Cys–Lys stapling for unprotected peptides via tunable linkers

Stapling has emerged as a transformative paradigm in peptide chemistry, enabling precise conformational control to endow peptides with augmented biophysical properties, including enhanced proteolytic stability and target-binding affinity. Although symmetric macrocyclization strategies have been investigated, non-symmetric stapling of native peptide scaffolds remains underexplored, owing to intricate synthetic challenges associated with achieving concurrent chemo- and site-selectivity. This limitation primarily stems from the requirement for orthogonal reactivity in modifying distinct proteinogenic residues while preserving native side-chain functionalities under biocompatible conditions. Herein, a non-symmetric stapling is disclosed for cysteine–lysine (Cys–Lys) crosslinking in unprotected peptides and proteins via unsymmetrically tunable linkers with high chemo-selectivity and regio-selectivity in a self-assembly manner, in which a library of seventeen stapling reagents with adjustable length, angle, flexibility, rigidity, and lipophilicity was comprehensively established for relay Cys–Lys ligation, facilitating the macrocyclization of intervening loops (6–30 amino acids) into 25–40-membered rings under physiologically compatible conditions. The conformationally restricted peptide displayed strengthened α-helicity, proteolytic stability, serum stability, and enhanced anti-bladder cancer activity, demonstrating the potential of this protocol for drug discovery.