Expedited Site-Selective Modification of Peptides and Proteins at N-Terminal Cysteine Facilitated by Imidazole

Site-selective modification of peptide and protein using commercially available or readily synthesized reagents remains a critical need in advancing the study and manipulation of biological processes. Herein, we present an imidazole-assisted strategy for the selective modification of N-terminal cysteine (NCys) residues in peptides and proteins using off-the-shelf acyl donors. Mechanistically, excess imidazole converts non-selective acyl donors into acyl imidazoles, which selectively and efficiently react with NCys residues. This approach operates under mild conditions and supports one-pot dual functionalization, as well as protein labeling on phages and live mammalian cells. Application to phage-displayed peptide libraries expanded the accessible chemical space and enabled the discovery of a bicyclic peptide binder with nanomolar affinity.