary
Magnesium participates in numerous enzymatic reactions in the
human body and is essential for the function of living organisms and
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J Biol Inorg Chem (2014) 19 (Suppl 2):S773–S777
has an essential role in maintaining the antioxidant system as well.
The aim of our study was to investigate the effect of magnesium on
element content in blood. Male Wistar rats were involved in the
experiments. The animals were divided into four groups, number I, II,
III and IV. In group I and II the rats were fed with normal diet, but in
latter they were treated with magnesium polygalacturonate additionaly (200 mg Mg/kg body weight ad libitum daily). The animals in
group III were fed with fat rich diet containing cholesterol (2.0 %),
sunflower oil (20 %) and cholic acid (0.5 %) added to the control diet.
The animals of group IV were fed with fat-rich diet and magnesium
polygalacturonate also. The rats were kept on the diets for 9 days. The
element concentration (Al, As, B, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, Li,
Mg, Mn, Ni, P, Pb, S, Si, Sn, Sr, Ti, V, Zn) of blood samples was
determined with ICP-OES after digestion with a mixture of concentrated nitric acid and hydrogen peroxide. The results show that the
concentration of several elements changed significantly in both
magnesium-treated groups (II, IV), nevertheless the alteration was
different in the control and hyperlipidemic groups (I, III). It has been
concluded that high amount of magnesium supplementation alters the
metal ion homeostasis in short-term experiment. Although some
favourable effects were found in the hyperlipidemic group with
magnesium polygalacturonate treatment, it should be noted that
supplementation with magnesium should be carried out carefully
especially in case of metabolic diseases.
References
1. Fagan TE, Cefaratti C, Romani A (2004) Am J Physiol Endocrin
Metab 286:E184
2. Lopez-Ridaura R, Willett WC, Rimm EB (2004) Diab Care 27:134
P5
Interaction of reduced and oxidized hepcidin with metal
ions
Dawid Płonka, Arkadiusz Bonna, Wojciech Bal
Department of Biophysics, Institute of Biochemistry and Biophysics,
Polish Academy of Sciences, ul. Pawinskiego 5a, 02-106 Warszawa,
Poland, [email protected]
Hepcidin, a recently discovered vertebrate hormone, is very important
for the iron metabolism. Its structure with eight cysteines forming
intramolecular bonds makes it similar to defensins and metallothioneins. This is consistent with hepcidin function. It was initially discovered
as a defensin like antimicrobial peptide, but in humans its primary
function is to control iron homeostasis in liver [1]. Moreover, hepcidin
is known to have a functional N-terminal Cu(II) and Ni(II) binding site
[2]. While hepcidin is generally thought to work as disulfide-bridged
peptide, none of these bonds is necessary for its function [3]. Our work
sheds more light on transition-metal binding properties of hepcidin.
We managed to chemically synthesize hepcidin with good purity
using Fmoc solid phase peptide synthesis. We used spectroscopic
methods to characterize metal binding properties of hepcidin in both
reduced (without S–S linkage) and oxidized state with respect to
Zn(II) and Cu(I) ions. Our results indicate specificity of binding, high
affinity and distinct stoichiometry, all suggesting the physiological
role for thiol-based metal ion binding by hepcidin.
Financial support by the Institute of Biochemistry and Biophysics
is gratefully acknowledged.
References
1. Nemeth E, Tuttle MS, Powelson J, Vaughn MB, Donovan A,
McVey Ward D, Ganz T, Kaplan J (2004) Science 306:2090–2093
J Biol Inorg Chem (2014) 19 (Suppl 2):S773–S777
2. Melino S, Garlando L, Patamia M, Paci M, Petruzzelli R (2006) J
Peptide Res 66:65–71
3. Nemeth E, Preza GC, Jung CL, Kaplan J, Waring AJ, Ganz T
(2006) Blood 107:328–333
P6
Productivity, metal accumulation capacity,
and hormetic response of the invasive metal tolerant
neophyte Buddleja davidii (summer lilac)
Dorde Topalovic, Helmut Brandl
Institute of Evolutionary Biology and Environmental Studies,
University of Zurich, Winter-thurerstrasse 190, CH-8057 Zurich,
Switzerland
The presence of heavy metals in soils can affect humans and ecosystems in many different ways. Adequate protection and restoration of
soils and ecosystems contaminated by heavy metals is needed. One
possible approach is to use metal tolerant plants which can extract and
accumulate heavy metals [1]. The principal aim of this project was to
measure the potential of the shrub Buddleja davidii for accumulation of
cadmium, lead, and zinc focusing on biomass production and metals
accumulated in different plant parts (roots, stems, leaves). To better
understand if there is also a biological mechanism that plants have
evolved in the past for improved accumulation of toxins, different
genotypes originating from different locations were included in the
study. Half of them were Buddleja davidii plant cuttings growing in
natural, non-contaminated soil in Grüningen (canton of Zurich) and
half were plant cuttings growing in metal-contaminated soil from a
landfill in Krauchtal (canton of Berne). In the greenhouse Buddleja
davidii cuttings from different genotypes and different sites, were
grown on standard soil in the presence of lead, zinc, and cadmium at
different concentrations to determine their influence on plant productivity in terms of biomass production. The results indicated that
Buddleja davidii is heavy metal tolerant able to accumulate different
heavy metals in both roots and shoots. No difference in plant productivity was observed between of treated and control plants indicating
that the metals present in the soil did not affect plant productivity in
any way. However, there were differences in the biomass production
between plants coming from different locations. Plants from the landfill
in Krauchtal produced more biomass than the cuttings from the noncontaminated area in Grüningen. In addition, strong indications of
hormetic effects (hormesis; growth stimulation at very low doses of
metals applied) regarding the biomass production were detected.
Overall, results showed that Buddleja davidii is a hyperaccumulating
plant with a great potential for phytoremediation use in the future.
S775
frequent allergen. The median prevalence of palladium allergy in
dermatitis and dental patients was found to be about 7 % [1].
Although many hypotheses have been proposed concerning the
mechanisms of development of metal-induced diseases, there is a high
possibility that in fact there are many yet unknown ways to develop
them. One of the probable mechanisms may also be related to
hydrolysis of peptide bond catalysed by palladium ions. The binding
of another metal—nickel to peptides and proteins and subsequent
metal-dependent hydrolysis of peptide bond occurs in the fragments
with amino acid sequence Ser/Thr-Xaa-His [2]. We found that this
kind of sequence can bind also Pd(II) ions, which leads to Pd(II)dependent hydrolysis of peptide bond in the same manner as observed
for Ni(II). Human annexins A1 and A2, proteins involved in the
regulation of immune system [3], carry the above mentioned amino
acid sequences making them favourable sites for palladium binding/
hydrolysis. Destruction of these proteins by Pd(II) may be one of the
mechanisms of allergy development. We found that peptides: AcGVGTRHKAL-am, Ac-TKYSKHDMN-am, Ac-KALTGHLEE-am
from annexin A1, Ac-SALSGHLET-am, Ac-KMSTVHEIL-am from
annexin A2 bind Pd(II) in a pH-dependent manner. Spectroscopic
measurements showed that pK values for Pd(II) complexation are in
the range from 5.6 to 6.4. It means that physiological pH (about 7.4)
favours the formation of hydrolytic complexes. The lowest values of
the half-time of decomposition of the peptides at pH 7.4 are for AcGVGTRHKAL-am: 2.4 and 1.2 h at 37 and 45 °C, respectively.
These data suggest that at least one region of annexin A1 is prone to
palladium-dependent hydrolysis in physiological conditions. Toxicological consequences of this finding will be discussed.
Financial support by Polish National Science Centre, Grant No.
DEC-2011/01/D/NZ1/03501 is gratefully acknowledged.
References
1. Faurschou A, Menne T, Johansen JD, Thyssen JP (2011) Contact
Dermat 64:185–195
2. Kopera E, Kre˛z_ el A, Protas AM, Belczyk A, Bonna A, WysłouchCieszyńska A, Poznański J, Bal W (2010) Inorg Chem 49:6636–6645
3. D’Acquisto F, Perretti M, Flower RJ (2008) Br J Pharmacol
155:152–169
P8
Novel glycoconjugated dipeptide for the treatment
of metal-related disorders
Francesco Bellia1, Giuseppa I. Grasso1, Graziella
Vecchio2, Giuseppe Arena2, Enrico Rizzarelli1,2
1
Reference
1. Sheoran,V, Sheoran AS, Poonia P (2012) Int J Earth Sci Eng
5:428–436
P7
Human annexins A1 and A2 are potential toxicological
targets for Pd(II) ions
Tomasz Fra˛czyk, Karolina Bossak, Arkadiusz Bonna,
Wojciech Bal
Institute of Biochemistry and Biophysics, Polish Academy
of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland
Palladium is a component of alloys used e.g. in jewelery, dental
materials and orthopedic implants. Palladium ions are released from
these materials. This is a substantial problem, as palladium is a
Institute of Biostructure and Bioimaging, CNR, Viale A. Doria 6,
95125 Catania, Italy, [email protected].
2
Department of Chemistry, University of Catania, Viale A. Doria 6,
95125 Catania, Italy
Carnosine is an endogenous dipeptide widely and abundantly distributed in muscle and nervous tissues of numerous animal species.
Many functions have been proposed for this compound, such as
antioxidant, antiaggregant, antiglycating agent and metal ion-chelator, especially for copper(II) and zinc(II). The administration of
carnosine provides benefits in Alzheimer’s disease and other neurodegenerative disorders. However, the main limitation on the
therapeutic use of carnosine on pathologies related to increased
oxidative stress and/or metal ion dyshomeostasis is associated with
the hydrolysis by the specific dipeptidase carnosinase. Several
attempts have been made to overcome this limitation. The glycoconjugation has been found to be a promising approach to protect
the dipeptide moiety in this respect. A number of glycoside
derivatives of carnosine have also been characterized in terms of
123
S776
their binding features for copper(II) [1–4]. Here, we report the
chemical and functional characterization a new carnosine derivative
with trehalose, a multifunctional sugar tested for the treatment of
Huntington’s disease, Parkinson disease and several tauopathies.
The copper(II) binding properties, as well as the antiaggregant and
antiglycating actions make the new carnosine conjugate a promising agent for the treatment of a wide class of degenerative
disorders.
Financial support by the Italian Ministry of Education, Universities and Research (MIUR) is gratefully acknowledged.
References
1. Grasso GI, Arena G, Bellia F, MacCarrone G, Parrinello M, Pietropaolo A, Vecchio G, Rizzarelli E (2011) Chem Eur J 17:9448–9455
2. Grasso GI, Bellia F, Arena G, Vecchio G, Rizzarelli E (2011) Inorg
Chem 50:4917–4924
3. Bellia F, Vecchio G, Rizzarelli E (2012) Amino Acids 43:153–163
4. Grasso GI, Arena G, Bellia F, Rizzarelli E, Vecchio G (2014) J
Inorg Biochem 131:56–63
P9
(5-Hydroxy-4-oxo-4H-pyran-2-yl)methyl
3-({[(5-hydroxy-4-oxo-4H-pyran-2yl)methoxy]carbonyl}amino)propanoate. Synthesis
and its complex formation study
Joanna I. Lachwicz1, Valeria M. Nurchi1, Guido
Crisponi1, Guadalupe J. Pelaez1, Piotr Stefanowicz2,
Maria A. Zoroddu3, Massimiliano Peana3
1
Dipartimento di Scienze Chimiche e Geologiche, University
of Cagliari, Cittadella Universitaria, 09042 Monserrato-Cagliari,
Italy, [email protected]
2
Faculty of Chemistry, University of Wrocław, ul. F. Joliot-Curie 14,
50-383 Wroclaw, Poland
3
Department of Chemistry and Pharmacy, University of Sassari, Via
Vienna 2, 07100, Sassari, Italy
The importance of iron chelators in medicine has significantly
increased in recent years [1]. Iron is essential for life but it, when in
excess, is also potentially more toxic than other trace elements. This is
because humans lack effective means to protect cells against iron
overload and because of the role of iron in generation of free radicals.
In order to protect patients from the consequences of iron toxicity, iron
chelating agents have been introduced in clinical practice. Unfortunately, the ideal chelator for treating iron overload in humans has not
been identified yet.Many tetradentate ligands have been investigated to
act as possible iron(III) chelators for oral use. They are most often
made up of dihydroxamic acids [2–6], but disphosphonates [7] and a
bis(3-hydroxy-4-pyridinone)-IDA derivative [8] have also been
described. The molecular weight of these ligands is generally below the
limit of 500 suggested by Lipinski for oral absorption and pFe values
are in the range of 18–21 log units. In order to completely saturate the
six coordination positions on ferric ion, the denticity of these ligands
requires formation of polynuclear species, which are invariably found
in these systems. In particular, the most common polynuclear complex
is the dimer Fe2L3, with a charge depending on ligand structure.
Kojic acid (5-hydroxy-2-(hydroxymethyl)-4-pyrone, HKa) is a
natural c-pyrone derivative, closely related to maltol, produced by
many species of Aspergillus and Penicillium moulds. Kojic acid,
recognized at first as an antibiotic substance, has antibacterial and
antifungal properties, and inhibits the formation of pigmented products in plant and in animal tissue, as well as the oxygen uptake when
o-dihydroxy- and trihydroxy phenols are oxidized by tyrosinase
[9–12]. It is used in food and cosmetics to preserve or ‘‘ameliorate’’
123
J Biol Inorg Chem (2014) 19 (Suppl 2):S773–S777
colors of substances: on cut fruits it prevents oxidative browning, in
seafood preserves pink and red colors and in cosmetics lightens the
skin.
Here we are going to present the one-pot efficient synthesis of a
new tetradentate chelator (5-hydroxy-4-oxo-4H-pyran-2-yl)methyl3({[(5-hydroxy-4-oxo-4H-pyran-2-yl)methoxy]carbonyl}amino)propanoate, and its complex formation studies with Fe(III), Al(III),
Cu(II) and Zn(II) metal ions. In order to present most accurate data
stability constants and of complex formation, in this work a variety of
techniques has been used: potentiometry, UV–Vis, NMR, ESI–MS
techniques.
Scheme of reaction of obtaining (5-hydroxy-4-oxo-4H-pyran-2yl)methyl 3-({[(5-hydroxy-4-oxo-4H-pyran-2-yl)methoxy]carbonyl}
amino)propanoate.
References
1. Crisponi G, Remelli M (2008) Coord. Chem. Rev. 252:1225
2. Caudle MT, Caldwell CD, Crumbliss AL (1995) Inorg Chim Acta
240:519
3. Nguyen-van-Duong MK, Guillot V, Nicolas L, Gaudemer A,
Lowry L, Spasojevic I, Crumbliss AL (2001) Inorg Chem 40:5948
4. Farkas E, Buglyo P, Enyedy TA, Gerlei VA, Santos AM (2002)
Inorg Chim Acta 339:215
5. Gaspar M, Telo JP, Santos MA (2003) Eur J Inorg Chem 22:4025
6. Farkas E, Buglyo P, Enyedy EA, Santos MA (2004) Inorg Chim
Acta 357:2451
7. Gumienna-Kontecka E, Silvagni R, Lipinski R, Lecouvey M,
Marincola FC, Crisponi G, Nurchi VM, Leroux Y, Kozlowski H
(2002) Inorg Chim Acta 339:111
8. Santos MA, Gama S, Gano L, Cantinho G, Farkas E (2004) Dalton
Trans 3772
9. Yabuta T (1924) J Chem Soc 125:575
10. Burdock FA, Soni MG, Carabin IG (2001) Reg Toxicol Pharmacol 33:80–101
11. Fukusawa R, Wakabayashi H, Natori T (1982) Japanese Patent
5740875
12. Kahn V, Ben-Shalom N, Zakin V (1997) J Agric Food Chem
45:4460–4465
P 10
Effect of salinomycin on the biodistribution of lead
and some essential metal ions in mice, subjected
to subacute lead intoxication
Juliana Ivanova1, Yordanka Gluhcheva2, Ekaterina
Pavlova2, Donika Dimova2
1
Department of Chemistry and Biochemistry, Physiology
and Pathopysiology, Faculty of Medicine, Sofia University ‘‘St.
Kliment Ohridski’’, 1 Kozjak Street, 1407 Sofia, Bulgaria,
[email protected].
2
Department of Experimental Morphology, Institute of Experimental
Morphology, Pathology and Anthropology with Museum, BAS,
Bulgaria Acad. Georgi Bonchev Str., bl. 25, 1113 Sofia, Bulgaria
Lead (Pb) is one of the most toxic metal ions released in the
environment as a result of human activity. It has been used in the
production of pigments, pottery glazes, batteries, etc. Pb is a
J Biol Inorg Chem (2014) 19 (Suppl 2):S773–S777
neurotoxic agent causing encephalopathy both in adults and children. When accumulated in the body Pb affects the function of
many organs in particular spleen, kidneys, brain. It disturbs the
function of hematopoietic system replacing Fe from the protoporphyrin ring. It has been reported that Pb mimics the biochemistry
of Ca, which has been considered as a primary mechanism for Pbinduced toxicity [1]. For the treatment of toxic metal intoxication a
therapy with chelating agents has been used. Recent studies have
demonstrated that monensin is much more effective than the traditional chelating agents in reducing Pb concentration in rats,
subjected to Pb intoxication [2]. Among the polyether ionophore
antibiotics salinomycin is the least toxic representative. To the best
of our knowledge there is still no data regarding the potential
application of this antibiotic as a chelating agent for the treatment
of toxic metal intoxication. Herein we present novel data demonstrating that salinomycin significantly decreases Pb content in the
organs of mice subjected to subacute Pb intoxication. No significant effect on essential metal homeostasis as a result of
salinomycin treatment is observed. Taken together these results
imply that salinomycin could be a potential chelating agent for the
treatment of subacute Pb intoxication.
Financial support by the Sofia University Fund for Scientific
Research is gratefully acknowledged (grant N 162/2014).
S777
binding properties by application of potentiometry, mass spectrometry (ESI–MS) and spectroscopic methods (CD, UV–Vis, EPR). These
results demonstrate an increase in complex stabilization when compared to linear fragments.
The modification of peptides by Dap branching was applied also in
the nuclear targeting peptide such as TAT(47–57). The fluorescence
microscopy as well as ICP-MS studies of cells treated with different
concentrations of designed complexes demonstrate the possibility of
application of Dap-based branched peptides as metal ions transport
platforms. This project was financed by Polish Foundation of Science
within the POMOST program (POMOST/2012-5/9).
References
1. Flora G, Gupta D, Tiwari A (2012) Interdiscip Toxicol 5:47–58
2. Pachauri V, Dubey M, Yadav A, Kushwaha P, Flora SJ (2012)
Food Chem Toxicol 50:4449–4460
P 11
The Cu(II) and Ni(II) binding properties of de novo
branched peptides based on 2,3-diaminopropionic acid
and their in vitro metal ions cellular trafficking features
Łukasz Szyrwiel1,2, Mari Shimura3, Junko Shirataki4,
Jozsef S. Pap5, Łukasz Szczułkowski2, Bartosz Setner6,
Zbigniew Szewczuk6, Wiesław Malinka2, Laurent
Chavatte1, Ryszard Łobinski1
1
CNRS/UPPA, LCABIE, UMR5254, Hélioparc.
2, Av. Pr. Angot, F-64053 Pau, France
2
Department of Chemistry of Drugs, Wrocław Medical University, ul.
Borowska 211, 50-552 Wrocław, Poland. [email protected]
3
Department of Intractable Diseases, 1-21-1 Toyama, Shinjuku-ku,
Tokyo 162-8655, Japan.
4
Inorganic Analysis Laboratories, Toray Research Center Inc., Otsu,
Shiga 520-8567, Japan.
5
Surface Chemistry and Catalysis Department, Centre for Energy
Research, HAS, 1525 Budapest 114, P.O. Box 49, Hungary.
6
Faculty of Chemistry, University of Wrocław, ul. F. Joliot-Curie 14,
50-383 Wrocław, Poland.
The de novo designed branched peptides based on the 2,3-diaminopropionic acid were investigated focusing on their Cu2? and Ni2?
123
J Biol Inorg Chem (2014) 19 (Suppl 2):S779–S814
DOI 10.1007/s00775-014-1162-1
POSTER PRESENTATION
Metals in medicine: metal-related diseases
P 20
Lipophilic platinum(II) complexes with dicarboxylates:
in vitro antiproliferative activity as well
as the mechanism of the suppression of tumour cells
growth
Kamil Hoffmann1, Ewa Maj2, Andrzej Wojtczak3, Joanna
Wietrzyk2, Iwona Łakomska1
1
Bioinorganic Chemistry Research Group, Faculty of Chemistry,
Nicolaus Copernicus University, Gagarina 7, 87-100 Toruń, Poland,
[email protected];
2
Ludwik Hirszfeld Institute of Immunology and Experimental
Therapy, Polish Academy of Sciences, Weigla 12, 53-114 Wrocław,
Poland;
3
Faculty of Chemistry, Nicolaus Copernicus University, Gagarina 7,
87-100 Toruń, Poland
Improving a therapeutic profile of existing platinum(II) complexes,
combination of 5,7-diterbutyl-1,2,4-triazolo[1,5-a]pyrimidine (dbtp)
as N-donor ligand and two types of leaving groups: malonate (mal)
(1) as well as cyclobutane-1,1-dicarboxylate (CBDC) (2) were used.
The complexes were characterized in depth by multinuclear magnetic
resonance spectroscopy (1H, 13C, 15N, 195Pt) and X-ray. The spectroscopical results indicate that the local geometry around the
platinum(II) center approximates a square-planar arrangement with
two monodentate nitrogen atom N3 of the dbtp and bidentate
dicarboxylate.
To estimate the permeability of [Pt(mal)(dbtp)2] and
[Pt(CBDC)(dbtp)2] through the cell membrane, their partition coefficients were calculated using shake-flask method. Both complexes
were much more lipophilic (log P = *2.0) than cisplatin (log P = 1.76) or carboplatin (log P = -1.48). Additionally, the therapeutic
potential of the obtained Pt(II) compounds was examined using
in vitro cytotoxicity experiments against two human cancer cell lines
i.e.: cisplatin-resistance breast (T47D) and lung adenocarcinoma
(A549). They exhibited improved cytotoxic activity against T47D cell
line, suggesting ability to overcome cisplatin resistance mechanism in
that tumour cell line. Furthermore, it ought to be highlighted that
presented platinum-based prodrugs, resulted to be over 36-times more
active than carboplatin against A549. Promising results encouraged to
analyze also the influence of platinum(II) complexes on cell cycle and
cell death (subG1) of A549 cell. It was demonstrated that (1,2) are
capable of arresting the cell cycle at the G0/G1 phase, whilst cisplatin
and carboplatin stopped the cells in G2/M stage, signifying the differences in the mechanism of the suppression of tumour cell growth.
Finally, in the quest for low–toxic platinum drugs, the antiproliferative in vitro activity against normal mouse fibroblast cells (Balb/3T3).
The title platinum (II) complexes were 1.5-fold less active than cisDDP, implying that they should be less toxic than the worldwide used
cisplatin.
Financial support by the National Science Center (DEC–2012/07/
N/ST5/00221) to K.H. is gratefully acknowledged.
P 21
Visible light-induced annihilation of human tumour
cells using novel platinum-porphyrin conjugates
Anu Naik, Riccardo Rubbiani, Gilles Gasser,
Bernhard Spingler
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland
Despite the extended use of porphyrin complexes in PDT [1,2], tetraplatinated porphyrins have so far not been studied for their
anticancer properties upon light irradiation [3]. This is in contrast to
ruthenated tetrapyridyl porphyrin complexes [4].
We would like to report about the synthesis of novel tetraplatinated
porphyrins as well as their photophysical characterization and in vitro
light-induced anticancer properties [5]. The quantum yield of 1O2 (U)
production upon light irradiation was found to be between 0.41 and 0.54.
The dark and light toxicity against human cancerous and non-cancerous
cell lines (MRC-5, HeLa, A2780 and CP70) was determined by the MTT
assay. IC50 values were obtained after 4 h incubation, a washing step,
followed by 15 min irradiation at either 420 nm (6.95 J cm-2) or
575 nm, (6.23 J cm-2) respectively. These platinum-porphyrin conjugates had only minor dark toxicity, however upon visible light irradiation,
IC50 values down to 19 ± 4 nM could be observed. These values correspond to an excellent phototoxic index (PI = IC50 dark/IC50 light) of
[5,000. After 4 h incubation in HeLa cells, incubation of a tetraplatinum-porphyrin conjugate led to a concentration of about 105 ng Pt in the
nucleus per mg protein in the cell. Strikingly, the use of this conjugate
increased the nuclear platinum content by more than 30-fold compared
with cisplatin. This is obviously only partially a consequence of the
porphyrin conjugate having 4 platinum centers versus one in cisplatin; the
main reason being that the conjugate is a more efficient platinum importer
into the cell nucleus than cisplatin.
Taken together, all these favourable characteristics imply that
tetraplatinated porphyrin complexes may be worth being explored as
novel PDT anti-cancer agents in vivo.
Financial support by the Forschungskredit (A.N.), the University
of Zurich, the Swiss National Science Foundation (Professorship No.
PP00P2_133568 to G.G) and the Novartis Jubilee Foundation (G.G
and R.R) is gratefully acknowledged.
References
1. MacDonald IJ, Dougherty TJ (2001) J Porphyrins Phthalocyanines
5:105–129
2. Agostinis P, Berg K, Cengel KA, Foster TH, Girotti AW, Gollnick
SO, Hahn SM, Hamblin MR, Juzeniene A, Kessel D et al (2011) CA
Cancer J Clin 61:250–281
3. Munakata H, Imai H, Nakagawa S, Osada A, Uemori Y (2003)
Chem Pharm Bull 51:614–615
4. Pernot M, Bastogne T, Barry NPE, Therrien B, Koellensperger G,
Hann S, Reshetov V, Barberi-Heyob M (2012) J Photochem Photobiol B 117:80–89
123
S780
J Biol Inorg Chem (2014) 19 (Suppl 2):S779–S814
5. Naik A, Rubbiani R, Gasser G, Spingler B (2014) Angew Chem Int
Ed 53 (in print)
P 22
Water-soluble 1,3,5-triazine platinum(II) complexes
as potential candidate of anticancer drugs
Yuet-Ting Lau, Chin-Wing Chan
School of Science and Technology, The Open University of Hong
Kong, 30 Good Shepherd Street, Homantin, Hong Kong
2,4-diamino-1,3,5-triazine and its derivatives have demonstrated
anticancer properties [1]. On the other hand, cisplatin, is the first
example of anticancer drug, which cross links double-strand DNA
and leads to DNA distortion and apoptosis of the tumor cell [2].
Derivatives or other platinum compounds, such as carboplatin, oxaliplatin and AMD473, were later synthesized and developed in
order to reduce the side effects of cisplatin [3]. The development of
platinum complex as anti-cancer drug goes unrelenting over the
years [4].
The water-soluble cis-diammine-bis-[2,4-diamino-6-(4-Pyridyl)1,3,5-triazine]platinum(II) tosylate [Pt(L1)2(NH3)2](OTs)2 (C1) was
synthesized from the ligation of 1,3,5-triazine derivative (L1) with
diammine platinum(II) complex (Figure 1). The gel mobility shift
assay had been shown a slight decrease in DNA mobility presenting
of C1. Besides, the disk diffusion test on ECC agar had been shown
C1 had the ability to inhibit the growth of E. coli.
H3N
NH3
Pt
N
Financial support by the following projects is gratefully
acknowledged: P207/11/0841, CZ.1.05/2.1.00/03.0058, LO1305 and
IGA_PrF_2014009.
2+
N
N
N
R1HN
N
N
Reference
1. Štarha P, Hošek J, Vančo J, Dvořák Z, Suchý P, Popa I, Pražanová
G, Trávnı́ček Z (2014) PLoS One 9:e90341
NHR2
N
N
NHR2
(X-)2
R1HN
X- = p-CH3-C6H4-SO3- R1/R2 = H, or C6H5
Ligand (L) 1: R1=R2=H; 2: R1=H, R2=C6H5; 3: R1=R2=C6H5
Figure 1: Structures of [Pt(L)2(NH3)2] (OTs)2 and
its phenyl substituted derivatives.
References
1. Brzozowski Z, Saczewski F, Gdaniec M (2000) Eur J Med Chem
35:1053–1064
2. Jamieson ER, Lippard SJ (1999) Chem Rev 99:2467–2498
3. Bakalova A, Varbanov H, Buyukliev R, Momekov G, Ferdinandov
D, Konstantinov S, Ivanov D (2008) Eur J Med Chem 43:958–965
4. Haxton KJ, Burt HM (2009) J Pharm Sci 98:2299–2316
P 23
Gold-coated maghemite nanoparticles for magnetic
delivery of antitumor platinum(II) complexes
Pavel Štarha, Kateřina Kubešová, Zdeněk Trávnı́ček
Department of Inorganic Chemistry, Regional Centre of Advanced
Technologies and Materials, Palacky University in Olomouc, 17.
listopadu 12, 77146 Olomouc, Czech Republic, [email protected]
The magnetic nanoparticles represent one of the crucial possibilities
of the targeted drug delivery, because their therapeutic application
123
could reduce the chemotherapeutic dose and consequently also negative side effects connected with the drug application. The
pharmacologically perspective systems should meet several
requirements, such as easy and reproducible preparation with good
yields, an effective response to the external magnetic field, high
stability and drug release, non-toxicity and significant therapeutic
action.
In this work we present the representatives of the maghemite
nanoparticles (&15 nm) functionalized by various cytotoxic platinum(II) complexes. The composites are based on gold-coated
maghemite covered by a sulphur-containing carboxylic acid (e.g.
thioctic acid) and functionalized by cisplatin or its analogues with
different N-donor ligands (e.g. 7-azaindoles [1]). The SEM and TEM
results showed that the products consist of well-dispersed nanoparticles, while EDS, ICP-MS and XPS proved the presence of the
platinum(II) complex within the prepared composites (see figure).
The biological experiments researching the stability and drug-release
under different conditions as well as in vitro cytotoxicity, were performed or are currently in progress and their results will be discussed
within the framework of the presentation.
P 24
Conjugates of cisplatin and cyclooxygenase inhibitors
as potent anti-tumour agents overcoming cisplatin
resistance
Wilma Neumann1, Brenda C. Crews2, Lawrence J. Marnett2,
Evamarie Hey-Hawkins1
1
Institute of Inorganic Chemistry, Universität Leipzig, Johannisallee
29, 04103 Leipzig, Germany;
2
Department of Biochemistry, Vanderbilt Institute for Chemical
Biology, Vanderbilt University School of Medicine, Nashville, TN
37232, USA
Platinum-based anti-tumour therapy is complicated by severe side
effects and intrinsic and acquired resistance of tumour cells. Implicated in cisplatin resistance is cyclooxygenase-2 (COX-2), a key
enzyme in the biosynthesis of prostaglandins. COX-2 is overexpressed in many tumours and plays a role in tumour initiation and
progression [1]. It is also associated with poor outcome in several
types of cisplatin-treated cancer [2]. Thus, COX inhibitors are used as
chemopreventive and adjuvant chemotherapeutic agents. Clinical
studies have shown synergistic effects when COX inhibitors are
administered in combination with various anti-tumour agents, such as
cisplatin [3]. However, the mechanism by which COX-2 is involved
in tumourigenesis is still mainly unknown, and also controversial
results have been reported. Prior studies of the influence of COX
inhibitors on the efficacy of anti-tumour agents have used combinatorial treatments resulting in potential discrepancies between clinical
J Biol Inorg Chem (2014) 19 (Suppl 2):S779–S814
and cell culture studies. Due to differential pharmacokinetics, delivery of the drugs to a tumour in vivo may fail to recapitulate
administration of the compounds to cells in culture. To address this
issue, we report the first covalently linked conjugates of cisplatin with
COX inhibitors [4]. Indomethacin or ibuprofen were coordinated at
cisplatin as axial ligands, resulting in platinum(IV) complexes.
Intracellular reduction allows these conjugates to act as prodrugs and
in a dual action mode upon cleavage. The covalent conjugation
ensures concerted transport of both drugs into tumour cells and may
promote enrichment of the complexes in COX-2-expressing tumours
[5]. The platinum(IV) complexes show highly increased cytotoxicity
compared to cisplatin and even overcome cisplatin-related resistance
in tumour cells. Furthermore, the indomethacin conjugate represents
the first highly potent COX-2-selective inhibitor containing a metal
centre. Furthermore, these conjugates provide tools for the elucidation
of the influence of COX inhibitors on the efficacy of platinum-based
anti-tumour agents.
Financial support by the Fonds der Chemischen Industrie (doctoral
grant for W.N.) and the Graduate School ‘Building with Molecules
and Nano-objects (BuildMoNa)’ funded by DFG as well as donation
of chemicals by Umicore AG & Co. KG are gratefully acknowledged.
References
1. Ghosh N, Chaki R, Mandal V, Mandal SC (2010) Pharmacol Rep
62:233
2. Stewart DJ (2007) Crit Rev Oncol Hematol 63:12
3. Hattori K, Matsushita R, Kimura K, Abe Y et al. (2001) Biol Pharm
Bull 24:1214
4. Neumann W, Crews BC, Marnett LJ, Hey-Hawkins E (2014)
ChemMedChem (in press)
5. Uddin MJ, Crews BC, Blobaum AL, Kingsley PJ, Gorden DL,
McIntyre JO, Matrisian LM, Subbaramaiah K, Dannenberg AJ, Piston
DW, Marnett LJ (2010) Cancer Res 70:3618
P 25
Ratiometric delivery of cisplatin and doxorubicin using
tumour-targeting carbon-nanotubes entrapping
platinum(IV) prodrugs
Siew Qi Yap1, Chee Fei Chin1, Jian Li2, Giorgia Pastorin2,3,4, Wee
Han Ang1
1
Department of Chemistry, National University of Singapore,
Singapore 117543, Singapore;
2
Department of Pharmacy, National University of Singapore,
Singapore 117543, Singapore;
3
NUS Graduate School for Integrative Sciences and Engineering,
Centre for Life Sciences (CeLS), Singapore 117456, Singapore;
4
NanoCore, Faculty of Engineering, National University
of Singapore, Singapore 117576, Singapore
Chemoresistance often occur after successive treatment with single
agent chemotherapy. Combination therapy, the administration of a
cocktail of different anticancer drugs has been employed to overcome
chemoresistance and improve the performance of single-agent chemotherapy. However, differences in pharmacokinetic profile of the
drugs often complicate treatment regimens. This can be overcome
using nanomaterials which allow simultaneous delivery of multiple
drugs to the targeted site. In order to deliver the exact stoichiometric
amount of cisplatin and doxorubicin, an inert platinum(IV) complex
was designed for entrapment in tumor-targeting multiwalled carbon.
Upon chemical reduction, equimolar of cisplatin and doxorubicin
S781
were released from the hydrophobic carrier thereby achieving synchronous delivery of these two mechanistically complementary drugs.
Reference
1. Chin CF, Yap SQ, Li J, Pastorin G, Ang WH (2014) Chem Sci (in
press)
P 26
On the hydrolysis of Pt(IV) pro-drugs with haloacetato
ligands in the axial positions
Raji Raveendran, Ezequiel Wexselblatt, Sawsan Salameh, Eylon
Yavin, Dan Gibson
Institute for Drug Research, School Of Pharmacy, The Hebrew
University of Jerusalem, Jerusalem, Israel, [email protected]
Platinum(II) based drugs are most widely used anticancer agents in
chemotherapy. Whilst they are effective, their use is limited by their
severe side effects and development of cellular resistance. In attempts
to overcome the drawbacks of Pt(II) based drugs, Pt(IV) complexes
were designed as prodrugs with the assumption that their high kinetic
inertness will minimize undesirable side reactions in the blood but
once inside the cancer cell they will be activated by reductive elimination, releasing the cytotoxic square-planar Pt(II) drugs. There were
reports that Pt(IV) complexes with either trifluoroaceate (TFA) [1] or
dichloroacetate (DCA) [2] axial ligands were potent anticancer
agents.
We recently reported that Pt(IV) complexes with either TFA or
DCA axial ligands can undergo rapid hydrolysis under physiological
conditions [3]. We now report on a systematic study on the hydrolysis
of Pt(IV) complexes with haloacetato ligands. Pt(IV) complexes with
axial TFA or DCA ligands can undergo hydrolysis and the rates of
hydrolysis increase with increasing pH consistent with the Sn1CB
mechanism. The half-lives for hydrolysis of the Pt(IV) complexes
with two TFA or DCA ligands at pH = 7 and 37 C range from
6–800 min which is short relative to the duration of cytotoxicity
studies that last 24–96 h. With two monochloroacetato (MCA) or
acetato axial ligands, hydrolysis is negligible. The rate of hydrolysis
depends primarily on the electron withdrawing strength of the axial
ligands but also upon the equatorial ligands.
Financial support of the Israel Science Foundation (grant to DG) is
gratefully acknowledged.
References
1. Khokhar AR, AlBaker S, Shamsuddin S, Siddik ZH (1997) J Med
Chem 40:112–116
2. Dhar S, Lippard SJ (2009) Proc Natl Acad Sci USA
106:22199–22204
3. Wexselblatt E, Yavin E, Gibson D (2013) Angew Chem Int Ed
52:6059–6062
123
S782
J Biol Inorg Chem (2014) 19 (Suppl 2):S779–S814
P 27
Photo-targeted platinum conjugates as selective
anticancer drugs
Marta Fernández1, Evyenia Shaili2, Alejandro González-Cantó1,
Gerard Artigas1, Carlos Sánchez1, Julie A. Woods3, Peter J.
Sadler2, Vicente Marchán1
1
Department of Organic Chemistry, University of Barcelona, Martı́ i
Franquès 1-11, E-08028 Barcelona, Spain, [email protected];
2
Department of Chemistry, University of Warwick, Coventry, CV4
7AL, UK; 3Photobiology, Ninewells Hospital, Dundee, DD1 9SY,
UK
The use of light provides a unique mechanism for triggering drug
release from delivery systems or for activating drugs at a desired
time and place. In this context, photoactivated Pt(IV) pro-drugs are
very attractive compounds since they can be selectively activated by
visible light to become highly active species towards a range of
cancer cell lines, including cisplatin-resistant cancer cells [1].
Despite the potential of photoactivatable metallodrugs for cancer
treatment, the application of the so-called targeted strategies to these
complexes can be used to improve their pharmacological properties
such as aqueous solubility, cell uptake and tumour selectivity to
further optimize their activity and reduce the occurrence of
unwanted adverse reactions [2–4]. Here, we report the synthesis,
characterization and phototoxicity of new conjugates in which Pt(IV)
complexes are covalently bound to tumour-targeting vectors based
on peptides or glycosides, either non-modified or caged with photoactivatable protecting groups.
Pt
Pt(IV)
complex
vector
Financial support from the Ministerio de Economı́a y Competitividad (CTQ2010-21567-C02-01, and the project RNAREG, grant
CSD2009-00080, funded under the programme CONSOLIDER INGENIO 2010), the Generalitat de Catalunya (2009SGR-208), ERC
(grant n8247450), and EPSRC, and stimulating discussions with EU
COST Action CM1105 are acknowledged.
References
1. Farrer NJ, Woods JA, Salassa L, Zhao Y, Robinson KS, Clarkson
G, Mackay FS, Sadler PJ (2010) Angew Chem Int Ed 49:8905–8908
2. Barragán F, López-Senı́n P, Salassa L, Betanzos-Lara S, Habtemariam A, Moreno V, Sadler PJ, Marchán V (2011) J Am Chem Soc
133:14098–14108
3. Barragán F, Carrion-Salip D, Gómez-Pinto I, González-Cantó A,
Sadler PJ, de Llorens R, Moreno V, González C, Massaguer A,
Marchán V (2012) Bioconjugate Chem 23:1838–1855
4. Grau-Campistany A, Massaguer A, Carrion-Salip D, Barragán F,
Artigas G, López-Senı́n P, Moreno V, Marchán V (2013) Mol Pharmaceutics 10:1964–1976
123
P 28
Synthesis and characterization of bis-maleimidefunctionalized platinum(IV) complexes for tumortargeted drug delivery
Josef Mayr1, Verena Pichler1, Petra Heffeter2,5, Orsolya
Dömötör3, Éva A. Enyedy3, Gerrit Hermann4, Diana Groza2,
Gunda Köllensperger4, Markus Galanski1, Walter Berger2,5,
Bernhard K. Keppler1,5, and Christian R. Kowol1,5
1
Institute of Inorganic Chemistry, University of Vienna, Waehringer
Strasse 42, A-1090 Vienna, Austria;
2
Institute of Cancer Research, Medical University of Vienna,
Borschkegasse 8a, A-1090 Vienna, Austria;
3
Department of Inorganic and Analytical Chemistry, University
of Szeged, Dóm tér 7, H-6720 Szeged, Hungary;
4
Department of Analytical Chemistry, University of Vienna,
Waehringer Strasse 38, A-1090 Vienna, Austria;
5
Research Platform ‘‘Translational Cancer Therapy Research’’,
University of Vienna, Waehringer Strasse 42, A-1090 Vienna, Austria
Metal-based cancer therapy using platinum(II) drugs, like cisplatin, is
essential in clinical practice. However, therapy is accompanied by severe
side effects and ineffectiveness due to development of resistance.
Therefore, current research focus is to specifically target the tumor tissue
exploiting their unique properties [1]. A well-known strategy is albuminbinding, since albumin is able to accumulate in the tumor tissue due to the
enhanced permeability and retention (EPR) effect.
Based on the above-mentioned knowledge, an albumin-targeting,
bis-maleimide-functionalized platinum(IV) complex (1) was synthesized in this study. As a reference compound, an analogues
succinimide-containing platinum(IV) complex (2), which lacks the
thiol affinity, was prepared. The very fast and quantitative binding of
1 towards simple thiols, like cysteine, was proven by RP-HPLC
studies, whereas 2 showed no binding affinity. The binding to albumin
was evaluated in detail by different analytical methods. In addition,
SEC–ICP–MS measurements using fetal calf serum revealed a high
selectivity of 1 for serum albumin. Finally, 1 and 2 were studied
in vivo in a syngeneic murine CT-26 colon cancer model. Both
compounds showed potent anticancer activity, however with significant higher activity and even disease stabilization for 1.
Financial support by the ‘‘Fonds der Stadt Wien für innovative
interdisziplinäre Krebsforschung’’ and TÁMOP 4.2.4. A/2-11-12012-0001 is gratefully acknowledged.
Reference
1. Galanski M, Keppler BK (2011) In: Kratz F, Senter P, Steinhagen
H (eds) Drug delivery in oncology. Wiley-VCH Verlag GmbH
Weinheim Germany pp 1605–1629
J Biol Inorg Chem (2014) 19 (Suppl 2):S779–S814
P 29
The development of novel HSP70-1 inhibitor molecules
as labile ligands for Pt anticancer compounds
Aoife McKeon1, Maria Morgan2, James Platts3, Darren Griffith1
1
Centre for Synthesis and Chemical Biology, Department
of Pharmaceutical and Medicinal Chemistry, Royal College
of Surgeons in Ireland, Dublin 2, Ireland;
2
Molecular & Cellular Therapeutics, Royal College of Surgeons
in Ireland, Dublin 2, Ireland;
3
Theoretical and Computational Chemistry Group, Cardiff
University, Wales, United Kingdom
Colorectal cancer is a major cause of death and disease worldwide.
Current treatment options depend on the stage of the cancer but can
generally include surgery, radiotherapy and chemotherapy. Oxaliplatin, a platinum (Pt)-based compound for example plays a very
important and well documented role in treating colorectal cancer [1].
The cytotoxicity of Pt drugs is attributed to multiple mechanisms but
primarily their ability to enter cells, hydrolyse and covalently bind
DNA, causing the formation of DNA adducts. These events can lead
to DNA damage responses and ultimately programmed cell death,
apoptosis. The clinical efficacy of Pt drugs is limited however by
drawbacks, such as toxicity, but primarily by the high incidence of
chemoresistance (intrinsic or acquired) [2]. Since many colorectal
cancers are intrinsically resistant to platinum-based therapies there is
an urgent need to develop novel and innovative therapeutic strategies
for combating colorectal cancer. The HSP70 family of heat shock
proteins are highly conserved molecular chaperones whose expression
is increased by cells in response to a variety of cellular stresses.
HSP70 is overexpressed in colorectal cancer, amongst other cancers,
and is associated with cancer progression, chemotherapy resistance
and poor prognosis as it is thought to provide cancer cells with a
survival advantage [3]. HSP70 is therefore an exciting and legitimate
anti-cancer target. Consequently, we wish to develop novel platinum
HSP70 inhibitor drug candidates as potential alternative treatments
for colorectal cancer. A summary of a molecular modeling study
undertaken to develop novel HSP70 inhibitor molecules as labile
ligands for Pt anticancer compounds using Vina and PLANTS will be
described.
This research was supported by Science Foundation Ireland under
Grant No. [12/IP/1305].
References
1. Kasparkova J, Vojtiskova M, Natile G, Brabec V (2008) Chem Eur
J 14:1300
2. Piccart MJ, Lamb H, Vermorken JB (2001) Ann Oncol 12:1195
3. Murphy ME (2013) J Carcinog 34:1181
P 30
Synthesis, characterization and biological evaluation
of platinum(II) compounds with carboxy derivatives
of boldine
Joan Villena2, Patricio G. Reveco1, Franz A. Thomet1
1
Department of Chemistry, Universidad Técnica Federico Santa
Marı́a, Avenida España 1680, Valparaı́so, Chile;
2
Faculty of Medicine, Universidad de Valparaı́so, Avenida Hontaneda
2664, Valparaı́so, Chile
Cisplatin has been employed worldwide during the last three decades
for the treatment of different kinds of cancer. To reduce the secondary
side effects of this drug, an enormous effort in the design of new
drugs has been carried out. The second and third generation analogues, carboplatin and oxaliplatin are now also used worldwide.
Our research group has been focused on the synthesis of new
platinum drugs, employing derivatives of the natural product boldine
S783
(I), as ligands [1]. Boldine has a powerful antioxidant and cytoprotective activity which was isolated from the bark of the endemic
Chilean boldo tree (Peumus boldus Molina). Previous work showed a
modulating effect on apoptosis induction of ovarian and leukemia
tumour cells by some natural products (synergistic in the case of
quercetin or anethole), when they were co-administered either with
cisplatin or oxaliplatin [2,3]. It was also observed, that the addition of
quercetin during cisplatin therapy, reduced the risk of drug induced
nephrotoxicity [3]. We are trying to evaluate the effect of using the
natural product as a ligand of the platinum analogue.
In vitro cytotoxic assay revealed that compounds IV and V are as
potent as the commercial drug oxaliplatin toward three human tumour
cells (MCF-7, PC-3 and HT-29) and that their activities are three to
four times lower than the analogous commercial drug over the epithelial human colon cell (CCD-841, a non-tumour cell). On the other
hand, compounds II and III exhibit no measurable activities ([100 lM)
toward all cell lines studied. In order to establish if the coordination of
the ligands II and III influence the cytotoxic activity of IV and V on
tumour vs non-tumour cells, further studies were developed.
This work was supported by CONICYT (Initiation Project No.
11121254).
References
1. Thomet FA, Pinyol P, Villena J, Reveco PG (2012) Inorg Chim
Acta 384:255–259
2. Nessa MU, Beale P, Chan C, Yu JQ, Huq F (2011) Anticancer Res
31:3789–3798
3. Kosmider B, Osiecka R (2004) Drug Develop Res 63:200–211
P 31
Syntheses, characterization and cytotoxicity assays
of novel binuclear Cu(II)–Pt(II), and correlated
mononuclear copper(II) complexes with oxindolimine
ligands
Esther Escribano1, Tiago Araújo Matias1, Koiti Araki1, Carol
PortelaLuz2, Fábio Marques2, Ana M. da Costa Ferreira1
1
Departamento de Quı́mica Fundamental, Instituto de Quı́mica,
Universidade de São Paulo, São Paulo, SP, Brazil;
2
Centro de Medicina Nuclear, Faculdade de Medicina, Universidade
de São Paulo, São Paulo SP, Brazil
The design, syntheses and characterization of novel mononuclear
copper(II) (species 1 and 2) and correlated binuclear heterometallic
Cu(II)–Pt(II) complexes (species 3 and 4) acting as DNA-targeting
agents are described. In addition to their spectroscopic characterization (UV/Vis, IR, EPR, mass spectrometry), the corresponding
formal redox potentials in DMF were determined. Cyclic voltammetry measurements showed that in all cases, one-electron quasireversible waves were observed, and ascribed to the formation of
corresponding copper (I) species, and to oxindolimine ligand
reduction. The Cu(II/I) redox potentials were similar (-0.86 V vs.
NHE), except for one of the mononuclear species, which showed a
123
S784
J Biol Inorg Chem (2014) 19 (Suppl 2):S779–S814
more negative E1/2 value (-1.2 V). The ligand reduction occurred
around -1.0 V. Furthermore, all complexes showed characteristic
EPR spectral profile and parameters with g|| [ g\ suggesting an
axially distorted environment around the copper(II) center [1]. By
complementary fluorescence studies, it was shown that glutathione
cannot reduce any of these complexes, under our experimental conditions (room temperature, phosphate buffer 50 mM, pH 7.4). Finally,
the cytotoxicity of each of these complexes was tested, in comparison
with cisplatin (species 5), towards murine B16F10 melanoma cells,
exhibiting low IC50 values, in the lM range, after 24 h incubation at
25 C, as shown in Table below. The obtained results indicate that
those complexes can be promising alternative antitumor species.
IC50 values (lM)
Complexes
1
2
3
4
light irradiation setup with three different LED sources (455, 528 and
618 nm), a controlled atmosphere, and temperature control, was
designed and used for testing the phototoxicity of a series of prodrugs
and liposome-supported prodrugs. The concept, experimental pitfalls,
and in vitro proof-of-concept data in several human cancer cell lines
will be shown and discussed.
Financial support by the European Research Council is gratefully
acknowledged.
Reference
1. Bonnet S, Limburg B, Meeldijk J, Klein Gebbink RJM, Killian JA
(2011) J Am Chem Soc 133:252–261; Goldbach E, Rodriguez-Garcia
I, van Lenthe JH, Siegler MA, Bonnet S (2011) Chem Eur J
17:9924–9929
5
B16F10 cells
1.98 ± 0.18 2.72 ± 1.06 0.63 ± 0.25 1.91 ± 0.20 2.88 ± 0.45
(24 h incubation)
Financial support by FAPESP, NAP-Redoxoma, and CEPIDRedoxoma is gratefully acknowledged.
Reference
1. Sakaguchi U, Addison AW (1979) J Chem Soc Dalton Trans 600–608.
P 32
Biological evaluation of light-activatable rutheniumbased anticancer prodrugs
Bianka Siewert1, Vincent H.S. van Rixel1, Azadeh Bahreman1,
Samantha L.H. Higgins1, Michael Heger2, Sven Askes1, Sylvestre
Bonnet1
1
Leiden Institute of Chemistry, University of Leiden, Einsteinweg 55,
2333 CC Leiden, The Netherlands.
2
Academic Medical Center, University of Amsterdam, Meibergdreef
9, 1105 AZ Amsterdam, The Netherlands
Innovative ideas are necessary to change the status quo in anticancer
treatment and overcome current limitations e.g. in chemotherapy. One
of such promising ideas is the activation of metal-based anticancer
prodrugs, also called photo-activatable chemotherapy (PACT). In this
approach, a Trojan horse-like prodrug is administered to tumorous
tissues followed by light irradiation of the tumor, which generates an
active form of the prodrug that selectively kills the irradiated cancer
cells.
P 33
Mixed monodentate tetracarboxylato platinum(IV)
complexes featuring a symmetric or unsymmetric
coordination sphere
Doris Hoefer1, Hristo P. Varbanov1, Markus Galanski1,
Alexander Roller1, Michael A. Jakupec1, Bernhard K. Keppler1,2
1
Department of Inorganic Chemistry, University of Vienna,
Waehringer Strasse 42, 1090 Vienna, Austria;
2
Research Platform ‘‘Translational Cancer Therapy Research’’,
University of Vienna, Waehringer Str. 42, 1090 Vienna, Austria
The advantageous pharmacological and chemical properties of platinum(IV) prodrugs turn them into promising candidates for anticancer
chemotherapy. They are capable to overcome limitations of currently
used platinum(II) cytostatics, which are mainly related to their toxicity and drug resistance mechanisms. In this work, symmetric and
unsymmetric platinum(IV) complexes with monodentate carboxylato
ligands were developed. In comparison to commonly used bidentate
carboxylato ligands in the equatorial position, monodentate ligands
are expected to confer an increased reactivity to the platinum(II)
species after reduction. The synthesized complexes exhibit a general
coordination sphere of [Pt(en)(OCOR)2(OCOR’)(OCOR’’)], where
the carboxylato ligands are represented by acetato and succinic acid
monoester ligands. Platinum(II) complexes have been synthesized
according to [1] and symmetrically and unsymmetrically oxidized
with corresponding peroxides to obtain platinum(IV) complexes,
which were further carboxylated with noncyclic anhydrides. The
complexes were investigated by elemental analysis, ESI–MS, FT-IR
and multinuclear (1H, 13C, 15N, 195Pt) NMR spectroscopy as well as
by X-ray crystallography in some cases. In addition, cytotoxic
properties were evaluated by means of the MTT colorimetric assay in
human cancer cell lines.
H2
N
OCOR'
OCOR
Pt
N
H2
OCOR
OCOR''
R = CH3, C4H 7O2
R' = CH 3, C4H7O 2, C5H 9O 2, C8H 15O 2
R'' = CH3, C4H 7O2, C5H9O 2, C8H15O 2
In this presentation we will show how the cytotoxic [Ru(tpy)(bpy)(OH)2]2? complex can be photochemically released by irradiation
of a prodrug protected by natural sulfur-containing ligands [1]. A
123
Reference
1. Rochon FD, Gruia LM (2000) Inorg Chim Acta 306:193–204
J Biol Inorg Chem (2014) 19 (Suppl 2):S779–S814
P 34
Metal element changes by the effect of cisplatin
administration in rats
Klára Szentmihályi1, Zoltán May1, Gábor Szénási2, Csaba
Máthé3, Andor Sebestény4, Mihály Albert5, Anna Blázovics6
1
Institute of Materials and Environmental Chemistry, Research
Centre for Natural Sciences of the HAS, 1117 Budapest, Hungary,
[email protected];
2
Institute of Pathophysiology, Semmelweis University, 1089
Budapest, Hungary, [email protected];
3
Department of Pulmonology, Semmelweis University, 1125
Budapest, Hungary, [email protected];
4
Laboratory Animal Science Unit, Faculty of Veterinary Science,
Szent István University, 1078 Budapest, Hungary,
[email protected];
5
Vetmed Laboratory Ltd. 1143 Budapest, Hungary,
[email protected];
6
Department of Pharmacognosy, Semmelweis University, 1085
Budapest, Hungary, [email protected]
Several platinum complexes, such as cisplatin, oxaliplatin, carboplatin are used. Generally serious problem is the excretion of essential
metal elements from the body during the treatment, while hardly any
data are available on the distribution and metabolism of nonessential
elements. Our aim was to study the concentration of both essential
and nonessential elements during the treatment with cisplatin. Male
Wistar rats (n = 20, 175–190 g) were randomly divided into 2 groups
(n = 10/group). The control group received 1 % methyl cellulose at
10 mL/kg body weight, p.o. by gastric gavage twice daily, and cisplatin was injected i.p. at a single dose of 6.5 mg/kg body weight.
Inductively coupled plasma optical emission spectrometry (ICP-OES)
was used for determination of Al, As, B, Ba, Ca, Co, Cr, Cu, K, Li,
Mg, Mn, Mo, Na, Ni, P, Pb, Pt, S, Sb, Si, Sn, Sr, V and Zn concentrations in the plasma, liver and kidney at 14 days after treatment.
Total scavenger capacity and diene conjugate content were also
determined in the liver. Elevated free radical reactions were observed
in the liver of the cisplatin-treated group, although redox balance did
not changed significantly. The concentrations of essential elements
were decreased in the plasma, kidney and liver, except for Fe, which
was elevated in the liver. The concentrations of non-essential elements were also changed, but mainly accumulation could be observed
especially for Al, Pb and Pt. According to the results of the study,
besides the excretion of essential elements, and the toxic effect of Pt
due mainly to induction of free radicals, the side-effects of increased
levels of non-essential elements have to be taken into consideration
during treatment with cisplatin.
P 35
Diverse cytotoxic behaviour of cisplatin and oxaliplatinderived complexes involving kinetin moiety
Radka Křikavová1, Lucie Hanousková1, Ján Vančo1, Zdeněk
Dvořák2, Zdeněk Trávnı́ček1
1
Regional Centre of Advanced Technologies and Materials,
Department of Inorganic Chemistry, Palacký University, 17. listopadu
12, 771 46 Olomouc, Czech Republic;
2
Regional Centre of Advanced Technologies and Materials,
Department of Cell Biology and Genetics, Palacký University,
Šlechtitelů 11, 783 71 Olomouc, Czech Republic,
[email protected]
Platinum-containing therapeutics (e.g. cisplatin, oxaliplatin) have
successfully been applied in medicine, however, have also shown
undesirable side-effects and their use is connected with resistance of
cancer cells [1]. The ongoing research in this field has proven that
simple modification in the structure of platinum-based drugs does not
S785
guarantee desirable activity of the resulting compounds even in the
first panel of testing, in vitro. For significant cytotoxicity, a suitable
combination of the leaving and carrier ligands in the studied molecule
is crucial.
In this work, N-donor carrier ligands (Ln) in the prepared dichlorido and oxalato complexes cis-[PtCl2(Ln)2] and [Pt(Ln)2(ox)]
were based on a plant hormone kinetin (N6-furfuryladenine), which is
non-toxic to human cells. Moreover, kinetin has been shown to be a
beneficial compound for cells, as it has been applied in medicinal
cosmetics ameliorating the signs of aging [2]. The herein reported
complexes were fully characterized and screened for their in vitro
cytotoxic activity, which identified only the dichlorido complexes as
cytotoxic. In order to address the contrasting behaviour of the two
groups of complexes, studies of hydrolysis and interactions with
sulphur-containing biomolecules were performed. The dichlorido
complexes were further tested on a panel of human cancer cells.
Notably, the results showed that the complexes are able to circumvent
cisplatin resistance in A2780cisR (IC50 & 3 lM), while also being
more cytotoxic against A2780 and HOS than cisplatin, and comparably active against MCF7 and G-361 human cancer cells.
Financial support is gratefully acknowledged (CZ.1.05/2.1.00/
03.0058; PrF_2014_009).
References
1. Alessio E (ed.) (2011) Bioinorganic Medicinal Chemistry Wiley
Weinheim Germany
2. Rattan SIS, Clark BFC (1994) Biochem Biophys Res Commun
201:665–672
P 36
Synthesis, purification, and characterization
of asymmetric Pt(IV) complexes that inhibit the Stat3
signal transduction pathway
James D. Hoeschele1, Emanuele Petruzzella1, Cristian Chirosca1
1
Chemistry Department, Eastern Michigan University, Ypsilanti, MI
48197, USA, [email protected]
The asymmetric Pt(IV) complex, fac-[PtCl3(NH3)2(NO2)] (CPA-7,
Figure 1), has been shown to control tumor cell growth by inhibiting the
Stat3 signal transduction pathway [1]. Although an improved synthesis
of CPA-7 was reported by Littlefield et al. [2], uncertainty as to the exact
nature of the reaction product(s) and the lack of the essential details of its
purification and purity warrant a reinvestigation of this system.
We present here (1) an optimized method of synthesis and purification
of this complex, (2) its complete characterization by spectroscopic (1H,
15
N & 195Pt-NMR & UV–Vis) and other analytical techniques (ESI–MS,
CV) and (3) the results of photo-stability studies in various media.
Additionally, we present a status report on the synthesis and
characterization of CPA-7 analogs of the general formula, fac[PtCl3A2(NO2)], wherein A2 represents two monodentate or one
bidentate primary alkyl and aromatic amine by replacing the NH3
groups in CPA-7. It is anticipated that the synthesis of these analogs
will lead to generally enhanced aqueous solubility, potentially
123
S786
J Biol Inorg Chem (2014) 19 (Suppl 2):S779–S814
allowing the Pt(IV) analogs of essentially insoluble Pt(II) precursors
to be evaluated as Stat3 inhibitors/antitumor agents.
R
Cl
N
R
NH3
N
Cl
N
X
R
N Pt
X
N
N
N
Cl
1
8
N
N
[Ru(bpy)3]2+
N
[Ru(bpy)3]2+
R
330
[Ru(R2bpy)2( -dpp)PtX2]2+
(b)
8
N
Ru
N
1
2+
R
N
N
R
Pt
NH3
R
N
Ru
N
(a)
2+
R
[Ru(R2bpy)2(dpp)]2+
R = H, X = Cl- (1) R = tert-Bu, X = Cl- (4)
R = H, X = Br- (2) R = tert-Bu, X = Br- (5)
R = H, X = I- (3) R = tert-Bu, X = I- (6)
R = H (7)
R = tert-Bu (8)
333
Magnetic field / mT
336 330
333
336
Magnetic field / mT
ESR spectra of DMPO (a) and TEMPOH (b)
irradiated with xenon lamp for 1 h.
NO2
References
1. Assi HH, Paran C, Vanderveen N, Savakus J, Doherty R, Petruzzella E, Hoeschele JD, Appelman H, Raptis L, Mikkelsen T,
Lowenstein PR, Castro MG (2014) J Pharm Exp Ther DOI:
10.1124/jpet.114.214619
2. Littlefield SL, Baird MC, Anagnostopoulou A, Raptis L (2008)
Inorg Chem 47:2798–2804
P 37
Antitumor activity of heterodinuclear ruthenium(II)–
platinum(II) complexes as photochemotherapeutic
agents
Takakazu Yano, Misaki Nakai, Yasuo Nakabayashi
Department of Chemistry and Materials Engineering, Kansai
University, 3-3-35 Yamate-cho, Suita, Osaka 546-8680, Japan,
[email protected]
Cisplatin (cis-[PtCl2(NH3)2]) has been one of the leading anticancer
drug for near 30 years. However, cisplatin has several drawbacks
such as toxicity and drug resistance. Ru(II)–polypyridine complexes
were proposed as potential antitumor substances with non-covalent
interactions and available for photodynamic therapy (PDT). In this
study, we have synthesized heterodinuclear Ru(II)–Pt(II) (1–6) and
mononuclear Ru(II) (7 and 8) complexes, and evaluated DNA
photocleavage ability. The interactions of these complexes with DNA
have been investigated by spectroscopic (UV–Vis, fluorescence, ESR)
and agarose gel electrophoretic methods. In addition, the cytotoxicity
of 1–8 were also determined using the MTT assay in Hela cell lines.
All the complexes can photocleave pBR322 DNA with visible light
radiation (xenon lamp, 300 W) through both •OH and 1O2 (1–6) and
1
O2 (7, 8) generation mechanisms [1]. The DNA photocleavage
ability of 1–6 is higher than that of 7 and 8. Furthermore, in the series
of 1–6 DNA photocleavage ability of 1–3 (R = H) is higher than that
of 4–6 (R = tert-Bu). 1 and 4 (X = Cl-) can bind covalently to DNA
through the dissociation of Cl- in low Cl- concentration (0–15 mM).
On the other hand, 2, 3, 5 and 6 interact with DNA by non-covalent
mode. 1–6 exhibit higher cytotoxicity compared to 7 and 8. Moreover,
4–6 are found to be more cytotoxic than 1–3, that is, 4–6 may be
expected to be applied to antitumor drugs that reduce the drawbacks
and increase the effects.
123
Reference
1. Gao F, Chao H, Zhou F, Chen X, Wei Y-F, Ji L-N (2008) J Inorg
Biochem 102:1050–1059
P 38
Cytotoxicity of palladium(II) complexes with 1,10phenanthroline derivatives and dithiocarbamate
as DNA intercalators
Masashige Fujii1, Hideaki Furusawa1, Kana Kondo1, Naho
Iizuka1, Misaki Nakai1, Takaji Sato2, Yoshiki Mino2, Yasuo
Nakabayashi1
1
Department of Chemistry and Materials Engineering, Kansai
University, 3-3-35, Yamate-cho, Suita, Osaka 564-8680.
2
Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara,
Takatsuki, Osaka 569-1094, Japan, [email protected]
Cisplatin (cis-[PtCl2(NH3)2]; cisPt) has been one of the leading
antitumor drug. However, dose-related nephrotoxicity and a variety
of toxic side effects are frequently observed with this drug. In
addition, some cancers have been reported to obtain resistance by
continuously administering cisPt (cisPt-resistant cancers). Therefore, much attention has been focused on the development of new
platinum complexes with decrease of the toxic side effects and
effectiveness for cisPt-resistant cancers. In this study, Pd(II) complexes with 1,10-phenanthroline derivatives (R-phen) and
dimethyldithiocarbamate (dmdt), [Pd(dmdt)(R-phen)]Cl, were synthesized and the interactions of these complexes with CT-DNA
investigated using competitive ethidium bromide (EtBr) studies. On
adding the complexes to CT-DNA pretreated with EtBr the fluorescence intensity of CT-DNA-bound EtBr decreased. The apparent
DNA binding constants (Kapp) estimated from relevant fluorescence
quenching data were Pd-1 [ Pd-2 [ Pd-3 [ Pd-4. Next, the cytotoxic activities of these complexes were determined by MTT assay
(IC50). Moreover, the partition coefficients of these complexes (log
Po/w) were also obtained by 1-octanol/water system. It is estimated
that the difference of IC50 values are attributed to the hydrophobicity
(log Po/w). In summary, it is concluded that the cytotoxic activities
of these complexes are proportional to the ability of intercalation
and hydrophobicity. It is particularly noteworthy that the cytotoxic
activity of Pd-2 was 16 times higher than that of cisPt for cisPtsensitive L1210 and 110 times higher than that of cisPt for cisPtresistant L1210.
J Biol Inorg Chem (2014) 19 (Suppl 2):S779–S814
R5
R4
R6
᧧
N
Pd
N
R2
-1
Complex 10 K app / M
log P o/w
S
N
R3
-6
Cl-
S
R1
R1 = R᧮ = R᧯ = R᧰ = R᧱ = R᧲ = H (Pd-1)
R1 = R6 = CH3 R2 = R3 = R4 = R5 = H (Pd-2)
R2 = R5 = CH3 R1 = R3 = R4 = R6 = H
(Pd-3)
R3 = R4 = CH3 R1 = R2 = R5 = R6 = H (Pd-4)
S787
IC50 / µM
L1210(0)a) L1210(cis Pt)b)
RFc)
Pd-1
2.8
-0.5
0.30 ± 0.05
0.28 ± 0.06
0.93
Pd-2
2.4
0.83
0.37 ± 0.02
0.17 ± 0.03
0.49
Pd-3
1.7
-0.27
2.0 ± 0.2
3.0 ± 0.4
1.5
Pd-4
0.93
-0.18
12 ± 1
15 ± 1
1.2
Pt-1d)
2.7
-0.78
0.53 ± 0.03
0.29 ± 0.13
0.55
cis Pte)
᧩
᧩
4.8 ± 0.3
19 ± 1
4.00
a) cisPt-sensitive L1210. b) cisPt-resistant L1210.
c) RF is defined as the relative ratio of IC50 values.
d) [Pt(dmdt)(phen)]+
e) Komeda S, et al. (2002) J Am Chem Soc 124:4738-4746
P 39
Photoactivatable ruthenium complex for cancer
therapy
Vanessa Pierroz1,2, Tanmaya Joshi1, Cristina Mari1, Lea
Gemperle1, Stefano Ferrari2, Gilles Gasser1
1
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland;
2
Institute of Molecular Cancer Research, University of Zurich,
Winterthurerstrasse 190, 8057 Zurich, Switzerland
The chemotherapeutical success of platinum-based anticancer agents
has driven the exploration of other transition metal complexes as new
metallodrugs displaying fewer side- effects. The best examples of
these are ruthenium-based compounds. Our work aims at developing
a robust mechanism for controlling the anticancer action from a
substitutionally-inert polypyridyl Ru(II) complex with very high
spatio-temporal resolution with light irradiation.
This bis(dppz)–Ru(II) complex showed cytotoxicity comparable to
that of cisplatin, targeted mitochondria, and impaired the mitochondrial membrane potential, leading to apoptosis [1]. Detailed structure–
activity relationship analyses on the active Ru(II) complex unraveled
the crucial role of the carboxylate group in the cytotoxic activity [2].
This fundamental study underpins the development of an efficient
substitutionally-inert metal complex-based prodrug candidate which
can selectively respond to activation by light, displaying a significant
increase in cytotoxicity against cervical and bone cancer cells upon
irradiation with UV-A light (2.58 J cm-2) [3].
Financial support by the Swiss National Science Foundation, the
University of Zurich (UZH), the Stiftung für Wissenschaftliche
Forschung of the UZH, the Stiftung zur Krebsbekämpfung, the
Huggenberger-Bischoff Stiftung and the UZH Priority Program is
gratefully acknowledged.
References
1. Pierroz V, Joshi T, Leonidova A, Mari C, Schur J, Ott I, Spiccia L,
Ferrari S, Gasser G (2012) J Am Chem Soc 134:20376–20387
2. Joshi T, Pierroz V, Ferrari S, Gasser G (2014) ChemMedChem.
doi:10.1002/cmdc.201400029
3. Joshi T, Pierroz V, Mari C, Gemperle L, Ferrari S, Gasser G (2014)
Angew Chem Int Ed. doi:10.1002/anie.201309576
P 40
Ru(II) polypyridyl complexes showing their potential
as novel anticancer PDT agents
Cristina Mari1, Vanessa Pierroz1,2, Riccardo Rubbiani1, Malay
Patra1, Stefano Ferrari2, Gilles Gasser1
1
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland, [email protected]; 2Insitute
of Molecular Cancer Research, University of Zurich, 8057 Zurich,
Switzerland
The treatment of cancer cells with a spatial and temporal control is
one of the most appealing feature of photodynamic therapy (PDT).
This innovative medical technique exploits the synergistic action of
light, oxygen and a non-toxic photosensitizer (PS). A lot of interest
and research has grown recently around this approach, since sideeffects due to the general toxicity of the drugs are minimized. On
the other hand, the PSs on the market displayed some other
drawbacks, as the prolonged light sensitivity induced in the patient.
Here we present an in-depth study in the photochemical and photobiological behaviour of six novel Ru(II) polypyridyl complexes
with strong DNA binding affinity. 1 and 2 showed the best phototoxic effect upon irradiation at 420 nm, with a dark/light toxicity
ratio of 150 and 40 respectively. The two complexes exhibited high
cellular uptake, together with an outstanding nuclear accumulation
(as confirmed by microscopy and AAS studies). Furthermore, the
ability to photocleave DNA at concentrations comparable with the
IC50 values upon irradiation and the very efficient binding to DNA
via intercalation (Kb * 10-6–10-7 M-1) suggested the involvement of DNA in the mechanism of phototoxic action of these
compounds.
