Shifting Native Chemical Ligation into Reverse through N!S Acyl Transfer Scheme 6. Neighboring group participation of the pendant carboxyl group may catalyze thioester hydrolysis.[24] the lower nucleophilicity of the MESNa sulfonate group can also account for its increased stability towards hydrol- ysis. Despite the favorable features of MBA, it was only poorly water soluble and displayed a tendency to sponta- neously form the g-thiolactone under the reaction condi- tions. Regardless of the thiol additive employed, the reaction conditions are generally acidic, and the range at which thioesters are formed is usually from pH 2 to pH 5.8, using 10 % v/v AcOH or 0.1 m Na phosphate buffer to control the pH respectively. However at or above pH 7, the peptide thioester is hydrolyzed, and when N!S acyl transfer was carried out in 1 % v/v TFA (pH 1) in the ab- sence of thiol additives, hydrolysis of the peptide across the Gly–Cys junction was observed. Although sodium phosphate buffer has been employed to maintain approxi- mately pH 5.8, increasing the ionic strength of the buffer (> 0.1 m) results in more rapid thioester hydrolysis. In 1.0 m Na phosphate, pH 5.8, the thioester is only a minor component of the reaction mixture and hydrolysis pre- dominates. The addition of tris-(2-carboxyethyl)phosphine (TCEP) to the reaction was found to increase the rate of thioester formation, such that at 50 8C the conversion to thioester was closer to that observed at 60 8C in the absence of TCEP.[20] The beneficial effect of TCEP is tentatively at- tributed to the prevention of disulfide bond formation by maintaining a reducing environment. However, since TCEP is known to cause the desulfurization of peptides, converting Cys to Ala (usually above pH 7),[25] and to cause the cleavage of peptides and proteins (above pH 7),[26] its use should be monitored carefully. Further- more, unless solutions of TCEP·HCl are neutralized before use they can dramatically lower the pH of the re- action. A significant additional improvement is observed in the rate of thioester formation when a precursor contains a C-terminal carboxylic acid, rather than a C-terminal car- boxamide (Figure 2). The reasons for this effect have not been investigated in detail. Intramolecular protonation of the scissile amide nitrogen by the carboxyl group is not unfeasible, although the reaction is already conducted at acidic pH (Scheme 7). Such protonation of the hydroxythiazolidine intermediate by the carboxyl group would result in a zwit- terionic intermediate. The S-peptide may also exist as a zwitterion and this might positively influence the amide/ S-peptide equilibrium where pKa 1 > pKa 2 (Scheme 7 b). Alternatively, thioester formation could be facilitated by sequential acyl-transfers involving participation of the carboxyl group (Scheme 7 c),[24b] although this would sug- gest that all C-terminal amino acids are inherently sus- ceptible to hydrolysis and so is extremely unlikely. 2.3. Thioester Formation vs. NCL In some instances the reaction shows a certain degree of dependence on the peptide concentration. We were keen to investigate whether NCL can compete with thioester formation, essentially ligating the released cysteine back to the peptide thioester, reforming the starting materi- al.[27] Initially we had considered that this would be un- likely because of the low pH at which thioester formation occurs and the presence of the vast excess (approximately 0.7 m) of MESNa. However, since thioester formation is often observed not to proceed to completion we could not disregard this possibility. To examine the competitive potential of NCL, increasing concentrations of d-cysteine were added into the thioester forming reaction and the amount of epimer formed was measured (Scheme 8). Since d-Cys–OH was added to an l-Cys–NH2 terminated peptide (6), ligation could be readily observed by both HPLC and mass spectrometry. The d-Cys “epimer” (8), formed by NCL, was only observed in significant amounts when the ratio of d-cysteine added to the original peptide Isr. J. Chem. 2011, 51, 885 – 899 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.ijc.wiley-vch.de 891