Novel Methods for the Chemical Synthesis of Insulin Superfamily Peptides and of Analogues Containing Disulfide Isosteres Published as part of the Accounts of Chemical Research special issue “Chemical Biology of Peptides”. Mohammed Akhter Hossain* and John D. Wade* The Florey Institute of Neuroscience and Mental Health and School of Chemistry, University of Melbourne, Melbourne, Victoria 3010, Australia CONSPECTUS: The insulin superfamily of peptides is ubiquitous within vertebrates and invertebrates and is charac- terized by the presence of a set of three disulfide bonds in a unique disposition. With the exception of insulin-like growth factors I and II, which are single chain peptides, the remaining 8 members of the human insulin superfamily are two-chain peptides containing one intramolecular and two intermolecular disulfide bridges. These structural features have long made the chemical synthesis of the peptides a considerable challenge, in particular, including their correct disulfide bond pairing and formation. However, they have also afforded the opportunity to develop modern solid phase synthesis methods for the preparation of such peptides that incorporate novel or improved chemical methods for the controlled introduction of both disulfide bonds and their surrogates, both during and after peptide chain assembly. In turn, this has enabled a detailed probing of the structure and function relationship of this small but complex superfamily of peptides. After initially using and subsequently identifying significant limitations of the approach of simultaneous random chain combination and oxidative folding, our laboratory undertook to develop robust chemical synthesis strategies in concert with orthogonal cysteine S-protecting groups and corresponding regioselective disulfide bond formation. These have included the separate synthesis of each of the two chains or of the two chains linked by an artificial C-peptide that is removed following postoxidative folding. These, in turn, have enabled an increased ease of acquisition in a good yield of not only members of human insulin superfamily but other insulin-like peptides. Importantly, these successful methods have enabled, for the first time, a detailed analysis of the role that the disulfide bonds play in the structure and function of such peptides. This was achieved by selective removal of the disulfide bonds or by the judicious insertion of disulfide isosteres that possess structurally subtle variations in bond length, hydrophobicity, and angle. These include lactam, dicarba, and cystathionine, each of which has required modifications to the peptide synthesis protocols for their successful placement within the peptides. Together, these synthesis improvements and the novel chemical developments of cysteine/cystine analogues have greatly aided in the development of novel insulin-like peptide (INSL) analogues, principally with intra-A-chain disulfide isosteres, possessing not only improved functional properties such as increased receptor selectivity but also, with one important and unexpected exception, greater in vivo half-lives due to stability against disulfide reductases. Such analogues greatly will aid further biochemical and pharmacological analyses to delineate the structure-function relationships of INSLs and also future potential drug development. ■ INTRODUCTION The then unexpected finding that the glycemic hormone, insulin, consists of not one but two peptide chains, A and B, linked by three disulfide bonds in a unique disposition was a milestone in protein chemistry and biochemistry. 1 The disulfide cross- bridging comprising an intra-A-chain and two interchain disulfide bonds was soon recognized to represent a signature structural feature (Figure 1A). 2 Early protein chemistry studies followed by genomic or expressed sequence tag database sequencing has led to the continuing identification of numerous insulin-like peptides in both vertebrates and invertebrates. 3-5 In Homo sapiens, the insulin superfamily consists of ten members: insulin, insulin-like growth factors (IGF) I and II, and a relaxin subfamily that comprises relaxin-1, -2, and -3 and four other insulin-like peptides (INSL 3, 4, 5, and 6). 3,5,6 IGFs I and II are unique in that they both possess a single peptide chain structure that is cross-linked by three disulfide bonds in the same disposition as for insulin. Whereas insulin and IGFs I and II interact with tyrosine kinase receptors, the members of the relaxin subfamily interact with G-protein-coupled receptors known as relaxin family peptide receptors. 7 The diversity of biological activity within the broad superfamily is striking, ranging from metabolic control through to gubernacular Received: June 9, 2017 Article pubs.acs.org/accounts © XXXX American Chemical Society A DOI: 10.1021/acs.accounts.7b00288 Acc. Chem. Res. XXXX, XXX, XXX-XXX