References
1. Dolmans D et al. (2003) Nature Rev Cancer 3:380–387
2. Mari C et al. (2014) Chem Eur J (submitted)
P 41
Synthesis and fluorescent properties of arene–
ruthenium(II) complexes
Elzbieta Budzisz1, Adam Pastuszko1, Bogumila Kupcewicz2
1
Department of Cosmetic Raw Materials Chemistry, Medical
University of Lodz, Muszynski Str. 1 90419 Lodz, Poland,
[email protected];
2
Department of Inorganic and Analytical Chemistry, Faculty
of Pharmacy, Collegium Medicum in Bydgoszcz, Nicholaus
Copernicus University in Torun, M. Sklodowskiej-Curie 9, 85-094
Bydgoszcz, Poland
In this study we designed and synthesized a series of novel halfsandwich organoruthenium(II) complexes with the general formula
[(g6-arene)RuLCl2] (where L = aminoflavone or amino-methylchrbenzene,
omone
derivatives
and
g6-arene = p-cymene,
hexamethylbenzene or mesitylene). The complexes were fully characterized by elemental analysis, MS, UV–Vis, IR and NMR
spectroscopy.
123
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J Biol Inorg Chem (2014) 19 (Suppl 2):S779–S814
References
1. Giannini F, Furrer J, Ibao AF, Süss-Fink G, Therrien B, Zava O,
Baquie M, Dyson PJ, Štěpnička P (2012) J Biol Inorg Chem
17:951–960
2. Giannini F, Furrer J, Süss-Fink G, Clavel CM, Dyson PJ (2013) J
Organomet Chem 744:41–48
3. Giannini F, Paul LEH, Furrer J, Therrien B, Süss-Fink G (2013)
New J Chem 37:3503–3511
The fluorescent properties of ligands and their complexes were
examined in series of solvents such as: nonpolar (chloroform), polar
aprotic (acetonitrile, DMSO, DMF) and polar protic (ethanol, methanol, glycerol and water). The behavior of complexes in solvents with
different polarity and viscosity was investigated in details by total
fluorescence spectroscopy (excitation-emission contour maps and 3-D
spectra) as well as by synchronous fluorescence spectroscopy SFS (at
different wavelength intervals). Ligands exhibit blue fluorescence,
whereas for their complexes with organoruthenium(II) blue to green
fluorescence was observed with Stokes’ shifts in range 40–150 nm.
The fluorescence excitation and emission spectra demonstrated significant solvatochromism of all the compounds. Additionally for some
complexes excitation-wavelength dependent emission was observed.
To describe the effect of polarity of solvents the Lippert-Mataga plot
(Stokes’ shift vs. polarizability of the solvent) was used.
Financial support from Medical University of Lodz (grant No
503/3-066-02/503-01 to E. Budzisz), grants No 502-03/3-066-02/50234-025 and 503/3-066-02/503-06-300 to A. Pastuszko.
P 42
Tuning the in vitro cell cytotoxicity of dinuclear arene
ruthenium trithiolato complexes: influence of the arene
ligand
Federico Giannini1, Lennart Geiser1, Lydia E. H. Paul1, Thomas
Roder1, Georg Süss-Fink2, Julien Furrer1
1
Department of Chemistry and Biochemistry, University of Berne,
Freiestrasse 3, 3012 Berne, Switzerland;
2
Institute of Chemistry, University of Neuchâtel, Avenue de
Bellevaux 51, 2000 Neuchâtel, Switzerland,
[email protected]
We recently synthesized thiophenolato-bridged p-cymene ruthenium
complexes of the type [(g6-p-MeC6H4Pri)2Ru2(SR)3]?, which are
highly cytotoxic against human ovarian cancer cells, the IC50 values
being in the nanomolar range [1–3]. Their exact mechanism of action
is still unclear, although the cytotoxicity of these complexes could be
to a certain extent correlated to the lipophilicity of the corresponding
thiophenol ligands [2–3].
In this contribution, the influence of the arene on the in vitro
cytotoxicity is investigated. For this purpose, a new series of eight
thiolato-bridged arene ruthenium complexes bearing 4-methylthiophenolato or 4-methylcoumarin-7-mercapto bridges and biphenyl,
5,8,9,10-tetrahydroanthracene, indane, and 1,2,3,4-tetrahydronaphtalene as arene ligands have been synthesized and studied for their
in vitro cytotoxicity.
123
P 43
Noncovalent DNA binding and nuclease activity
of mixed-ligand ruthenium(II) complexes
Naho Iizuka, Misaki Nakai, Yasuo Nakabayashi
Department of Chemistry and Materials Engineering, Kansai
University, 3-3-35 Yamate-cho, Suita, Osaka 564-8680, Japan,
[email protected]
Metal complexes are ideal templates for the design of DNA-interactive systems. In addition to a variety of binding modes, metal
complexes that reversibly bind to DNA are becoming of increasing
interest. In this study, to design novel anticancer drugs, two type
ruthenium(II) complexes cis-[Ru(bpy)2ClL]? and cis-[Ru(bpy)2L2]2?
(bpy = 2,20 -bipyridine, L = 1,6-diaminohexane (1,6-dahx), 1-aminohexane (1-ahx)) were synthesized, and the interactions of these
complexes with DNA were experimentally explored. The ligand 1,6dahx possesses the potential to form hydrogen-bonding with suitable
DNA functionalities, which would be expected to enhance the DNA
binding affinity significantly. Moreover, the hydrogen-bonding of 1,6dahx with the intrastrand nucleobases may exhibit high levels of DNA
sequence-specific recognition. The emission spectra have been used
to probe the interaction of the present ruthenium(II) complexes with
calf thymus DNA (CT-DNA) and artificial DNA ([poly(dG-dC)]2
(GC), [poly(dA-dT)]2 (AT)). The emission intensity decreased on
addition of GC to 1 and 2, indicating that 1 and 2 covalently bind at
guanine residues. In contrast, the enhancement of emission intensity
was observed on addition of AT to 1 and 2. Hence, both 1 and 2
possess no specificity for base-pair binding. The emission intensity
increased by factors of 8.7 and 1.3 on addition of AT to 3 and 4,
respectively. In addition, the binding of 3 to AT led to a blue shift of
the emission maximum (Dk = 49 nm). These findings clearly indicate that 3 possesses the specificity for AT base-pair binding. In the
AT-rich regions of the minor groove, 3 is stabilized by hydrogenbonding to the N3 atoms of adenine and/or O2 atoms of thymine
residues. The emission intensity showed about 1.7 times enhancement
at a [CT-DNA]/[3] ration of 20:1, whereas the emission spectra of 4
were little affected by the addition of CT-DNA. The chemical
nuclease activity in the presence of H2O2 follows the order:
1 [ 2 & 3 [ 4. 1 and 3 possessed hydrogen-bonding ability show
more efficient cleavage activity.
J Biol Inorg Chem (2014) 19 (Suppl 2):S779–S814
P 44
Studies on anticancer properties of molecularly
targeted multifunctional ruthenium(II) and iridium(III)
complexes
Cai-Ping Tan, Rui-Rong Ye, Liang He, Zong-Wan Mao, LiangNian Ji
MOE Key Laboratory of Bioinorganic and Synthetic Chemistry,
School of Chemistry and Chemical Engineering, Sun Yat-sen
University, Guangzhou, [email protected]
Cisplatin is one of the most effective anticancer drugs in clinic.
However, the applications of platinum-based anticancer agents are
hindered by their intrinsic and acquired drug resistance, side effects,
and limited spectrum of activity. Recently, other transition metal
complexes, such as RuIII/II and IrIII complexes, show great potential as
alternatives to platinum-based drugs for anticancer therapy [1].
The major focus of our research is on the rational design of
molecularly targeted multifunctional metallo-anticancer agents. Most
of the traditional anticancer drugs exert their activities by causing
DNA damage or disturbances in mitotic apparatus. Molecularly targeted anticancer strategies offer tremendous hope for greater
anticancer activity, fewer side effects as well as personalized therapy
by inhibiting or activating cancer-specific biomolecules. On the other
hand, as compared with organic molecules, metal complexes are
endowed with many unique features, including photophysical, photochemical, redox and magnetic properties, which can be utilized to
construct multifunctional platforms for cancer therapy.
The molecular targets that we investigate mainly include histone
deacetylases (HDACs) [2], cyclin-dependent kinases (CDKs) [3–5]
and the mammalian target of rapamycin (mTOR). The structural–
activities relationships for inhibition of the targets by ruthenium(II)
and iridium(III) complexes are studied at the molecular level. Additionally, the information on the mechanism of cell death induced by
these complexes is investigated in detail [2–8].
References
1. Muhammad N, Guo Z (2014) Curr Opin Chem Biol 19:144–153
2. Ye RR, Ke ZF, Tan CP, He L, Ji LN, Mao ZW (2013) Chem Eur J
19:10160–10169
3. Tan CP, Hu S, Liu J, Ji LN (2011) Eur J Med Chem 46:1555–1563
4. He L, Liao SY, Tan CP, Ye RR, Xu YW, Zhao M, Ji LN, Mao ZW
(2013) Chem Eur J 19:12152–12160
5. He L, Liao SY, Tan CP, Lu YY, Xu CX, Ji LN, Mao ZW, (2014)
Chem Commun. doi:10.1039/C4CC01461H
6. Tan CP, Lai S, Wu S, Hu S, Zhou L, Chen Y, Wang M, Zhu Y,
Lian W, Peng W, Ji LN, Xu A (2010) J Med Chem 53:7613–7624
7. Tan CP, Wu S, Lai S, Wang M, Chen Y, Zhou L, Zhu Y, Lian W,
Peng W, Ji LN, Xu AL (2011) Dalton Trans 40:8611–8621
S789
8. Tan CP, Lu YY, Ji LN, Mao ZW (2014) Metallomics. doi:
10.1039/C3MT00225J
P 45
Oxicams as bioactive ligand systems in anticancer
RuII(p-cymene) complexes
Farhana Aman1,2, Muhammad Hanif1, Adnan Ashraf,2, Waseeq
Ahmad Siddiqi2, Stephen Jamieson3, Christian G. Hartinger1
1
School of Chemical Sciences, University of Auckland, Private Bag
92019, Auckland 1142, New Zealand;
2
Department of Chemistry, University of Sargodha, Sargodha-40100,
Pakistan;
3
Auckland Cancer Society Research Centre, University of Auckland,
Private Bag 92019, Auckland 1142, New Zealand,
[email protected]
Bioorganometallic chemistry provides an excellent platform to
incorporate drug-like properties in molecules. In recent years, metal
arene complexes have been extensively studies for the design of
metallodrugs. The most successful examples are RAPTA-C and
RM175 [1]. Despite small difference in their structures, both exhibited contrasting biological activity and modes of action. RM175
displayed in vitro anticancer activity similar to that of cisplatin; while
RAPTA-C inhibits metastasis in vivo. As evident from X-ray crystallographic experiments, RM175 prefers to bind to DNA while
RAPTA-C has high affinity to histone proteins of the nucleosome core
particle [2]. This demonstrates that a small structural variation can
dramatically alter the reactivity of compounds with cellular targets
and hence alter their biological activity. Coordination of a biologically active ligand to a metal center may result in enhanced activity due
to synergetic effects. The use of ligands with anti-inflammatory properties may improve the bioactivity of the Ru arene scaffold. As oxicams
are known for their anti-inflammatory properties, herein we report the
synthesis and characterization of RuII(p-cymene) complexes of oxicamderived ligands. Their hydrolytic stability, reactivity with biomolecules
and in vitro antiproliferative activity will be discussed.
Financial support by the University of Auckland, the Austrian
Science Fund (MH) and the Higher Education Commission of Pakistan (FA) is gratefully acknowledged.
References
1. Hartinger CG, Nolte MN, Dyson PJ (2012) Organometallics
31:5677–5685
2. Adhireksan Z, Davey GE, Campomanes P, Groessl M, Clavel CM,
Yu H, Nazarov AA, Yeo CHF, Ang WH, Dröge P, Rothlisberger U,
Dyson PJ, Davey CA (2014) Nat Commun 5. doi:
10.1038/ncomms4462
123
S790
J Biol Inorg Chem (2014) 19 (Suppl 2):S779–S814
P 46
Polynuclear Rh(III), Ir(III) and Ru(II) organometallic
complexes: synthesis and biological evaluation
as anticancer agents
P 47
A new complex of Ru(III) with N-(2pyridyl)salicylideneimine: DNA binding properties
and activity against Staphylococcus aureus
Andrew R. Burgoyne1, Gregory S. Smith1, Mervin Meyer2
1
Chemistry Department, University of Cape Town, South Africa;
2
Biotechnology Department, University of Western Cape, South
Africa
Cancer is one of the major diseases of the modern world. Traditional
chemotherapeutic approaches have experienced an increasing rise in
resistance, thus new strategies to target resistance are being explored.
The discovery of cisplatin by Rosenberg and the work by others have
pioneered medicinal chemistry and have paved the way in drug design
to offer compounds with a broad spectrum of biological activities.
Recently, particularly promising anticancer activities have been
shown by Rh(III), Ir(III) [1] and Ru(II) complexes [2], with polynuclear complexes showing increased potencies over analogous
mononuclear complexes. Recently, a series of polynuclear polyester
complexes, based on monodentate ligands, were synthesized in our
group that exhibited moderate anticancer activities, in ovarian cancer
cells [3].
This presentation reports on the preparation and spectroscopic
characterization of new monomeric and trimeric Rh(III), Ir(III) and
Ru(II) polyester complexes, Figure 1, based on bidentate ligands.
Several model mononuclear analogues have been prepared and their
structures determined by single crystal X-ray diffraction analysis. The
in vitro cytotoxicities of the ligands and complexes were established
on A2780 and A2780cisR, human ovarian carcinoma cells, and
human non-tumorous skin cells, KMST-6.
Adnan Zahirović1, Sabaheta Bektaš2, Ilda Graca1, Maida Puška1,
Emir Turkušić1, Emira Kahrović1
1
Department of Chemistry, Faculty of Science, Zmaja od Bosne
33-35, 71000 Sarajevo, Bosna and Herzegovina,
[email protected];
2
Institut for Public Health of Canton Sarajevo, 71000 Sarajevo,
Bosnia and Herzegovina
The design and study of new drugs is permanently huge challenge for at
least two reasons: (i) the cancer has not been defeated (ii) bacteria
strains rapidly develop resistance to existing drugs. Primarily due to the
ability of Ru(III) complex to bind DNA as one of key targets, new
complexes of ruthenium are subject of growing interest. We present
here the ability of a new complex, bis[N-(2-pyridyl)salicylideneimineONN]ruthenium(III) chloride, to bind CT DNA and in vitro activity
against Staphylococcus Aureus (MRSA). The complex was characterized based on mass spectrometry, infrared and electronic spectra, cyclic
voltammetry. Spectrophotometric titration of Ru(III) complex with
increasing concentration of CT DNA at 287 nm showed 3 nm red shift
and decrease of absorption indicating an intercalative mode of binding
(Kb = 3.61 9 104 M-1) as the most probable. Titration of DNA with
increasing concentration of Ru(III) compound at 260 nm resulted in
close value of constant binding (Kb = 3.90 9 104 M-1). Ru(III)
compound has shown significant activity against community-acquired
methicillin-resistant Staphylococcus aureus (CA-MRSA), hospital
acquired methicillin-resistant Staphylococcus aureus (HA-MRSA),
methicillin-sensitive Staphylococcus aureus (MSSA) reaching about
76 % of vancomycin activity, the reference antibiotic.
O
X = C, Y = H
X = N, Y = X
X = C, Y = OH
Y
N
M = Rh, Ar = Cp*
M
M = Ir, Ar = Cp*
Ar
Cl
M = Ru, Ar = p-Cy
O
Figure 1 Polyester complexes
References
1. Iavicoli I, Cufino V, Corbi M, Goracci M, Caredda E, Cittadini A,
Bergamaschi A, Sgambato A (2012) Toxicol Vitro 26:963–969
2. Scolaro C, Chaplin AB, Hartinger CG, Bergamo A, Cocchietto M,
Keppler BK, Sava G, Dyson PJ (2007) Dalton Trans 5065–5072
3. Chellan P, Land KM, Shokar A, Au A, An SH, Taylor D, Smith PJ,
Riedel T, Dyson PJ, Chibale K, Smith GS (2014) Dalton Trans
43:513–526
123
A
+
N
O
N
N
Ru
O
N
λ
Financial support by Federal Ministry of Education and Science,
Bosnia and Herzegovina, Project No.05-39-4390-1/13 is gratefully
acknowledged.
J Biol Inorg Chem (2014) 19 (Suppl 2):S779–S814
P 48
Synergistic effect of nitric oxide and singlet oxygen
originated from light irradiation on nitrosyl ruthenium
complex as potentiation for photodynamic therapy.
Kinetic, photobiological and cytotoxicity studies
Roberto S. da Silva, Ioyanne C. B. Ramos, Lilian Franco, Juliana
Uzuelli, Joicy Santamalvina
Pharmacy School, University of Sao Paulo, Av do Cafe s/n, Ribeirao
Preto, SP, Brazil, [email protected]
Nitric oxide (NO) is an important biological messenger. It has been
implicated in many physiological processes, including cardiovascular control, neuronal signaling, defense against microorganism and
tumors. It can trigger pro- and antitumor responses depending on the
concentration of this molecule (NO) in biological system. The
antitumor effect is pronounced when there are high levels of NO in
tumor cells, which make a challenge develop compounds that can
deliver NO under external stimulation. This work describes synthesis, kinetic, photochemical and photobiological studies of some
nitrosyl ruthenium complex type [Ru(phthalocyanine-R)NO]n? and
[RuL5NO]—antibody used as NO deliver system. Cell viability
decreased to 12 % for Ru–NO–IgG compound while aqueous
[RuL5NO]2? was found 85 %. It may be related to the NO release
in an appropriate target to kill cancer cell. Apoptosis was described
as the main biological mechanism for the nitrosyl ruthenium complex. It is also being performed in our laboratories a chemical
investigation on phthalocyanine ruthenium compounds as nitric
oxide releasers. One of this complex is [Ru(pc-R)NO(NO2)]
(I) (where pc-R = phthalocyanine-(COO)n) as putative system to
improve photodynamic therapy (PDT). Once phthalocyanine compounds are widely used in PDT due to their capacity of singlet
oxygen generation, cytotoxicity assays against cancer cell lines were
evaluated as well. The compound was able to inhibit cellular viability when irradiated at 660 nm, compared with the treatment
without photo stimulus. The cytotoxicity is depend on the charge o
the ruthenium complex. Similar studies was also conduced with
Quantum dot coupled to nitrosyl ruthenium (QD-Ru) system. The
generated QD-Ru was able to produce NO by photoinduced eletron
transfer as well singlet oxygen by energy transfer. The cell viability
were found between 10–25 % with 5 J/cm of potency in ligh irradiation. The cell death is mainly attributed to the apoptosis
mechanism. The initial studies suggested us the NO production
increases the sensitivity of the cells to singlet oxygen. The synergistic effect of NO and 1O2 may improve Photodynamic Therapy.
Acknowledgments: FAPES, CNPq, CAPES and photochem NAP.
P 49
Interaction of ruthenium(II) polypyridyl complexes
with some sulfur- and nitrogen-donor ligands
Ana Rilak1, Ralph Puchta2, Živadin D. Bugarčić1
1
Faculty of Science, University of Kragujevac, R. Domanovića 12, P.
O. Box 60, 34000 Kragujevac, Serbia, [email protected];
2
Institute for Inorganic Chemistry, Department of Chemistry
and Pharmacy, University of Erlangen-Nürnberg, Egerlandstrasse 1,
91058 Erlangen, Germany
In the last few decades, a large interest has grown in ruthenium
polypyridyl complexes as a possible alternative to the use of classical
platinum chemotherapy. We have recently developed a series of new,
water-soluble, monofunctional Ru(II) complexes with meridional
geometry of the general formula mer-[Ru(L3)(N–N)X][Y]n, (where
L3 = 2,20 :60 ,200 -terpyridine (tpy) or 40 -chloro-2,20 :60 ,200 -terpyridine
(Cl-tpy); N–N = 1,2-diaminoethane (en), 1,2-diaminocyclohexane
(dach) or 2,20 -bipyridine (bpy); X = Cl or dmso-S; Y = Cl, PF6 or
S791
CF3SO3; n = 1 or 2, depending on the nature of X) were synthesized.
With the aim of gaining insight into the possible interactions between
S-containing amino acids and N-containing heterocycles with Ru(II)
complexes, we have studied the ligand substitution reactions of two
Ru-tpy complexes, [Ru(Cl-tpy)(en)Cl]Cl (1) and [Ru(Cl-tpy)(dach)Cl]Cl (2) with S-containing ligands such as thiourea (Tu), Lcysteine (L-Cys) and L-methionine (L-Met), and with N-containing
ligands such as pyrazole (Pz), 1,2,4-triazole (Tz) and pyridine (Py).
The kinetics and thermodynamics of the investigated substitution
reactions were established quantitatively by UV–Vis spectrophotometry and NMR spectroscopy. The kinetic studies were supported
also by theoretical calculations.
Financial support by the Ministry of Education and Science of the
Republic of Serbia, project No. 172011 is gratefully acknowledged.
Reference
1. Rilak A, Bratsos I, Zangrando E, Kljun J, Turel I, Bugarčić ŽD,
Alessio E (2014) Inorg Chem (submitted)
P 50
Investigations on the cytotoxicity, cellular uptake
and thioredoxin reductase inhibition of gold(I) alkynyl
complexes
Vincent Andermark, Ingo Ott
Institute of Medicinal and Pharmaceutical Chemistry, Technische
Universität Braunschweig, Beethovenstraße 55, 38106 Braunschweig,
Germany
Besides the well-known use in catalysis, gold(I) complexes are also
known for their luminescent properties as wells as for their potential
as cytotoxic agents [1–3]. Recent work of our group has also shown
that compounds out of a series of gold(I) alkynyl phosphane complexes showed anti-angiogenic properties in zebrafish embryos and
inhibited thioredoxin reductase, a selenocysteine containing enzyme
that is crucial for cell proliferation, in the low-nanomolar range.
Additionally these complexes were cytotoxic against MCF-7 breast
cancer and HT-29 colon carcinoma cells making them a very promising substance class for tumor therapy [4].
However, a pitfall of this type of organometallics is its poor solubility in aqueous medium caused by the lipophilic
triphenylphosphane ligand (see figure). This work presents a new
series of novel gold(I) alkynyl phosphane complexes, for which we
used the most promising alkynyl ligand of the former work, and
modified the phosphane ligand to improve solubility.
After successful synthesis the novel gold(I) alkynyl complexes were
tested for their antiproliferative properties using HT-29 adenocarcinoma cells and MDA-MBA-231 breast cancer cells. Additional
experiments that were carried out with these compounds were the
determination of the cellular uptake into HT-29 colon carcinoma cells
using high-resolution continuum source AAS as well as the investigation of the inhibiting potential against isolated thioredoxin reductase.
O
Au P
lead-structure
Financial support by the COST Action CM1105 and Deutsche
Forschungsgemeinschaft (DFG) is gratefully acknowledged.
123
S792
References
1. Hashmi ASK (2007) Chem Rev 107:3180–3211
2. Arcáu J, Andermark V, Aguiló E, Gandioso A, Moro A, Cetina M,
Lima J C, Rissanen K, Ott I, Rodriguez L (2014) Dalton Trans
43:4426–4436
3. Ott I (2009) Coord Chem Rev 253:1670–1681
4. Meyer A, Bagowski C P, KokoschkaM, Stefanopoulou M, Alborzinia H, Can S, Vlecken D H, Sheldrick W S, Wölfl S, Ott I (2012)
Angew Chem Int Ed 51: 8895–8899
P 51
Fluorescent organometallic gold(I) N-heterocyclic
carbene complexes: synthesis and biological activities
Benoı̂t Bertrand1,2, Andreia de Almeida1, Evelien P. M. van der
Burgt1, Anna Citta3, Alessandra Folda3, Ewen Bodio2, Michel
Picquet2, Maria Pia Rigobello3, Pierre Le Gendre2, Angela
Casini1
1
Pharmacokinetics, Toxicology and Targeting, Research Institute
of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713
AV Groningen, The Netherlands;
2
Institut de Chimie Moléculaire de l’Université de Bourgogne, UMR
6302 CNRS Université de Bourgogne, Dijon, France ;
3
Department of Chemical Biology, University of Padua, Viale G.
Colombo 3, 35121 Padova, Italy
In modern times, gold complexes have been investigated for their
activity against tuberculosis, and several gold containing anti-arthritis
drugs have subsequently entered the market and remain in clinical use
today. Both gold(I) and gold(III) complexes have also appeared in the
last years as promising candidates for new possible anticancer agents
[1]. However, the identification of the actual biological targets for
those compounds, as well the determination of their distribution in
tissues, cells and subcellular compartments still remains a challenge.
Among the various strategies to achieve metal compounds imaging in
biological environments, fluorescence microscopy is certainly one of
the most explored, and an increasing number of publications has
appeared reporting on bifunctional metal compounds bearing fluorescent moieties for both therapeutic and imaging applications (so
called theranostic agents) [2,3]. Within this frame, we have synthetized new gold(I)–NHC complexes bearing a fluorescent coumarin
moiety, and characterized their photophysical properties. The compounds were tested as possible anticancer agent against several
human tumor cell lines and a model of healthy cells in vitro. Moreover, they were proved to be potent inhibitors of thioredoxin
reductase, a seleno-enzyme whose inhibition can lead to apoptosis via
mitochondria-related pathways [4]. Finally, we imaged the compounds’ uptake into cancer cells using fluorescence microscopy
techniques.
References
1. Bertrand B, Casini A (2014) Dalton Trans 43:4209–4219
2. Kelkar SS, Reineke TM (2011) Bioconjugate Chem 22:1879–1903
3. Tasan S, Zava O, Bertrand B, Bernhard C, et al (2013) Dalton Trans
42:6102–6109
123
J Biol Inorg Chem (2014) 19 (Suppl 2):S779–S814
4. Bindoli A, Rigobello MP, Scutari G, Gabbiani C, Casini A, Messori
L (2009) Coord Chem Rev 253:1692–1707
P 52
Synthesis and structural characterization of gold(III)
complexes with nitrogen-containing heterocycles
Miloš I. Djuran1, Beata War_zajtis2, Biljana Ð. Glišić1, Niko S.
Radulović3, Urszula Rychlewska2
1
Department of Chemistry, Faculty of Science, University
of Kragujevac, R. Domanovića 12, 34000 Kragujevac, Serbia;
2
Faculty of Chemistry, Adam Mickiewicz University, Grunwaldzka 6,
60-780 Poznań, Poland; 3Department of Chemistry, Faculty
of Science and Mathematics, University of Niš, Višegradska 33,
18000 Niš, Serbia
Aromatic nitrogen-containing heterocycles represent an important
class of ligands in coordination, supramolecular and bioinorganic
chemistry [1]. These ligands have been used for the synthesis of
different mononuclear and dinuclear gold(III) complexes, showing remarkable stability under physiological conditions and
relevant cytotoxic activity toward different human tumor cell
lines [2]. Considering the importance of gold(III) heterocyclic
complexes, we have recently investigated the reactions between
KAuCl4 and three diazine ligands (az), pyridazine, pyrimidine
and pyrazine [3]. It was showed that regardless of different
stoichiometric ratio of the reactants, reactions of [AuCl4]- with
the above mentioned ligands lead to the formation of mononuclear complexes of the general formula [AuCl3(az)], in which the
corresponding diazine acts as a monodentate ligand. This study
provided the first crystal structures of [AuCl3(az)] complexes,
allowed to establish the correspondence between basicity of the
diazine ligand and its ability to engage the uncoordinated nitrogen atom in intermolecular interactions, and demonstrated the
inherent helicity of the investigated molecules in crystals [3]. As
a continuation of our ongoing interest towards the coordination
chemistry of gold(III) with nitrogen-containing heterocyclic
ligands, herein we report the synthesis, NMR spectroscopic and
X-ray crystallographic characterization of two mononuclear
gold(III) complexes with monodentate coordinated heterocycles,
phenazine and quinoxaline, as well as spectroscopic characterization of the dinuclear gold(III) complex with bridging 4,40 bipyridine ligand. We found that the formation of mononuclear
gold(III) complexes with monodentate coordinated diazines and
phenazine or quinoxaline has resulted from the strong electronwithdrawing effect of Au(III) ion. However, this effect has not
been manifested in the reaction of Au(III) ion with 4,40 -bipyridine (2:1 molar ratio, respectively), which finally lead to the
dinuclear {[AuCl3]2(l-4,4-bipyridine)} complex. The structural
properties of gold(III) complexes with the above mentioned
nitrogen-containing heterocycles have been discussed in terms on
the nature of coordinated ligand.
This work was funded in part by the Ministry of Education, Science and Technological Development of the Republic of Serbia
(Project No. 172036).
References
1. Sumby CJ (2011) Coord Chem Rev 255:1937–1967
2. Ott I (2009) Coord Chem Rev 253:1670–1681
3. War_zajtis B, Glišić BÐ, Radulović NS, Rychlewska U, Djuran MI
(submitted)
J Biol Inorg Chem (2014) 19 (Suppl 2):S779–S814
S793
P 53
Toward the elaboration of new gold-based optical
theranostics for in vivo imaging
P 54
Vitamin B12-gold(III) conjugates for the selective
delivery of chemotherapeutics into tumor cells
Pierre-Emmanuel Doulain1, Semra Tasan1, Richard Decréau1,
Catherine Paul2, Pierre Le Gendre1, Franck Denat1, Christine
Goze1, Ewen Bodio1
1
Institut de Chimie Moléculaire de l’Université de Bourgogne, UMR
6302 CNRS, Université de Bourgogne, 9 avenue A. Savary, 21078
Dijon, France, [email protected];
2
EPHE Cancer Immunotherapy Laboratory; EA7269, University
of Burgundy, Dijon, F-21000, France
Since the pioneer discovery of cisplatin for biological applications
by Rosenberg in the 1960s, metal complexes have become the most
currently investigated and used class of compounds in cancer chemotherapy [1]. Gold-based derivatives gave very promising results
as anticancer agents [2]. One challenge is to understand their
mechanism of action to improve the efficiency and to limit the side
effects of such compounds. To deal with this issue, we have drawn
our inspiration from theranostics: we attached a fluorophore on
metal-based complexes to be able to track them in vitro. More
precisely, we recently developed three metal-containing BODIPYphosphine compounds based on Ru, Os and Au. This first series of
complexes showed promising results: interesting IC50 in several
cancer cell lines, especially for the Au derivative, and the possibility
to follow the compounds in vitro by optical imaging [3]. In the
present study, we decided to improve the gold(I) complex for it to be
suitable for in vivo studies in small animals. First, the conjugation
was extended on the BODIPY core, which enables the displacement
of the absorption and emission wavelength of the compound to the
near infrared region, and then we worked on the introduction of
small biovectors for targeting them selectively on several cancer cell
lines (Scheme 1). The synthesis and the photophysical studies of the
different targeted systems will be discussed. The biological studies
will then be presented and compared with the first described compounds.
Rebecca Pigot1, Marjorie Sonnay2, Roger Alberto2, Felix Zelder2,
Luca Ronconi1
1
School of Chemistry, National University of Ireland Galway,
University Road, Galway, Ireland, [email protected].;
2
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland
Some gold(III)–dithiocarbamato complexes have recently shown
promising antitumor activity, both in vitro and in vivo, together with
negligible systemic and organ toxicity [1], although selective tumor
targeting is still a major issue.
In order to maximize the impact on cancer cells and minimize
side-effects, our latest approach focuses on complexes with tumor
targeting properties provided by the coordination of biologicallyactive ligands, such as vitamin B12 (cyanocobalamin). Vitamin B12 is
an essential nutrient with very low availability. Therefore, rapidly
dividing tumor cells, requiring higher amounts of nutrients and energy
for cell proliferation, show increased demand of vitamin B12 compared to healthy ones [2]. Such avidity of cyanocobalamin can, thus,
be exploited for the site-specific delivery of drugs into the tumor by
binding vitamin B12 (carrier) to an anticancer agent (chemotherapeutics) [3].
We here report on the conjugation of gold(III)–dithiocarbamato
derivatives to the 50 -ribose of vitamin B12 [4] aimed at combining
the anticancer properties and favorable toxicological profile of the
gold analogues previously reported with an improved tumor selectivity provided by the conjugated cobalamin acting as delivery
carrier, so as to achieve biomolecular recognition and tumor targeting.
increased
demand
of vitamin B12 in
tumor cells (carrier)
chemoprotective function
(dithiocarbamate)
O
O
Br
S
C
B12
C
H2
N
CH3
labile sites
Au
C
S
Br
anticancer core (metal)
Financial support by the National University of Ireland Galway
(CoS Scholarship to R.B.) and the COST Action CM1105 (STSM
Grant to R.B.) is gratefully acknowledged.
References
1. (1965) Nature 205:698–699; (2003) Curr Opin Chem Biol
7:481–489
2. (2011) Curr Top Med Chem 11:2647–2660
3. (2013) Dalton Trans 42:6102–6109
References
1. Nagy EM, Ronconi L, Nardon C, Fregona F (2012) Mini Rev Med
Chem 12:1216–1229
2. Collins DA, Hogenkamp HPC, O’Connor MK, Naylor S, Benson
LM, Hardyman TJ, Thorson LM (2000) Mayo Clin Proc 75:568–580
3. Ruiz-Sánchez P, König C, Ferrari S, Alberto R (2011) J Biol Inorg
Chem 16:33–44
4. Zhou K, Zelder F (2010) Angew Chem Int Ed 49:5178–5180
123
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P 55
Synthesis and preliminary biological investigation
of novel NHC gold(I) complexes as potential anticancer
agents
Caroline M. Gallati, Matthias Plangger, Irene Würtenberger,
Ronald Gust
Department of Pharmaceutical Chemistry, CCB-University
of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria
Platinum complexes are widely used in cancer treatment. However,
the use of these drugs is limited by severe side effects and the
development of resistance during the therapy. Therefore, new metal
centers have been investigated to replace platinum. Among these
metals, gold has raised particular interest and several complexes of
gold(I) and (III) have been synthetized and tested against tumor cells
[1].
Of all the possible ligands for gold complexes, N-heterocyclic
carbenes (NHCs) are especially promising because of their extremely
strong bond to gold(I), leading to complexes with high stability [2,3].
Thus, a new NHC ligand based on an imidazole core bearing a
substituted phenyl ring and a methoxy-pyridine ring in positions 4 and
5, respectively, was designed. This carbene precursor is structurally
related to a compound proven to be a Janus kinase (JAK) inhibitor [4].
In several solid tumors and most hematopoietic tumors the JAK/STAT
pathway is overactivated, suggesting JAK inhibitors as potential
anticancer agents [5]. Therefore, including a ligand whose structure is
related to a JAK inhibitor could be expected to increase the anticancer
activity of the newly designed gold complexes. A versatile pathway to
synthesize the imidazolium ligands has been developed, allowing the
preparation of a series of derivatives with various substituents. The
corresponding gold(I) complexes were prepared and characterized.
Preliminary investigations of on cytotoxicity and cellular uptake
provided promising results.
References
1. Che C-M, Wai-Yin Sun R (2011) Chem Commun 47:9554–9560
2. Liu W, Gust R (2012) J Med Chem 55:3713–3724
3. Rubbiani R, Ott I (2010) J Med Chem 53:8608–8618
4. Thompson JE, et al (2002) Bioorg Med Chem Lett 12:1219–1223
5. Seavey M, Dobrzanski P (2012) Biochem Pharmacol
83:1136–1145
P 56
Titanocene-gold compounds inhibiting AKT
and MAPKAP kinases block renal cancer growth
in vitro and in vivo
Jacob Fernández-Gallardo1, Benelita T. Elie1, Florian
Sulzmaier2, Mercedes Sanaú3, Joe W. Ramos2, Maria Contel1,2
1
Department of Chemistry, Brooklyn College and The Graduate
Center, The City University of New York, Brooklyn, NY, 11210,
USA;
2
Cancer Biology Program, University of Hawaii Cancer Center,
University of Hawaii at Manoa, Honolulu, HI 96813, USA;
3
Departamento de Quı́mica Inorgánica, Universidad de Valencia,
Burjassot, Valencia, 46100, Spain
We and others have reported on the cytotoxic effects of gold(I)–
titanocene compounds (such as 1[1] and 2[2]) on ovarian [1,2] and
prostate [2] cancer cell lines. One of the main problems of these
complexes is the expected dissociation of the cyclopentadienyl groups
in physiological media. This implies that the heterometallic compound will break down into two monometallic components before
123
J Biol Inorg Chem (2014) 19 (Suppl 2):S779–S814
reaching the target tumor in vivo defeating the purpose of using a
single molecule with two different cytotoxic metals.
In order to overcome this problem we have used titanocene
derivatives containing gold(I) fragments that are not directly bound to
the Cp rings. These compounds have been significantly more effective
than monometallic titanocene and gold (I) analogues in renal cancer
cell lines indicating a synergistic effect of the resulting heterometallic
species. They were also markedly more active than cisplatin and titanocene Y. We will report on initial mechanistic studies in vitro
coupled with studies of their inhibitory properties on a panel of 34
kinases of oncological interest. We have found that the compounds
inhibit AKT and MAPKAP kinases with a high selectivity for
MAPKAP3 (IC50 = 91 nM). In vivo studies on mice with human
renal cancer xenografts indicate that the compounds can be excellent
candidates for further development as potential renal cancer chemotherapeutics.
Ph Ph
Cl
Cl
Ti
P Au Cl
Ph Ph
P
Au
P
Ph Ph
Ti
Cl
Cl
PF6
Ti
Cl
Cl
P Au Cl
Ph Ph
1
2
Financial support by the National Cancer Institute (US) grant
1SC1CA182844 (M.C.) and NIGMS (US) grant RO1GM088266-A1
(J.W.R.) is gratefully acknowledged.
References
1. Casini A, et al. (2011) Inorg Chem 50:9472–9480
2. Contel M, et al. (2011) Inorg Chem 50:11099–11110
P 57
A glimpse into molecular mechanism of phenolato
titanium(IV) anticancer complexes
Maya Miller1, Ori Braitbard2, Yoav Smith3, Jacob Hochman2,
Edit Y. Tshuva1
1
Institute of Chemistry, The Hebrew University of Jerusalem, 91904
Jerusalem, Israel;
2
Alexander Silberman Institute of Life Science, The Hebrew
University of Jerusalem, 91904 Jerusalem, Israel;
3
Genomic Data Analysis Unit, Hebrew University Medical School,
91220 Jerusalem, Israel
In the past decades titanium complexes have emerged as potential
anticancer drugs, with two derivatives reaching the clinical trials:
titanocene dichloride and budotitate. Due to instability in biological
environment, difficulties in identifying the active species and in
resolving the mechanism of action for these classes of complexes, a
new class of titanium compounds, based on aminophenolato ligands,
was developed. This group of complexes exhibits high cytotoxicity
towards various cancer cell lines, negligible toxicity towards primary
murine cells, high hydrolytic stability, and identified hydrolysis products that are both stable and biologically active.
This study aims to probe into the mechanism(s) of action of the
phenolato titanium(IV) complexes. Since these complexes exhibit C2
or C1 symmetry, rendering them chiral, the examination of isolated
enantiomers is essential both for medicinal use and for gaining
J Biol Inorg Chem (2014) 19 (Suppl 2):S779–S814
mechanistic insights. We found that for complexes based on bipyrrolidine chiral moiety, generally the racemic mixtures were inactive
whereas the pure enantiomers exhibited similarly high cytotoxic
activity. This observation supports the assumption on the involvement
of polynuclear hydrolysis product as the active species as well as the
premise that the biological target is chiral.
To further explore possible molecular mechanism(s) of action for
this class of compounds, gene chip array, western blotting, ELISA
and FACS methods were applied on leading salan complexes. Gene
chip array studies suggested involvement of the following pathways:
(1) Systemic Lupus Erythematosus; (2) Disruption to cell cycle; (3)
p53 signaling; (4) ECM receptor interaction. Studies of flow cytometry revealed growth arrest in G-1 of the cell cycle within 24 h.
ELISA and western blotting revealed increasing levels of apoptotic
related proteins, p53 and p21, throughout exposure for 48 h, as well
as the expression of caspase3, caspase9 and MDM2 proteins.
Collectively, the present findings are consistent with interaction of
phenolato Ti(IV) complexes with DNA and initiation of a DNA
damage response culminating in cell cycle arrest and apoptosis. This
pathway resembles those of chemotherapeutic drugs that exert their
effect through DNA interstrand crosslinking and double strand breaks.
This research was funded by the European Research Council under
the European Community’s Seventh Framework Programme (FP7/
2007-2013)/ERC Grant agreement (No. 239603)
Reference
Miller M, Tshuva EY (2014) Eur J Inorg Chem 9:1485–1491
P 58
Combination of anti-cancer salan TiIV complexes
with cisplatin: synergistic effects
Nitzan Ganot, Edit Y. Tshuva
The Institute of Chemistry, The Hebrew University of Jerusalem,
91904 Jerusalem, Israel
The significant drawbacks of cisplatin, namely its high toxicity and
development of drug-resistance, initiated an extensive search for other
metals that can lead to anti-cancer activity. TiIV complexes showed
promising cytotoxic activity, and two TiIV based complexes reached
clinical trials. Nevertheless, these complexes have failed clinical trials
due to instability in aqueous environment. Our group designed a new
family of anti-cancer TiIV complexes based on salan ligands, which
showed high cytotoxic activity toward numerous cancer cell lines and
enhanced stability in aqueous environment.
Combination therapy is a very common method in clinical treatment of cancer. By combining two drugs or more, the doses that are
required to reach the desired effect are reduced, and consequently
their side effects and toxicity are reduced as well.
Herein, combinations of salan TiIV complexes with cisplatin are
presented. Non-covalent combination of salan TiIV complexes and
cisplatin often showed a synergistic behavior, depending on the
substitution on the salan ligand, the ratio of the combined drugs, the
type of the treated cancer cell lines, and the schedule of administration. Additionally, attempts are made to covalently combine salan
TiIV complexes with cisplatin and with steroids. Such combined
complexes may enhance the cytotoxicity as well as the selectivity of
the complexes to particular cell types. Achievements in these directions will be discussed.
This research was funded by the European Research Council under
the European Community’s Seventh Framework Programme (FP7/
2007-2013)/ERC Grant agreement (No. 239603).
Reference
Ganot N, Redko B, Gellerman G, Tshuva EY (2014) (submitted)
S795
P 59
Highly active antitumor titanium(IV) phenolato
complexes: the influence of structural factors
on complex performance
Avia Tzubery, Edit Y. Tshuva
The Institute of Chemistry, The Hebrew University of Jerusalem,
91904 Jerusalem, Israel
The anticancer activity of the inorganic compound cisplatin and its
worldwide use in the pharmaceutical industry initiated a new field of
research, aimed at finding new inorganic complexes of other metals
that may lead to improved anticancer drugs. Among others, Ti(IV)
complexes titanocene dichloride and budotitane demonstrated high
cytotoxic activity but eventually failed clinical trials due to their low
hydrolytic stability. We previously introduced cytotoxic Ti(IV)
complexes based on salan ligands that demonstrated high activity
along with exceptional hydrolytic stability.
Herein we examine the influence of structural aspects on the
complex reactivity and stability. We will present the first transTi(IV) complexes of high cytotoxicity based on salen ligands
[1–2]. These complexes are highly active towards various cancer
cell lines, with improved hydrolytic stability relative to that of the
known Ti(IV) complexes. Structure–activity relationships will be
discussed. Additionally, we will present salalen-Ti(IV) complexes,
which are half salan-half salen, with a fac-mer binding mode that
gives a C1 symmetry. The effect of this geometry on the properties and performance of the Ti(IV) complexes will also be
addressed.
This research was funded by the European Research Council under
the European Community’s Seventh Framework Programme (FP7/
2007-2013)/ERC Grant agreement (No.239603).
References
1. Tzubery A, Tshuva EY (2011) Inorg Chem 50:7946–7948
2. Tzubery A, Tshuva EY (2012) Inorg Chem 51:1796–1804
P 60
Highly effective and hydrolytically stable
vanadium(V) phenolato antitumor agents:
development, analysis and mechanistic investigation
Lilia Reytman, Edit Y. Tshuva
Institute of Chemistry, The Hebrew University of Jerusalem, 91904
Jerusalem, Israel
In the field of anti-cancer chemotherapeutic research, numerous metal
compounds are being investigated worldwide in order to resolve the
limited activity range and severe toxicity of the antitumor drug cisplatin. One of the most promising metals studied today is vanadium.
Vanadium compounds were found to exert favorable properties for
use in therapy, but the complicated aquatic chemistry of vanadium
compounds impeded the potential of vanadium as an antitumor agent.
Therefore, the development of compounds with improved hydrolytic
stability is essential for therapeutic utilization. In previous work we
developed a new family of vanadium(V) complexes with tetradentate
diamino bis(phenolato) ‘‘salan’’ ligands with favorable cytotoxic
activity. Nevertheless, theses complexes exhibited mils water stability. Appreciable cytotoxic activity was measured for an isolated
hydrolytsis product that was found to exhibit a dimeric structure with
no labile ligands [1].
Herein we developed a family of oxo-vanadium(V) complexes
with pentadentate diamino tris(phenolato) ligands, which do not
contain any labile ligands, and therefore exhibit remarkable resistance towards hydrolysis. Importantly, these compounds display
exceptional cytotoxic activity, higher than that of cisplatin by up to
123
S796
two orders of magnitude, which is preserved during incubation in
DMSO for several weeks [2]. These properties, along with promising in vivo results for a representative complex, encourage further
development and investigation regarding the mechanism of action of
this family of complexes. Preliminary evidence of possible cellular
pathways of these complexes will be discussed, as well as structure–
activity studies concerning different substituents on the phenolato
rings.
This research was funded by the European Research Council under
the European Community’s Seventh Framework Programme (FP7/
2007-2013)/ERC Grant agreement (No. 239603).
References
1. Reytman L, Braitbard O, Tshuva EY (2012) Dalton Trans
41:5241–5247
2. Reytman L, Tshuva EY (in preparation)
P 61
Redox properties of iron sucrose
Leila Mahmoudi, Reinhard Kissner
Department of Chemistry and Applied Biosciences, ETH Zurich,
Vladimir-Prelog-Weg 2, 8093 Zurich, Switzerland,
[email protected]
Iron sucrose is a Fe(III) oxide hydroxide colloid stabilized by a layer
of sucrose molecules. Some preparations are used in the treatment of
iron deficiency anemia [1]. We applied cyclic voltammetry and
polarography to investigate their redox properties. The Fe(II) content
of iron sucrose is routinely determined by a polarographic method,
because it is thought to cause toxic effects [1] in therapeutic
applications.
In the potential range of 100–1600 mV vs. Ag/AgCl, two major
current waves were detected which increased and changed shape
with every cycle. The first broad wave can safely be assigned to the
reduction of iron(III) to iron(II). The second wave is conventionally
representing iron(II) to iron(0) reduction [2,3]. Because of the
2-electron transfer reaction, the integral of the second wave should
be strictly 2 times higher than the first. However, we found a variable ratio depending on exposition time of the electrode to the
solution. We conclude that the second wave is also mainly caused
by iron(III) to iron(II) reduction, namely of Fe(III) that is located
deep inside the particle, compared to the Fe(III) species at the
particle surface which should produce the first wave. The figures
below show the current evolution in time on a gold (left) and a
mercury (right) electrode.
J Biol Inorg Chem (2014) 19 (Suppl 2):S779–S814
P 62
Interaction of hybrid polyoxometalates with biological
targets
Mauro Carraro, Gloria Modugno, Valeria Zamolo, Marcella
Bonchio
Department of Chemical Sciences, University of Padova, Via
Marzolo, 1 35131 Padova, Italy, [email protected]
Polyoxometalates (POMs) are discrete and polyanionic metal-oxides
with potential applications in medicine. Due to their redox and
structural properties, indeed, they can affect electron transport within
the cells and form adducts with macromolecules, via electrostatic
interactions and hydrogen bonds. This behaviour has shown to be
useful to denature proteins and inhibit enzymes, leading to antiviral,
antitumoral and antibacterial activities [1].
In this field, POMs interaction with biological substrates seems to
be favoured, in terms of delivery, stability and biological activity, by
the presence of organic domains [2].
The presentation will thus describe:
(i) The synergistic antibacterial effect of POMs and chitosan,
forming hybrid nanoaggregates (see figure) [3];
(ii) Cell delivery and tracking of fluorescent hybrid POMs;
(iii) The recognition capabilities by a biotinylated POM, for which
spectroscopic and surface plasmon resonance techniques were used to
explain the nature of the interaction with avidin.
Financial support by the MIUR–FIRB prot. RBAP11ETKA is
gratefully acknowledged.
References
1. Hasenknopf B (2005) Front Biosci 10:275–287
2. Yang HK, Cheng YX, Su MM, Xiao Y, Hu MB, Wang W, Wang Q
(2013) Bioorg Med Chem Lett 23:1462–1466
3. Fiorani G, Saoncella O, Kaner P, Altinkaya SA, Figoli A, Bonchio
M, Carraro M (2014) J Clust Sci 25:839–854
P 63
EuroTracker dyes: very bright europium complexes
for live cell imaging
References
1. Toblli JE, Cao G, Oliveri L, Angerosa M (2009) Arzneimittelforschung 59:76–190
2. Lingane JJ (1946) J Am Chem Soc 68:2448–2453
3. Meites L (1951) J Am Chem Soc 73:3727–3731
123
James W. Walton, David Parker
Department of Chemistry, Durham University, South Road, Durham,
DH1 3LE, UK, [email protected]
Several exceptionally bright europium complexes have been prepared
that exhibit excellent cell uptake behaviour and distinctive localisation
profiles within various mammalian cell types. Each complex localises
in specific organelles within the cell, including the mitochondria,
lysosomes and the endoplasmic reticulum, and is visualised by fluorescence microscopy (Figure). The long luminescence lifetimes of
these complexes ([1 ms) allow for time-gated spectral imaging and
make them an attractive alternative to commercial fluorescent dyes.
J Biol Inorg Chem (2014) 19 (Suppl 2):S779–S814
The advantages of lanthanide complexes over traditional fluorescence dyes for live cell imaging include: a large Stokes’ shift
that minimizes self-quenching; an optical signal that conveys
information on the local environment through emission spectral
form and a long luminescence lifetime allowing time-gated
detection that overcomes problems with autofluorescence of
biomolecules.
The complexes reported have very high molar extinction coefficients (e = 55–60,000 M-1 cm-1) and quantum yields (u * 50 %),
resulting in exceptionally bright (e 9 u) compounds. They are nontoxic (IC50 [ 100 lM), show high levels of cellular uptake and
exhibit selective organelle staining depending on the nature of the
macrocyclic co-ordinating ligand.
In summary, this new class of ‘‘EuroTracker’’ dyes as cellular
stains offer several improvements over the classical fluorescence
organic dyes.
X
N
N
Eu
P
Me
O
N
N
Z
Y
O
Me
O
O
P
X
Z
N
N
R3O
Y
Z
O
Me
HYDROLYSES IN WATER
N
WATER-STABLE
N
N
O
N
O
N
N
N
O
N
N
N
N
N R
N
R R
O N
O Tc O
Tc
N
R N
THF, 25°C
N
N
Tc
N
THF, 25°C
29% (R = Me)
33% (R = Et)
O
O
55%
N
N
O
N
R R
References
1. Hock SJ, Schaper L-A, Herrmann WA, Kuhn FE (2013) Chem Soc
Rev 42:5073–5089
2. Braband H, Kückmann TI, Abram U (2005) J Organomet Chem
690:5421–5429
3. Benz M, Spingler B, Alberto R, Braband H (2013) J Am Chem Soc
135:17566–17572
P 67
Does length really matter? Structure–luminescence
relationships in EuIII and TbIII complexes
OR1
Z
S797
Y
P
Z
O
X
OR2
Z
References
1. Walton JW, Maury O, Parker D et al (2013) Chem. Commun
49:1600
2. Butler SJ, Lamarque L, Pal R, Parker D (2014) Chem Sci 5:1750
P 64
Synthesis of water stable {M(V)O2}1-NHC Complexes
(M 5 Re, 99Tc)
Michael Benz, Henrik Braband, Roger Alberto
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland, [email protected]
99m-Technetium (99mTc) is the main nuclide used for diagnosis in
nuclear medicine due to its almost ideal properties. Therefore, the
search for novel ligand systems suitable for application with 99mTc is
of great interest in this field of research. Recently, the scope of
N-heterocyclic carbenes (NHCs) has been extended from catalytic
application to the field of bioinorganic chemistry and metals in
medicine. In this context, the NHC chemistry of technetium came into
our research focus. However, 99Tc-NHC complexes are scarce [1].
While {Re(V)O2}? complexes, which contain monodentate NHCs, are
hydrolytically stable, the corresponding {Tc(V)O2}?-NHC complexes
show rapid hydrolysis in the presence of trace amounts of H2O [2].
We present novel synthetic pathways for the synthesis of water stable
{M(V)O2}?-NHC complexes [3]. The key link for these general
procedures is [M(V)O(glyc)2]- (M = Re, 99Tc; glyc = ethylene glycolato). The high water stability of the products allows conversion of
the {M(V)O2}? core into {M(V)OCl}2? with HCl as the H? and Clsource. The remarkable stability and pH-controllable reactivity of the
new complexes underline the potential of NHCs as stabilizing ligands
for 99Tc complexes and pave the way for the first 99mTc-NHC complexes in the future.
Lena J. Daumann, Elisabeth Fatila, David S. Tatum, Kenneth N.
Raymond
Department of Chemistry, University of California, Berkeley, CA
94720-1460, USA
Luminescent lanthanides have attracted attention for their unique
photophysical properties making them a vital tool for modern
medicinal applications such as diagnostic immunoassays for the
detection of cancer markers, blood processing and drug monitoring. In
the Raymond group, one current interest is the sensitization of the
luminescent visible emission from TbIII and EuIII. To achieve high
overall quantum yields, we utilize the 2-hydroxyisophthalamide
(IAM) or 1-hydroxypyridin-2-one (1,2-HOPO) chelate groups as
antennas, respectively [1] Herein we report a large group of octadentate and tetradentate ligands based on the 1,2-HOPO and IAM
moieties with various modifications to not only improve quantum
yields, but also increase water solubility and stability in aqueous
solution. An example of one set of modifications we have made, is
varying the tether length in the tetradentate 1,2-HOPO ligands. We
will point out geometric and electronic factors contributing to high
quantum yields in EuIII and TbIII complexes. Examples of the ligands
that have been prepared are shown in the figure below. Financial
support by the Alexander von Humboldt Foundation and a grant from
the Department of Energy are gratefully acknowledged.
R
O
R
H
H N
N
O
O
Eu
Tb
O
4
O
HN
IAM
O
O
H2 N
H2N
H2N
H2N
HO
N
H
N
n
4
O
O
1,2-HOPO
H2N
H2N
H2N
O
OH
H
N
N
N
NH2
NH2
H2 N
NH2
H2N
NH2
H2N
NH2
NH2
NH2
H2N
H2N
O
S
N
H
NH2
S
S
H 2N
NH2
NH2
NH2
NH2
S
H2N
S
NH2
NH2
S
H2 N
H2N
H
N
N
N
H
NH2
NH2
Reference
1. Moore EG, Samuel APS, Raymond KN (2009) Acc Chem Res
42:542–552
123
S798
P 68
DNA sensing by lanthanide-binding peptides
Christelle Gateau1,2, Alexandra Botz1,2, Laetitia Ancel1,2, Colette
Lebrun1,2, Pascale Delangle1,2
1
Univ. Grenoble Alpes, INAC, SCIB, RICC F-38000 Grenoble,
France;
2
CEA, INAC, SCIB, Laboratoire de Reconnaissance Ionique et
Chimie de Coordination F-38054 Grenoble, France
DNA is a fascinating biomolecule, which is storing and dispensing
genetic data required for life. Therefore, molecules that bind to DNA
are extremely useful as biochemical tools to detect DNA both in vitro
and inside the cell. Time-resolved luminescence of lanthanides is
particularly attractive for applications of these ions as luminescent
DNA probes, since it allows to eliminate the background natural
fluorescence. Lanthanides-binding peptides are especially promising
molecules for biological applications, since they provide high
hydrophilicity and solubility in water to the probe.
In the lab, we demonstrated that hexapeptides containing unnatural
amino acids bearing aminodiacetate side chains and a sequence Pro-Gly
give stable lanthanide-peptide complexes at physiological pH. Introduction of aromatic moieties, which act both as a lanthanide sensitizer
and DNA binding group allows the detection of double stranded DNA
thanks to the time-resolved luminescence of the lanthanide ion.
Here we presented the rational design, preparation and evaluation
of these new DNA sensing lanthanide binding peptides.
This research was supported by the ‘‘Région Rhone-Alpes’’ and
the Labex ARCANE (Grant ANR-11-LABX-0003-01).
References
1. Ancel L, Gateau C, Lebrun C, Delangles P (2013) Inorg Chem
52:552–554
2. Niedzwiecka A, Cisnetti F, Lebrun C, Gateau C, Delangle P (2012)
Dalton Trans 41:3239–3247
3. Cisnetti F, Gateau C, Lebrun C, Delangle P (2009) Chem Eur J
15:7456–7469
123
J Biol Inorg Chem (2014) 19 (Suppl 2):S779–S814
P 69
Structures and biotransformations of potent insulinenhancing VIVO complexes with picolinate derivatives
Tanja Koleša-Dobravc1, Elzbieta Lodyga-Chruscinska2, Marzena
Symonowicz2, Daniele Sanna3, Anton Meden1, Franc Perdih1,
Eugenio Garribba4
1
Faculty of Chemistry and Chemical Technology, University
of Ljubljana, Aškerčeva cesta 5, SI-1000 Ljubljana, Slovenia, and CO
EN–FIST, Dunajska cesta 156, SI-1000 Ljubljana, Slovenia,
[email protected];
2
Institute of General Food Chemistry, Technical University of Lodz,
ul. Stefanowskiego 4/10, Lodz, Poland;
3
Istituto CNR di Chimica Biomolecolare, Trav. La Crucca 3, I-07040
Sassari, Italy;
4
Dipartimento di Chimica e Farmacia, and Centro Interdisciplinare
per lo Sviluppo della Ricerca Biotecnologica e per lo Studio della
Biodiversità della Sardegna, Università di Sassari, Via Vienna 2,
I-07100 Sassari, Italy, [email protected]
The potential use in the therapy of type 2 diabetic patients is one of
the most important applications of vanadium in medicine [1,2].
Promising insulin-like effects have also been found in the case of
VIVO picolinato complex and its derivatives [3].
Three new VIVO compounds with the 5-cyanopyridine-2-carboxylic
acid,
3,5-difluoropyridine-2-carboxylic
acid
and
3-hydroxypyridine-2-carboxylic acid have been synthesized and
characterized by X-ray, EPR and DFT methods. Their interactions
with the blood proteins apo-transferrin and albumin have also been
studied by EPR spectroscopy.
Studies in the solid state and in solution have revealed cis-octahedral structure of all three compounds with the solvent or
monodentate ligand in equatorial position cis to the VIVO moiety,
while the arrangement of the bidentate picolinate ligands is variable.
In the solid state OC-6-23 and/or OC-6-24 arrangements of the
complexes have been determined, but in the solution partial isomerization into OC-6-42 complexes has been observed. In the presence of
protein apo-hTf at physiological pH complexes decompose and the
majority of VIVO2? ions binds to the protein, while in the presence of
HSA mixed species with ligands can also be formed.
O
N
V
O
O
O
L
N
O
V
N
L
N
O
V
N
O
L
N
O
O
O
N
OC-6-23
OC-6-24
OC-6-42
X
O
J Biol Inorg Chem (2014) 19 (Suppl 2):S779–S814
S799
Financial support by the Slovenian Research Agency is gratefully
acknowledged. We also thank the EN–FIST Centre of Excellence for
the use of the Supernova diffractometer.
References
1. Shechter Y, Goldwaser I, Mironchik M, Fridkin M, Gefel D (2003)
Coord Chem Rev 237:3–11
2. Sakurai H, Yoshikawa Y, Yasui H (2008) Chem Soc Rev
37:2383–2392
3. Katoh A, Matsumura Y, Yoshikawa Y, Yasui H, Sakurai H (2009) J
Inorg Biochem 103:567–574
P 70
Silver(I) bis(norharmane) compounds: synthesis, 3D
structures and cytostatic properties
Rais Ahmad Khan1, Khalid, Al-Farhan1, Andreia de Almeida2,
Ali Alsalme1, Angela Casini2, Mohamed Ghazzali1, Jan Reedijk1,3
1
Department of Chemistry, College of Science, King Saud University,
P.O. Box 2455 Riyadh 11451, Kingdom of Saudi Arabia;
2
Research Institute of Pharmacy, University of Groningen, Antonius
Deusinglaan 1, 9713 AV Groningen, The Netherlands;
3
Leiden Institute of Chemistry, Leiden University, P.O. Box 9502,
2300 RA Leiden, The Netherlands
Silver coordination compounds are well known as protectors of the
open skin to prevent bacterial infections. They are hardly toxic for
human beings, so the question has risen whether such compounds,
when the proper ligands are present, can be investigated for their
cytotoxic activity [1]. So we have chosen a less well-known ligand
that in addition to the nitrogen metal binding site has a remote H-bond
donor group which may establish additional hydrogen bonds. The
ligand norharmane (9H-Pyrido[3,4-b]indole, abbreviated as Hnor; see
Figure below) is a mixed-ring heterocyclic compound that belongs to
an alkaloid family called b-carbolines (bCs).
We report on four new Ag coordination compounds with this
ligand. As counter ions we have chosen: NO3- (potential metal
H
N
N
coordination), BF4-, ClO4- and PF6- (decreasing tendency to coordinate) also to see whether the NH ligand would bind
(intermolecularly) to the anion. In the case of the first three anions the
compounds have the formula [Ag(Hnor)2](anion), while the compound with the PF6- anion resulted to be [Ag(CH3CN)(Hnor)2](PF6),
with a weakly coordinated CH3CN (Ag–N 259.2 vs 217.0 pm). See
figure below for the hexafluoridophosphate.
These four Ag compounds were tested for their antiproliferative
activities in human ovarian (A2780) and lung (A549) cancer cell
lines. Surprisingly, only the perchlorate-containing derivative shows
promising activity in the selected cancer cells.
Financial support from the Distinguished Scientists Fellowship
Program (DSFP), King Saud University, is gratefully acknowledged.
Reference
1. Reedijk J (2009) Eur J Inorg Chem 2009:1303–1312
P 71
Zn(II)-triggered cellular uptake and nuclear
localization of a near-infrared fluorescent probe
Sandra G. König, Roland Krämer
Institute of Inorganic Chemistry, University of Heidelberg, Im
Neuenheimer Feld 274, 69120 Heidelberg
The distribution of Zn(II) ions in tissues is remarkably specific.
Amongst others, zinc-rich tissues include the central nervous system,
prostate, pancreas, and breast cancer tissue [1]. In contrast, Zn(II)
levels are dramatically decreased in prostate cancer tissue [2]. Fluorescent probes for endogenous zinc sensing are promising tools for
early detection of, for example, prostate cancer [3].
In earlier studies, our group has demonstrated that the attachment of terpyridine (tpy) to peptide nucleic acids results in
significantly elevated cellular uptake of these PNA-tpy conjugates
in presence of Zn(II) [4]. We have now developed a novel nearinfrared probe (NIR-Z) consisting of a fluorophore and a terpyridine moiety. While NIR-Z is poorly internalized in the absence of
Zn(II), we observed a significant enhancement of cellular uptake
for Zn(II)-complexes of NIR-Z as well as an influence on the
intracellular distribution [5].
This probe might allow tissue-selective labeling and even the
detection of potential malignancies. Its advantageous near-infrared
absorption and emission that allow imaging with low background
fluorescence from endogenous fluorophores as well as its high
extinction coefficient and sufficient quantum yield render the
probe a highly interesting candidate for potential in vivo applications.
123
S800
Financial support by the University of Heidelberg and the Foundation of German Business (sdw) is gratefully acknowledged.
References
1. Qian W-J, Gee KR, Kennedy RT (2003) Anal Chem
75:3468–3475; Margalioth EJ Schenker JG, Chevion M (1983)
Cancer 52:868–872
2. Franklin RB, Feng P, Milon B, Desouki MM, Singh KK, Kajadaczy-Balla A, Bargasra O, Costello LC (2005) Mol Cancer 4:32
3. Gosh SK, Kim P, Zhang X, Yun S–H, Moore A, Lippard SJ,
Medarova Z (2010) Cancer Res 70:6119–6127
4. Füssl A, Schleifenbaum A, Göritz M, Riddell A, Schultz C, Krämer
R (2006) J Am Chem Soc 128:5986–5987
5. König SG, Krämer R (2014) in preparation
P 72
Novel multi-functional metallodrug candidates
as potential cancer therapeutics
Ziga Ude1, Sara Sersen2, Iztok Turel2, Celine J Marmion1
1
Centre for Synthesis and Chemical Biology, Department
of Pharmaceutical and Medicinal Chemistry, Royal College
of Surgeons in Ireland, Dublin 2, Dublin, Ireland;
2
Faculty of Chemistry and Chemical Technology, University
of Ljubljana, Askerceva 5, 1000 Ljubljana, Slovenia
The first platinum-based anti-cancer chemotherapeutic, cisplatin,
was granted clinical approval in 1978. Yet, surprisingly to date,
only two further platinum drugs have gained full global approval
namely carboplatin and oxaliplatin. Although hugely successful, the
widespread application and efficacy of platinum drugs are hindered
by their toxic side effects, limited activity against many human
cancers and susceptibility to acquired drug resistance. As a consequence, many investigations have been conducted into trying to
identify new molecular targets beyond DNA which may present
unique opportunities for therapeutic exploitation. In recent years,
histone deacetylase (HDAC) enzymes have been identified as novel
cancer targets, the inhibition of which suppresses tumour cell
proliferation. Our group has designed and synthesised novel anticancer bifunctional platinum drug candidates which possess both
DNA binding and HDAC inhibitory activity [1–4]. Building on this
research, we have been adding to this library of compounds
including the development of novel ruthenium HDAC inhibitor
derivatives. In doing so, our ultimate aim is to develop novel drug
candidates that may overcome the drawbacks associated with
classical platinum drugs. A summary of our results to date will be
described.
This material is based upon works supported by the Science
Foundation Ireland under Grants No. [07/RFP/CHEF570] and [11/
RFP.1/CHS/3094]. We also gratefully acknowledge the Programme
for Research in Third Level Institutions (PRTLI), administered by the
HEA for funding. We thank also colleagues in COST CM1105 for
fruitful discussions and collaborations.
References
1. Griffith D, Morgan MP, Marmion CJ (2009) Chem Commun
44:6735–6737
2. Griffith D, Parker JP, Marmion CJ (2010) Anticancer Agents Med
Chem 10:354–370
3. Brabec V, Griffith D, Kisova A, Kostrhunova H, Lenka Z,
Marmion CJ, Kasparkova J (2012) Mol Pharmaceutics 7:1990–1999
123
J Biol Inorg Chem (2014) 19 (Suppl 2):S779–S814
4. Parker JP, Nimir H, Griffith D, Duff B, Chubb AJ, Brennan MP,
Morgan MP, Egan DA, Marmion CJ (2013) J Inorg Biochem
124:70–77
P 73
Coordination abilities of the alloferon 1 mutants
containing two histidine residues towards copper(II)
ions
Agnieszka Matusiak1, Mariola Kuczer1, El_zbieta Czarniewska2,
Grzegorz Rosiński2, Teresa Kowalik-Jankowska1
1
Faculty of Chemistry, University of Wrocław, Joliot-Curie 14,
50–383 Wrocław, Poland;
2
Department of Animal Physiology and Development, Institute
of Experimental Biology, Adam Mickiewicz University, Umultowska
89, 61–614 Poznań, Poland
Alloferon is a tridecapeptide H1GVSGH6GQH9GVH12G isolated
from the bacteria-challenged larvae of the blow fly Calliphora vicina
[1]. Alloferon is believed to have similar therapeutic use to interferons and interferon inducers including but not limited to treatment of
interferon-sensitive viral and cancer diseases. Alloferon is practically
non-toxic, has no teratogenic, embryotoxic or mutagenic properties as
has been shown in advanced preclinical studies [1]. Many essential
metal ions act as the important factor influencing the structure of
natural and synthetic oligopeptides, and as a consequence, they may
have a critical impact on their biological activity. It was found that
alloferon causes apoptosis in hemocytes of Tenebrio molitor [2].
Also, heavy metals are known to be typical stimuli to trigger apoptosis in vertebrate and invertebrate cells [3].
In this study the copper(II) complexes of the (H6,9A), (H6,12A)
and (H9,12A) mutants of alloferon 1 were performed by the combined
application of potentiometric equilibrium, spectroscopic (UV–Visible, CD, EPR) and MS methods in solution. The CuL complex with
{NH2,NIm-H1,NIm-H6,9or12} binding site dominates in wide 4.5–7.5
pH range. At physiological pH 7.4 are present in equilibrium the CuL
and CuH-1L {NH2,N-,CO,NIm-H6,9or12}complexes. The first amide
nitrogen deprotonation is suppressed by the coordination of the H1
residue and the formation of the macrochelate by the H6, H9 or H12
residues.
The induction of apoptosis in vivo in T. molitor cells by the
ligands and their Cu(II) complexes was studied. The biological results
show that Allo(H6,9A), Allo(H6,12A) and Cu(II)-Allo(H9,12A) show
weaker proapoptotic activity than alloferon 1. The Cu(II) complexes
of the Allo(H6,9A) and Allo(H6,12A) were practically inactive.
These results may confirm our suggestion that an important role in the
biological activity of Cu(II) complexes with alloferon play histidine
residues at position 1 and 6 through which a macrochelate is created
[4,5].
References
1. Chernysh S, Kim SI, Bekker G, Pleskach VA, Filatova NA, Anikin
VB, Platonov VG, Bulet P (2002) PNAS 99:12628–12632
2. Kuczer M, Czarniewska E, Rosiński G (2013) Regul Pept
183:17–22
3. Habbeebu SS, Liu J, Klaassen CD 1998 Toxicol Apel Pharmacol
149:203–209
4. Kuczer M, Błaszak M, Czarniewska E, Rosiński G, Kowalik-Jankowska T (2013) 52:5951–5961
5. Matusiak A, Kuczer M, Czarniewska E, Rosiński G, KowalikJankowska T J Inorg Biochem (submitted for publication)
J Biol Inorg Chem (2014) 19 (Suppl 2):S779–S814
P 74
Novel bio-inorganic tools for ROS detection in living
cells
Arjan Geersing1, Monique G. P. van der Wijst2, Gerard Roelfes1
1
Stratingh Institute for Chemisty, University of Groningen,
Nijenborgh 4, 9747 AG, The Netherlands, [email protected];
2
Department of Pathology and Medicinal Biology, University
Medical Center Groningen, University of Groningen, Hanzeplein 1,
9713 GZ, The Netherlands
Free radicals and other Reactive Oxygen Species (ROS) are important
components in many physiological and pathological processes in the
living cell [1]. Still much is unknown about the roles of ROS and
oxidative damage in cellular processes. Elevated concentrations of
ROS can cause oxidative stress which severely impacts the cellular
function by damaging important cellular components [2]. The goal of
this project is to design novel catalytic tools for the detection and
manipulation of ROS and oxidative stress in both healthy as well as
cancer cells with a particular focus on the sub-cellular environment.
