Shifting Native Chemical Ligation into Reverse through N!S Acyl Transfer Scheme 2. Schematic thioester formation through N!S acyl shift, and some new routes to peptide thioesters using a) the cysteinyl pro- line ester (CPE) method;[10] b) N-alkyl cysteine;[9h,i] c) N-sulfanylethylanilides (SEAlide);[9f,11] d) bis(2-sulfanylethyl)amide peptides (BSEA);[9c–e] e) N-methyl enamides, R3 = linker.[9g] Species depicted in square brackets are not isolated. In our efforts to explore a scalable method for Fmoc- based SPPS of glycopeptide thioesters, we opted to first examine the cysteinylprolylester (CPE) method, reported by Kawakami and Aimoto.[10] We felt that this method was particularly attractive because the thioester was formed post-SPPS, allowing the precursors to be isolated in high yield and easily characterized. Additionally, since there was no requirement for specialized reagents or link- ers, all aspects of the thioester production could be easily monitored. We first prepared a short erythropoietin (EPO)-derived model peptide that was adorned with C-terminal CPE se- quence (1, Scheme 3).[12] Upon exposure to typical reac- tion conditions (N-cysteinyl peptide, 2 % w/v MESNa, pH 7) we were disappointed to find that little reaction had occurred. Based on previous experience[13] and addi- tional reports,[14] we predicted that the internal cysteine residue might, in some way, be influencing the process. When the peptide was resynthesized with the internal cys- teine residue Acm protected, we found that the reaction was capable of proceeding as originally proposed, to form the Ala thioester. In order to “rescue” our non-Acm-pro- tected peptide we decided to forcibly form the thioester by heating 1 to 60 8C in 30 % v/v 3-mercaptopropionic acid (MPA), as previously reported by Hojo[15] for mer- captomethyl prolyl esters. However, when the sample was heated we found that an alternative major product, 2, emerged which corresponded to the Gly–MPA thioester, where the peptide had undergone thiolysis at the internal cysteine site. This result was initially surprising since we were unaware of any solely chemical method reported to bring about selective peptide or protein backbone cleav- age with concomitant thioester formation. The fact that thiolysis appeared to have taken place se- lectively across the Gly–Cys junction (no smaller frag- ments could be observed) suggested that participation from the neighboring cysteine thiol, in a retro-NCL type process, was in operation (Scheme 4). Presumably any peptide or protein sample that contains cysteine could be a substrate, and indeed fully reduced erythropoietin Isr. J. Chem. 2011, 51, 885 – 899 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.ijc.wiley-vch.de 887