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Topic: Ligation and Bioconjugation
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1
Introduction
“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
Recently, the advent of various methods has revolutionized the field of ligation and bioconjugation. The
selective site modifications of proteins, labeling and the molecular imaging have enabled chemists,
biochemists and biologists to gain more insight into biological processes in living systems.
2
The criteria to perform 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 reaction must not be water sensitive.
The starting materials and products must be inert to various biological functional groups and
enzymatic digestions.
The starting materials and products be must stable in physiological conditions and in
oxidizing/reducing environment.
The reactions should proceed under mild conditions. Heat, pressure, and high reaction
concentrations should not be required.
The inherent biological functions in the biological systems must not be altered after
ligation/bioconjugation.
The starting materials, products, and the waste must not pose toxicity to the bio organisms.
The reaction should be as efficient as possible. A lot of biomolecule are extremely expensive and
should not be used in excess.
Working with such molecules (from 1000 Da to 100 000 Da or more) implies working in really diluted
conditions, thus the reaction must be extremely fast.
The graph below is a good illustration of what are the current ligation/bioconjugation reaction rates in diluted
conditions and why very fast reaction are needed.
1
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Types of ligation/bioconjugaitions
3.1 Linkages Containing Thioethers
The first modifications of proteins and peptides were based on the inherited functional groups of the amino
acid sidechains. The thiol of the cysteines and the amino groups of the lysines are the most often modified
moieties. The unique reactivity of the thiol function of the cysteine makes it a privileged site for site-specific
modifications. But after tryptophan, cysteine is the second rarest amino acid. However in natural systems
lysine is incorporated 3 times more often than cysteine (Proteins 1988, 4, 99-122.).
3.1.1 Reactions between Thiolates and Haloacetamides
In 1972, free cysteines were detected through the reaction with iodoacetamides. (Gurd Methods Enzymol.,
1972, 25, 424.). Iodoacetamide derivatives were used to attach polyethylene glycol (PEG) side chains,
fluorophores and inert, insoluble materials (for immobilization) to proteins.
Sippel J. Histochem. Cytochem., 1981, 29, 314
2.1.2 Reactions between Thiolates and Maleimides
Michael additions of thiolates to maleimides are used frequently to attach fluorophores to proteins. The
maleimidocaproyl (MIC) building block can be attached to the N-terminus of a peptide by standard amide
bond formation. Subsequently the free thiol moiety of a cysteine of another biomolecule can attack the unsaturated carbonyl derivative and form a thioether bond. The problem with this linkage is that the imido
group of maleimide conjugates tends to hydrolyze spontaneously.
2
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Schwarzer ACIE, 2008, 47, 10030
3.1.3 Disulfide bridge bioconjugation
Here, the nucleophilic/electrophilic properties of sulfur are used to modify an unprotected nucleophilic
cysteine with an activated electrophilic disulfide bridge. Via this method glycosides and other biomolecules
can be introduced in the presence of an azide moiety that, can be used to further modify the peptide.
Davis Nature, 2007, 446, 1105
3.1.4 Radical-Induced Thioether Formation (Sulfo-click)
Thiols and alkenes can react to form thioethers. Reaction can be promoted photochemically or with radical
initiator (AIBN).
Kunz ACIE 2007, 46, 5226
3.2 Linkages Containing Amide Bonds
Traditional amide bond formation involves three steps: (1) Activation: C-terminal carboxylate groups are first
activated by coupling reagents. (2) Addition: free N-terminal amines attack the activated carboxyl groups. (3)
Elimination: activated groups are eliminated and amide bonds formed. This methodology is widely used in
both academia and industry. Various coupling reagents and conditions have been designed and reported.
However, for coupling of peptide fragments, this methodology raises several issues: (1) Peptide fragments
have to be fully protected. (2) Sensitive  positions are easily epimerized during activation and deprotection
steps. (3) Hydrophobic aggregations occur when peptide chains grow longer. (4) A large amount of waste is
generated. In contrast, through different reaction mechanisms, chemoselective peptide ligation reactions
address most of the problems above.
