Peptide Backbone Cleavage and Transamidation via Thioester-to-Imide Acyl Transfer

Cysteine thioesters are involved in a myriad of central biological transformations due to their unique reactivity. Despite their well-studied properties, we discovered an unexpected transamidation reaction of cysteine thioesters that leads to peptide backbone cleavage. <i>S</i>-Acylcysteine-containing peptides were found to spontaneously fragment by cleavage of the amide bond in the <i>i</i>-1 position to the acylated cysteine residue at pH 8-10. We present compelling evidence of a mechanism involving a central reversible thioester-to-imide acyl transfer step. The discovered transamidation reaction was found to be highly sequence dependent and to occur in peptides containing post-translational modifications (PTMs) such as cysteine S-acetylation and S-palmitoylation as well as in peptide-peptide branched thioesters, mimicking class I intein splicing. Thus, the inherent reactivity of peptide backbones containing <i>S</i>-acylcysteine residues should represent a starting point for investigation of endogenous protein behavior and may serve as a foundation for the discovery of mild new peptide and protein transformations.