This will give rise to new insights into the behavior of cancer cells
compared to healthy cells under oxidative stress. Ideally suited for this
purpose are the bio-inspired iron oxidation catalysts based on N4Py
ligands (See Figure) [3]. This transition metal catalyst can convert
H2O2 and O2 into highly reactive oxygen species (hROS), which may
include diffusible radicals such as OH, or iron-based species such as
FeIIIOOH or FeIVO, that can easily be detected and are capable of
generating controlled oxidative stress inside the (sub-)cellular environment. Current research focuses on the covalent attachment of a
fluorophore to the Fe(II)-N4Py catalyst, in order to track the ligand
inside living cells using con-focal microscopy. This will give more
insight into the mode of action of Fe(II)-N4Py and its role in cell death.
S801
P 75
Improved reaction conditions for the synthesis of novel
KP1339 derivatives and preliminary investigations
on their anticancer potential
Paul-Steffen Kuhn1, Verena Pichler1, Alexander Roller1, Michael
Jakupec1,2, Wolfgang Kandioller1,2, Bernhard K. Keppler1,2
1
Institute of Inorganic Chemistry, University of Vienna, Waehringer
Str.42, 1090 Vienna, Austria, [email protected];
2
Research Platform ‘‘Translational Cancer Therapy Research’’,
University of Vienna, Waehringer Str. 42, 1090 Vienna, Austria
Due to the very promising results of Na[RuCl4(1H-indazole)2]
(NKP1339) in clinical studies [1], the chemistry of Keppler-type
ruthenium(III) coordination compounds is still of large scientific
interest [2]. By applying a new acid free method on the way to
this type of coordination compounds, six different complexes of
the general formula (cation)-trans-[RuCl4(azole)2], where (cation) = tetrabutylammonium (Bu4N–) (1, 2), sodium (3, 4), azolium
(5, 6) and azole = 1-methyl-indazole (1–3), 1-ethyl-indazole (4–6),
have been synthesized. The new synthetic method allows the
introduction of acid-sensitive or -labile ligands and can therefore
facilitate the future development of ruthenium(III) based anticancer
agents. All synthesized complexes have been characterized by
elemental analysis, electrospray ionization (ESI) mass spectrometry, IR-, UV–Vis- and NMR spectroscopy. Furthermore, the
influence of the alkyl substituent at indazole ligand on the stability
in aqueous media as well as on the biological activity in three
human cancer cell lines (CH1, A549 and SW480) will be discussed.
Solv
N
N
N
N
N Fe N
N
N
N
N
References
1. Winterbourn CC (2008) Nat Chem Biol 4:278
2. Dickinson BC, Sriikun D, Chang CJ (2010) Curr Opin Chem Biol
14:50
3. a) Lubben M, Meetsma A, Wilkinson EC, Feringa BL, Que Jr. L
(1995) Angew Chem Int Ed 34:1512; b) Roelfes JG, Branum ME,
Wang L, Que Jr. L, Feringa BL (2000) J Am Chem Soc 122:11517; c)
Li Q, van den Berg TA, Feringa BL, Roelfes JG (2010) Dalton Trans
39:8012
Financial support by the University of Vienna is gratefully
acknowledged.
References
1. Thompson DS (2012) J Clin Oncol 30:suppl abstr 3033
2. Levina A, Mitra A, Lay PA (2009) Metallomics 1:458
123
S802
P 76
Surfactant-mediated activation of the lead anticancer
ruthenium compound KP1019 by encapsulation
into polymeric nanoparticles
Britta Fischer1, Petra Heffeter2,3, Kushtrim Kryeziu2,3, Lars
Gille4, Samuel M. Meier1,3, Walter Berger2,3, Christian R.
Kowol1,3, Bernhard K. Keppler1,3
1
Institute of Inorganic Chemistry, University of Vienna, Waehringer
Str. 42, 1090 Vienna, Austria;
2
Institute for Cancer Research and Comprehensive Cancer Center,
Medical University Vienna, Borschkegasse 8a, 1090 Vienna, Austria;
3
Research Platform ‘‘Translational Cancer Therapy Research’’,
University of Vienna and Medical University of Vienna, Vienna,
Austria;
4
Molecular Pharmacology and Toxicology Unit, Department
of Biomedical Sciences, University of Veterinary Medicine Vienna,
Veterinaerplatz 1, 1210 Vienna, Austria
KP1019 is the lead anticancer ruthenium(III) compound and has been
successfully tested in a clinical phase I [1]. Since, it is characterized by
quite low stability in aqueous solution especially at physiological pH,
nanoparticles would offer the possibility to circumvent these limitations
[2]. Therefore, highly reproducible poly(lactic) acid (PLA) nanoparticles of KP1019 were prepared by a single oil-in-water (o/w) emulsion.
During storage the color of these nanoparticles changed from brown to
green. Cytotoxicity measurements comparing different aged nanoparticles revealed that the color change is associated with a 20-fold
increased activity compared to ‘‘free’’ KP1019. A reaction between the
used surfactant Tween 80 and KP1019 was identified as the cause of the
green color. Kinetic studies using UV–Vis, ESI–MS and ESR spectroscopy indicated a coordination of Tween 80 to KP1019, and
furthermore the color change was found to correlate with a reduction
of the Ru(III) by Tween 80. The results provide a first approach to
stabilize a biologically active Ru(II) species of KP1019 in aqueous
solution and the nanoparticles probably can be used to selectively
generate this activated species in the tumor tissue via passive targeting.
Financial support by the exploratory focus ‘‘Functionalized
Materials and Nanostructures’’ of the University of Vienna is gratefully acknowledged.
123
J Biol Inorg Chem (2014) 19 (Suppl 2):S779–S814
References
1. Hartinger CG, Zorbas-Seifried S, Jakupec MA, Kynast B, Zorbas
H, Keppler BK (2006) J Inorg Biochem 100:891–904
2. Brannon-Peppas L, Blanchette JO (2012) Adv Drug Delivery Rev
Ed 64:206–212
P 77
Synthesis, characterization and anticancer activity
of (thio)pyrone-derived organometallic complexes
Melanie Schmidlehner1, Verena Pichler1, Alexander Roller1,
Michael A. Jakupec1,2, Wolfgang Kandioller1,2, Bernhard K.
Keppler1,2
1
Institute of Inorganic Chemistry, University of Vienna, Waehringer
Str.42, 1090 Vienna, Austria, [email protected];
2
Research Platform ‘‘Translational Cancer Therapy Research’’,
University of Vienna, Waehringer Str. 42, 1090 Vienna, Austria
Non-platinum tumor-inhibiting metal and organometallic complexes
have gained increasing interest in recent years. One promising
approach is the coordination of organometallic fragments to bioactive
molecules. Pyrones are known for their bioavailability, favorable
toxicity profile and high affinity towards metal ions. This intensively
studied ligand system can be easily converted to the respective thiopyrone which offer a potential S,O coordination motif. It has already
been shown that thiopyrone-based Ru(II) complexes provide an
increased stability under physiological conditions and a different
biomolecule interaction profile compared to the pyrone analogues. In
addition, these organometallics exhibit promising cytotoxicity in vitro
[1,2] and are currently investigated in vivo.
The (thio)pyrone scaffold was modified via Mannich reaction
utilizing morpholine, N-methylpiperazine and piperidine in order to
expand the (thio)pyrone library and to investigate the influence on
cytotoxicity of the corresponding metal complexes. Due to the
emerging interest in Rh(III) Cp* and Os(II)–arene complexes, the
corresponding organometallics were synthesized and characterized by
means of 1D and 2D NMR spectroscopy, elemental analysis, ESI–MS
and if possible by X-ray diffraction analysis. The anticancer potential
was examined by means of the colorimetric MTT assay. The stability
in aqueous solution was studied via 1H NMR spectroscopy.
Financial support by the University of Vienna and the Johanna
Mahlke née Obermann Foundation is gratefully acknowledged.
J Biol Inorg Chem (2014) 19 (Suppl 2):S779–S814
References
1. Kandioller W, Kurzwernhart A, Hanif M, Meier SM, Henke H,
Keppler B, Hartinger CG (2011) J Organomet Chem 696:999–1010
2. Kandioller W, Hartinger CG, Nararov AA, Kuznetsov ML, John R,
Bartel C, Jakupec MA, Arion VB, Keppler BK (2009) Organometallics 28:4249–4251
P 78
A cobalt-based strategy for tumor targeting of EGFRinhibitors
Claudia Karnthaler-Benbakka1, Diana Groza2, Kushtrim
Kryeziu2, Verena Pichler1, Alexander Roller1, Walter Berger2,3,
Petra Heffeter2,3, Christian R. Kowol1,3, Bernhard K. Keppler1,3
1
Institute of Inorganic Chemistry, University of Vienna, Waehringer
Straße 42, 1090 Wien, Austria;
2
Institute of Cancer Research, Medical University of Vienna,
Borschkegasse 8A, 1090 Wien, Austria;
3
Research Platform ‘‘Translational Cancer Therapy Research’’,
University of Vienna and Medical University of Vienna, Wien,
Austria
During the last decades the development of receptor tyrosine-kinase
inhibitors was a major step forward in cancer treatment. However,
despite its success, the therapy is limited by strong adverse effects and
resistance development [1,2]. Aim of the here presented study was the
design of novel epidermal growth factor receptor (EGFR) inhibitors
which are specifically activated in malignant tissue and, thus, are
expected to show reduced side effects. To this end, a cobalt(III)-based
prodrug strategy was used, which allows targeted release of an active
EGFR inhibitor triggered by hypoxia in the tumor tissue [3]. New
inhibitors with bis-chelating moieties were synthesized and tested for
their EGFR-inhibitory potential. The most promising candidate was
subsequently coordinated to Co(III) and the biological activity of this
complex was tested in cell culture under hypoxic vs. normoxic conditions. Indeed, hypoxic activation and subsequent EGFR inhibition could
be proven. The anticancer activity of the new complex was tested in
xenograft models revealing potent anticancer activity also in vivo.
Summarizing, this study shows, that Co(III)-based tumor-targeting
represents a promising strategy to improve EGFR inhibitor treatment.
Financial support by the Fonds der Stadt Wien für innovative
interdisziplinaere Krebsforschung and the COST Action CM1105 is
gratefully acknowledged.
References
1. Li T, Perez-Soler R (2009) Targ Oncol 4:107–119
2. Zükin M (2012) Rev Assoc Med Bras 58:263–268
3. Ott I, Gust R (2007) Arch Pharm Chem Life Sci 340:117–126
S803
P 79
Fluorophore ATCUN complexes: dual probes for DNA
cleavage
Christian Wende, Nora Kulak
Institute of Chemistry and Biochemistry, Freie Universität Berlin,
Fabeckstr. 34/36, 14195 Berlin, Germany, [email protected]
The knowledge about the amino terminal Cu(II)- and Ni(II)-binding
motif (ATCUN) has expanded from a small metal binding site in proteins
like certain species of albumins to efficient ligands for artificial nucleases [1–3]. In the last years several newly designed amino acid
sequences based upon the ATCUN motif have been demonstrated to act
as highly selective, fluorescent chemosensors for Cu(II) ions [4]. Based
on findings of Imperiali and co-workers we have recently synthesized
some fluorophore-ATCUN peptides for investigation of their nuclease
activity and fluorescence properties [5]. We could show that the Cu(II)induced fluorescence quenching can partly be recovered when the
Cu(II)–peptide complex undergoes oxidative DNA cleavage involving
Cu(I) species. Therefore, our new approach might allow detecting
Cu(II)-dependent DNA cleavage not only by commonly used gel electrophoresis but also via fluorescence spectroscopy.
O O
O
N
N
Cu2+
fluorophore
N
H2
N
N
H
OH
H
N
O
O
NH2
OH
N
H
0.05 mM ligand
0.05 mM complex
0.05 mM complex (after incubation)
Financial support by the Deutsche Forschungsgemeinschaft (DFG)
is gratefully acknowledged.
References
1. Peters Jr T (1960) Biochim Biophys Acta 39:546–547
2. Harford C, Sarkar B (1997) Acc Chem Res 30:123–130
3. Kimoto E, Tanaka H, Gyotoku J, Morishige F, Pauling L (1983)
Cancer Res 43:824–828
4. Zheng Y, Gattás-Asfura KM, Konka V, Leblanc RM (2002) Chem
Commun 2350–2351
5. Torrada A, Walkup GK, Imperiali B (1998) J Am Chem Soc
120:609–610
P 80
Amphiphilic metal complexes of cyclen derivatives
for improved protein cleavage via particle formation
Chrischani Perera, Nora Kulak
Freie Universität Berlin, Institute of Chemistry and Biochemistry,
Fabeckstr. 34/36, 14195 Berlin, Germany, [email protected]
Cleavage of pathogenic proteins, as they appear in amyloidogenic
diseases like Alzheimeŕs disease, Parkinson’s disease and type 2
diabetes mellitus, is a therapeutic option for their treatment. The
moderate protein cleavage activity of cyclen complexes is known
123
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J Biol Inorg Chem (2014) 19 (Suppl 2):S779–S814
since the nineties and enhancement of this activity and targeting to
pathogenic proteins are under investigation ever since [1].
Formation of micelles and vesicles from amphiphilic Zn(II) cyclen
derivatives has already been proven as potent strategy for DNA cleavage
[2]. Our goal is to improve the protease activity of Cu(II) and Co(III)
complexes of cyclen derivatives by a similar strategy, i.e. the selfassembly to micelles. The influence of mono N-alkylation and -carboxylation of the cyclen ligand on the protein cleavage activity and
particle formation behaviour is described in this work.
Financial support by the Studienstiftung des deutschen Volkes is
gratefully acknowledged.
References
1. Armitage B, Schister GB (1997) J Photochem Photobiol
66:164–170
2. Koch T, Ropp JD, Sligar SG, Schuster GB (1993) J Photochem
Photobiol 58:554–558
3. Hormann J, Perera C, Deibel N, Lentz D, Sarkar B, Kulak N (2013)
Dalton Trans 42:4357–4360
X
HN
N
self
assembly
protein
cleavage
N
NH
P 82
HSP70 as a metallodrug target
C16H33
=
X= H, COOH
= Co(III), Cu(II)
Financial support by the FU Focus Area NanoScale is greatfully
acknowledged.
References
1. Jang B-B, Lee K-P, Min D-H, Suh J (1998) J Am Chem Soc
120:12008–12016
2. Gruber B, Kataev E, Aschenbrenner J, Stadlbauer S, König B
(2011) J Am Chem Soc 133:20704–20707
P 81
Copper complexes of novel anthraquinone-substituted
cyclen derivatives for DNA cleavage
Jan Hormann, Nora Kulak
Freie Universität Berlin, Institute of Chemistry and Biochemistry,
D-14195 Berlin Germany, [email protected]
The photolytic cleavage of plasmid DNA by anthraquinone derivatives has been known for years. Such derivatives are promising
candidates for future anti-cancer therapeutics and especially for those
against skin cancer [1, 2]. We have recently designed three new water
soluble copper(II) complexes that link the anthraquinone moiety with
the well studied artificial nuclease Cu(II)-1,4,7,10-tetraazacyclododecane, Cu(II)cyclen [3]. We were able to show that these systems
can cleave plasmid DNA under irradiation at 365 nm as effectively or
even more effectively than anthraquinone itself. The anthraquinone
moiety increases DNA affinity and enhances the oxidative cleavage
activity, as well. To gain further insight into the redox processes
involved in the cleavage mechanism we performed electrochemical
studies, indicating synergic effects might exist between the anthraquinone moiety and the copper centre for some of the complexes.
Aoife McKeon1, Maria Morgan2, Darren Griffith1
1
Centre for Synthesis & Chemical Biology, Department
of Pharmaceutical and Medicinal Chemistry, Royal College
of Surgeons in Ireland (RCSI) Dublin 2, Ireland, [email protected].;
2
Molecular & Cellular Therapeutics, RCSI, Dublin 2, Ireland
Cancer is a major cause of death and disease worldwide. Over the past
30 years platinum (Pt) compounds have played a very important and
well documented role in treating cancer. The cytotoxicity of Pt drugs
is attributed to multiple mechanisms but primarily their ability to
form DNA adducts. The clinical efficacy of Pt drugs is limited though
by toxicity and chemoresistance (intrinsic or acquired)[1]. Since
many cancers are intrinsically resistant to Pt-based therapies there is
an urgent need to develop novel and innovative therapeutic strategies
for combating cancer.
HSP70 is a stress-inducible chaperone, which maintains protein
homeostasis during normal cell growth but during a stress response is
overexpressed and binds to and stabilises its protein substrates. It is
overexpressed in colorectal and prostate cancer amongst other cancers, and is associated with cancer progression, chemotherapy
resistance (including against cisplatin) and poor prognosis as it is
thought to provide cancer cells with a survival advantage by conferring protection against apoptosis, influencing senescence and
inhibiting autophagy for example. In addition given HSP70 is overexpressed in cancer cells relative to normal cells this effect should be
selective. Inhibition of HSP70 is therefore an exciting and legitimate
anti-cancer target.
Consequently, we wish to develop novel Pt HSP70 inhibitor
drug candidates as potential alternative treatments for colorectal
and prostate cancer. A summary of results to date will be described.
This research was supported by Science Foundation Ireland under
Grant No. [12/IP/1305]
References
1. Barry NPE, Sadler PJ (2013) Chem Commun 49:5106–5131
2. Murphy ME (2013) Carcinogen 34:1181–1188
123
J Biol Inorg Chem (2014) 19 (Suppl 2):S779–S814
P 83
New carbohydrate-bearing 8-hydroxyquinoline
compounds as multifunctional chelators of copper(II)
and zinc(II) ions
Valentina Oliveri1, Giuseppa I. Grasso2, Francesco Attanasio2,
Francesco Bellia2, Graziella Vecchio1
1
Dipartimento di Chimica, University of Catania, Viale A. Doria 6,
95125 Catania, Italy;
2
Instituto di Biostrutture e Bioimmagini, CNR, Viale A. Doria 6,
95125 Catania, Italy, [email protected]
Mounting evidence suggests a pivotal role of metal imbalances in
protein misfolding and amyloid diseases. As such, metal ions represent a promising therapeutic target and therefore, the synthesis of
chelators that also contain complementary functionalities to combat
the multifactorial nature of neurodegenerative diseases is a highly
topical issue Recent investigations have rekindled interest in
8-hydroxyquinolines (OHQs) as therapeutic agents for cancer, Alzheimer’s disease and other neurodegenerative disorders. We have
recently demonstrated that glycosylation is a versatile and powerful
strategy for improving drug features including solubility, pharmacokinetics, drug targeting, and biological activities [1–4]. Here, we
report several OHQ glycoconjugates whose multifunctional properties
are highlighted, including their Cu(II) and Zn(II) binding abilities,
and antioxidant and metal-induced antiaggregant capacity. Glucose,
trehalose and cyclodextrin were the carbohydrates of choice because
of their interesting properties such as antioxidant, antiaggregant and
inclusion abilities.
We thank FIRB_RINAME and PON01_01078 for financial
support.
References
1. Oliveri V, Puglisi A, Viale M, Aiello C, Sgarlata C, Vecchio G,
Clarke J, Milton J, Spencer J (2013) Chem Eur J 19:13946–13955
2. Oliveri V, Giuffrida ML, Vecchio G, Aiello C, Viale M (2012)
Dalton Trans 41:4530–4535
3. Oliveri V, Viale M, Caron G, Aiello C, Gangemi R, Vecchio G
(2013) Dalton Trans 42:2023–2034
4. Oliveri V, Attanasio F, Puglisi A, Spencer J, Sgarlata C, Vecchio G
(2014) Chem Eur J. doi:10.1002/chem.201402690
P 84
Antimicrobial properties of Cu-based nanoparticles:
interaction with DNA, ROS production and lipid
peroxidation
Kleoniki Giannousi, Anastasia Pantazaki, Catherine DendrinouSamara
Department of Chemistry, Aristotle University of Thessaloniki, 54124
Thessaloniki, Greece, [email protected]
The abuse of antimicrobial drugs has led to increasing number of
infections associated with antibiotic-resistance microbes. By using
water as a solvent or typical solvothermal synthesis and in the presence of surfactants, copper based nanoparticles (Cu-based NPs) were
formed, while synthesis control is achieved to tune their composition,
size and shape. The higher biological activity of the NPs combined
with lower applicable doses could give rise to the next generation of
antimicrobials, namely nano-antimicrobials.
Herein, we solvothermally prepared Cu-based NPs of different
composition and sizes capped with the non ionic surfactants tetraethylene glycol, polyethylene glycol 1000, polysorbate 20 and
oleylamine. She antimicrobial activity of the synthesized Cu, Cu/
Cu2O, Cu2O and CuO NPs has been screened against Gram-positive
(Staphylococcus aureus, Bacillus cereus), Gram-negative bacteria
S805
(Escherichia coli, Xanthomonas campestris, Bacillus subtilis) and
fungus (Saccharomyces cerevisiae). The results clearly indicated that
the composition of the NPs was the main factor affecting their performance, since Cu2O NPs found with enhanced antimicrobial effect
and specificity against the Gram-positive strains. In an attempt to
further explore their mechanism of action, we studied their interaction
with DNA and Cu-based NPs found to induce pDNA, ds CT-DNA
and fungal DNA degradation in a dose-dependent manner. The ROS
production and lipid peroxidation have also been verified, while ionic
contribution to the bactericidal activity of NPs cannot be supported as
the released ions found below the value of inhibiting bacterial growth.
The research has been co-financed by the European Union and
Greek national funds through the Operational Program ‘‘Education
and Lifelong Learning’’ of the National Strategic Reference Framework—Research Funding Program: Thales. Investing in knowledge
society through the EU Social Fund.
Reference
1. a) Giannousi K, Lafazanis K, Arvanitidis J, Pantazaki A, Dendrinou-Samara C (2014) J Inorg Biochem 133:24–32; b) Giannousi K,
Avramidis I, Dendrinou-Samara C (2013) RSC Adv 3:21743–21752
P 85
Cystic fibrosis: new molecular imaging tools
Vera F. C. Ferreira1, Bruno L. Oliveira1, João D. G. Correia1,
Isabel Santos1, Carlos M. Farinha2, Filipa F. Mendes1
1 2
C TN-Center of Nuclear Sciences and Technologies, Instituto
Superior Técnico, Universidade de Lisboa, Campus Tecnológico e
Nuclear, Estrada Nacional 10, km 139.7, 2695-066 Bobadela- LRS,
Portugal;
2
BioFig-Center for Biodiversity, Functional and Integrative
Genomics, Faculdade de Ciências, Universidade de Lisboa, Campo
Grande, 1749-016 Lisboa, Portugal
Cystic Fibrosis (CF) is caused by mutations in the cystic fibrosis
transmembrane conductance regulator (CFTR) gene, which encodes
the CFTR protein, a chloride channel expressed in the apical membrane of epithelial cells in the airways, pancreas, intestine and exocrine
glands. Therapies based in drugs that correct the trafficking or gating
defects of CFTR (termed correctors or potentiators, respectively) are
emerging. Although the ultimate endpoints to assess the efficacy of
pharmacological correction would be the benefits upon the clinical
phenotype, there is no available methodology to detect the presence of
normal (or corrected) CFTR at the membrane in living organisms.
Molecular Imaging can be the solution, since it allows the in vivo
non-invasive visualization of a target molecule by virtue of its interaction with an imaging probe. Single-photon emission computed
tomography (SPECT) and positron emission tomography are the most
sensitive imaging modalities available, and allow early disease diagnosis and follow up of therapy. So, the aim of this work is the
development of non-invasive radiolabelled imaging probes for the
detection of CFTR at the plasma membrane of human cells. The probe
reported in this communication was based on a CFTR inhibitor known
to interact specifically with CFTR at the region of the channel pore.
123
S806
The 99mTc radioisotope, used in SPECT, was the chosen radionuclide
due to its physical properties, low cost and easy availability. The
CFTR inhibitor was radiolabelled with the fac-[99mTc(CO)3]? core,
using the bifunctional chelator (BFC) approach. This strategy involved
a three-component system constituted by a high affinity biomolecule
(CFTRinh), a radiometal (99mTc) and a BFC, designed to bind both to
the radiometal and the biomolecule. Cellular studies in human bronchial epithelial cells expressing wt-CFTR were performed and the
amount of radiolabelled probe that can interact with CFTR assessed.
To evaluate if the metal complex of CFTRinh still maintained its ability
to interact with CFTR, a non-radioactive surrogate rhenium complex
was synthesized and its inhibitory efficacy was assessed through a
functional assay in cells expressing CFTR. In the future, these types of
probes may have the potential to be used on SPECT imaging to assess
early therapy response in drug evaluation.
Acknowledgments: This work was funded by grant EXPL/BIMMEC/0115/2012 (FCT, Portugal). FCT is also acknowledged for the
PhD Fellowship (SFRH/BD/38753/2007 to B Oliveira and for the
FCT Investigator grant to F Mendes. The authors would like to thank
the Cystic Fibrosis Foundation Therapeutics for providing the CFTR
inhibitor through the Chemical Compound Distribution Program.
P 86
DNA binding and cytotoxic activity of Zn(II) and Cu(II)
complexes with new bis(thiosemicarbazone) derivatives
António Paulo, Inês Rodrigues, Maria P. C. Campello, Goreti
Morais, Vera Ferreira, Filipa Mendes, Isabel Santos, Sofia Gama
Centro de Ciências e Tecnologias Nucleares-C2TN, Instituto Superior
Técnico, Universidade de Lisboa, Campus Tecnológico e Nuclear,
Estrada Nacional 10, km 139.7, 2695-066 Bobadela, LRS-Portugal
Bis(thiosemicarbazone) complexes of Zn(II) and Cu(II) have received
considerable attention in the design of metallodrugs, either as anticancer therapeutics or diagnostic radiopharmaceuticals. Moreover, the
availability of several medically relevant copper radioisotopes (e.g.
62
Cu, 64Cu and 67Cu) makes them potentially useful tools for cancer
theranostics, profiting from a versatile chemical modification of the
bis(thiosemicarbazone) framework and a stable coordination of radiocopper ions. The mechanism involved in the anticancer activity of
Zn(II) and Cu(II) bis(thiosemicarbazonates) is not fully understood.
However, it has been shown in a few studies that there is an accumulation of the complexes in the nucleus of tumor cells, indicating
that DNA could be a potential target of their action. In this context,
we have embarked in the synthesis of Zn(II) and Cu(II) complexes
with new bis(thiosemicarbazone) chelators, symmetrically functionalized with protonable cyclic amines of the piperidine and morpholine
type (Fig. 1). We have hypothesized that the presence of the cyclic
amine groups could enhance the DNA affinity of the compounds and,
together with the planarity of the metallic center, could promote some
selectivity towards different types of DNA (e.g. G-quadruplex vs.
duplex DNA). In this communication, we report the DNA binding and
cytotoxic activity studies of these new M(II)-bis(thiosemicarbazone)
complexes, performed with the aim of assessing their usefulness in
the design of metal-based drugs for cancer theranostics.
123
J Biol Inorg Chem (2014) 19 (Suppl 2):S779–S814
R
N N
N
n
N
H
S
MII
N N
S
N
H
N
n
Fig. 1
The authors would like to acknowledge FCT (EXCL/QEQ-MED/
0233/2012, PTDC/QUI-QUI/114139/2009, SFRH/BPD/29564/2006
grant to SGama and FCT Investigator Grant to FMendes) and COST
CM1105 Action for financial support.
Reference
1. Dilworth JR, Hueting R (2012) Inorg Chim Acta 389:3–15
P 87
Biological evaluation of zinc(II) chlorido complexes
with O6-substituted 9-deazahypoxanthine derivatives
Jana Gáliková1, Jan Hošek1, Zdeněk Dvořák2, Zdeněk Trávnı́ček1
1
Regional Centre of Advanced Technologies and Materials,
Department of Inorganic Chemistry, Faculty of Science, Palacký
University, 17. listopadu 12, CZ-771 46 Olomouc, Czech Republic,
[email protected];
2
Regional Centre of Advanced Technologies and Materials,
Department of Cell Biology and Genetics, Faculty of Science,
Palacký University, Šlechtitelů 11, CZ-783 71 Olomouc, Czech
Republic
In this work, a series of new zinc(II) complexes with the general
formula [Zn(Ln)2Cl2] involving O6-substitued 9-deazahypoxanthine
derivatives (Ln) were biologically evaluated for their in vitro
cytotoxic and immunomodulating activity. The present study
showed that the compounds exhibited no cytotoxic effect on the
human prostate (PC3, LNCaP), ovarian carcinoma (A2780) and
monocytic leukemia (THP-1) cancer cell lines up to the concentration of IC50[50 lM, and IC50[10 lM, respectively. The effect
of these complexes to influence the activity of inflammatoryrelated zinc-dependent matrix metalloproteinase (MMP-2) and to
affect the secretion of pro-inflammatory cytokine IL-1b was
determined using the lipopolysaccharide-activated macrophage-like
THP-1 cell model. The ability of the complexes to attenuate IL-1b
production was not observed. On the other hand, these complexes
were able to increase the total amount of MMP-2 protein and
significantly elevate the level of the active form of this protease.
Increased activity of MMP-2 could be beneficial during wound
healing, rheumatoid arthritis or repairing of injured nervous system
due to the ability of this protease to remove some pro-inflammatory cytokines, promote neovascularisation and organise tissue
remodelling [1–3].
J Biol Inorg Chem (2014) 19 (Suppl 2):S779–S814
S807
This work was supported by the Fundamental Research Funds for
the Central Universities and the Research Funds of Renmin University of China (no. 10XNJ011).
References
1. Chen H, Ikeda-Saito, M, Shaik S (2008) J Am Chem Soc
130:14778–14790
2. Du WH, Syvitski R, Dewilde S, Moes L, La Mar GN (2003) J Am
Chem Soc 125:8080–8081
P 89
pH-Specific structural speciation studies in biologically
relevant binary Cr(III)-hydroxycarboxylic acid systems
We acknowledge the financial support (CZ.1.07/2.3.00/30.0004
and CZ.1.05/2.1.00/03.0058).
References
1. Nguyen M, Arkell J, Jackson CJ (2000) J Biol Chem
275:9095–9098
2. Le NTV, Xue M, Castelnoble LA, Jackson CJ (2007) Front Biosci
12:1475–1487
3. Verslegers M, Lemmens K, Van Hove I, Moons L (2013) Prog
Neurobiol 105:60–78
P 88
Interaction of azide with human neuroglobin and H64Q
mutant investigated by solution NMR and molecular
dynamics simulation
Bingbing Zhang1, Xuesong Wang1,2, Haili Li2, Jia Xu1, Lianzhi
Li2,*, Weihong Du1
1
Department of Chemistry, Renmin University of China, Beijing
100872, People’s Republic of China;
2
School of Chemistry and Chemical Engineering, Liaocheng
University, Liaocheng, Shandong Province, 252059, China
Neuroglobin (Ngb), a new member of hemoprotein family, can
reversibly bind some small ligands and take part in many biological
processes such as signal transduction and hypoxic adaptation. Ngb
presents hexacoordinated heme, either in ferrous or ferric forms,
having the distal His64(E7) as the internal sixth ligand of heme iron.
To explore the mechanism of exogenous ligand binding to hexacoordinated Ngb and the effect of distal key residue His64 on the
binding property, two proteins including HNgb and the mutant
[H64Q] HNgb were used in the present work, and their interactions
with azide were studied by solution NMR. Further, the protein
conformation was analyzed through molecular dynamics (MD)
simulation. The results showed that the mutant protein had better
binding affinity with azide than that of wild type HNgb, which
revealed that the mutation of His64 influence the binding property
of exogenous ligand. Comparing 1H NMR spectra of HNgb (a),
HNgbN3(b), [H64Q] HNgb (c), [H64Q] HNgbN3 (d), [H64Q] MNgb
(e) [1], [H64Q] MNgbCN (f), MNgb (g) and MNgbCN (h) [2] may
find that different exogenous ligands result in remarkable pseudocontact shifts of heme methyl protons, implying distinct magnetic
axial orientation in these proteins and corresponding complex systems. In accordance with those from NMR observation, the
influences of both azide binding and H64Q mutation on heme
electronic structure and heme pocket conformation were reflected by
the MD analysis as well.
Catherine Gabriel, Athanasios Salifoglou
Department of Chemical Engineering, Aristotle University
of Thessaloniki, Thessaloniki 54124, Greece,
[email protected]
Chromium is abundantly present on the earth’s crust. Its use includes
a) industrial processes in tanneries, cement industries, plating and
alloying industries, corrosive paints [1], doping [2] of advanced
materials for the modification of the efficiency and lifetime of the
photorefractive signals (i.e. the ‘‘memory’’-type signals in connection
with hologram recording) [3], heterogeneous catalysts, electrochromic devices and, more recently, in gas sensors [4], and b) direct or
indirect involvement in plants, animals, and humans [5].
In a widely diverse coordination environment, through which
Cr(III) develops its activity, a field of avid research activity has
emerged. In this field of research, the role(s) of Cr(III) in biological
systems is inevitably associated through its aqueous chemistry with
lipids, proteins, and amino acids free in the cytosol or as components
of peptides and lipid membrane structures. In all such cases, soluble
and bioavailable forms of Cr(III) promote (bio)chemical activity, thus
reflecting the complexity of the aqueous speciation in binary and
ternary systems present in biological media. In view of chromium’s
involvement in cellular processes, thereby directly or indirectly
affecting the physiology of organisms with often deleterious consequences, research efforts have targeted a) the aqueous structural
speciation of Cr(III), with metal-complexing carboxylate-containing
low molecular mass physiological ligands, and b) the study of the
physicochemical properties of arising species potentially bioavailable
and eliciting interactions with cellular biotargets.
In our quest to probe and comprehend interactions of chromium
with ligands often involved in chemistries of toxic and biologically
significant processes, we have looked into the aqueous structural
speciation of the binary system of Cr(III)-heida (2-hydroxyethyliminodiacetic acid). Synthesis in aqueous media led to the
isolation of three new complexes. The complexes were characterized
by elemental analysis, spectroscopic, structural, thermal, EPR and
magnetic susceptibility studies. The physicochemical properties of the
new species project the fundamental features of the interaction of
Cr(III) with (O,N)-containing bio-substrates potentially involved in
toxic effects manifested at the cellular level.
Financial support ‘‘IJ! Fellowships of Excellence for Postgraduate
studies in Greece—Siemens Program’’ is gratefully acknowledged.
References
1. Ramos RL, Martinez AJ, Coronado GRM (1994) Water Sci
Technol 30:191.
2. Pèter À, Szakács O, Fö1dvári I, Bencs L, Munoz AF (1996) Mater
Res Bull 31:1067
3. Fö1dvári I, Pèter À, Powell RC, Taheri B (1995) Opt Mater 4299
4. Moseley T, Norris JOW, Williams E (eds) (1991) Techniques and
mechanisms in gas sensing. I.O.P. Publishing, Bristol Adam Hilger
Series on Sensors.
123
S808
5. Bae W-C, Lee H-K, Choe Y-C, Jahng D-J, Lee S-H, Kim S-J, Lee
J-H, Jeong B-C (2005) J Microbiology 43:21–27
P 90
The therapeutic properties of VO(dmpp)2
as demonstrated by in vivo studies in type 2 diabetic GK
rats
N. Domingues1,3,#, J. Pelletier3,#, C.-G. Ostenson3, M. M. C.
A. Castro1,2
1
Department of Life Sciences, Faculty of Sciences and Technology
and
2
Coimbra Chemistry Centre and Center for Neurosciences and Cell
Biology, University of Coimbra, Coimbra, Portugal,
[email protected];
3
Department of Molecular Medicine and Surgery, Karolinska
Institutet, Stockholm, Sweden
#
these authors contributed equally to this work
This work aims at confirming the therapeutic properties already
demonstrated by the bis(1,2-dimethyl-3-hydroxy-4-pyridinonato)oxidovanadium (IV), VO(dmpp)2 [1,2] in non-obese type 2 diabetic
Goto-Kakizaki (GK) rats. An in vivo study was carried out, treating
Wistar (W) and GK rats during 21 days with a daily dose of
VO(dmpp)2 (44 lmol/kg). This study showed that, VO(dmpp)2 does
not affect the normal increase of body weight of both W and GK rats,
after 8 days of treatment ameliorates glycaemia in GK rats
(8.4 ± 0.3 mM vs 10.1 ± 0.2 mM in GK control, P \ 0.001) but
does not interfere with glucose levels in W rats. Moreover, after
21 days of treatment, it improves the glucose intolerant profile of GK
rats (13.1 ± 0.5 mM/min vs 20.6 ± 0.7 mM/min in GK control,
P \ 0.001), despite no increase of plasma insulin levels during oral
glucose tolerance test (OGTT). The glucose intolerance is also
ameliorated in GK rats submitted to an acute treatment (single dose of
44 lmol/kg 30 min before the glucose load) although better results
were obtained with the chronic one. Western blotting was used to
clarify the mechanism of action at the molecular level, and the results
revealed that in W and GK rats VO(dmpp)2 significantly promotes
IRS2 (P \ 0.05) and p-AKT expression (P \ 0.001 and P \ 0.05,
respectively, relative to the respective controls) and in the diabetic
animals reduces the increase of PTP1b expression (P \ 0.001, relative to GK treated with placebo).
The anti-diabetic properties of VO(dmpp)2 showed in GK rats
through its effects on glycaemia and OGTT and explained by its
interaction with proteins of the insulin signaling cascade [3] corroborate previous data and suggest the possible use of this compound in
the therapy of type 2 diabetes.
Financial support by Swedish Research Council, Swedish Diabetes
Association and Karolinska Institutet is acknowledged as well as the
synthesis of VO(dmpp)2 by João Costa Pessoa and his Post-doc student Somnath Roy at the Technical University of Lisbon, Portugal.
J.Pelletier was supported by a Föreningen för diabetesforskningens
främjande grant and N.Domingues by an Erasmus grant during her
stay in Karolinska Institutet.
References
1. Passadouro M, Metelo AM, Melao AS, Pedro JR, Faneca H,
Carvalho E, Castro MMCA (2010) J Inorg Biochem 104:987–992
123
J Biol Inorg Chem (2014) 19 (Suppl 2):S779–S814
2. Metelo AM, Perez-Carro R, Castro MM, Lopez-Larrubia P (2012) J
Inorg Biochem 115: 44–49
3. Domingues N, Pelletier J, Ostenson C-G, Castro MMCA (2014) J
Inorg Biochem 131:115–122
P 91
Synthesis, spectral characterization and antimicrobial
activity of Co(II) and Hg(II) complexes of 1,3-bis(1Hbenzimidazol-2-yl)-2-oxapropane
Aydin Tavman1, Adem Cinarli1, Demet Gürbüz1, A. Seher
Birteksöz Tan2
1
Department of Chemistry, Faculty of Chemistry, Istanbul University,
34320, Avcilar, Istanbul, Turkey, [email protected];
2
Department of Pharmaceutical Microbiology, Faculty of Pharmacy,
Istanbul University, 34452, Beyazit, Istanbul Turkey
Bis-benzimidazoles are strong chelating agents coordinating through
both of the C=N groups nitrogen atoms. In addition, the benzimidazole ring system is present was clinically approved anthelmintics,
antiulcers, antivirals and antihistamines [1]. Recently, there have been
reports on benzimidazole derivatives exhibiting antitumor and antimicrobial properties and acting as thrombopoietin receptor agonists
[2,3].
In this study, Co(NO3)2 and Hg(NO3)2 complexes of 1,3-bis(1Hbenzimidazol-2-yl)-2-oxapropane were synthesized and characterized
by elemental analysis, molar conductivity, magnetic moment, TGA,
FT-IR, ESI–MS, fluorescence spectroscopy. The complexes are 1:1
electrolytes and they have 1:1 M:L ratio. According to the magnetic
moment data (leff = 1.26 BM) there is M–M interaction in the Co(II)
complex. The complexes fluoresce although weaker compared to the
ligand.
In addition, antibacterial activities of the compounds were evaluated using the disk diffusion method against six bacteria and
Candida albicans. The Hg(II) complex shows superior activity toward
S. epidermidis and E. coli whereas the Co(NO3)2 complex, [Co(L)(NO3)(H2O)2]NO3 (Fig. 1), showed weak activity toward all of the
microorganisms.
H
N
H
N
O
N
N
Co
H2O
+
NO 3
-
OH2
O
+
N O
O
Fig. 1. The suggested structure of the Co(II) complex.
Financial support by Istanbul University is gratefully
acknowledged.
References
1. Harrell CC, Kohli P, Siwy Z, Martin CR (2004) J Am Chem Soc
126:15646–15647
2. Sun T, Li K, Lai Y, Chen R, Wu H (2010) Acta Cryst E66:m1058
J Biol Inorg Chem (2014) 19 (Suppl 2):S779–S814
S809
3. Chang CM, Kulkarni MV, Chen CH, Wang CH, Sun CM (2008) J
Comb Chem 10:466–477
P 92
New photoactivatable CO-releasing molecules
of manganese with imidazoline/benzimidazole ligands
Elvan Üstün1, Serpil Demir2, İsmail Özdemir2, Ulrich
Schatzschneider3
1
Department of Chemistry, Ordu University, Cumhuriyet Yerleşkesi
52200 Ordu, Türkiye;
2
Department of Chemistry, İnönü University, 44280 Malatya,
Türkiye;
3
Institut für Anorganische Chemie, Julius-Maximilians-Universitat
Würzburg, Am Hubland D-97074 Würzburg, Germany
Metal carbonyl complexes are the systems of choice to exploit the
beneficial effects of CO for therapeutic applications in medicine [1].
Also photoactivatable CO-releasing molecules (photo-CORMs) are
important part of these researches [2]. Imidazolines/benzimidazoles
are such bioactive compounds with anti-inflammatory and antihypertensive action [3].Therefore; we combined these two kinds of
bioactive molecules and synthesized four molecules shown in
Scheme. The selected molecules allow of making a comparison with
CO-releasing properties of imidazoline and benzimidazoles.
All molecules were fully characterized by NMR, IR MS and
elemental analysis. Currently we are investigating the long-term
stability, electronic absorption spectra and CO-releasing properties of
these complexes.
CH3
N
CH3
N
CO
N
N
Mn
OC
CO
CH3
N
CO
N
CH3
H3C
N
Mn
N
OC
CO
CH3
N
CO
CO
N
CH3
H3C
N
N
N
OC
CO
N
Mn
Mn
N
OC
CO
N
This work was supported by ‘‘The Scientific and Technological
Research Council of Turkey (TÜBİTAK) within project 112T320’’.
References
1. Johnson TR, Mann BE, Clark JE, Foresti R, Green CJ, Motterlini R
(2003) Angew Chem Int Ed 42:3722–3729
2. Schatzschneider U (2011) Inorg Chim Acta 374:19–21
3. Schlenk M, Ott I, Gust R, (2008) J Med Chem 51:7318–7322
P 93
Antiproliferative effect of polyoxometalates is induced
by ROS-mediated DNA damage in human cancer cells
B. González, V. Ortiz, A. Bachmann, E. Ruiz, J.M. GutiérrezZorrilla, A. Bonnin, S. Martı́n, N. Valle
Universidad Francisco de Vitoria, Biotechnology Department, Ctra.
M-515, Pozuelo-Majadahonda, km 1,800, Pozuelo de Alarcón
(Madrid), Spain, [email protected]
Polyoxometalates (POMs) are discrete oligomeric anions of early
transition metals, such tungsten (W), molybdenum (Mo) and vanadium (V) oxides. Various biological effects of POMs have been
studied, like their antitumor, antiviral and antibacterial activities [1].
We studied the mechanism involved in the antitumoral effect of two
polyoxometalates, (NHi3Pr)4[Mo8O26] (OCTA) and the commercial
polyoxometalate (NH4)6[Mo7O24]4H2O (HEPTA) [2] in a variety of
cancer cell lines.
The POMs have been tested against five types of human cancer
cell lines and the results showed that POMs promoted morphological
changes and repressed the proliferation of all the cancer cell lines
studied in a dose-dependent manner. We have observed that these
POMs dramatically induced cell cycle arrest in G2/M phase and
caused a remarkable DNA damage, DNA double-strand breaks (DSB)
which are potentially lethal lesions for the cells. We found an increase
of histone H2AX phosphorylation (cH2AX) after treatment, which
provides a sensitive probe of the induction of DSBs in nuclear foci,
and DNA fragmentation, analyzed by Comet assay. POMs treatment
also led to an increased intracellular reactive oxygen species (ROS)
formation.
These results show that POMs induce an increased in intracellular
ROS formation, that causes oxidative stress and DNA damage, which
leads to G2/M cell phase arrest and proliferation inhibition. These
observations provide an evidence for a new anticancer mechanism of
POMs and suggest a potential therapeutic strategy, targetly the dysfunctional redox regulation in cancer cells. This therapy may tackle
the classical problem of intra-tumor heterogeneity and be widely
applicable to a wide range of tumors.
Financial support by Universidad Francisco de Vitoria is gratefully
acknowledged.
References
1. Yamase T (2005) J Mater Chem 15:4773
2. Ogata A, Yanagie H, Ishikawa E, Morishita Y, Mitsui S, Yamashita
A, Hasumi K, Takamoto S, Yamase T, Eriguchi M (2007) Brit J
Cancer 98:399
P 94
Synthesis and kinetic of metalation
of 2,3,7,8,12,13,17,18-octakis(propyl), N,N,N’,N’tetramethylamino porphyrazines
and 2,3,9,10,16,17,23,24-octa substituted
phthalocyanine
David Isabirye1, Fanyana Mtunzi2, Temitayo Aiyelabola1,3
Department of Chemistry, North –West University, Mafikeng
Campus, South Africa;
2
Department of Chemistry, Vaal University of Technology, South
Africa;
3
Department of Chemistry, Obafemi Awolowo University Ile-Ife,
Nigeria
Tetrapyrrole macrocyclic compounds such as phthalocyanine and
porphyrazines and their metal complexes have found usage in medicine as potential photosensitisers for photo dynamic therapy in the
treatment of cancer and tumour cells, as well as biomedical imaging
agent [1]. Tuning of their properties may be achieved by the modification of their peripheral substituents and the incorporation of varied
metal ions in their central cavity [1]. Peripheral functionalities may be
symmetrical or unsymmetrical. Unsymmetrical substituents include
the porphyrazine-phthalocyanine hybrid which combines the electronic character and extended p electron system of porphyrazines and
phthalocyanines [2].
In this regard three tetrapyrrole macrocyclic compounds
2,3,7,8,12,13,17,18-octakis(propyl)porphyrazine 1a, N,N,N’,N’-tetramethylamino porphyrazine hybrid 2a and 2,3,9,10,16,17,23,24octa substituted phthalocyanine 3a were synthesized and characterized using elemental analysis, FTIR, 1H, 13C NMR and UV–Vis
spectroscopic techniques. Kinetics of their metalation with Cu(II) and
Co(II) was studied and are been reported for the first time. It is
suggested that deformation of the ring, which is a function of their
peripheral functionalities, is essential for effective coordination of the
ligands.
123
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J Biol Inorg Chem (2014) 19 (Suppl 2):S779–S814
RO
R
N
N
N
O
N
N
N
OR
N
O
O
N
N
N
N
N
O
OR
N
N
N
(2a)
OR
R=N(Me)2
(3a)
R=
Financial support by the North West University for a Post-doctoral
Fellowship award and MARSI (Focus Group) NWU is gratefully
acknowledged by T.A
References
1 Tuncer S, Koca A, Gül A, Avcıata U (2012) Dyes Pigments
92:610–618
2 Forsyth TP, Williams DBG, Montalban AG, et al. (1998) J Org
Chem 63:331–336
P 95
Assessment of the anti-cancer activity of the copper
complexes with imidazole and pivalato ligands
Sylwia Godlewska1, Anna Dolega1, Ewa Augustin,2, Katarzyna
Baranowska1, Harald Kelm3
1
Department of Inorganic Chemistry, Gdańsk University
of Technology, Narutowicza 11/12, 80-233 Gdańsk Poland,
[email protected];
2
Department of Biochemistry and Pharmaceutical Technology,
Gdańsk University of Technology, Narutowicza 11/12, 80-233
Gdańsk Poland;
3
Technische Universität Kaiserslautern, Erwin-Schrödinger Strasse
54, 67663 Kaiserslautern, Germany
Despite the development of science and technology progress so far there
have not been found effective drugs for cancer. Many coordination
compounds were investigated due to their antitumor potential. The most
known cis-diamminedichloroplatinum(II) is used as anti-cancer therapeutic agent. The copper complexes are the coordination compounds
possessing anti-tumour properties. Their capability to kill cancer cells is
mainly linked to the induction of oxidative stress [1]. Among various
copper complexes tested as potential anti-cancer drugs, strong antitumor
activity was found in the bis(acetato)bis(imidazole)copper(II) complex.
In the 50 % inhibition dose (ID50) of the cell growth tests using the mouse
cancer cell line B16 melanoma, the cytotoxic effect of this compound
(20 ng/mL) was equivalent to that of the therapeutic drug cis-DDP (8 ng/
mL) and better than that of mitomycin C (100 ng/mL) [2]. Previously
investigated copper complexes with imidazole ligand showed moderate,
concentration dependent cytotoxic effects [3]. We have synthesized and
investigated the anti-cancer effect of copper (II) complex with (4(5)methylimidazole) and pivalato ligands. The compound is more lipophilic
than bis(acetato)bis(imidazole)copper(II) so we have expected greater
cytotoxic activity than in case of previously investigated compound. The
investigation has been limited due to small solubility of the examined
compound in ethanol. The effect of the compound on cell line growth is
considerable.
Financial support by rector of Gdańsk University of Technology is
gratefully acknowledged.
123
References
1. Tardito S, Marchiò L (2009) Curr Med Chem 16:1325–1348
2. Tamura H, Imai H (1987) J Am Chem Soc 109:6870–6871
3. Godlewska S, Jezierska J, Baranowska K, Augustin E, Dołe˛ga A
(2013) Polyhedron 65:288–297
RO
OR
(1a)
OR
N M N
O
O
N
RO
N M N
N M N
N
N
R
P 96
Antitumor potential of copper complexes
with mitochondrion as the cellular target
Xiaoyong Wang
State Key Laboratory of Pharmaceutical Biotechnology, School
of Life Sciences, Nanjing University; State Key Laboratory
of Analytical Chemistry for Life Science, Nanjing University,
Nanjing 210093, People’s Republic of China. [email protected]
Copper complexes are promising antitumor agents for their redox
properties and low toxicity. In this study, the antitumor potential of
copper(II) complexes derived from triphenylphosphonium derivatives
was investigated. Triphenylphosphine (TPP) was introduced into the
complexes for its mitochondrion-targeting ability and lipophilic
character. The complexes are able to cross the cytoplasmic and
mitochondrial membranes of tumor cells and influence the mitochondrial membrane potential more keenly than anticancer drug
cisplatin. The cytotoxicity of the complexes was tested on MCF-7,
HeLa, Skov-3, A549 and cisplatin-resistant A549R tumor cells. The
complexes are more cytotoxic against these cells than cisplatin; particularly, they are highly effective against cisplatin-resistant tumor
cells. The complexes interact strongly with DNA via an intercalation
stabilized by electrostatic force, and display a significant cleavage
activity towards supercoiled pBR322 DNA and cellular DNA through
an oxidative mechanism. The cytotoxicity of the complexes seems to
arise from a multiple mechanism of action, including the modification
of DNA conformation, generation of reactive oxygen species, scission
of DNA strands, and dissipation of mitochondrial membrane potential. This study demonstrates that copper complexes with
mitochondrion-targeting group could be efficient antitumor agents
free of drug resistance to cisplatin.
Financial support by the National Natural Science Foundation of
China (No. 21271101) is gratefully acknowledged.
References
1. Pathania D, Millard M, Neamati N (2009) Adv Drug Deliv Rev
61:1250–1275
J Biol Inorg Chem (2014) 19 (Suppl 2):S779–S814
2. Zhou W, Wang XY, Hu M, Zhu CC, Guo ZJ (2014) Chem Sci. doi:
10.1039/C4SC00384E
P 97
Cobalt-coordination and oxidation state
in a heterobimetallic complex
Marcel Swart1,2, Abril C. Castro1
1
Institut de Quı́mica Computacional i Catàlisi & Departament de
Quı́mica, Universitat de Girona, 17071 Girona, Spain,
[email protected];
2
Institució Catalana de Recerca i Estudis Avançats (ICREA), Pg.
Lluı́s Companys 23, 08010 Barcelona, Spain
In 2010, a new iron-oxygen species was reported [1], in which an
iron-oxygen complex was capped by a Sc3?-moiety. This new species
represents a series of complexes where a Lewis acid is binding to an
metal-bound oxygen or nitrogen. Through extensive molecular
modelling [2] of FeIV-oxo, FeIII-oxo and FeIII-hydroxo complexes and
the new species, it was shown unambiguously that the oxidation state
of iron in this Lewis-acid capped metal-bound oxygen system is ?3,
coinciding with water as secondary axial ligand to scandium. Here we
report our results [3] for a related heterobimetallic complex where
two proposals [4,5] have been brought forward for the oxidation state
of cobalt and its coordinating ligands (see Figure). We have studied
both of them and other possible scenarios in the same rigorous
computational setup.
Financial support by ICREA, MICINN, MINECO and GenCat is
gratefully acknowledged.
References
1. Fukuzumi S, Morimoto Y, Kotani H, Naumov P, Lee YM, Nam W
(2010) Nature Chem 2:756–759
2. Swart M (2013) Chem Commun 49:6650–6652
3. Castro AC, Swart M (2014) submitted
4. Pfaff FF, Kundu S, Risch M, Pandian S, Heims F, Pryjomska-Ray I,
Haack P, Metzinger R, Bill E, Dau H, Comba P, Ray K (2011) Angew
Chem Int Ed 50:1711–1715
5. Lacy DC, Park YJ, Ziller JW, Yano J, Borovik AS (2012) J Am
Chem Soc (2012) 134:17526–17535
P 98
Peptide-based chelating drugs in diagnosis
and treatment of Alzheimer disease
Ana B. Caballero1, Cristina Fuster1, Ernesto Nicolás1, Jordi
Garcı́a1, Patrick Gamez1,2
1
University of Barcelona, Martı́ i Franquès 1-11, 08028 Barcelona,
Spain, [email protected]; 2Institució Catalana de Recerca i
Estudis Avançats (ICREA), Passeig Lluı́s Companys 23, 08010
Barcelona, Spain
Alzheimer’s disease (AD) is a devastating neuro-degenerative disorder
characterized by the progressive and irreversible loss of memory
S811
followed by complete dementia. Despite the disease’s high prevalence
and great economic and social burden, an explicative aetiology or viable
cure is still not available. Currently available therapeutics for AD only
alleviate its symptoms [1]. The AD-affected brain suffers from metal-ion
homeostasis (metallostasis), resulting in redistribution of metals into
inappropriate compartments. This metal disorder gives rise to the production of amyloid-b aggregates (SPs) and oxidative stress, which are
two associated signs of AD pathology. To date, all clinical trials targeting
amyloid b have failed; however, some clinical trials targeting metal
interactions with amyloid b have all shown benefit for patients [2]. In
addition, recent data indicate that metals play a role more upstream in the
disease process than previously thought and might provide new targets
for pharmacotherapy [3]. Consequently, targeting metals probably represents a tractable avenue for an AD-modifying therapy.
In this context, early detection of copper ion and the recuperation of
its normal trafficking in the brain represent an attractive new therapeutic approach in AD. With the aim of developing biocompatible
chelating drugs with sensing properties, we present the synthesis of a
series of natural and non-natural fluorescent peptides and preliminary
studies on their binding and selectivity towards copper(II).
Financial support by the University of Barcelona and the Ministerio de Economı́a y Competitividad (MINECO) of Spain (Project
CTQ2011-27929-C02-01) is kindly acknowledged. ABC and PG are
thankful to COST Actions CM1003 and CM1105.
References
1. Nazem A, Mansoori GA (2011) Insciences J 1:169–193
2. Faux NG, Ritchie CW, Gunn A, Rembach A, Tsatsanis A, Bedo J,
Harrison J, Lannfelt L, Blennow K, Zetterberg H, Ingelsson M,
Masters CL, Tanzi RE, Cummings JL, Herd CM, Bush AI (2010) J
Alzheimers Dis. 20:509–516
3. Lei P, Ayton S, Finkelstein DI, Spoerri L, Ciccotosto GD, Wright
DK, Wong BX, Adlard PA, Cherny RA, Lam LQ, Roberts BR, Volitakis I, Egan GF, McLean CA, Cappai R, Duce JA, Bush AI (2012)
Nat Med 18:291–295
P 99
The impact of synthetic analogs of histidine
on copper(II) coordination properties to an albuminlike peptide
Izabela A. Zawisza, Mariusz Mital, Agnieszka PolkowskaNowakowska, Arkadiusz Bonna, Wojciech Bal
Institute of Biochemistry and Biophysics, Polish Academy
of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland,
[email protected]
The purpose of our project was to obtain peptidomimetics possessing
Cu(II) binding properties, which would be useful for biomedical
applications. In this context we used potentiometry, molecular modelling, UV–Vis and CD spectroscopies to characterize the Cu(II)
binding properties of pentapeptide analogs of the N-terminal
sequence of histatin 5. The peptides investigated had a general
sequence DSXAK-am (am stands for N-terminal amide), with X
including His and its three synthetic analogues, (4-thiazolyl)-L-alanine (1),(2-pyridyl)-L-alanine (2), and (pyrazol-1-yl)-L-alanine (3).
The heterocyclic nitrogens present in these analogs were significantly
more acidic than that of the His imidazole.
We found that DSXAK-am peptides were able to bind Cu(II) and
form 4 N complexes in a cooperative fashion, with similar affinities.
These results indicate that acidic heterocyclic amino acids provide a
viable alternative for histidine in peptidomimetics designed for metal
ions binding.
123
S812
J Biol Inorg Chem (2014) 19 (Suppl 2):S779–S814
5. Hasnain SS, Murphy LM, Strange RW, Grossmann JG, Clarke AR,
Jackson GS, Collinge J (2001) J Mol Biol 311:467–473
6. Shearer J, Soh P (2007) Inorg Chem 46:710–719
7. Badrick AC, Jones CE (2009) J Inorg Biochem 103:1169–1175
P 101
Binding of Cu21 to the islet amyloid polypeptide (IAPP)
This work was supported in part by the project ‘‘The impact of
synthetic modifications of the histidine side chain on coordination
properties of analogs of the N-terminal pentapeptide of Histatin 5
towards copper(II) ions.’’ (Grant No. DEC-2012/05/N/ST5/01499)
financed by National Science Centre.
P 100
Probing Cu1 binding properties of two toxic mutants
of the human prion protein fragment
Aleksandra Hecel1, Marek Luczkowski1, Daniela Valensin2
1
Faculty of Chemistry, University of Wroclaw, F. Joliot-Curie 14,
50383 Wroclaw, Poland, [email protected];
2
Department of Chemistry, University of Siena, via A. de Gaspari 2,
53100 Siena, Italy
Prion proteins are responsible for the transmissible spongiform
encephalopathies (TSEs), which is a group of fatal and infectious
neurodegenerative diseases that affect human and diverse animal
species [1]. Prion diseases result in associated with accumulation of
abnormal protein aggregates in diffuse synaptic plaques that result in
neurodegeneration [2]. Prion protein (PrP) exist in at least two conformational states, the normal cellular form (PrPC) and an abnormal
infective form (PrPSc) having higher content of b-sheet structure than
the native protein [3]. Human Prion Protein (hPrPC) is able to
sequester transition metal ions like Cu2? with binding atoms allocated
within octarepeat domain, a part of unstructured N-terminal domain
or outside it to His-96 and His-111 residues of so called ‘‘toxic’’
domain [4,5]. Study on some electrochemical reports suggested that
Cu(I) binding site was located in the region between residues 90 and
114, and X-ray techniques were employed to show that Cu(I) was
coordinated via S, N and O ligands [6]. Using spectroscopic techniques find that the PrP91–124 region encompassing His-96 and His111 can bind a Cu(I) ion in a favourable tetrahedral environment
comprising His-96, His-111, Met-109 and Met-112, but mutation of
histidine residues reduce the Cu(I) affinity of PrP fragment [7]. Two
peptidic analogues of the PrP91–115 fragment were synthesized with
His-96 (PrP91–115 H111A) and His-111 (PrP91–115 H96A) residues
substituted by alanine residues. In this work we have used Ag(I) to
probe the interactions of Cu(I) with peptidic analogues of histidine
residues. Our aim was to evaluate the binding mode, coordination
geometry and affinity of the studied analogues.
Financial support by the National Science Centre (NCN 2011/01/
B/ST5/03936) is gratefully acknowledged.
References
1. Collinge J (2001) Annu Rev Neurosci 24:519–550
2. Shimure H, Hattori N, Kubo SJ (2000) Nat Genet 25:302–305
3. Imai Y, Soda M, Takahashi R (2000) J Biol Chem
275:35661–35664
4. Jackson GS, Murray I, Hosszu LLP, Gibbs N, Waltho JP, Clarke
AR, Collinge J (2001) Proc Natl Acad Sci USA 98:8531–8535
123
Jacob Andersen-Usaj1, Amalie C. Bonde1, Jessica Pihl1, MarieLouise F. Thomsen1, Miki Shavit1, Jeppe T. Pedersen2, Kaare
Teilum3, Peter W. Thulstrup1, Lars Hemmingsen1
1
Department of Chemistry, University of Copenhagen,
Universitetsparken 5, 2100 Cph, Denmark;
2
Department of Pharmacy, University of Copenhagen,
Universitetsparken 2, 2100 Cph, Denmark;
3
Department of Biology, University of Copenhagen, Ole Maaløes Vej
5, 2200 Cph, Denmark
Metal ions may induce protein aggregation and amyloid formation
associated with several medical disorders, for example Alzheimer’s
disease [1,2]. Similarly, islet amyloid polypeptide (IAPP) may be
involved in the etiology of type II diabetes, as amyloid plauqes rich
in IAPP are often found in the pancreas of type II diabetics [3].
However, the knowledge compiled on metal ion–IAPP interactions
[4–7] is much less than for A-b and Alzheimer’s disease. In contrast to human IAPP (hIAPP), the corresponding peptide from rat
(rIAPP) does not appear to aggregate, probably due to the differences in the amino acid sequences displayed below. In terms of
metal ion binding capacity, the R18H substitution from rIAPP to
hIAPP implies that hIAPP may display stronger binding of metal
ions such as Zn2? and Cu2?, and this indeed appears to be the case
for Cu2? [4], although literature data are scarce. In this work we
aim to explore the fundamental properties of the interaction of
Cu(II) with IAPP, i.e. to determine the dissociation constant of the
metal ion–peptide complex, and to identify amino acids coordinating to the metal ion at physiological pH, with a particular focus
on the role of His18. To this end we compare the binding of Cu2?
to rIAPP and the R18H variant of rIAPP.
Financial support by the Lundbeck Foundation, the Danish
Chemical Society and the University of Copenhagen is gratefully
acknowledged.
References
1. Faller P (2009) ChemBioChem 10:2837–2845
2. Pedersen JT, Teilum K, Heegaard NHH, Østergaard J, Adolph H–
W, Hemmingsen L (2011) Angew Chem Int Ed 50:2532–2535
3. Cooper GJS (1994) Endocr Rev 15:163–201
4. Lee EC, Ha E, Singh S, Legesse L, Ahmad S, Karnaukhova E,
Donaldson RP, Jeremic AM (2013) Phys Chem Chem Phys
15:12558–12571
5 Kállay C, Dávid A, Timári S, Nagy EM, Sanna D, Garribba E,
Micera G, De Bona P, Pappalardo G, Rizzarelli E, Sóvágó I (2011)
Dalton Trans 40:9711–9721
6 Yu Y-P, Lei P, Hu J, Wu W–H, Zhao Y-F, Li Y-M (2010) Chem.
Commun 46:6909–6911
7 Abedini A, Raleigh DP (2005) Biochemistry 44:16284–16291
J Biol Inorg Chem (2014) 19 (Suppl 2):S779–S814
P 102
Islet amyloid polypeptide (IAPP)—Structural effects
of Zn21
Miki Shavit1, Amalie Carnbring Bonde1, Jessica Pihl1, MarieLouise F. Thomsen1, Jeppe T. Pedersen1, Jacob Andersen-Usaj1,
Kaare Teilum2, Lars Hemmingsen1, Peter W. Thulstrup1
1
Department of Chemistry, University of Copenhagen,
Universitetsparken 5, 2100 Copenhagen, Denmark;
2
Department of Biology, University of Copenhagen, Ole Maaløes Vej
5 2200 Copenhagen, Denmark, [email protected]
Aggregation of Islet amyloid polypeptide (IAPP), a peptide hormone
secreted by the pancreatic b cells, cause increased b cell apoptosis and loss
of pancreatic mass, together with the buildup of amyloid deposits, in type 2
diabetes[1]. As binding of metal ions may induce aggregation and oligomerization of several other amyloidogenic proteins, among them
amyloid b peptides [2,3] it is of interest to investigate the structural effects
of metal ions on IAPP. We investigate the effects of the Zn2? ion, being
highly prevalent in vivo, with concentrations ranging up to mM during costorage with IAPP in the b-granules prior to secretion [4]. The effects of
Zn2? on IAPP fibrillation have been described previously [5,6], though
much remains to be elucidated regarding the specifics of the metal ion–
peptide interaction. We explore the structural effects of Zn2? through a
CD spectroscopic analysis. Also, via NMR spectroscopy the role of
individual side-chains for the metal ion interaction is explored. Previous
investigations on human IAPP indicated His 18 as important for IAPP’s
ability to bind Zn2? [5]. Here, In order to work with a non-fibrillating
system, we as an initial study work with the truncated rat IAPP 9–37
sequence, comparing it with the corresponding R18H variant.
Financial support by Kemisk Forenings Rejsefond, travel grant
given by The Danish Chemical Society, is gratefully acknowledged.
References
1. Leonardi O, Mints G, Hussain MA (2003) Eur J Endocrinol
149:99–102
2. Pedersen JT, Teilum K, Heegaard NH, Østergaard J, Adolph HW,
Hemmingsen L (2011) Angew Chem Int Ed 50:2532–2535
3. Faller P (2013) ChemBioChem 10:2837–2845
4. Foster MC, Leapman RD, Li MX, Atwater I (1993) Biophys J
64:525–532
5. Brender JR, Hartman K, Nanga RPR, Popovych N, de la Salud Bea
R, Vivekanandan S, Marsh ENG, Ramamoorthy A (2010) J Am Chem
Soc 132:8973–8983
6. Salamekh S, Brender JR, Hyung S, Nanga RPR, Vivekanandan
S, Ruotolo BT, Ramamoorthy A (2011) J Mol Biol 410:294–306
P 103
Metal ion catalyzed oxidation of a human prion
fragment
Gizella Csire1, Csilla Kallay2, Lajos Nagy3, Katalin Varnagy1,
Imre Sovago1
1
Department of Inorganic and Analytical Chemistry, University
of Debrecen, H-4032 Debrecen, Hungary.
[email protected];
2
MTA-DE Homogeneous Catalysis and Reaction Mechanisms
Research Group, H-4032 Debrecen, Hungary;
3
Department of Applied Chemistry, University of Debrecen, H-4032
Debrecen, Hungary
Metal-catalyzed oxidation (MCO) can lead to damage of bio-molecules and this is implicated in oxidative stress, biological aging and
neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease [1]. MCO of proteins is mainly a site-specific process
in which only one or a few amino acids at the metal-binding sites of
the protein are preferentially oxidized. The amino acid residues of
S813
histidine and methionine have been proposed to play important roles
in metal mediated oxidative stress. Histidine oxidation predominantly
forms oxo-histidine [2], methionine oxidation forms methionine
sulfoxide and, under extreme conditions, sulfone [3].
Oxidation of Hu-PrP(103–112) fragment (Ac-SerLysProLys
ThrAsnMetLysHisMet-NH2, SKPKTNMKHM) and its systematically
synthesised mutants (SKPKTNAKHA, SKPKTNAKHM, SKPKT
NMKHA) were studied. Potentiometric and spectroscopic techniques
(UV–Vis, CD and EPR) were used to study the speciation, and bonding
details of copper(II) complexes, the oxidation of the peptide in the
presence of copper(II) ions were studied by HPLC–ESI–MS.
Only 1:1 complexes are formed at any copper(II) ion to ligand
ratios. The histidine residue is the anchoring binding site and the
successive deprotonation and coordination of amide functions takes
place toward the N-termini. In the case of peptides containing
Met109, the thioether donor atom of methionine residue may be
equatorially involved in the binding. Cu(II)/hydrogen peroxide system as oxidizing agent were applied at pH 7.4 where 2 N and 3 N
complexes are formed. Oxidation of the peptides leaded the cleavage
of peptide bonds. Oxidation products were identified by MS/MS.
This research was supported by the European Union and the State of
Hungary, co-financed by the European Social Fund in the framework of
TAMOP 4.2.4.A/2-11-1-2012-0001 ‘National Excellence Program’.
References
1. Stadtman ER, Berlett BS (1998) Drug Metab Rev 30:225–243
2. Kowalik-Jankowska T, Ruta M, Wisniewska K, Lankiewicz L,
Dyba M (2004) J Inorg Biochem 98:940–950
3. Hoshi T, Heinemann S (2001) J Physiol (London) 531:1–11
P 104
Cu ligands with high selectivity over Zn to combat
Alzheimer’s disease
Christelle Hureau1,2, Sabrina Noël1,2, Amandine Conte-Daban1,2,
Adam Day1,2, Emmanuel Gras 1,2, Peter Faller1,2
1
CNRS; Laboratoire de Chimie de Coordination, Toulouse, France ;
2
Université de Toulouse, UPS, INPT; LCC; Toulouse, France,
[email protected]
The aetiology of Alzheimer’s disease is linked to the aggregation of
the amyloid-b peptide, a central event of the so-called amyloid cascade. The intervention of Cu and Zn ions in the amyloid cascade is
widely acknowledged. Cu can (i) form oligomeric aggregates considered as the most toxic species of the aggregation process and (ii) be
involved in Reactive Oxygen Species (ROS) production due to its
redox ability [1]. Because these two effects are deleterious, Cu is a
target of choice for therapeutic approaches based on chelation [2,3].
Recent results on Cu(II) [4,5] and Cu(I) removal and impact on ROS
production and Ab aggregation will be described as well as the
interplay of Zn in such processes [6]. Several strategies such as Cu(II)
and Cu(I) ligation or use of bi-functional ligands encompassing an Ab
recognition moiety complexes will be illustrated.
