MODULATION OF REACTIVITY IN NATIVE CHEMICAL LIGATION THROUGH THE USE OF THIOL ADDITIVES

In native chemical ligation, an unprotected peptide alpha-carboxy thio ester is reacted with a second peptide containing an N-terminal cystei ne residue. It was anticipated that addition of thiophenol to a native chemical ligation reaction would keep cysteine side chains reduced, c atalyze the reversal of unproductive thioester formation, and generate a more reactive phenyl thioester through thiol exchange. Several mode l 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 convert ed to the less activated benzyl alpha-thioester through the addition o f an excess of benzyl mercaptan; similarly, a peptide containing the b enzyl alpha-thioester group was converted to a more reactive phenyl al pha-thioester by addition of thiophenol. Even a weakly activated pepti de-alpha-thioester was converted to a substantially more reactive spec ies, as demonstrated by the conversion of peptide-COS-CH2COOH to pepti de-(COS)-C-alpha-phenyl. The utility of in situ transthioesterificatio n in native chemical ligation reactions was demonstrated by model synt heses of the 110-residue barnase polypeptide chain. The use of thiophe nol as an additive in the ligation gave clean, rapid reaction to form the desired amide-linked product in high yield. The in situ transthioe sterification process is broadly applicable to the total chemical synt hesis of proteins by native chemical ligation.