An Efficient Thiol‐ene Mediated Protocol for Thiolated Peptide Synthesis and On‐Resin Diversification

Abstract The unique nucleophilic and redox properties of the sulfhydryl group render it highly useful as a synthetic handle for the diversification of peptide structure, including macrocyclization, ligation, and bioconjugation. Herein, a sequential acyl‐thiol‐ene/ S ‐deacetylation protocol for selectively installing thiol residues onto bioactive peptides on‐resin is demonstrated. Through judicious placement of appropriate unsaturated residues, the hydrothiolation/ S ‐deacetylation protocol offers a novel synthetic strategy to investigate the structure‐activity relationship of disulfide‐containing peptides displaying different ring sizes. Furthermore, a new and generally applicable fluorescent labeling strategy is introduced to facilitate direct on‐resin conjugation without intermediate purification steps. These new methods provide a robust and versatile platform for peptide macrocyclization and bioconjugation, with broad applications in peptide synthesis and chemical biology.