Andrew G. Roberts, Adam M. Levinson, Michael Oschmann, Adam H. Trotta, Artem Shvartsbart, Ting Wang, Samuel J. Danishefsky
In an effort to develop new methods for the synthesis of peptide therapeutics, our laboratory studies the utility of thiocarboxylic acids in amide bond forming reactions. Their activation by reaction with an isonitrile generates a presumed electrophilic intermediate termed, thio-formimidate carboxylate mixed anhydride, which can serve as a C-terminal acyl donor when intercepted by competent nucleophiles. Intriguingly, thiocarboxylic acids react with high chemoselectivity under mild conditions in the presence of carboxylic acids and other native side-chain functionality. Previously, we showcased the potential of this amide forming reaction for the synthesis of short peptides, N-methylated peptides and macrocyclic peptides. We reasoned that the mild nature of this activation method could enable bimolecular ligations to occur between large (>50-mer), minimally protected peptidyl partners, even in dilute solutions (2 to 10 mM). Various representative examples of this isonitrile-mediated peptide ligation will be presented with a focus on strategies to address reactive side-chain orthogonality (i.e. Cys, Lys) and opportunities for controlled peptide modification. Notably, the ligation method does not employ cysteine, thiol-surrogates or auxiliaries—rendering the strategy direct, versatile and complementary to native chemical ligation (NCL) for the preparation of long peptidyl sequences. In combination with NCL strategies, this method has enabled chemical syntheses of granulocyte colony-stimulating factor (G-CSF, 174-mer), Kirsten rat sarcoma protein (KRAS, 166-mer) and studies toward bovine pancreatic trypsin inhibitor (BPTI, 58-mer), as well as several other commercial peptide therapeutics.