Download Lecture 1: Key Concepts in Stereoselective Synthesis

OC VI (HS 2015)
Bode Research Group
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Topic: Ligation and Bioconjugation
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1.
Introduction
Macromolecules, especially peptides up to 30-40 amino acids can reliably synthetized by solid phase peptide
synthesis. Longer sequences can be accessed with fragment coupling methods. The first possibility is to
couple partially protected peptide fragments followed by the removal of protecting groups. This strategy is
limited by the low solubility of the fragments and by the possible epimerization of the activated C terminus.
To overcome these limitations new approaches were reported. These are called chemoselective ligations of
unprotected peptides or macromolecules.
“The term chemoselective ligation refers to the coupling of two mutually and uniquely reactive functional
groups in aqueous environment or in the presence of biological material. Thus, even among a multitude of
potentially reactive functional groups, two chemoselective partners will react only with each other.”
Bertozzi Trends Biotech. 1998, 16, 506
The ligation reactions can be used for the chemical synthesis macromolecules or for the
modification/labeling of biomolecules. On the scheme above A and B functional groups are chemoselective
reaction partners.
2.
The criteria to perform chemoselective ligation/bioconjugation in biological systems
Ligation and bioconjugation are essentially used to bind high molecular mass molecules together in aqueous
media or biological systems. To fulfill these challenges, ligation/bioconjugation reactions have to respect
certain criteria:




The functionalities of the ligation reaction must react selectively with each other under mild
conditions
The reaction must yield covalent bonds and no or harmless side products like water or carbon
dioxide
The reactants must be stable before the ligation reaction and not toxic
The reaction must proceed with a reasonable rate
Bode ACS Chem. Biol. 2015, 10, 1026
The graph on the right illustrates the limitations of the
current ligation reactions. The low solubility of
macromolecules requires working in diluted
conditions (μM concentrations). To achieve good
yields under reasonable time at these conditions fast
reactions are required.
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3.
Types of ligation reactions
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3.1 Ligation at cysteine junctions
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The first type of protein ligation reactions are based on the unique reactivity of the thiol function on the side
chain of cysteine. This allows the chemoselective ligation of unprotected protein fragments. Unfortunately
cysteine is the second rarest amino acid in natural proteins.
Lau Justus Liebigs Ann. Chem. 1953, 583, 129
3.1.1
Native Chemical Ligation (NCL)
In 1994 Kent and co-workers reported the reaction of unprotected peptides with thioesters on the N peptide
and cysteine on the C peptide.
Kent Science 1994, 266, 776
The method can be applied to unprotected peptide fragments, and importantly, additional cysteine residues
do not interfere with the overall reaction, since the irreversibile S -> N shift occurs uniquely on the N terminal
of the cysteine residue.
NCL is a highly chemoselective reaction and forms native amide bonds. The reactions proceed at low (mM)
concentration and in neutral buffered aqueous solutions. NCL even tolerates denaturing reagents during the
hydrophobic peptide synthesis. Compared with solution phase peptide synthesis (<10 amino acids) and solid
phase peptide synthesis (< 40 amino acids), NCL can generate peptides/small proteins up to around 200
amino acids.
Kent JACS 1997, 119, 4325
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Example for applied NCL:
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Total Synthesis of HIV-1 protease (203 amino acids)
Kent ACIE 2007, 46, 1667
The major limitation of NCL is the scarcity of cysteine in natural peptide sequences (< 2%). Substituting
other amino acids with cysteine at ligation sites can cause problems and is not regarded as a non-influential
replacement.
3.1.2
Chemical modifications of NCL products
Methods have been developed to transform Cys to Ala.
Dawson JACS 2001, 123, 526
Danishefsky ACIE 2007, 46, 9248
Dawson reported catalytic methods to convert cysteine to alanine. Danishefsky and co-workers elaborated
an independent approach using radical reactions for the conversion of cysteine to alanine. (TCEP = tris(2carboxyethyl)phosphine and AIBN = 2,2’-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride).
In 2013 Danishefsky and co-workers reported the chemical synthesis of Erythropoietin. Erythropoietin is a
signaling glycoprotein (166 amino acids).
Danishefsky Science 2013, 342, 1357
3.1.3
Auxiliary methodes
The usage of thiol-auxiliaries overcomes the problem of the
necessity for cysteine in NCL. Auxiliary groups are facilitating
the ligation and can be removed without the modification of
any amino acid residue of the peptide product.
