Shifting Native Chemical Ligation into Reverse through N!S Acyl Transfer Scheme 8. The extent to which NCL competes with thioester formation can be examined by addition of increasing concentrations of d- Cys during thioester formation.[27] efficiency of the reaction is significantly compromised at lower temperatures.[12] The search for optimized reagents and conditions is far from complete. The discovery that small changes in the structure of thiol additive can dramatically affect the out- come, as well as the user friendliness, of the reaction is extremely encouraging. Furthermore, small changes in precursor structure (i.e., carboxyl vs, carboxamide) can also significantly affect the rate of thioester formation. The increased reactivity of carboxyl-terminated substrates is supported by several HPLC, LC-MS, and 13C NMR la- belling experiments and suggests that additional “engi- neering” of the cysteine residue or Xaa–Cys motif should be possible. 3. “There and Back Again”: Application to Cyclic Peptide Synthesis Cyclic peptides are highly prized materials, since the con- formationally constrained structures can offer enhanced binding to targets, increased stability to proteolysis, and enhanced bioavailability.[28] Although they are of consid- erable interest as therapeutics, they can be difficult to prepare by synthetic or biological means.[29] A practical consequence of the lower reactivity of Xaa–Cys–NH2 peptides, compared with Xaa–Cys–OH peptides, is that the formation of cyclic amide products from carboxyl-terminated precursors should be favored. Since the cyclic products result in the formation of an in- ternal Xaa–Cys motif at the expense of a terminal Xaa– Cys motif, the preference for cyclic products could be even more pronounced. Previous studies suggested that NCL only competes with thioester formation significantly at high concentrations of added cysteine.[27] However, we considered that the high effective Cys concentration in an intramolecular NCL reaction might render cyclization feasible. Interestingly, the successful synthesis of cyclic peptides of biological origin is most often facilitated by inteins,[30] which utilize the same peptide rearrangement process that we would aim to employ.[31] Recently we tested this hypothesis with some short, 9– 14 residue antimicrobial peptides derived from mouse b- defensin, DefB14 The ability of defensins to kill a broad range of microbes has evolved over millennia, and their precise mechanism of bacterial cell surface disruption re- mains a matter for debate. We discovered that a small de- fensin fragment retains much of the antimicrobial activi- ty.[32] and sought to improve its stability through head-to- tail cyclization. In one example (9, Scheme 9) we initially examined thioester formation/cyclization at approximately pH 2 (employing 10 % v/v AcOH as solvent) and found that, although the thioester accumulated, only a small degree of cyclization took place. Upon increasing the reaction pH to 5.8 we found that the cyclic peptide 10 emerged from the reaction as the major product (Scheme 9).[32b] We anticipated that raising the pH might reduce the pro- pensity for N!S acyl transfer, but a compensatory in- crease in the nucleophilicity of the N-terminal cysteine could render NCL more effective. An interesting feature of this reaction is that both retro-NCL and NCL appear to proceed in the same reaction vessel yet, under the re- action conditions, are working in concert to produce the cyclic peptide. 13C NMR analysis of the reaction mixture does not suggest that the thioester intermediates 11 or 12 accumulate to a significant extent at approximately pH 5.8. S-carboxamidomethylated derivatives of the cyclic pep- tides derived from b-defensins retained much of their original activity but, disappointingly, their stability in serum was not significantly enhanced. In contrast we found that the antimicrobial activity of the mirror image cyclic peptide (ent-10, P. aeruginosa, MBC = 5.6 mm) re- mained largely unchanged in 10 % serum (Figure 3). Con- sequently, we believe this cyclic mirror image peptide serves as an excellent lead “scaffold” from which new an- timicrobial agents could be developed. Although cyclic peptides ultimately emerge from the reaction as major products, it is not obvious why this is the case since the reaction is inherently reversible. It is unlikely that the relative stabilities of Xaa–Cys–NH2 and Xaa–Cys–OH alone account for the outcome. Peptides terminating in an Xaa–Cys–NH2 motif still undergo cycli- zation albeit less efficiently, suggesting that the internal nature of the cyclic amide bond within the product is also important. Since water appears to be the best solvent for the reaction, it is possible that the more compact and hy- drophobic nature of the cyclic product also inhibits rever- sion to linear species by excluding water from the reac- tion site. Furthermore, the conformational constraints im- posed by cyclization could present an additional obstacle for the internal Cys, preventing it from attaining an ap- propriate geometry for N!S acyl transfer. Isr. J. Chem. 2011, 51, 885 – 899 2011 Wiley-VCH Verlag GmbH & Co. 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