826 Mol. BioSyst., 2013, 9, 826--833 This journal is c The Royal Society of Chemistry 2013 Cite this: Mol. BioSyst., 2013, 9, 826 Synthetic cysteine surrogates used in native chemical ligation Clarence T. T. Wong, Chun Ling Tung and Xuechen Li* Native chemical ligation (NCL) has become the method of choice in synthesizing large or cyclic peptides/proteins. To overcome the limitation of NCL requiring N-terminal cysteine to mediate ligation, a strategy involving thiol-mediated ligation followed by desulfurization has been developed and advanced to realize peptide ligation at other amino acid sites, including Phe, Val, Leu, Thr, Lys, Pro and Gln. The syntheses of these mercapto-containing unnatural amino acids used as cysteine surrogates will be discussed in this review article. Introduction Over the past several decades, the methods in synthesizing polypeptides have been greatly advanced. 1 Owing to Merrifield’s solid phase peptide synthesis (SPPS), the chemical synthesis of peptides has become a routine practice in most research laboratories. 2 However, SPPS cannot meet the increasing need for the synthetic large-sized polypeptides and even proteins, as it is more suitable for the target peptide chains of a length below B50 amino acids. Therefore, the strategies 3 involving solid phase peptide synthesis followed by the in-solution fragment coupling have been introduced to facilitate the chemical synthesis of large polypeptides. Among all the in-solution fragment condensation reactions, the most successful method is native chemical ligation (NCL) developed by Kent et al. in 1994. 4 It involves a chemoselective reaction between a side-chain unprotected peptide segment with a C-terminal thioester and a second side-chain unprotected peptide segment with a N-terminal cysteine residue (Scheme 1a). The distinct nucleo- philicity of the thiol group on cysteine is capable of initiating a highly specific transthioesterification with the peptide thioester in the presence of all the side-chain unprotected functional groups. Then, an S-to-N acyl transfer reaction produces the more stable amide product, enabling the formation of the natural peptidic bond (Xaa–Cys) (Xaa represents any natural amino acid) at the ligation site. NCL has allowed many proteins, including those carrying posttranslational modifications, to be Department of Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong, China. E-mail: xuechenl@hku.hk Clarence T. T. Wong Clarence T. T. Wong obtained his BSc in Biochemistry from The Chinese University of Hong Kong and received his PhD in 2012 in Biological Sciences from The Nanyang Technological University of Singapore under the supervision of Prof. James P. Tam. He is currently working in the group of Dr X. C. Li at The University of Hong Kong, appointed as a post- doctoral fellow in 2012. His current research interests include design and characterization of bioactive peptides and the development of methodology in cyclic peptide synthesis. Chun Ling Tung Chun Ling Tung received his BSc in Chemistry and Biology from the University of Indianapolis, USA. He started his PhD in 2011 in The University of Hong Kong under the supervision of Dr X. C. Li. His current research concentrates on the peptide chemistry and post-translational modifications of peptides and proteins. Received 12th October 2012, Accepted 10th December 2012 DOI: 10.1039/c2mb25437a www.rsc.org/molecularbiosystems Molecular BioSystems REVIEW Published on 12 December 2012. Downloaded by University of Hong Kong Libraries on 02/12/2013 14:13:54. View Article Online View Journal | View Issue