Shifting Native Chemical Ligation into Reverse through N!S Acyl Transfer rived from the d-Ala terminated peptide was identical to that of the epimeric product observed when using l-Ala. The d-Ala-derived thioester had also epimerized to a sig- nificant extent. No similar phenomenon was observed when using peptides terminating in His–Cys, or Cys– Cys.[17] What also became apparent from these studies was that some species could not be easily observed by HPLC when using MPA as the reagent to effect thioester forma- tion. MPA has a tendency to precipitate from the reaction mixture upon extended reaction time, and additional MPA-derived peaks are observed in the HPLC trace as the reaction progresses. Furthermore, the acidity of MPA appeared incompatible with Asp residues, as also report- ed by Hojo,[15] and so it became clear that new reagents and reaction conditions would need to be investigated. 2. N !S Acyl-Transfer in Native Peptide Sequences 2.1. Some Peptide Features That Affect N!S Acyl-Transfer in Native Peptide Sequences So far, formation of thioesters via N!S acyl transfer at Cys-terminated peptides has been shown to proceed more efficiently when a peptide possesses a His, Gly, or Cys residue adjacent to the C-terminal Cys.[12] It is proba- bly not a coincidence that synthetic peptide thioesters adorned with these residues at the C-terminus are also known to react fastest in NCL reactions.[18] When Xaa– Cys motifs occur within a peptide sequence, thioester for- mation can potentially occur more widely, but often with reduced efficiency. Consequently, while Ala–Cys-termi- nated peptides can undergo slow thioester formation under normal conditions (with accompanying epimeriza- tion), this reaction appears extremely inefficient when the Ala–Cys motif is situated internally. An obvious limitation is that when additional especially labile sites exist within a peptide, these Cys residues must be protected to prevent unwanted cleavage, or the adja- cent residue (e.g., Gly) must be substituted (e.g., with Ala). Although we have observed faster thioester-forming rates when the scissile site is at the C-terminus of a pep- tide, rather than situated internally, and prepared as a C- terminal carboxylic acid rather than a carboxamide,[17] this difference in rate is not sufficient to ensure that reac- tion occurs selectively at the C-terminus. As a conse- quence, it is a priority to discover new reagents, additives, reaction conditions, or C-terminal motifs, such that the process can be improved, although it would be ideal if the optimal Xaa–Cys motif could be genetically encoded such that biologically derived proteins could be pro- cessed. Asp–Cys sequences were poorly tolerated when heated to 60 8C in MPA, and substrate peptides can undergo complete hydrolysis at the Asp residue over prolonged reaction times.[19] This undesirable reaction can be com- pletely abolished if the conservative substitution from Asp to Glu is made, although this is not practical for large proteins. Forming thioesters in Asp-containing pep- tides at pH 5.8 is currently a compromise solution since performing the reaction at this pH reduces hydrolysis at Asp. A further current limitation is that long peptides are often particularly prone to precipitation or aggregation upon periods of prolonged heating, so for the reaction to be widely applicable to recombinant samples, it is essen- tial that reaction conditions are developed that allow thioester formation to proceed at lower temperatures. The reaction can be conducted in 6 m guanidine·HCl, but while the samples may remain in solution throughout the experiment, thioester formation can remain inefficient. While there are clearly still significant obstacles to overcome, several short (10–15 residues) peptide thioest- ers can be routinely prepared, using regular peptide se- quences as thioester precursors, in good yield. Using thio- esters derived from this novel approach, we have already assembled biologically active samples of human b-defen- sin 3 (hBD3),[20] and the iron-regulatory peptide hepci- din.[21] The simplicity with which the precursors can be prepared has propelled us forward to investigate satisfac- tory solutions to the current limitations, since they do not appear overtly intractable. 2.2. Optimal Conditions and Reagents In order to initiate optimization studies we needed a useful handle with which reaction progress under varying conditions could be compared. The previous work of Aimoto[22] and Danishefsky[8b] suggested that employing a 13C-1 labelled Gly–Cys motif would be useful, owing to the significant difference in 13C NMR chemical shift of the carbonyl carbon upon conversion from 13CONHR (approx. 170 ppm) to 13COSR (approx. 200 ppm). Useful- ly, hydrolysis (13CO2H) could also be observed (Figure 1).[20] Interestingly, the 13C NMR spectrum appeared free of any intermediates, suggesting that N!S acyl transfer is rate-determining. However, although the S-peptide has not been isolated, it appeared essentially indistinguishable from the thioester product by 13C NMR, and it is unclear to what extent it accumulates during the reaction. A reac- tion that appears complete by 13C NMR can return signif- icant quantities of starting material following HPLC, sug- gesting that the S-peptide exists in solution and reverts to amide upon purification.[22] These findings then suggest, that the transthioesterification, rather than the N!S acyl shift, can determine the rate of the reaction. Both steps would be affected by changes in pH, and the precise mechanistic details of the reaction require a more thor- ough investigation. Isr. J. Chem. 2011, 51, 885 – 899 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.ijc.wiley-vch.de 889