Synthesis of Bis-urea-Bridged Cyclopeptides via Stapling of Unprotected Peptides

This work establishes a chemoselective peptide stapling method via bis-urea bridge formation between native amino groups, using diisocyanates as linchpin reagents. This protocol achieves macrocyclization across 19- to 41-membered rings through either lysine-lysine or N-terminus-lysine cross-linking, while preserving sensitive residues including Arg, His, Trp, Tyr, Ser, Glu and Cys. Evaluation of the resulting stapled peptides revealed improved membrane permeability and increased stability against both chemical and proteolytic degradation. Representative stapled peptides demonstrated potent cytotoxicity in several cancer cell lines and induced dose-dependent apoptotic responses in selected tumor models.