Macrocyclization of Native Peptides through (Thio)urea Crosslinking of Two Amines

Cyclopeptides have emerged as promising candidates for developing bioactive molecules and drugs. However, owing to the scarcity of efficient cyclization strategies for natural peptides, the development of novel and diverse cyclopeptides remains a formidable challenge. In this study, a variety of novel macrocyclic peptides featuring (thio)urea bridges were developed through the site-selective Lys-Lys crosslinking of native peptides, employing CDI/TCDI as carbonylation reagents. In particular, the dual thiourea-bridged macrocyclic peptides were further synthesized by using a one-pot two-step multicomponent macrocyclization. These strategies enabled the chemoselective macrocyclization of peptides bearing diverse nucleophilic residue into novel (thio)urea-bridged macrocyclic analogs, as exemplified by the successful transformation of bioactive peptides including RGD, Anoplin, and HHC-36. Biological evaluation demonstrated that the cyclopeptides, particularly the thiourea- and bis-thiourea-bridged analogs, exhibit a greater than 10-fold enhancement in target binding affinity, potent antitumor activity, as well as improved membrane permeability and stability. Molecular docking elucidates the structural origin of the enhanced bioactivity, revealing their superior target engagement through reinforced molecular interactions.