Bode Nature, 2011, 480, 471
3
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3.2.1 Native Chemical Ligation (NCL): Thioester vs Cysteine
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Kent Science, 1994, 266, 776
NCL is a highly chemoselective reaction. It proceeds with unprotected peptides with various functional
groups on the side chains, even additional cysteine residues. The reactions proceed at low (mM)
concentration and in neutral buffered aqueous solutions. NCL even tolerates denaturing reagents during the
hydrophobic peptide synthesis (Kent JACS, 1997, 119, 4325). Compared with solution phase peptide
synthesis (<10 amino acids) and solid phase peptide synthesis (< 50 amino acids), NCL can generate
peptides/small proteins up to around 200 amino acids.
Example: 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. Methods have been developed to transform Cys to Ala, dehydro-Ala, Ser, Met analogue…etc.
Dawson JACS, 2001, 123, 526
Danishefsky ACIE, 2007, 46, 9248
3.2.1.1 Replacement of Cysteines in NCL
3.2.1.1.1 via Histidines (His ligation)
It’s known that histidine participates in enzymatic acryl transfer process in biological systems. To mimic
processes in nature, cysteines are substituted with histidines in His ligation. This ligation runs in acidic
conditions and peptides cannot posess other nucleophilic side chains (e.g. Lys).
4
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Tam TL, 1997, 38,3
3.2.1.1.2. via Selenocysteines (Sec ligation)
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
Protein Pept. Lett., 2005, 12, 757
3.2.1.1.3 via Homocysteines (Met ligation)
Tam Biopolymers 1998, 46, 319
3.2.1.2 Replacement of Cysteines with Thiol-Containing Auxiliaries in NCL
The usage of thiol-auxiliaries overcomes the problem of the necessity for cysteine in NCL. This methodology
is still under developed because the size of the auxiliaries influences the reaction rates and the choice of the
amino acids at the ligation site is still limited. Also, the ease of removing the auxiliary is very important for
practical applications.
Danishefsky ACIE, 2006, 45, 4116
5
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3.2.3 Ketoacid Hydroxylamine Ligation (KAHA Ligation)
Bode ACIE, 2006, 45, 1248
Bode ACIE, 2012, 51, 513
Bode Chem. Sci., 2011, 2, 1976
KAHA ligation is highly chemoselective, proceeds with unprotected peptides with various functional groups
on the side chains, and does not need coupling reagents. Practical synthesis of ketoacid and hydroxylamine
modified amino acids has been developed. The only by-products are CO2 and water. The ligation site can be
between any amino acid residues in the peptide.
More recently a variant of the KAHA ligation using 5-oxaproline has been reported, which proceeds faster
and affords a natural amide bond and homoserine side chain at the ligation site.
H2 N
CONH2 NH3+CONH2
COOH COOH NH2+
PQGTVKWFNAEKGFGFIAPEDGSADVFVH
FmocHN
OH NH3+
COOH
O
H
N
OH
O
H
N
+
N
H
O
NH3+
NH2
OH NH
O
EIQGSGFRTLEENQKVEFEVGQSPKGPQATGVRTI
OH
O
COOH
COOH NH3+ COOH
COOHOH
CONH2 NH
H2 N
HO
CONH2COOH CONH2 CONH2 OH
NH2
cspA (2-31) a-ketoacid
cspA (32-67) oxaproline
15 mM, 8:2 DMSO:H2O
0.1 M oxalic acid
60 °C, 24 h, 53% isolated yield
+
COOH COOH NH2
CONH2 NH3 CONH2
FmocHN
OH NH3+
COOH
+
H
N
N
H
COOH
NH2
OH NH
O
EIQGSGFRTLEENQKVEFEVGQSPKGPQATGVRTI
OH
O
CONH2 NH
H 2N
HO
T32T§
NH3+
COOH NH3 COOH
COOHOH
O
H
N
PQGTVKWFNAEKGFGFIAPEDGSADVFVH
H 2N
OH
+
CONH2COOH CONH2 CONH2 OH
NH2
cspA (2–67)
Bode ACIE, 2012, 51, 5114
3.2.4 Serine and threonine ligation
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
unprotected peptide
N,O-benzylidine acetal cleavage
TFA/H2O 10 min
OH
Li PNAS, 2013, 110, 6657
6
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3.2.5 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
4
The techniques listed below are more often (but not always) used for labeling experiments. While still of
great importance in such reactions one of the reagents quite often is used in excess. 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
(Classical) Staudinger Reaction
Staudinger and Meyer discovered that azides can be reduced to amines by triarylphosphines in 1919.
