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Theoretical analysis of the detailed mechanism of native chemical ligation reactions.

The method of native chemical ligation between an unprotected peptide α-thioester and an N-terminal cysteine-peptide to give a native peptide in aqueous solution is one of the most effective peptide ligation methods. In this work, a systematic theoretical study was carried out to fully understand the detailed mechanism of ligation. It was found that for the conventional native chemical ligation reaction between a peptide thioalkyl ester and a cysteine in combination with an added aryl thiol as catalyst, both the thiol-thioester exchange step and the transthioesterification step proceed by an anionic concerted S(N)2 displacement mechanism, whereas the intramolecular rearrangement proceeds by an addition-elimination mechanism, and the rate-limiting step is the thiol-thioester exchange step. The theoretical method was then extended to study the detailed mechanism of the auxiliary-mediated peptide ligation between a peptide thiophenyl ester and an N-2-mercaptobenzyl peptide in which both the thiol-thioester exchange step and intramolecular acyl-transfer step proceed by a concerted S(N)2-type displacement mechanism. The energy barrier of the thiol-thioester exchange step depends on the side-chain steric hindrance of the C-terminal amino acid, whereas that of the acyl-transfer step depends on the side-chain steric hindrance of the N-terminal amino acid.