Macmillan ACIE 2006, 45, 7668
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3.2 Ligation at Selenocysteines site (Sec ligation)
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Selenols have similar reactivity to thiols, but selenols are more acidic and more nucleophilic than
corresponding thiols. The ligation proceeds fast and in high yield. The selenol-incorporated products have
important applications in spectroscopic structural investigation, and mechanistic studies. 77Se NMR
measurement is feasible with high molecular weight proteins.
Hilvert Helv. Chim. Acta. 2001, 84, 1197
Hondal Protein Pept. Lett. 2005, 12, 757
3.3 Homocysteine ligation (Met ligation)
Tam Biopolymers 1998, 46, 319
Homocystein is the homolog of cysteine and its thiomethylether is the natural amino acid methionine. This
approach allows to perform ligation at methionine sites since the reactivity of the homocysteine and cysteine
are similar.
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3.4 Ketoacid Hydroxylamine Ligation (KAHA Ligation)
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In 2006, Bode et al. reported the decarboxylative condensation of N-hydroxylamines with ketoacids to yield
amide bonds chemoselectively.
Bode ACIE 2006, 45,1248
This ligation allows the chemoselective coupling of large unprotected peptides.
Depending on the hydroxylamine, the KAHA ligation reactions are divided into two mechanistically distinct
categories.
3.4.1
Type I KAHA ligation
In Type I ligations O-unsubstitued hydroxilamines are used and the methode is suittable for the synthesis of
small peptides but dose not work well in aqueous media.
The mechanism of type I KAHA ligation was extensively studied and the pathway below was proposed.
Bode ACIE 2012, 51, 513
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Despite the limitations of the Type I KAHA ligation, the method was successfully applied to the synthesis of
the human GLP-1 (7-21) protein.
Bode Chem. Sci. 2011, 2, 1976
3.4.2
Type II KAHA ligation
In Type II ligations O-substitued hydroxylamines are used succesfully in the aqueous media to form peptides
with more than 100 residues. Typically 5-oxaproline, a cyclic hydroxylamine, is employed in these types of
ligation reactions. The use of oxaproline results in a homoserine residue at the ligation site.
Bode ACIE 2012, 51, 5114
The reaction pathway of type II KAHA ligation supported by oxaproline proceeds through an ester
intermediate which can be converted to the corresponding amide product.
Bode ACIE 2014, 53, 12244
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The formation of the ester as major product can be explained by the intramolecular addition of the alcohol
side chain of the homoserine residue.
KAHA ligation with 5-oxaproline made possible the total chemical synthesis of small proteins from
Mycobacterium, the prokaryotic-ubiquitin-like protein (Pup, 63 residues).
Bode ACIE 2012, 51, 5114
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3.5 Potassium Acyltrifluoroborate (KAT) Ligation
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Recently the Bode group identified the potassium acyl trifluoroborates (KATs) as a novel type of reaction
partners of hydroxylamines to afford amides in chemoselective ligation reactions.
Bode ACIE 2012, 51, 5683; Bode ACIE 2014, 53, 7604
The KATs react selectively with O-substitued hydroxylamines and form amide bonds. The KAT ligations are
suitable to conjugate unprotected peptides with large molecules (other peptides or polymers) in aqueous
media.
Bode JACS 2014, 136, 5611
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3.6 Serine and threonine ligation
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Recently, Prof Li has developed a new method for peptide synthesis generating a native bond at a serine or
threonine residue. In this case the elctrophile - thioester is replaced with a salicylaldehyde (SAL) derived
ester. The reaction proceeds by the formation of a N,O-benzylidine acetal that is subsequently cleaved in
acidic media to give the native amide bond and the conserved stereocenter.
HO
O
H
unprotected peptide
O
R
H 2N
SAL ester
CHO
O
unprotected peptide
OH
H
unprotected peptide
Ser or Thr
N
O
unprotected peptide
OH
R
OH
O
H
unprotected peptide
R
N
H
OH
N,O-benzylidine acetal cleavage
TFA/H2O 10 min
unprotected peptide
OH
Li PNAS 2013, 110, 6657
3.7 Isopeptide chemical ligation ICL
The concept of the NCL can also be used to form isopeptide bonds. An isopeptide bond is an amide bond
between the carboxyl/amine moiety of an amino acid side chain and the amine/carboxyl moiety of another
amino acid located on a side chain of another peptide. A classic isopeptide bond is the one between the
carboxyl terminus of a protein and the amino group of a lysine residue of another protein.
