Review D. Macmillan et al. Figure 1. 13C NMR analysis of a Gly 13C-1 labelled Gly–Cys motif undergoing thioester formation.[20] 13C NMR spectroscopy, followed by LC-MS to confirm the product distribution, became a useful way to examine thioester formation under a varying reaction condi- tions.[20] Upon screening a small selection of water-soluble thiols, sodium 2-mercaptoethanesulfonate (MESNa) quickly emerged as the better thiol additive. Not only did it have good solubility in water and was free of the stench, it also caused less hydrolysis of the thioester prod- uct than MPA. Moreover, unlike MPA, MESNa elutes long before the peptides, allowing less complicated HPLC analysis and product isolation. Furthermore, when thio- ester formation was attempted on a peptide containing a Ser–Cys motif using MPA at 60 8C, no thioester product was formed. When the same reaction was performed at a higher temperature, a 1 : 1 ratio of thioester to starting material was observed, along with further condensation of the peptide with MPA to form an ester with the hy- droxyl of the serine.[12] It has not yet been reinvestigated, but such side reactions would not be expected when using MESNa. The reason for the increased stability towards hydroly- sis could originate from the fact that the sulfonate moiety of MESNa (pKa = 2) would be deprotonated in the pH range of the reaction (pH 2–6) (Scheme 5), whereas MPA would be considerably protonated. The more negatively charged MESNa thioester may stabilize the product against nucleophilic attack by water. This explanation, in addition to the known acid- or base-promoted degradation of acetylated b-hydroxy thiols,[23] also accounts for the poor stability of thioesters derived from b-mercaptoethanol or dithiothreitol. Interestingly the homologous thioacids 2-mercaptoace- tic acid (MAA, pKa = 3.7), 3-mercaptopropionic acid (MPA, pKa = 4.3), and 4-mercaptobutyric acid (MBA, pKa ~ 4.7) each displayed very different behavior during thio- ester formation, despite the similarities in pKa. With MAA as the thiol additive, only transient thioester forma- tion was observed, followed by rapid hydrolysis. With MPA, hydrolysis only appeared slowly (after 24 h), and when using MBA no hydrolysis was observed. In cases where hydrolysis occurred, it was observed only after sig- nificant thioester formation, and so it seemed likely that the thioester product, and not the amide starting material, was undergoing hydrolysis. This was supported by the ob- servation that the starting material appeared completely stable to heating in AcOH (pKa 4.76). These observations could be explained by participation of the pendant car- boxylic acid, with anhydride formation accelerating hy- drolysis (Scheme 6). Although hydrolysis of thioesters via anhydride inter- mediates is uncommon, hydrolysis of the anhydride formed by attack of a nearby nucleophilic carboxyl group within an active site of 3-hydroxyisobutyryl-CoA hydro- lase on its substrate has been observed.[24a] Consequently, Scheme 5. Dissociation of MPA and MESNa thioesters may account for the observed increase in stability of MESNa thioesters towards hy- drolysis. 890 www.ijc.wiley-vch.de 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Isr. J. Chem. 2011, 51, 885 – 899