123
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References
1. Hureau C (2012) Coord Chem Rev 256:2164–2174
2. Rodriguez–Rodriguez C, Telpoukhovskaia M, Orvig C (2012)
Coord Chem Rev 256:2308–2332
3. Noël S, Cadet S, Gras E, Hureau C (2013) Chem Soc Rev
42:7747–7762
123
J Biol Inorg Chem (2014) 19 (Suppl 2):S779–S814
4. Noël S, Perez F, Ladeira S, Sayen S, Guillon E, Gras E, Hureau C
(2012) J Inorg Biochem 117:322–325
5. Jensen M, Canning A, Chiha S, Bouquerel P, Pedersen JT,
Østergaard J, Cuvillier O, Sasaki I, Hureau C, Faller P (2012) Chem
Eur J 18:4836–4839
6. Day A, Conte-Daban A, Faller P, Hureau C (manuscript in preparation)
J Biol Inorg Chem (2014) 19 (Suppl 2):S815–S832
DOI 10.1007/s00775-014-1163-0
POSTER PRESENTATION
Metal–nucleic acid interactions
P 110
Metal-mediated base pairs with 6-substituted purines:
a combined experimental and computational approach
Indranil Sinha1,2, Celia Fonseca Guerra3, F. Matthias
Bickelhaupt3,4, Jens Müller1,2
1
Westfälische Wilhelms-Universität Münster and
NRW Graduate School of Chemistry, Corrensstraße 28/30, 48149,
Münster, Germany;
3
Department of Theoretical Chemistry and Amsterdam Center
for Multiscale Modeling (ACMM), Scheikundig Laboratorium der
Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The
Netherlands;
4
Institute of Molecules and Materials, Radboud University Nijmegen,
Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands
The use of DNA as sequence-specific, self-assembling building
blocks has become a promising promenade for the construction of
bio-inspired nanoarchitectures. In this context, metal-mediated base
pairs have evolved as a convenient and versatile method for the sitespecific functionalization of nucleic acids with metal ions. In metalmediated base pairs, the hydrogen bonds present in natural base pairs
are formally replaced by coordinative bonds to metal ions. Hence,
these transition metal ions can be site-specifically introduced into the
oligonucleotide sequence and are located inside the duplex along the
helical axis [1].
Purine derivatives are of great interest for metal-mediated base
pairs because of their structural similarity to the natural nucleobases.
We performed a systematic study of a family of purine-based artificial
nucleosides with an additional donor moiety attached to the C6
position. Incorporation of these nucleosides into DNA led to a variety
of new metal-mediated base pairs [2]. In principle, different coordination modes are feasible (Watson–Crick edge vs. Hoogsteen edge,
resulting in antiparallel vs. parallel-stranded DNA). The experimental
conditions (ionic strength, pH) can be used to pre-select the conformation of the DNA. Computational studies were used to elucidate the
most stable and preferred conformation as well as the kinetics of the
formation of the metal-mediated base pairs.
Financial support by GSC-MS, NWO-CW, NWO-EW, COST
CM1105 is acknowledged.
References
1. Scharf P, Müller J (2013) ChemPlusChem 78:20–34; Johannsen S,
Megger N, Böhme D, Sigel RKO, Müller J (2010) Nat Chem
2:229–234; Megger DA, Fonseca Guerra C, Hoffmann J, Brutschy B,
Bickelhaupt FM, Müller J (2011) Chem Eur J 17:6533–6544
2. Sinha I, Kösters J, Hepp A, Müller J (2013) Dalton Trans 42:16080–
16089
2
P 111
Electron density misattributions in RNA
crystallographic structures: Mg21 or anions
in ribozymes?
Luigi D’Ascenzo1, Pascal Auffinger1
1
Architecture et Réactivité de l’ARN, Université de Strasbourg,
Institut de Biologie Moléculaire et Cellulaire du CNRS, 15 rue René
Descartes, 67084 Strasbourg, France
Magnesium cations are considered to be essential for structure and
function of RNA systems and were supposed to govern the catalytic
mechanism of all ribozyme types. This paradigm has evolved in the
last decade. Divalent metal ions were confirmed as catalysts in selfsplicing ribozymes but displaced by nucleobases in self-cleaving
ones. In order to understand the role of Mg2? cations in their catalytic
mechanism, reliable crystal structures are required. Yet, the attribution of electron densities to ionic species is not always straightforward
and can lead to quite disturbing misattributions [1]. We present a case
of misattribution that was shown to take place at high concentrations
of either Cl- or SO42- ions and that has been associated with several
ribozyme crystal structures. These systematic errors are associated
with electron densities located at &3.2 Å (Cl-) or &3.8 Å (SO42-)
from electropositive sites such as –NH2 groups (see figure). These
results question further the participation of divalent metals in selfsplicing ribozymes. Such misattribution errors might be avoided
through a better understanding of anion-binding properties to nucleic
acids [1,2].
123
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References
1. Auffinger P, Bielecki L, Westhof E (2004) Structure 12:379–388
2. D’Ascenzo L, Auffinger P (2014) in: Methods in Molecular Biology. Nucleic acids crystallography: Methods and Protocols Ennifar E
(ed.) Humana Press in press.
P 112
Condensation properties of antitumor dinuclear Ru(II)
arene complexes with aliphatic linkers: relation to DNA
binding and cytotoxicity
Olga Novakova1, Martina Stankova1, Jaroslav Malina1,
Bernhard K. Keppler2, Viktor Brabec1
1
Institute of Biophysics, Academy of Sciences of the Czech Republic,
CZ-61265 Brno, Czech Republic. [email protected];
2
University of Vienna, Institute of Inorganic Chemistry, Austria
Ruthenium(II) organometallic complexes have been gaining popular
interest as potential anticancer agents. Biological effects of a number
of these compounds are connected with their binding to nuclear DNA.
The resulting DNA damage triggers downstream effects including
inhibition of replication and transcription, cell cycle arrest, and
apoptosis or necrosis. Structures of new, water-soluble dinuclear
Ru(II) arene compounds with long flexible linkers make it possible
to predict that they can also condense DNA. These complexes,
in which two {(g6-p-isopropyltoluene)RuCl[3-(oxo-jO)-2-methyl-4pyridinonato-jO4]} units are linked by aliphatic chains of different
length [(CH2)n (n = 4, 6, 8, 12)] were found to exert promising
cytotoxic effects in human cancer cells. A pronounced influence of
the spacer length on the in vitro anticancer activity was found.
DNA condensation and cross-linking by these dinuclear Ru(II)
arene compounds were examined by various methods of biophysics,
biochemistry and molecular biology. The complexes bind DNA
forming intrastrand and interstrand cross-links in one DNA molecule.
These dinuclear complexes also form specific DNA lesions which can
efficiently cross-link proteins to DNA. Very interesting phenomenon
specific for the DNA binding of these dinuclear Ru(II) arene complexes is that they form interduplex cross-links that are tethered by
ruthenium–DNA bonds. In accordance with the ability of dinuclear
Ru(II) arene complexes to cross-link two DNA duplexes, the results
of the present work demonstrate that these dinuclear complexes also
condense DNA. The concept for the design of agents based on dinuclear Ru(II) arene complexes with sufficiently long linkers between
two Ru centers may result in new compounds which exhibit a variety
of biological effects and can be also useful in nucleic acids research.
Financial support by the Czech Science Foundation (13-08273S) is
gratefully acknowledged.
References
1. Novakova O, Nazarov AA, Hartinger ChG, Keppler BK, Brabec V
(2009) Biochem Pharmacol 77:364–374
2. Mendoza-Ferri MG, Hartinger ChG, Mendoza MA, Groessl M, Egger
AE, Eichinger RE, Mangrum JB, Farrell NP, Maruszak M, Bednarski PJ,
Klein F, Jakupec MA, Nazarov AA, Severin K, Keppler BK (2009) J
Med Chem 52:916–925
P 113
X-ray crystal structure of an octameric RNA duplex
and different divalent and trivalent metal ions
Michelle F. Schaffer1, Joachim Schnabl1, Bernhard
Spingler1, Guanya Peng2, Vincent Olieric2, Roland
K.O. Sigel1
123
J Biol Inorg Chem (2014) 19 (Suppl 2):S815–S832
1
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland;
2
Swiss Light Source at Paul Scherrer Institute, 5232 Villigen,
Switzerland
The principle of charge neutralization and electrostatic condensation
require cations to overwhelm the repulsive forces of the negatively
charged backbone of RNA to adopt its three-dimensional structure
[1,2]. A precise structural knowledge of RNA-metal ion interaction is
crucial to understand the role of metal ions in the catalytic or regulatory activity of RNA [1,3]. In our study we use an octameric RNA
duplex as a model system to investigate the coordination of various
metal ions to specific binding sites and to understand the interactions
between metal ions and RNA.
We were able to solve the crystal structure of the octameric RNA
duplex in presence of six different di- and trivalent metal ions and
could extend the knowledge of the influence of metal ions for conformational changes in RNA structure
The results reveal the strong influence of cations for a more
compact RNA structure, although the kind of metal ions employed
has structurally no particular influence. We considered different
parameters to carefully assign the positions of the metal ions and
suggest two prevalent positions in the investigated octameric RNA
structures. One is located at the phosphate backbone, the second
cation is in the centre of the RNA, interacting by a particular innersphere binding to O4 of uracil in presence of calcium, cobalt and
copper. In addition our study reveals for the first time a RNA structure
associated with copper.
Financial support by the ERC, the Swiss National Science Foundation and the University of Zurich is gratefully acknowledged.
References
1. Sigel RKO (2005) Eur J Inorg Chem 12:2281–2292
2. Freisinger E, Sigel RKO (2007) Coord Chem Rev 251:1834–1851
3. Pyle AM (2002) J Biol Inorg Chem 7:679–690
P 114
Dissecting the role of cations in RNA tertiary structure
formation by single-molecule fluorescence
Sebastian L. B. König, Danny Kowerko, Mokrane
Khier, Roland K. O. Sigel
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland. [email protected]
RNA folding and function are largely dependent on the presence of
cations. Using single-molecule Förster Resonance Energy Transfer
(smFRET) and intron–exon recognition sequences of the self-splicing
group II intron Sc.ai5c as a model system (IBS1*/d30 EBS1*, see
Figure), we have dissected the influence of eleven M2? ions along the
extended Irving-Williams series on RNA–RNA interaction [1,2].
Rigorous analysis of the thermodynamics and the kinetics reveals that
the metal ion acts as a cofactor in both the docking and the undocking
reaction. Excellent agreement with the characteristic metal ion complex stabilities along the extended Irving-Williams series shows that
RNA/RNA docking depends on ionic strength and relies on nitrogen
or carbonyl coordination, while undocking chiefly depends on the
disruption of specific cation-phosphate bonds (see Figure) [2].
This study shows for the first time that RNA/RNA interaction
correlates with the intrinsic coordination chemistry of the metal ion
involved. It is further the first application of smFRET in a systematic
characterisation of cation-dependent nucleic acid structure formation.
Financial support by the European Research Council (MIRNA N°
259092), the Swiss National Science Foundation and the University
of Zurich (FK-57010302 and FK-13-108) is gratefully acknowledged.
J Biol Inorg Chem (2014) 19 (Suppl 2):S815–S832
S817
P 116
Peptide nucleic acids: superior FRET-labels for single
molecule studies of RNA structure and folding
Anita G. Schmitz, Susann Zelger-Paulus, Philipp
Anstaett, Gilles Gasser, Roland K. O. Sigel
References
1. Kruschel D, Skilandat M, Sigel RKO (2014) RNA 20:295–307
2. Sigel H, Griesser R (2005) Chem Soc Rev 34:875–900
P 115
Structure and stability of the human BCL2 RNA
G-quadruplex
Alicia Domı́nguez-Martı́n, Roland K. O. Sigel
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland. [email protected]
Guanine-quadruplexes (G4) are structures formed within guanine-rich
DNA or RNA sequences when four guanine bases are associated
through cyclic Hoogsteen hydrogen bonds forming planar G-quartets.
These quartets stack onto each other resulting in a right-handed
helical conformation stabilized by the presence of metal ions [1].
Recently, the in cellulo existence of RNA G-quadruplex structures in
mRNAs has been evidenced [2]. For example, RNA G4 present in the
50 -unstranslated region of the mRNA of the BCL2 (B Cell Lymphoma/leukemia-2) proto-oncogene was shown to down-regulate the
expression of BCL2 proteins over expressed in several types of
human cancers [3,4]. In this context, the study of this RNA G4
sequence is of great relevance for future novel anticancer therapy.
We evaluated the thermal stability of the BCL2 RNA G4 through a
series of UV melting experiments. CD spectroscopy has also been used
to probe the G-quadruplex folding topology based on well-known
patterns in CD spectra. G-quadruplex formation and structure was
observed by monitoring the imino proton region of 1H NMR spectra.
CD and NMR titrations clearly show the stabilization of the structure
upon addition of KCl. However, time evolution experiments suggest
the existence of more than one G-quadruplex stable conformation.
Financial support by the Fundacion Ramon Areces (A.D.M.), the
Swiss National Science Foundation (R.K.O.S.), within the COST
Action CM1105 (Swiss State Secr. Res. Innov. to R.K.O.S.) and by
the University of Zurich is gratefully acknowledged.
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland. [email protected]
We are interested in studying the dynamic folding behaviour of large
RNAs via single molecule Förster Resonance Energy Transfer
(smFRET). One of the systems that we are investigating is the group II
intron of Saccharomyces cerevisiae, a catalytically active ribozyme
[1]. A very challenging and crucial step is the labelling of such large
RNA constructs with fluorophores. To this end, the current state-of-art
technique is to add short complementary DNA-sequences that carry
the fluorophores. However, the binding strength of these labels is
generally fairly weak and heavily dependent on the conditions. More
efficient labelling techniques are therefore of great interest. Peptide
nucleic acid (PNA) is a non-natural analogue of DNA. It has been used
extensively due to its superior binding strength towards DNA and
RNA. So far, the influence of PNA on the folding behaviour of flexible
RNA constructs has not been sufficiently investigated and it remains
unclear if it can be used as an unbiased probe in smFRET studies.
Herein, we present first experimental evidence, that indeed the binding
strength of PNA based labels is superior to DNA based ones. Furthermore, the catalytic activity of the group II intron is similar with
both labels, as well as with unlabelled RNA. smFRET studies show
comparable general trends, but also differences between DNA- and
PNA-labelled constructs. These differences have implications for the
interpretation of the RNA structure and its flexibility [2].
Financial support by an ERC starting Grant 2010 (259092-MIRNA to R. K. O. S.), within the COST Action CM1105, and the
University of Zurich is gratefully acknowledged.
References
1. Steiner M, Karunatilaka KS, Sigel RKO, Rueda D (2008) Proc
Natl Acad Sci USA 105:13853–13858
2. Schmitz AG, Zelger-Paulus S, Gasser G, Sigel RKO (2014)
submitted
P 117
Reversible stabilization of transition metal-binding
DNA G-quadruplexes
David M. Engelhard1, Roberta Pievo2, Guido H.
Clever1
References
1. Halder K, Hartig JS (2011) Met Ions Life Sci 9:125–139
2. Biffi G, Di Antonio M, Tannahill D, Balasubramanian S (2014) Nat
Chem 6:75–80
3. Bugaut A, Balasubramanian S (2012) Nucleic Acids Res 40:4727–
4741
4. Adams JM, Cory S (1998) Science 281:1322–1326
1
Institute for Inorganic Chemistry, Georg-August-University
of Göttingen, Tammannstraße 4, 37077 Göttingen, Germany.
[email protected]
2
Max Planck Institute for Biophysical Chemistry, Am Fassberg 11,
37077 Göttingen, Germany
Among the secondary structure motives of DNA, G-quadruplexes are now intensely studied as research indicates that
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G-quadruplex formation is preventing human telomere elongation, and therefore shortening cancer cell lifetimes, as well as
participating in gene expression of oncogenes [1]. G-quadruplexes self-assemble from guanine-rich oligonucleotides by
Hoogsteen base pairing. Differences in strand orientation and
connecting loops of the oligonucleotides give rise to a high
diversity of topologies, one of many remarkable aspects which
render G-quadruplexes valuable as tools in the fields of diagnostics and DNA nanotechnology [2]. In this respect it is
interesting to gain control over topology formation and the
stability of G-quadruplex assemblies, e.g. by regulation of deand renaturation, as well as to incorporate non-biogenic functionalities (e.g. fluorescence or magnetism).
We synthesized tetramolecular G-quadruplexes with a terminal ‘‘metal base-tetrad’’, which self-assemble from Guaninerich oligonucleotide strands. Each strand is modified with a
covalently bound pyridine unit, together capable of transition
metal coordination. The formation of the G-quadruplexes in
buffer solution, together with their ability to reversibly coordinate transition metal ions, was monitored by UV–VIS and CD
spectroscopy, as well as gel electrophoresis and EPR studies
[3].
Currently we are extending this strategy to more complex
G-quadruplex metal base-tetrad constructs with a focus on metal
interactions and quadruplex topology.
J Biol Inorg Chem (2014) 19 (Suppl 2):S815–S832
population for each conformational state depending on the mutated
nucleobases [3]. Although essential for the folding process, Mg2?,
even at high concentration, cannot compensate the presence of the
mutations. From the information obtained by the different mutations
we understand the importance of different tertiary contacts. We can
interpret them and obtain the timeline from unfolded, via intermediates, to the folded form of the ribozyme.
Financial support by the European Research Council (to R.K.O.S.)
and the University of Zurich is gratefully acknowledged.
References
1. Pyle AM (2010) Crit Rev Biochem Mol Biol 45:215–232
2. Steiner M, Rueda D, Sigel RKO (2009) Angew Chem Int Ed 48:
9739–9742
3. König SLB, Hadzic M, Fiorini E, Börner R, Kowerko D, Blanckenhorn WU, Sigel RKO (2013) PLoS ONE 8:e84157
P 119
Aromatic-ring stacking between indole and nucleobase
residues. The effect of bridge formation
Astrid Sigel, Helmut Sigel
References
1. Collie GW, Parkinson GN (2011) Chem Soc Rev 40:5867–5892
2. Davis JT (2004) Angew Chem Int Ed 43:668–698
3. Engelhard DM, Pievo R, Clever GH (2013) Angew Chem Int Ed
52:12843–12847
P 118
From bulk to single molecule RNA studies: Point
mutations reveal specific intra domain interactions
essential for group II intron ribozyme folding pathway
Erica Fiorini, Danny Kowerko, Richard Börner
and Roland K. O. Sigel
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland. [email protected]
Group II introns belong to the class of self-splicing ribozymes and are
genetic elements found in the genome of bacteria, plants and lower
eukaryotes [1]. These RNAs are active upon formation of specific
long-range tertiary interactions that define a precise conformation
influenced by co-factors such as Mg2? [2]. We study the folding
pathway of the Sc. ai5c group II intron through point mutations in the
RNA sequence in positions responsible for inter-domain docking.
Combining bulk activity assays and single molecule Fluorescent
Resonance Energy Transfer (smFRET) experiments we test the effect
of these mutations on the catalytic activity and folding pathway of this
ribozyme. In both, bulk and single molecule experiments, different
mutations have distinct kinetic effects on the activity and the folding.
In particular smFRET allowed us to quantify the differences in the
123
Department of Chemistry, Inorganic Chemistry, University of Basel,
Spitalstrasse 51, CH-4056 Basel, Switzerland.
[email protected]
Aromatic-ring stacking is prominent among the noncovalent interactions occurring in biosystems; they are important, e.g., for the
formation of protein-nucleic acid adducts and they are crucial for
selectivity [1]. We consider here the interactions between the amino
acid tryptophan, H(Trp)±, and the nucleotides adenosine 50 -monophosphate, AMP2-, or cytidine 50 -monophosphate, CMP2-. The
corresponding stability constants are listed in the Table (1H NMR;
D2O; 27 °C; I = 0.1–0.15 M, KNO3 [1]). Clearly, adduct formation
Entry
(T)(N)
-1
K(N)
(T)(N) (M )
1
(Trp)(AMP)3-
2.24 ± 0.58
2
[H(Trp)](AMP)2-
6.83 ± 1.62
3
(Trp)(CMP)3-
0.14 ± 0.05
4
[H(Trp)](CMP)2-
0.77 ± 0.42
is more pronounced between H(Trp)± and AMP2- than between
Trp- and AMP2-. The repulsion between the –COO- (Trp)- and
the –PO32- (AMP2-) groups is expected to be small due to their
distance. Hence, the main reason for the enhanced stability of the
[H(Trp)](AMP)2- adduct is that in H(Trp)± the amino group
carries a proton and thus an interaction between the –NH3? group
and the –PO32- unit of AMP2- occurs, giving rise to an ionic
bridge (or hydrogen bond) between the indole and adenine residues forming the stack. This bridge enhances the adduct stability
J Biol Inorg Chem (2014) 19 (Suppl 2):S815–S832
(entry 2) by a factor of ca. 3 and thus facilitates the indoleadenine recognition. The analogous observation is made with the
CMP2- systems (entries 3, 4) though these adducts are considerably less stable than those formed with AMP2- due to the
smaller size of the pyrimidine compared with the adenine residue.
With the AMP2- systems, three different isomers may form with
H(Trp)±, which are in equilibrium with each other [1]: the
stacked form [H(Trp)AMP]2st , the adduct with a sole ionic
interaction [H(Trp)AMP]2ii , and the ‘‘closed’’ isomer in which
the stack is bridged by the intramolecular NH3?/PO32- interaction, [H(Trp)AMP]2cl ; their formation degrees are 33, 15, and
53 %, respectively [1]. These calculations are based on the values
in the Table and on the stability constant of the ionic adduct,
KAMP
[H(Trp)]AMP/ii = 1.0 [1]. Evidently, the ionic bridge (or hydrogen
bond) can be replaced by a bridging metal ion that coordinates
glycinate-like to Trp- and also to the phosphate group of AMP2-.
With Ni2? as bridging metal ion (with Zn2? or Cu2? precipitation
occurred), the ‘‘closed’’ isomer in the intramolecular equilibrium,
open closed, amounts to about 57 %; with CMP2- the closed
ternary complex forms only to about 11 % (possibly not at all)
[2]. Overall it is no surprise to find that protein-nucleic acid
interactions occur in Nature. among others, on the basis of Trp/
indole-AMP/adenine stacks (see, e.g., [1]).
Supported by the Department of Chemistry of the University of
Basel.
References
1. Sigel A, Operschall BP, Sigel H (2014) J Biol Inorg Chem 19: I
Bertini memorial issue
2. Yamauchi O, Odani A, Masuda H, Sigel H (1996) Met Ions Biol
Syst 32: 207–270
P 120
Chemical modifications of coenzyme B12 allow
to investigate the interaction mechanism of the B12-btuB
riboswitch system
Anastasia Musiari, Sofia Gallo, Roland K.O. Sigel
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland, [email protected]
The btuB riboswitch is a bacterial RNA sequence able to control gene
expression of a protein involved in the B12 transport by specifically
binding coenzyme B12, its natural metabolite [1]. Coenzyme B12 can
interact with this large RNA through different moieties. Our research
focuses on the understanding of this binding mechanism, investigating the influence of B12 derivatives differently modified. Previous
works already demonstrated the importance of the adenosyl moiety
for the affinity of the metabolite to the RNA, meanwhile the corrin
ring is determinant for the correct structural rearrangement of the
riboswitch [2,3]. Moreover, recent X-ray structures of B12-riboswitches highlighted the presence of a specific binding pocket for the
adenosyl moiety, meanwhile the great hydrogen-bonding potential of
coenzyme B12 is not fully exploited [4,5].
To continue this structural study and to elucidate the role of a
correct H-bonding and electrostatic network for the B12-RNA interaction, we synthesized new B12 derivatives modified on the corrin
ring sidechains b and e. In both coenzyme B12 and vitamin B12, many
primary amide sidechains are protruding from the corrin ring. We
modified these sidechains to study how the presence of a carboxylic
group or secondary/tertiary amidic groups influences the structural
rearrangement of the btuB riboswitch. Both vitamin B12 and coenzyme B12 derivatives have been synthesized and tested. To investigate
S819
the impact of these chemical modifications, we exploited in-line
probing assays.
The experiments performed in this work confirmed the importance
of the adenosyl moiety to get a high binding affinity to the riboswitch.
Indeed coenzyme B12 derivatives show an affinity in the nanomolar
range, meanwhile vitamin B12 derivatives have an affinity in the
micromolar range. Chemical modifications on the sidechain e seem to
affect the structural rearrangement of the RNA but not the affinity.
Meanwhile the presence of a secondary amide on the sidechain
b increases the affinity to the riboswitch and leads to differences in the
cleavage pattern of the RNA. The presence of a negative charge in the
corrin ring sidechains usually decreases the binding affinity and leads
to differences in the RNA structural rearrangement.
Financial support by the European Research Council (ERC starting grant 2010 259092-MIRNA to R.K.O.S.), the Swiss State
Secretariat for Education Research and Innovation (COST Action
CM1105) and the University of Zurich is gratefully acknowledged.
References
1. Nahvi A, Sudarsan N, Ebert MS, Zou X, et al. (2002) Chem Biol
9:1043–1049
2. Gallo S, Oberhuber M, Sigel RKO, Kräutler B (2008) ChemBioChem 9:1408–1414
3. Gallo S, Mundwiler S, Alberto R, Sigel RKO (2011) Chem Commun
47:403–405
4. Peselis A, Serganov A (2012) Nat Struct Mol Biol. 19:1182–1184
5. Johnson JE Jr, Reyes FE, Polaski JT, Batey RT (2012) Nature
492:133–137
P 121
Gene regulation on the RNA level. The B12 dependent
btuB riboswitch studied with single molecule FRET
Richard Börner, Michelle Schaffer, Sofia Gallo, Roland
K.O. Sigel
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland. [email protected]
The btuB riboswitch is a promising candidate for understanding gene
regulation at the RNA level [1,2]. This B12 specific RNA is encoded
in the 50 -untranslated region (UTR) of the btuB gene encoding a
coenzyme B12 (AdoCbl) transporter found among other bacteria,
especially in E. coli. Upon binding of B12, a conformational switch of
the btuB aptamer occurs, thus inhibiting the expression of the cellular
B12 transporter (compare Figure). This controls the uptake of AdoCbl
and ensures its constant level in the cytosol. RNA as polyanionic
polymer is known to form secondary structures as well as tertiary
contacts upon binding of mono and divalent metal ions. The folding
of the btuB is therefore strongly dependent on the metal ion concentration [3]. In particular, the presence of Mg2? is essential for the
formation of different conformational states and thus for the functional switch of the aptamer upon cofactor binding.
Herein, we use Förster resonance energy transfer on a single
molecule level (smFRET) to characterize the conformational
states and the folding kinetics of the aptamer region of the btuB
riboswitch. We developed a modified sequence of the btuB riboswitch labelled with the Cy3–Cy5 fluorophore pair and biotin
for surface immobilization. Preliminary folding studies on the
bulk level ensure for best in vitro folding conditions. Inline
probing experiments test the switching behaviour and control for
the activity of the FRET construct. Both will allow for thorough
comparisons with former bulk studies [1–3]. The investigation of
the btuB riboswitch on the single molecule level will complement our experiments. Thereby, we will especially focus on the
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influence of AdoCbl and the function of Mg2? for folding and
switching to propose a first kinetic model for the btuB
riboswitch.
Financial support by the University of Zurich, the European
Research Council (to R.K.O.S.), the Swiss National Science Foundation and the Swiss State Secretariat for Education and Research
(COST Action CM1105) is gratefully acknowledged.
J Biol Inorg Chem (2014) 19 (Suppl 2):S815–S832
hand, the intercalation can be affected due to small distortions of the
overall structure, or due to a direct interaction of the HgII ions with
the metal complex resulting in altered electronic properties of the
metal complex. Furthermore, we observed a strong difference in the
intercalative properties to the different DNA models between the K
and D enantiomers.
Financial support by the University of Zurich and within the
COST Action CM1105 is gratefully acknowledged.
References
1. Takezawa Y, Shionoya M (2012) Acc Chem Res 45:2066–2076
2. Scharf P, Müller J (2013) ChemPlusChem 78:20–34
3. Kondo J, Yamada T, Hirose C, Okamoto I, Tanaka Y, Ono A
(2014) Angew Chem 126: 2417–2420
4. Lim M H, Song H, Olmon E D, Dervan E E, Barton J K (2009)
Inorg Chem 48:5392–5397
References
1. Winkler WC, Breaker RR (2003) ChemBioChem 10:1024–1032
2. Perdrizet GA, Artsimovitch I, Furman R, Sosnick TR, Pan T (2012)
Proc Natl Acad Sci USA 109:3323–3328
3. Choudhary PK, Sigel RKO (2014) RNA 20:36–45
P 122
Influence of HgII on the intercalation
of [Ru(bpy)2(dppz)]21 to DNA
Bhaumik S. Dave, Silke Johannsen, Roland K. O. Sigel
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland. [email protected]
DNA, due to its robust structural features and unique self-assembly
properties, is a very attractive molecule for application in nanotechnology and medicinal technology. Replacing the hydrogen bonds
between the complementary nucleobases by coordinative bonds to
transition-metal ions generates the so called metal-mediated base
pairs [1]. One of the most interesting applications is the usage of these
metal-modified biomolecules as building block for nanowires. Metalmodified nucleic acids are expected to possess better conductive
properties than their natural counterparts due to the functionalization
with metal ions along the helical axis [2]. The best studied metal
mediated base-pair is the Thymine-HgII-Thymine base pair. In this
case the rather unstable thymine–thymine mismatch is strongly stabilised by the coordination of one HgII ion between the two opposite
N3 nitrogens of the thymine–thymine base pair. In the absence of HgII
ions, the mismatch region adopts an unusual non-helical fold that
upon addition of HgII converts into a stable B-helical conformation
[3].
In this study we use the metallointercalator [Ru(bpy)2(dppz)]2? to
observe small structural deviations between a natural, T–T mismatched and a HgII modified 17 bp long DNA. The emission
properties of the [Ru(bpy)2(dppz)]2? complex, a well-known DNA
Light-switch, are known to be strongly dependent on the intercalation
site [4]. We characterised the binding interaction of the racemic
[Ru(bpy)2(dppz)]2? complex and the separated D and K enantiomers
using spectroscopic techniques like UV–VIS, CD, Fluorescence and
NMR. Our first results show that the emission is significantly influenced by the presence of HgII ions within the DNA helix. The much
lower emission observed for the HgII-modified DNA compared to the
natural and T–T mismatched sequences can be twofold. On the one
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P 123
Structural investigation of the D1jfext region of group
II intron Sc.ai5c
Serranda Gashi, Maria Pechlaner, Roland K. O. Sigel
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland. [email protected]
Group II introns are self-splicing RNAs and belong to the class of
large ribozymes [1]. They share a common ancestry with the
eukaryotic spliceosome and have possible applications in biotechnology and gene therapy raising the interest in their structure and
mechanism. Group II introns have a highly conserved secondary
structure comprising six domains around a central wheel that folds
into a complex three-dimensional structure in the presence of Mg2?
ions [2]. Domain 1 (D1) and domain 5 (D5) of this large ribozyme
serve as the minimal catalytically active structure [3]. Both, folding
and catalytic activity is only achieved in the presence of Mg2? ions.
The first folding step is carried out by a small region within D1, a
three-way junction containing the so called jf element (D1jf). This
jf element provides also the platform for D5 docking, forming the
catalytic core of the group II intron. We have recently solved the
NMR solution structure of the D1jf region from the yeast mitochondrial group II intron Sc.ai5c in the presence of Mg2? ions [4].
However, under our experimental conditions we could not achieve D5
docking probably due to false intradomain interactions [4]. In this
project we are interested to investigate the extended D1jf region
(D1jfext) including the neighbouring coordination loop. The presence of the coordination loop is expected to disrupt the intradomain
interaction and thus favouring the D1jf/D5 docking. To reach our
final goal we first focus on the structural characterization of the
D1jfext region in solution by NMR spectroscopy. We started using a
57 nt long construct containing both, the j region and the coordination loop. Preliminary results confirmed that the three-way junction is
stabilized by Mg2? ions [4], while interestingly the coordination loop
seems to adopt a defined structure already in the absence of Mg2?
ions.
Financial support by the Swiss National Science Foundation
(RKOS) and the University of Zurich is gratefully acknowledged.
References
1. Sigel RKO (2005) Eur J Inorg Chem 2005: 2281–2292
2. Steiner M, Karunatilaka KS, Sigel RKO Rueda D (2008) Proc Natl
Acad Sci USA 105:13853–13858
3. Michels WJJ, Pyle AM (1995) Biochemistry 34: 2965–2977
4. Donghi D, Pechlaner M, Finazzo C, Knobloch B, Sigel RKO
(2013) Nucl Acids Res 41:2489–2504
J Biol Inorg Chem (2014) 19 (Suppl 2):S815–S832
P 124
Following inter- and intramolecular dynamics of single
encapsulated RNA molecules by FRET spectroscopy
Mélodie C.A.S. Hadzic, Sebastian L.B. König, Danny
Kowerko, Roland K.O. Sigel
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland. email: [email protected]
Single molecule FRET (Förster resonance energy transfer) is a stateof-the-art technique to investigate molecular dynamics revealing
sparse spatial configurations and kinetic heterogeneities hidden in
bulk experiments [1]. In bimolecular reactions, direct surface
immobilisation of one reactive element allows long observation time
but might bias the authenticity of the response. Co-encapsulation in
surface-tethered vesicles ensures both free diffusion and constant
proximity of the reactants. After experimental procedure optimisation, we co-encapsulated in a 1:1 ratio a previously studied RNA
duplex (fluorophore-labelled EBS1* and IBS1*) and followed the
successive association/dissociation kinetics over time by 2-colour smFRET depending on the Mg2? concentration. As encapsulation permits to keep both RNAs in focal position—regardless if the duplex is
formed or dissociated—we could alternatively collect the constant
Cy5 emission from direct excitation by alternating laser pulses.
Hereby, we detected a new type of event unknown from previous
surface tethered studies, in which all fluorescence is quenched
simultaneously. Significance tests based on the evaluation of the
cross-sample variability [2], reveal a new conformation displaying
Cy3–Cy5 distance lower than 2 nm.
Financial support from the ERC Starting Grant (to R.K.O. Sigel),
and the Forschungskredit of the University of Zürich (to M.C.A.S
Hadzic) is gratefully acknowledged.
References
1. König SLB, Kowerko D., Sigel RKO (2013) CHIMIA 67: 240–243
2. König SLB, Hadzic MCAS, Fiorini E, Börner R, Kowerko D, Sigel
RKO (2013) PLoS ONE 8: e84157
P 125
Biologically relevant RNA G-quadruplex structure
studied at the single-molecule level
Helena Guiset Miserachs, Roland K. O. Sigel
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland. [email protected]
Guanine-rich nucleic acid sequences have the tendency to aggregate
into non-canonical, K?-sensitive helical structures, known as
G-quadruplexes [1]. These sequences are highly enriched in regulatory regions of DNA and RNA, which suggests that they play a role
in vivo [2, 3]. RNA G-quadruplexes, more stable than their DNA
counterparts and easier to form (as RNA is more often single stranded
in the cell), offer new possibilities as novel antitumor targets. Such
structures are often located in regulatory, non-coding regions of the
messenger RNAs (mRNAs) of oncogenes. We chose NRAS (Neuroblastoma RAS viral oncogene homolog), an oncogene that is
S821
overexpressed in some types of leukemias and melanomas. It contains
a 18-nt G-quadruplex sequence in the 50 untranslated region of its
mRNA, which has been shown to be stable and inhibit translation
in vitro [4]:
50 -GGGAGGGGCGGGUCUGGG-30
We are working in setting up a system to visualize the NRAS
RNA molecules individually, via single-molecule Förster Resonance
Energy Transfer (smFRET). We are interested in elucidating the
dynamics and kinetics of the RNA G-quadruplex formation and dissociation, as well as observing possible folding or unfolding
intermediates.
Financial support within the COST Action CM1105, by an ERC
Starting Grant 2010 (R.K.O.S.), a UZH Forschungskredit Grant 2013
(H.G.M.), by the Swiss National Science Foundation (R.K.O.S.) and
by the University of Zurich is gratefully acknowledged.
References
1. Williamson JR (1994) Annu Rev Biophys Biol Struct 23:703–730
2. Maizels N (2006) Nature Struct Mol Biol 13:1055–1059
3. Bugaut A, Balasubramanian S (2012) Nucleic Acids Res 40:4727–
4741
4. Kumari S, Bugaut A, Huppert JL Balasubramanian S (2007) Nat
Chem Biol 3:218–221
P 126
The effect of metal ions on nucleic acids interactions,
seen at the single molecule level
Mokrane Khier, Sebastian L.B. König, Danny
Kowerko1, Roland K.O. Sigel
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland. [email protected]
The coined role of RNA in the ‘‘modern dogma’’ and the increasing
appreciation of RNA for biotechnological and medical applications
demands more than ever a clear picture and fundamental understanding of how RNA folds and acts.
In our study, single molecule Förster Resonance Energy
Transfer (smFRET) has been applied to characterize the effect of
metal ions on an RNA tertiary contact: an RNA hairpin, known as
EBS1* (Exon Binding Site 1), interacting with its cognate (Intron
Binding Site 1). The cognate was either an RNA (IBS1*) or DNA
(dIBS1*) fragment [1]. Our results revealed that the two interactions differ slightly in the conformation of their bound state, in
a qualitative agreement with the NMR results performed on the
same system [2]. In parallel, the thermodynamic and kinetic
analysis shows that the affinity of EBS1* toward the RNA cognate is almost two orders of magnitude higher than its affinity
toward the DNA one, in an independent manner from the nature
of metal ions in the solution. However, in the case of EBS1*–
IBS1* interaction, the presence of Mg2? leads to a pronounced
heterogeneity in the kinetic of this interaction due to the presence
of at least two kinetic rates describing the dissociation process, a
finding that was not observed for the same interaction in the
presence of high amount of K? nor in the case of EBS1*–dIBS1*
in the presence of both Mg2? and high amount of K?.
Based on our results and previous work that had pointed out at
least three potential binding site for metal ions in EBS1*–IBS1*
structure [3], we propose a model for the interaction where the kinetic
heterogeneity seen in the presence of Mg2? results from the different
combination of occupying the three binding pockets within EBS1*–
IBS1* structure.
Financial support by the European Research Council (MIRNA N°
259092), the Swiss National Science Foundation to Roland. K. O.
Sigel and the University of Zurich (FK-570110302 to Sebastian L.
123
S822
B. König and FK-13-108 to Danny Kowerko) are gratefully
acknowledged.
References
1. Su LJ, Qin PZ, Michels WJ, Pyle AM (2001) J Mol Biol 306: 655–
668
2. Skilandat M, Sigel RKO submitted
3. Kruschel D, Skilandat M, Sigel RKO (2014) RNA 20:295–307
P 127
Thermodynamics of the formation of metal-mediated
base pairs
Kristina Schweizer1, Bart Jan Ravoo2, Jens Müller1
1
Institut für Anorganische und Analytische Chemie, Westfälische
Wilhelms-Universität Münster, Corrensstrasse 28/30, 48149 Münster,
Germany. [email protected]; 2Organisch-Chemisches Institut,
Westfälische Wilhelms-Universität Münster, Corrensstrasse 40,
48149 Münster, Germany
The introduction of metal-mediated base pairs is a convenient method
for the site-specific functionalization of nucleic acids [1]. Hence, the
base pair within a nucleic acid double helix is no longer mediated by
hydrogen bonds but rather by coordinative bonds to a central metal
ion.
Artificial nucleosides, in particular azole-based nucleosides
(azole = imidazole, triazole, tetrazole) have proven to be excellent
building blocks for Ag(I)-modified DNA [2]. The association constants for the formation of azole complexes outside the DNA context
are known [2a]. Hence, the constants for the formation of Ag(I)mediated azole base pairs within a DNA double helix were determined with isothermal titration calorimetry. Through a combination
of CD- and UV-spectroscopy and isothermal titration calorimetry we
demonstrated that DNA double helices comprising two neighbouring
artificial imidazole-Ag(I)-imidazole base pairs incorporate the
Ag(I) ions in a cooperative fashion (see illustration) [3]. Other systems, e.g. oligonucleotides with two non-consecutive artificial
imidazole-Ag(I)-imidazole base pairs, were studied as well.
Financial support by the DFG (SFB 858) is gratefully
acknowledged.
J Biol Inorg Chem (2014) 19 (Suppl 2):S815–S832
3. Petrovec K, Ravoo BJ, Müller J (2012) Chem Commun
48:11844–11846
P 128
A metal-mediated base pair with a triazole-based
tridentate ligand
Stefanie Litau, Jens Müller
Institut für Anorganische und Analytische Chemie, Westfälische
Wilhelms-Universität Münster, Corrensstr. 28/30, 48149 Münster,
Germany. [email protected]
The incorporation of artificial base pairs into nucleic acid double
helices is a convenient method for the site-specific functionalization
of these self-assembling biomolecules with metal ions [1]. We have
established various nucleic acid systems with metal-mediated base
pairs derived from azole ligands [2, 3]. Moreover, we recently
developed a metal-mediated base pair with a trigonal planar coordination geometry of the central transition metal ion. This geometry
was achieved by combining one bidentate and one monodentate
ligand [4]. The triazole-based ligand used in that context was the first
of an entire family of ‘‘click’’ nucleosides [5]. The latest addition to
this family is a metal-mediated base pair with a triazole-based tridentate ligand and a complementary monodentate nucleoside. The
synthesis of the new artificial nucleoside and the metal-binding
properties of oligonucleotides containing the respective base pair will
be reported.
Financial support by the Deutsche Forschungsgemeinschaft
(TRR61) is gratefully acknowledged.
References
1. Scharf P, Müller J (2013) ChemPlusChem 78:20–34
2. Johannsen S, Megger N, Böhme D, Sigel RKO, Müller J (2010)
Nat Chem 2:229–234
3. Kumbhar S, Johannsen S, Sigel RKO, Waller MP, Müller J (2013)
J Inorg Biochem 127:203–210
4. Richters T, Müller J (2014) Eur J Inorg Chem 2014:437–441
5. Richters T, Krug O, Kösters J, Hepp A, Müller J (2014) Chem Eur
J (doi: 10.1002/chem.201402221)
References
1. a) Scharf P, Müller J (2013) ChemPlusChem 78:20–34, b) Megger
DA, Megger N, Müller J (2012) Met Ions Life Sci 10:295–317
2. a) Müller J, Böhme D, Lax P, Morell Cerdà M, Roitzsch M (2005)
Chem Eur J 11:6246–6253, b) Böhme D, Düpre N, Megger DA,
Müller J (2007) Inorg Chem 46:10114–10119, c) Johannsen S,
Megger N, Böhme D, Sigel RKO, Müller J (2010) Nat Chem 2:
229–234
123
P 129
Hit to lead: SAR studies of cyclometalated
benzimidazole and dimethylbenzylamine platinum
group metals anticancer compounds
Jose Ruiz1, Gorakh Yellol1, Ana Zamora1, Jyoti Yellol1,
Sergio Perez1, Antonio Donaire1, Venancio Rodrı́guez1,
Alicia Buceta1, Natalia Cutillas1, Patrick J. Bednarski2,
Christoph Janiak3, Vera Vasylyeva3
J Biol Inorg Chem (2014) 19 (Suppl 2):S815–S832
1
Department of Inorganic Chemistry and Regional Campus
of International Excellence (Campus Mare Nostrum), University
of Murcia and Instituto Murciano de Investigación Biosanitaria,
Campus de Espinardo, E-30071 Murcia, Spain.
2
Pharmaceutical & Medicinal Chemistry, Institute of Pharmacy,
Ernst-Moritz-Arndt University Greifswald, 17487 Greifswald,
Germany.
3
Institut für Anorganische Chemie und Strukturchemie Universität
Düsseldorf, D-40204 Düsseldorf, Germany
Organometallic compounds with properties somewhat intermediate
between classical inorganic and organic drugs have recently been
considered as promising alternatives in medicinal chemistry. These
are relatively lipophilic and can be endowed with a huge variety of
functionalized organic ligands with very specific reactivities. Due to
the exciting results from previous studies of dimethylbenzylamine
(DMBA) Pt complexes and some smart phenyl-benzimidazole Ru(II)
and Ir(III) derivatives [1], various sets of small libraries have been
synthesized focusing on different aspects of the core molecules
benzimidazole and DMBA to modulate anticancer activity: (i) to
modulate the lipophilicity by the N-substitution (R2) with different
alkyl and aryl groups; (ii) for SAR studies- different function group
substitutions (R1) such as EDG, EWG, alkyl, aryl groups etc.; (iii)
exploring the aromaticity of the ligand- substituting one phenyl group
by two to four aromatic ones; (iv) linking to heterocyclic moieties
resulting bis-heterocyclic compounds; and, (v) producing different
combinations of secondary ligands attached to the metal (L1 and L2)
like chloro, p-cymene, penta-methylcyclopentadiene, phosphines and
small heterocycles.
Financial support by the European Union Seventh Framework Programme—Marie Curie COFUND (FP7/2007–2013) under U-IMPACT
Grant Agreement 267143, the Spanish Ministerio de Economı́a y
Competitividad and FEDER (Project SAF2011-26611), by Fundación
Séneca (Project 15354/PI/10) and COST ACTION CM1105.
S823
little is known on the effects of platinum drugs on RNA structure and
biology [2–4].
In order to understand how platinum drugs affect RNA
structure we use as a model RNA a 27 nucleotide long construct
derived from the mitochondrial group II intron ribozyme Sc.ai5c
[5]. As platination agent we use oxaliplatin which is FDA
approved anticancer drug that is used in clinics worldwide [6].
Gel electrophoresis mobility shift assays were used to identify
the best experimental conditions to obtain monoplatinated RNA
in high yield and separate it from the unreacted RNA. The
platinated adducts collected from the gels were then further
purified and isolated by HPLC. The characterization of the pure
monoplatinated adducts is now in progress using a combination
of different techniques including mass spectrometry, UV–Vis, CD
and NMR spectroscopy.
Financial support by the Swiss National Science Foundation
(Ambizione fellowship PZ00P2_136726 to DD), by the University of
Zurich and within the COST Action CM1105 is gratefully acknowledged.
References
1. Alderden RA, Hall MD, Hambley TW (2006) J Chem Educ
83:728–734
2. Chapman EG, Hostetter AA, Osborn MF, Miller AL, DeRose VJ
(2011) Met Ions Life Sci 9:347–377
3. Chapman EG, De Rose VJ (2010) J Am Chem Soc 132:1946–1952
4. Hostetter AA, Chapman EG, De Rose VJ (2009) J Am Chem Soc
131:9250–9257
5. Donghi D, Pechlaner M, Finazzo C, Knobloch B, Sigel RKO
(2013) Nucleic Acids Res 41:2489–2504
6. Ibrahim A, Hirschfeld S, Cohen MH, Griebel DJ, Williams GA,
Pazdur R (2004) The Oncologist 9:8–12
P 131
Recognition of an RNA three-way junction by a de novo
designed drug
Susann Zelger-Paulus1, Siriporn Phongtongpasuk2,
Michael J. Hannon2, Roland K. O. Sigel1
1
Reference
1. Yellol GS, Donaire A, Yellol JG, Vasylyeva V, Janiak C, Ruiz J
(2013) Chem Commun 49:11533–11535
P 130
Purification and isolation of platinated RNA
Marianthi Zampakou, Daniela Donghi
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland. [email protected]
Nucleic acids are an important potential drug target and we are currently investigating the way in which platinum anticancer drugs
interact with RNA. These drugs are normally thought to exert their
activity upon covalent binding to DNA purine nitrogens [1]. However
in recent years potential alternative binding partners have been
explored, including RNA [2]. The latter has many important functions
in vivo and the disruption of these processes can have serious consequences [3]. It has already been reported that some RNA dependent
activities are inhibited upon administration of platinum drugs, but still
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland. [email protected].
2
School of Chemistry, University of Birmingham, Edgbaston,
Birmingham B152TT, UK
RNA is a very exciting biomedical target because of its high structural
diversity and the ability to regulate essential processes during RNA
maturation. Ribosomal RNA, the hammerhead or introns of premRNA are only few examples of catalytically active RNAs called
ribozymes. Structure and function of such ribozymes are inextricably
linked with each other. The complex architecture of RNA is represented by countless bulges, loops, helices or junctions. One special
structural element is the Y-shaped three way junction (3WJ) which
can take form of a perfectly shaped or a bulged junction. The
detection and intercalation of such 3WJs by small molecules is of
high importance since various RNA processes could be controlled this
way. We demonstrate the recognition and stabilization of an perfect
RNA 3WJ by a designed cylinder-shaped drug ([Fe2L3]4?) through
native gel studies [1]. In addition, we can illustrate that even bulged
3WJs are stabilized by the iron cylinder. A competition assay with
analogous RNA and DNA sequences and the composition of RNA/
DNAs constructs showed that the 3WJ formation induced by the
cylinder is sequence dependent. In summary, we demonstrate that the
recognition and formation of an RNA 3WJ by the iron cylinder is a
very good approach of how drug design should be faced and offers
high potential towards medical applications.
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Financial support within the COST Action CM1105, from the Swiss
State Secretariat for Education and Research, the University of Zurich,
and the University of Birmingham are gratefully acknowledged.
Reference
1. Phongtongpasuk S, Paulus S, Schnabl J, Sigel RKO, Spingler B,
Hannon MJ, Freisinger E (2013) Angew Chem Int Ed 52:11513–11516
P 132
Rhenium(I)-dppz complexes as potential RNA binding
agents
Elena Alberti1, Michael P. Coogan2, Daniela Donghi1
1
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland. [email protected];
2
Department of Chemistry, Faraday Building, Lancaster University,
Bailrigg, Lancaster, LA1 4YB, United Kingdom
There is an increasing interest in the development of small molecules
as structure-selective binding agents for bioimaging and along this
line luminescent metal complexes have been extensively studied due
to their suitable photophysical, spectroscopic and electrochemical
properties [1, 2]. Interestingly, while their interaction with DNA is
widely studied, a small amount of information is available to date on
their RNA binding properties.
Re(I)-dppz complexes belong to the large class of d-block lumophores which includes also Ru(II) and Ir(III) and their interaction with
DNA has been already studied some years ago [3]. Besides their
binding properties to CT-DNA, it was also shown that their cellular
uptake varies upon changing their axial ligand. It has been proved that
these changes do not affect their optical properties but result in a
different cell localization including RNA-rich regions [3]. Even if it is
well known that Re(I)-dppz complexes are capable of binding nucleic
acids via metallo-intercalation, the field is still rather unexplored,
especially on their RNA interaction. For these reasons we decided to
investigate the interaction of different mononuclear rhenium complexes with several RNA models containing the most common
secondary structural features. Both the effects of varying the RNA
model and the axial ligand of the Re(I)-dppz complexes are evaluated
to rationalize the structural origin of interaction preferences.
We are currently investigating the changes of the optical properties of
one of these complexes upon interaction with a 27 nucleotide construct
by means of UV/Visible and fluorescence spectroscopy. This study
enables us to evaluate the effect of the structural features contained in the
model, e.g. internal and terminal loops on the binding properties.
Moreover, as the NMR structure in solution of this RNA sequence is
available [4], preliminary NMR studies have been performed to determine the site of interaction. Further NMR studies are in progress to
investigate RNA structural changes upon rhenium complex binding.
Financial support by the Swiss National Science Foundation
(Ambizione fellowship PZ00P2_136726 to DD), by the University of
Zurich (including the Forschungskredit FK-13-107 to DD) and within
the COST Action CM1105 is gratefully acknowledged.
123
J Biol Inorg Chem (2014) 19 (Suppl 2):S815–S832
References
1. Coogan MP, Fernández-Moreira V (2014) Chem Commun 50:384–
399
2. Fernández-Moreira V, Thorp-Greenwood FL, Coogan MP (2010)
Chem Commun 46:186–202
3. Thorp-Greenwood FL, Coogan MP, Mishra L, Kumari N, Rai G,
Saripella S (2012) New J Chem 36:64–72
4. Donghi D, Pechlaner M, Sigel RKO unpublished
P 133
The CPEB3 ribozyme pseudoknot is tied
up by magnesium(II)
_
Miriam Skilandat, Magdalena-Rowińska-Zyrek,
Roland K. O. Sigel
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland. [email protected]
The CPEB3 ribozyme is the only small self-cleaving ribozyme that
has been shown to be highly conserved in mammalian genomes [1]. It
is hypothesized to be evolutionary related to the HDV (hepatitis delta
virus) ribozyme since both have the nested double-pseudoknot fold
and the catalytic chemistry in common [1, 2]. However, there is to
date neither an explanation for this relation nor any knowledge about
the role of the CPEB3 ribozyme in the cell.
In this study we focus on the structural effects of metal-ion binding to
CPEB3, which is known to use Mg(II) for its catalytic mechanism. We
present the first results in elucidating the structure of CPEB3 in solution,
obtained by NMR spectroscopy. We demonstrate that, in the absence of
multivalent metal ions, the three major helices P1, P2 and P4 of CPEB3
and thus also the P1-P2 pseudoknot are formed according to the proposed secondary structure [1]. There is evidence for several Mg(II)
binding sites within CPEB3. Binding of Mg(II) leads to a compaction of
the ribozyme and to the formation of additional base pairs and stacking
interactions in the ribozyme core, thereby indicating the formation of the
internal loop P3 and possibly the second pseudoknot.
These results are prove of the remarkable similarity of the CPEB3and HDV fold and shed light on the important role of Mg(II) for
formation of the nested double-pseudoknot motif.
Financial support by the Swiss National Science Foundation
(RKOS), the University of Zurich and a Marie Curie fellowship (No.
PIEF-GA-2012-329700; MRZ) is gratefully acknowledged.
J Biol Inorg Chem (2014) 19 (Suppl 2):S815–S832
References
1. Salehi-Ashtiani K, Lupták A, Litovchick A, Szostak JW (2006)
Science 313:1788–1792
2. Webb C-HT, Lupták A (2011) RNA Biology 8:719–727
P 134
G4-DNA vs. B-DNA binding of Schiff base transition
metal complexes
Giampaolo Barone, Alessio Terenzi, Riccardo
Bonsignore, Angelo Spinello, Anna Maria Almerico,
Antonino Lauria
Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e
Farmaceutiche, Università di Palermo, Viale delle Scienze, Ed. 17,
90128 Palermo, Italy. [email protected]
The competitive binding of nickel(II), copper(II) and zinc(II)
complexes toward B- and G4-DNA was addressed through
spectroscopic titrations and rationalized by computational investigations, consisting of molecular dynamics simulations followed
by density functional theory/molecular mechanics (DFT/MM)
calculations [1]. The experimental DNA binding studies clearly
highlight the selectivity of the compounds, in particular the
nickel(II) complex, toward G4-DNA from both h-Telo and c-myc.
Moreover, the compounds show biological activity against HeLa
and MCF-7 cancer cell lines. Remarkably, the experimental
DNA-binding affinity trend of the three metal complexes,
obtained from the DNA-binding constants as DG° = -RT ln(Kb),
is reproduced by the Gibbs formation free energy calculated by
DFT/MM for the DNA-binding complexes, in the implicit water
solution.
Financial support by the University of Palermo is gratefully
acknowledged.
S825
The CPEB3 ribozyme is a small, highly conserved, mammalian, self
cleaving, non-coding RNA located in the second intron of the cpeb3
gene [1]. Most of the available knowledge about this ribozyme is
based on comparative studies with the HDV (Hepatitis Delta Virus)
ribozyme; both are predicted to fold into a similar pseudoknot
structure and use a self-cleavage mechanism which employs a catalytic site cytosine [1, 2].
In this study, we focus on understanding the impact of Mg2? ions
on the structure of the CPEB3 ribozyme. Several direct metal ion
counting techniques show that there are at least three specific binding
sites for this metal and NMR spectroscopy precisely shows their
localisation. In order to distinguish an outer- from an innersphere
Mg2? binding, [Co(NH3)6]3? is used as a spectroscopic probe for
[Mg(H2O)6]2? [3].
The importance of these metal binding sites is underlined by cotranscriptional cleavage studies, which show the impact of single
mutations of the predicted metal binding sites on the catalytical
activity of the ribozyme.
Financial support by the Marie Curie IEF (no. PIEF-GA-2012329700 to MRZ) and the Swiss National Science Foundation (to
RKOS) is gratefully acknowledged.
References
1. Salehi-Ashtiani K, Luptak A, Litovchick A, Szostak JW (2006) Science
313:1788–1792.
2. Webb C, Riccitelli NJ, Ruminski DJ, Luptak A (2009) Science 326:
953–932
3. Skilandat M, Rowinska-Zyrek M, Sigel RKO (2014) J Biol Inorg
Chem DOI:10.1007/s00775-014-1125-6.
Reference
1. Lauria A, Bonsignore R, Terenzi A, Spinello A, Giannici F, Longo
A, Almerico AM, Barone G (2014) Dalton Trans 43:6108–6119
P 135
NMR localization of Mg21 ions in the mammalian
CPEB3 ribozyme
Magdalena Rowińska-Żyrek, Miriam Skilandat,
Roland K. O. Sigel
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland. [email protected]
P 136
Proton and nitrogen-15 NMR spectroscopic studies
of AgI-mediated C–C base-pairs
Takenori Dairaku1, Itaru Okamoto2, Kyoko Furuita3,
Shuji Oda1, Daichi Yamanaka1, Hidetaka Torigoe4,
Tetsuo Kozasa4 Yoshinori Kondo,1 Akira Ono2, Chojiro
Kojima3, Vladimı́r Sychrovský5, Yoshiyuki Tanaka1
1
Graduate School of Pharmaceutical Sciences, Tohoku University,
Sendai 980-8578, Japan;
2
Faculty of Engineering, Kanagawa University, Yokohama,
Kanagawa 221-8686 Japan;
3
Institute for Protein Research, Osaka University, Suita, Osaka
565-0871, Japan;
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4
Faculty of Science, Tokyo University of Science; Shinjuku-ku,
Tokyo 162-8601, Japan;
5
Institute of Organic Chemistry and Biochemistry, Academy
of Sciences of the Czech Republic, Flemingovo nám. 2, 16610, Praha
6, Czech Republic
The structure of AgI-mediated Cytosine–Cytosine base-pairs (C–AgI–
C) in DNA duplex was not clearly understood. Our previous 1H NMR
spectroscopic studies of DNA duplex with a single C–C mismatch
revealed that the stoichiometry between C–C mismatch and AgI-ion
was 1:1 [1, 2]. In 1H NMR spectra of a self-complementary DNA
duplex1: d(50 -ATAATAATAAACTTTATTATTAT-30 )2 [2] and nonself-complementary DNA duplex2: d(50 -TTAATAATATACT
TAATTATAAT-30 )/d(50 -TTAATAATATACTTAATTATAAT-30 ),
the H5 and H6 signals of the cytosine residues were down-field
shifted by *0.2 ppm upon the formation of the C–AgI–C base-pair.
Also, this characteristic down-field shift was found in 1H NMR
spectra of cytosine mononucleoside [2]. Furthermore, in 15N NMR
spectra of 15N-labeled cytosine mononucleoside, the N3 signal of the
cytosine was drastically up-field shifted by *22 ppm upon the
binding with AgI-ion in conjunction with *0.2 ppm down-field shifts
for H5 and H6 resonances [2]. These data indicated that the binding
site of AgI-ion is N3 of cytosine. In addition, degree of down-field
shifts for H5 and H6 resonances in the cytosine nucleoside
(*0.2 ppm) were the same as those of DNA duplexes. This similarity
in chemical shift perturbations between cytosine and cytosine residues in the DNA duplexes suggested that AgI-binding site in DNA
duplexes were also N3 of cytosine residues. Because 15N NMR
spectroscopy was employed as a powerful tool for determining
chemical structures of metal-mediated base-pairs [3, 4], we will also
discuss 15N NMR spectroscopic data for DNA duplexes.
References
1. Ono A, Cao S, Togashi H, Tashiro M, Fujimoto T, Machinami T,
Oda S, Miyake Y, Okamoto I, Tanaka Y (2008) Chem Commun
4825–4827
2. Torigoe H, Okamoto I, Dairaku T, Tanaka Y, Ono A, Kozasa T (2012)
Biochimie 94:2431–2440
3. Tanaka Y, Oda S, Yamaguchi H, Kondo Y, Kojima C, Ono A
(2007) J Am Chem Soc 129:244–245
4. Johannsen S, Megger N, Böhme D, Sigel RKO, Müller J (2010) Nat
Chem 2:229–234
P 137
A luminescent purine-based ligand for metal-mediated
base pairs
Soham Mandal, Jens Müller
Institut für Anorganische und Analytische Chemie & The Graduate
School of Chemistry, Westfälische Wilhelms-Universität Münster,
Corrensstr. 28/30, 48149, Münster, Germany. [email protected]
DNA, the self assembling supramolecule, plays a pivotal role in
chemical evolution using its capacity to store and transfer genetic
information, its optimized hybridization properties and highly specific
molecular recognition, resulting in diversified applications.
Introducing transition metal ions in DNA by exploiting metalmediated base pairs offers a promising and versatile bottom-up
strategy for its site-specific functionalization [1, 2]. Applicability is
furthermore extended by replacing natural nucleobases by metalmediated non-natural base pairs aiming to expand the genetic four
letter code. This allows formation of a duplex with one or more metal-
123
J Biol Inorg Chem (2014) 19 (Suppl 2):S815–S832
mediated base pairs interspersed between natural ones, helices with a
long stretch of metalated base pairs and duplexes with metal arrays in
a sequence-controlled pre-defined order [3]. Hence, metal-based
properties can be introduced into modified nucleic acids [2]. This
broadens the potential application of DNA as a functionalized nanoscale object.
We report for the first time the use of a luminescent bidentate
purine-based moiety for an application in metal-mediated base pairs.
Its synthesis and detailed characterization, including the formation of
model complexes, will be presented. Moreover, first attempts towards
its incorporation into different DNA sequences and the metal-binding
properties of the resulting nucleic acids will be reported.
Financial support by the Graduate School of Chemistry (GSC MS)
is gratefully acknowledged.
References
1. Müller J (2008) Eur J Inorg Chem 3749–3763
2. Scharf P, Müller J (2013) ChemPlusChem 78:20–34
3. Johannsen S, Megger N, Böhme D, Sigel RKO, Müller J (2010) Nat
Chem 2:229–234
P 138
Insights into the structural determinants
for the formation of fluorescent silver nanoclusters
on DNA, RNA and DNA-RNA chimeras
Peter W. Thulstrup1, Pratik Shah2, Seok Keun Cho2,
Morten J. Bjerrum1, Seong Wook Yang2
1
Department of Chemistry, University of Copenhagen,
Universitetsparken 5, 2100 Copenhagen, Denmark. [email protected];
2
UNIK Center for Synthetic Biology, Department of Plant
and Environmental Sciences, University of Copenhagen,
Thorvaldsensvej 40, 1871 Frederikberg, Denmark
Recently, silver nanoclusters (AgNCs) bound to nucleic acids have
become a highly promising group of fluorescent probes, displaying an
intense response in the visible and near-infrared spectral regions.
Through variation of the nucleic acid composition numerous different
properties can be achieved, for instance to allow for the detection of
micro RNA in solution [1]. It is known that probe design is complex,
as not only base sequence but nucleic acid conformation is important
[2]. RNA has been shown to host fluorescent AgNCs [3], and here we
find that the emission properties are strongly dependent on the
backbone composition in a comparison of two probe systems using
both DNA, RNA and DNA-RNA chimera sequences as the scaffold
for AgNC formation. One probe sequence shows strongly enhanced
J Biol Inorg Chem (2014) 19 (Suppl 2):S815–S832
fluorescence with RNA and is subdued with DNA, and another probe
acts in the opposite way. In addition to the fluorescence properties, the
systems are characterized through capillary electrophoresis and circular dichroism spectroscopy. This is one of the first studies of RNAbased probes and it seems clear that the choice of backbone will be an
important parameter in the design of new fluorescent probes based on
AgNCs.
Financial support by the ‘‘Centre for Synthetic Biology’’ (Grant
09-065274) is acknowledged, and we thank Thomas Günter-Promosky for use his fluorimeter.
S827
Reference
1. Sigel RKO, Sigel H (2010) Acc Chem Res 43:974–984
P 140
DNA-interacting photoswitchable metal complexes
based on dithienyl-cyclopentene ligands
Andreu Presa1, Guillem Vázquez1, Ivana Borilovic1,
Olivier Roubeau2, Patrick Gamez1,3
1
References
1. Yang SW, Vosch T (2001) Anal Chem 83:6935–6939
2. Shah P, Rorvig-Lund A, Chaabane SB, Thulstrup PW, Kjaergaard
HG, Fron E, Hofkens J, Yang SW, Vosch T (2012) ACS Nano
6:8803–8814
3. Schultz D, Gwinn E (2001) Chem Commun 47:4715–4717
P 139
Stabilisation and nucleic acids binding of a 13 ion:
the aluminium/cacodylate/RNA system
Tarita Biver1, Natalia Busto2, Begoña Garcı́a2, Matteo
Lari1, José-Maria Leal2, Hector Lozano2, Fernando
Secco1, Marcella Venturini1
1
Department of Chemistry and Industrial Chemistry, University
of Pisa, Via Risorgimento 35, 56126 Pisa, Italy. [email protected];
2
Department of Chemistry, University of Burgos, Plaza Misael
Bañuelos s.n., 09001 Burgos, Spain
Metal ions influence the three-dimensional architecture and function of
nucleic acids, can induce folding of nucleic acids strands, or even can
aid catalytic mechanism in ribozymes. Therefore, investigations of
metal ion binding to specific sites, and of the ability to stabilize local
motifs or particular non-canonical structures are of primary interest [1].
We have recently found that Mg(II) and Ni(II) cations are able to
induce quadruplex and triplexes formation starting from duplex
poly(rA)poly(rU) under still unexplored high ions concentrations. We
have now extended our research to the study of the less explored class
of tervalent metal ions. The interaction between aluminium(III) and
poly(rA) nucleic acid, both in the form of single or double strand, has
been analysed. The Al3?/poly(rU) system has been also investigated for
comparison purposes. At the pH range needed for these experiments
(pH = 5–7) the stability of Al3? in solution is guaranteed by the
cacodylate buffer that also complexes the metal ion. It is shown that the
binding occurs indeed, with features that differ on passing from
poly(rA) to poly(rA)poly(rA). The fast process of the metal ion binding
to RNA is studied by means of the initial rate analysis in different
reactants conditions and a binding mechanism is proposed (figure, left).
This enables the main binding species to be individuated. In the case of
poly(rA) a slow cooperative aggregation of the single strands is found
to occur (figure, right), that is favoured by the presence of the aluminium ion. The different binding features will be discussed.
Financial support by Obra Social ‘‘La Caixa’’ is gratefully
acknowledged.
Departament de Quı́mica Inorgànica, Facultat de Quı́mica,
Universitat de Barcelona, Martı́ i Franquès 1-11, 08028 Barcelona,
Spain. [email protected];
2
Instituto de Ciencia de Materiales de Aragón (ICMA), CSIC
and Universidad de Zaragoza, Plaza San Francisco s/n, 50009
Zaragoza, Spain;
3
Institució Catalana de Recerca i Estudis Avançats (ICREA), Passeig
Lluı́s Companys 23, 08010 Barcelona, Spain
Dithienylethene-based compounds of the type illustrated in the figure
below are well known for their potential use as molecular switches, as
they undergo reversible ring closure upon irradiation with UV or visible
light [1–3], giving rise to their contraction or expansion, respectively.
Surprisingly, their use for biological applications has not yet been
extensively studied. Here we present a new series of photoswitchable
coordination compounds obtained from both symmetrical and unsymmetrical dithienylcyclopentene ligands. The open and closed forms of
these metal complexes not only exhibit interesting optical properties, but
also show clearly distinct DNA-interacting behaviours.
Financial support by the Ministerio de Economı́a y Competitividad (MINECO) of Spain (Project CTQ2011-27929-C02-01). COST
Action CM1105 is kindly acknowledged.
References
1. Feringa BL (ed) Molecular Switches Wiley–VCH Weinheim 2001
2. Irie M (2000) Chem Rev 100:1685–1716
3. Hanazawa M, Sumiya R, Horikawa Y, Irie M (1992) J Chem Soc
Chem Commun 206–207
P 141
Targeting DNA structures using metallosupramolecular
complexes
Rosa F. Brissos1, David Aguilà1, Mohanad Darawsheh1,
Leoni Barrios1, Olivier Roubeau2, Guillem Aromı́1,
Patrick Gamez1,3
1
Departament de Quı́mica Inorgànica, Facultat de Quı́mica,
Universitat de Barcelona, Martı́ i Franquès 1-11, 08028 Barcelona,
Spain. [email protected];
2
Instituto de Ciencia de Materiales de Aragón (ICMA), CSIC
and Universidad de Zaragoza, Plaza San Francisco s/n, 50009
Zaragoza, Spain;
123
S828
3
Institució Catalana de Recerca i Estudis Avançats (ICREA), Passeig
Lluı́s Companys 23, 08010 Barcelona, Spain
The molecular recognition of DNA duplex in the major groove is a
topical strategy to develop therapeutic agents (antigene compounds), taking into account that the major groove is the binding
site of proteins playing a key role in replication, transcription or
recombination. These recognition processes occur via specific
hydrogen-bonding (donor/acceptor) contacts with the edges of the
base pairs [1]. Here, we report on the design and preparation of
metallosupramolecular complexes, i.e. metallohelicates obtained
from b-diketone ligands (see figure below), which are able to target
specific DNA locations and/or conformations. The biological
activity, namely the potential interaction of these new compounds
with the DNA major groove, has been examined using UV–Vis
spectroscopy, fluorescence dye-displacement techniques, circular
dichroism (CD), gel electrophoresis and Atomic-Force Microscopy
(AFM).
Financial support by the Ministerio de Economı́a y Competitividad (MINECO) of Spain (Project CTQ2011-27929-C02-01). COST
Action CM1105 is kindly acknowledged.
J Biol Inorg Chem (2014) 19 (Suppl 2):S815–S832
i Innovació del Govern Balear (project 23/2011, FEDER funds) for
financial support.
Table 1. Energetic (interaction energies in kcal/mol) and
geometric parameters (anion to ring centroid distances in
Å) computed for several anion-p complexes. 6R and 5R
stands for six- and five-membered rings, respectively
Anion-p interaction
Distance
Energy
Bromide/(N1CytH)C6
[2]
3.58
-21.7
Chloride/(N6AdeH)C10
[3]
3.48
-15.6
Chloride/(N6AdeHC10)(N6AdeHC10)
[3]
3.45
-25.2
(ZnCl3)Cl/(AdeH)2C3, N1 protonated, 6R
[4]
3.58
-77.8
(ZnCl3)Cl/(AdeH)2C3, N1 protonated, 5R
[4]
3.32
-83.7
(ZnCl3)Cl/(HypH)2C3, N7 protonated, 6R
(HgCl3)Cl/(AdeH)2C3, N1 protonated, 6R
[4]
[4]
3.20
3.29
-82.4
-83.3
References
1. Fiol JJ, Barceló-Oliver M, Tasada A, Frontera A, Terrón A, Garcı́aRaso A (2013) Coord Chem Rev 257:2705–2715
2. Barceló-Oliver M, Baquero BA, Bauzá A, Garcı́a-Raso A, Terrón
A, Mata I, Molins E, Frontera A (2012) CrystEngComm
14:5777–5784
3. Garcı́a-Raso A, Albertı́ FM, Fiol JJ, Lagos Y, Torres M, Molins E,
Mata I, Estarellas C, Frontera A, Quiñonero D, Deyà PM (2010) Eur J
Org Chem 2010:5171–5180
4. Garcı́a-Raso A, Albertı́ FM, Fiol JJ, Tasada A, Barceló-Oliver M,
Molins E, Escudero D, Frontera A, Quiñonero D, Deyà PM (2007)
Inorg Chem 46:10724–10735
Reference
1. Lusby PJ (2010) Annu Rep Prog Chem, Sect A Inorg Chem
106:319–339
P 142
Anion-p interactions in biological molecules derived
from purine and pyrimidine bases
Angel Terrón, Miquel Barceló-Oliver, Juan J. Fiol,
Angel Garcı́a-Raso, Antonio Frontera
Department of Chemistry, Universitat de les Illes Balears, Campus
UIB, 07122 Palma de Mallorca, Spain
Long chain N-alkyl substituted purines and pyrimidines have
been used as biological models. We summarize the results from
the characterization of the bases and their coordination chemistry focusing the attention on the anion-p interactions. The
results are obtained from X-ray diffraction studies of wellcharacterized molecules, and theoretical studies based on the
experimental data [1]. Some of the selected examples are
indicated in Table 1.