The Isopetide Chemical Ligation (ICL) follows the principle of the NCL: a thioester is required as well as a
“cysteine like” moiety.
The synthesis of polyubiquitinated protein is a good example of this ICL methodology, where the “cysteine
like” moiety is a -mercaptolysine. After the ligation is done, a simple desulfurization affords the native
isopeptide bond.
Brik PNAS 2013, 110, 17726
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4. Ligations and modifications of peptides and proteins with chemoselective reactions
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The techniques listed below are more often (but not always) used for labeling experiments. While still of
great importance, one of the reagents is quite often used in excess in such reactions. Such reactions still
need to be highly chemoselective but as one of the component is in excess the rate of these reactions are
not as essential as for the ligation reactions, which are used to synthesize peptides and proteins.
4.1 Amide Bond Formation via Azido Groups
4.1.1
(Non-traceless) Staudinger Ligation
The non-traceless azide-phosphine ligation was first introduced in 2000 by Bertozzi group. The reaction
proceeds via a cyclisation between the generated aza-ylide and an ester placed in ortho position.
Considering, that phosphine and azide are chemically orthogonal to most functional groups found in nature,
this method was a long sought solution to investigate cellular processes. The advantages of Staudinger
ligation over copper-catalyzed Click reactions are that one can avoid the use of toxic copper salts however
the reaction is much slower and often does not proceed to full conversion.
(Review: Bräse Chem. Soc. Rev., 2011, 40, 4840).
,
Bertozzi Science 2000, 287, 2007
It is possible to label enzymes using this ligation in a living cell. Attaching biotin to a peptide is a common
method to prepare derivatives because biotin readily binds to streptavidin, which is a 60 kDa bacterium
protein, and this non-covalent interaction is one of the strongest known noncovalent forces. Attached
streptavidin is usually modified with fluorophores (imaging), antibodies (Western blot analysis) and
polystyrene (capturing proteins).
Ploegh ACS Chem. Biol. 2006, 1, 713
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4.1.2 Traceless Staudinger Ligation
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Although the Staudinger ligations work fine even in a cell, a modification forming amide bond without the
unnatural phosphine oxide is appreciated. In this version of the Staudinger ligation the forming phosphine
oxide is excised from the target molecule
Raines Org. Lett. 2000, 2, 1939
Bertozzi Org. Lett. 2000, 2, 2141
4.1.3
Staudinger-phosphite Reaction
When phosphite reacts with an azide component, after the formation of trialkyl phosphorimidate it loses an
O-bound moiety (instead of hydrolysis) and forms a pentavalent phosphorus compound. The phosphite
starting material is easy to access, the reaction proceds under mild aqueous conditions and the purification
of products is easy. This reaction can be used for protein-labelings.
Hackenberger and coworkers demonstrated that the Staudinger-phosphite reaction is not only suitable to
attach PEG side chains to proteins, which could reduce their immunogenicity and increase their half-life,
bioavailability and water solubility but also they liberated the protein using light. This kind of system could be
utilized for targeted release of therapeutic peptide.
Hackenberger Chem. Sci. 2010, 1, 596
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4.2 Oxime ligation
Ketones and aldehydes react with hydroxylamines to form oximes or with hydrazides to form hydrazones.
The equilibrium favros the imine due to the -effect of the adjacent heteroatom. The reaction kinetics is
largely dependent on pH of the reaction mixture, and the high reaction rate is observed under the acidic
conditions (pH 4-5). Anilines are often used as catalysts to speed up the reaction. This reaction can be used
for site-specific modification of peptides. In the example below, ubiquitin-peptide conjugates were prepared
by the oxime ligation. The conjugates were used as nonhydrolyzable probes to investigate the affinities of
deubiquitinating enzymes for K48 and K63 topoisomer mimics.
Dawson ACIE 2006, 45, 7581
Ovaa JACS, 2010, 132, 8834
4.3 Linkages Made by Cycloaddition
4.3.1 1,3-Dipolar Cycloaddition ([3 + 2] Cycloaddition)
1,3-Dipolar cycloadditions between an azide and an alkyne have been extensively studied by Rolf Huisgen
since 1950s and therefore sometimes this reaction is termed as Huisgen cycloaddition.
