Peptide thioester synthesis via an auxiliary-mediated N-S acyl shift reaction in solution

The 4,5-dimethoxy-2-mercaptobenzyl (Dmmb) group attached to a main chain amide in a peptide is easily transformed into an S-peptide via an intramolecular N-S acyl shift reaction under acidic conditions, and the S-peptide produces a peptide thioester through an intermolecular thiol-thioester exchange reaction. In order to develop a method for efficiently preparing peptide thioesters based on the N-S acyl shift reaction, the factors involved in this process were analyzed in detail. The general features of the transformation at the Dmmb group attached amide bond in a trifluoroacetic acid (TFA) solution and the generation of a peptide thioester were examined by C-13-NMR spectral measurements, reversed-phase (RP) HPLC analyses, mass measurements, and amino acid analyses. The methoxy group of the Dmmb group was not essential for the N-S acyl shift reaction, but played a role in stabilizing the thioester form. The addition of water to the TFA solution accelerated the N-S acyl shift reaction mediated by the Dmmb group and also suppressed the acid-catalyzed cleavage of the Dmmb group. A peptide thioester was produced from the S-peptide via an intermolecular thiol-thioester exchange reaction with minimal epimerization of the amino acid residue that constituted the thioester bond. Undesirable side reactions, such as the hydrolysis of the thioester bond and an S-N acyl shift reaction occurred during the synthetic process, which is a subject of further investigation.