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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 13 123 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] 123 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 123 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 123 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 123 S704 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. 123 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 123 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. 123 S708 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]. 123 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 123 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 123 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 S714 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 123 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 S716 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]. S719 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 123 S720 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. 123 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. S721 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 123 S722 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] 123 J Biol Inorg Chem (2014) 19 (Suppl 2):S713–S724 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 S723 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 123 S724 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. 123 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 S726 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 123 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 S727 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 S728 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 130–172 123 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 S730 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. 123 S732 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. S735 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 123 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 123 S738 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 123 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 473:55–60 123 S742 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 2014:742–749 123 S744 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 S746 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. 123 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 S750 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. 123 S752 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 123 S754 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 123 S756 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 123 J Biol Inorg Chem (2014) 19 (Suppl 2):S749–S764 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 123 S758 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 123 J Biol Inorg Chem (2014) 19 (Suppl 2):S749–S764 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 123 S760 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 S762 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 S764 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). 123 S768 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 123 S770 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 123 S772 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 123 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 123 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 S788 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 S794 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 S804 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 S810 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 S814 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 S816 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 123 S818 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 123 S820 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 123 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. 123 S824 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; 123 S826 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 S834 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. 123 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 S858 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 S860 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 123 S868 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 123 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 S870 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 123 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 134:20–24 123 J Biol Inorg Chem (2014) 19 (Suppl 2):S867–S874 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 S876 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. S877 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 S878 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 References 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. 123 J Biol Inorg Chem (2014) 19 (Suppl 2):S875–S880 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 S880 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