Modulation of Reactivity in Native Chemical Ligation through the Use of Thiol Additives

Abstract

In native chemical ligation, an unprotected peptide α-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−α-thioesters were treated with an excess of a competing thiol to investigate their susceptibility to thiol exchange: a highly activated 3-nitro-4-carboxybenzyl α-thioester was smoothly converted to the less activated benzyl α-thioester through the addition of an excess of benzyl mercaptan; similarly, a peptide containing the benzyl α-thioester group was converted to a more reactive phenyl α-thioester by addition of thiophenol. Even a weakly activated peptide−α-thioester was converted to a substantially more reactive species, as demonstrated by the conversion of peptide−αCOS-CH2COOH to peptide−αCOS-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.