Suppressing the epimerization of endothioamide peptides during Fmoc/t-Bu-based solid phase peptide synthesis

Despite a number of intriguing utilities associated with thioamide-containing peptides and proteins in the context of biophysics, pharmacology and chemical biology, it has hitherto remained as one of the underexplored territories of peptidomimetics. The synthesis of long mono to multiply substituted endothioamide peptides is invariably accompanied with severe epimerization, oxoamide formation and various other undesired side reactions, resulting in messy product profiles. This has completely restrained their use as novel chemical tools for biological studies. During the chain elongation of an N-terminally located thioamide peptide using the Fmoc/t-Bu chemistry, it becomes vulnerable to the repetitive basic treatments as required for such chemistry. The incompatibility of thioamide moiety with bases as well as strong coupling reagents leads to epimerization as well as other side reactions due to its nucleophilicity, resulting in the loss of the stereochemical identity of the thioamidated amino acid residue. An easy-to-implement and efficient protocol to synthesize long (>10-mer) endothioamide peptides, significantly suppressing epimerization and other side reactions using 10% piperidine/dimethylformamide for 1min, is reported herein. The novelty of the protocol is shown through the efficient synthesis of a number of 10-12-mer mono to multiply thioamide-substituted peptides with broad substrate scopes. The utility of the protocol in the context of protein engineering and chemical protein synthesis is also shown through the synthesis of a thioamide version of the 16-mer peptide from the B1 domain of protein G. Such a protocol to synthesize long endothioamide peptides would open up avenues toward engineering and accessing novel thiopeptide and thioprotein-based chemical tools, the synthesis of which had been a serious hurdle thus far. Copyright (c) 2016 European Peptide Society and John Wiley & Sons, Ltd