Efficient Amino‐Sulfhydryl Stapling on Peptides and Proteins Using Bifunctional NHS‐Activated Acrylamides

Abstract

Widely used reagents in the peptide functionalization toolbox, Michael acceptors and N‐hydroxysuccinimide (NHS) activated esters, are combined in NHS‐activated acrylamides for efficient chemoselective amino‐sulfhydryl stapling on native peptides and proteins. NHS‐activated acrylamides allow for a fast functionalization of N‐terminal cysteines (k2=1.54±0.18×103 M−1 s−1) under dilute aqueous conditions, enabling selectivity over other nucleophilic amino acids. Additionally, the versatility of these new bioconjugation handles was demonstrated in the cross‐linking of in‐chain or C‐terminal cysteines with nearby lysine residues. NHS‐activated acrylamides are compatible with the use of other cysteine selective reagents, allowing for orthogonal dual‐modifications. This strategy was successfully applied to the late‐stage functionalization of peptides and proteins with a PEG unit, fluorescent probe, and cytotoxic agent. The level of molecular control offered by NHS‐activated acrylamides is expected to promote amino‐sulfhydryl stapling technology as a powerful strategy to design functional bioconjugates.

Recent advances in protein modifications techniques for the targeting N‐terminal cysteine

“…In order to improve the NHS ester reaction kinetics, and consequently, expand the scope of this reaction, Gois' group also explored the orthogonality of NHS-activated acrylates. [75] The ability of acrylates to efficiently and rapidly react with cysteine has previously been…”

“…All images in part C were reprinted with permission from (ref. [75]). Copyright (2021) Wiley-VCH GmbH demonstrated by Bernardes' group.…”

“…In order to improve the NHS ester reaction kinetics, and consequently, expand the scope of this reaction, Gois' group also explored the orthogonality of NHS‐activated acrylates. [ 75 ] The ability of acrylates to efficiently and rapidly react with cysteine has previously been demonstrated by Bernardes' group. [ 6,27 ] Therefore, the authors leveraged the advantages of NHS esters and the excellent reaction kinetic profile of acrylates to construct a bifunctional reagent (Figure 5C).…”

Fast and Selective Reaction of 2-Benzylacrylaldehyde with 1,2-Aminothiol for Stable N-Terminal Cysteine Modification and Peptide Cyclization

“…The reaction rate can be greatly accelerated using benzaldehyde bearing an ortho-boronic acid substituent to promote formation of a boronated thiazolidine, though aryl boronates can be subject to rapid oxidation by endogenous hydrogen peroxide in living systems . In addition, the reaction between 2-cyanobenzothiazole (CBT) and Cys, which is the last step of the synthesis of d -luciferin, has been developed into a useful ligation strategy for protein labeling and functional macrocycle construction. ,, Recently, N -hydroxysuccinimide activated acrylamide has been designed and synthesized for both the functionalization of N-terminal Cys and the cyclization of in-chain or C-terminal Cys and nearby lysine (Lys) residues . Monosubstituted cyclopropenone (CPO) has also been exploited for selective N-terminal Cys modification with a reaction rate (3.0 M –1 s –1 ) comparable to that for the CBT-cysteine reaction (9.19 M –1 s –1 ). , In addition, the reaction of 2-((alkylthio)­(aryl)­methylene) malononitrile (TAMM) and 1,2-aminothiol have been developed recently as a novel bioorthogonal reaction (4.2 M –1 s –1 ) for site-specific protein modifications and peptide cyclization .…”

“…20,21,25 Recently, N-hydroxysuccinimide activated acrylamide has been designed and synthesized for both the functionalization of N-terminal Cys and the cyclization of inchain or C-terminal Cys and nearby lysine (Lys) residues. 30 Monosubstituted cyclopropenone (CPO) has also been exploited for selective N-terminal Cys modification with a reaction rate (3.0 M −1 s −1 ) comparable to that for the CBTcysteine reaction (9.19 M −1 s −1 ). 20,31 In addition, the reaction of 2-((alkylthio)(aryl)methylene) malononitrile (TAMM) and 1,2-aminothiol have been developed recently as a novel bioorthogonal reaction (4.2 M −1 s −1 ) for site-specific protein modifications and peptide cyclization.…”