Financial support by the Universitat de les Illes Balears is gratefully acknowledged. A.F. thanks the DGICYT of Spain (projects
CTQ2011-27512/BQU and CONSOLIDER INGENIO 2010
CSD2010-00065, FEDER funds) and the Direcció General de Recerca
123
P 143
Synthesis, characterization and DNA interaction study
of some copper(II) complexes with substituted
terpyridine ligands
Amparo Caubet1, Jordi Grau1, Patrick Gámez1,2,
Olivier Roubeau3
1
Departament de Quı́mica Inorgànica, Universitat de Barcelona, Martı́
i Franquès 1-11 08028 Barcelona, Spain. [email protected];
2
Institució Catalana de Recerca i Estudis Avançats (ICREA), Passeig
Lluı́s Companys 23, 08010 Barcelona, Spain; 3Instituto de Ciencia de
Materiales de Aragón (ICMA), CSIC y Universidad de Zaragoza,
Plaza San Francisco s/n, 50009 Zaragoza Spain
Cisplatin and related platinum based antitumor drugs are widely
used to treat cancer. However, their administration is limited due
to toxic side effects, such as neurotoxicity, emetogenesis and
nephrotoxicity, and many tumors display intrinsic or acquired
resistance against these drugs [1]. Therefore, it is of great interest
to develop new anticancer drugs based on other transition-metal
complexes.
Copper complexes provide a potential alternative. Among the
essential metal ions in the human body, Cu2? is third in abundance
after Fe3? and Zn2?, and it plays very important roles in several
biological processes. Also many copper(II) complexes have been
reported to efficiently cleave DNA by an oxidative or hydrolytic
mechanism [2].
J Biol Inorg Chem (2014) 19 (Suppl 2):S815–S832
In this communication we report the synthesis and characterization
of some copper(II) compounds with substituted 2,20 :60 ,200 -terpyridine
ligands. The interaction between the ligands and their metal complexes with DNA has been studied by UV–Vis spectroscopy titrations,
fluorescent indicator displacement and electrophoretic mobility.
Financial support by the Ministerio de Economı́a y Competitividad of Spain (Project CTQ2011-27929-C02-01) is gratefully
acknowledged.
S829
monitoring the conversion of supercoiled UX174 plasmid DNA to
nicked circular and linear DNA. All the Cu(II) complexes tested
present a high artificial nuclease activity. Their cytotoxic properties
were also tested on A2780 and A2780cisR cell lines. All the new
mixed-ligand Cu(II) complexes presented a higher cytotoxicity when
compared with cisplatin, and were able to significantly overcome
cisplatin resistance.
The authors would like to acknowledge FCT (PTDC/QUI–QUI/
114139/2009, SFRH/BPD/29564/2006 grant to SGama and FCT
Investigator Grant to FMendes) and COST CM1105 Action for
financial support.
References
1. Ghosh K, Kumar P, Tyagi N, Singh UP, Goel N (2011) Inorg Chem
Commun 14:489–492
2. Sigman DS, Graham DR, Daurora V, Stern AM (1979) J Biol
Chem 254:12269-12272
3. Loganathan R, Ramakrishnan S, Suresh E, Riyasdeen A, Akbarsha
MA, Palaniandavar M (2012) Inorg Chem 51:5512–5532
References
1. Galanski M, Jakupec MA, Keppler BK (2005) Curr Med Chem 12:185–
211
2. Li GY, Du KJ, Wang JQ, Liang JW, Kou JF, Hou XJ, Ji LN, Chao
H (2013) J Inorg Biochem 119:43–53
P 144
New mixed-ligand Cu(II) complexes acting as ‘‘selfactivating’’ chemical nucleases
Inês Rodrigues1, Filipa Mendes1, Elisa Palma1, Isabel
Correia2, Fernanda Carvalho2, Isabel C. Santos1,
Fernanda Marques1, Isabel Santos1, António Paulo1,
Sofia Gama1
1
Centro de Ciências e Tecnologias Nucleares-C2TN, Instituto
Superior Técnico, Universidade de Lisboa, Campus Tecnológico e
Nuclear, Estrada Nacional 10, km 139.7, 2695-066 Bobadela, LRSPortugal;
2
Centro de Quı́mica Estrutural, Instituto Superior Técnico,
Universidade de Lisboa, Avenida Rovisco Pais 1, 1049-001 Lisboa,
Portugal
There has been considerable interest in the recent years for the
development of DNA cleaving reagents aiming at their application in
biotechnology and medicine [1]. Transition metal ions show diverse
structural features, variable oxidation and spin states and redox
properties in different complexes, offering plenty of opportunities to
discover novel artificial nucleases.
Among the first row transition elements, copper has got a special
interest in this regard since the discovery of the first chemical nuclease
by Sigman et al. [2]. Copper has high affinity for the nucleobases and
copper complexes possess biologically accessible redox properties [1].
Several copper complexes have been proposed as potential anticancer
substances and cancer inhibiting agents, as they demonstrate
remarkable anticancer activity and show general toxicity lower than
platinum compounds [3]. Very recently, mixed ligand copper(II)
complexes were found to exhibit prominent anticancer activity by
inducing apoptosis, binding strongly and cleaving DNA [3].
In order to develop novel artificial nucleases, several mixed
bipyridine-terpyridine Cu(II) complexes were synthesized. The ability
of the ligands and Cu(II) complexes to cleave DNA was evaluated by
P 145
Site-specific covalent post-transcriptional labeling
of oligonucleotides for the study of single molecules
Igor Oleinich, David Egloff, Eva Freisinger
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland. [email protected]
Single molecule spectroscopy has been used intensively to investigate
mechanisms in living cells [1]. This technique requires the site-specific functionalization of native biomolecules while retaining their
structures and functions. Currently, our research focuses on a new
labeling strategy for the site-specific functionalization of long oligonucleotides [2, 3]. The newly developed strategy is based on a
modular system consisting of three parts: a short oligonucleotide
recognition sequence, a reactive group that is specifically designed to
generate the respective functional group, as well as a linker between
recognition sequence and reactive group.
The basic principle of this approach relies on the annealing of the
reactive strand with the target oligonucleotide to position the reactive
group close in space to the target nucleotide. Following, if need be,
the activation of the reactive group, generally first a cross-linked
duplex is formed, which upon cleavage of the reactive group or the
linker results in the new functionality that can be used to attach, e.g., a
fluorescent dye. Among the new functional groups that we were able
to introduce so far are alkine, aldehyde, and thiol groups.
Financial support by the University of Zurich, the Swiss National
Science Foundation (EF), and the COST CM1105 Action is gratefully
acknowledged.
References
1. Wang Z, Zhang K, Shen Y, Smith J, Bloch S, Achilefu S, Wooley KL,
Taylor J-S (2013) Org Biomol Chem 11:3159–3167
2. Oleinich IA, Freisinger E (2014) to be submitted
3. Egloff D, Freisinger E (2014) submitted
123
S830
P 146
Interaction of Pd21 complexes of 2,6-disubstituted
pyridines with nucleoside 50 -monophosphates
Oleg Golubev, Tuomas Lönnberg, Harri Lönnberg
University of Turku, Department of Chemistry, FIN-20014 Turku,
Finland. [email protected]
To learn more about the underlying principles of metal-ionmediated recognition of nucleic acid bases, PdCl? complexes of
six 2,6-disubstituted pyridines, viz. pyridine-2,6-dicarboxamide,
its N2,N6-dimethyl and N 2,N 6-diisopropyl derivatives, 6-carbamoylpyridine-2-carboxylic
acid,
6-aminomethylpyridine-2carboxamide and its N2-methyl derivative, were prepared and
their interaction with nucleoside 50 -monophosphate (NMP) was
studied by 1H NMR spectroscopy in D2O at pH 7.2. The
binding sites within the nucleobases were assigned on the basis
of Pd2? induced changes in chemical shifts of the base moiety
proton resonances. The mole fractions of NMPs engaged in
mono- or dinuclear Pd2? complexes were determined at various
concentrations by comparing the intensities of the aromatic and
anomeric protons of the complexed and uncomplexed NMPs.
Some of the pyridine complexes showed moderate discrimination between the NMPs.
P 147
Recognition of alternative DNA and RNA structures
by bimetallic triple-stranded ferro-helicates
Jaroslav Malina1, Peter Scott2, Viktor Brabec1
1
Institute of Biophysics, Academy of Sciences of the Czech Republic,
v.v.i., Kralovopolska 135, CZ-61265 Brno, Czech Republic;
2
Department of Chemistry, University of Warwick, Gibbet Hill Road,
Coventry CV4 7AL, UK. [email protected]
Metal-templated helicates commonly comprise three bis(bidentate)
NN—NN ligands wrapped in a helical array around two metal ions
[often iron(II)]. They are of a similar size and shape to natural biomolecule recognition units, such as a-helices or zinc fingers that
recognize the major groove of the DNA. There is a hope that they can
be developed into useful molecules in medicinal chemistry as a nonpeptide mimetics. Indeed, promising anticancer and antimicrobial
activity has been reported for some helicates [1,2] and it has been
hypothesized that binding to DNA is responsible for this behavior [1].
Unfortunately, the helicates have remained difficult to use in the
medicinal arena because they contain mixtures of isomers, are
insoluble, or are too difficult to synthesize. Recently, it has been
reported a new strategy to prepare optically pure, water-stable,
functionalized metallo-helical assemblies that have been called
‘flexicates’ [3, 4]. The experiments revealed that in addition to specific interactions with DNA, flexicates exhibit very promising
antimicrobial activity against MRSA (methicillin-resistant S. aureus)
and E. coli, alongside low toxicity towards the nematode worm C.
elegans [4]. Flexicates have been also shown to have comparable
activity to cisplatin against the cell lines MCF7 and A2780 and *5fold higher activity against the cisplatin-resistant A2780cis [5].
J Biol Inorg Chem (2014) 19 (Suppl 2):S815–S832
We have been investigating interactions of the two classes of
iron(II) flexicates with alternative DNA and RNA structures
regarded as potential drug targets via a range of biophysical techniques. The results show that L1a flexicates have significantly
enhanced binding and stabilizing properties towards DNA threeway and four-way junctions and also towards DNA and RNA
bulges.
References
1. Richards AD, Rodger A, et al. (2009) Int J Antimicrob Agents
33:469–472
2. Pascu GI, Hotze ACG, et al. (2007) Angew Chem Int Ed 46:
4374–4378
3. Howson SE, Scott P, et al. (2011) Dalton Trans 40:10268–
10277
4. Howson SE, Bolhuis A, et al. (2012) Nat Chem 4:31–36
5. Brabec V, Howson SE, et al. (2013) Chem Sci 4:4407–4416
P 148
NMR study of metal ion interactions with the D1 j-f
region of a group II intron
Simona Bartova, Maria Pechlaner, Daniela Donghi,
Roland K. O. Sigel
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland. [email protected]
Self-splicing group II introns are highly structured RNA molecules, containing a characteristic secondary and catalytically
active tertiary structure, which is formed only in the presence of
Mg2? [1, 2]. This divalent metal ion initiates the first folding step
governed by the j-f element of domain 1 (D1), which also provides the platform for other domain docking [3, 4]. Mg2? can
coordinate to RNA via inner or outer-sphere interactions [5]. In
order to better characterize these interactions, other metal ions
can be used to mimic Mg2?, namely [Co(NH3)6]3? for outersphere interaction and Cd2? for inner- and outer-sphere interactions, having also a higher affinity.
We performed a detailed multinuclear NMR study of Cd2? and
[Co(NH3)6]3? interactions with the j-f region of the group II intron
ribozyme Sc.ai5c from baker’s yeast [4]. The aim was to confirm
the stabilization of the j-f region induced by these metal ions, as
well as to characterize their binding sites. Accordingly, several 1D
(1H, 31P, 113Cd) and 2D ([1H, 13C]-HSQC, 2J-[1H, 15N]-HSQC)
NMR experiments were recorded to map the spectral changes upon
addition of different amounts of the metal ions. Our NMR data
reveal a strong interaction of Cd2? with G1N7 and proved the
macrochelate formation of the corresponding phosphate group and
G1N7. Other interactions of Cd2? were detected in the GAAAtetraloop, the j region and around the three-way junction.
[Co(NH3)6]3? interaction was also confirmed by multinuclear NMR
spectroscopy. Together with our recently published data on Mg2?
interaction [4] we now present a much better understanding of Mg2?
binding to D1 j-f.
Financial support by the Swiss National Science Foundation (to DD
and RKOS) and the University of Zurich is gratefully acknowledged.
References
1. Pyle AM (2002) J Biol Inorg Chem 7:679–690
2. Sigel RKO (2005) Eur J Inorg Chem 2281–2292
3. Waldsich C, Pyle AM (2007) Nat Struct Mol Biol 14:37–44
4. Donghi D, Pechlaner M, Finazzo C, Knobloch B, Sigel RKO (2013)
Nucleic Acids Res 41:2489–2504
5. Donghi D, Sigel RKO (2012) Methods Mol Biol 848:253–273
123
J Biol Inorg Chem (2014) 19 (Suppl 2):S815–S832
P 149
Anti-cancer effects of Pt(IV) complex in cisplatinresistant ovarian cancer cells
Takao Tobe, Karin Shimizu, Miki Motoyama, Koji
Ueda, Yoshinori Okamoto, Nakao Kojima
Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempakuku, Nagoya 468-8503, Japan
Platinum(IV) [Pt(IV)] complexes are an anti-cancer prodrug to be activated via reduction by which they exert Pt(II)-based drug activities in
cells including the target cancer cells. Pt(IV) complexes show less
adverse effects such as renal toxicity because of their low reactivity with
biological molecules compared to a typical anti-cancer drug cisplatin.
This different reactivity prompted us to investigate the effectiveness of
Pt(IV) on cisplatin-resistant cancer cells with its mechanisms. This study
aims to elucidate the anti-cancer effects of Pt(IV) complex.
In this study we have investigated cis-diammine-tetrachloroPt(IV) [cis-Pt(IV)] and cisplatin of the cytotoxicity against human
ovarian cancer cell lines (A2780), and its cisplatin-resistant subline
(A2780cis), with their influx/efflux patterns using ICP-MS. Pt–DNAcrosslink formation was also determined by agarose gel electrophoresis, in which the crosslink was quantified by decrease of ethidium
bromide staining of DNA.
Cisplatin completely failed to decrease the survival of A2780cis
cells at a concentration that killed A2780, whereas cis-Pt(IV) exerted
cytotoxicity in both cell lines. Pt–DNA-crosslink formation assay
showed cis-Pt(IV) bound to DNA only in the presence of reductant
such as glutathione (GSH) or ascorbic acid, indicating cisplatin production. However, it is also known excess amount of GSH suppress
DNA binding of cisplatin. Indeed, intracellular GSH level in
A2780cis cells was about 3 times higher as compared with A2780
cells; therefore cisplatin would be inactivated in A2780cis cells.
These findings suggest that cis-Pt(IV) is actually reduced to cisplatin
in GSH-rich environment and then induce cytotoxicity by somehow
escaping from GSH-dependent inactivation. Pt(IV)-specific subcellular distribution might contribute this paradoxical advantageous
effect of GSH on cis-Pt(IV). Intracellular accumulation of Pt was
reduced by competing a related transporter CTR1 by copper in cisPt(IV) treatment, whereas cytotoxicity was not suppressed. Therefore
CTR1 is involved in the intake of Pt complexes including cis-Pt(IV),
but the alternative pathway(s) could be responsible for anti-cancer
effect of Pt(IV) complexes.
S831
since this reduces or even favors the interaction of the metabolite with
the negatively charged RNA backbone. This simple consideration has
been recently questioned by the discovery of a riboswitch that targets
a negatively charged fluoride anion. This fluoride sensing riboswitch
was found to activate the expression of genes that encode fluoride
transporters. Besides targeting the small fluoride anion with good
efficiency, this riboswitch has also remarkable halide selectivity,
since no chloride binding was observed in presence of KCl. The X-ray
structure of the fluoride riboswitch evidenced that the fluoride is the
central unit keeping together a small cluster of three Mg2? cations,
whose coordination sphere is completed by either oxygen atoms of
phosphate groups, and by water molecules, one of them acting as a
bridge between two of the Mg2? ions.
The peculiar halide selectivity and the structure of the fluoride
riboswitch provoke a series of questions: (i) Is the small Mg2?/F-/
phosphate/water cluster at the center of the riboswitch a stable entity
on its own? (ii) Considering that water molecules bridging metal
cations are known to be quite acidic, which is the acidity of the
bridging water of the central cluster of the fluoride riboswitch? (iii)
Which is the origin of the halide selectivity? In this contribution we
provide a clear explanation to the above three fundamental questions,
based on static and dynamic density functional theory calculations.
P 151
Nano-arrays of DNA-binding cylinders
Jenifer C. White, Michael J. Hannon
Department of Chemistry, University of Birmingham, Birmingham,
B15 2TT, England. [email protected]
Metallo-supramolecular helicates are able to bind to DNA via a
number of interactions. The di-nuclear supramolecular cylinders;
[Fe2L3]4? and [Ru2L3]4? (L = C25H20N4), have a size and shape
comparable to those of zinc fingers [1]. Consequently, it is able to fit into
the major groove of B-DNA and bind at the heart of DNA fork structures to induce structural change. These interactions mean the cylinder
has the potential to act as a potent apoptotic and cytostatic agent [2, 3].
It is aimed to combine this supramolecular chemistry with nanotechnology, by attaching the helicate to the surface of gold
nanoparticles, AuNPs. In doing so, it is hoped to enhance the interactions currently observed with DNA, ultimately with the aim of
causing hindrance to DNA replication as a result of enhanced coiling,
resulting in a more potent anticancer therapy.
In an attempt to achieve this, triple stranded helicates were synthesised with varying functionality in an attempt to attachment the
cylinders to the AuNP surface. The use of surfactant coated nanoparticles and the electrostatic interactions between cationic cylinders
and anionic AuNPs have also being investigated.
Supramolecular helicates have been functionalised using the novel
use of click chemistry. These new structures are shown to bind to
DNA, inducing a structural change, hence the use of click chemistry
will be investigated to add functionality to the surface of AuNPs [4].
P 150
Stability and halide selectivity of the fluoride riboswitch
explained
Mohit Chawla1, Romina Oliva2, Luigi Cavallo1
1
King Abdullah University of Science and Technology, KAUST
Catalysis Center, Thuwal, 23955-6900, Saudi Arabia;
2
University ‘‘Parthenope’’ of Naples, Department of Sciences
and Technologies Centro Direzionale Isola C4, 80143 Naples, Italy
Riboswitches are short mRNA segments deputed to control gene
expression in response to the selective binding of a metabolite. Currently, over 20 classes of riboswitches are known, targeting
metabolites that are either neutral or positively charged molecules,
123
S832
References
1. Hannon MJ, Moreno V, Prieto MJ, Moldrheim E, Sletten E, Meistermann I, Isaac CJ, Sanders KJ, Rodger A (2001) Angew Chem Int Ed
40:879–884
2. Hotze ACG, Hodges NJ, Hayden RE, Sanchez-Cano C, Paines C,
Male N, Tse M-T, Bunce CM, Chipman JK, Hannon MJ (2008) Chem
Biol 15:1258–1267
123
J Biol Inorg Chem (2014) 19 (Suppl 2):S815–S832
3. Khalid S, Hannon MJ, Rodger A, Rodger PM (2006) Chem Eur J
12:3493–3506
4. Zhou Y, Wang S, Zhang K, Jiang X (2008) Angew Chem Int Ed
47:7454–7456
J Biol Inorg Chem (2014) 19 (Suppl 2):S833–S852
DOI 10.1007/s00775-014-1164-z
POSTER PRESENTATION
Metalloproteins: structure and function
P 160
Biologically relevant or an artifact? The copper binding
site in wheat metallothionein
Katsiaryna Tarasava, Eva Freisinger
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland. [email protected]
Metallothioneins (MTs) are small cysteine-rich proteins and are
suggested to be involved in metal tolerance and homeostasis due to
their ability to bind metal ions through the thiol groups of cysteine
residues. The early cysteine-labeled MT Ec-1 from Triticum aestivum
(common wheat) hosts two metal binding domains, c and bE, coordinating two and four divalent d10 metal ions, respectively (Fig.) [1].
Ec-1 is currently assumed to play a role in zinc homeostasis, as it is
forming well defined species with Zn(II) upon in vitro reconstitution
[1]. Nevertheless, the isolation of Ec-1 directly from the wheat germ
revealed the presence of some amounts of copper [2]. The simultaneous binding of both Zn(II) and Cu(I) has been reported for a
number vertebrate MTs [3], and in many cases, these additionally
coordinated Cu(I) ions have a function, e.g. the prevention of coppermediated aggregation of amyloid b-aggregates in the human MT2A
subtype [4]. Hence it cannot be excluded that also Cu(I) binding to a
defined site in the Ec-1 MT could exhibit a specific function. Our data
suggest specific coordination of the first Cu(I) ion to the N-terminal cEc-1 domain. It replaces one zinc ion, and this binding is not affected
by the second domain, i.e. b-Ec-1. Increasing amounts of Cu(I) were
added to the separate c-Ec-1 domain and spectral changes monitored
with UV/Vis, circular dichroism, and fluorescence spectroscopy. The
same spectra result regardless if the apo-protein is used in the titration
study or the metal-loaded and hence pre-folded Zn2- or Cd2-c-Ec-1
forms, suggesting the formation of very similar cluster structures.
Together with the reported occurrence of copper containing Ec-1
in vivo our results provide evidence about the possible functional
importance of Cu(I)-species of wheat MT.
Financial support from the Swiss National Science Foundation
(EF) and the Forschungkredit 2013 from the University of Zurich
(KT) are gratefully acknowledged.
References
1. Peroza EA, Schmucki R, Güntert P, Freisinger E, Zerbe O (2009) J
Mol Biol 387:207–218.
2. Leszczyszyn O, Schmid R, Blindauer CA (2007) Proteins 68:
922–935.
3. Chen P, Onana P, Shaw C, Petering D (1996) Biochem J 317:
389–394.
4. Chung R, Howells C, Eaton E, Shabala L, Zovo K, Palumaa P,
Sillard R, Woodhouse A, Bennett W, Ray S, Vickers J, West A (2010)
PLoS One 5:1–11.
P 161
Investigation of the metal ion binding properties,
folding, and structure of unique histidine-rich
metallothioneins
Jelena Habjanič, Katsiaryna Tarasava, Eva Freisinger
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland. [email protected]
Metallothioneins (MTs) are intracellular cysteine-rich proteins with a
low molecular weight of roughly 5–12 kDa. They are able to coordinate various transition metal ions with a d10 electron configuration,
including the essential metal ions ZnII and CuI, but also the toxic
metal ions CdII and HgII. The exact mechanism and the precise
function of MTs is still a matter of debate, but is assumed that they
have a role in metal homeostasis, provide protection against metal
toxicity, and have redox capabilities and hence can act against oxidative stress.
Bacterial metallothioneins have been known since the mid
1980s but until today only the structure of the MT SmtA from
Synechococcus elongatus has been explored, and for the first time
it was shown that not only cysteine but also histidine residues can
contribute to metal ion coordination in MTs [1]. Studies enabled
by recent advances in genomic sequencing revealed that a number
of different Pseudomonas fluorescens strains, which are gram
negative aerobic bacteria, contain MT sequences that are unusually rich in His residues. At the same time they show rather
conserved Cys distribution patterns consisting of an N-terminal
CxCxxCxC motif, a central YCC/SxxCA stretch as well as an
C-terminal Cxxxx(x)CxC part. The largest differences between
these sequences are the number of His residues as well as their net
charge which have a major influence on the properties of the
proteins.
We focus our study on the metal ion binding abilities, the metallation pathway as well as on the structure and folding of these MTs.
The methods applied include UV–Vis, (magnetic) circular dichroism,
fluorescence, atomic absorption, and extended X-ray absorption fine
edge structure spectroscopy combined with a number of analytical
methods. Analysis of the three-dimensional structure and the metaldependent folding with NMR spectroscopy will be a major part of this
study.
Financial support by the Alfred-Werner Foundation (JH) and the
Swiss National Science Foundation (EF) is gratefully acknowledged.
Reference
1. Blindauer CA, Harrison MD, Parkinson JA, Robinson AK, Cavet J,
Robinson NJ, Sadler PJ (2001) Proc Natl Acad Sci USA
98:9593–9598.
123
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J Biol Inorg Chem (2014) 19 (Suppl 2):S833–S852
P 162
Towards the structure–function characterization
of a metal binding plant metallothionein from Musa
acuminata (banana)
Jovana Jakovleska, Eva Freisinger
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland. [email protected]
We study the structure and metal coordination properties of metal
complexes formed by a plant MT from the subfamily MT3 found in
Musa acuminata (banana), baMT3, in more detail. As previous studies
in the group indicate that baMT3 can coordinate three divalent metal
ions in two separate domains [1], the metal complexes of the separate
N- and C-terminal parts of baMT3 with Zn(II) and Cd(II), but also with
Cu(I) and Ag(I) are studied separately. Besides analyzing the native
sequence of baMT3, there is also work on a homologue of the C-terminal part, in which the potentially metal ion coordinating amino acid
histidine is mutated to a cysteine residue (H46C-baMT3). This enables
us to study the relevance of non-cysteine residues for the metal ion
binding properties of MTs.
Furthermore, with the help of potentiometric pH titrations, the
thermodynamic stabilities and properties of the metal complexes of
the separate domains as well as of the full-length protein are
investigated.
Financial support from the Swiss National Science Foundation
(EF) and the CMSZH Graduate School is gratefully acknowledged.
Reference
1. Freisinger E (2007) Inorg Chim Acta 360:369–380.
P 163
Investigating a dominant Zn2Cd species of the fungal
metallothionein Neclu_MT2
Jens Loebus, Aleksandar Salim, Gerd-Joachim Krauss,
Dirk Dobritzsch, Eva Freisinger
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland. [email protected]
The aquatic fungus Heliscus lugdunensis was isolated from a heavily
metal ion polluted spring of an ancient metal ore dump, containing,
besides others, 25 lM CdII and 30 mM ZnII ions. The H. lugdunensis
strain 4-4-2 growing under these austere conditions expresses two
metallothioneins Neclu_MT1 and Neclu_MT2, which share the same
primary amino acid sequence except for a Ser ? Thr exchange.
Neclu_MT1 has been shown to be the first CdII-specific MT through:
(i) protein induction studies showing an exclusive CdII-inducibility of
the gene product and (ii) a different metal cluster stoichiometry
observed in case of CdII (Cd3) and ZnII (Zn2) metallation at physiologically relevant protein concentrations [1]. However, the 24 amino
acid long, 8 Cys and 1 His containing MT Neclu_MT2 was not
studied so far.
Employing optical, chiro-optical and NMR spectroscopy along
with mass spectrometry, we investigated in vitro the stoichiometries and the stability of the ZnII and CdII metal clusters formed as
well as the metallation pathways of Neclu_MT2 and compared the
results with those obtained for Neclu_MT1. As the Ser ? Thr
exchange is induced by a switch of only one base pair, the
occurrence of a dominant ZnCd2 species exclusive to Neclu_MT2
is discussed in respect to evolutionary adoption regarding metal
ion specificity.
Financial support by the Swiss National Science Foundation (E.F.)
is gratefully acknowledged.
123
Reference
1. Loebus J, Leitenmaier B, Meissner D, Braha B, Krauss GJ, Dobritzsch D, Freisinger E (2013) J Inorg Biochem 127:253–260.
P 164
X-ray crystallographic structure analyses
and characterization of a blue copper protein,
pseudoazurin, Met16Ile variant
Kana Gunji1, Takahide Yamaguchi1, Akiko
Takashina1, Masaki Unno1,2, Takamitsu Kohzuma1,2
1
Institute of Applied Beam Science, Ibaraki University, Mito, Ibaraki,
310-8512, Japan.
2
Frontier Research Centre for Applied Atomic Sciences, Ibaraki
University, Tokai, Ibaraki, 319-1106, Japan
Pseudoazurin (PAz) from Achromobacter cycloclastes functions as
an electron donor to nitrite reductase and nitrous oxide reductase.
The copper atom is coordinated by His40 and His81 imidazole N
atoms of, Cys78 thiolate S atom, and Met86 sulfide S atom with
distorted tetrahedral geometry. The Met16 residue is located in the
vicinity of His81 and weakly interacted to the active site [1], and
several Met16 mutated PAz were studied to shed light on the role
of weak interaction on the electronic structure of blue copper
protein.
Met16Ile PAz was expressed, purified, and crystallized to add
further knowledge into the effect of Met16 moiety. Very recently, we
have reported the Met16Ile PAz gives almost pure rhombic EPR [2].
The crystal structures of oxidized and reduced Met16Ile PAz were
determined with 1.4 and 1.7 Å resolution at pH 7.5 and pH 4.5,
respectively. Overall structure of Met16Ile PAz was almost identical
to the structure of WT with an RMS value of 0.34 Å. The Cu-SMet86
bond length of Met16Ile PAz is 2.52 Å, which is shorter than that in
the structure of WT (2.87 Å). The Cu-NHis40 bond length at pH 4.5 is
significantly shorter by 0.15 Å than the bond length at pH 7.5. The
flip out of His6 imidazole ring (pKa = 5.9) was also observed. The
flip out of the His6 imidazole may induce the structural changing at
the active site.
References
1. Abdelhamid RF, Obara Y, Uchida Y, Kohzuma T, Dooley DM,
Brown DE, Hori H (2007) J Biol Inorg Chem 12:165–173.
2. Gast P, et al. (2014) J Inorg Biochem (in press).
J Biol Inorg Chem (2014) 19 (Suppl 2):S833–S852
P 165
Efficient oxidation and destabilization of a Zn(Cys)4
zinc finger by singlet oxygen
Lebrun Vincent1, Tron Arnaud2, Scarpantonio Luca2,
Lebrun Colette3, Ravanat Jean-Luc4, Latour JeanMarc1, McClenaghan Nathan2, Sénèque Olivier1
1
UMR 5249, LCBM/PMB, Univ. Grenoble Alpes/CNRS/CEA, 38054
Grenoble Cedex 9, France.
2
ISM, Univ. Bordeaux/CNRS, 33405 Talence Cedex, France.
3
SCIB, iNAC/SCIB/RICC, CEA, 38054 Grenoble Cedex 9, France.
4
SCIB, iNAC/SCIB/LAN, CEA, 38054 Grenoble Cedex 9, France
Singlet oxygen (1O2), the lowest excited state of molecular oxygen, is
one of the most reactive ROS. Despite its occurrence in all aerobic
organisms and its ability to severely damage nucleic acids and proteins
[1], its biological chemistry has been neglected in non-photosynthetic
organisms. In addition, it has been demonstrated that living organisms
can mount a defense system against 1O2, suggesting a specific detection
pathway. Yet, the cellular and molecular mechanisms involved in this
detection remains poorly understood. Cysteine plays a central role in
redox biology, and it is well known for reacting rapidly with singlet
oxygen, yielding a several oxidation products (disulfides, thiosulfinates,
sulfinates, sulfonates) [2]. However, in many proteins, the sulfur atom of
cysteine is bound to a metal, modulating its reactivity, as exemplified by
zinc fingers. Found in a large number of proteins through the entire living
world, these sites contains two, three or four cysteines bound to a ZnII
ion, providing structural stabilization essential for its folding [3]. Nevertheless, rising examples of reactive zinc fingers led to the emergence of
the hypothesis of a new role for them: oxidative stress sensors, via a
redox switch mechanism [4, 5]. Because the literature related to the
reactivity of zinc fingers toward singlet oxygen is extremely scarce, we
have studied the interaction of 1O2 and zinc finger model peptides to
obtain accurate chemical measurements relevant to biological molecules.
We will report on the reactivity of a treble clef zinc finger model
Zn(Cys)4 toward 1O2. Measurement of the reaction rate and identification
of the oxidation products allow us to discuss the possibility such oxidation
to occur in cells, and their consequence on zinc finger’s stability.
References
1. Davies MJ (2005) Biochim Biophys Acta 1703:93–109.
2. Devasagayam TPA, Sundquist AR, Di Mascio P, Kaiser S, Sies H
(1991) J Photochem Photobiol B 9:105–106.
3. Andreini C, Banci L, Bertini I, Rosato A (2006) J Proteome Res 5:196.
4. Kröncke K-D, Klotz L-O (2009) Antioxid Redox Signal
11:1015–1027.
5. Ilbert M, Graf PCF, Jakob U (2006) Antioxid Redox Signal 8:835–846.
P 166
The structure of octaheme cytochrome c sulfite
reductase MccA from W. succinogenes reveals
a CX15CH heme c binding motif and a novel active site
geometry
Bianca Hermann1, Melanie Kern2, Jörg Simon2, Oliver
Einsle1
S835
metabolic pathways, particularly in the nitrogen and sulfur cycle.
MCCs have been shown to have a broad substrate spectrum including
the six-electron reduction of nitrite to ammonia and sulfite to sulfide
as well as the oxidation of hydroxylamine [1–3].
MccA from W. succinogenes harbours eight heme c groups per
monomer, including an unusual CX15CH motif [4]. Its activity towards
sulfite is significantly higher than that reported for other MCCs and
siroheme-containing dSir proteins [2, 5]. At the same time it is the only
sulfite-reducing enzyme known to date that does not react with nitrite.
By means of X-ray crystallography we were able to solve the
´
structure of MccA to a maximum resolution of 2.2 Å with different
substrates and intermediates of sulfite reduction bound at an active
site with an unprecedented architecture. This enables us to propose a
reaction mechanism that greatly differs in some aspects from the
proposed mechanism in dSir proteins and provides a rationale for the
absence of nitrite reductase activity.
References
1. Einsle O, Messerschmidt A, Stach P, Bourenkov GP, Bartunik HD,
Huber R, Kroneck PMH (1999) Nature 400:476–480.
2. Simon J, Kroneck PMH (2013) Adv Microb Phys 62:45–117.
3. Lukat P, Rudolf M, Stach P, Messerschmidt P, Kroneck PMH,
Simon J, Einsle O (2008) Biochemistry 47:2080–2086.
4. Hartshorne RS, Kern M, Meyer B, Clarke TA, Karas M, Richardson DJ, Simon J (2007) Mol Microbiol 64:1049–1060.
5. Kern M, Klotz MG, Simon J (2011) Mol Microbiol 82:1515–1530.
P 167
A covalent mechanism at an unusual reactive thiolateligated heme
Daniel B. Grabarczyk1,3, Paul E. Chappell2, Ian J.
McPherson3, Kylie A. Vincent3, Susan M. Lea2, Ben C.
Berks1
1
Department of Biochemistry, Oxford University, South Parks Road,
OX1 3QU Oxford, UK.
2
Dunn School of Pathology, Oxford University, South Parks Road,
OX1 3QU Oxford, UK.
3
Inorganic Chemistry Laboratory, Department of Chemistry, Oxford
University, South Parks Road, OX1 3QR Oxford, UK
TsdA is a diheme cytochrome c which reversibly catalyzes the oxidation of
thiosulfate to tetrathionate. This enzyme is important in gut microbiology
and the biogeochemical sulfur cycle. Catalysis is expected to involve an
unusual cysteine/histidine ligated heme. By solving the first structure of
this enzyme by X-ray crystallography to 1.3 Å resolution we have been
able to dissect the catalytic mechanism at this heme. We have shown that
the heme-ligating thiolate is indeed reactive and can dissociate from the
heme to be covalently modified with alkylating reagents. Additionally, a
covalent reaction intermediate can be detected by acid-quenching the
enzyme mid-reaction, or by performing stalled half-reactions. We have
characterised these cysteine adducts, their formation and their stability by a
combination of X-ray crystallography, mass spectrometry, various spectroscopic techniques and protein film electrochemistry. We propose a
tentative catalytic mechanism based on our data.
1
Institute for Biochemistry, University of Freiburg, Albertstrasse 21,
79104 Freiburg, Germany. [email protected]
2
Department of Biology, Microbial Energy Conversion
and Biotechnology, Technische Universität Darmstadt,
Schnittspahnstrasse 10, 64287 Darmstadt, Germany
The family of multiheme cytochromes c (MCC) comprises diverse
electron carriers and redox enzymes that play key roles in several
P 168
Structures and properties of blue copper protein,
pseudoazurin Thr36X variants
Risa Aoki1, Akiko Takashina1, Masaki Unno1,2,
Takamitsu Kohzuma1,2
123
S836
1
Institute of Applied Beam Science, Ibaraki University, 2-1-1
Bunkyo, Mito, Japan.
2
Frontier Research Center for Applied Atomic Science, Ibaraki
University, Tokai, Japan
Pseudoazurin (PAz) from Achromobacter cycloclastes is a type 1
copper protein, which functions as an electron carrier in denitrification process [1]. The copper atom at the active site is coordinated by
two histidine imidazoles (His40 and His81), one cysteine thiolate
(Cys78), and one methionine sulfide (Met86) with a distorted tetrahedral geometry [2]. PAz shows intense absorption band around at
450 and 600 nm due to Cys (S-)?Cu2+ charge transfer. The structure
and functional role of weak interaction in the vicinity of the active site
of PAz have been studied [3]. The Thr36 is located in the back loop
region involving copper coordinated His40, and the hydroxyl group of
Thr36 forms hydrogen bond with His6 imidazole group. The protonation/deprotonation of His6 have been proposed to regulate the
electron transfer reaction of PAz [2].
The Thr36X variants of pseudoazurin, Thr36Arg and Thr36Lys
were constructed and purified to add more pages into the structural
and functional features of weak interaction in blue copper protein.
The spectroscopic, electrochemical, and X-ray crystallographic
characterization of Thr36X mutant protein have been performed.
The ratio of the molar absorption coefficient at 460 and 600 nm,
e*460/e*600 of WT, Thr36Arg, and Thr36Lys were 0.46, 0.31 and
0.34, respectively. The significantly smaller e*460/e*600 ratio for
Thr36Arg and Thr36Lys were observed, which reflect that the active
site structures of Thr36Arg and Thr36Lys have more axial character
than WT PAz. The reduction potential of the Thr36Arg and Thr36Lys
variants were 30–50 mV higher than that of wild type PAz. The
reduction potential shift into higher region may due to the introduction of positively charged residue closed to the active site.
The X-ray crystal structure analyses of Thr36Arg and Thr36Lys
variants showed that the overall structures were almost identical to
WT PAz. The Cu-SMet86 bond lengths in both of the variant proteins
were observed to be 2.8 and 2.5 Å suggesting the two different active
site structures, axial and rhombic.
References
1. Averill BA (1996) Chem Rev 96:2951–2964.
2. Kohzuma T, Dennison C, McFarlane W, Nakashima S, Kitagawa T,
Inoue T, Kai Y, Nishio N, Shidara S, Suzuki S, Sykes AG (1995) J
Biol Chem 270:25733–25738.
3. Abdelhamid RF, Obara Y, Uchida Y, Kohzuma T, Dooley DMB,
Hori DE (2007) J Biol Inorg Chem 12:163–173.
4. Inoue T, Nishio N, Suzuki S, Kataoka K, Kohzuma T, Kai Y (1999)
J Biol Chem 274:17845–17852.
P 169
The metalloprotein HbpS from Streptomycetes
specifically interacts with iron, heme and cobalamin
Darı́o Ortiz de Orué Lucana1, Ina Wedderhoff1,
Andrew Torda2, Sergey Fedosov3
1
Applied Genetics of Microorganisms, Department of Biology/
Chemistry, University of Osnabrueck, Barbarastr. 13, 49067
Osnabrueck, Germany.
2
Centre for Bioinformatics, Hamburg University, Bundesstr. 43,
20146, Hamburg, Germany.
3
Department of Engineering Science, Aarhus University, Gustav
Wieds Vej 10C 8000 Aarhus C, Denmark
The extracellular protein HbpS acts as an accessory protein of the
two-component system SenS-SenR from the cellulose degrader
Streptomyces reticuli. HbpS-SenS-SenR is involved in the protection
123
J Biol Inorg Chem (2014) 19 (Suppl 2):S833–S852
of this soil bacterium against the toxic effects of oxidative stress and
is conserved in many other Actinobacteria [1]. Analysis of the 3D
crystal structure and biochemical studies revealed that HbpS assembles as an octamer [2]. Further spectroscopic analyses showed that
HbpS specifically binds iron ions, heme [3] and aquacobalamin.
Based on 3D crystal structures, structural alignments, sequence
comparisons, mutagenesis, and comparative biochemical investigations, we identified the coordination sites for iron, heme and
aquacobalamin in HbpS, and demonstrated that each site differs from
each other. While the physiological relevance of iron- and hemebinding by HbpS has been analysed in detail, the in vivo role of the
HbpS-aquacobalamin complex remains to be elucidated.
References
1. Wedderhoff I, Kursula I, Groves MR, Ortiz de Orué Lucana D
(2013) PLoS One 8:e71579.
2. Siedenburg G, Groves MR, Ortiz de Orué Lucana D (2012) Antioxid Redox Signal 16:668–677.
3. Ortiz de Orué Lucana D, Bogel G, Zou P, Groves MR (2009) J Mol
Biol 386:1108–1122.
P 170
An artificial monooxygenase?
Laurianne Rondot, Adeline Jorge Robin, Christine
Cavazza, Caroline Marchi-Delapierre, Stéphane
Ménage
Laboratoire de Chimie et Biologie des métaux. UMR 5249 Université
Joseph Fourier, CNRS et CEA, CEA-Grenoble-17, Avenue des
Martyrs, 38054 Grenoble. [email protected]
Enantiopurity of drugs is still a need, but nowadays, lots of processes are
still leading to a mixture of both enantiomers. In this context, the BioCe
team of the LCBM is working on the development of new systems able
to oxidize substrates selectively. Another need is the protection of the
environment. We so decide to work with dioxygen as the oxidant and
artificial metalloenzymes as catalysts in order to follow most of the rules
of « green chemistry » . Recently, during our studies on the oxidation
mechanism of aromatic substrates using the crystallo-kinetic methodology, we designed an artificial metalloenzyme by anchoring an
inorganic iron complex into the NikA protein, a Ni transport protein in
E. coli [1]. The X-rays studies revealed that the artificial metalloenzyme
was able to hydroxylate two times the aromatic intramolecular substrate
using only dioxygen and DTT. After the two catalysis cycles, the catalysts was inactivated by the coordination of the just created hydroxyl
moiety to the iron disabling the oxidation of an exogenous substrate.
These promising results lead us to modify the inorganic complex
by synthesizing new ligands free of oxidation moieties. We then
needed to verify the ability of the new complexes to activate dioxygen. Without substrate in the reaction medium, mass spectrometry
experiments showed the presence of various oxidated moieties upon
the ligand backbone leading us to conclude that the modified catalysts
were still able to activate dioxygen. Moreover, these oxidations did
J Biol Inorg Chem (2014) 19 (Suppl 2):S833–S852
S837
not show inhibition of the activity of the complex, contrary to the
parent complex. Cristallo-kinetic studies have been conducted on
crystals of the new artificial metalloenzymes showing a single oxidation reaction on the ligand backbone when placed in a DTT/O2
medium in the absence of an exogenous substrate.
In order to transform these artificial metalloenzymes into artificial
mono-oxygenases we then had to demonstrate their ability to transfer
the activated oxygen atom to a substrate of interest. The poster will
present our latest results in this field.
References
1. Goetzl S, Jeoung JH, Hennig SE, Dobbek H (2011) J Mol Biol
411:96–109.
2. Dau H, Liebisch P, Haumann M (2003) Anal Bioanal Chem
376:562–583.
3. Leidel N, Chernev P, Havelius K, Schwartz L, Ott S, Haumann M
(2012) J Am Chem Soc 134:14142–14157.
Reference
1. Cavazza C, Bochot C, Rousselot-Pailley P, Carpentier P, Cherrier
MV, Martin L, Marchi-Delapierre C, Fontecilla-Camps JC, Menage S
(2010) Nat Chem 2:1069.
P 172
Radical-SAM enzymes with two FeS clusters, RimO
and TYW1: Using spectroscopy to assign
the interactions and function of each cluster
Thibaut Molle1, Velavan Kathirvelu1, Ricardo GarciaSerres1, Martin Clemancey1, Jean-Marc Latour1,
Vincent Maurel1, Serge Gambarelli1, Farhad
Forhouar2, John F. Hunt2, Marc Fontecave3, Etienne
Mulliez1, Mohamed Atta1
P 171
Cobalt redox and ligation changes in cobalamine
systems revealed by X-ray absorption spectroscopy
and density functional theory
Peer Schrapers1, Sebastian Goetzl2, Ramona Kositzki1,
Sandra E. Hennig2, Holger Dau1, Holger Dobbek2,
Michael Haumann1
1
Department of Physics, Free University Berlin, Arnimallee 14,
14195 Berlin, Germany. [email protected]
2
Department of Biology, Humboldt University Berlin, Philippstr. 13,
10115 Berlin, Germany
Corrinoid-cofactor containing proteins are prominently involved in COx
converting reactions in bacteria, which are inspiring for potential renewable fuel applications. During the reductive acetyl-CoA (WoodLjungdahl) pathway, the cobalt ion in protein-bound cobalamine is
changing its oxidation state to transfer a methyl group from a methyltransferase/methyltetrahydrofolate complex to the reduced [NiNi]–[4Fe4S]
cluster of acetyl-CoA synthase [1]. Refinement of metal–ligand bond
lengths in the crystal structures here was attempted by X-ray absorption
spectroscopy (XAS) analysis [2]. We investigated different states of the
cobalamine cofactor in isolated corrinoid-iron-sulfur protein (CoFeSP), in
CoFeSP in complex with its reductive activator (RACo), and in cobalamine in solution by Co-XAS. K-edge energy shifts suggested mainly
Co(II) to Co(III) redox transitions, but the expected binding of additional
ligands at the Co was difficult to identify by EXAFS. This was remedied
by analysis of the pronounced pre-edge absorption changes due to core-tovalence electronic transitions using density functional theory calculations
[3], which explained the spectral changes by electronic structure variations
in response to ligand binding, thereby leading to specific models for the
cobalt sites in the different states. We show that electronic transition
analysis by XAS-DFT facilitates assignment of redox changes and ligand
binding in hetero-macrocycle systems.
Financial support by the DFG (grants Ha3265/2-2,/3-1, and/6.1),
the BMBF (grant 05K14KE1), and within Unicat (CoE Berlin) is
gratefully acknowledged.
1
Laboratoire de Chimie et Biologie des Métaux, iRTSV, UMR 5249,
CEA/CNRS/UJF, CEA-Grenoble, Grenoble, France.
[email protected].
2
Department of Biological Sciences, Columbia University, NewYork, NY, USA 3Collège de France, Paris, France
The two enzymes TYW1 and RimO share sequence homology,
similar folds, and use radical chemistry through S-Adenosy-LMethionine (SAM) cleavage. The reactions that they catalyze are,
however, quite different. TYW1 inserts a C–C moiety in a methylguanosine during a tRNA modification [1], while RimO is a
methylthiotransferase whose function as a true catalyst has been
recently highlighted [2]. In both cases the two [4Fe-4S] clusters are in
close vicinity, 11 Å apart in M. janaschii TYW1 [3] and only 8 Å
apart in T. maritima RimO [2]. The space between the clusters has
been proposed to be the enzyme’s catalytic site and to serve as a
docking cavity for substrates and co-substrates. Using a combination
of spectroscopies, we have been able to identify specific interactions
between each cluster and individual amino-acids or substrates [4], and
to assess the effect of the other cluster on such interactions. On the
basis of these assignments, catalysis mechanisms are proposed for
both enzymes.
References
1. Young AP, Bandarian V (2011) Biochemistry 50:10573–10575.
2. Forouhar F, Arragain S, Atta M, Gambarelli S, Mouesca J-M,
Hussain M, Xiao R, Kieffer-Jacquinod S, Jayaraman S, Acton TB,
Montelione GT, Mulliez E, Hunt JF, Fontecave M (2013) Nat Chem
Biol 9:333–341.
3. Suzuki Y, Noma A, Suzuki T, Senda M, Senda T, Ishitani R,
Nureki O (2007) J Mol Biol 372:1204–1214.
123
S838
4. Perche-Letuvee P, Kathirvelu V, Berggren G, Clemancey M, Latour
J-M, Maurel V, Douki T, Armengaud J, Mulliez E, Fontecave M, GarciaSerres R, Gambarelli S, Atta M (2012) J Biol Chem 287:41174–41185.
P 173
Two new C-type cytochromes found in cyanobacteria:
Tll0287 and PsbV2
Thanh-Lan Lai1, Miwa Sugiura2, Michihiro Suga3,
Jian-Ren Shen3, Alain Boussac1
1
Department of life sciences, CEA, Institute of biology
and technologies of Saclay, Laboratory of fundamental mechanisms
in bioenergetics, 91140 Gif sur Yvette, France.
2
University of Ehime, Matsuyama, Japan.
3
Presto, Japan Science and Technology Agency, Japan
The photosystem II (PSII) catalyses the light-driven water oxidation.
The catalytic site named Oxygen Evolving Complex (Mn4CaO5) accumulates oxidizing equivalents. This cluster is localized on the D1
protein. The mechanism of water oxidation is not completely understood
while the PSII structure was obtained at 1.9 Å in Thermosynechococcus
vulcanus in 2011 [1]. The efficiency of the electron transfer steps is
modulated by the nature of the interaction between key amino acids and
the cofactors. The D1 protein can be encoded by three different psbA
genes (psbA1/psbA2/psbA3) in the wild-type thermophilic cyanobacterium Thermosynechococcus elongatus. The amino acids sequences of
these three D1 proteins are not identical; it differs from 21 to 35 amino
acids. It has been shown that these three genes are expressed differently
according to the environment of the cells. Some functional differences
between PSII of these three proteins have been found [2]. A new
C-type cytochrome Tll0287 has been discovered in the mutant
expressing only the gene psbA2 and cultivated in aerobic conditions
[3]. PsbV2, another C-type cytochrome was discovered in the wild type
cells. Its crystallographic structure was resolved at 1.5 Å [4]. New
results on the expression, purification, structural and biophysical
characterizations of those two C-type cytochromes of cyanobacteria
found in the laboratory will be presented.
Financial support by the program Bioenergy from the CEA Saclay
is gratefully acknowledged.
References
1. Umena Y, Kawakami K, Shen J-R, Kamiya N (2011) Nature
473:55–60.
2. Sugiura M, Azami C, Koyama K, Rutherford A, Rappaport F,
Boussac A (2013) BBA 1837:139–148.
3. Boussac A, Koyama K, Sugiura M (2013) BBA 1827:1174–1182.
4. Suga M, Lai T-L, Sugiura M, Shen J-R, Boussac A (2013) FEBS
Letters 587:3267–3272.
P 174
Metal sensing in a bacterial cell: a network
that differentiates metals
Cecilia Piergentili, Andrew W. Foster, Carl J.
Patterson, Rafael Pernil, Deenah Osman, Nigel J.
Robinson
School of Biological and Biomedical Sciences, Durham University,
South Road, DH1 3LE, Durham, UK
Bacterial metal-sensor proteins bind metal ions and repress, derepress
or activate the transcription of operons that encode metal-specific
efflux pumps, metal transport proteins, metal-sequestering proteins
123
J Biol Inorg Chem (2014) 19 (Suppl 2):S833–S852
and often the metal-responsive transcriptional regulator itself. In this
way the correct concentration of a particular transition metal in the
cell is maintained. Emerging evidence suggests that the actions of
metal-sensors are interconnected such that the detection of metals
in vivo is a shared function of the cell’s set of sensors. Determination
of relative and absolute metal affinities in vitro is not always sufficient
to fully explain which metals each sensor detects inside the cell.
An illustrative example is given by metal sensing in Synechocystis.
InrS is a Ni(II)-responsive, CsoR/RcnR-like, DNA-binding transcriptional-repressor of nrsD [1]. InrS has a KDNi(II) tighter than those
of the other metal sensors in Synechocystis. In addition, Cu(I) and
Zn(II) also impair InrS-binding to the nrsD operator-promoter in vitro
but InrS responds to these metals only during the initial transient
upshift (1 h) while, after 48 h, Zn(II)–sensor ZiaR continues to
respond. InrS KZn(II) is comparable to the sensory-sites of ZiaR so the
relative zinc affinity does not determine the final detection threshold
for Zn(II). The key parameter here is the coupling free energy DGMC
InrS•DNA
, which is sufficiently high for Ni(II), to trigger sensing, but
insufficient for Zn(II) (and potentially copper).
The same organism presents the Mer-like transcriptional activator
CoaR, which detects surplus Co(II) to regulate Co(II) efflux. CoaR
has KCo(II) weaker than 7 9 10-8 M which is weaker than ZiaR, Zur
and InrS [2]. Yet, after 48 h exposure to maximum non-inhibitory
[Co(II)], CoaR responds in vivo, where the two Zn(II) sensors do not,
despite their tighter KCo(II) and despite Co(II) triggering allostery in
ZiaR and Zur in vitro. The specific detection of Co(II) by CoaR is
enigmatic. CoaR is membrane associated via a domain with homology to precorrin isomerase, an enzyme which catalyses a reaction
required during the biosynthesis of B12. The pathway for B12 biosynthesis is also known to be membrane associated in other bacteria
with intermediates channeled between enzymes, suggesting putative
mechanisms by which Co(II) is channeled to CoaR.
In conclusion, these studies highlight the merit in comparing the properties of multiple sensors for a given metal as opposed to the typical approach
of comparing the properties of a single sensor for multiple metals.
References
1. Foster AW, Pernil R, Patterson CJ Robinson NJ (2014) Mol
Microbiol (in press).
2. Patterson CJ, Pernil R, Dainty SJ, Chakrabarti B, Henry CE, Money
VA, Foster AW, Robinson NJ (2013) Metallomics 5:352–362.
P 175
Design of a novel type of enzymatic control in NColE7based zinc finger nucleases
Eszter Németh1, Chris Oostenbrink2, Béla Gyurcsik1
1
Department of Inorganic and Analytical Chemistry, University
of Szeged, Dóm tér 7., 6720 Szeged, Hungary.
2
University of Natural Resources and Life Sciences, Institute
of Molecular Modeling and Simulation, Muthgasse 18,1190 Wien,
Austria
Zinc-finger nucleases (ZFN) became useful tools for targeted cleavage of genomic DNA. The majority of these chimeric enzymes use
the FokI nuclease domain as their hydrolytic unit, fused to the specific
DNA-binding zinc-fingers. Since the DNA binding properties can be
redesigned, ZFN-s provide a promising tool as therapeutic agents for
monogenetic diseases. However, the cytotoxic side-effect of the
presently available ZFN-s prevents such applications.
Our research is focused on the design of a safely controlled ZFN
being inactive in the absence of the substrate of specific sequence,
while binding to the target DNA induces its active conformation.
Instead of the commonly used FokI domain, we fused NColE7 to zinc
fingers. NColE7 is a nonspecific metallonuclease, part of the defence
J Biol Inorg Chem (2014) 19 (Suppl 2):S833–S852
mechanism of E. coli. Its special structural feature is that its Zn2+binding C-terminal catalytic domain (HNH-motif) cooperates with the
N-terminus of the protein during the enzymatic reaction [1].
In our approach, the zinc-fingers were inserted between the N- and
C-terminal parts of NColE7. Computational methods including
database search, protein stability estimation and MD simulations were
carried out to design the linkers, as well as the division of NColE7 to
C-and N-terminal units. The genes encoding the four designed proteins have been constructed and the proteins were expressed in E. coli
cells. The DNA-binding affinity, specificity and nuclease activity of
the proteins were studied. Two of the designed models are shown in
the figure below. The zinc fingers are in blue, while the C-terminal
and N-terminal part of NColE7 are in green and red, respectively.
Reference
1. Czene A, Németh E, Zóka IG, Jakab-Simon NI, Körtvélyesi T,
Nagata K, Christensen HEM, Gyurcsik B (2013). J Biol Inorg Chem
18:309–321.
P 176
Understanding the inhibition mechanism of chlorite
dismutase from the nitrite-oxidizing bacterium
Candidatus Nitrospira defluvii
Stefan Hofbauer1, Clemens Gruber1, Irene Schaffner1,
Katharina F. Pirker1, Christa Jakopitsch1, Axel
Sündermann2, Chris Oostenbrink2, Paul G.
Furtmüller1, Christian Obinger1
1
Department of Chemistry, Division of Biochemistry, 2Department
of Material Sciences and Process Engineering, Institute of Molecular
Modeling and Simulation, VIBT-Vienna Institute of BioTechnology,
BOKU-University of Natural Resources and Life Sciences,
Muthgasse 18, A-1190 Vienna, Austria
Chlorite dismutases (Clds) are oligomeric heme b-dependent oxidoreductases capable of catalyzing the conversion of toxic chlorite
(ClO2-) into chloride and dioxygen. Anthropogenic chlorite is a
serious environmental concern that pollutes groundwater, surface
waters and soils. In order to use Clds for bioremediation it is
important to know its stability, substrate specificity, reaction and
inhibition mechanism. Cld from the mesophilic nitrite-oxidizing
bacterium ‘‘Candidatus Nitrospira defluvii’’ (NdCld) is a catalytically
efficient (kcat/KM = 6.0 9 105 M-1 s-1) homopentamer of high stability and redox thermodynamics similar to a dimeric Cld from a
different phylogenetic lineage. However, NdCld suffers from early
self-inhibition [1]. Wild-type NdCld and variants having the only
charged distal side residue (Arg173) exchanged by Ala or Lys were
produced in E. coli and characterized [2].
In detail we probed the impact of HOCl-traps on the enzyme’s
activity to better understand its inhibition pattern and further analyzed
oxidative modifications of the protein matrix and the heme co-factor.
The proposal that HOCl is an intermediate in enzyme turnover that is
able to leave the active site (also supported by Molecular Dynamics
S839
Simulations) and irreversibly inactivates the protein in the course of
reaction was underlined by increased O2-generation and chlorite
degradation activity in the presence of the HOCl-trap methionine and
was quantified by chlorination of MCD and APF by UV–Vis stoppedflow and fluorescence spectroscopy, respectively. Enzyme modifications caused by the HOCl-release were identified with MS and EPR
spectroscopy. Data are discussed with respect to the proposed reaction
mechanism and solved X-ray structures of Cld.
Our research was supported by the Austrian Funding Agency
(FWF-doctoral program BioToP-Biomolecular Technology of Proteins, W1224 and the stand alone project P25270).
References
1. Hofbauer S, Schaffner I, Furtmüller PG, Obinger C (2014) Biotechn J 9:461–473.
2. Hofbauer S, Gysel K, Bellei M, Hagmüller A, Schaffner I, Mlynek
G, Kostan J, Pirker KF, Daims H, Furtmüller PG, Battistuzzi G,
Djinovic-Carugo K, Obinger C (2014) Biochemistry 53:77–89.
P 177
Mechanism of reaction of chlorite with mammalian
peroxidases
Christa Jakopitsch1,, Katharina F. Pirker1, Jörg
Flemmig2,3, Stefan Hofbauer 1, Denise Schlorke2, Paul
G. Furtmüller1, Jürgen Arnhold2,3, Christian Obinger1
1
Department of Chemistry, University of Natural Resources and Life
Sciences, Muthgasse 18, 1190 Vienna, Austria.
2
Institute for Medical Physics and Biophysics, Medical Faculty,
University of Leipzig, Haertelstr 16-18, 04107 Leipzig, Germany.
3
Translational Centre for Regenerative Medicine, University
of Leipzig, Philipp-Rosenthal-Str. 55, 04103 Leipzig, Germany
In plant-type heme peroxidases chlorite mediates the two-electron
oxidation of ferric enzymes to compound I, thereby releasing hypochlorous acid. Furthermore, chlorite acts as one-electron reductant of
both compound I and compound II forming chlorine dioxide [1].
In contrast both human myeloperoxidase (MPO) and bovine lactoperoxidase (LPO) cannot utilize chlorite to form chlorinating
species. Moreover, both peroxidases are rapidly and irreversible
inactivated by chlorite accompanied by a decrease in conformational
and thermal stability, partial heme bleaching and free iron release.
A detailed analysis of the reaction of all biological relevant redox
intermediates with chlorite revealed that chlorite serves as efficient
one-electron donor for compounds I and II and that inactivation takes
place at the ferric state. The kinetics of inactivation consists of two
phases: (i) binding of chlorite to ferric enzymes resulting in formation
of a MPO-chlorite-high-spin complex and (ii) heme degradation.
These findings are discussed with respect to differences in active
site architecture and heme modification between plant peroxidases
and their mammalian counterparts. In contrast to plant peroxidases the
heme in LPO and MPO is covalently attached to the protein via two
ester-linkages. In MPO in addition a sulfonium linkage is formed
between the heme and the protein.
Inactivation of myeloperoxidase by chlorite can be of physiological and medical relevance since MPO is involved in the regulation of
immune functions and chlorite-based drugs are used as wound-healing agents (OxoferinTM) or applied as intravenous infusion in patients
with chronic-inflammatory diseases (WF10). Comparing effects of
pure chlorite with the chlorite-containing drug OxoferinTM nearly
identical kinetics of its interaction are observed and it also mediates
heme bleaching. To what extent the interaction of the chlorite-containing drugs with MPO are responsible for the observed therapeutic
effects of these drugs remains unknown.
123
S840
References
1. Jakopitsch C, Spalteholz H, Furtmüller PG, Arnhold J, Obinger C
(2008) J Inorg Biochem 102:293–302.
2. Jakopitsch C, Pirker KF, Flemmig J, Hofbauer S, Furtmüller PG,
Schlorke D, Arnhold J, Obinger C (2014) J Inorg Biochem
135:10–19.
P 178
Investigating the relation between structure
and reaction mechanism of eukaryotic catalaseperoxidase
Bernhard Gasselhuber1, Andrea Nicolussi1, Xavi
Carpena2, Marcel Zamocky1, Christa Jakopitsch1, Paul
G. Furtmüller1, Ignacio Fita2, Christian Obinger1
1
Department of Chemistry, Division of Biochemistry, VIBT-Vienna
Institute of BioTechnology, BOKU-University of Natural Resources
and Life Sciences, A-1190 Vienna.
2
Institute for Research in Biomedicine (IRB Barcelona) and Institut
de Biologia Molecular de Barcelona (IBMB) from Consell Superior
d’Investigacions Cientı́fiques (CSIC), Parc Cientı́fic, 08028
Barcelona, Spain
Bifunctional catalase-peroxidases (KatGs; EC 1.11.1.21) have raised
considerable interest since they are the only known heme peroxidases
with substantial catalatic (i.e. hydrogen peroxide dismutating) activity.
Phylogenetic analysis showed the distribution of KatGs in both prokaryotic and eukaryotic organisms. Eukaryotic KatGs can be located
either intra-(KatG1) or extracellularly [1]. The secreted representatives
can be found almost exclusively in pathogenic organisms (mainly
fungi), where they seem to be involved in degradation of reactive
oxygen species released by the attacked plant during infection [2].
Recent reports showed an enhanced thermostability of eukaryotic
KatG2 compared to previously investigated prokaryotic representatives.
An available high resolution crystal structure of the extracellular KatG
from the so-called ‘rice blast fungus’ Magnaporthe grisea (world’s most
dreaded rice pathogen) renders this metalloenzyme (MagKatG2) as an
interesting target for structural and mechanistic studies [3].
This work focuses on structural studies of wild-type MagKatG2
together with variants of the unique so-called covalent Trp-Tyr-Met
adduct. This active site modification seems to be responsible for the
unique bifunctional activity of catalase-peroxidases. Here we correlate
structural with mechanistic studies and present newly obtained high
resolution crystal structures of the heme enzyme at different pH-values
and oxidation states. Moreover, molecular dynamics simulations under
different conditions support the experimental data. We present the
dramatic consequences both on structure and activity when the KatGtypical Trp-Tyr-Met adduct is disrupted in mutant proteins.
Detailed elucidation of the mechanism of the bifunctional activity
of KatG will help (i) to understand its biological role as well as (ii) to
design inhibitors by (semi-)rational approaches for modern pest and
crop management.
Our research was supported by the Austrian Funding Agency
(FWF-doctoral program BioToP-Biomolecular Technology of Proteins, W1224).
References
1. Zamocky M, Gasselhuber B, Furtmüller PG, Obinger C (2012)
Arch Biochem Biophys 525:131–141.
2. Tanabe S, Ishii-Minami N, Saitoh KI, Otake Y, Kaku H, Shibuya N,
Nishizawa Y, Minami E (2011) Mol. Plant Microbe Interact 24:163–171.
3. Zamocky M, Garcia-Fernandez MQ, Gasselhuber B, Furtmüller
PG, Loewen PC, Fita I, Obinger C, Carpena X (2012) J Biol Chem
287:32254–32262.
123
J Biol Inorg Chem (2014) 19 (Suppl 2):S833–S852
P 179
Mimicking the molybdopterin system by synthesis
of molybdenum-Pterin complexes
Ivan Trentin, Carola Schulzke
Institut für Biochemie-Bioinorganic chemistry, University
of Greifswald, D17487 Greifswald, Germany. [email protected]
The pteridine family comprises a very important chapter of the
chemistry and the biology of the 20th century. Particularly pterins, a
subclass of this family, fulfil a variety of roles in biology including
being pigments, toxins, redox cofactors and C1 transfer cofactors. The
term pterin refers to a substituted pteridine by a keto group at the
position 4 and an amino group at position 2 (Fig); this particularly
structure is present in a rather complicated molecule called molybdopterin, which, by coordinating to molybdenum, forms the
molybdenum cofactor (MoCo) (Fig). The main purpose of this project
is the study of differently substituted pterin-dithiolene moieties bound
to molybdenum (Fig) in order to obtain a better understanding of
structure–function relationships and the high instability of this very
important part of all molybdenum dependent oxidoreductases.
In fact, despite having shown that the third ring of mpt does not
electronically influence the active site metal strongly if at all [1], we are
not convinced that it is actually negligible. Bearing a keto, two amine
and one amide group, it is potentially reactive and more importantly it is
able to take part in a substantial number of hydrogen bonds.
Financial support of the European Research Council is gratefully
acknowledged (project: MocoModels).
Molybdenum Cofactor (MoCo)
Pterin
O
L1
L2
Mo
O
N
HN
H2 N
O
N
HN
N
H2N
N
H
N
S
N
H
O
S
O
O
P
O
1
O
2
L , L = O, OR, SR, Cl
O
L1
Mo
O
HN
H2 N
N
H
N
N
H
L2
S
S
R
R = H, CH3, tert-butyl, Ph
Reference
1. Ryde U, Schulzke C, Starke K (2009) J Biol Inorg Chem
14:1053–1064.
P 180
Affinity capillary electrophoresis to investigate metal
ion interactions with the protein AtHIRD11
and the proline-rich peptide CBPep
Markus Nachbar1, Mona Mozafari1, Hassan Alhazmi1,
Lutz Preu1, Hassan Albishri2, Deia Abd El-Hady2,3,
Sami El Deeb1,4, Sabine Redweik1, Hermann Wätzig1
1
Institute of Medicinal and Pharmaceutical Chemistry, TU
Braunschweig, Beethovenstrasse 55, 38106 Brunswick, Germany.
J Biol Inorg Chem (2014) 19 (Suppl 2):S833–S852
2
Chemistry Department, Faculty of Science, King Abdulaziz
University, 80203 Jeddah, Saudi Arabia.
3
Chemistry Department, Faculty of Science, Assiut University,
71516-Assiut, Egypt.
4
Department of Pharmaceutical Chemistry, Al-Azhar UniversityGaza, Gaza, Palestine
The conformations of proteins do not only depend on the pH, temperature
and the ionic strength of the surrounding solution, but are also influenced
by the containing ions. These ions can change the conformation by binding
to specific motifs at the protein e.g. the EF hand motif or by unspecific
electrostatic attraction between a cation and negatively charged amino
acids. These changes in the conformation can be observed using mobility
shift affinity capillary electrophoresis. When ions bind to a protein its
electrophoretic mobility is altered, so does the time until it is detected. The
influence of various ions was determined by using mobility ratios of the
EOF-marker and the protein to prevent effects of migration time shifts
which are not related to interactions. The difference of the mobility ratio of
the protein with the ligand (Ri) and without the ligand (Rf) was normalised
to Rf (DR/Rf) [1]. The result gives an idea about the change in the overall
charge of the protein-ion-complex and also the strength of the interaction.
During the experiments various metal ions e.g. Mn2+, Cu2+ and Ba2+
as well as complexes of metal ions which have a low solubility at
physiological pH (e.g. Fe3+) were tested to determine their ability to
interact with different proteins such as ovalbumin, bovine serum albumin, the dehyadrin AtHIRD11 and a proline-rich proteolytic peptide
obtained from galectin-3 digestion (CBPep). The values for DR/Rf were
obtained with relative standard deviations below 1 % [n = 6 each].
The conformation changes of AtHIRD11 when certain transient
metal ions bind to the peptide [2] have been confirmed by mobility
shift affinity capillary. Additional interactions between this protein
and other metal ions could be identified.
In aqueous solution, the analysis of CBPep did not show the strong
interactions with calcium ions that have been obtained by mass
spectrometry in the vacuum state [3]. Only very weak interactions
towards other metal ions were found.
References
1. Redweik S, Xu Y, Wätzig H (2012) Electrophoresis 22:3316–3322.
2. Hara M, Kondo M, Kato T (2013) J Exp Bot 6:1615–1624.
3. Lehmann WD, Wei J, Hung CW, Gabius HJ, Kirsch D, Spengler B.
Kübler D (2006) Rapid Commun Mass Spectrom 16:2404–2410.
P 181
Investigation of a prokaryotic peroxidase
with a covalently bound heme
b from the cyanobacterium Lyngbya sp. PCC 8106
Andrea Nicolussi1, Markus Auer1, Georg Schütz1,
Clemens Gruber1, Katharina Pirker1, Marcel
Zamocky1,2, Paul G. Furtmüller1, Christian Obinger1
1
Department of Chemistry, Division of Biochemistry, VIBT-Vienna
Institute of BioTechnology, BOKU-University of Natural Resources
and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria.
2
Institute of Molecular Biology, Slovak Academy of Science, 84551
Bratislava, Slovakia
Heme peroxidases catalyze the oxidation of various organic and
inorganic substrates by hydrogen peroxide. Several heme peroxidase
(super)families have evolved independently during evolution. Representatives from the peroxidase-cyclooxygenase superfamily include
mammalian peroxidases, which are important players in the innate
immune system. These enzymes eliminate invaders by producing
reactive and oxidizing antimicrobial hypohalous acids [1]. Recently,
S841
homologous peroxidases were also detected in prokaryotic organisms.
