An N ‐acyl Donor for Serine/Threonine Ligation Involving a Direct N ‐to‐ N Acyl Shift

Developing precise peptide modification protocols is crucial for peptide‐based drug development. While chemical ligation addresses this, conventional methods often face difficulties such as side reactions due to the high electrophilicity of S ‐ or O ‐acyl donors. Here we developed an N ‐(2‐formylphenyl)‐hydroxamate (HDX) ester as an N ‐acyl donor for chemical ligation involving a direct N ‐to‐ N acyl shift, eliminating the need for S ‐ or O ‐acyl compounds. Mechanistic studies revealed kinetic selectivity between substrates and the reversibility of the acyl shift, suggesting the possibility achieving chemoselectivity through kinetic and thermodynamic control. In addition, an HDX ester was applied to the structural modification of polymyxin derivatives, demonstrating that HDX esters can be useful for the modification of complex peptides. These findings provide a blueprint for designing N ‐acyl donors for chemical ligation, accelerating the advancement of peptide‐based therapeutics.