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

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.