Novel Methods for the Chemical Synthesis of Insulin Superfamily Peptides and of Analogues Containing Disulfide Isosteres

Abstract

The insulin superfamily of peptides is ubiquitous within vertebrates and invertebrates and is characterized 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.

Chemical synthesis of the crustacean insulin‐like peptide with four disulfide bonds

“…In our previous study, we used DPDS in trifluoromethanesulfonic acid (TfOH)/TFA solution for deprotecting MeOBn group . However, this reaction conditions were quite similar to those for the removal of Bu t group reported previously . In order to remove MeOBn group specifically, the reaction conditions were examined.…”

Chemical synthesis of N‐glycosylated insulin‐like androgenic gland factor from the freshwater prawn Macrobrachium rosenbergii

“…Among insulin‐superfamily peptides, the positions of six cysteine residues are well conserved, and these residues form three disulfide bonds. Chemical syntheses of insulin‐superfamily peptides have been achieved by many research groups, and the synthetic method is well established …”

Bridged bicyclic peptides: Structure and function

“…The triple cross‐linked peptide dimer motif ( 3 ) has many different topological subsets, and is nearly always encountered as a multiply disulfide‐linked system (e.g., insulin family peptides, conotoxins). Such peptides will not be covered in this review: the topology of such peptides are interconvertible, some of which are not bridged bicycles, and further these systems have been extensively reviewed elsewhere …”

“…As described in the introduction, multiply cystine‐linked bicyclic peptides will not be covered in this review as these systems can be topologically unstable and have been extensively reviewed elsewhere . Nevertheless, modified cross‐links have been developed to solve the issue of disulfide scrambling in such peptides.…”