Non‐symmetric Stapling of Tyrosine‐Containing Peptides Promoted by Intramolecular Non‐Covalent Interactions

Nonsymmetric stapling of peptides that connects two distinct endogenous residues offers a valuable tool for expanding the structural diversity of peptide macrocycles. However, its development remains challenging due to the high demand for chemo‐ and site‐selectivity while minimizing competing intermolecular peptide crosslinking. In this study, we reported a non‐covalent interaction‐promoted strategy for nonsymmetric stapling of tyrosine‐containing peptides by employing ethenesulfonyl fluoride (ESF) as a bifunctional crosslinker. Once covalently anchored to peptide, the sulfonyl fluoride motif is activated by non‐covalent interactions, which triggers an intramolecular SuFEx click reaction with tyrosine to afford peptide macrocycles. This approach efficiently crosslinks diverse residue pairs, including Cys–Tyr, His–Tyr, and Pro–Tyr. Notably, the protocol is scalable and can be performed in solid‐phase synthesis, providing robust access to tyrosine‐bridged peptide macrocycles with excellent diversity. Nanomolar binding affinity of nonsymmetrically stapled RGD peptide to integrin was also demonstrated.