Disulfide Click Reaction for Stapling of S‐terminal Peptides

Abstract A disulfide click strategy is disclosed for stapling to enhance the metabolic stability and cellular permeability of therapeutic peptides. A 17‐membered library of stapling reagents with adjustable lengths and angles was established for rapid double/triple click reactions, bridging S‐terminal peptides from 3 to 18 amino acid residues to provide 18‐ to 48‐membered macrocyclic peptides under biocompatible conditions. The constrained peptides exhibited enhanced anti‐HCT‐116 activity with a locked α‐helical conformation (IC 50 =6.81 μM vs. biological incompetence for acyclic linear peptides), which could be unstapled for rehabilitation of the native peptides under the assistance of tris(2‐carboxyethyl)phosphine (TCEP). This protocol assembles linear peptides into cyclic peptides controllably to retain the diverse three‐dimensional conformations, enabling their cellular uptake followed by release of the disulfides for peptide delivery.