Review D. Macmillan et al. Scheme 3. Exposure to CPE peptide 1 to 30 % v/v 3-mercaptopropionic acid at 60 8C for 48 h allowed the peptide thioester 2 to be isolat- ed in 86 % yield. (EPO), a 166-residue protein containing four cysteines (Cys7, Cys 29, Cys33, and Cys 161), was shown to under- go cleavage to afford thioesters. Surprisingly, only two thioesters could be observed, corresponding to residues [Ala1–Gly28]–COSCH2CH2CO2H, and [Ala1–His32]– COSCH2CH2CO2H. There was no evidence for cleavage at the Ile6–Cys7 junction or the Ala160–Cys161 junction, and this led us to predict that some selectivity might be achievable in thioester formation across Xaa–Cys junc- tions. When 66 KDa bovine serum albumin (BSA), con- taining over 30 cysteine residues, was subjected to similar MPA treatment, this protein underwent significant and complex fragmentation in only 1 h. These observations led us to the realization that pep- tides simply equipped with a C-terminal cysteine should be substrates for thioester formation. The fact that the re- action occurs at all is presumably because the N!S acyl shift is facilitated by enhanced protonation of the liberat- ed amino group below pH 7.[16] Further model reactions confirmed that thioester formation could be selective, particularly across Gly–Cys, His–Cys, and Cys–Cys junc- tions, and that in these cases the products do not appear to undergo epimerization of the C-terminal residue (Table 1). In substrates where the Xaa of an Xaa–Cys motif was a b-branched amino acid, there was no appreciable thioest- er formation. It is noteworthy that alternative Xaa–Cys motifs such as Ala–Cys, Ser–Cys, and Phe–Cys all ap- peared capable of forming thioesters under more forcing conditions, though the potential for epimerization of the terminal amino acid was not explored in detail. In one ex- ample, epimerization of the Ala residue appeared to have occurred to a significant extent (> 10 %). The presumed epimeric product was confirmed by synthesis of the d- Ala-terminated peptide and subjecting it to identical re- action conditions. The retention time of the thioester de- Scheme 4. Thioester synthesis through N!S acyl transfer. Table 1. Peptides designed to test for selectivity in MPA-mediated fragmentation.[12] Peptide sequence H–AENITTGCAEHC–NH2 H–AENITTGC(Acm)AEHC–NH2 H–AENITTICAEHC–NH2 H–AENITTICAEGC–NH2 H–AENITTGCAEGC–NH2 H–AENITTGCAECC–NH2 H–AENITTGCAEIC–NH2 888 www.ijc.wiley-vch.de X8C cleavage X12C cleavage X12C : X8C calc. m/z obs. m/z calc. m/z obs. m/z 793.3 793.3 849.4 849.4 793.3 793.3 793.3 793.5 n/o n/o n/o 793.5 793.5 793.5 1233.5 1304.5 1289.5 1209.5 1153.4 1199.4 1209.5 1233.6 1304.6 1289.7 1209.7 1153.6 1199.6 n/o 1:1 > 9:1 > 9:1 > 9:1 1:1 1:1 1:9 Isolated 11 mer yield % 28 39 60 n.d. 26 28 n.d. 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Isr. J. Chem. 2011, 51, 885 – 899