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

Widely used reagents in the peptide functionalization toolbox, Michael acceptors and N-hydroxysuccinimide (NHS) activated esters, are combined NHS-activated acrylamides for efficient chemoselective amino-sulfhydryl stapling on native peptides proteins. allow a fast 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, versatility these new bioconjugation handles was demonstrated cross-linking in-chain or C-terminal with nearby lysine residues. compatible use cysteine selective reagents, allowing orthogonal dual-modifications. This strategy successfully applied to late-stage proteins PEG unit, fluorescent probe, cytotoxic agent. The level molecular control offered by is expected promote technology as powerful design functional bioconjugates.