Inert to almost all biological functionalities and possessing a small size, azides and alkynes hardly disturb
protein conformations and functions of the biological system. The triazoles have similarities to the peptide
bond in the atom planarity and electronic properties. Also, the great stability of triazoles and resistance to
oxidation and reduction makes Huisgen cycloaddition very attractive.
Maarseveen OL 2006, 8, 919
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4.3.2 Copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC)
The concerted Huisgen cycloaddition has a high activation barrier (~26 kcal/mol for methyl azide and
propyne) and requires high temperature and pressure for the reaction to proceed. Therefore, it had largely
remained as an academic teaching tool until Sharpless and Meldal independently discovered the Cucatalyzed azide-alkyne cycloaddition. Cu(I) catalyst dramatically accelerates the rate of the reaction and
gives 1,4-substituted triazole as the only product. The ligands are often used for the reaction in order to
protect Cu(I) from oxidation and/or to accelerate the reaction.
Initial reports: Fokin & Sharpless ACIE 2002, 41, 2596; Meldal JOC 2002, 67, 3057
Mechanism: Fokin Science 2013, 340, 457
Protein transferrin was attached to the polyvalent cowpa mosaic virus scaffold
Finn Bioconjugate Chem. 2005, 16, 1572
One of the drawbacks of the CuAAC is the cell toxicity of the copper reagents. The copper-chealating azide
substrates showed higher reactivity and revealed the reduced cell toxicity. In the exapmle below, the azide
substrates were designed to form strong, active copper complexes, and the reaction could be regarded as a
bimolecular system.
Taran ACIE 2014, 53, 5872
4.3.3 Strain-promoted azide-alkyne cycloaddition (SPAAC)
The great kinetic properties, chemoselectivity, and high yields make CuAAC one of the most useful
reactions. However, the toxicity of metals to disturb metabolic balance in living organisms greatly limits their
applications in biological systems.
To increase the reaction rates of Huisgen cycloaddition without metal catalysts, the Bertozzi group applied
strained cyclooctynes to promote the [3 + 2] cycloaddition. The ring strain for cyclooctyne is around
18 kcal/mol. Smaller cyclic alkynes are too unstable for practical use. The sp-hybridized bond angle in
cyclooctynes is around 160, which is close to the distorted bond angles in the transition states, resulting in
the great rate acceleration.
Bertozzi Chem. Soc. Rev. 2010, 39, 1272
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In vivo imaging in developing zebrafish
Bertozzi Science 2008, 320, 664
At the early stage of the reaction development, the reaction rate was rather slow (10 –3 to 10–2 M–1 s–1). The
modification of the substrate structure led to the excellent improvement of the reaction rate (ca. 1 M–1 s–1).
Bertozzi JACS 2012, 134, 9199
4.3.4
Alternative 1,3-dipolar cycloaddition reactions
4.3.4.1 Nitrones and cyclooctynes
One-pot three step site-specific modification of the protein, IL-8 (in situ generation of nitrones)
Boons and Delft ACIE 2010, 49, 3065
4.3.4.2 Nitrile oxides and norbornenes
Nitrile oxides are good electrophiles and the angular strain of norbornene ring is about 6 kcal/mol.
DNA modification
Carell OL 2009, 11, 2405
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4.3.4.3 Azides and oxanorbornadienes
Alkenes have also been used in [3 + 2] cycloadditions with azides. While instead of giving triazoles, they give
triazolines, which are relatively unstable. (The cycloadditions of alkynes and azides forming aromatic triazole
have G~ - 60 kcal/mol.)
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The Rutjes group circumvented the problem of unstable triazolines by using the oxanorbornadiene with an
azide. After cycloaddition, a furan was extruded through a retro Diels-Alder and gave a triazole instead.
Rutjes ChemBioChem. 2008, 9, 1805
4.3.4.4 Nitrile imines and alkenes
Lin group developed a photochemical 1,3-dipolar cycloaddition between nitrile imines and alkenes. The
ligation product, pyraoline, is fluorescent and enables monitoring of the reaction in vivo. The reaction can
occur at higher wavelength (365 nm) to minimize the damage to living systems caused by he irradiation.
Simple unactivated alkenes can be used without problems.
Mechanims:
Photoinduced cycloaddition in bacterial cells
Lin JACS 2008, 130, 9654
Recently, Lin group has reported the synthesis of the spirocyclic alkenes as extremely reactive yet stable
substrates for the photoclick reaction. The specific modification of superfolder GFP (sfGFP) was achieved
with the excellent reaction rate (k > 10,000 M–1 s–1).
