Ferricyanide-mediated direct ligation of peptide hydrazides in neutral water

Abstract

Hydrazide-based native chemical ligation has emerged as one of the most widely used methods in modern chemical protein synthesis, but its traditional two-step, dual-environment workflow complicates operations and limits small-scale reactions. Here we present a ferricyanide-mediated system that enables chemoselective activation of peptide hydrazides and their ligation with N-terminal cysteine peptides in a single neutral buffer. This approach obviates the need for pH adjustment and intermediate isolation, thereby reducing labour, minimizing operational errors and expanding compatibility across a broader range of synthetic scales. We validate this method via the streamlined synthesis of biochemically or pharmaceutically relevant targets, including the antimicrobial protein Dptb, D-enantiomer of human interleukin-8 and glycosylated histone H4. In addition, this versatile chemistry can be repurposed for rapid one-step peptide cyclization and efficient C-terminal functionalization of recombinant proteins. Collectively, ferricyanide-mediated hydrazide ligation establishes a robust and generalizable platform for chemical protein synthesis.