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.