Review DOI: 10.1002/ijch.201100084 Shifting Native Chemical Ligation into Reverse through N S Acyl Transfer ! Derek Macmillan,*[a] Anna Adams,[a] and Bhavesh Premdjee[a] Abstract : Peptide thioester synthesis by N!S acyl transfer is being intensively explored by many research groups the world over. Reasons for this likely include the often straight- forward method of precursor assembly using Fmoc-based chemistry and the fundamentally interesting acyl migration process. In this review we introduce recent advances in this exciting area and discuss, in more detail, our own efforts to- wards the synthesis of peptide thioesters through N!S acyl transfer in native peptide sequences. We have found that several peptide thioesters can be readily prepared and, what’s more, there appears to be ample opportunity for fur- ther development and discovery. Keywords: acyl-transfer · native chemical ligation · peptides · protein modifications · thioester 1. Introduction The importance of peptides and proteins in biology and medicine has inspired chemists to consider methods that bring about their synthesis for over a century.[1] In most popular modern methods the peptide thioester, whether produced explicitly or transiently, plays a prominent role in the assembly of proteins and their analogues using con- vergent coupling strategies.[2] Native Chemical Ligation (NCL)[2b] has proved particularly useful, since it provides access to native proteins by coupling peptide thioesters with an additional component which usually, but not nec- essarily,[3] contains an N-terminal cysteine. Synthetic chemists have optimized efficient routes to peptide thio- esters using tert-butyloxycarbonyl (Boc)-based methods of solid phase peptide synthesis (SPPS) and, until more recently, 9-fluorenylmethoxycarbonyl (Fmoc)-based methods lagged behind.[4] This is because thioesters them- selves are not generally stable to the basic reaction condi- tions commonly employed for Fmoc removal and so a less direct path to thioesters is often required. Despite this obstacle, researchers have sought to develop efficient methods for thioester synthesis using Fmoc-based chemis- try. The milder reaction conditions employed are general- ly considered more compatible with a variety of post- translational modifications and the additional, and some- times chemically fragile, functionality embedded within various molecular probes.[4b] Using Fmoc-based SPPS, the thioester is usually isolat- ed following chain assembly and activation of safety- catch resins,[5] formed after release of the otherwise fully protected peptides,[6] or formed from N-acyl urea termi- nated peptides[7] (Scheme 1). Each method appears to have its own advantages and disadvantages with respect to chemical yield, ease of monitoring, and user friendli- ness, but those described above currently appear the most reliable for the “routine” formation of long (> 25 amino acid residues) peptide thioesters. 1.1. N!S Acyl Transfer Recently, a number of methods that explore thioester for- mation via O!S[8] or N!S[9] acyl shift have been report- ed. Methods that involve N!S acyl shift seem particular- ly attractive since the nature of the acyl transfer-facilitat- ing “device” can be simplified to cysteine and cysteine analogues or derivatives (Scheme 2). Furthermore, in some cases (Scheme 2 a, 2 c, and 2 d) a thioester precursor can be used directly in ligation experiments, producing the thioester only transiently as with N-acyl urea termi- nated peptides. In most cases N!S acyl shift precursors contain a b- amino thiol motif. Since the equilibrium (depicted in Scheme 2) between the amide form of the peptide and the less stable thioester form (S-peptide) is expected to reside far to the left, this basic motif is “activated” by a number of attached groups. In each case the added group is believed to destabilize the scissile amide bond, making it more prone to thiolysis, and this is achieved in a number of ways. Generally: [a] D. Macmillan, A. Adams, B. Premdjee Christopher Ingold Laboratories, Department of Chemistry University College London 20 Gordon Street, London WC1H 0AJ, UK phone: + 44 (0)20 7679 4684 e-mail: d.macmillan@ucl.ac.uk Isr. J. Chem. 2011, 51, 885 – 899 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 885