This might suggest that unspecific cellular protection was already
necessary in early stages of evolution.
In the present work we investigated a novel bacterial heme peroxidase from the cyanobacterium Lyngbya sp. PCC 8106 (LspPOX).
This prokaryotic oxidoreductase has a very high sequence homology
to the mammalian peroxidases myeloperoxidase (MPO), lactoperoxidase (LPO), eosinophil peroxidase (EPO), and thyroid peroxidase
(TPO) [2]. We have heterologously expressed LspPOX in E. coli and
characterized it by a broad set of biochemical and biophysical techniques. Interestingly, the prosthetic group of LspPOX is covalently
bound to the protein (like MPO, EPO, LPO and TPO). This is a
unique feature for a bacterial peroxidase. We could demonstrate that
formation of these covalent bonds occurs autocatalytically, triggered
by the presence of hydrogen peroxide. This posttranslational modification modifies the catalytic properties as well as enhances the
conformational and thermal stability [3]. Our results are discussed
with respect to the well known mammalian counterparts.
Our research was supported by the Austrian Science Fund FWF
(doctoral program BioToP-Biomolecular Technology of Proteins,
W1224).
References
1. Zámocký M, Jakopitsch C, Furtmüller PG, Dunand C, Obinger C
(2008) Proteins 72:589–605.
2. Bernroitner M, Zámocký M, Furtmüller PG, Peschek GA, Obinger
C (2009) J Exp Bot 60:423–440.
3. Auer M, Gruber C, Bellei M, Pirker KF, Zámocký M, Kroiss D,
Teufer SA, Hofbauer S, Soudi M, Battistuzzi G, Furtmüller PG,
Obinger C (2013) J Biol Chem 288:27181–27199.
P 182
Molecular peculiarities of hybrid B heme peroxidases
Marcel Zamocky1,2, Katharina F. Pirker1, Bernhard
Gasselhuber1, Paul G. Furtmüller1, Katarina
Chovanova2, Jana Harichova2, Christian Obinger1
1
Department of Chemistry, Division of Biochemistry, VIBT-Vienna
Institute of BioTechnology, BOKU-University of Natural Resources
and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria.
2
Institute of Molecular Biology, Slovak Academy of Sciences, SK84551 Bratislava, Slovakia
Heme peroxidases are key enzymes of hydrogen peroxide metabolism. They are represented by three main superfamilies and two minor
families. Hybrid heme peroxidases are phylogenetically viewed as
turning points of the largest known peroxidase-catalase superfamily
[1] and might possess interesting mechanistic peculiarities. Hybrid B
peroxidases are extracellular fungal enzymes produced and secreted
mainly from various phytopathogens and soil fungi. Their expression
is rather constitutive when comparing the normal growth with the
presence of 10 mM H2O2 or peroxyacetic acid and slightly induced
by the occurrence of Cd2+ as revealed by qRT-PCR. In some HyBpox
representatives the conserved peroxidase domain is fused to a carbohydrate binding WSC domain. Here the hybrid B peroxidase from
the phytopathogenic fungus Magnaporthe grisea (MagHyBpox1)
was recombinantly produced and secreted from the methylotrophic
yeast Pichia pastoris. The affinity purified recombinant protein
MagHyBpox1 reveals a Soret maximum at 409 nm, Q bands at 535
and 570 nm, CT band at 625 nm with a purity number of 0.53.
EPR analysis suggests a mixture of high- and some low-spin species in the ferric state (Figure). The dissociation constant of the
low-spin cyanide complex (KD) is 6.6 lM. We report steady-state
and presteady-state kinetic data of the peroxidase activity and
123
S842
discuss the findings with those from well studied and phylogenetically related peroxidases.
Our research was supported by the Austrian Funding Agency FWF
with research projects P23855 and W1224 (doctoral program BioToPBiomolecular Technology of Proteins) and by the Slovak Grant
Agency VEGA with grant 2/0021/14.
J Biol Inorg Chem (2014) 19 (Suppl 2):S833–S852
4. Bı́ró L, Hüse D, Bényei AC, Buglyó P (2012) J Inorg Biochem
116:116–125.
5. Bı́ró L, Godó A, Bihari ZS, Garribba E, Buglyó P (2013) Eur J
Inorg Chem 2013:3090–3100.
P 184
Cu binding and oxidation kinetics characterization
of the multi-subunit Mn(II, III) oxidizing multicopper
oxidase, Mnx, from Bacillus sp. PL-12
Cristina N. Butterfield, Kelly N. Chacón, Ninian J.
Blackburn, Bradley M. Tebo
Reference
1. Zamocky M, Gasselhuber B, Furtmüller PG, Obinger C (2014) Cell
Mol Life Sci (submitted CMLS-D-14-00176).
P 183
Interaction between half-sandwich type [Ru(g6-pcym)(H2O)3]2+ with potential proliferative effect
and multihistidine containing oligopeptides
Zsolt Bihari1, Zoltán Nagy2, Péter Buglyó1
1
University of Debrecen, Faculty of Science and Technology,
Department of Inorganic and Analytical Chemistry, Egyetem tér 1,
H-4032, Debrecen, Hungary. [email protected]
2
University of Debrecen, Faculty of Science and Technology,
Department of Colloid and Environmental Chemistry, Egyetem tér 1,
H-4032, Debrecen, Hungary
In continuation of our recent work highlighting the interaction
between half-sandwich type ruthenium complexes with potential
antiproliferative activity and various small ligands with biological
relevance [1–5], the complex formation between [Ru(g6-pcym)(H2O)3]2+ and short oligopeptides containing histidine amino
acids at various positions in the peptide chain—Ac-HAHH-NH2 and
Ac-HAHAH-NH2 was studied to model the metal ion binding via the
surface histidine residues of high molecular mass serum components,
albumin and transferrin. In the metal ion—oligopeptide systems the
rather slow reactions prevented the characterization of the complex
formation processes using pH-potentiometry. Mass spectrometric,
NMR and UV–Vis results support the presence of various complexes
with different protonation degree in solution. This contribution will
also summarize the results obtained for the selectivity of the various
imidazole nitrogens within one oligopeptide ligand.
The authors thank members of the EU COST Action CM1105
for motivating discussions. The research was supported by the EU
and co-financed by the European Social Fund under the project
ENVIKUT (TÁMOP-4.2.2.A-11/1/KONV-2012-0043) and Richter
Gedeon Talentum Foundation. The work was also supported by
the Campus Hungary Scholarship Program (TÁMOP 4.2.4. B/211/1-2012-0001).
References
1 Buglyó P, Farkas E (2009) Dalton Trans 38:8063–8070.
2 Bı́ró L, Farkas E, Buglyó P (2010) Dalton Trans 39:10272–10278.
3 Bı́ró L, Farkas E, Buglyó P (2012) Dalton Trans 41:285–291.
123
Institute of Environmental Health, Oregon Health and Science
University, 3181 SW Sam Jackson Park Rd, Portland OR, 97239,
USA. [email protected]
In addition to chemical and physical forces, many global geochemical
cycles, such as the redox cycling of manganese, are driven by bacteria. Bacterial Mn(II) oxidation and subsequent Mn(IV) mineral
formation is widespread and diverse. Direct enzymatic Mn oxidation
presents novel Mn chemistry in that Mn is the substrate of the reaction
instead of the catalytic cofactor. Until recently, however, there has
been no protein available to investigate. The heterologously purified
Mn oxidase (Mnx) from marine Bacillus sp. PL-12 is made up of the
multicopper oxidase (MCO) MnxG, and two hypothetical proteins
MnxE and MnxF. Mnx binds Cu and oxidizes both Mn(II) and
Mn(III), generating Mn(IV) oxide minerals that resemble those found
on the Bacillus spore surface. Here we describe the characterization
of Cu binding and substrate flexibility and present direct spectroscopic and kinetic evidence to verify the presence of the canonical
MCO Cu types. Additionally, we describe the kinetics of the oxidation by Mnx of Mn(II) and the more typical MCO substrates Fe(II),
2,20 -azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) and 2,
6-dimethoxyphenol (2,6-DMP). Interestingly, Mn oxidation is the
only reaction to take on an allosteric sigmoidal trend in lieu of
Michaelis–Menten. This distinct trend may point towards a unique
mechanism for this enzyme to catalyze the two direct electron
transfers of Mn(II, III) oxidation. Elucidating the course of Mn oxidation within Mnx will unlock an unexplored corner of Mn chemistry
and could begin to explain how and perhaps why bacteria oxidize Mn.
P 185
Characterization of novel metallo-b-lactamases
from the B3 subgroup
Manfredi Miraula1,2, Gerhard Schenk2, Nataša Mitić1
1
Department of Chemistry, National University of Ireland-Maynooth,
Maynooth, Co. Kildare, Ireland.
2
School of Chemistry and Molecular Biosciences, The University
of Queensland, Brisbane, QLD 4072, Australia
Metallo-b-lactamases (MBLs) are a family of Zn(II)-dependent
enzymes that are very effective in inactivating most of the commonly used b-lactams antibiotics. They have emerged as a major
threat to global health care. Recently, we identified two novel
MBL-like proteins in a sequence data base search in the marine
organisms Novosphingobium pentaromativorans (MIM-1) and
Simiduia agarivorans (MIM-2), respectively [1]. We have
expressed these putative MBLs in Escherichia coli and purified
them to homogeneity. Their catalytic properties were determined
with a number of representative b-lactam antibiotics (i.e. penams,
penems and cephalosporins) and confirm that MIM-1 and MIM-2
J Biol Inorg Chem (2014) 19 (Suppl 2):S833–S852
are indeed MBLs. The enzymes are Zn(II)-dependent but activity
can be reconstituted with other transition metal ions (i.e. Co(II)
and Mn(II)). Isothermal titration calorimetry in combination with
catalytic assays was employed to monitor metal ion binding.
Structural and spectroscopic studies to probe the mechanism of
action of MIM-1 and MIM-2 are in progress. Although these
enzymes are less effective than MBLs associated with known
pathogens their occurrence in organisms that have not yet been in
contact with the human population may indicate a potential future
threat to global health, in particular when humans keep changing
the natural environment.
Reference
1. Miraula M, Brunton CS, Schenk G, Mitic N (2013) Am J Mol Biol
3:198.
P 186
Alkaline transition mechanism of cytochrome c’
from Alcaligenes xylosoxidans NCIMB11015
Akiko Takashina1, Michael Tiedemann2, Masaki
Unno1,3, Martin Stillman2, Takamitsu Kohzuma1,3
1
Department of Institute of Applied Beam Science, Ibaraki
University, 2-1-1 Bunkyo, Mito, Ibaraki, Japan.
2
Department of Chemistry, University of Western Ontario, Western
Ontario, Canada.
3
Frontier Research Center for Applied Atomic Sciences, Ibaraki
University, Tokai, Ibaraki, Japan
Cytochrome c’ (cyt c’) is found in many photosynthetic bacteria and
denitrifying bacteria as a c-type cytochrome [1]. The protein structure of
Cyt c’ is a typical four a-helices bundle, which binds to the heme prothetic
group in the C-terminal region. The unusual magnetic properties of ferric
cyt c’ from photosynthetic bacteria at neutral pH have been interpreted as a
quantum mechanical admixture of an intermediate-spin (S = 3/2) and a
high-spin (S = 5/2) states, and the spin state of Cyt c’ is well known to be
changeable by pH [1]. The EPR and electronic absorption spectra of Cyt c’
were markedly dependent upon pH. Alkaline spectroscopic transition has
been observed in many cytochromes c’. The electronic absorption spectrum of Cyt c’ shows typical low-spin spectrum under highly alkaline pH
conditions [2]. Moreover Cyt c’ indicates ionic-strength dependent structure rearrangements. The high spin state of the protein decreases
accompanied with the increment of intermediate spin state at low ionicstrength [3]. The alkaline transition mechanism of Cyt c’ from Alcaligenes
xylosoxidans (AxCyt c’) was studied. We would like to report electrospray
ionization mass (ESI–MS), magnetic circular dichroism (MCD), resonance Raman spectroscopic studies, and the first X-ray crystallographic
analyses of the alkaline transited cytochrome c’ will be reported.
Four different pH dependent species, 6C-HS, HS I, HS II, and 6C-LS
have been identified from the MCD spectra and ESI–MS of AxCyt c’.
The high resolution X-ray crystallographic structures of AxCyt c’ at pH
6.0 and 10.4 were determined with 1.10 Å and 1.48 Å resolution,
respectively. The space group of AxCyt c’ crystals made at pH 6.0 and
10.4 were both P6522. The bond length between heme iron and NHis120
atom of AxCyt c’ at pH 10.4 was estimated to be 0.1 Å longer than that
observed in the pH 6.0 structure. The heme plane became more planar
configuration at pH 10.4. The crystal structures of AxCyt c’ and related
spectroscopic properties will also be provided.
References
1. Weiss R, Gold A, Terner J (2006) Chem Rev 106:2550.
2. Yoshimura T (1985) Biochemistry 25:2436.
3. Kohzuma T, Suzuki S (1996) Mol Cryst Liq Cryst 286:95.
S843
P 187
Geometry and electronic structures of axial/rhombic
site in pseudoazurin Met16 variants: XAS and DFT
investigations
Takahide Yamaguchi1, Junko Yano2, Vittal K.
Yachandra2, Robert K. Szilagyi3, Takamitsu Kohzuma1
1
Institute of Applied Beam Science, Ibaraki University, Mito, Ibaraki,
310-8512, Japan.
2
Lawrence Berkeley National Laboratory, Berkeley, CA, 94720,
USA.
3
Department of Chemistry and Biochemistry, Montana State
University, Bozeman, MT USA and Department of Analytical
Chemistry, University of Pannonia, Veszprem, Hungary
Pseudoazurin (PAz) functions as an electron donor to nitrite reductase
utilizing Type 1 copper site [1]. The spectroscopic and electrochemical properties of PAz are significantly affected by the mutation
of Met16 located at the second coordination sphere of Type 1 copper
site [2]. The EPR spectra demonstrated the population of ‘‘axial’’ and
‘‘rhombic’’ site are influenced by the interaction with mutated amino
acid at Met16 position [3].
The geometric and electronic structures of Type 1 copper sites in
PAz Met16 variants were probed by multi-edge X-ray absorption
spectroscopic (XAS) measurements. X-ray absorption near edge
structure (XANES) at the Cu K-edge showed the increase of the Cu
effective nuclear charge as indication of less covalent bonding in the
axial site. The extended X-ray absorption fine structure (EXAFS) was
used to obtain Cu-ligand distances including Cu-SMet (2.48 Å), CuSCys (2.16 Å), and average Cu-NHis (1.95 Å) for the pure rhombic site
of Met16Val. The Cu-SCys bond is characterized by S K-edge XANES
in WT, Met16Phe and Met16Val variants to have 38 ± 8, 39 ± 6 and
30 ± 10 % S covalency, respectively.
In order to extend the experimental results beyond local structural
information, a 89 atoms computational model was generated from the
1.35 Å resolution structure of WT PAz. DFT calculations were carried out using a systematic series of hybrid density functionals. The
structures were optimized with various structural constraints. The
optimization of Cu-ligand distances with keeping the a- and b-C
atoms positions fixed allowed for the understanding of the origins of
experimental Cu-L distances from EXAFS and atomic spin densities
from XANES using validated electronic structural methods [4].
Comparison of the WT and Met16Ile variant of PAz with the pure
rhombic site [5] enabled us to expand the scope of our investigations
toward the non-covalent outer sphere environment and generate
realistic structural models for the axial and rhombic sites. Differences
in these models generated an experimentally hypothesis regarding
protein dynamics involving the 16th amino acid in regulating geometric and electronic structure of the Type 1 Cu site.
References
1. Averill BA (1996) Chem Rev 96:2951–2964.
2. Kohzuma T, et al. (2010) J Inorg Chem 104:250–260.
3. Kohzuma,T, et al. (2007) J Biol Inorg Chem 12:165–173.
4. Szilagyi RK, Solomon EI (2002) Curr Opin Chem Biol 6:250–258.
5. Kohzuma T, et al. (unpublished results).
P 188
Mechanistic studies on a new functional dimeric
chlorite dismutase
Irene Schaffner1, Nicola Flego2, Georg Mlynek3,
Marzia Bellei4, Stefan Hofbauer1, Gianantonio
123
S844
Battistuzzi4, Kristina Djinovic-Carugo3, Giulietta
Smulevich2, Paul G. Furtmüller1, Christian Obinger1
1
Department of Chemistry, Division of Biochemistry, VIBT-Vienna
Institute of BioTechnology, BOKU-University of Natural Resources
and Life Sciences, Vienna, Austria.
2
Dipartimento di Chimica ‘‘Ugo Schiff’’, Università di Firenze, Via
della Lastruccia 3-13, I-50019 Sesto Fiorentino (FI), Italy.
3
Department for Structural and Computational Biology, Max F.
Perutz Laboratories, University of Vienna, Campus Biocenter 5,
A-1030 Vienna, Austria.
4
Department of Chemistry, University of Modena and Reggio Emilia,
via Campi 183, 41100 Modena, Italy
Chlorite dismutases (Clds) are heme b containing enzymes which were
discovered in chlorate- and perchlorate-reducing bacteria [1] but are
found in many other bacterial and archaeal phyla [2]. Reduction of
(per)chlorate leads to generation of chlorite, which is a strong oxidant
and has cell-damaging effects. Cld protects the organism from the
accumulation of harmful chlorite by degrading it to chloride and
dioxygen (1). This catalytic function turns Cld into a highly interesting
enzyme for bioremediation as the serious environmental pollutants
chlorate and chlorite are used as bleaching agents in the textile, pulp
and paper industries, as disinfectants, etc. Additionally, Cld is extremely interesting from a biochemical point of view as it is the only
known enzyme system which efficiently catalyzes O–O bond formation
beside photosystem II. In order to apply the enzyme industrially it is
important to gain knowledge about its structural and functional properties. Phylogenetically, Clds divide into two clades, clade 1 consisting
of multimeric Clds and clade 2 consisting of dimeric representatives.
Whereas clade 1 is relatively well investigated, just one dimeric Cld (of
Nitrobacter winogradskyi, NwCld) has been characterized so far [3].
To gain further insight into the general reaction mechanism and differences between clade 1 and 2 chlorite dismutases, we chose a
homodimeric Cld from Cyanothece sp. PCC 7425 (CCld) for comprehensive characterization. We succeeded in solving the X-ray crystal
structure of the enzyme at a resolution of 1.3 Å. Together with electronic absorption and resonance Raman spectroscopy studies as well as
spectroelectrochemical measurements, these data provide detailed
mechanistic information and enable us to discuss aforementioned
questions on the basis of significant experimental results.
References
1. van Ginkel CG, Rikken GB, Kroon AG, Kengen SW (1996) Arch
Microbiol 166:321–326.
2. Maixner F, Wagner M, Lücker S, Pelletier E, Schmitz-Esser S,
Hace K, Spieck E, Konrat R, Le Paslier D, Daims H (2008) Environ
Microbiol 10:3043–3056.
3. Mlynek G, Sjöblom B, Kostan J, et al. (2011) J Bacteriol
193:2408–2417.
P 189
Electrochemistry of cytochromes P450 in surfactant
films: ‘‘tunable’’ catalysts?
Andrew K. Udit, Michael G. Hill
Department of Chemistry, Occidental College, 1600 Campus Road,
Los Angeles CA 90041, USA
The cytochromes P450 perform demanding oxidation reactions regioand stereospecifically under physiological conditions. Harnessing
P450 activity in vitro for commercial use has proven difficult, largely
due to inadequately reproducing the in vivo electron transfer
machinery. Electrochemical methods continue to present an attractive
means for addressing this electron transfer problem; specifically,
surfactant-P450 films on carbon electrodes have shown particular
123
J Biol Inorg Chem (2014) 19 (Suppl 2):S833–S852
promise. Despite rapid electron transfer (* 200 s-1) and good longterm stability, these films show poor catalytic activity. To investigate
the nature of electrode-bound P450, comparative electrochemical
studies were performed using P450 from Bacillus megaterium (BM3,
fully soluble), and mammalian microsomes (2B4, membrane associated) confined within didodecyldimethylammonium bromide
(DDAB) surfactant films on graphite electrodes [1]. Electronic
absorption spectra of P450-DDAB films on silica slides revealed
absorption maxima at 418 nm, characteristic of low-spin, six-coordinate, water-ligated FeIII heme in P450. The FeIII/II and FeII/I redox
couples (E1/2) of substrate-free P450 measured by cyclic voltammetry
were similar for both enzymes, approximately -0.23 and -1.02 V
(vs. SCE) at 21 C. Catalysis experiments indicated dioxygen
reduction by the films, however substrate oxidation was not observed.
From the variation of E1/2 with temperature the entropy and enthalpy
changes that accompany heme reduction for 2B4 (-151 J/mol-K and
-46 kJ/mol) and BM3 (-163 J/mol-K and -47 kJ/mol) were
determined. Similar measurements were less for the six-coordinate
low-spin, imidazole-ligated enzymes (-59 J/mol-K and -18 kJ/mol
for 2B4, -73 J/mol-K and -21 kJ/mol for BM3), consistent with
minimal nuclear reorganization (imidazole remains bound, unlike
water). That similar thermodynamic parameters were observed for
both enzymes, which show marked differences in solution, alludes to
the strong effect exerted by the surfactant environment on P450. This
in turn suggests that electrochemical parameters and enzyme activity
may be tuneable by appropriate choice of surfactant film.
Financial support by the donors of the American Chemical Society
Petroleum Research Fund and the Research Corporation is gratefully
acknowledged.
Reference
1. Hagen KD, Gillan JM, Im S–C, Landefeld S, Mead G, Hiley M,
Waskell LA, Hill MG, Udit AK (2013) J Inorg Biochem 129:30–34.
P 190
Structure–function relationship of zinc enzymes
by a computational method
Toshiaki Taura, Yuki Kato, Masayo Goto
Group of Chemistry, School of Information Science and Technology,
University of Aichi Prefecture, Ibaragabasama 1522-3, 480-1198
Nagakute, Japan. [email protected]
We studied on the relationship between structures and functions of
zinc enzymes using a computational method, an artificial neural
network. Enzymes having zinc ions generally function as hydrolase
(Hy), oxidoreductase (Ox), transferase (Tr), lyase (Ly), isomerase (Is)
and ligase (Li). However, their quantitative structure–function relationships are not fully established. Concerning the structures of
metalloenzymes, their crystal structures determined by X-ray analysis
are opened for a use as Protein Data Bank (PDB) files. Therefore, we
tried to predict functions of zinc enzymes described above using their
PDB structural data. We used coordination numbers of nitrogen,
oxygen and sulfur, bond lengths, bond angles, and so on as eight input
parameters for six types of function on the neural network processing.
Although a number of crystal structures of iron and copper
enzymes have also been determined, metalloenzymes containing iron
or copper almost have porphyrin like structures or functions concerning redox reactions. Therefore, we focus on zinc enzymes in this
J Biol Inorg Chem (2014) 19 (Suppl 2):S833–S852
work. As data sets, we collected structural data for 8 input parameters
from 237 PDB structures of Zn enzymes which contain one metal
(Zn) ion in one enzyme structure (structures containing two or three
metal ions and structures in which a metal ion is surrounded by only
water molecules are eliminated). The number of PDB structures for
zinc enzymes reported in each year is shown in the figure.
A computational method, a neural network is widely used as a
pattern-recognition analysis. After training data sets containing eight
input parameters (structural data) for two types of function, we could
predict nearly 100 % of right functions from structural data for Ox-Tr,
Ox-Is, Tr-Ly, Tr-Is, Ly-Li, Is-Li data sets. We could predict functions
for other data set which was newly obtained, and also tried to predict
functions for 3, 4, 5 and 6 types of data set.
Zn enzyme
600
500
400
Hydrolase
Oxidoreductase
Transferase
300
200
100
Lyase
will be deposited to a public strain bank (RIKEN BioResource Center
(BRC), Japan).
References
1. Lin MT, Sperling, LJ, Frericks Schmidt HL, Tang M, Samoilova
RI, Kumasaka T, Iwasaki T, Dikanov SA, Rienstra CM, Gennis RB
(2011) Methods 55:370–378.
2. Iwasaki T, Fukazawa R, Miyajima-Nakano Y, Baldansuren A,
Matsushita S, Lin MT, Gennis RB, Hasegawa K, Kumasaka T,
Dikanov SA (2012) J Am Chem Soc 134:19731–19738.
P 192
Probing the structural properties and dynamics
of the heme-based sensor protein YddV by timeresolved step-scan FTIR spectroscopy
Andrea Pavlou1, Markéta Martı́nková2, Toru Shimizu3,
Eftychia Pinakoulaki1
1
Isomerase
Ligase
0
P 191
A set of Escherichia coli amino acid auxotrophic
expression host strains for selectively labelled
metalloenzyme research
Risako Fukazawa1, Myat T. Lin2, Yoshiharu MiyajimaNakano1, Shinichi Matsushita1, Sylvia K. Choi3,
Amgalanbaatar Baldansuren4, Sergei A. Dikanov4,
Robert B. Gennis2,3, Toshio Iwasaki1
1
S845
Department of Biochemistry and Molecular Biology, Nippon
Medical School, 1-1-5 Sendagi, Tokyo 113-8602, Japan. [email protected] 2Department of Biochemistry, University
of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
3
Center for Biophysics and Computational Biology, University
of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
4
Department of Veterinary Clinical Medicine, University of Illinois
at Urbana-Champaign, Urbana, IL 61801, USA
Specific contributions of particular residues in the reaction mechanisms
and/or folding dynamics of a metalloenzyme of interest can be best
addressed by using magnetic resonance (e.g. NMR and EPR) and
vibrational (e.g. FTIR and resonance Raman) spectroscopic techniques,
often aided by X-ray crystallographic analysis. In this work, we report a
set of Escherichia coli C43(DE3) and/or BL21(DE3) based amino acid
auxotrophic expression host strains suitable for these studies. Currently,
our E. coli expression host strain collection may be used for selective
15
N and/or 13C labelling of 17 different amino acids, except for serine,
aspartate and glutamate. These strains may be applied for producing
recombinant archaeal and bacterial metalloproteins when a plasmid
carrying extra copies of tRNA genes for E. coli rare codons is incorporated. Examples of our successful application of some of these
strains in 2D pulsed EPR (HYSCORE) analysis of 15N or 13C amino
acid-labelled iron-sulfur proteins will be presented in the poster.
Our E. coli auxotrophic expression host strains are presently free
for academic use, under the terms that they are not for resale and that
one should not provide these strains to any third parties without
permission (note that they are ‘‘living modified organisms (LMO)’’
according to Article 18 and Article 20 of Cartagena Protocol on
Biosafety to the Convention on Biological Diversity). These strains
Department of Chemistry, University of Cyprus, P.O. Box 2037,
1678 Nicosia, Cyprus.
2
Department of Biochemistry, Faculty of Science, Charles University
in Prague, Prague, Czech Republic.
3
Department of Cell Biology, Shantou University Medical College,
Shantou 515041, China
YddV from Escherichia coli is a globin-coupled heme-based oxygen
sensor protein displaying diguanylate cyclase activity [1]. In this work
we have employed Fourier transform infrared (FTIR) and timeresolved step-scan (TRS2)-FTIR spectroscopy to probe the structural
properties and dynamics of the heme site of YddV. The YddV-CO
adducts of the wild type sensor domain protein as well as the L65T,
L65 M, Y43 W, Y43F and Y43A mutants have been studied. The
TRS2-FTIR photodissociation experiments reveal monophasic kinetics for CO rebinding to the heme in wild type sensor domain YddV. In
addition, our data provide evidence that Leu65 controls the ligand
entry pathway to the heme site. Protein conformational motions upon
ligand photodissociation and rebinding will be discussed.
Financial support by the University of Cyprus is gratefully
acknowledged.
References
1. Kitanishi K, Kobayashi K, Kawamura Y, Ishigami I, Ogura T,
Nakajima K, Igarashi J, Tanaka A, Shimizu T (2010) Biochemistry
49:10381–10393.
2. Pinakoulaki E, Yoshimura H, Daskalakis V, Yoshioka S, Aono S,
Varotsis C (2006) Proc Natl Acad Sci USA 103:14796–14801.
P 193
Ligand mutagenesis of TthNEET, a thermophile
homolog of mitoNEET
Toshio Iwasaki1, Emi Hagiuda1, Risako Fukazawa1,
Yoko Hayashi-Iwasaki2, Tairo Oshima2, Kazuya
Hasegawa3, Takashi Kumasaka3
1
Department of Biochemistry and Molecular Biology, Nippon
Medical School, 1-1-5 Sendagi, Tokyo 113-8602, Japan.
[email protected] 2Institute of Environmental Microbiology,
Kyowa Kako Co., Tokyo 194-0035, Japan.
3
Japan Synchrotron Radiation Research Institute (JASRI/SPring-8),
Hyogo 679-5198, Japan
MitoNEET is a mitochondrial outer membrane [2Fe-2S](His)1(Cys)3
protein, initially identified as a potential pharmacological and clinical
123
S846
target of the insulin sensitizing drug, pioglitazone, for the treatment of
type 2 diabetes. The mitoNEET superfamily is distributed among
various organisms including some archaea, bacteria and eukaryal
mitochondria, although the in vivo function of this new protein
superfamily remains unclear.
We have previously identified an extreme thermophile homolog of
mitoNEET, TthNEET, from Thermus thermophilus HB8 and determined its structure at 1.8 Å resolution. In this study we report the
structural and physiological characterization of the TthNEET variant
containing an engineered [2Fe-2S](Cys)4 cluster by ligand mutagenesis, and discuss a possible role of TthNEET in the thermophile cells.
Supported in part by JSPS-NSF International Collaborations in
Chemistry (ICC) project grant and by Nagase Science and Technology Foundation.
References
1. Iwasaki T, Samoilova RI, Kounosu A, Ohmori D, Dikanov SA
(2009) J Am Chem Soc 131:13659–13667.
2. Kounosu A, Iwasaki T, Baba S, Hayashi-Iwasaki Y, Oshima T,
Kumasaka T (2008) Acta Cryst F64:1146–1148.
P 194
The role of dimerisation and protein–protein
interactions for P450 aromatase (CYP19) proteins
in biomimetic membranes
Slavica Praporski1, C. Jo Corbin2, Nicholas
Hatzirodos3, Dario Mizrachi4, Raymond J. Rodgers3,
Alan J. Conley2, Lisandra L. Martin1
1
School of Chemistry, Monash University, Clayton, Victoria, 3800,
Australia. [email protected] 2School of Veterinary Medicine,
University of California, Davis, USA.
3
Obstetrics and Gynaecology, The University of Adelaide, S.A.,
5005, Australia.
4
Chemical and Biomolecular Engineering, Cornell University, Ithaca,
NY 14853, USA
Cytochrome P450aromatase is a membrane bound enzyme that
synthesizes estrogens. This reaction involves electron delivery from
NADPH via cytochrome P450 reductase (CPR). Little is known as
to how the P450arom performs the aromatase reaction or how it
associates with CPR in the endoplasmic reticulum (ER). In humans,
there is only one P450arom, however pigs have three isozymes. The
catalytic efficiency of one of these, the porcine gonadal P450arom,
is much lower than the human and the porcine placental isozyme
despite the high amino acid sequence homologies.
We have used in vivo and in vitro techniques to study the interaction of P450arom proteins and lipid membranes. FRET (Forster
Resonance Energy Transfer) studies explored the protein–protein
interactions in the ER. A quartz crystal microbalance (QCM) was
used to measure the mass of protein(s) binding to a lipid membrane
and in association with the Western blot data the stoichiometry for
P450arom and CPR was determined. QCM also provides information
about the conformational and structural organization of proteins in the
lipid membrane. In silico calculations were also used to further probe
the human and porcine P450arom.
Our FRET studies showed that the human P450arom forms dimers
in vivo. The QCM showed tight binding of all recombinant P450arom
enzymes examined to the lipid membranes. The reconstituted
P450arom: CPR complexes exhibited good catalytic turnover. However, the human P450arom ‘associated’ very differently with the lipid
membrane than the porcine gonadal P450arom. The rate of porcine
P450arom binding was most influenced by the amount of CPR
present. Thus, despite their high homology the structural organization
123
J Biol Inorg Chem (2014) 19 (Suppl 2):S833–S852
of each enzyme within the membrane is very different. The solvation
energy of the respective human and porcine gonadal P450arom dimer
also differs, indicating that dimerization may influence the mechanism of P450arom function.
References
1. Corbin CJ, Trant JM, Conley AJ (2001) Mol Cell Endocrin
172:115–124.
2. Praporski S, Ng S, Nguyen AD, Corbin CJ, Mechler A, Zheng J,
Conley AJ, Martin LL (2009) J Biol Chem 284:33224–33232.
P 195
Towards better understanding of metal specificity in dimetal carboxylate proteins
Vivek Srinivas, Julia J. Griese, Martin Högbom
Stockholm Center for Biomembrane Research, Department
of Biochemistry and Biophysics, Stockholm University, SE-106 91
Stockholm, Sweden. [email protected]
Reduction of ribonucleotides to deoxyribonucleotides required for DNA
synthesis is catalyzed by ribonucleotide reductase (RNR), which is the
only enzyme known to do so. The Escherichia coli RNR is a dimer of two
distinct homodimers namely the R1 and R2 subunits. The R1 subunit
holds the redox active cysteine residue, the substrate-binding site and the
allosteric effector region, whereas the R2 subunit holds a di-iron site,
which upon oxygen activation generates a single radical, to be transported to the R1 subunit for catalysis [1]. Recently a new class of RNR
was found to hold a heterodinuclear Mn–Fe metal center instead of the
classical di-iron cofactor [2, 3]. This discovery shows that the metal
affinity, specificity and the redox tuning of the metal centers are carefully
regulated in each of the RNR classes. In this study we try point mutational
studies on class I E. coli RNR to identify the participating elements in its
metal coordination sphere affecting its metal affinity and specificity.
Financial support is provided by the Swedish Research Council,
the Swedish Foundation for Strategic Research, the Knut and Alice
Wallenberg Foundation and BioStruct X.
References
1. Nordlund P, Reichard P (2006) Annu Rev Biochem 75:681–706.
2. Hogbom M, et al. (2004) Science 305:245–248.
3. Jiang W, et al. (2007) Science 316:1188–1191.
P 196
Structure and function of heme-binding proteins
in heme-uptake systems of gram-positive bacteria
Norifumi Muraki1, Yasunori Okamoto1,2, Takashi
Hayashi2, Shigetoshi Aono1
1
Institute for Molecular Science and Okazaki Institute for Integrative
Bioscience, National Institutes of Natural Sciences, 5-1 Higashiyama,
Myodaiji, Okazaki 444-8787, Japan.
2
Department of Applied Chemistry, Graduate School of Engineering,
Osaka University, Suita, Osaka 565-0871, Japan. [email protected]
Some bacteria, especially pathogenic bacteria, can be used heme (iron
protoporphyrin complex) as an iron source, which have several heme
uptake systems. An ABC-type heme transporter system is widely used
for heme acquisition, which consists of an ATP-binding protein, heme
permease, and heme-binding protein (substrate-binding protein). In
this work, we have studied the structural and functional relationships
of two heme-binding proteins, HupD and HmuT in Listeria monocytogenes and Corynebacterium glutamicum, respectively. HupD and
J Biol Inorg Chem (2014) 19 (Suppl 2):S833–S852
HmuT act as a heme-binding protein in the heme acquisition systems,
HupDGC and HmuTUV for Listeria monocytogenes and Corynebacterium glutamicum, respectively. Heme captured in HupD/HmuT
is transferred into cytoplasm by the ABC-type HupDG/HmuUV heme
transporter, but the detailed molecular mechanisms of heme transport
in these systems remain to be elucidated.
HupD binds heme with 1:1 binding ratio of heme to protein.
Heme-bound HupD shows the absorption peaks at 412, 538, and
575 nm and a rhombic EPR signals of g = 3.2 and 2.1 in the ferric
form, which are typical for bis-His ligated heme proteins. Sitedirected mutagenesis revealed that His105 and His259 are the axial
ligands of the heme in HupD. Though the heme captured in HupD is
in a 6-coordinate state, it can form CO-bound heme upon the reaction
of ferrous HupD and CO. The association constant of heme is
determined to be 6.45 9 10-9 M-1 for HupD.
We have determined the crystal structure of heme-bound HmuT
that captures heme with His141 and Tyr240 as the axial ligands, as
shown in the Figure below. The global fold of HmuT from C. glutamicum (CgHmuT) is similar to that of the periplasmic heme-binding
protein HmuT from Yersinia pestis (YpHmuT). However, hemebinding properties are different from each other, i.e. CgHmuT binds
only one heme though YpHmuT binds two stacked hemes. We will
discuss the detailed structural and functional properties of CgHmuT.
P 197
Structural and functional study of R2-like ligandbinding oxidases
Hugo Lebrette, Julia J. Griese, Martin Högbom
Stockholm Center for Biomembrane Research, Department
of Biochemistry and Biophysics, Stockholm University, SE-106 91
Stockholm, Sweden
Ribonucleotide reductase R2 proteins belong to the ferritin-like superfamily and utilize dinuclear metal centers to generate a free radical
involved in the synthesis of deoxyribonucleotides [1]. Further analyses
of R2 homologues have led to the discovery of a novel group of R2-like
proteins, namely R2-like ligand-binding oxidases (R2lox), with a completely different function. Recently solved crystal structures of these
proteins [2, 3] show that, although the R2-protein fold is conserved, this
latter is remodeled in order to accommodate an unexpected ligand, in
interaction with a dinuclear Mn/Fe cluster. Moreover, this metal center
activates oxygen and catalyzes the formation of an ether cross-link in the
protein scaffold. However, until now, the physiological substrate and
reaction catalyzed are unknown. Using X-ray crystallography and
complementary approaches, the aim of our project is to provide new
insights into the function of this novel group of proteins.
Financial support was provided by the Swedish Research Council,
the Swedish Foundation for Strategic Research and the Knut and
Alice Wallenberg Foundation and BioStruct X.
References
1. Nordlund P, Reichard P (2006) Annu Rev Biochem 75:681–706.
2. Andersson CS, Högbom M (2009) Proc Natl Acad Sci USA
106:5633–5638.
S847
3. Griese JJ, Roos K, Cox N, Shafaat HS, Branca RM, Lehtiö J,
Gräslund A, Lubitz W, Siegbahn PE, Högbom M (2013) Proc Natl
Acad Sci USA 110:17189–17194.
P 198
Biochemical characterization and structural aspects
of recombinant human peroxidasin 1 and truncated
peroxidasin 1 variants
Martina Paumanna-Page, Monika Soudi, Eva
Edenhofer, Paul G. Furtmüller, Christian Obinger
Department of Chemistry, Division of Biochemistry, BOKUUniversity of Natural Resources and Life Sciences, Muthgasse 18,
A-1190 Vienna, Austria
Peroxidasins are heme-containing oxidoreductases representing subfamily 2 of the peroxidase-cyclooxygenase superfamily [1]. They are
found in invertebrates and vertebrates including mammals. These multidomain proteins are comprised of a peroxidase domain with homology
to mammalian peroxidases (subfamily 1) and additional leucine-rich
repeat domain(s), immunoglobulin domains and a carboxy terminal VonWillebrand factor C domain [2], which are all known to be important for
cell adhesion and protein–protein interaction.
Several physiological roles are under discussion. Only recently it has
been reported that basement membrane associated human peroxidasin 1
(hsPXD01) is involved in the formation of a distinct covalent sulfilimine
cross link between two particular amino acid residues in the collagen IV
protein network providing structural integrity. Hypohalous acids (HOBr
and/or HOCl) produced by hsPXDN01 were demonstrated to drive the
formation of this unique covalent bond that had never been identified in
a biomolecule before [3]. Hypohalous acids play an important role in
innate host defense but are also known to contribute to tissue injury
during oxidative stress and inflammation. Therefore the presence of
adhesive domains in hsPXD01 suggests that the production of reactive
oxidants in the collagen IV network is a targeted and highly specific
posttranslational protein modification. Participation of hsPXD01 in
immune defense has also been proposed [4].
The modelled structures of hsPXD01 domains will be discussed in
regards to the successful expression of several truncated hsPXD01
variants in HEK293 cells. The oligomeric structure, spectral features
of the ferric and ferrous state as well as complex formation with
various ligands and enzymatic activity will be presented and related to
the full length protein.
References
1. Zamocky M, Jakopitsch C, Furtmüller PG, Dunand C, Obinger, C
(2008) Proteins 72:589–605.
2. Soudi M, Zamocky M, Jakopitsch C, Furtmüller PG, Obinger, C
(2012) Chem Biodivers 9:1776–1793.
3. Bhave G, Cummings CF, Vanacore RM, Kumagai-Cresse C, EroTolliver IA, Rafi M, Kang JS, Pedchenko V, Fessler LI, Fessler JH,
Hudson BG (2012) Nat Chem Biol 8:784–790.
4. Li H, Cao Z, Moore DR, Jackson PL, Barnes S, Lambeth JD,
Thannickal VJ, Cheng G (2012) Infect Immun 80:2528–2537.
P 199
Metalloprotein manipulation for metal biosensors
and bio-recovery
Wei Wei, Xiangzhi Liu, Peiqing Sun, Jing Zhao
State Key Laboratory of Pharmaceutical Biotechnology, Institute
of Chemistry and BioMedical Sciences, School of Life Sciences,
Nanjing University, Nanjing 210093, People’s Republic of China
123
S848
Heavy metal pollution has become an increasing global environmental
concern. Considerable efforts have thus focused on developing facile
approaches for heavy metal remediation. By studying metalloproteins,
especially MerR family proteins from bacteria, we reported the development of a whole-cell based gold biosensor that allows the selective
detection of gold ions by naked-eyes. Furthermore, a bacterial surface
display system was engineered for the enrichment and recovery of gold
ions via a newly identified gold(I)-specific binding protein. Such systems are advantageous over conventional methods in terms of
sensitivity, robustness and reusability [1].
Recently, we also have developed highly selective heavy metal
bioremediation methods that detect and absorb lead ions with high
selectivity. Our approach takes advantage of MerR family proteins for
their high selectivity in recognizing specific heavy metals. By engineering the specific microbial heavy metal-binding MerR protein with
the cell surface display system and BioBrick system, we have
developed a general strategy that lays the foundation for generating a
series of engineered bacteria for selective bio-detection and bioremediation of different heavy metals. We demonstrate the utility of the
engineered bacteria for protecting Arabidopsis thaliana seed germination from the toxicity of lead ions [2].
J Biol Inorg Chem (2014) 19 (Suppl 2):S833–S852
was evidentiated a reaction intermediate, most probably a Fe(III)-OCl
adduct, which rapidly is transformed to ferryl.
Acknowledgements: the work shown here has been supported by
the Romanian Ministry for Education and Research (grants PN-II-IDPCE-2012-4-0488).
References
1. Maitra D, Byun J, Andreana PR, Abdulhamid I, Saed GM, Diamond MP, Pennathur S, Abu-Soud HM (2011) Free Radic Biol Chem
51:364–373.
2. Maitra D, Byun J, Andreana PR, Abdulhamid I, Diamond MP, Saed
GM, Pennathur S, Abu-Soud HM (2011) Free Radic Biol Chem
51:374–386.
3. Gebicka L, Banasiak E (2012) Toxicol Vitro 26:924–929.
P 201
Hemoglobin and hemerythrin based blood substitutes
Florina Scurtu1, Denisa Hathazi1, Augustin Mot1,
Anetta Vaida1, Eva Fischer-Fodor2, Grigore Damian3,
Donald Kurtz4, Vlad Toma1,5, Anca Farcas1,5, Ioana
Roman5, Radu Silaghi-Dumitrescu1
1
References
1. Wei W, Liu X, Sun P, Wang X, Hong Z, Hong M, Mao Z-W Zhao J
(2014) Environ Sci Technol 48:3363–3371.
2. Wei W, Zhu T, Wang Y, Yang H, Hao Z, Chen P, Zhao J (2012)
Chem Sci 3:1780–1784.
P 200
Hypochlorite activation at the heme iron center
of hemoglobin
Cristina Bischin1, Radu Silaghi-Dumitrescu1
1
Department of Chemistry, Babes-Bolyai University, 11 Arany Janos
Street, Cluj-Napoca 400028, Romania. [email protected]
Hypochlorous acid (HClO), generated by myeloperoxidase using
chloride and hydrogen peroxide as substrate, is one of the most
powerful biological oxidizing and chlorinating agents. A mechanistic
link is expected to be between high HClO, which appear in pathological conditions, and higher free Fe level [1]. It has been shown that
this molecule can easily penetrate the red blood cells leading to the
destruction of one of the most abundant proteins found here, hemoglobin (Hb). There is evidence that an excess of HClO induces similar
absorption changes with those observed with an excess of H2O2. Some
studies revealed that the reaction between HClO and oxy- and metHb
involved the formation of the strong ferryl intermediate (Fe(IV) = O),
followed by heme degradation and protein aggregation. It has been
reported that HClO not only binds to heme iron but also destroys free
heme iron by the interaction with tetrapyrrole ring [2, 3].
In the present work, we investigated the reaction between oxi- and
met-Hb with HClO. Using stopped-flow technique, for the first time
123
Faculty of Chemistry and Chemical Eng., Babes-Bolyai University,
Cluj-Napoca, Romania 2Ion Chiricuta Cancer InstituteComprehensive Cancer Center, Cluj Napoca, Romania 3Faculty
of Physics, Babes-Bolyai University, Cluj-Napoca, Romania
4
Department of Chemistry, University of Texas at San Antonio, USA
5
Institute of Biological Research, Cluj-Napoca, Romania
Blood substitutes are chemical or biochemical preparations aimed at being
used in transfusions with the sole role of enhancing oxygen transport by the
patient’s blood (as opposed to the more complex functions that blood normally achieves, such as transport or defense) [1]. Hemoglobin can only be a
reasonable blood substitute candidate once its side-reactions can be controlled/reduced. Such reduction has been among others been achieved so far
via interprotein crosslinking or derivatization with polyethylene glycol,
thereby generating particles of large molecular weight/volume, which (1)
have increased stability in the bloodstream and (2) avoid homogenous close
contact between hemoglobin and endothelium, thereby reducing the
hemoglobin-NO reactivity [1]. Another approach has been to genetically
modify human hemoglobin; mutations have been identified that drastically
reduce reactivity towards NO and/or peroxides by modifying amino acids
gating access of small molecules towards the heme site [2].
We have recently proposed an alternative, whose active ingredient is
also a protein specialized in oxygen transport, but of non-heme nature:
hemerythrin (Hr). Hr is found in marine worms and bacteria and employs
a di-ferrous center to bind oxygen; the resulting adduct is formally
described as Fe(III)-Fe(III)-OOH, i.e. diferricperoxo. This mode of
binding oxygen has the remarkable advantage of (1) not featuring a
superoxide ligand (unlike Hb), which drastically reduces the reactivity
towards NO, and (2) exhibiting stability towards peroxide (unlike Hb).
Recent results will be shown, illustrating how both Hb-based and Hrbased preparations can transport oxygen and elicit reasonable response
from human cell cultures (HUVEC, lymphocytes) [3–5].
Financial support from the Romanian Ministry for Education and
Research (grant PCE 488/2012) is gratefully acknowledged.
References
1. Alayash AI (2004) Nat Rev Drug Discov 3:152–159.
2. Reeder B, Grey M, Silaghi-Dumitrescu R, Svistunenko D, Bulow
L, Cooper C, Wilson M (2008) J Biol Chem 283:30780–30787.
3. Hathazi D, Mot A, Vaida A, Scurtu F, Lupan I, Fischer-Fodor E,
Damian G, Kurtz Jr. D, Silaghi-Dumitrescu R (2014) Biomacromolecules (in press).
J Biol Inorg Chem (2014) 19 (Suppl 2):S833–S852
4. Fischer-Fodor E, Mot A, Deac F, Arkosi M, Silaghi-Dumitrescu R
(2011) J Biosci 36:215–221.
5. Scurtu F, Zolog O, Iacob B, Silaghi-Dumitrescu R (2014) Artif
Cells Nanomed Biotechnol 42:13–17.
P 202
IR spectroelectrochemical study of ligand binding
to the metal centres of nitrogenase
Pathinan Paengnakorn1, Philip A. Ash1, Karamatullah
Danyal2, Lance C. Seefeldt2, Kylie A. Vincent1
1
Department of Chemistry, University of Oxford, South Park Road,
Oxford, OX1 3QR, UK. [email protected]
2
Department of Chemistry and Biochemistry, Utah State University,
0300 Old Main Hill, Logan, UT 84322, USA
This poster describes a new approach for studying ligand binding to
nitrogenase, the metalloenzyme responsible for biological fixation of
dinitrogen to ammonia. Although there are high resolution structures
of the MoFe protein and its metallo-clusters, there are still many
missing pieces in our understanding of the catalytic mechanism.
Investigating the binding of substrates and substrate analogues to the
iron-molybdenum cofactor active site of nitrogenase (‘FeMoco’) is
challenging because of the specialised electron transfer pathway for
this enzyme. Most substrates of nitrogenase bind only at reduced
levels of the MoFe protein, and its reduction requires electron transfer
from the partner Fe protein coupled with ATP hydrolysis.
Here we make use of an infrared spectroelectrochemical approach
[1] together with a system of low-potential redox mediators to electrochemically activate ligand binding to nitrogenase.
K.A.V. and P.A.A. are grateful for financial support from the
European Research Council (ERC) (EnergyBioCatalysis-ERC-2010StG-258600). P.P. is grateful for financial support from Thai government scholarship and Jesus College, Oxford.
S849
In addition to the mutation towards the Cys ligand to the type I
copper by Ser, a highly conserved Glu residue involved in the
proton transfer pathway in CueO is substituted by Ala at the
aim of studying dioxygen reduction mechanism of multicopper
oxidases. Differing from the wild type enzyme and every
mutant prepared hitherto, Cys500Ser/Glu506Ala CueO is in the
dual resting form to give a new band at ca. 400 nm in addition
to the weakened 330 nm band due to a probable modification in
the structure of the trinuclear copper center. Reactions of the
reduced mutant with dioxygen resulted in a slower production
of the intermediate I (peroxide intermediate) compared to
Cys500Ser CueO. In addition, the life-time of the intermediate I
was not very long. As a result of the present double mutation it
is found that the conserved Glu residue located in the hydrogen
bond network leading from bulk water to the trinuclear copper
center plays a dual role in the binding of dioxygen and its
conversion to water.
Fig. 1 Hydrogen bong network involving Glu506 in CueO to
assist the binding of dioxygen and its conversion to water at the
trinuclear copper center by transporting protons from bulk water,
and mutational modifications of the network with Asp, Ala, Gln,
and Ile.
Reference
1. Komori F, Kajikawa T, Kataoka K, Higuchi Y, Sakurai T (2013)
Biochem Biophys Res Commun 438: 686–690.
Reference
1. Healy AJ, Ash PA, Lenz O, Vincent KA (2013) Phys Chem Chem
Phys 15:7055–7059.
P 203
Dual effect of the mutational modification of the proton
transfer pathway and a type I Cu ligand on properties
and function of the trinuclear copper center in CueO
Takeshi Sakurai, Takao Kajikawa, Kunishige Kataoka
Graduate School of Natural Science and Technology, Kanazawa
University, Kakuma, Kanazawa 920-1192, Japan
P 204
Protein-solvent interaction and metal substitution
of membrane type I matrix metalloproteinase
Elena Decaneto1,2, Markus Knipp1,2, Hideaki Ogata1,
Martina Havenith2, Wolfgang Lubitz1
1
Max Plank Institute for Chemical Energy Conversion, Mülheim
an der Ruhr, Germany; 2Physical Chemistry II, Ruhr University
Bochum, Germany. [email protected]
Matrix metalloproteinases (MMPs) are zinc dependent endopeptidases that play a major role in the degradation and remodeling of
extracellular matrix (ECM) substrates, wound healing, bone
resorption etc. More than 20 members of the MMP family are
expressed in human tissue, many of which have been associated
with common diseases such as atherosclerosis, arthritis, and cancer. After decades of searching for specific MMP inhibitors, a
candidate that meets pharmaceutically required specificity and
affinity is not in sight.
123
S850
123
Department of Cellular and Molecular Medicine, University
of Leuven, O&N1 Herestraat 49-box 901, 3000 Leuven, Belgium.
[email protected] 2Department of Chemistry,
University of Leuven, 3001 Leuven, Belgium.
3
IMEC, 3001 Leuven, Belgium
Protein Phosphatase 1 (PP1) is a major protein Ser/Thr phosphatase in
eukaryotic cells [1]. Its activity depends on two metal ions in the catalytic site, which were identified as manganese in recombinant PP1 [2],
but their identity in native PP1 is unknown. In this study, Total
reflection X-Ray Fluorescence (TXRF) was used to detect stoichiometric levels of zinc and sub-stoichiometric levels of iron in native PP1.
The observed difference in metal identity provides an explanation for
the striking dissimilarities that are found between recombinant PP1 and
native PP1 with respect to their activity and substrate specificity. Furthermore, the detected iron levels were significantly reduced when PP1
was pre-treated with Inhibitor 2 (I2), confirming a previously postulated
model that implicates I2 in the inactivation of PP1 by the removal of
one of its catalytic-site metals [3].
Zn
Fe
1
0,9
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
Mn
I2
ith
w
ed
ea
t
etr
Department of Physics, Free University Berlin, Arnimallee 14,
14195 Berlin, Germany. [email protected] 2Department
of Biochemistry and Biophysics, Stockholm University, Svante
Arrhenius vag 16c, 10691 Stockholm, Sweden
Enzymes containing a dimetal-carboxylate cofactor (DMC) perform
numerous crucial functions in nature, most prominent are O2 activation
reactions. In a recently discovered ligand-binding oxidase (GkR2loxI)
from Geobacillus kaustophilus the cofactor can be of the [FeFe] or
[MnFe] type and oxidation of initial metal(II)2 centers by O2 leads to highvalent cofactor species and to an amino acid crosslink close to the active
site [1]. Crystal structures show the similarity of GkR2loxI to the R2
subunit of ribonucleotide reductases, but are affected by X-ray photoreduction (xpr) of the metal centers [2, 3]. Therefore, we studied GkR2loxI
by X-ray absorption spectroscopy (XAS) at the Fe and Mn K-edges. This
has revealed structural changes during rapid xpr at the initially oxidized
[FeFe] and [MnFe] sites as well as metal–ligand bond lengths and metal–
metal distances for the (II)2 and (III)2 states. Combination of parameters
from XAS and crystallography [1] thus resulted in models for the [FeFe]
and [MnFe] cofactors in GkR2loxI, which differ with respect to the
configurations of metal-bridging oxides and carboxylate ligands from the
classic DMC sites in R2 proteins [3]. XAS facilitates in silico reversion of
xpr effects in crystallographic data of DMC enzymes to obtain the
structures of high valent cofactor intermediates.
Financial support within the German-Swedish Röntgen-Angström
Cluster and by the DFG (grants Ha3265/2-2 & 6-1 and Unicat CoE
1
pr
1
P 206
TXRF analysis of native PP1 and the PP1-Inhibitor 2
complex
Ewald Heroes1,2, Jens Rip3, Luc Van Meervelt2, Stefan
De Gendt3, Monique Beullens1, Mathieu Bollen1
PP
1
P 205
Fine structure and redox changes at [FeFe] and [MnFe]
cofactors in a ligand-binding oxidase studied by X-ray
absorption spectroscopy
Ramona Kositzki1, Julia J. Griese2, Martin Högbom2,
Michael Haumann1
References
1. Griese JJ, Roos K, Shafaat HS, Branca RM, Lehtiö J, Gräslund A,
Lubitz W, Siegbahn PE, Högbom M (2013) Proc Natl Acad Sci USA
110:17189–17194.
2. Sigfridsson K, Leidel N, Chernev P, Popovic-Bijelic A, Gräslund
A, Haumann M (2013) J Biol Chem 288:9648–9661.
3. Leidel N, Popovic-Bijelic A, Havelius K, Chernev P, Voevodskaya
N, Gräslund A, Haumann M (2012) Biochim Biophys Acta
1817:430–444.
PP
1
References
1. Grossman M, Born B, Heyden M, Tworowsky D, Fields GB, Sagi I,
Havenith M (2011) Nat Struct Mol Biol 18:1102–1108.
2. Garmer DR, Krauss M (1993) J Am Chem Soc 115:10247–10257.
3. Bertini I, Fragai M, Lee YM, Luchinat C, Terni B (2004) Angew
Chem Int Ed 43:2254–2256.
Berlin) is gratefully acknowledged.
molar ratio of
metal ions to PP1
Application of the novel technique of THz absorbance spectroscopy [1] demonstrated for the case of membrane type I matrix
metalloproteinase (MT1-MMP or MMP14) that the coupling between
solvent water dynamics and protein motion plays a crucial role for the
enzymatic activity. To further establish this novel, far reaching concept, further experiments for the detailed understanding of the
protein-solvent interaction are being conducted. Thus, a strong
declining influence of salt on the enzymatic activity, even at physiological concentrations was observed. In contrast, addition of various
proteins as co-solvents, mimicking the molecular crowding in the
ECM, led to an increase in enzymatic activity, suggesting that the
influence on enzymatic activity is imparted through changes in the
water dynamics rather than electrostatic potential.
To get spectroscopic access to the active site, Zn(II) was exchanged
by Co(II) (S = 3/2), still maintaining the enzymatic activity. Observation of the d–d transitions by UV–Vis and MCD spectroscopy revealed
the presence of a distorted tetrahedral or pentacoordinated complex in
accordance with previous suggestions [2, 3]. Co(II)-MT1-MMP
substituted will be investigated with EPR and NMR spectroscopy.
This work was supported by the Cluster of Excellence RESOLV
(EXC 1069) funded by the Deutsche Forschungsgemeinschaft and by
the Max Planck Society.
J Biol Inorg Chem (2014) 19 (Suppl 2):S833–S852
J Biol Inorg Chem (2014) 19 (Suppl 2):S833–S852
References
1. Heroes E, Lesage B, Görnemann J, Beullens M, Van Meervelt L,
Bollen M (2013) FEBS J 280:584–595.
2. Dancheck B, Ragusa MJ, Allaire M, Nairn AC, Page R, Peti W
(2011) Biochemistry 50:1238–1246.
3. Hurley TD, Yang J, Zhang L, Goodwin KD, Zou Q, Cortese M,
Dunker AK, DePaoli-Roach AA (2007) J Biol Chem
282:28874–28883.
P 207
Structural and functional insight into trafficking
of copper in the cell
Peep Palumaa1, Lucia Banci2, Ivano Bertini2, Simone
Ciofi-Baffoni2, Kairit Zovo1
S851
hydrogenase small subunit, HupS, by heterologous expression as a
fusion protein, which we denote f-HupS [1]. The purified protein has
been characterized by UV–visible and EPR spectroscopy. This was
the first isolation of a NiFe-hydrogenase small subunit in the absence
of the large subunit. EPR spectroscopy data showed that f-HupS
contains two distinguishable 4Fe4S clusters, that became EPR active
after reduction with dithionite, and one 3Fe4S cluster which is visible
in EPR after oxidation with ferricyanide.
In the present study our aim was to investigate the accessibility of
the FeS clusters in HupS to exogenous electron donation, by utilizing
photogenerated Ru(I)(bpy)3 as reductant [2]. We followed the
reduction of the fusion protein f-HupS by observing FeS-cluster EPR
signals, after a varying number of laser flashes in the presence of
Ru(bpy)3 and the sacrificial electron donor Na-ascorbate (see Figure).
Financial support by The Knut and Alice Wallenberg Foundation
and The Swedish Energy Agency is gratefully acknowledged.
1
Department of Gene Technology, Tallinn University of Technology,
Akadeemia tee 15, 12618 Tallinn, Estonia. [email protected]
2
Magnetic Resonance Center CERM, University of Florence, Via
Luigi Sacconi 6, 50019, Sesto Fiorentino, Florence, Italy
Copper trafficking in cellular cytoplasm and mitochondrial intermembrane space is substantially assisted by various copper
chaperones. Copper chaperones compose a specific class of proteins
assuring safe handling and specific delivery of potentially harmful
copper ions to variety of essential copper proteins like Cu-ATP-ases,
Cu, Zn-superoxide dismutase and cytochrome c oxidase. Metallation
of Cu-ATP-ases is performed with copper efflux chaperone Atx1
(yeast) or Hah1 (human). Cu,Zn-SOD is metallated with copper
chaperone for SOD-Ccs. Metallation of cytochrome c oxidase is
apparently the most complicated task of copper delivery as it requires
highest number of assisting proteins, such as Cox11, Cox17, Sco1,
Sco2, Cox19 and Cox23. Copper chaperones compose structurally
heterogeneous class of proteins, which can exist in multiple metalloaded as well as oligomeric forms. Moreover, many copper chaperones (Ccs, Cox17, Sco1) exist in various oxidative states and
participate in redox catalysis, connected with their functioning.
Analysis of the metal-binding properties of copper chaperones and
their partners allowed us to establish affinity gradients determining
copper transport in the cell.
References
1. Banci L, Bertini I, Cantini F, Kozyreva T, Massagni C, Palumaa P,
Rubino JT, Zovo K (2012) Proc Natl Acad Sci USA
109:13555–13560.
2. Banci L, Bertini I, Ciofi-Baffoni S, Kozyreva T, Zovo K, Palumaa P
(2010) Nature 465:645–648.
3. Banci L, Bertini I, Ciofi-Baffoni S, Hadjiloi T, Martinelli M, Palumaa P (2008) Proc Natl Acad Sci USA 105:6803–6808.
P 208
Laser flash photolysis induced electron transfer
in the isolated uptake hydrogenase subunit HupS
studied by EPR spectroscopy
Ann Magnuson, Patrı́cia Raleiras, Leif Hammarström,
Stenbjörn Styring
Department of Chemistry, Ångström, Uppsala University, Box 523,
SE-75120 Uppsala, Sweden. [email protected]
The filamentous and heterocystous cyanobacterium Nostoc punctiforme ATCC 29133 expresses the uptake hydrogenase HupSL in
heterocysts under nitrogen-fixing conditions. We isolated the uptake
References
1. Raleiras P, Kellers P, Lindblad P, Styring S, Magnuson A (2013) J
Biol Chem 888:18345–18352.
2. Streich D (2013) Doctoral thesis, Acta Universitatis Upsaliensis,
Uppsala, ISBN 9789155486587.
P 209
X-ray structural studies of metal-binding to human
serum transferrin: metal-induced conformational
changes and biocoordination of metals
Tsz-Pui Lai1, Minji Wang1, Li Wang1,2, Hongzhe Sun1
1
Department of Chemistry, The University of Hong Kong, Pokfulam
Road, Hong Kong, People’s Republic of China.
[email protected] 2School of Chemistry, Central China Normal
University, Wuhan, People’s Republic of China
Human serum transferrin (hTf) is an indispensable serum protein
primarily responsible for carrying ferric ions Fe(III) inside the human
circulatory system [1]. Metal-induced conformational change of the
protein is a momentous process for specific transferrin-receptor
(TfR)-transferrin (hTf) recognition, leading to cellular uptake of ferric
ions through a ‘receptor-mediated endocytosis’ [1]. It has been
reported that hTF is capable of binding various multivalent metals
such as Bi(III), Ga(III), In(III), Ru(III), Ti(IV), Yb(III), Dy(III), Pt(II),
etc., and may play an critical role in delivery of metallodrugs [1,2]. In
addition to the conventional bicarbonate, other anions such as oxalate
and malonate, may serve as alternative synergistic anions to stabilize
metal coordination [1]. However structural evidence of hTf binding to
other metal ions and synergistic anions as well as the resulting conformational changes to hTf are still currently lacking.
123
S852
We had previously reported a diferric-hTf (FeNFeC-hTf) crystal
structure (2.8 Å) and a bismuth-bound hTf (BiNFeC-hTf) crystal
structure (2.4 Å, which revealed a ‘partially-opened’ conformation in
the N-lobe while maintaining a ‘closed’ conformation in the C-lobe
[3]. In the current study, we have solved a monoferric-hTf (FeC-hTf)
(2.7 Å), a mono-ytterbium(III)-hTf (YbC-hTf) (2.5 Å) and a monovanadium(IV)-hTf (VN-hTf) (3.15 Å) structure. In the former two
structures, we observe metal-binding in C-lobe only but not the
N-lobe, causing the N-lobe to adopt an opened conformation and
C-lobe to a closed conformation. We observed that malonate serves as
a synergistic anion in the metal site. In contrast, vanadium binds to the
N-lobe only but not the C-lobe in the form of a oxovanadium species,
causing the metal-bound N-lobe to adopt a ‘partially-opened’ conformation but ‘opened’ conformation in the C-lobe. No synergistic
anion is present for metal binding in this structure.
123
J Biol Inorg Chem (2014) 19 (Suppl 2):S833–S852
These X-ray structures justify the role of hTf as a metal ion
mediator [1] and coordination geometry of metals and conformational
changes of hTf can be revealed. Our structures may also provide an
insight into the intermediate conformations of hTf during metal
binding and release.
Financial support by the Research Grants Council of Hong Kong
(705310P, HKU6/11G) is gratefully acknowledged.
References
1. Sun H, Li H, Sadler PJ (1999) Chem Rev 99:2817.
2. Qian ZM, Li H, Sun H, Ho K (2002) Pharmacol Rev 54:561–587.
3. Yang N, Zhang H, Wang M, Hao Q, Sun H (2012) Sci Rep 2:999.
J Biol Inorg Chem (2014) 19 (Suppl 2):S853–S866
DOI 10.1007/s00775-014-1165-y
POSTER PRESENTATION
Bioinspired and biomimetic systems
P 220
The mechanism of catechol intradiol dioxygenation
by bio-inspired non-heme iron(III) complexes
P 222
The first metal complex with unidentate N1-purine
ligand
Robin Jastrzebski1, Matthew G. Quesne2, Bert M. Weckhuysen1,
Sam P. de Visser2, Pieter C. A. Bruijnincx1
1
Inorganic Chemistry and Catalysis, Debye Institute
for Nanomaterials Science, Utrecht University, Universiteitsweg 99,
3584 CG Utrecht, The Netherlands
2
The Manchester Institute for Biotechnology and School of Chemical
Engineering and Analytical Science, The University of Manchester,
131 Princess Street, Manchester M1 7DN, United Kingdom
Intradiol catechol dioxygenases are an important class of enzymes in
the catabolism of aromatic substrates by microorganisms. These
enzymes selectively cleave the aromatic ring of catechols with oxygen to obtain derivatives of cis,cis-muconic acid [1]. Bio-mimetic and
bio-inspired iron(III) complexes also catalyse this reaction and we
have recently shown this to be a viable renewable route to adipic acid
[2]. Obtaining a better mechanistic understanding of these bioinspired systems is important both to develop more active catalysts
and to gain new insight in the enzymatic mechanism.
Here, we report on our combined computational and experimental investigation of the mechanism of catechol intradiol
dioxygenation by iron(III) complexes of tris(2-pyridylmethyl)amine,
which is the best bio-inspired system reported so far based on both
activity and selectivity. Based on DFT calculations, oxygen binds
the iron(III) center at a vacant site generated by partial dissociation
of the catecholic substrate (Figure 1a). This then directs oxygen to
attack the substrate carbon to form a bridging peroxide intermediate,
which was found to be the rate-determining step. Comparison with
experimental rate data of a number of different complexes showed
excellent agreement between the calculated barrier height and the
observed reactivity (Figure 1b). The influence of the electronic
structure and relevance to the enzymatic mechanism will also be
discussed.
Marina Serrano-Braceras1, Duane Choquesillo-Lazarte2, Alicia
Domı́nguez-Martı́n1,3, Esther Vı́lchez-Rodrı́guez1,4, Alfonso
Castiñeiras3, Josefa Marı́a González-Pérez1, Juan NiclósGutiérrez1
1
Dep. Inorg. Chem., Fac. Pharmacy, Univ. Granada, 18071 Granada,
Spain. [email protected]
2
LEC-IACT, CSIC-UGR, Avda. de las Palmeras 4, 18100 Armilla,
Granada, Spain
3
Dep. of Chemistry, Univ. of Zurich, Winterthurerstrasse 190, 8057
Zurich, Switzerland
4
Dep. Inorg. Chem., Fac. Pharmacy, Univ. Santiago de Compostela,
15782 Santiago de Compostela, Spain
Adenine is the most versatile purine ligand due to its ability to
combine proton tautomerism or ionization within different metal
binding patterns (MBP). To better understand this feature an interesting approach is to investigate the MBP of deaza- and aza-adenines
as well as isomers of adenine [1]. As part of our program, we report
the structure of the compounds [Cu(p3F)(H(N9)pur)(H2O)] (1, see
Figure) and {[Cu(l2-p3F)(Hhyp)Cu(p3F)(Hhyp) (H2O)]7.25H2O
(2), where p3F is p-(trifluoromethylbenzyl)iminodiacetate(2-) ligand
and H(N9)pur or H(N9)hyp represents the most stable tautomers of
purine or hypoxanthine, respectively. The Cu(II) centres have a 4 + 1
coordination, p3F exhibits the expected mer-NO2 conformation and
Hpur [1,2] or Hhyp [3] supplies their N1 or N9 atom among the four
closest Cu(II)-donor set. Previous works refer to the MBP N9H(N7)pur, l2-N1,N7-H(N9)pur and l2-N7,N9-H(N1)pur, using three
different tautomers of purine. Hence, compound 1 is the first metal
complex reported with unidentate N1-purine. That increases the
versatile behaviour of MBP for Hpur. On the other hand, 2 is the first
dinuclear complex where the chelating ligand conformation is related
to the MBP of Hhyp [3].
References
1. Vaillancourt FH, Bolin JT, Eltis LD (2006) Crit Rev Biochem Mol
Biol 41:241–267
2. Jastrzebski R, Weckhuysen BM, Bruijnincx PCA (2013) Chem
Commun 49:6912–6914
References
1. Domı́nguez-Martı́n A, Brandi-Blanco MP, Matilla-Hernández A,
El Bakkali H, Nurchi VM, González-Pérez JM, Castiñeiras A, NiclósGutiérrez J (2013) Coord Chem Rev 257: 2814–2838
123
S854
2. Nockeman P, Schulz F, Naumann D, Meyer G (2005) Z Anorg Allg
Chem 631:649–653
3. Patel DK, Choqesillo-Lazarte D, Domı́nguez-Martı́n A, BrandiBlanco MP, González-Pérez JM, Castiñeiras A, Niclós-Gutiérrez J
(2011) Inorg Chem 50:10549–10551
P 223
Pyrazole based ligand scaffolds provide second sphere
hydrogen bonding: Substrate coordination
by bimetallic complexes
Natascha Bruckner1, Sebastian Dechert1, Joanna Galezowska2,
Franc Meyer1
1
Institute for Inorganic Chemistry, Georg-August-University
Göttingen, Tammannstraße 4, 37077 Göttingen, Germany.
