Modulation of reactivity in native chemical ligation through the use of thiol additives

In native chemical ligation, an unprotected peptide alpha-carboxy thioester is reacted with a second peptide containing an N-terminal cysteine residue. It was anticipated that addition of thiophenol to a native chemical ligation reaction would keep cysteine side chains reduced, catalyze the reversal of unproductive thioester formation, and generate a more reactive phenyl thioester through thiol exchange. Several model peptide-alpha-thioesters were treated with an excess of a competing thiol to investigate their susceptibility to thiol exchange: a highly activated 3-nitro-4-carboxybenzyl alpha-thioester was smoothly converted to the less activated benzyl alpha-thioester through the addition of an excess of benzyl mercaptan; similarly, a peptide containing the benzyl alpha-thioester group was converted to a more reactive phenyl alpha-thioester by addition of thiophenol. Even a weakly activated peptide-alpha-thioester was converted to a substantially more reactive species, as demonstrated by the conversion of peptide-COS-CH2COOH to peptide-(COS)-C-alpha-phenyl. The utility of in situ transthioesterification in native chemical ligation reactions was demonstrated by model syntheses of the 110-residue barnase polypeptide chain. The use of thiophenol as an additive in the ligation gave clean, rapid reaction to form the desired amide-linked product in high yield. The in situ transthioesterification process is broadly applicable to the total chemical synthesis of proteins by native chemical ligation.