Therefore azides can be considered as synthons for amines. First, azide reacts promptly with
triarylphosphine to form aza-ylide (iminophosphorane) and loses one equivalent of N2. This aza-ylide can
react with a variety of electrophiles. In case of water, the product is an amine and a phosphine oxide.
Staudinger Helv. Chim. Acta, 1919, 2, 635
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: Schepers and Bräse
Chem. Soc. Rev., 2011, 40, 4840).
7
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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
4.1.2 Traceless Staudinger Ligation
Although the Staudinger ligations work fine even in a cell, a modification forming amide bond without the
unnatural phosphine oxide is appreciated. The result of this ligation is similar to the NCL. In addition peptides
without cysteine can be prepared.
Raines Org. Lett., 2000, 2, 1939
Bertozzi Org. Lett., 2000, 2, 2141
8
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Hackenberger developed a chemoselective peptide cyclization procedure based on traceless Staudinger
ligation. A borane-protected phosphinothiol was used, and borane protecting group could be removed under
acidic or basic condition.
Hackenberger ACIE, 2008, 47, 5984
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
4.1.4 Thioacid/Azide Amidation
In this reaction azide and thioacid react forming an amide bond. The reaction proceeds through a [3+2]
cycloaddition. One can use polar and unpolar organic solvents and even water. With this reaction one can
prepare also N-acyl sulfonamides, which can serve as a protecting group. This procedure works in the
presence of acid- and base-sensitive protecting groups. And after the ligation the product can be modified
via two different pathways.
9
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Williams Org. Lett., 2006, 8, 823
4.2 Linkages Containing Carbon-Nitrogen Single Bonds
4.2.1 Mannich Peptide Conjugation
Tyrosine has a phenolic side chain, which shows nucleophilic properties. Owing to amphiphilicity, tyrosine is
very common amino acid on the surface of proteins, making it an ideal target for selective bioconjugation.
One procedure is based on a Mannich-type coupling reaction, where a phenol moiety reacts with an iminium
salt (formed from anilin and formaldehyde).
Francis JACS, 2004, 126, 15942
4.2.2 Ligation by Reductive Amination
This is also a Cys-free ligation method. In this example, a monosaccharide of horseradish peroxidase (HRP)
enzyme was oxidized by periodate. An Amine-containing antibody molecule reacts with an aldehyde to form
the labile Schiff base intermediate, which is reduced to a secondary amine linkage.
Hermanson Bioconjugate Techniques (Second Edition), 2008 Elsevier, pp 800
4.2.3 Ligation by Cyclization
The Fukase group applied the method developed by Katsumura for PET imaging. Unsaturated (E)-ester
aldehydes were reacted with lysine functional groups and formed imine intermediates. After
azaelectrocyclization, it gave 1, 2-dihydropyridines as products.
10
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Fukase ACIE, 2008, 47, 102
4.3. Linkages Containing Carbon-Nitrogen Double Bonds
Ketones and aldehydes react with amines to form imines, which can react with aminooxy compounds
(hydroxylamine) to form oximes or with hydrazide compounds to form hydrazones. This is a transimination
and owing to the -effect of adjacent nitrogens the equilibrium favors the imine. Aniline can speed up the
reaction, catalytically, and the reaction proceeds under less acidic conditions. This reaction sequence can be
used for site-specific modification of a peptide. Owing to the absence of ketones and aldehydes from cell
surfaces, they are unique chemical reporters.
