Acetohydroxamic acid-assisted peptide hydrazide ligation for chemical protein synthesis

We here report a novel acetohydroxamic acid-assisted peptide hydrazide ligation (APHL) strategy for chemical protein synthesis. With acetohydroxamic acid serving as a non-thiol additive, sodium nitrite-activated peptide hydrazides are in situ converted to reactive peptide O-acyl hydroxamates, which undergo efficient and direct ligation with N-terminal cysteine-containing peptides. This thiol-additive-free approach is compatible with one-pot ligation-desulfurization and ligation-oxidative folding. The utility of this strategy is demonstrated by the expedient synthesis of site-specifically lactylated histone H3 (H3K18la) and the disulfide-rich neurotoxin Calciseptine. Our study expands the repertoire of hydrazide-based ligation and provides a practical, operationally straightforward tool to accelerate chemical protein synthesis.