In 2007, we developed an N-alkylcysteine (NAC)-assisted thioesterification method, in which the NAC in the C-terminus of the peptide is used as an N- to S-acyl migration device.¹  This method realizes the efficient synthesis of peptide thioester by the Fmoc method, which is useful to obtain peptide thioesters containing acid-sensitive moieties, such as carbohydrates and phosphate group.

In the NAC method, it usually takes about 24 h for the thioesterification to complete. The rate-determining step in this reaction is the N- to S-acyl migration step. Thus, if this equilibrium is shifted to the thioester, the overall rate of the thioesterification reaction would be enhanced. In this presentation, we examined the effect of the N-alkyl moiety of the NAC on the equilibrium between the amide and the thioester form.

Various N-alkylcysteines were synthesized from S-trityl-L-cysteine by the reductive amination followed by the removal of Trt group. The obtained cysteine derivatives were then reacted with a peptide thiophenyl ester to give peptides having various N-alkylcysteines at their C-termini. The acyl shift reaction of these peptides was monitored by the RPHPLC. The results of the analysis of the equilibrium, and the use of the N-alkylcysteine to the native chemical ligation via the in situ thioester formation with the external thiol will be discussed.