A one-step, resin-compatible Cys-Trp cross-linking system opens a new avenue for stapled peptide therapeutics

Protein-protein interactions (PPIs) are attractive yet challenging drug targets, motivating the development of conformationally constrained peptides such as stapled α-helices. Here we report a one-pot cysteine-tryptophan (Cys-Trp) cross-linking strategy that enables direct formation of Trp-indole CH sulfenylation staples concomitant with global side-chain deprotection and resin cleavage. Application of this approach afforded a Cys-Trp-stapled analogue of estrogen receptor-α (ERα) activity regulator synthetic peptide (ERAP), designed to disrupt a pathogenic PPI implicated in breast cancer. The resulting stapled ERAP, stERAP(C-W), exhibited potent and sustained antiproliferative activity comparable to that of the original stERAP. Moreover, stereochemical modification at the stapling residues was readily achieved, and circular dichroism (CD) analyses revealed a systematic decrease in α-helicity upon D-amino acid substitution. Overall, this study establishes Cys-Trp stapling as a concise and versatile platform for generating bioactive stapled peptides with tunable conformational properties.