Thiol–Ene Photoclick Chemistry for Efficient Bicyclic Peptide Synthesis and Genetically Encoded Peptide Library

Abstract Bicyclic peptides have emerged as privileged scaffolds in chemical biology and drug discovery owing to their high target affinity, enhanced metabolic stability, and rigid conformational structure. Conventional bicyclization strategies often face challenges related to efficiency and chemoselectivity, which can limit their broader applicability. Here, we present the first application of thiol–ene photoclick chemistry for the bicyclization of unprotected peptides, employing TAIC as a trifunctional crosslinker. This photochemical approach enables rapid, high‐yield, and highly selective cyclization under mild, biocompatible conditions and is compatible with all proteinogenic amino acids. The reaction reaches completion within 6 minutes and is fully compatible with phage display, allowing high‐throughput generation and selection of genetically encoded bicyclic peptide libraries. Using this platform, we identified two submicromolar ligands targeting cyclophilin A, both of which showed substantially improved binding affinity compared to their linear counterparts. These findings establish thiol–ene photoclick chemistry as a robust and versatile platform for the discovery of conformationally constrained peptide ligands, particularly suited for addressing therapeutically challenging protein–protein interactions.