[email protected]
2
Department of Inorganic Chemistry, Faculty of Pharmacy, Wroclaw
Medical University, Borowska 211A, 50-556 Wroclaw, Poland
The synthesis of model systems is of great interest for understanding
metalloenzyme active site features, and for developing biomimetic
catalysts. Compartmental ligands based on 3,5-disubstituted pyrazolate bridges are well suited for generating highly preorganized
bimetallic complexes (figure left) with tuneable metal–metal separations in the range 3.5–4.5 Å [1]. Appending second sphere functions
for H-bonding interactions may enhance, in a biomimetic approach,
the activation of bound substrates or the stabilisation of catalytic
intermediates [2]. To this end we and Borovik et al. prepared pyrazolate-based ligand scaffolds such as H5L featuring amide groups in
the periphery of the bimetallic pocket [3]. The coordination chemistry
of these ligands (e.g. the dicopper(II) complex shown) and their
properties compared to the parent systems lacking the amide groups
will be presented.
Financial support by the Deutsche Forschungsgemeinschaft (IRTG
1422, Metal Sites in Biomolecules, Structure, Regulation and
Mechanism) is gratefully acknowledged.
References
1. Klingele J, Dechert S, Meyer F (2009) Coord Chem Rev
253:2698–2741
2. Shook RL, Borovik AS (2010) Inorg Chem 49:3646–3660
3. (a) Ng GK-Y, Ziller JW, Borovik AS (2011) Inorg Chem
50:7922–7924, (b) Graef T, Galezowska J, Dechert S, Meyer F (2011)
Eur J Inorg Chem 2011:4161–4167
123
J Biol Inorg Chem (2014) 19 (Suppl 2):S853–S866
P 224
Bulky N,N,O ligands in mononuclear non-heme iron
enzyme mimics
Emma Folkertsma, Marc-Etienne Moret, Robertus J.M. Klein
Gebbink
Organic Chemistry and Catalysis, Utrecht University,
Universiteitsweg 99, 3584CG Utrecht
Iron-based metallo-enzymes, commonly found in nature, are well
known for their ability to catalyse a wide range of reactions in
which molecular oxygen is activated. The superfamily of mononuclear non-heme iron enzymes is found to share a common
structural motif in which the active site contains an iron center
that is facially coordinated by two histidines and one carboxylato
group (see Figure). We have a particular interest in this so-called
2-His-1-carboxylate facial triad because of its unique reactivity
scope including selective cis-dihydroxylation, oxidative ring
cleavage, and oxidative C–N bond formation and ring closure
[1,2].
By strictly mimicking the active site of these iron enzymes we
attempt to develop new catalysts with a high activity and selectivity,
and in addition hope to learn about reaction mechanisms that may also
be operative in the enzymes. Furthermore, the use and development of
new iron-based catalysts is favourable above many other metals
because iron is cheap and non-toxic.
New types of N,N,O ligands designed during earlier studies in
our group have, e.g., allowed us to build models for enzymes
involved in catechol cleavage [3]. Unfortunately, the use of the
parent ligand mainly resulted in [FeL2]0/2+ complexes in which all
six coordination sites around iron are occupied and which,
accordingly, display hardly any catalytic activity [4,5]. In order to
overcome the formation of these coordinatively saturated homoleptic complexes, we recently developed more bulky variations of
the N,N,O ligand. The use of these bulky ligands resulted in lowcoordinate 1:1 ligand-to-iron complexes and one of the most
structurally faithful enzyme mimics to date. We will present the
design and synthesis of the new bulky N,N,O ligands and the
corresponding iron complexes.
References
1. Koehntop KD, Emerson JP, Que L (2005) J Biol Inorg Chem
10:87–93
2. Bruijnincx PCA, Klein Gebbink RJM (2008) Chem Soc Rev
37:2716–2744
3. Bruijnincx PCA, Klein Gebbink RJM, et al. (2007) J Am Chem Soc
129:2275–2286
4. Bruijnincx PCA, Klein Gebbink RJM, et al. (2008) Chem Eur J
14:1228–1237
5. Moelands MAH, Klein Gebbink RJM, et al. (2013) Inorg Chem
52:7394–7410
J Biol Inorg Chem (2014) 19 (Suppl 2):S853–S866
P 225
Dioxygen activation at new binuclear iron complexes
Anne Schober, Sebastian Dechert, Serhiy Demeshko, Franc
Meyer
Georg August University Göttingen, Institute for Inorganic
Chemistry, Tammanstr. 4, 37077 Göttingen, Germany
Non heme diiron enzymes perform remarkable catalytic reactions in
nature especially for the oxidation or oxygenation of various substrates.
The diiron core in e.g. soluble Methane Monooxygenase (sMMO)
provides a total of four electrons in order to reduce dioxygen and generate
highly reactive high-valent iron-oxo intermediates that can attack C–Hbonds [1, 2] With the aim of finding functional analogues for such diiron
sites many attempts were made towards stabilizing the two metal ions
with chelating ligands bearing carboxylate or nitrogen containing donor
groups. Very promising in stabilizing high valent diiron(IV) intermediates
has been lately the Tris(pyridylmethyl)amine (TPA) motif [3].
In our approach two iron atoms are bridged by a pyrazole building
block with varying side arms containing carboxylates, imidazole- or
pyridyl-residues [4]. Here we report a family of new pyrazolate
ligands that provide two pentadentate binding pockets, leaving a
reactive site between two bound metal ions.
The synthesis of these ligands scaffolds, the characterization and
the properties of their diiron(II) complexes and the binding and activation of dioxygen by these complexes will be reported.
Financial support for this project by the Deutsche Forschungsgemeinschaft (IRTG 1422: Metal Sites in Biomolecules, Structures,
Regulation and Mechanisms) is gratefully acknowledged.
References
1. Whittington DA, Lippard SJ (2001) J Am Chem Soc 123:827–838
2. Merkx M, Kopp DA, Sazinsky MH, Blazyk JL, Müller J, Lippard
SJ (2001) Angew Chem Int Ed 40:2782–2807
3. Xue G, Wang D, De Hont R, Fiedler AT, Shan X, Münck E, Que L
(2007) Proc Natl Acad Sci USA 104:20713–20718
4. Burger B, Dechert S, Grosse C, Demeshko S, Meyer F (2011)
Chem Commun 47:10428–10430.
P 226
Proton channels and structured peptides for H2
oxidation electrocatalysts to mimic hydrogenase
Wendy J. Shaw1, Sheri Lense2, Arnab Dutta1, John A.S. Roberts1,
Bojana Ginovska-Pangovska1
1
Catalysis Science, Pacific Northwest National Laboratory, Richland,
WA, 99354, USA.
2
University of Wisconsin, Oshkosh,Oshkosh, WI, USA
Proton channels are essential to the high rates and low overpotentials
of enzymes such as hydrogenase. In this work, we attach a proton
channel consisting of multiple proton relays to Ni(PR2 NR0
2 ) molecular
H2 oxidation electrocatalyst, to investigate its role in enhancing rates
or lowering overpotentials [1]. Proton channels consist of a second
sphere pendant amine and COOH groups from amino acids or small
functional groups in the outer coordination sphere [1]. The complexes
operate in water or methanol as both solvent and base, with rates up to
50 s-1 and overpotentials as low as 100 mV [1]. Additional components in the outer coordination sphere optimize the active site to
further enhance catalysis to *200 s-1 with 1 atm H2 and 140,000 s1
at 133 atm H2 [2]. Further enhancements may be achieved by
S855
additional proton relays or outer coordination sphere elements, positioned by a structured peptide based scaffold. The incorporation and
rate enhancements of a Ni(PR2 NR0
2 ) due to a structured b-hairpin
scaffold will be discussed [3, 4].
Financial support by the US Department of Energy, Basic Energy
Sciences Early Career Award is gratefully acknowledged.
References
1. Dutta A, Lense S, Engelhard M, Roberts JAS, Shaw WJ (2013) J
Am Chem Soc 135:18490–18496
2. Dutta A, Roberts JAS, Shaw WJ (2014) Angew Chem (submitted)
3. Reback M, et al. (2014) Chem Eur J (in press)
4. Reback M, et al. (2014) ACS Catalysis (submitted)
P 227
Synthesis and characterisation of the metal-binding
properties of a tripodal peptide ‘prototype’
Ágnes Dancs1, Nóra Veronika Nagy2, Dávid Árus1, Zsuzsanna
Darula3, Tamás Gajda1,4
1
Department of Inorganic and Analytical Chemistry, University
of Szeged, Dóm tér 7, 6720 Szeged, Hungary
2
Institute of Molecular Pharmacology, Research Centre for Natural
Sciences, Hungarian Academic of Sciences, Pusztaszeri út 59-67,
1025 Budapest, Hungary
3
Proteomics Research Group, Biological Research Center, Hungarian
Academy of Sciences Temesvári krt 62, 6726 Szeged, Hungary
4
Bioinorganic Chemistry Research Group of the Hungarian Academy
of Sciences, University of Szeged, Szeged, Hungary
One of the most important goals of recent bioinorganic research is the
developement of metal complexes with low molecular weigth but
effective catalytic activity and selectivity similar to native enzymes.
Functionalized tripodal ligands are probable compounds of biomimetic catalysts, since their special structure allows to form rigid,
preorganised metal-binding sites reminding to the active centres of
metalloenzymes.
Here we report the synthesis and solution equilibrium study of a
new tripodal tren (tris(2-aminoethyl)amine) based pseudopeptide
ligand (trenHis3) containing histidine as metal-binding moiety. The
preparation of the ligand was accomplished by solvent-phase peptide
synthesis (DCC, HOBt coupling agents), the product was purified by
preparative HPLC. Protonation steps of the ligand was followed by
1
H NMR spectroscopy and pH potentiometry. We also present the
complex formation properties of the ligand with copper(II) and zinc(II) ions, and the oxidoreductase mimicking activity of the examined
complexes. Stability constants were calculated based on potentiometric and CD measurements, structural information of the
complexes was gained by UV–Vis, CD, NMR, EPR and MALDI–
TOF–MS measurements.
In presence of both metal ions, various complexes were found with
MHxL and M3HxL2 compositions. Around the neutral pH, depending
on the copper(II)-to-ligand ratio, CuL and Cu3L2 complexes are the
dominant species with bis-histamine type coordination. Further deprotonations of these complexes take place under more alkaline pH
conditions than linear peptide-copper(II) complexes, which is due to
the increased stability of the bis-histamine type structure. The major
deprotonated zinc(II) complex Zn3H–4L2 shows slow ligandexchange processes, the related 1H NMR spectrum indicates the
presence of more than eight different chemical environment for the
imidazole rings. This can be due to the presence of either slow
exchanging conformational isomers, or chemical exchange between
different oligomers.
Copper(II)-ligand complexes were tested in di-tertbutyl-catechol
oxidase and superoxide dismutase model reactions as enzyme mimicking
catalysts: they were found to possess considerable catalytic activity.
123
S856
This work was supported by the Hungarian Scientific Research
Fund (OTKA K101541).
P 228
Room temperature catalyst for toluene aliphatic C–H
bond oxidation: Tripodal tridentate copper complex
immobilized in functionalized mesoporous silica
nanoparticles
Chih-Cheng Liu, Tien-Sung Lin, Chung-Yuan Mou
Department of Chemistry, National Taiwan University, Taipei 106,
Taiwan. [email protected]
A tripodal tridentate histidine-like copper(II) complex, CuImph
(Imph = bis(4-imidazolyl methyl)benzylamine), was synthesized to
mimic the catalytic behavior of native copper enzyme, such as performing the aliphatic C–H bond activation at mild condition. The
stability and catalytic activity of the formation of copper-dioxygen
complex are moderate under room temperature. However, by
immobilizing the model complex in the nano-channels of functionalized mesoporous silica nanoparticles (MSNs), we observed high
stability and good reactivity at ambient temperature. The enhancements are attributed to the following factors: (1) nanochannels which
provides confined space and optimizes product yield, (2) rigid silica
framework which mimics the protein skeleton, (3) aluminum substituted silica framework which introduces weak surface acidity (Lewis
acid, like H-ZSM-5), promotes the turnover number of the reaction,
and increases the loadings of the positively charged model complex,
and (4) TP (3-trihydroxysilyl)propyl-methylphosponate modified
MSN framework which provides negatively charged surface to
increase the loading of the complex. We propose the mimic system
promotes the formation of bis-l-oxo species ([{CuIIIImph}2(lO22)2]) at ambient temperature. The stable bis-l-oxo species @MSN
samples show high reactivity and selectivity for toluene aliphatic C–H
bond oxidation, which converts toluene initially to benzyl alcohol,
and further oxidized to the major product benzaldehyde as a kinetic
consecutive reaction. Over-oxidation to benzoic acid is not observed,
but is the norm in present industrial high temperature process. The
catalytic system can be fully recovered even after several cycles just
like the catalytic behavior of native enzyme. This is an unprecedented
catalytic system that can perform the oxidation of toluene to benzaldehyde at room temperature with high efficiency and stability which
should have potential industrial applications.
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J Biol Inorg Chem (2014) 19 (Suppl 2):S853–S866
Reference
1. Fang YC, Lin HC, Hsu I-J, Lin TS, Mou CY (2011) J Phys Chem C
115:20639–20652
P 229
Metal ion binding of TACH-based multidentate
tripodal ligands
Attila Szorcsik1, Ferenc Matyuska2, Ágnes Dancs2, Tamás Gajda2
1
MTA-SZTE Bioinorganic Chemistry Research Group, Dóm tér 7.,
H-6720 Szeged, Hungary
2
Department of Inorganic and Analytical Chemistry, University
of Szeged, Dóm tér 7., H-6720 Szeged, Hungary
The development of low molecular weight artificial enzymes
operating by similar activity, selectivity and mechanism to native
proteins, is one of the most important research direction of modern
bioinorganic chemistry. In recent years our research group focused
on the metal complexes of tripodal ligand derivatives which
enclose the metal ion(s), similarly to metalloenzymes.
The coordination chemistry of simple tripodal ligands (e.g. 1,3,5cis,cis-triaminocyclohexane, tach) is now well established. By
derivatization of this tripodal platform it is possible to alter and
optimize the steric properties for controlled reactivity at the metal
centres, i.e. to create metal complexes for small molecule activation
used in biomimetic chemistry. The fine tuning of the properties and
reactivity of the metal centres can be obtained in different levels, even
with relatively simple multidentate tripodal ligands. In the present
work we report the preparation and metal ion coordination of two new
triaminocyclohexane-based tripodal ligands, which possess quite
different coordination properties. The metal–ligand interaction was
studied in solution with pH-potentiometry, UV–VIS, EPR, NMR
spectroscopy.
The ligand L1 (N,N0 ,N00 -tris(3-pyridylmethyl)-1,3,5-cis,cis-triaminocyclohexane 3pyrtach) provides a 3N metal binding sites for the
metal ions, while the binding pocket created by the pyridine rings
may promote the substrate binding by hydrophobic/H-bridging
interactions. Accordingly, in the presence of copper(II) and zinc(II),
3N coordinated ML complexes are formed in the neutral pH range. At
higher pH hydroxido complexes are formed, which are active catalysts of phosphodiester hydrolysis.
The 6N metal binding site of L2 (N,N,N-tris(5-pyrazolylmethyl)1,3,5-cis,cis-triamino-cyclohexane, paztach) fully enclose the metal
ions, and the remaining pyrrole nitrogens create a preorganized
allosteric binding site for another metal ion. Both zinc(II) and copper(II) form highly stable ML complexes around pH 7. In the
copper(II) containing system at higher pH the trinuclear Cu3H–
4(paztach)2 complex is formed, which is the only species at pH 10
even in the equimolar solution, indicating its very high stability. The
X-ray crystal structure of Cu3H–4(paztach)2(ClO4)2 indicates, that the
two outer copper(II) are coordinated by five nitrogens in a square
pyramidal geometry, while the central copper is bound to four
deprotonated pyrrolic nitrogens with a tetrahedral geometry. Due to
the reac-tivity of the unsaturated tetrahedrally coordinated copper(II),
the complex Cu3H–3(paztach)2 is highly active catecholase mimic. It
is worth to mention, that the maximal catalytic activity is observed at
surprisingly low pH (*6).
J Biol Inorg Chem (2014) 19 (Suppl 2):S853–S866
This work was supported by the Hungarian Scientific Research
Fund (OTKA K101541).
P 230
Factors affecting the design of biomimetic ferrichrome
analogues
Evgenia Olshvang1, Elzbieta Gumienna-Kontecka2, Agnieszka
Szebesczyk2, Henryk Kozłowski2, Yitzhak Hadar3, Abraham
Shanzer1
1
Department of Organic Chemistry, The Weizmann Institute
of Science, Rehovot, Israel.
2
Faculty of Chemistry, University of Wrocław, Wrocław, Poland
3
Department of Plant Pathology and Microbiology, Faculty
of Agriculture Food and Environment, The Hebrew University
of Jerusalem, Rehovot, Israel.
Iron is an essential element for virtually all life forms. However, in the
oxidizing earth atmosphere it exists as insoluble ferric (Fe(III)) polymers. To overcome iron deficiency, microorganisms have developed a
unique acquisition system. This system consists of: (i) secreted iron
chelators, called siderophores, which bind and solubilize iron, and (ii)
specific outer membrane receptors, that deliver the siderophore-iron
complex into the microbial cell by active transport.
The Biomimetic Chemistry approach, followed in our laboratory,
aims at reproducing or mimicking the functions of natural products
rather than their detailed structure. A library of new biomimetic
ferrichrome analogues was prepared. The cyclic hexapeptide of the
natural ferrichrome was replaced by symmetric tripodal triamine or
alternatively by triacid template. The template was extended with
variety of amino acids as well as succinic acid, and terminated by a
hydroxamate chelating unit. Both structural and stereo-isomers (L-AA
and D-AA enantiomers) have been prepared. The isomers differ in
their length, and the location of the external residue, with respect to the
coordinated metal binding site.
The effect of structural preferences and iron complex stability on
bacterial growth promotion in two gram negative bacterial strains:
Escherichia coli and Pseudomonas putida, will be presented and
discussed.
The differences in Fe(III) uptake in model bacteria, can indicate
the optimization steps for obtaining advanced analogues, and reflect
the structural differences of the microbial uptake system in the different strains.
P 231
Functional ribonucleotide reductase enzyme models
Miklós István Szávuly, József Kaizer, Gábor Speier
Department of Chemistry, University of Pannonia, 8200 Veszprém,
Hungary. [email protected]
Dioxygen activation by non-hem diiron enzymes occurs in a number
of metabolically important transformations including the conversion
of ribonucleotides to deoxyribonucleotides by ribonucleotide reductase (RNR) [1], the formation of unsaturated fatty acids by fatty acid
S857
desaturases, the biosynthesis of antibiotics (CmlA, CmlI). In general,
O2 activation is thought to be initiated by the binding of O2 to the
diiron(II) center to form a peroxidodiiron(III) intermediate that in turn
converts to the oxidizing species. Peroxidodiiron(III) intermediates
with visible features between 600–750 nm have been identified for
RNR R2 [2].
The complexes have been isolated from the reaction of different
non-symmetric bidentate N-donor ligands and FeII salts in acetonitrile,
and have been characterized by X-ray crystallography and several
spectroscopic techniques [3] (Scheme 1). They are suitable catalyst for
oxidation of 2,6-di-tert-butylphenol and 2,4-di-tert-butylphenol with
H2O2 as the oxidant, where the in situ formed peroxidodiiron(III)
intermediates were isolated as key species, as found for RNR R2. The
peroxidodiiron(III) intermediate undergoes O–O bond scission to
generate a high-valent oxidant capable of X–H bond cleavage.
Financial help of TÁMOP-4.1.1.C-12/1/KNOV-2012-0017/is
greatly acknowledged.
N
(L1)
H
N
H
N
N
N
N
(L2)
N
S
N
N
(L3)
1 [FeII(L1)3](CF3SO3)2
2 [FeII(L2)3](CF3SO3)2
3 [FeII(L3)3](CF3SO3)2
References
1. Nordlund P, Reichard P (2006) Annu Rev Biochem 75:681–706
2. Pap JS, CransWick MA, Balogh-Hergovich É, Baráth G, Giorgi M,
Rohde GT, Kaizer J, Speier G, Que L Jr (2013) Eur J Inorg Chem
22–23:3858–3866
3. Pap JS, Draksharapu A, Giorgi M, Browne WR, Kaizer J, Speier G
(2014) Chem Commun 50:1326–1329
P 232
Towards criteria necessary for designing successful
siderophore mimic
Agnieszka Szebesczyk1, Jenny Besserglick2, Evgenia Olshvang2,
Abraham Shanzer2, El_zbieta Gumienna-Kontecka1
1
Faculty of Chemistry, University of Wrocław, 14 F. Joliot-Curie str.,
50-383 Wrocław, Poland. [email protected]
2
Weizmann Institute of Science, 234 Herzl Street, Rehovot 76100,
Israel
To bind iron (III) ions bacteria and fungi have evolved the ability to
secrete small molecules called siderophores, along with receptors able
to recognize and transport Fe(III)-siderophore complexes. As some
virulent bacteria can recognize not only their own compounds, but
also those secreted by other species, it is very important to determine
the factors affecting the process of recognition. Additionally, investigation of mechanisms of iron uptake may provide to novel paths of
antibiotic action through ‘‘Trojan horse’’ strategy.
In our research analogs of ferrichrome, one of hydroxamic siderophores, were investigated. Hexapeptide ring, difficult in synthesis,
was replaced by three longer arms [1], which consist of amino acid
(Xaa) and are terminated by retro-hydroxamic group (Figure). A is the
apical site, to which antibiotic or fluorescent molecule might be bound.
The key step to understanding the relationship between structure
and function of siderophores is the determination of coordination
characteristics of artificial siderophores with Fe(III) ions. Coordination properties may be modified by elongation of arms or location of
additional groups nearby the binding ones.
In the physicochemical studies we focused on formation and stability of iron complexes. We have investigated coordination
properties of ligands with different arms length and with substituents
nearby the retro-hydroxamic groups. The binding properties of biomimetic compounds are similar to those of natural ferrichrome.
123
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J Biol Inorg Chem (2014) 19 (Suppl 2):S853–S866
Moreover, in bacterial growth promotion studies these analogs were
able to transport iron ions into the cells of Pseudomonas putida.
Financial support by the Polish National Science Centre (research
grant UMO-2011/03/B/ST5/0) is gratefully acknowledged.
N
N
OH
HO
HO
Xaa
N
Xaa
Xaa
O
O
(CH2)2
(H2C)2
(H2C)2
O
O
O
O
A
Reference
1. Shanzer A, Libman J in: Handbook of Microbial Iron Chelates;
Winkelmann G, Ed.; CRC: Boca Raton, FL, 1991; pp 309–338
P 233
Targeted protein surface sensors: A general approach
for tracking protein structural changes and binding
interactions
Yael Nissinkorn, Leila Motiei, and David Margulies
Department of Organic Chemistry, Weizmann Institute of Science,
Rehovot 76100, Israel. [email protected]
Conventional approaches for detecting protein conformational changes
in their natural environment involve labeling the proteins with genetically encoded fluorescent proteins: a donor and an acceptor. Although
this approach has been successfully applied for tracking protein
structural changes, it is limited to detecting proteins that undergo significant changes in the distances between their N’ and C’ termini.
The aim of my research is to create a novel class of fluorescent
molecular sensors that can sense dynamic changes on protein surfaces.
This novel approach will allow one to track changes in the conformations of various proteins including changes that result from binding
interactions. Our molecular design integrates an environmentally
sensitive fluorophore, a protein surface binder, and a genetically targeted molecule on a single molecular platform. As a proof-of-concept,
we applied these probes to detect changes in the conformation of
Calmodulin (CaM) upon binding to calcium ions, small molecules, and
protein binding partners. When bound to calcium, CaM exposes a
large hydrophobic cleft on its surface, which leads to a change in the
environment of the solvatochromic fluorophore and to enhanced
fluorescence emission. Binding of a small molecule inhibitor or a
protein binding partner for this cleft causes the protein to fold and
conceal the hydrophobic cleft, which leads to decreased fluorescence
emission. The high specificity of the sensor-CaM interaction should
allow one to perform the assay in biological mixtures.
P 234
Computational investigations of potential water
oxidation catalysts
Florian H. Hodel, Marcella Iannuzzi-Mauri, Sandra Luber, Jürg
Hutter
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland
Employing ab initio methods, we tried to calculate electronic and
structural properties of a polyoxometalate (POM) used as a catalyst in
oxidative water splitting [1]. We did this in vacuum, as well as in solution, using QM/MM for the latter. With the same methods we employed
for the POM alone, we also looked at various aggregates of the photosensitizer Tris(bipyridine)ruthenium(II) with the POM in an attempt to
not only explore the actual structure of such an aggregate, but also the
possible influences of the sensitizer on the redox properties of the POM.
For a cobalt-cubane [2], another water oxidation catalyst, we are
trying to determine the free energy change and the reaction barrier for
a ligand exchange reaction where acetate would be replaced by water.
We use the Nudged Elastic Band method to obtain a rough overview
and Metadynamics to further investigate the free energy surface.
Financial support by the University of Zurich is gratefully
acknowledged.
References
1. Car PE, Guttentag M, Baldridge KK, Alberto R, Patzke GR (2012)
Green Chem 14:1680–1688
2. Evangelisti F, Güttinger R, Moré R, Luber S, Patzke GR (2013) J
Am Chem Soc 135:18734–18737
P 235
Synthesis of a chiral, polydentate ligand system setting
out from L-cysteine and investigation of its nickel
complexes
Dana S. Warner, Stefan Mebs, Beatrice Braun, Christian
Limberg
Department of Chemistry, Humboldt-Universität zu Berlin, BrookTaylor-Str. 2, 12489 Berlin, Deutschland
Cysteinate is one of the most abundant amino acid ligands found
within the active sites of metalloproteins. For example the nickel ions
in acetyl coenzyme A synthase (ACS) or in the [NiFe] hydrogenase
are exclusively coordinated by cysteinate units [1]. In attempts to
prepare structural or functional model compounds for the active
123
J Biol Inorg Chem (2014) 19 (Suppl 2):S853–S866
center of the ACS, polydentate thiolates have been employed to
mimic cysteinate ligand environments [2, 3].
In our work, we set out from protected natural L-cysteine to synthesize a 2,5-diketopiperazine [4]. Reduction with NaBH4/TiCl4 leads
to N,N0 -dimethyl-(2R,5R)-bis-(sulfanylmethyl) piperazine, LH2, the
thiol functions of which electronically resemble cysteine. Hence, LH2
represents a novel, chiral ligand precursor to mimic Ni(Cys)n sites [5].
Deprotonation of LH2 with NaOMe followed by treatment with
NiBr2(dme) led to the isolation of a diastereomerically pure nickel
complex [LNi]2. In [LNi]2 each Ni center is coordinated by two
thiolate and one amino donor functions, and these entities dimerise to
give [LNi]2. Cyclic voltammetry indicated the possibility of
NiII ? NiIII oxidation for this complex [5]. [LNi]2 may be regarded
as a structural model for the A cluster within the ACS, which also
contains two Ni centers bridged by cysteinate functions, as some of
the metric data compare well.
By the conversion of [LNi]2 with nucleophiles like KSEt or KCN,
the Ni-(l2-S)-Ni bridges are cleaved to afford planar mononuclear
products, which represent interesting precursor compounds to set out
from for biomimetic or bioinspired studies.
S859
the equilibrium between the two species depends on both solvent and
temperature and that the l-thiolate complex forms under kinetic control,
whereas the disulfide complex is the most stable species. This is the first
time that the energies involved in this equilibrium are quantified, which
is done both experimentally (VT-NMR) as theoretically (DFT). The
DFT calculations and the experimental findings give a consistent view
that is further supported by X-ray crystal structures [2].
References
1. Itoh S, Nagagawa M, Fukuzumi S (2001) J Am Chem Soc
123:4087–4088
2. Ording-Wenker ECM, Van der Plas M, Siegler MA, Bonnet S,
Bickelhaupt FM, Fonseca Guerra C, Bouwman E (2014) submitted
References
1. Darnault C, Volbeda A, Kim EJ, Legrand P, Vernède X, Lindahl P,
Fontecilla-Camps JC (2003) Nat Struct Biol 10:271
2. Linck RC, Spahn CW, Rauchfuss TB, Wilson SR (2003) J Am
Chem Soc 125:8700
3. Krishnan R, Riordan CG (2004) J Am Chem Soc 126:4484
4. Iannotta D, Castellucci N, Monari M, Tomasini C (2010) Tetrahedron Lett 51:4558
5. Warner DS, Mebs S, Limberg C (2013) Z Anorg Allg Chem
639:1577
P 236
Thermodynamics of the CuII l-thiolate and CuI
disulfide redox equilibrium: A combined experimental
and theoretical study
Erica C. M. Ording-Wenker1, Martijn van der Plas1, Maxime A.
Siegler2, F. Matthias Bickelhaupt3, Célia Fonseca Guerra3,
Elisabeth Bouwman1
1
Leiden Institute of Chemistry, Leiden University, P.O. Box 9502,
2300 RA Leiden, The Netherlands.
2
Department of Chemistry, John Hopkins University, Baltimore,
Maryland 21218, USA.
3
Department of Theoretical Chemistry, VU University Amsterdam,
De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands
The ability of copper to cycle between CuII and CuI oxidation states
makes it a suitable metal for active sites in a wide range of enzymes,
giving it a pivotal role in many biological processes. A ligand system
was reported wherein minor changes in the ligand result in a drastic
change in the type of copper compound formed [1]. Either a CuII lthiolate complex, which bears similarity to the biological CuA site, or
a CuI disulfide complex is formed. We have investigated this redox
equilibrium to find out when this biologically-relevant CuII l-thiolate
species forms and whether we can tune this equilibrium in a controllable fashion. With two newly synthesized ligands, we found that
P 237
One-electron oxidized Cu(II)-di(phenolate) complexes;
Relationship between electronic structures
and reactivities
Yuichi Shimazaki
College of Science, Ibaraki University, 2-1-1 Bukyo, Mito, 310-8512
Japan
The Cu(II)–phenoxyl radical formed during the catalytic cycle of
galactose oxidase (GO) attracted much attention, and the structures
and properties of a number of metal–phenoxyl radical complexes
have been studied. Some of functional model system of GO have been
reported previously that the Cu complexes showed the oxidation of
primary alcohols to aldehydes, and formation of the Cu(II)–phenoxyl
radical species was revealed in the catalytic cycle. As an extension of
the studies on one-electron oxidized Cu(II)–phenolate species, we
synthesized one-electron oxidized square-planar Cu(II) complexes of
5- and 6-membered chelate salen-type ligands and characterized the
valence state and reactivity of one-electron oxidized complexes.
The neutral and one-electron oxidized Cu(II) six-membered chelate
1,3-Salcn complexes have been investigated and compared with the fivemembered chelate 1,2-Salcn complexes (Fig. 1). Further, we have been
characterized one-electron oxidized salophen-type complexes, [Cu(salophen)]+ (H2salophen = N,N0 -bis(3,5-di-tert-butylsalicylidene)-1,2diaminobenzene) and its methoxy derivatives, [Cu(MeO-salophen)]+
and [Cu(salophen-(MeO)2)]+. Cyclic voltammetry of all Cu(II) complexes showed two reversible redox waves and their potentials are very
similar in the range of 0.3–0.5 V for the first reversible wave. Reaction of
the complexes with 1 equivalent of AgSbF6 afforded the oxidized complexes. The electronic structures of the oxidized complexes were
different; [Cu(1,2-Salcn)]SbF6 has a Cu(III)-phenolate ground state and
valence tautomerisation between Cu(III)-phenolateand Cu(II)-phenoxyl
radical in CH2Cl2 solution at room temperature, while [Cu(1,3-Salcn)]SbF6 has the phenoxyl radical species whose electron is relatively
localized on one phenolate moiety in the molecule. The solid state
structures of [Cu(salophen)]+ and [Cu(MeO-salophen)]+ can be
assigned to relatively localized Cu(II)-phenoxyl radical complexes, while
[Cu(salophen-(MeO)2)]+ is the diiminobenzene radical complex. On the
123
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J Biol Inorg Chem (2014) 19 (Suppl 2):S853–S866
other hand, [Cu(salophen)]+ in solution showed a different electronic
structure from that of the solid sample, the radical electron being delocalized over the whole p-conjugated ligand. The reactivity of oxidized
complexes with benzyl alcohol was also studied. Quantitative conversion
of benzyl alcohol to benzaldehyde was observed, while the reaction
mechanisms were different dependent upon the electronic structures.
These differences will be discussed based on the electronic structure difference.
References
1. Bradshaw B, Dinsmore A, Collison D, Garner CD, Joule JA (2001)
J Chem Soc Perkin Trans 1 3232–3238
2. Taylor EC, Dötzer R (1991) J Org Chem 56:1816–1822
3. Ryde U, Schulzke C, Starke K (2009) J. Biol. Inorg. Chem
14:1053–1064
P 239
Photosystem II like water oxidation mechanism
in a bioinspired tetranuclear manganese complex
P 238
Synthesis of pyrazine-pyrane-dithiolene ligands
mimicking specific features of molybdopterin
Claudia Schindler, Carola Schulzke
Department of Biochemistry, University of Greifswald, FelixHausdorff-Strasse 4, 17487 Greifswald, Germany
Molybdopterin (MPT) dependent enzymes are part of nearly any
known organism on earth ranging from ancient archaea over plants to
modern human beings.
While the biological synthesis of MPT is well understood, a chemical
synthetic pathway is very challenging and is still being studied.
The aim of the project is the synthesis of model compounds for the
molybdopterin depending cofactor, which are able to catalyse oxygen
atom transfer reactions and which can be incorporated into the apoenzyme.
The focus of our research is the development of ligand precursors
that address the dithiolene function, the pyrane ring and the adjacent
pyrazine ring and their coordination with especially molybdenum but
also tungsten to understand the influence of various structural units on
the stability, the catalytic properties or the redox potential.
Financial support of the ERC (European Research Council) is
gratefully acknowledged.
O
NH
H2N
S
H
N
N
L1 L
2
Mo O
S
N
H
O
P
HO
OH
O
H
N
N
H
123
S
Mo
S
O
L1
L2
References
1. Umena Y, Kawakami K, Shen JR, Kamiya, N (2011) Nature
473:55–60
2. Karlsson EA, Lee BL, Åkermark, T, Johnston EV, Kärkäs MD, Sun
J, Hansson Ö, Bäckvall JE, Åkermark B (2011) Angew Chem Int Ed
50:11715–11718
3. Kok B, Forbush B, McGloin M (1970) Photochem Photobiol
11:457–475
molybdopterin
O
O
Rong-Zhen Liao, Markus D. Kärkäs, Bao-Lin Lee, Björn
Åkermark, Per E. M. Siegbahn
Department of Organic Chemistry, Arrhenius Laboratory, Stockholm
University, SE-10691 Stockholm, Sweden. [email protected],
[email protected]
Photosystem II is the only nature photosynthetic apparatus that catalyze water oxidation to harvest dioxygen utilizing a Mn4Ca cluster
in the oxygen-evolving complex (OEC) [1]. Extraordinary efforts
have been devoted into the synthesis of multinuclear manganese
complex to mimic the properties of OEC. The most encouraging
breakthrough in this field came with the synthesis of a tetranuclear
manganese complex that enables water oxidation in homogeneous
phosphate buffer with single-electron oxidant [Ru(bpy)3]3+ [2].
Hybrid DFT calculations have been performed to investigate the
water oxidation mechanism of this bioinspired manganese catalyst.
We propose an oxygen-evolving complex like water oxidation
mechanism, in which oxygen evolution occurs through the Kok cycle
(S0 to S4) [3] and the crucial O–O bond formation takes place in the
formally MnIVMnIVMnIVMnV state (known as S4 in OEC) by direct
coupling of a MnIV-bound terminal oxygen radical and a di-Mn
bridged oxo group. All other mechanisms, including coupling of
oxygen radical and bridging oxo at other states (S2 and S3) in the
tetranuclear manganese complex and water attack on MnIV-oxygen
radical in the dinuclear manganese complex, are found to be associated with higher barrier.
Financial support by the Wenner-Gren Foundation, the Swedish
Research Council and the Knut and Alice Wallenberg Foundation is
gratefully acknowledged.
target model compound
P 240
Photochemical CO2-reduction catalyzed by monoand dinuclear phenanthroline-extended tetramesityl
porphyrin complexes
Corinna Matlachowski, Matthias Schwalbe
Institut für Chemie, Humboldt-Universität zu Berlin, Brook-TaylorStr. 2, 12489 Berlin, Germany
The catalytic reduction of CO2 to CO or into liquid fuels represents a
crucial challenge due to the global warming problem and the fossil
fuel scarceness. This energy demanding process is favorably driven
using sunlight as energy source [1, 2].
The construction of heterodinuclear complexes (or photochemical
molecular devices-PMDs) [3] is of great importance, as they use
J Biol Inorg Chem (2014) 19 (Suppl 2):S853–S866
energy derived from solar light to drive a catalytic reaction. PMDs
consist of a light harvesting unit, often a Ru(bpy)2+
2 fragment, a linker
and a reaction centre at which the substrate transformation (e.g.
carbon dioxide reduction) takes place. The linker plays a crucial role
as it should allow for guided electron transfer towards the reaction
center and possess possible electron storage capacity.
Fujita et al. could show that iron and cobalt porphyrins are suitable
for the photochemical CO2-reduction [4]. We like to develop their
system further and use mono- and dinuclear complexes with a phenanthroline-extended tetramesityl porphyrin ligand (H2-1) [5]. This
fragment
ligand can selectively coordinate a Ru(tbbpy)2+
2
(tbbpy = 4,40 -di-tert-butyl-2,20 -bipyridine) while any other metal can
reside in the porphyrin cavity. The coordination of a ruthenium
fragment decreases the reduction potentials, which should thermodynamically be more suitable for efficient CO2-reduction.
Thorough catalytic investigation on M-1(-Ru) compounds
(M = 2H, Cu, Pd, Co, FeCl) was done in DMF saturated with CO2
and in the presence of triethylamine (TEA) as sacrificial electron
donor.
References
1. Armaroli N, Balzani V (2007) Angew Chem Int Ed 46:52–66
2. Federsel C, Jackstell R, Beller M (2010) Angew Chem Int Ed
49:6254–6257
3. Inagaki A, Akita M (2010) Coord Chem Rev 254:1220–1239
4. Dhanasekaran T, Grodkowski J, Neta P, Hambright P, Fujita E
(1999) J Phys Chem A 103:7742–7748
5. Matlachowski C, Schwalbe M (2013) Dalton Trans 42:3490–3503
P 241
Synthetic approaches towards the models of sulfite
oxidase
Yulia B. Borozdina1, Nicolas Chrysochos1, Muhammad Zubair2,
Carola Schulzke1,2
1
Institute of Biochemistry, Ernst-Moritz-Arndt University, FelixHausdorff-Straße 4, 17487 Greifswald, Germany
2
Trinity College, Dublin University, College Green, Dublin 2,
Ireland. [email protected]
Molybdenum cofactor, Moco, presents in a number of redox enzymes,
such as pyridoxal oxidase, formate dehydrogenase, pyrine hydroxylase, sulphite oxidase (SO), etc. essential for metabolism of all
aerobic organisms [1]. After the structure of highly conserved
molybdopterin (MPT) ligand was established by Rajagopalan et al.
[2], that discovery marked a new wave in the model chemistry of
molybdenum- and tungsten-containing enzymes. Continuing our
interest in monodithiolene complexes as simplified structural variants
of SO, we have considered reactions leading to formation of such
compounds in situ. In our group it was found that some pyrazine
based bisdithiolene complexes undergo stepwise oxidative ageing
either in water solutions (fast) or in the solid state (slow). The progress of such reactions could be monitored by UV–Vis or mass
spectroscopy (the later preferably for the solid powders).
Other intriguing aspects that deserve, in our opinion, a detailed
study, are the structural features that control the geometry and, consequently, the catalytic activity of a model compound. For instance, it
S861
was shown that the bite angle of the stabilizing ligand and dihedral
angle of a dithiolene had a crucial influence on the oxidation state of
the molybdenum active site [3]. Thus, we designed a number of
complexes in attempt to approach the redox potentials of SO by
tuning the geometry of the models.
Financial support by ERC is gratefully acknowledged.
References
1. Stiefel EI (1993) Molybdenum Enzymes, Cofactors and Model
Systems, Am Chem Soc Washington DC 1–18
2. Leimkühler S, Wuebbens MM, Rajagopalan KV (2011) Coord
Chem Rev 255:1129–1144
3. Mitra J, Sarkar S (2013) Inorg Chem 52:3032–3042
P 242
Ligand architectures as factors affecting
the thermodynamic and redox stability of metal ion–
siderophore complexes
Etelka Farkas1, Orsolya Szabó2, István Pócsi3
Department of Inorganic and Analytical Chemistry, University
of Debrecen, Egyetem tér 1, 4032 Debrecen, Hungary.
[email protected]
2
MTA-DE Homogenous Catalysis and Reaction Mechanisms
Research Group, Debrecen, Hungary
3
Department of Microbiology and Biotechnology, University
of Debrecen, Hungary
The role of siderophores in iron uptake of microorganisms is essential. They are able to bind iron(III) ion with very high selectivity. In
the most cases, there is identity in the number of chelating functions
of the different siderophore molecules on one side, but large versatility in their global architecture on the other side is seen. The effect
of this versatility of the ligand structures on the metal binding ability
and especially on the metal-ion selectivity of hydroxamate based
siderophores have been studied in our lab during the past few years.
In the present work, four trihydroxamate-based siderophores, the
acyclic desferrioxamine B (DFB) and its exocyclic counterpart
desferricrocin (DFCR) as well as the acyclic desferrocoprogen (DFR)
together with its endocyclic analogous, N0 ,N00 ,N¢¢¢-triacetylfusarinine
C (TAF) have been involved into the investigation.
Numerous results for interaction with different metal ions have
been determined and published previously with the two acyclic
molecules, DFB and DFC [1–3], but even the acid–base characterization of the two cyclic siderophores, DFCR and TAF, have been
performed in the present work. The results obtained for Fe(III),
Mn(II)/(III) and Co(II)/(III) complexes with the four siderophores
were compared with each other. The comparison showed that
although, significant architecture-determined differences in metal
binding ability and also in redox stability of these natural compounds
were found, but identities were also observed. For example surprisingly, all of them were found to form higher stability complexes with
Co(III) compared to Fe(III). Based on this, a conception for disturbance of the iron-uptake of microorganisms by kinetically inert
Co(III)-siderophore complexes seems to have reality. Since many
diseases are initiated by microorganisms, this possibility is interesting, indeed.
Financial supports by the TAMOP 4.2.2.A-11/1/KONV-20120043 Joint European-Hungarian Research Fund and COST M1105 are
gratefully acknowledged.
References
1. Farkas E, Bátka D, Kremper G, Pócsi I (2008) J Inorg Biochem
102:1654–1659
2. Szabó O, Farkas E (2011) Inorg Chim Acta 376:500–508
3. Farkas E, Szabó O (2012) Inorg Chim Acta 392:354–361
123
S862
P 243
Evaluation of the antioxidant, antihypertensive
and antitumor activity of a Cu(II) and Irbesartan (Irb)
complex [Cu(Irb)2(H2O)] (CuIrb): synthesis,
characterization and DFT study
Marı́a S. Islas1, Carlos A. Franca1, Luis Lezama2, Teófilo Rojo2,
Mercedes Griera Merino3, Marı́a A. Cortes3, Laura Calleros3,
Manuel Rodriguez Puyol3, Evelina G. Ferrer1, Patricia A.
M. Williams1
1
Centro de Quı́mica Inorgánica (CEQUINOR/CONICET/UNLP)Universidad Nacional de La Plata. 47 y 115, 1900. La Plata,
Argentine. [email protected]
2
Universidad del Paı́s Vasco, Departamento de Quı́mica Inorgánica,
Facultad de Ciencia y Tecnologı́a, Apdo. 644, 48080, Bilbao, Spain
3
Departamento de Biologı́a de Sistemas, Universidad de Alcalá,
Campus Universitario, 28871, Alcalá de Henares, Madrid, Spain
Irbesartan is an angiotensin II receptor antagonist used mainly for the
treatment of hypertension [1]. Based on the fact that in some cases
metal complexes displayed improved therapeutic properties compared
to the parent drugs [2], we have synthesized a solid coordination
Cu(II) complex with Irb and CuCl2 in ethanolic solution.
Two different conformers of CuIrb, depending on the pH value of
the preparative have been obtained; one of them was blue and insoluble in all tested solvents, and the other green one, was soluble in
ethanol and DMSO. Both conformers were characterized by means of
elemental analysis, thermal decomposition measurements, UV–vis,
diffuse reflectance, infrared, Raman and EPR spectroscopies. Furthermore, the two possible structures for CuIrb have been determined
by means of the density functional theory (DFT). Besides, we have
calculated the theoretical vibrational frequencies which allowed us to
make comparisons with the experimental FTIR and Raman spectral
data. Solution assays were performed only with the soluble conformer
of CuIrb and stability studies under biological conditions and ethanolic
solutions have been performed. The free radical scavenging capacities
were measured on ABTS, DPPH, peroxyl radicals, and superoxide
anion. While Irb was not able to scavenge any of these free radicals,
CuIrb showed antioxidant activity against DPPH radicals.
Moreover, we have tested the activity of the complex in different
systems in vitro. The antihypertensive effects have been tested by the
analysis of the inhibition of the activity of angiotensin II (Ang II) to
reduce the planar cell surface area in human mesangial cells pretreated with Irb, CuIrb, and CuCl2. Surprisingly, the inhibition of the
contraction induced by AngII under CuIrb treatment has been
increased (18 %), compared to the parent drug.
On the other hand, we have tested the antitumor activity by means
of MTT and cell cycle assays, in three prostate cancer cell lines:
LNCaP, PC3 and DU145. We did not observe significant differences
compared to basal conditions in concentrations up to 100 lM.
References
1. Burnier M (2001) Circulation 103:904–912
2. Farell N (1989) Transition Metal Complexes as Drugs and Chemotherapeutic Agents
P 244
CO releasing molecules as precursors for manganese
based oxygen evolving complexes
Ulf Sachs, Tobias Fischer, Selina Leone, Hans-Martin Berends,
Philipp Kurz
Institute for Inorganic and Analytical Chemistry, Albert-LudwigsUniversity Freiburg, Albertstr. 21, 79104 Freiburg, Germany. [email protected]
123
J Biol Inorg Chem (2014) 19 (Suppl 2):S853–S866
Despite the interest in mimicking the Mn4CaOx cluster of the
oxygen evolving complex (OEC) of natural photosynthesis, only few
multinuclear complexes with more than two manganese atoms have
been investigated so far [1].
Stabilizing the multinuclear manganese cluster in different oxidation states, that are needed for the oxidation of water, seems to be a
very important part in this challenge.
To find an inexpensive and easy way to obtain multinuclear
manganese complexes is the challenge that led to the question if it is
possible to use well studied carbon monoxide releasing molecules
(CORMs) as precursors for defined molecular MnxOy clusters.
Although there have been a lot of studies about CORMs and their
potential use in medical therapy in the last couple of years [2], it has
not been investigated yet what happens to the complex after the release
of the CO molecules. Previous studies in our group by H. M. Berends
[3] indicate that the manganese will be oxidized, first to MnII and then
further, therefore the objective of the recent studies is to find out if
multinuclear manganese complexes can be achieved through UV
radiation, electrochemical oxidation or chemical oxidation.
References
1. Kanady JS, Tran R, Stull JA, Lu L, Stich TA, Day MW, Yano J,
Britt RD, Agapie T (2013) Chem Sci 4:3986–3996
2. Romao CC, Blättler WA, Seixas JD, Bernardes GJL (2012) Chem
Soc Rev 41:3571–3583
3. Berends H-M, Kurz P (2012) Inorg Chim Acta 380:141–147
P 245
Alleviating photoinhibition in cyanobacteria
by biomineralization-based biological shellization
Wei Xiong, Xurong Xu, Ruikang Tang
Department of Chemistry, Zhejiang University, Hangzhou, Zhejiang
310027, China
Photoinhibition has long been considered to be a main factor for limiting photosynthetic biomass, which has attracted a great deal of interest
for its potential role in the development of renewable energy sources for
decades. Here we report a biomimetic method for encapsulating individual cyanobacteria cells under physiologically mild, biocompatible
conditions, inspired by the biosilicification of diatoms. Further, we
show that biomimetic encapsulation of cyanobacterial cells with silica
greatly alleviates high light stress, which means enhancing efficiency of
solar energy utilization. Our present findings open a gate to the formulation of the encapsulation of other photosynthetic cellular,
subcellular or molecular entities that could contribute to utilizing solar
energy in nearby future. These results may also have implications in
silicification studies, biosensors and bioreactors.
P 246
Binding site preference of ZnII and CuII in cyclic
pseudo-peptides promotes phosphoester hydrolysis
activity
Peter Comba1, Annika Eisenschmidt1, Lawrence R. Gahan2,
Graeme R. Hanson3, Nina Mehrkens1, Michael Westphal1
1
Universität Heidelberg, Anorganisch-Chemisches Institut, Im
Neuenheimer Feld 270, 69120 Heidelberg, Germany.
[email protected]
2
School of Chemistry and Molecular Biosciences and
3
Centre for Advanced Imaging, The University of Queensland, St.
Lucia 4072, Australia
Patellamides are naturally produced by ascidiae, and their biological
role is not fully understood to date. To shade light into the putative
biological activity of complexes formed by the binding of transition
metal ions, model complexes were prepared from the cyclic pseudo-
J Biol Inorg Chem (2014) 19 (Suppl 2):S853–S866
peptides depicted below. The dinuclear complexes of cyclic peptides
with copper(II) were not only shown to function as carbonic anhydrase
mimics [1] with up to kcat = 7.3 9 103 s-1 (uncatalyzed: 3.7 9 102 -1
s ) but also to exhibit catalytic activity for phosphoester hydrolysis
[2]. While for the ligands H4pat1 and H4pat2 the phosphoester
hydrolysis activity shows high rates for copper(II) complexes, no
catalytic activity could be observed for the corresponding zinc(II)
complexes. Surprisingly however, complexes of zinc(II) with H4pat4
catalyze the phosphoester hydrolysis. Supported by NMR studies, we
presume that these experimental findings indicate differing binding
modes at physiological pH values, namely (A) the amide and (I) the
imidazole site (Figure). This is believed to lead to significant changes
of the pKa values of the water bound to the metal ions. In this study we
investigated this hypothesis by means of MM- as well as QM-methods.
S863
bacterial cells followed by the ultracentrifugation. Photoinduced
methane oxidation was carried out by the light irradiation to the
mixture containing thylakoid, membrane fraction from M. trichosporium OB3b, NAD+ and methane. The amount of methanol was
apparently higher with light irradiation than in the dark condition.
This result indicates that selective oxidation of methane to methanol
can be achieved by the combination of thylakoid and membrane
fraction from M. trichosporium OB3b under light irradiation.
I
A
Reference
1. Ito H, Mori F, Tabata K, Okura I, Kamachi T (2014) RSC Adv
4:8645–8648
References
1. Comba P, Gahan LR, Hanson GR, Maeder M, Westphal M (2014)
Dalton Trans 43:3144
2. Comba P, Gahan LR, Hanson GR, Westphal M (2012) Chem.
Commun 48:9364
3. Comba P, Dovalil N, Gahan LR, Hanson GR, Westphal M (2014)
Dalton Trans 43:1935
P 248
Methane hydroxylation using light energy
by the combination of thylakoid and methane
monooxygenase
Hidehiro Ito1, Fumiya Mori2, Kenji Tabata2, Ichiro Okura2,
Toshiaki Kamachi2
1
Education Academy of Computational Life Sciences, Tokyo Institute
of Technology, 2-12-1, Ookayama, Meguro-ku, Tokyo, 152-8550,
Japan
2
Department of Bioengineering, Tokyo Institute of Technology, 2-121, Ookayama, Meguro-ku, Tokyo, 152-8550, Japan
Enzymatic reactions have many specific features, such as highly
controlled regio-, stereo- and substrate-specificity under very mild
conditions. Because of these favourable features, enzymes are used in
industrial applications. One of the enzyme groups, oxidoreductases
show prominent features such as selective oxygenation of alkane and
so on, but the requirement of coenzyme, such as NAD(P)H, limits the
application of this enzyme group to the industrial applications.
In this work, thylakoid membrane, on which photoreduction taken
place is used as NAD(P)H regeneration system. By using thylakoid,
the electrons needed for oxidation of methane catalyzed by membrane-bound methane monooxygenase (pMMO) can be obtained from
inexhaustible water and solar energy. Here, we established a photoinduced hydroxylation system by the combination of thylakoid from
spinach for photoreduction of NAD(P)+ with pMMO from Methylosinus trichosporium OB3b for the catalyst of methane oxidation.
Thylakoid was prepared from spinach leaves. Membrane fraction
from M. trichosporium OB3b was isolated by the sonication of the
P 249
Evaluation of photolysis, antibacterial, and adsorption
abilities onto the biomimetic synthesized calcium
phosphate compound
Mayumi Minamisawa1, Kyoko Suzuki2, Shoichiro Yoshida3
1
Department of Chemistry, Education Center, Chiba Institute
of Technology, Shibazono, Narashino, Chiba 275-0023, Japan.
[email protected]
2
Yokohama City University School of Medicine, Yokohama, Japan
3
Tokyo college of medico-pharmaco technology, Tokyo, Japan
We have been studying the functionalization [1] of waste biomass
which is easily available globally and which can be regenerated
during a human life cycle. Animals or plants are well known to
take in and accumulate elements required for life support.
Importantly, their work lowers environmental load and is carried
out without the need to introduce expensive raw materials. We
succeeded in using fowl droppings to yield biomimetic calcium
phosphate compounds that are less crystalline have attracted
attention as a potential functional material [2]. In this study, we
report the purification technology of the air pollutants such as
NOx, SOx, and bacteria, using the synthesised compounds. The
calcium phosphate compound has attracted attention as one of the
air cleaning materials, e.g., exhaust filters and car ventilation [3],
adsorbents or separators of proteins [4], and catalysts [5]. We
have investigated the adsorption of NO2, SO2, and bacteria, onto
the synthesized calcium phosphate compound. High adsorptive
capabilities were observed for all synthesized calcium phosphate
compounds (Table 1). Furthermore, the effects of photocatalytic
decomposition and antibacterial also been suggested for the synthesized calcium phosphate compounds. Hence, the biomimetic
synthesized calcium phosphate compound from fowl droppings
which is a disposal resources existing around the world may
provide new options as a cheap and highly functional material in
clean air technology.
123
S864
J Biol Inorg Chem (2014) 19 (Suppl 2):S853–S866
Financial support by Chiba Institute of Technology is gratefully
acknowledged.
Table 1 Adsorption-concentration of NOx or SOx in air mixture Tetra
Pak (5L) on the various adsorbents (5 g each/at room temperature for
24 h)
Sample
NOx, SOx gas
adsorbed ppm
(vol. ppm)/
5 g (sample)
Pore characteristic
SSA/m2 g-1
(specific
surface area)
Synthesized compounds
from fowl droppings
NO2
SO2
A
95.5
41.0
17.10
B
98.0
41.0
64.43
HAD
95.0
41.0
10–30
b-TCP
95.0
41.0
Commerical
References
1. Minamisawa M, Minamisawa H, Yoshida S, Takai N (2005) Green
Chem 7:595–601
2. Minamisawa M, Yoshida S, Uzawa A Powder Technol 230 (2012)
20–28
3. Hiraide T (1993) Ceramics 28:642–646
4. Jang SH, Min BG, Jeong YG, Lyoo WS, Lee SC (2008) J Hazard
Mater 152:1285–1292
5. Legeros RZ, Lin S, Rohanizadeh R (2003) J Mat Sci Mat Med
14:201–209
P 250
Biomimetic catalysts based on metalloporphyrin MOFs
Arkaitz Fidalgo-Marijuan1, Gotzone Barandika2, Begoña Bazán1,
Miren Karmele Urtiaga1, Edurne S. Larrea1, Marta Iglesias3,
Marı́a Isabel Arriortua1
1
Department of Mineralogy and Petrology, University of the Basque
Country (UPV/EHU), Barrio Sarriena s/n, 48940 Leioa, Spain
2
Department of Chemistry, University of the Basque Country (UPV/
EHU), Paseo de la Universidad 7, 01006 Vitoria-Gasteiz, Spain
3
Institute of Materials Science of Madrid-CSIC, Sor Juana Inés de la
Cruz 3, Cantoblanco, 28049 Madrid, Spain
During the past years, a great effort has been devoted to the anchoring
of catalysts into MOFs in order to achieve heterogeneous catalysts
[1]. In this sense, an innovative approach consists on using metalloporphyrins as coordination-network synthons mimicking their natural
catalytic activity in order to reproduce it in the solid state [2].
The work herein presented explores the activity of l-O-[FeTCPP]2nDMF (TCPP = meso-tetracarboxyphenylporphyrin; n & 16)
and [CoTPPS0.5(bipy)(H2O)2]6H2O [3] (TPPS = meso-tetrasulfonatophenylporphyrin, bipy = 4,40 -bipyridine) compounds as
heterogeneous catalysts on oxidation and acetylation reactions of
different organic substrates [4].
This work has been financially supported by the Ministerio de
Ciencia e Innovación (MAT2010-15375), the Gobierno Vasco (Basque University System Research Groups, IT-630-13) and the UPV/
EHU (UFI 11/15), which we gratefully acknowledge. Technical and
human support provided by SGIker (UPV/EHU, MICINN, GV/EJ,
ESF) is gratefully acknowledged. A. Fidalgo-Marijuan thanks the
UPV/EHU for funding.
123
References
1. Whittington CL, Wojtas L, Larsen RW (2014) Inorg Chem
53:160–166
2. Zhang Z, Zhang L, Wojtas L, Eddaoudi M, Zaworotko MJ (2012) J
Am Chem Soc 134:928–933
3. Fidalgo-Marijuan A, Barandika G, Bazán B, Urtiaga MK, Arriortua
MI (2013) CrystEngComm 15:4181–4188
4. Fidalgo-Marijuan A (2014) PhD Thesis, Leioa, Spain
P 251
Adduct formation of aromatic biomolecules with Pt(II)aromatic diimine-polyaminopolycarboxylate complexes
Tatsuo Yajima1, Atsushi Ito1, Gabriella Munzi2, Carmelo
Sgarlata2, Yasuo Nakabayashi1, Tadashi Shiraiwa1, Giuseppe
Arena2, Osamu Yamauchi1,3
1
Faculty of Chemistry, Materials and Bioengineering, Kansai
University, Suita, Japan.
2
Department of Chemical Science, University of Catania, Catania,
Italy.
3
Department of Chemistry, School of Science, Nagoya University,
Nagoya, Japan. [email protected]
Non-covalent interactions such as electrostatic interactions, hydrogen bonds and aromatic ring stacking interactions play important
roles in biological systems for molecular recognition. Metal-coordinated aromatic diimine ligands such as 1,10-phenanthroline
(phen) interact to aromatic moieties with stacking interactions as
seen in [CuII(phen)(Trp)] (Trp = tryptophan), and interactions with
uncoordinated aromatic molecules lead to formation of adducts
[1–3].
We studied syntheses and characterizations of binuclear complexes of PtII(phen) moieties bound to EDTA and its analogs.
[PtCl2(phen)] reacted with EDTA, 1,3-diaminopropane-N,N,N’,N’tetraacetate (PDTA), and 1,4-diaminobutane-N,N,N’,N’-tetraacetate
(BDTA) to form [Pt2(phen)2(EDTA)] (1), [Pt2(phen)2(PDTA)] (2),
and [Pt2(phen)2(BDTA)] (3), respectively, where the Pt(phen) units
are bound to the both ends of EDTA, etc. with a 3N1O donor set, as
revealed by X-ray crystal structure analyses. Adduct formations of the
complexes with aromatic biomolecules such as indoleacetate and
5-hydroxytryptamine (serotonin) were studied by 1H NMR measurements and calorimetry methods.
Financial support by the Kansai University is gratefully
acknowledged.
J Biol Inorg Chem (2014) 19 (Suppl 2):S853–S866
S865
sphere metal complexes. In contrast, H4app (not protonated) gives the
inner sphere complex 5 where the Cu(II)-N3(H4app) bond cooperates
with the intra-molecular interligand N9-HO(carboxyl) interaction.
The efficient substitution of H4app in [Cu(H2EDTA)(H2O)] points out
the consequences of the N7/C8 translocation in adenine [2].
References
1. Domı́nguez-Martı́n A, Brandi-Blanco MP, Matilla-Hernández A,
El Bakkali H, Nurchi VM, González-Pérez JM, Castiñeiras A, NiclósGutiérrez J (2013) Coord Chem Rev 257:2814–2838
2. Domı́nguez-Martı́n A, Choquesillo-Lazarte D, Dobado JA, Martı́nez-Garcı́a MP, Lezama L, González-Pérez JM, Castiñeiras A,
Niclós-Gutiérrez J (2013) Inorg Chem 52:1916–1925
P 254
N2O reduction at a mixed-valent {Cu2S} core
References
1. Yamauchi O, Odani A, Hirota S (2001) Bull Chem Soc Jpn
74:1525–1545
2. Yamauchi O, Odani A, Takani M (2002) J Chem Soc Dalton Trans
3411–3421
3. Yajima T, Maccarrone G, Takani M, Contino A, Arena G, Takamido R, Hanaki, M, Funahashi Y, Odani A, Yamauchi O (2003)
Chem Eur J 9:3341–3352
P 252
The outer/inner sphere dichotomy in the reaction
of some adenine-like N-ligands
with [Cu(H2EDTA)(H2O)] chelate
Delara Mansoori1,3, Duane Choquesillo-Lazarte2, Alicia
Domı́nguez-Martı́n1,4, Valeria M. Nurchi3, Alfonso Castiñeiras5,
Guido Crisponi3, Josefa M. González-Pérez1, Juan NiclósGutiérrez1
1
Deparment of Inorganic Chemistry, Faculty of Pharmacy, University
of Granada, 18071 Granada, Spain.
2
Laboratorio de Estudios Cristalográficos, IACT, CSIC-UGR, Avda.
de las Palmeras 4, 18100 Armilla, Granada, Spain.
3
Dipartimento di Scienze Chimiche e Geologiche, Cittadela
Universitaria, 09042 Monserrato, (CA) Italy. [email protected].
4
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland.
5
Department of Inorganic Chemistry, Faculty of Pharmacy,
University of Santiago de Compostela, 15782 Santiago de
Compostela, Spain
Reactions of the ‘acidic’ chelate [Cu(H2EDTA)(H2O)] with adenine
(Hade) [1] or other purine-like bases [1] open possibilities to protontransfer and/or coordination reactions to give outer/inner sphere
complexes. The outer sphere complex (H2ade)[Cu(HEDTA)(H2O)]
2H2O build ‘ion-pairs’ formed by N1-HO and N6-HO interactions
with both O-atoms of the same HEDTA3-carboxyl group. Pairs of
‘ion-pairs’ p-stack their H2ade? ions. Here we report the structures of
(1),
(H27deaA)[Cu(HE(H27azain)[Cu(HEDTA)(H2O)]2H2O
DTA)(H2O)]3H2O (2), (6,7-H2ddA)[Cu(HEDTA)(H2O)]2H2O
(3), (H9Meade)[Cu(HEDTA)(H2O)]H2O (4) and [Cu(H2EDTA)(H4app)] (5), being 7-azaindole (H7azain), 7-deazaadenine (H7deaA),
6,7-dideazaadenine (6,7-HddA), 9-methyladenine (9Meade) and
7-deaza-8-azaadenine (H4app), respectively. Compounds 1-4 are outer
Charlène Esmieu1, Maylis Orio2, Laurent Le Pape1, Colette
Lebrun3, Jacques Pécaut3, Stéphane Torelli1, Stéphane Ménage1
1
Laboratoire de Chimie et Biologie des métaux. UMR 5249
Université Grenoble Alpes-Grenoble France, CNRS et CEA-CEA
DSV-iRTSV-17, Avenue des Martyrs, 38054 Grenoble.
[email protected].
2
Université des Sciences et Technologies de Lille-Laboratoire de
Spectrochimie Infrarouge et Raman-Bâtiment C5-UMR CNRS
8516-59655 Villeneuve d’Ascq Cedex.
3
Laboratoire de Reconnaissance Ionique et Chimie de CoordinationUMR E3 Université Joseph Fourier et CEA-SCIB/INAC–CEA
Grenoble–17, Avenue des Martyrs, 38054 Grenoble
Since 22 years, environmental impacts of Nitrous oxide (N2O) are
known (Kyoto protocol). N2O is indeed a potent greenhouse agent
and a dominant ozone depleting molecule [1]. It is released by biological processes like in bacterial respiration, nitrification and
denitrification pathways. However it is also produced by the industry
and its rising atmospheric concentration is mainly due to anthropogenic activities. In recognition of these facts, the need of remediation
processes has stimulated a lot of interest, particularly through the use
of transition metal catalysts. Moreover, in nature, one metalloenzyme
(N2O-reductase) cleanly converts N2O in N2 via a unique tetranuclear
l4-sulfido copper center named Cuz [2]. A mechanism for N2O
reduction has been proposed and involves an N2O binding between
two of the four copper ions [3]. Today, there is no active system that
degrades this gas under mild conditions.
Following a bioinspired approach related to the previous hypothesis, our team is seeking to reproduce CuZ reactivity. Herein, we
report the synthesis of novel binuclear copper complexes capable of
N2O reduction. These compounds, synthesized via an original
method, contain a thiophenolate ligand with N-coordinating atoms
[4]. These new complexes have been spectroscopically and theoretically characterized, and present a {Cu2S}2? mixed-valent motif
where the delocalization depends on the organic ligand. The reactivity
toward N2O depends of the presence of a labile site, which potentially
indicates the coordination of this weak substrate. In a biomimetic
approach, we are also able to synthesize a new tetranuclear copper (II)
complex with the same starting ligand which presents a new kind of
reactivity versus O2.
References
1. Ravishankara AR, et al. (2009) Science 326:123–125
2. Einsle O, et al. (2012) Biol Chem 393:1067–1077
3. Solomon EI, et al. (2007) J Am Chem Soc 129:3955–3965
123
S866
J Biol Inorg Chem (2014) 19 (Suppl 2):S853–S866
4. Torelli S, et al. (2010) Angew Chem Int Ed 49:8249–8252
2
P 255
Investigation of a novel biomimetic Co(II)-based cubane
water oxidation catalyst
Dinuclear metal centers are ubiquitous at the active sites of metallohydrolases. They uniformly comprise two first row transitional
metal ions that are positioned in close proximity. To better understand
the function-structure relationships of the first and the second coordination spheres [1], we combined supramolecular chemistry with
inorganic model complexes. The suprmolecular dinuclear complexes,
Zn2L [2], Cu2L [3] and Co2L [4] that have dual intramolecular
cyclodextrins (CDs) were synthesized and exploited as functional
mimics of phosphate esterases. Our work indicated, by combining
metal complexes with b-CDs, the properties and functions of metal
complexes were positively changed, reflected in enhancement of
catalytic activity, increase of substrate specificity and activation of
reactive species.
Financial support by the National Natural Science Foundation of
China (Nos. 21172274, 21231007, 21121061 and J1103305), National
High-Tech Research and Development Programme of China (863
Program No. 2012AA020305), the Ministry of Education of China
(Nos., 20100171110013 and 313058), the National Basic Research
Program of China (973 Program No. 2014CB845604) and the Fundamental Research Funds for the Central Universities.
Sandra Luber, Fabio Evangelisti, Robin Güttinger1, René Moré,
Greta R. Patzke
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland. [email protected]
Solar energy is an inexhaustible energy source for a long-term
solution to the global energy consumption. The storage of large
amounts of light energy can be achieved by conversion into
chemical energy saved in biomass. Artificial photosynthesis permits
the splitting of water into hydrogen and oxygen using solar light
and is thus a very promising and sustainable strategy to meet the
increasing worldwide need for clean energy. However, water oxidation remains the bottleneck of the overall photochemical water
splitting process.
Very recently, the first Co(II)-based cubane water oxidation catalyst (WOC) has been presented [1]. This WOC excels through
hitherto unprecedented structural analogies to the oxygen-evolving
complex of photosystem II. We will present results of our joined
computational and experimental approach for the investigation of its
structure and activity.
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland. [email protected]
Reference
1. Evangelisti F, Güttinger R, Moré R, Luber S, Patzke GR (2013) J
Am Chem Soc 135:18734–18737
P 256
Supramolecular Dinuclear Phosphate Esterase Mimics
Meng Zhao1,2, Liang-Nian Ji1, Zong-Wan Mao1
1
MOE Key Laboratory of Bioinorganic and Synthetic Chemistry,
School of Chemistry and Chemical Engineering, Sun Yat-sen
University, Guangzhou 510275, China. [email protected].
123
References
1. Zhao M, Wang HB, Ji LN, Mao ZW (2013) Chem Soc Rev
42:8360–8375
2. Zhao M, Zhang L, Chen HY, Wang HL, Ji LN, Mao ZW (2010)
Chem Commun 46:6497–6499
3. Zhao M, Wang HL, Zhang L, Zhao C, Ji LN, Mao ZW (2011)
Chem Commun 47:7344–7346
4. Zhao M, Xue SS, Jiang XQ, Ji LN, Mao ZW submitted
J Biol Inorg Chem (2014) 19 (Suppl 2):S867–S874
DOI 10.1007/s00775-014-1166-x
POSTER PRESENTATION
Bioorganometallic chemistry
P 260
Lighting-up red luminescence by a blue transition metal
complex of a chlorophyll catabolite
Chengjie Li1, Markus Ulrich1, Xiujun Liu1, Klaus
Wurst2, Bernhard Kräutler1
P 261
Biological evaluation of easy-to-prepare metallocene
derivatives
Jeannine Hess1, Malay Patra1, Anna Leonidova1,
Vanessa Pierroz1,2, Stefano Ferrari1,2, Gilles Gasser1
1
1
Institute of Organic Chemistry and Centre of Molecular Biosciences,
University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria.
2
Institute of General, Inorganic and Theoretical Chemistry, University
of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria
Chlorophyll breakdown is commonly associated with the appearance of
the fall colors [1]. When they were first identified, they were revealed to
be colorless linear, bilane-type tetrapyrroles that were classified as
‘‘nonfluorescent’’ chlorophyll catabolites (NCCs) [2]. Recently, colored chlorophyll catabolites were discovered in senescent leaves,
named yellow chlorophyll catabolites (YCCs) and pink-red catabolites
(PiCCs) [3]. A YCC could be prepared by chemical oxidation of an
NCC [4]. Here, we report the effective partial synthesis of PiCC from
YCC, which occurs via a blue zinc complex of PiCC: the first example
of chlorophyll catabolite binding metal ion. The crystal structure of
PiCC potassium salt was obtained and will be reported also. PiCC was
found to be a high affinity ligand for zinc-ions. Binding of Zn-ion to the
PiCC leads to the intensive red fluorescence of Zn-PiCC.
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland. [email protected].
