Review D. Macmillan et al. Figure 2. Reaction profiles of Xaa–Cys-terminated peptides prepared as C-terminal carboxylic acids and as C-terminal carboxamides, upon exposure to 10 % w/v MESNa in 10 % v/v AcOH at 60 8C.[17] Note: 3 c corresponds to a peptide sequence where the His-Cys motif is relocat- ed to the N-terminus of the model peptide, and thioester formation is inferred by observation of the N-cysteinyl peptide. Scheme 7. The presence of the carboxyl group appears to accelerate thioester formation, possibly by a) stabilization of the hydroxythiazo- lidine intermediate, b) increased pKa of the a-amino group (pKa 1 > pKa 2) favoring the S-peptide, c) through neighboring group participa- tion. was more than 1 : 1. Although in this study the difference in rates of thioester formation between carboxyl- and car- boxamide-terminated peptides was not considered, the re- sults clearly indicated that competing NCL occurs under the reaction conditions, but only becomes significant when d-Cys is added in large excess. From these results we concluded that, although thioester formation is rever- sible, the limited amount of “free” cysteine available ( 1 equiv) under normal reaction conditions precludes sig- nificant interference from NCL. Nagaike and colleagues reported the rate of N!S acyl transfer and thioester exchange to be improved by the use of microwave irradiation.[15] We have found little ad- vantage of microwave irradiation over conventional heat- ing for the formation of peptide thioesters at 60 8C.[20] The reaction can proceed with slower rates at lower tempera- tures such as 40 8C, which can reduce the hydrolysis of Asp-containing peptides sufficiently to allow for the de- sired thioester formation on these peptides; however the 892 www.ijc.wiley-vch.de 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Isr. J. Chem. 2011, 51, 885 – 899