A Chlorooxime-Mediated Amidination of N-Terminus for Late- Stage Modifications of Unprotected Peptides

Late-stage modification of unprotected peptides at amine functionality has emerged as a powerful tool for modulating peptide structure and functions. However, existing methods often suffer from the challenge of controlling chemo- and site-selectivity due to the competition between the amine at the N-terminus and lysine side chain. Herein, we report a chlorooxime-mediated strategy for N-terminus-selective amidination of unprotected peptides that outcompetes lysine ε-amines. This strategy enables efficient synthesis of amidine-containing peptides carrying valuable payloads under physiological conditions, enabling late-stage derivatization of various peptides and peptide-based drugs such as semaglutide and tirzepatide. In addition, this protocol was applied as a general platform for diverse transformations, including dual-amidination, head-to-side chain macrocyclization, aminosaccharide conjugation, and protein labeling. Furthermore, the amidine motif was utilized as a chemical trigger for site-specific amide cleavage, leading to N-terminus deletion, highlighting its potential for developing peptide sequencing technology.