2
Institute of Molecular Cancer Research, University of Zurich,
Winterthurerstrasse 190, 8057 Zurich, Switzerland
Simple and efficient synthetic modifications of metallocenes are
crucial tools for the construction, for example, of new pharmacologically relevant organometallic compounds. Small pharmacophores
can indeed play a pivotal role for drug uptake, intracellular accumulation and can therefore determine the bioactivity of a drug
molecule. We have recently reported three simple but useful synthetic
procedures to prepare metallocene derivatives: (1) a single-step threecomponent condensation reaction for the efficient syntheses of various metallocenyl thioamides [1]; (2) a novel synthetic pathway for
trifluoromethylthio-substituted metallocenes, which does not involve
the use of toxic mercury(II)-based reagents [2]; (3) an improved
synthesis of aminometallocenes that adheres with the basic green
chemistry guidelines [3,4].
S
N
H
M
SCF3
X
R`
(1)
(2)
M
M
X = CH 2NR2, Br, I
M = Fe, Ru
(3)
NH2
Financial support by the Austrian National Science Foundation
(FWF, projects P-19596 and I-563) is gratefully acknowledged.
References
1. Matile P, Hörtensteiner S, Thomas H, Kräutler B (1996) Plant
Physiol 112:1403–1409
2. Kräutler B, Hörtensteiner S (2013) In: Ferreira GC, Kadish KM,
Smith KM, Guilard R (eds) Handbook of porphyrin science, vol 28.
World Scientific Publishing, USA, pp 117–185
3. Ulrich M, Moser S, Müller T, Kräutler B (2011) Chem Eur J
17:2330–2334
4. Moser S, Ulrich M, Müller T, Kräutler B (2008) Photochem
Photobiol Sci 7:1577–1581
M
References
1. Patra M, Hess J, Konatschnig S, Spingler B, Gasser G (2013)
Organometallics 32:6098–6105
2. Hess J, Konatschnig S, Morard S, Pierroz V, Ferrari S, Spingler B,
Gasser G (2014) Inorg Chem 53:3662–3667
3. Leonidova A, Pierroz V, Rubbiani R, Heier J, Ferrari S, Gasser G
(2014) Dalton Trans 43:4287–4294
4. Gasser G, Leonidova A, Joshi T, Nipkow D, Frei A, Penner J-E,
Konatschnig S, Patra M (2013) Eur Pat Appl EP13157319
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P 264
Vectorized ferrocenes and titanocenes with biologically
active steroids
Enrique Meléndez, José Vera, Xiomara Narváez, José
Carmona, Li Ming Gao
Department of Chemistry, University of Puerto Rico, Department
of Chemistry Box 9019 Mayagüez, 00681, Puerto Rico
A series of functionalized ferrocenes and titanocenes with estrogens
and androgens vectors were synthesized and characterized by spectroscopic methods. Functionalized metallocenes with hormones as
pendant groups (substitution on the Cp rings) is expected to enhance
the selectivity to target more effectively hormone dependent cancers.
We have selected these steroids as molecular carriers (vectors) to
specifically deliver and accumulate the anticancer drug into the target
organs (breast, ovarian and prostate). The antiproliferative activity of
these metallocenes was initially studied on hormone dependent breast
cancer cell line MCF-7 and colon cancer cell line HT-29. Some of
these vectorized metallocenes showed enhanced antiproliferative
activity on MCF-7 cell line. Docking studies on the estrogen receptor
alpha suggest that ferrocenoyl 17b-hydroxyestra-1,3,5(10)-trien-3olate (estradiol ferrocenoylate) docks into the estrogen binding pocket
and this explain the high activity this complex has on MCF-7. On the
other hand, 16-ferrocenylidene-3b-hydroxy androstan-17-one showed
high antiproliferative activity on MCF-7 but it seems to follow a
different mechanism of action compared to the estradiol derivative.
Financial supports by the University of Puerto Rico and the
National Institute of Health are gratefully acknowledged.
References
1. Gao LM, Vera JL, Matta J, Meléndez E (2010) J Biol Inorg Chem
15:851–859
2. Vera J, Gao LM, Santana A, Matta J, Meléndez E (2011) Dalton
Trans 40:9957
P 265
Evaluation of novel ruthenium(II)- and rhodium(III)organosilane thiosemicarbazone complexes
as antiparasitic agents
Muneebah Adams1, Kelly Chibale1,2, Lubbe Wiesner3
and Gregory S. Smith1
1
Department of Chemistry, University of Cape Town (UCT), Cape
town, South Africa. [email protected].
2
Institute of Infectious Disease and Molecular Medicine, UCT, cape
town, South Africa.
3
Division of Clinical Pharmacology, UCT, Medical School, South
Africa
Parasitic diseases are rife in developing regions where the climate
provides suitable conditions for replication and transmission. Malaria
(an infectious disease) and trichomoniasis (a common STD) have
become problematic due to the emergence of treatment-resistant
strains.[1,2] Metals are recognised for their medicinal properties and
have been introduced into organic systems with the idea that they may
either aid with transportation into, or accumulation of the compound
within, the active site. Metal ions allow for the addition of ancillary
ligands which may lend properties such as lipophilicity or hydrophilicity to the complex as a whole. Therefore, the promising effects
displayed by metal complexes in the treatment of parasitic diseases
[e.g. Ferroquine (malaria)] motivated research into designing other
metal-containing systems as potential treatments. Thiosemicarbazones (TSC’s), known for a range of pharmacological properties (e.g.
antiparasitic), are metal chelating ligands that have been studied in
our group [3]. Organosilane compounds are also of interest as they
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J Biol Inorg Chem (2014) 19 (Suppl 2):S867–S874
generally exhibit enhanced pharmacological activity when compared
with their non-silicon counterparts [4]. Therefore, in this study TSC’s
were combined with an organosilane moiety to explore compounds
with increased potency and decreased susceptibility to resistant
strains. In this presentation, the synthesis of organosilane TSC’s and
their complexes will be discussed, as well as their evaluation against
plasmodium falciparum and trichomonas vaginalis.
References
1. Burrows JN, Chibale K, Wells TNC (2011) Curr Top Med Chem
11:1226–1254
2. Schwebke JR, Burgess D (2004) Clin Microbiol Rev 17:794–803
3. Chellan P, Stringer T, Shokar A, Dornbush PJ, Vazquez-Anaya G,
Land KM, Chibale K, Smith GS (2011) J Inorg Biochem 105:1562–1568
4. Franz AK, Wilson SO (2013) J Med Chem 56:388–405
P 266
Transition metal complexes of a natural chlorophyll
catabolite
Xiujun Liu, Chengjie Li, Markus Ulrich, Bernhard
Kräutler
Institute of Organic Chemistry and Centre of Molecular Biosciences,
University of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria
Chlorophyll breakdown is easily seen in fall leaves and in ripening
fruit by their characteristic colour changes [1]. In senescent leaves,
colourless, ‘nonfluorescent’ bilane-type catabolites typically accumulate, classified as NCCs and DNCCs [2]. NCCs were considered to
represent the ‘final’ tetrapyrrolic products of chlorophyll breakdown
[3]. These ‘late’ forms of chlorophyll catabolites accumulate in the
vacuoles [3]. Recently, some colored chlorophyll catabolites were
also discovered, named yellow chlorophyll catabolites (YCCs) and
pink chlorophyll catabolites (PiCCs) [4]. Accumulation of heavy
metals, such as Zn, Cd, Ni, Cu, Hg, has been detected in plants [5, 6].
Here, we report several transition metal complexes of PiCC. Binding
of these metal ions to the PiCC is rapid. Formation of complexes
M-PiCC by coordination of transition metal-ions to PiCC could be a
biologically significant capacity of chlorophyll catabolites. This may
give such chlorophyll catabolites unsuspected physiological functions
in plants, e.g. as toxins against pathogens or in ‘heavy metal transport
and detoxification’.
Financial support by the Austrian National Science Foundation (FWF,
projects P-19596 and I-563) is gratefully acknowledged.
J Biol Inorg Chem (2014) 19 (Suppl 2):S867–S874
References
1. Kräutler B (2008) Photochem Photobiol Sci 7:1114–1120
2. Kräutler B, Hörtensteiner S (2013) In: Ferreira GC, Kadish KM,
Smith KM, Guilard R (eds) Handbook of porphyrin science, vol 28.
World Scientific Publishing, USA, pp 117–185
3. Kräutler B, Matile P (1999) Acc Chem Res 32:35–43
4. Ulrich M, Moser S, Müller T, Kräutler B (2011) Chem Eur J
17:2330–2334
5. Rascio N, Navari-Izzo F (2011) Plant Sci 180:169–181
6. Krämer U (2010) Annu Rev Plant Biol 61:517–534
P 267
Development of a new class of fluorescence sensors
for biologically relevant thiophilic and toxic heavy
metals based on the combination of dithiolene ligands
with electron deficient fluorophores
Ashta Ch. Ghosh, Carola Schulzke
Institut für Biochemie, Ernst-Moritz-Arndt-Universität Greifswald,
Greifswald, Germany. [email protected]
Metal ions play critical roles in many physiological and pathological
processes in living organisms. Heavy metals such as iron, copper and
zinc are among the most abundant transition metals in cellular systems
holding an outstanding biological importance because of their presence
in the structures of numerous enzymes and proteins but excessive levels
can be damaging. Other heavy metals such as mercury and arsenic,
which have no known vital or beneficial effect on organisms and their
accumulation over time in the bodies of animals and human beings can
cause serious illness. To investigate the distribution of essential metals
in physiological or excessive concentrations or that of toxic metals in
cellular systems, research has been focused on the development of
fluorescence sensors [1] targeting the endosomal/lysosomal system.
The aim of this study is developing a new class of fluorescence sensors
based on the combination of dithiolene ligands, which are frequently
used in oxidoreductase model complexes [2], with electron deficient
fluorophores. The dithiolene ligands are particularly good in establishing a connection between a coordinated metal and a fluorophore
since the bis-thiol group has a high affinity for heavy metals and in
addition has a non-innocence character (i.e. the ability to directly participate in redox reactions and to buffer high oxidation states of the
coordinated metal). Based on this receptor design approach, we have
developed a fluorophore-spacer-receptor system by using namely three
types of spacer between dithiolene and fluorophore: peptide bond, diimide bond and triazole ring that show fluorescence enhancement/
quenching in the presence of metal ions since the thiol group has a high
affinity for heavy metals and due to its non-innocence character The
resulting ligand systems have been tested with respect to their coordination behavior towards different metals (Cu, Fe, Mo, W, Hg, As, Cd),
which are known to be easily coordinated by dithiolenes to evaluate their
possible use as markers/sensors for these biological or toxic metals.
References
1. Formica M, Fusi V, Giorgi L, Micheloni M (2012) Coord Chem
Rev 256:170–192
2. Schulzke C (2011) Eur J Inorg Chem 2011:1189–1199
S869
P 268
Structure and biological activity of organoruthenium
complexes with b-diketonates
Sara Seršen1, Jakob Kljun1, Walter Berger2, Iztok
Turel1
1
Faculty of Chemistry and Chemical Technology, University
of Ljubljana, Aškerčeva 5, 1000 Ljubljana, Slovenia.
2
Department of Medicine I, Institute of Cancer Research, Medical
University Vienna, Vienna, Austria
In recent years, ruthenium-based molecules have emerged as promising antitumor and antimetastatic agents with potential uses in
platinum-resistant tumours [1, 2]. Properties that make these complexes theoretically suitable for medicinal use are their slow ligand
exchange kinetics, access to multiple oxidation states, and the ability
to mimic iron in binding to certain biological molecules and hence
accessible transport inside the cell.
A series of half-sandwich ruthenium(II) complexes containing
j2(O,O)-4,4,4-trifluoro-(aryl)-1,3-butanedionate ligands have been
synthesized and completely characterized. Some of these coordination
compounds have already shown good catalytic properties for ortho
arylation via C–H activation of 2-phenylpyridine molecule [3]. The
whole series of compounds was tested for activity towards several
cancer cell lines and uncovered particular sensitivity of an ovarian
carcinoma and an osteosarcoma cell model. Some interesting structure–activity relations were observed leading to preferential cytostatic
or apoptosis-inducing activities. These data might help to create more
suitable ruthenium anticancer complex for the treatment of this deadly
disease.
Financial support by junior researcher grant for S.S. and the project J1-4131 of the Slovenian Research Agency (ARRS). We thank
the EN?FIST Centre of Excellence, Dunajska 156, 1000 Ljubljana,
Slovenia, for use of the SuperNova diffractometer. The study was
supported by COST action CM1105.
References
1. Barry NP, Sadler PJ (2013) Chem Commun 49:5106–5131
2. Antonarakis ES, Emadi A (2010) Cancer Chemother Pharm 66: 1–9
3. Seršen S, Kljun J, Požgan F, Štefane B, Turel I (2013) Organometallics 32:609–616
123
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P 269
DNA-based catalytic cyclopropanation in water
Ana Rioz-Martı́nez, Jens Oelerich, Gerard Roelfes
Department of Chemistry, Stratingh Institute for chemistry,
University of Groningen, Nijenborgh 4, 9747 AG Groningen, The
Netherlands. [email protected]
DNA-based asymmetric catalysis represents a powerful tool for the
preparation of chiral compounds in water [1]. This novel concept is
based on the use of hybrid catalysts, which comprise a transition
metal complex (a metal ion coordinated to a non-chiral ligand which
is able to bind to DNA) bound to DNA. In this way, the reaction
occurs in close proximity to the DNA, allowing chirality transfer and
subsequent formation of one of the enantiomers of the reaction
product [2]. These hybrid catalysts have been exploited in many
Lewis acid catalytic enantioselective reactions, such as Diels–Alder,
(oxa)-Michael addition, Friedel–Crafts, syn-hydration of enones and
fluorinations [1]. Recently, the catalytic scope of DNA-based asymmetric catalysis has been expanded beyond Lewis acid catalysis when
it was applied successfully in a Cu(I) catalysed intramolecular cyclopropanation of a-diazo-b-keto sulfones [3]. Up to now, all the
examples of DNA-based catalysis described use copper complexes in
combination with DNA. However, alternative metal catalysts can be
used as well. Cationic porphyrins (e.g., meso-tetrakis-(N-methyl-4pyridyl)-porphyrin (TMpyP4)), are well known ligands that can bind
through p-stacking and electrostatic interactions to DNA. Especially
the interactions of these porphyrins with G-quadruplexes have been
described [4]. Expanding the scope of DNA-based asymmetric
catalysis to other reactions, substrates and novel hybrid catalysts are
crucial challenges in our research. Here, we propose the DNA-based
catalytic cyclopropanation in water catalysed by iron porphyrin/salmon testes DNA hybrids.
Financial support by the Ramón Areces Foundation is gratefully
acknowledged.
References
1. Boersma AJ, Megens RP, Feringa BL, Roelfes G (2010) Chem Soc
Rev 39:2083–2092
2. Roelfes G, Feringa BL (2005) Angew Chem Int Ed 44:3230–3232
3. Oelerich J, Roelfes G (2013) Chem Sci 4:2013–2017
4. Pradines V, Pratviel G (2013) Angew Chem Int Ed 52:2185–2188
P 270
Halogenated gold(I) NHC complexes and their
biological evaluation
Claudia Schmidt, Ingo Ott
Institute of Medicinal and Pharmaceutical Chemistry, Technische
Universität Braunschweig, Beethovenstr. 55, 38106 Braunschweig,
Germany
N-heterocyclic carbene metal complexes have already shown antiproliferative effects on tumor cell lines and have proven their
potential as anticancer drugs. Thioredoxin reductase (TxrR) is an
example for a relevant enzyme, which protects the cells against
oxidative stress and apoptosis. It is upregulated in carcinoma cells and
represents a possible target in anticancer therapy. Especially
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J Biol Inorg Chem (2014) 19 (Suppl 2):S867–S874
gold(I) containing compounds inhibit TxrR in vitro, due to the high
affinity of gold to selenocysteine moieties. A well-known drug is
auranofin, which is established in the current antirheumatic therapy,
shows antiproliferative effects and a remarkable TxrR inhibition
[1–5].
New halogenated mono- and bis-NHC-gold(I)-complexes of the
imidazole-, benzimidazole- or phenylimidazole-type were synthesized, purified and characterized. Afterwards they were tested
against tumorigenic HT-29 colon carcinoma cells, MCF-7 breast
carcinoma cells, MDA-MB-231 breast carcinoma cells and nontumorigenic RC-124 human kidney cells. They all showed cell
growth inhibition with IC50 values in the submicromolar range.
Especially the activities of the bis-NHC-complexes were comparable
to auranofin.
The potency of TxrR inhibition of these gold(I) derivatives were
determined and cell uptake studies were performed using high resolution continuum source atomic absorption spectroscopy for the
quantification of the intracellular amount.
References
1. Hickey JL (2008) J Am Chem Soc 130:12570–12571
2. Ott I (2009) Coord Chem Rev 253:1670–1681
3. Rubbiani R, Ott I (2011) J Med Chem 54:8646–8657
4. Liu W, Gust R (2013) Chem Soc Rev 42:755–773
5. Hackenberg F, Tacke M (2013) Organometallics 32:5551–5560
P 271
Synthesis, characterization and cytotoxicity of (g6p cymene) ruthenium(II) complexes of a-amino acids
Folake A. Egbewande1, Lydia E. H. Paul 1, Bruno
Therrien2, Julien Furrer1
1
Department of Chemistry and Biochemistry, University of Berne,
Freiestrasse 3, CH-3012 Berne, Switzerland.
[email protected].
2
Institut de Chimie, Université de Neuchâtel, Avenue de Bellevaux
51, CH-2000 Neuchâtel, Switzerland
Arene ruthenium complexes of a-amino acids, obtained by mixing
aqueous solutions of [(g6-p-cymene)RuCl2]2 in the presence of
AgCF3SO3with various amino acids, have been studied at 37 °C using
NMR spectroscopy and electrospray ionization mass spectrometry
(ESI–MS). Presumably, complexes with the general formula [(g6-pcymene)Ru(AA)2]n+ and bridged complexes with the general formula
[((g6-p-cymene)Ru)2(l-AA)2(l-OH)]+ are formed together with the
expected bi- and tridentate chelate complexes. All complexes are
highly cytotoxic, with IC50 values ranging from 0.16 to 19.8 lM.
Interestingly, all complexes exhibit selectivity towards A2780 versus
A2780cisR cells, indicating a distinct mechanism of action, different
from that of many previously reported cytotoxic ruthenium complexes. No direct correlation between the kinetics of formation and
the cytotoxicity could be evidenced, suggesting that other physicochemical parameters such as the stability and ligand exchange
kinetics may play an important role in their biological activity [1].
J Biol Inorg Chem (2014) 19 (Suppl 2):S867–S874
Reference
1. Egbewande FA, Paul LEH, Therrien B, Furrer J (2014) Eur J Inorg
Chem 2014:1174–1184
S871
platinum(IV) prodrug complexes [1, 2]. However, the existing strategy centred on platinum(IV) complexes with symmetrical axial
ligands does not fully exploit the vast potential of this class of prodrugs. We therefore adapted this strategy to access asymmetric Pt(IV)
complexes with contrasting axial ligands through sequential acylation. By modifying the characteristics of each of the axial ligand, it is
now feasible to control the physical and chemical properties of
resultant platinum(IV) prodrug complex [3]. To that end, we report a
library of finely-tuned asymmetric platinum(IV) complexes and their
efficacy against a panel of human cancer cell lines in vitro.
Financial support by the National University of Singapore and
Solvay Singapore is gratefully acknowledged.
P 272
New rhenium complexes for carbon monoxide
photorelease
Elham Kianfar, Günther Knör
Institute of Inorganic Chemistry, Johannes Kepler University Linz,
Altenbergerstrasse 69, 4040 Linz, Austria
Carbon monoxide has come a long way from just being considered as a
silent killer and an environmental pollutant to an important molecular
messenger involved in several physiological processes. Recently,
important progress has been made towards developing photoactive COreleasing moieties (PhotoCORMs) for possible therapeutic applications
[1]. Here, the synthesis and characterization of new rhenium tricarbonyl
diimine complexes of the type fac-Re(1,2-diimine)(CO)3Cl, carrying
bis-(phenylimino)acenaphtene ligands (BIAN) 1–3 is reported [2].
Among these novel complexes, a special focus will be given to the
deeply coloured water-soluble organometallic rhenium(I) derivative 3,
which offers highly desirable features for potential biomedical and catalytic applications such as light-controlled release of carbon monoxide
(PhotoCORMs) [3], and homogeneous photocatalytic CO2 reduction.
CO liberated from the irradiated solutions of CORMs 1–3 has been
quantified in the headspace gas using FTIR and GC analysis.
Financial support of this work by the Austrian Science Fund
(FWF- Project 25038: Functional Light Responsive Metal Carbonyl
Systems) is gratefully acknowledged.
References
1. Wilson JJ, Lippard SJ (2011) Inorg Chem 50:3103–3115
2. Ang WH, Pilet S, Scopelliti R, Bussy F, Juillerat-Jeanneret L,
Dyson PJ (2005) J Med Chem 48:8060–8069
3. Chin CF, Tian Q, Setyawati MI, Fang W, Tan ESQ, Leong DT,
Ang WH (2012) J Med Chem 55:7571–7582
P 274
Copper, zinc, and lead solubilization from foundry sand
and uptake by Penicillium expansum
Carlotta Fabbri, Helmut Brandl
References
1. Rimmer RD, Pierri AE, Ford PC (2012) Coord Chem Rev
256:1509–1519
2. Kianfar E, Monkowius U, Portenkirchner E, Knör G (2014) Z
Naturforsch (in press)
3. Kianfar E, Monkowius U, Knör G (2013) Bioinorg React Mechanism 9: S1–S105
P 273
Expanding the scope of finely tuned asymmetric
platinum(IV) complexes
Chee Fei Chin, Thng Agnes, Siew Qi Yap, Wee Han
Ang
Department of Chemistry, National University of Singapore, 3
Science Drive 3, Singapore 11754, Singapore. [email protected]
Structure activity relationship studies have previously established a
link between the nature of axial ligands and the efficacy of anticancer
Institute of Evolutionary Biology and Environmental Studies,
University of Zurich, Winter-thurerstrasse 190, 8057 Zurich,
Switzerland
In nature, microorganisms can act geological agents and, therefore,
mediate biogeochemical processes such as e.g., metal solubilization
from solid matrices (minerals, rocks, ores), metal speciation as well
as immobilization. Generally, microbes are able to mobilize metals
from solids by the formation of acids (protons), by oxidation and
reduction reactions; and by the excretion of complexing agents or
ligands [1]. In the present work, a fungal strain (Penicillium expansum) has been investigated for its ability of Cu, Zn, and Pb
solubilization from a solid matrix (foundry sand) through the formation of organic acids and its subsequent accumulation in the
fungal mycelium. The fungus was grown at 25 °C for up to
2 months in Petri dishes filled with potato-dextrose agar medium
enriched. Metal-containing foundry sand containing mainly (in
g/kg) Al (31.4), Cu (5.6), Pb (1), Sn (0.3), Zn (5.3) was added at
final concentration of 30 g/L during the preparation of the Petri
dishes. After cultivation and mycelial growth, the biomass was
digested using nitric acid and hydrogen peroxide followed by total
metal content analysis by atomic absorption spectroscopy. P. expansum was able to solubilize and accumulate Cu from foundry
123
S872
sand at a recovery rate of 65 % assuming a efficient metal accessibility. In contrast, Zn—besides Cu also an essential metal for
fungal metabolism—showed a much lower accumulation compared
to Cu (maximum recovery B20 %. Pb—as non-essential metal in
the metabolism of fungi—was accumulated from foundry sand at
high percentage (60 %) by P. expansum. Our results show the
potential of P. expansum in effectively mobilizing Cu and Pb,
which might be of future importance considering the recovery of
valuable metals from solid wastes for a recycling and re-use as
secondary resource.
Financial support by the University of Zurich through the ‘‘Forschungskredit’’ gratefully acknowledged.
Reference
1. Brandl H, Faramarzi MA (2006) China Particuol 4:93–97
P 275
Reactivity of cyclometalated ruthenium and osmium
complexes towards oxidoreductases
Omar Saavedra-Dı́az, Ricardo Cerón-Camacho, Ronan
Le Lagadec
Instituto de Quı́mica, Universidad Nacional Autónoma de México,
Circuito Exterior, Ciudad Universitaria, 04510 México D.F., Mexico.
Mexico. [email protected]
Our group has been studying the interactions between group 8 metals
and oxidoreductases, with a special emphasis on bio-sensing applications [1, 2]. In particular, we have prepared series of structurally
similar complexes of the general formula [M(C * N)x(N * N)3-x]m+
(M = Ru, Os; C * N = o-2-phenylpyridinato; N * N = 2,20 bipyridine; x = 0–3), covering almost a 2 V potential range. The
effects of the successive replacement of nitrogen atoms by r-bound
sp2 carbon atoms on the reactivity towards redox-active enzymes have
been explored. For instance, we found that, depending on their oxidation state and reduction potentials, the complexes can act as
competitive and non-competitive inhibitors or activator of glucose
oxidase from Aspergillus niger [3]. On the other hand, the secondorder rate constants for the one-electron transfer between the organometallic species and compounds I and II of horseradish peroxidase
have been evaluated, and have also been found to greatly depend on
the reduction potentials of the complexes. Finally, theoretical docking
simulations have been performed in order to get a better understanding of the systems [4].
Financial support by DGAPA (PAPIIT Project IN204812) and
CONACyT (Project 153151) is gratefully acknowledged.
References
1. Cerón-Camacho R, Hernández S, Le Lagadec R, Ryabov AD
(2011) Chem Commun 47:2823–2825
2. Ryabov AD, Cerón-Camacho R, Saavedra-Diaz O, Denardo M,
Ghosh A, Le Lagadec R, Collins T (2012) Anal Chem 84:9096–9100
3. Saavedra-Dı́az RO, Le Lagadec R, Ryabov AD (2013) J Biol Inorg
Chem 18:547–555
4. Cerón-Camacho R, Le Lagadec R, Kurnikov IV, Ryabov AD
(2014) J Inorg Biochem 134:20–24
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P 276
Nitric oxide reaction with oxy-heme models containing
trans 1-methyl imidazole ligand
Astghik A. Hovhannisyan, Tigran S. Kurtikyan
Molecule Structure Research Center of the Scientific
and Technological Center of Organic and Pharmaceutical Chemistry
NAS, 0014 Yerevan, Armenia. [email protected]
Five-coordinate high-spin iron(II) porphyrinates have been of great
interest as models of deoxyhemoglobin and deoxymyoglobin. In
addition, they are also found in many other heme proteins, such as
reduced cytochrome P450, chloroperoxidase, and horseradish peroxidase. Interaction of dioxygen with them leads to the formation of
6-coordinate oxygen complexes which represent the models of oxyhemoglobin and oxymyoglobin. The NO dioxygenation reaction
promoted by oxyhemoglobin and oxymyoglobin is considered as a
major pathway leading to the scavenging of the bioregulatory molecule nitric oxide in mammalian systems [1].
In this presentation we show that co-condensation of Fe-porphyrins and 1-MeIm onto a low temperature substrate makes it possible to
construct a system mimicking the active center of deoxyhemoglobin
and deoxymyoglobin, which has a proximal 1-methyl imidazole
ligand Fe(Por)(1-MeIm) (Por-meso-tetraphenyl- and meso-tetra-ptolyl-porphyrinatodianions). The interaction of this system with O2 at
low temperatures leads to the formation of 6-coordinate Fe(Por)(1MeIm)(O2) complexes (Scheme).
We then monitored the interactions of these dioxygen complexes
with NO while warming up the layered solids from LN2 temperature
to RT. FTIR spectral measurements, supported by using 18O2 and
15
NO isotopomers,reveal the formation of six-coordinate nitratocomplexes with trans 1-Meim ligand already at very low temperatures
(80-120 K) (Scheme).
It will also be demonstrated that the further transformations of this
system upon warming depends on the experimental conditions.
The financial support of NFSAT (Project YSSP 13-48) is gratefully acknowledged.
O
N
O
O
R
R
N
N
R
Fe
N
N
N
R
O2
LT
R
N
N
R
N
R - phenyl, p-tolyl
O
R
Fe
N
N
NO
R 80-110K R
N
R
N Fe
R
N
CH3
N
O
N
N
R
N
N
CH3
CH3
Reference
1. Su J, Groves JT (2010) Inorg Chem 49:6317–6329
P 277
Naphthoquinone derivatives as a bioactive ligand
scaffold for the development of organometallic
metallodrugs
Carmen M. Hackl1, Michael Jakupec1,2, Wolfgang
Kandioller1,2, Christian G. Hartinger3, Bernhard K.
Keppler1,2
1
University of Vienna, Institute of Inorganic Chemistry, Waehringer
Str. 42, 1090 Vienna, Austria.
2
Research Platform ‘‘Translational Cancer Therapy Research’’,
University of Vienna, Waehringer Str. 42, 1090 Vienna, Austria.
3
University of Auckland, School of Chemical Sciences, 23 Symonds
Str., Auckland 1010, New Zealand
Naturally occurring naphthoquinones such as vitamin K and its
derivatives constitute an important group of bioactive compounds [1].
J Biol Inorg Chem (2014) 19 (Suppl 2):S867–S874
[1,4]-Naphthoquinones are known to be enzyme inhibitors and show
antibacterial, anticancer, anti-proliferative and anti-inflammatory
activity. The mode of action imparted by [1,4]-naphthoquinones
mostly relies on their ability to accept one or two electrons to form
radical anion species. The presence of electron-donating or -accepting
substituents modulates the redox properties, which enables the compound to generate radicals such as hydrogen peroxide or superoxide
that can cause damage of cancer cells (reactive oxygen species, ROS)
[2]. The coordination of [1,4]-naphthoquinone derivatives to organometallic ruthenium and osmium moieties leads to complexes of the
well-known ‘‘piano-stool’’ configuration. Important characteristics
such as anticancer activities, pharmacological properties, cellular
uptake and biomolecule interactions can be fine-tuned due to the
structural diversity and especially the substitution options in position
3. The impact of the modification at position 3 on the redox behavior
of the corresponding complexes was investigated by cyclic voltammetry measurements and preliminary biological results will be
discussed.
References
1. Tandon VK, Singh, RV, Yadav DB (2004) Bioorg Med Chem Lett
14:2901–2904
2. Klaus V, Hartmann T, Gambini J, Graf P, Stahl W, Hartwig A,
Klotz L (2010) Arch Biochem Biophys 496:93–100
P 278
New vanadium complexes as optical probes to detect
Cys sulfenic modifications in PTEN
Agostino Cilibrizzi, Juliet Collins, Rudiger Woscholski,
Robin Leatherbarrow, Ramon Vilar
Institute of Chemical Biology, Department of Chemistry, Imperial
College London, Exhibition Road, London SW7 2AZ, UK.
[email protected]
Over the past few decades, a large number of proteins and enzymes
have been identified wherein chemoselective oxidation of cysteine
residues by reactive oxygen species (ROS) acts as a mechanism to
alter or regulate normal cellular functions [1]. Generally, sulfenic acid
(SOH) formation is the first event in the oxidative process and it has
been widely reported as an important post-translational modification
(PTM) found in several oxidative stress-related diseases, such as
cancer and neurodegenerative disorders [2]. Phosphatase and tensin
homologue deleted on chromosome 10 (PTEN) is a member of the
protein tyrosine phosphatase family known as an important tumor
suppressor whose function includes significant roles in oxidative
damage-associated pathologies [3]. According to recent studies, the
switch-off nature of SOH modification functions as a reversible
means to regulate PTEN function as well as a marker of initial protein
S873
damage. In this scenario, chemical probes for SOH detection could
represent promising new tools for elucidating PTEN signaling pathways and regulatory mechanisms that involve thiol oxidation and
redox regulation. This evidence led us to design, synthesize, and
biologically evaluate a new series of vanadium complexes as optical
probes towards modified PTEN (i.e. its sulfenic acid form), with the
ultimate aim to detect and optically visualize the SOH oxidative
modification on this enzyme by fluorescence methods. In previous
studies we identified VO-OHpic, a vanadyl complex which displays
remarkable affinity for PTEN inhibiting its activity in a dose dependent manner [4]. Thus, we have worked towards modifying VOOHpic by the introduction of coumarin- or naphthalenesulfonic-based
fluorophores and dimedone (5,5-dimethyl-1,3-cyclohexane-dione)
moiety, a cyclic ß-diketone which selectively reacts with sulfenic
acids [1], in order to reach both optical visualization and SOH
detection of oxidativeliy-damaged PTEN. Combining the three targeting modules (VO-OHpic + dimedone + fluorophore), a library of
novel ‘bridged-bipicolinic vanadyl complexes’ has been synthesized
and its biological evaluation is ongoing to confirm the success of our
targeting strategy for the development of new complexes as versatile
tools to be easily modified for structure–activity relationship (SAR)
analysis towards PTEN sulfenic oxidative damage investigation.
Financial support by the EPSRC (UK) through the Proxomics Project
(http://www.proxomics.ac.uk/) is gratefully acknowledged.
References
1. Paulsen CE, Carroll KS (2013) Chem Rev 113:4633–4679
2. Paulsen CE, Carroll KS (2010) ACS Chem Biol 5:47–62
3. Salmena L, Carracedo A, Pandolfi PP (2008) Cell 133:403–414
4. Rosivatz E, Matthews JG, McDonald NQ, Mulet X, Ho KK, Lossi
N, Schmid AC, Mirabelli M, Pomeranz KM, Erneux C, et al. (2006)
ACS Chem Biol 1:780–790
P 279
Design of catalytic anticancer drugs: NADH
regeneration by Rh(III) half-sandwich complexes
Abraha Habtemariam, Joan Soldevila-Barreda
and Peter J. Sadler
Department of Chemistry, University of Warwick, Coventry, UK
CV4 7AL. [email protected]
The coenzyme pair NAD+/NADH is involved in many biological
processes, such as regulation of energy metabolism and redox balance, DNA repair and transcription, and immunological functions.
Disruption of the NAD+/NADH ratio can lead to cell death [1], and
offers new possibilities for the design of catalytic drugs. Ru(II) arene
complexes [(g6-arene)Ru(L)Cl]PF6 (arene = hexamethylbenzene,
p-cymene, bip, bn; L = ethylenediamine (en), N-(2-aminoethyl)-4(trifluoromethyl)benzenesulfonamide (TfEn)) catalyze the reduction
of NAD+ to 1,4-NADH via transfer hydrogenation with formate as
hydride source under physiological conditions.[2,3].
We describe here a series of [(CpX)Rh(NŃ)Cl]n+ complexes where
X
Cp = pentamethylcyclopentadiene (Cp*), 1-phenyl-2,3,4,5-tetraor
1-biphenyl-2,3,4,5methylcyclopentadiene
(CpXPh)
tetramethylcyclopentadiene (CpXPhPh), and NN0 = ethylendiamine
(en) or N-(2-aminoethyl)-4-(trifluoromethyl)benzenesulfonamide
(TfEn) as bidentate ligands. The complexes are capable of regenerating NADH. The en analogue displayed higher catalytic activity for
the reduction of NAD? than the TfEn analogues. The catalytic
activity of the Rh(III) complexes was further improved by the use of
123
S874
extended CpX rings. Mechanistic studies suggested a catalytic cycle
similar to that proposed for the Ru(II) arene analogues.
Acknowledgements: We thank ERDF/AWM (Science City), EPSRC, and ERC for their support of this work.
123
J Biol Inorg Chem (2014) 19 (Suppl 2):S867–S874
References
1 Lin S-J, Guarente L (2003) Curr Opin Cell Biol 15:241–246
2 Yan YK, Melchart M, Habtemariam A, Peacock AFA, Sadler PJ
(2006) J Biol Inorg Chem 11:483–488
3 Soldevila-Barreda JJ, Bruijnincx PCA; Habtemariam A, Clarkson
GJ, Deeth, RJ Sadler, PJ (2012) Organometallics 31:5958–5967
J Biol Inorg Chem (2014) 19 (Suppl 2):S875–S880
DOI 10.1007/s00775-014-1158-x
POSTER PRESENTATION
New methods and tools for bioinorganic chemistry
P 290
Regression-based analysis of thermal melting curves
Danny Kowerko, Sebastian L. B. König, Roland K. O. Sigel
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland, [email protected]
Extracting melting temperatures and thermodynamic (stability)
parameters (DH, DS and DG) from temperature-dependent absorption
profiles is a well-established for nucleic acids since the 1960s and 70s.
However, analysis of thermal melting curves typically suffers from
oversimplification and/or bias due to subjectivity. For example,
melting temperatures are often erroneously determined from the first
derivative of the thermal melting curve and thermodynamic parameters determined through van’t Hoff analysis often involves manual
baseline selection, followed by arbitrary selection of a subset of data
points [1, 2].
In this work, well-known models describing intra- and intermolecular thermal melting reactions are summarized, their
equations simplified and validated as a method to objectively
analyze thermal melting data. In particular, melting temperature
and thermodynamic parameters are obtained by fitting the whole
experimental dataset to the appropriate model, i.e. by regressionbased analysis. Hence, experimental and simulated data are rigorously analyzed, reporting for the first time on fit- and
instrument-specific limitations of the method. Finally, metal ion
dependent studies of an RNA hairpin with a 7nt complementary
strand (see Figure) are used to demonstrate the power and accuracy of this method [3, 4].
Financial support by the European Research Council (MIRNA No
259092) and the University of Zurich (FK-57010302 and FK-13-108)
is gratefully acknowledged
References
1. Mergny JL, Lacroix L (2003) Oligonucleotides 13:515–537
2. König SLB, Huppert JL, Sigel RKO, Evans ACE (2013) Nucleic
Acids Res 41:7453–7461
3. Kowerko D, König SLB, Skilandat M, Kruschel D, Cardo L,
Sigel RKO (submitted)
4. Kowerko D, König SLB, Sigel RKO (to be submitted)
P 292
Avoiding slips in the determination of uncaging
quantum yields of organic and organometallic
bio-active compounds
Philipp Anstaett, Anna Leonidova, Gilles Gasser
Department of Chemistry, University of Zurich, Winterthurerstrasse
190, 8057 Zurich, Switzerland
Protecting groups, which are cleavable by light irradiation, can be
used to inactivate bio-active compounds. Upon irradiation the compounds are re-activated (‘‘uncaged’’), thereby triggering their
biological function. This principle has been extensively used in
chemical biology to study the spatio-temporal responses of a system
to the presence of a chemical stimulus [1].
The uncaging quantum yield is an important physico-chemical
parameter for the uncaging process and depends on the caged
compound as well as irradiation conditions. Typically, it has been
determined by an indirect method referencing to caged phosphate,
for which the quantum yield was determined directly before [2].
However, we found that this technique can lead to incorrect
results as minor differences in the wavelength composition of the
light source leads to greatly different quantum yields of caged
phosphate. We developed a new method for evaluating the
uncaging efficiency in the UV-A range [3]. The technique has
been applied to investigate the photochemical properties of various organic and organometallic single- as well as two-photon
uncaging compounds.
This work was supported by the Swiss National Science
Foundation (Professorship No PP00P2_133568 and Research
Grants No 200021_129910 and No 200020_146776 to G.G.), the
University of Zurich (G.G.) and the Stiftung für Wissenschaftliche
Forschung of the University of Zurich (G.G.). The authors thank
Klemens Koziol and Peter Hamm for help with two-photon
uncaging experiments.
References
1. Brieke C, Rohrbach F, Gottschalk A, Mayer G, Heckel A (2012)
Angew Chem Int Ed 51:8446–8476
2. Walker JW, Reid GP, McCray J.A, Trentham DR (1988) J Am
Chem Soc 110:7170–7177
3. Anstaett P, Leonidova A, Gasser G (2014) (submitted)
123
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P 293
Targeting biomolecules and living cells by luminescent
ruthenium(II) Schiff base complexes
Julian Walther, Dumitru Arian, Roland Krämer
Department of Inorganic Chemistry, University of Heidelberg, Im
Neuenheimer Feld 270, 69120 Heidelberg, Germany
A novel Ru(II) complex, [Ru(bpy(CHO)2)3](PF6)2 (bpy(CHO)2: 2,2’bipyridine-4,4’-dicarboxaldehyde), is a precursor to easily accessible
luminescent ruthenium(II) Schiff base complexes for the detection of
biomolecules [1]. Reaction with primary amines gives fast and neat
access to complexes of the type [Ru(bpy(CHNR)2)3](PF6)2, a noteworthy matter of fact as the design of functionalized ruthenium(II)
complexes usually involves a challenging synthesis. A series of
complexes with high positive overall charge (R = ammonium substituent) and strong luminescent properties was applied to the detection
of the polyanionic biomolecule heparin, an important anticoagulant
drug. These complexes also accumulate in the phospholipid membrane
of living cells (see fluorescence microscopy figure). Simple Schiff base
functionalization chemistry offers access to a large number of structurally diverse ruthenium(II) probes that become readily available for
testing the response and selectivity toward biomolecules and cellular
substructures, with regard to the development of novel bioassays.
Financial support by the University of Heidelberg is gratefully
acknowledged.
J Biol Inorg Chem (2014) 19 (Suppl 2):S875–S880
importance to assess the molecular and electronic structure and the
stability of complexes under near physiological conditions. In case of
paramagnetic metal complexes (e.g. CuII, VIVO) EPR spectroscopy is
one of the most powerful methods to accomplish this task. At room
temperature, pH-dependent EPR spectra are recorded using a circulating system to perform an in situ titration with a strong base
solution. In the ‘‘two-dimensional’’ spectrum analysis, beside the
magnetic field, the measured spectra are also treated as a function of
the pH/concentration, and are analyzed simultaneously by fitting the
formation constants along with the EPR parameters for all the paramagnetic species. [2] This method was used to investigate several
TSC–CuII equilibrium systems, 3-methyl-(S)-pyrrolidine-2-carboxylate-2-formylpyridine thiosemicarbazone (L-Pro-FTSC) among
them, which was found to have enhanced water solubility permitted
us to study its complexation in pure water [3]. The stoichiometry and
stability of the formed species were investigated by pH-potentiometry
and UV–vis spectroscopy, as well. UV–vis and EPR spectroscopic
data indicate that L-Pro-FTSC acts in solution as a pentadentate
ligand via a [NPro, Npy, N, S-, COO
ðaxialÞ ] donor set, building up a
square-pyramidal monocomplex with CuII. The proved Topo IIa
inhibition potential in combination with excellent water solubility of
this complex is a sound basis for the further development of anticancer copper(II) thiosemicarbazonate complexes.
Financial support by OTKA PD103905 and TÁMOP 4.2.4. A/211-1-2012-0001 ‘‘National Excellence Program’’.
References
1. Yu Y, Wong J, Lovejoy DB, Kalinowski DS, Richardson DR
(2006) Clin Cancer Res 12:6876
2. Rockenbauer A, Szabó-Plánka T, Árkosi Zs, Korecz L (2001) J Am
Chem Soc 123:7646–7654
3. Bacher F, Enyedy ÉA, Nagy NV, Rockenbauer A, Bognár GM,
Trondl R, Novak MS, Klapproth E, Kiss T, Arion VB (2013) Inorg
Chem 52:8895–8908
P 295
Affinity capillary electrophoresis (ACE) for fast
prediction of protein selectivity to metal ions
Reference
1. Walther J, Arian D, Krämer R (2014) (in preparation)
P 294
Electron paramagnetic resonance (EPR) spectroscopy
to determine structural and solution equilibrium data
of copper(II) complexes with anticancer prospects
Nóra Veronika Nagy1, Éva Anna Enyedy2, Christian R. Kowol3,
Felix Bacher3, Tamás Kiss2, Antal Rockenbauer1, Vladimir B.
Arion3
1
Institute of Molecular Pharmacology, Research Centre for Natural
Sciences, Hungarian Academy of Sciences, Magyar Tudósok körútja
2, H-11117 Budapest, Hungary, [email protected].
2
Department of Inorganic and Analytical Chemistry, University
of Szeged, Dóm tér 7, H-6720 Szeged, Hungary.
3
Institute of Inorganic Chemistry, University of Vienna, Währinger
Strasse 42, A-1090 Vienna, Austria
Thiosemicarbazone (TSC)-based compounds are very promising
candidates for antitumor drugs, which form stable complexes with
transition metal ions resulting in versatile pharmacophores [1]. For
complexes with potential biological relevance, it is of crucial
123
Hassan A. AlHazmi1, Markus Nachbar1, Sami El Deeb1,2, Deia El
Hady3,4, Hassan M. AlBishri3, Hermann Wätzig1
1
Institute of Medicinal and Pharmaceutical Chemistry, University
of Braunschweig, Germany.
2
Department of Pharmaceutical Chemistry, Al-Azhar UniversityGaza, Gaza, Palestine.
3
Chemistry Department, Faculty of Science-North Jeddah, King
Abdulaziz University, Jeddah, Saudi Arabia.
4
Chemistry Department, Faculty of Science, Assiut University,
71516-Assiut, Egypt
Developing organometallic complexes is still growing for the
treatment of different disorders such as cancer and infection. In
fact, these complexes are pro-drugs, and have the role to transfer
metal ions to target binding sites [1]. Therefore, investigations of
metal ion behavior and their interaction with a desirable binding
site are important prior to the development of a new pro-drug.
Hence, techniques to reliably investigate these interactions are
urgently needed. Recently, Affinity Capillary Electrophoresis
(ACE) has been provided as an important tool for this purpose [2,
3]. In a recent study, the performance of this technique has been
improved by applying an appropriate rinsing protocol. Using 0.1 M
EDTA in the rinsing protocol became mandatory to desorb the
metal ion from the capillary wall and prevent the influence of EOF
changes on the precision and accuracy of results. ACE can now be
performed in approximately 10 min including rinsing procedures.
J Biol Inorg Chem (2014) 19 (Suppl 2):S875–S880
An excellent precision, corresponding to RSD % of \1.0 % was
achieved. The influence of different metal ions, including alkali
metals (Li?, Na?), alkaline earth metal (Ba2?), group IIIa (Al3?,
Ga3?), heavy metals (La3?, Pd2?, Ir3?, Ru3?, Rh3?, Pt4?, Os3?,
Au3?, Au?, Ag?, Cu2?, Fe2?, Fe3?, Co2?, Ni2?, Cr3?, V3?) and
the metal-containing anionic complexes MoO42- and SeO32- were
investigated by ACE, giving deep insight into the functional
interactions between these species and biomolecules. The selectivity of certain proteins (human and bovine serum albumins,
ovalbumin, b-lactoglobulin and myoglobin) to these metal ions has
been predicted. Hence, ACE technique could become first choice
for in vitro studies of protein–metal interactions.Financial supports
by the University of Jazan, Cultural Bureau of Saudi Arabia in
Berlin and University of King Abdulaziz are gratefully
acknowledged.
References
1. Romero-Canelón I, Sadler PJ (2013) Inorg Chem 52:12276–12291
2. AlHazmi HA, Nachbar M, El Deeb S, Abd El-Hady D, AlBishri
HM, Wätzig H (submitted to Electrophoresis)
3. El Deeb S, Wätzig H, El-Hady D Trends Anal Chem (2013)
48:112–131
P 296
A novel sequential extraction method for fractionation
of vanadium in mineral soils
Yu-Hui Xu1, Helmut Brandl1, Jen-How Huang2
1
Institute of Evolutionary Biology and Environmental Studies,
University of Zurich, Winterthurerstrasse 190, 8057 Zurich,
Switzerland, [email protected].
2
Department of Environmental Sciences, University of Basel,
Bernoullistasse 30, CH-4056 Basel, Switzerland
Vanadium (V) is a trace element essential to human beings and animals e.g. insulin-mimetic activity, but numerous reports have also
demonstrated the carcinogenicity and toxicity of V at higher concentrations. The major sources of V in the surface environment are
the combustion of fossil fuels and industrial wastes. Although V
pollution does not pose an immediate threat to ecosystems on a global
scale, high V levels may cause local environmental hazards. The
bioavailability and mobility of V in the terrestrial environment is
strongly dependent on the associations of V with solid phases, but is
only little known on the sequential distribution of V in soils. We
developed and tested two sequential extraction procedures (SEP) for
V in soils. Finally, a novel eight-step SEP has been established. The
eight-step SEP takes the advantage of describing more completely
and in more detail the V binding phases in following pools: (1) water
soluble V; (2) exchangeable V; (3) organic matter bound V; (4) V
associated with Mn oxides; (5) V associated with very poorly crystalline Fe and Al (hydr)oxides; (6) V associated with poorly
crystalline Fe and Al (hydr)oxides; (7) V associated with crystalline
Fe and Al (hydr)oxides; (8) residual V. The reproducibility and
adaptability of the proposed eight-step SEP has been verified by the
results of total seven soils with different V concentration and geocharacterizations. The median recovery is 85 %. The partition of V
among the eight fractions is (%, medians and ranges): (1) 0.8
(0.1–1.1); (2) 0.8 (0.1–3.9); (3) 7.8 (0.3–27); (4) 1.9 (0.2–9.3); (5) 6.0
(1.7–22); (6) 8.3 (3.3–17); (7) 32 (19–44); (8) 38 (23–45). The eightstep SEP is dependable and novel for the analysis of V sequential
distribution especially the phases correlated with metal oxides in
mineral soils. An application of the proposed SEP to the seven soils
results in clues on the relatively low mobility of V in terrestrial
environments. It also indicates the potential effect of anthropogenic
activities on the bioavailability and mobility of V in soils.
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P 297
Probing the mechanism of acrylamide formation
by HPLC-FTIR and Raman spectroscopy
Aristos Ioannou1, Andri Ioannou2, Eftychia Pinakoulaki2,
Constantinos Varotsis1
1
Department of Environmental Science and Technology, Cyprus
University of Technology, 95 Eirinis Str., P.O. Box 50329, 3603
Lemesos, Cyprus.
2
Department of Chemistry, University of Cyprus, P.O. Box 20537,
1678 Nicosia, Cyprus
The combination of HPLC with an Infrared or a Raman spectrometer
results in a powerful instrument that allows the detection and characterization of chemical species/food ingredients. The chemical
mechanism(s) for acrylamide formation in heated foods is not known.
Several plausible mechanistic routes have been suggested, involving
reactions of carbohydrates, proteins/amino acids, lipids and probably
also other food components as precursors. With the data and
knowledge available today it is not possible to point out any specific
routes, or to exclude any possibilities. Most probably a multitude of
reaction mechanisms are involved, depending on food composition
and processing conditions. Acrolein is one strong precursor candidate.
Current data indicate that the Maillard reaction might be an important
reaction route for the acrylamide formation, but also lipid degradation
pathways to the formation of acrolein should be considered. Acrylamide is a reactive molecule and it can readily react with various
other components in the food. The actual acrylamide level in a specific food product is therefore probably reflecting the balance between
ease of formation and potential for further reactions in that food
matrix. We will present the HPLC-FTIR and Raman data of the
acrylamide formation in model food systems.
Financial support by the European Regional Development Fund
and the Republic of Cyprus through the Research Promotion Foundation (Grant, YGEIA/TROFH/0311(BIE)/04) is gratefully
acknowledged.
P 298
CuO/ZnO nanocomposite for colorimetric detection
of cysteine
M. Šimšı́ková1, J. Čechal1,2, A. Zorkovská3, M. Antalı́k4,5,
T. Šikola1,2
1
CEITEC BUT, Brno University of Technology, Technická 10, 616
69 Brno, Czech Republic.
2
Institute of Physical Engineering, Brno University of Technology,
Technická 2, 616 69 Brno, Czech Republic.
3
Institute of Geotechnics, SAS, Watsonova 45, 043 53 Košice,
Slovakia.
4
Department of Biochemistry, Faculty of Science, P.J. Šafárik
University, Šrobárova 2, 041 54 Košice, Slovakia.
5
Department of Biophysics, Institute of Experimental Physics, SAS,
Watsonova 47, 040 01 Košice, Slovakia
Cysteine (Cys) plays an important biological role in human body,
such as protein synthesis, detoxification, and metabolism of various
biochemicals. Cys acts as the regulator in some diseases, as well.
Deficiency of cysteine involves hematopoiesis decrease, leucocyte
loss, and psoriasis. On the other hand, altered level of cysteine has
been implicated in hyperhomocysteinemia, the risk factor of numerous diseases, including the cardiovascular diseases, osteoporosis,
Alzheimer’s and Parkinson’s diseases, and AIDS [1].
Due to the important role of Cys in biological systems, great
attention has been paid to the detection with high sensitivity and
selectivity. Several analytical techniques, such as high-performance
liquid chromatography (HPLC), electrophoresis, electrochemistry,
123
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J Biol Inorg Chem (2014) 19 (Suppl 2):S875–S880
spectroscopy, and colorimetric detection have been extensively
investigated [2–6]. The last of these, the colorimetric assay, is widely
developed because the ability to detect Cys by naked-eye, without the
aid of any advanced instruments.
The CuO, ZnO, and CuO/ZnO nanomaterials have been used to
detection for various compounds, i.e. glucose, cholesterol, and ethanol. We have discovered a new type of biosensoring application of
CuO/ZnO nanocomposite based on the color change of nanocomposite in the presence of cysteine. The results showed that Cys can be
detected with concentration as low as *40 lM with high sensitivity
and selectivity against other amino acids. The advantage of this
detection method is its simplicity and no any other assistant reactions
and organic solvents are required.
limited sensitivity. Moreover, many other analytical tools fail for the
same metal ions because of the closed electronic shell structure.
Here we present an experimental NMR approach that is one billion
times more sensitive than conventional NMR spectroscopy. The
increase in sensitivity is achieved by recording the anisotropic
emission of b-particles in the decay of highly spin-polarized nuclei
[1, 2]. b-NMR may be applied to many chemical elements and we
aim to study e.g. Mg2?, Ca2?, Cu? and Zn2?. The technique has
been applied in solid-state physics [3, 4] but not yet in biological
inorganic chemistry. Although there are still technological challenges to overcome, b-NMR spectroscopy holds considerable
promise as a novel very sensitive technique in biological inorganic
chemistry.
References
1. Zhang M, Yu M, Li F, M. Zhu, M. Li, Gao Y, Li L, Liu Z, Zhang
J, Zhang D, Yi T, Huang C (2007) J Am Chem Soc 129:10322–10323
2. Ivanov AR, Nazimov IV, Baratova LA (2000) J Chromatogr A
870:433–442
3. Chen G, Zhang LY, Wang J (2004) Talanta 64:1018–1023
4. Ge S, Yan M, Lu J, Zhang M, Yu F, Yu J, Song X, Yu S (2012)
Biosens Bioelectron 31:49–54
5. Rusin O, Luce NNS, Agbaria RA, Escobedo JO, Jiang S, Warner
IM, Dawan FB, Lian K, Strongin RM (2004) J Am Chem Soc
126:438–439
6. Xiao W, Hu H, Huang J (2012) Sensor Actuat B 171–172:
878–885
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1. Matthias E, Olsen B, Shirley DA, Templeton JE, Steffen RM
(1971) Phys Rev A4:1626–1658
2. Gottberg A, Stachura M, Kowalska M, Bissell ML, Arcisauskaite
V, Blaum K, Helmke A, Johnston K, Kreim K, Larsen FH, Neugart R,
Neyens G, Szunyogh D, Thulstrup PW, Yordanov DT, Hemmingsen
L (2014) (manuscript in preparation)
3. Mansour AI, Morris GD, Salman Z, Chow KH, Dunlop T, Jung J,
Fan I, MacFarlane WA, Kiefl RF, Parolin TJ, Saadaoui H, Wang D,
Hossain MD, Song Q, Smadella M, Mosendz O, Kardasz B, Heinrich
B (2007) Physica B 401–402:662–665
4. Salman Z, Wang D, Chow KH, Hossin MD, Kreitzman SR, Keeler
TA, Levy CDP, MacFarlane WA, Miller RI, Morris GD, Parolin TJ,
Saadaoui H, Smadella M, Kiefl RF (2007) Phys Rev Lett 98:167001
P 299
b-NMR: a novel spectroscopic technique in biological
inorganic chemistry?
P 300
Surface plasmon resonance imaging and fluorescence
spectroscopy solutions to explore molecular interactions
A. Gottberg1,2, M. Stachura1,3, M. Kowalska1, M.L. Bissell4,
V. Arcisauskaite3, K. Blaum5, A. Helmke6, K. Johnston7,
K. Kreim5, F.H. Larsen8, R. Neugart9, G. Neyens4, D. Szunyogh10,
P.W. Thulstrup3, D.T. Yordanov5, L. Hemmingsen3
1
CERN, Geneva, Switzerland, [email protected]; 2Instituto
de Estructura de la Materia, CSIC, Serrano 113bis, 28006 Madrid,
Spain.
3
Kemisk Institut, Københavns Universitet, Thorvaldsensvej 40, 1871
Frederiksberg C, Denmark, [email protected].
4
Instituut voor Kern- en Stralingsfysica, KU Leuven, Celestijnenlaan
200 D, B-3001 Leuven, Belgium.
5
Max-Planck-Institut für Kernphysik, Saupfercheckweg 1, 69117
Heidelberg, Germany.
6
Humedics GmbH, Marie-Elisabeth-Lüders-Str. 1, 10625 Berlin,
Germany.
7
Universität des Saarlandes, Experimentalphysik, 66123 Saarbrucken,
Germany.
8
Institut for Fødevarevidenskab, Københavns Universitet,
Rolighedsvej 30, 1958 Frederiksberg C, Denmark.
9
Institut für Kernchemie, Universität Mainz, Fritz-Straßmann-Weg 2,
55128 Mainz, Germany.
10
Szervetlen és Analitikai Kémiai Tanszék, Szegedi
Tudományegyetem, Dom ter 7, 6720 Hungary
Nuclear magnetic resonance (NMR) spectroscopy is a powerful and
versatile technique, which allows for elucidation of protein structure
and dynamics. However, several biologically relevant metal ions,
such as Mg2?, Ca2?, Cu? or Zn2? are problematic in conventional
NMR spectroscopy due to the lack of suitable stable isotopes and
Frank Birke, Cecile Schuhmacher, Rainer Nehm
HORIBA Jobin Yvon GmbH, Scientific, Hauptstr. 1, 82008
Unterhaching, Germany, [email protected]
Proteins play an essential role in the functioning of living organisms.
Their activity is mostly controlled by other molecules that bind to
proteins in order to stimulate or activate them. As a result, the analysis
of binding events or conformational changes using biophysical
techniques (label-based and label-free) is being routinely used in
scientific research.
Fluorescence spectroscopy is a standard technique used in Life
Sciences for the characterization of biomolecules. More precisely,
Förster fluorescence resonance energy transfer (FRET) is a useful tool
for the determination of intra- and inter-molecular distances. It is
based on the non-radiative energy transfer from an excited donor
fluorophore to an acceptor fluorophore. It can be used to analyze
molecular interactions at the single-molecule level.
Surface Plasmon Resonance (SPR) is a label-free technique used
to follow the binding of molecules onto ligands immobilized on a
biochip surface by monitoring changes of the refractive index.
Molecular interactions are followed in real-time, providing information on kinetic processes (association and dissociation rates), affinity
and specificity. Surface Plasmon Resonance imaging (SPRi) is adding
an imaging capacity to SPR. It combines the strength of SPR to
monitor label-free biomolecular interactions to the throughput of
microarrays. It is thus possible to measure several dozen to several
hundred interactions in parallel (multiplexing).
A technical overview of both techniques will be given and illustrated by recent applications in the field of chemical biology.
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P 301
Investigating the impact of ROS on the toxicity
of plumbagin and its copper complexes
Veronika F. S. Pape1,2, Eva A. Enyedy3, Anna Lovrics1,
Zsuzsanna Nagy1, Nora Kucsma1, Aron Szepesi1, Miklós Geiszt4,
Michael Wiese2, Gergely Szakács1
1
Institute of Enzymology, Research Centre for Natural Sciences,
Hungarian Academy of Sciences, H-1117 Budapest, Hungary;
2
Department of Pharmaceutical Chemistry, Rheinische FriedrichWilhelms Univeristät, D-53121 Bonn, Germany.
3
Department of Inorganic and Analytical Chemistry, University
of Szeged, H-6720 Szeged, Hungary.
4
Department of Physiology, Semmelweis University, Faculty
of Medicine, H-1094 Budapest, Hungary
Plumbagin is a naturally occurring naphtoquinone used in traditional
Chinese medicine. The antitumor activity of this compound is mediated through reactive oxygen species (ROS) formation induced by its
copper complexes [1, 2]. Herein we investigate the solution equilibrium of the Cu(II)-plumbagin system together with the antitumor
potential of the ligand and its Cu(II) complexes on the uterine sarcoma cell line MES-SA.
The most frequently used method to measure intracellular ROS is
based on the dye 2’7’-dichlorofluorescein diacetate (DCFDA), which
is oxidized by ROS to the fluorescent Dichlorofluorescein DCF after
intracellular cleavage to DCFH. Despite being cheap and enabling the
detection of a broad range of ROS, this method has certain limitations. Several intracellular enzymes, metal ions or complexes may
influence the ROS-mediated oxidation of DCFH. Furthermore the dye
can undergo a light induced autoxidation via its semiquinone radical.
[3, 4] Therefore we also used a label-free measurement system based
on the genetically encoded fluorescent indicator HyPer, which specifically measures intracellular hydrogen peroxide levels [5].
Using Hyper-transfected cells, we confirm that plumbagin
increases intracellular H2O2 levels. To further analyze the role of
ROS and intracellular redox cycling, measurements were repeated in
the presence of the ROS-scavenger N-acetylcysteine. Together with
the toxicity data our results suggest a complex mechanism of action,
involving ROS-mediated cell-killing and activation by reduction
through intracellular redox cycling [6].
We wish to acknowledge financial support by ERC (StG-260572),
Hungarian Academy of Sciences (Momentum) and TÁMOP 4.2.4.
A/2-11-1-2012-0001.
References
1. Kumar KS, et al. (2008) Austral Asian J Cancer 7:65–72
2. Nazeem S, et al. (2009) Mutagenesis 24:413–418
3. Gomes A, et al. (2005) J Biochem Biophys Methods 65:45–80
4. Kalyanaraman B, et al. (2012) Free Radic Biol Med 52:1–6
5. Belousov VV, et al. (2006) Nat Methods 3:281–286
6. Kowol CR, et al. (2012) J Biol Inorg Chem 17:409–423
P 302
Infrared spectroscopy coupled with protein film
electrochemistry to study hydrogenase inhibition
by p-acid ligands
Min-Wen Chung, Benjamin Aucott, Suzannah Hexter, Philip A.
Ash, Fraser A. Armstrong, Kylie A. Vincent
University of Oxford, Department of Chemistry, Inorganic Chemistry
Laboratory, South Parks Road, Oxford, OX1 3QR, UK, [email protected]
Hydrogenases are of great interest because they utilise abundant and
inexpensive metals, Fe or Ni–Fe, in their active sites to catalyse
hydrogen cycling—highly relevant to enzyme-based or bio-inspired
S879
technologies. The Fe in the active site is in a biologically unusual
ligand environment coordinated by CO and CN-. These small molecule ligands have intense infrared absorption features and serve as good
probes to monitor the coordination state and electronic environment of
the active site. Infrared spectroscopy has therefore become a standard
approach for characterising hydrogenases isolated from different
organisms [1], but to date the spectroscopic results have not been
correlated with states of the active site generated during catalysis. We
have developed a new approach that allows direct electrochemical
control of hydrogenase, immobilised on a carbon particle electrode, to
be combined with in situ IR spectroscopic measurements. Efficient
solution flow in our spectroelectrochemical cell means that we can
introduce inhibitors or substrates during an experiment [2]. Here, probe
the reactivity of two hydrogenases, Hyd-1 (O2-tolerant enzyme) and
Hyd-2 (O2-sensitive enzyme) from E. coli, by addressing reactivity
with p-acid ligands are reported. We also compare the observed active
site CO and CN- stretching frequencies with DFT calculations by
building a cluster model, which is useful for further studies on how the
inhibitors coordinate to the active site.
This research was supported by European Research Council grant
EnergyBioCatalysis-ERC-2010-StG-258600 and M.-W.C. is grateful
for a Clarendon Scholarship from the University of Oxford.
References
1. De Lacey AL, Fernández VM, Rousset M, Cammack R (2007)
Chem Rev 107:4304
2. Healy AJ, Ash PA, Lenz O, Vincent KA (2013) Phys Chem Chem
Phys 15:7055
P 303
Thermally induced spin-crossover
in Fe(III)diethyldithiocarbamate studied by X-ray
spectroscopy and quantum chemical calculations
Stefan Mebs, Peer Schrapers, Ramona Kositzki, Michael
Haumann
Department of Physics, Free University Berlin, Arnimallee 14, 14195
Berlin, Germany, [email protected]
The spin state plays a crucial role in the reaction mechanism of a
wealth of transition metal complexes. Sulfur-coordinated covalent iron
sites are prominently involved in hydrogen catalysis in hydrogenase
enzymes. In such systems, spin-crossover may occur at cryogenic
temperatures, which could obscure the functional electronic configuration at room temperature. The spin sensitivity and selectivity of X-ray
absorption and emission spectroscopy (XAE) in principle facilitate
monitoring of spin state changes [1–3], but further studies are required
to firmly establish spin crossover detection. The effects of the low
temperature induced high- to low-spin transition in Fe(III)diethyldithiocarbamate were studied by XAE at the Fe K-edge. The spin
crossover was clearly detectable in the Kb and valence-to-core (Kb2,5)
emission lines, in the core-to-valence (pre-edge) absorption spectra., as
well as in the Fe–S bond lengths from EXAFS of the powder compound. Analysis by a Bolzmann population model yielded an energy
difference of *50 meV. However, the shape of the temperature curves
may suggest the involvement of vibrational modes of the molecule in
123
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J Biol Inorg Chem (2014) 19 (Suppl 2):S875–S880
normalized emission
the spin crossover. Density functional theory calculations supported the
XAE data and yielded the spin density distributions for the states.
These results further establish XAE as a viable tool for characterization
of electronic structures of metal centers in (bio)inorganic chemistry at
cryogenic and ambient temperatures.
MH acknowledges financial support by the DFG (grants Ha3265/
2-2/3-1, and/6.1), the BMBF (grant 05K14KE1 within the RöntgenAngström Cluster), and Unicat (CoE Berlin).
1,3
(267K - 8K)
T [K]
267
230
190
150
110
70
30
8
7040
7050
7060
emission energy / eV
References
1. Leidel N, Hsieh C, Chernev P, Sigfridsson K, Darensbourg M,
Haumann M (2013) Dalton Trans 42:7539–7554
2. Leidel N, Chernev P, Havelius K, Schwartz L, Ott S, Haumann M
(2013) J Am Chem Soc 134:14142–14157
3. Leidel N, Chernev P, Havelius K, Ezzaher S, Ott S, Haumann M
(2012) Inorg Chem 51:4546–4559
P 304
Novel metrological tool for assessment nanoand micro-sized organic/inorganic materials by field
flow fractionation
Haruhisa Kato, Ayako Nakamura
National Metrology Institute of Japan (NMIJ), National Institute
of Advanced Industrial Science and Technology (AIST), Tsukuba
Central 5, Higashi 1-1-1, Tsukuba, Ibaraki, 305-8565 Japan,
[email protected]
The number of studies related to ‘‘size’’ of nano- and micro-sized
materials has grown rapidly since the size is crucial to developing
nano- and micro-scale technologies and related to many of the
physicochemical properties and functions of these materials.
In colloidal suspensions of nano- and micro-sized organic/inorganic materials, fractionation methods such as field-flow fractionation
(FFF) are attractive method as elution techniques wherein nanoparticles, microparticles, and macromolecules are separated by their
physicochemical properties [1–3]. For example, a flow FFF method
with a multi-angle light scattering detector gives a more accurate size
distribution for nanoparticles than do dynamic light scattering methods since the values determined by ensemble methods strongly
depend on the instrument and analytical procedure used. On the other
hand, a centrifugal FFF method with inductively coupled plasmamass spectrometry is used in environmental analysis such as assessment of colloidal micro-sized materials in rivers.
Herein, a novel metrological tool that is based on field-flow
fractionation (FFF) separation system, a separation system hyphenated a flow FFF and a centrifugal FFF, was investigated as a hybrid
fractionation method for nano- and micro-organic/inorganic materials
[4]. The investigated hyphenated flow FFF and centrifugal FFF system shows higher performance than using either FFF method alone,
namely, hyphenated FFFs system is able to separate wider size range
than flow-FFF and improves the relaxation step of centrifugal-FFF for
small and low density materials. The investigated system should also
serve as useful tool for multi-dimensional separation of micro-
123
organic/inorganic materials. This research should facilitate the higher
precision analysis of nano- and micro-organic/inorganic materials and
become a practical method for achieving new production possibilities
for nano-, micro-, and bio-materials in industrial and biological
research.
This work was supported by the Ministry of Economy Trade and
Industry (METI).
References
1. Kato H, Nakamura A, Takahashi K, Kinugasa S (2009) Phys Chem
Chem Phys 11:4946–4949
2. Kato H, Nakamura A, Takahashi K, Kinugasa S (2012) Nanomaterials 2:15–30
3. Kato H, Nakamura A, Noda N (2014) Mater Express 4:144–152
4. Kato H, Nakamura A (2014) Anal Methods (in press)
P 305
Characterization of size and size distribution of nanoand micro-organic/inorganic materials: a comparison
of scanning electron microscopy, dynamic light
scattering, and flow field-flow fractionation
with multiangle light scattering methods
Ayako Nakamura, Haruhisa Kato
National Metrology Institute of Japan (NMIJ), National Institute
of Advanced Industrial Science and Technology (AIST), Tsukuba
Central 5, Higashi 1-1-1, Tsukuba, Ibaraki, 305-8565 Japan,
[email protected]
Methods for the accurate determination of the size and size distribution of nano- and micro-sized organic/inorganic materials are
essential for nano- and biotechnology. Among the methods available,
flow field-flow fractionation with multiangle light scattering and UV
absorption detectors (FFFF-MALS-UV) is considered to be a more
effective method than field emission scanning electron microscopy
(FE-SEM) and dynamic light scattering (DLS) for determining the
size and size distribution of nanomaterials [1]. However, the raw
values of size and size distribution obtained using these three methods
are number-, volume-, and z-averaged, respectively. In order to
compare the size and size distribution determined using different
measurement methods, it is necessary to transform the raw values into
the same dimensionality of length.
In this study, we transformed the raw values obtained using the
above mentioned three methods into the same dimensionality and
found that the results for averaged size and size distribution were
qualitatively similar despite the differences between the measurement
methods. However, even though the obtained values were transformed
to have the same dimensionality, the values obtained using FFFFMALS-UV were found to be weighted more heavily by larger particles
than those obtained using FE-SEM. Furthermore, the results obtained
using DLS were weighted more heavily by larger particles than those
obtained using FFFF-MALS-UV. This result was due to the observed
physicochemical phenomena utilized by these two measurement
methods, namely, UV absorption in the case of FFFF-MALS-UV and
light scattering intensity in the case of DLS [2]. Our finding plays an
important role and aspects in producing a new application of nano- and
biotechnology in research of functional materials.
Financial support from the Nanotechnology Material Metrology
Project of the New Energy and Industrial Technology Development
Organization (NEDO) is gratefully acknowledged.
References
1. Kato H, Nakamura A, Takahashi K, Kinugasa S(2012) Nanomaterials 2:15–30
2. Kato H, Nakamura A, Noda N (2014) Mater Express 4:144–152