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Lin JACS 2014, 136, 4153
4.4 Inverse electron-demand Diels-Alder reaction
The normal Diels-Alder reaction usually requires EWG-activated dienophiles (e.g. maleimides). Those
dienophiles are not suitable because of their Michael acceptor properties to various nucleophiles commonly
found in biological systems.
4.4.1 trans-Cyclooctenes and tetrazines
Bioorthogonal reactions between tetrazines and trans-cyclooctenes proceed very fast in water
(k = 102-104 M–1s–1) without any catalyst and produce N2 as a sole byproduct. The fast kinetics result from
the ring strain of trans-cyclooctenes (16 kcal/mol). However, there is also a concern of the isomerization of
the double bond from trans to cis over time. Also cyclooctenes and tetrazines are large in size and may
complicate the metabolic or enzymatic incorporation into biomolecules.
Fox JACS 2008, 130, 13518
4.4.2 Norbornenes and tetrazines
This cycloaddition is based on the inverse electron-demand Diels-Alder reaction of tetrazines to
norbornenes, followed by retro-Diels-Alder elimination of molecular nitrogen to afford the expected
dihydropyridazines and their regioisomers. Although the reaction kinetics is slower (k = 10–1-10 M–1 s–1)
compared to the reaction with trans-cyclooctenes, norbornenes are bench stable.
16
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Hilderbrand Bioconjugate Chem. 2008, 19, 2297
4.4.3 Cyclopropenes and tetrazines
Functionalized cyclopropenes were recently found to be good substrates for IED-DA reactions with
tetrazines. Cyclopropenes are stable yet reactive and compatible with various metabolic pathways sue to
their small size. The reaction rate is, however, slower than that of the reaction with trans-cyclooctenes.
Prescher JACS 2012, 134, 18638
4.5 Metal-mediated bioconjugation reactions
4.5.1 Ru-mediated cross-metathesis
Cross-metathesis appears as an attractive method to install modifications onto biomolecules through the
formation of a stable C=C bond. Ruthenium-catalyzed metathesis is a remarkable reaction for its high
selectivity and tolerance toward different functional groups. However, the reactions are usually run with
organic co-solvents. In the case below, the method was applied to the serine protease subtilisin Bacillus
lentus (SBL).
Davis JACS 2008, 130, 9642
4.5.2 Pd-mediated Suzuki-Miyaura cross-coupling
The Suzuki-Miyaura cross-coupling reaction is an emerging tool for bioconjugations. The reaction can be
utilized such as for modification of the protein surface and DNA. This bioconjugation method does not
require organic solvents.
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Davis JACS 2009, 131, 16346
4.6 Mutually orthogonal bioconjugation
A new challenge in bioconjugation areas is identification of the mutually orthogonal bioconjugation reactions.
Those reactions allow the simultaneous monitoring of the multiple biomolecules in a single biological system.
- Some cyclooctynes are reactive toward tetrazines, and they cannot be used for the multilabeling purpose.
However, the judicious choice of the substrate structure allowed double [3 + 2] cycloaddition simultaneously
in the cell imaging by taking advantage of the difference in reaction rates. The substituents on the tetrazine
was critical. Hilderbrand demonstrated the simultaneous labeling of two different cancer cell types.
N NH
NH 2
Me
5
no reaction observed
N
R
O
N
N R
N
(2.1 ± 0.2) M -1s-1
N N N
R
N
N
R
R
O
O
Me
Me
N
N
N
N
(210 ± 10) M -1s-1
HO
N
NH
HO
5 NH 2
5 NH 2
(0.0064 ± 0.002) M -1s-1
HO
N
N
N
R = PEG 4COOH
* Buffer pH 7.4, 37 °C
R
Hilderbrand ACIE 2012, 51, 920
- Houk and Prescher have recently reported the unique reactivity of the substituted cyclopropenes. 1,3disubstituted cyclopropenes showed high reactivity toward tetrazines to undergo the IED-DA reaction while
3,3-disubstituted cyclopropenes did not react with tetratines. Instead, 3,3-disubstituted cyclopropenes
showed the facile reaction with nitrile imines (generated by photolysis) to undergo the 1,3-dipolar
cycloaddition. They tested the mutual orthogonality by labeling the model protein, BSA (Scheme below).
Houk & Prescher JACS 2013, 135, 13680
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