Dawson ACIE, 2006, 45, 7581
4.4 Linkages Made by Cycloaddition
4.4.1 1,3-Dipolar Cycloaddition ([3 + 2] Cycloaddition)
1,3-Dipolar cycloadditions between an azide and an alkyne have been extensively studied majorly 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 biological system functions. The cyclo-adducts, 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
11
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4.4.2 Metal Catalyzed [3 + 2] Cycloaddition
4.4.2.1 Copper(I)-Catalyzed [3 + 2] Cycloaddition (CuAAC)
The concerted Huisgen cycloaddition has a high activation barriers (~26 kcal/mol for methyl azide and
propyne) and requires high temperatures and pressures for the reactions to proceed. Therefore, it had
largely remained as an academic teaching tool until Sharpless and Meldal independently discovered the Cu
(I) catalyzed azide-alkyne 1,3-dipolar cycloaddition. Cu (I) catalyst dramatically accelerates the rate of the
reaction and gives 1,4-substituted triazole as the only product.
Sharpless ACIE, 2001, 40, 2004
Fokin Chem. Soc. Rev. 2010, 39, 1302
Mechanism:
Protein transferrin was attached to the polyvalent cowpa mosaic virus scaffold
Finn Bioconjugate Chem., 2005, 16, 1572
4.4.2 Strain Promoted [3 + 2] Cycloaddition
The great kinetic properties, chemoselectivity, and high yields make metal-mediated 1,3-dipolar
cycloaddition of azides and alkynes 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.
4.4.2.1 Strain Promoted [3 + 2] Cycloaddition via Cyclic Alkynes
Between Alkynes and Azides
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. Although the reaction proceeds slower (k = 10-3-1 M-1S-1) than Cu(I)-cat [3 + 2]
cycloaddition, it gives a sufficient rate for in vivo labeling.
Bertozzi Chem. Soc. Rev., 2010, 39, 1272
12
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In vivo imaging in developing zebrafish
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Bertozzi Science, 2008, 320, 664
By manipulating intrinsic kenetic properties, double [3 + 2] cycloaddition was used simultaneously in the cell
imaging.
Hilderbrand ACIE, 2012, 51, 920
Recently, Houk group reported a calculation of different cycloadditions and according to these results,
rational design was proposed.
Houk, JACS, 2012, 134, 17904
13
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4.4.3.1 Between Cyclooctynes and Nitrones
One-pot three step site-specific modification of protein, IL-8
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Boons and Delft ACIE, 2010, 49, 3065
4.4.3.2 Between Norbornenes and Nitrile Oxides
Nitrile oxides are good electrophiles and the angular strain of norbornene ring is about 6 kcal/mol.
DNA modification
Carell OL, 2009, 11, 2405
4.4.4 Strain Promoted [3 + 2] Cycloaddition via Cyclic Alkenes
4.4.4.1 Between Oxanorbornadienes and Azides
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.)
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
4.4.4.2 Photoinduced [3 + 2] Cycloaddition
The Lin group developed a photochemical 1,3-dipolar cycloaddition between diaryl tetrazoles 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.
14
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Mechanims:
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Photoinduced cycloaddition in bacterial cells
Lin JACS, 2008, 130, 9654
4.5 Invers-Demand Diels-Alder Reaction ([4 + 2] Cycloaddition)
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.
4.5.1 Between 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
trans-cyclooctene ring strain (16 kcal/mol). However, there is also a concern of isomerization of the double
bond from trans to cis over time. Also cyclooctenes and tetrazines are large groups and may complicate the
metabolic or enzymatic incorporation into biomolecules.
Fox JACS, 2008, 130, 13518
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4.5.2 Between 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.
Hilderbrand Bioconjugate Chem. 2008, 19, 2297
4.6 Ligations by Metathesis
Cross-metathesis appears as an attractive method to install biomolecule modifications through formation of a
stable C=C bond. Ruthenium-catalyzed metathesis is a remarkable reaction for its high selectivity and
tolerance of different functional groups. However, the reactions are usually run in organic solvents.
Davis JACS, 2008, 130, 9642
16