Insulin remains indispensable in the treatment of diabetes, but its use is hampered by its narrow therapeutic index. Although advances in peptide chemistry and recombinant DNA-based macromolecule synthesis have enabled the synthesis of structurally optimized insulin analogues, the growing epidemics of obesity and diabetes have emphasized the need for diabetes therapies that are more efficacious, safe and convenient. Accordingly, a broad set of drug candidates, targeting hyperglycaemia plus other disease abnormalities, is now progressing through the clinic. The development of an insulin therapy that is responsive to glucose concentration remains an ultimate goal, with initial prototypes now reaching the proof-of-concept stage. Simultaneously, the first alternatives to injectable delivery have progressed to registration.

Insulin remains a challenging synthetic target due in large part to its two-chain, disulfide-constrained structure. Biomimetic single chain precursors inspired by proinsulin that utilize short peptides to join the A and B chains can dramatically enhance folding efficiency. Systematic chemical analysis of insulin precursors using an optimized synthetic protocol identified a 49 amino acid peptide named DesDi, which folds with high efficiency by virtue of an optimized structure and could be proteolytically converted to bioactive two-chain insulin. In subsequent applications, we observed that the folding of the DesDi precursor was highly tolerant to amino acid substitution at various insulin residues. The versatility of DesDi as a synthetic insulin precursor was demonstrated through the preparation of several alanine mutants (A10, A16, A18, B12, B15), as well as ValA16, an analog that was unattainable in prior reports. In vitro bioanalysis highlighted the importance of the native, hydrophobic residues at A16 and B15 as part of the core structure of the hormone and revealed the significance of the A18 residue to receptor selectivity. We propose that the DesDi precursor is a versatile synthetic intermediate for the preparation of diverse insulin analogs. It should enable a more comprehensive analysis of function to insulin structure than might not be otherwise possible through conventional approaches.

The synthesis of insulin has inspired fundamental advances in the art of peptide science while simultaneously revealing the structure-function relationship of this centrally important metabolic hormone. This review highlights milestones in the chemical synthesis of insulin that can be divided into two separate approaches: (i) disulfide bond formation driven by protein folding and (ii) chemical reactivity-directed sequential disulfide bond formation. Common to the two approaches are the persistent challenges presented by the hydrophobic nature of the individual A-chain and B-chain and the need for selective disulfide formation under mildly oxidative conditions. The extension and elaboration of these synthetic approaches have been ongoing within the broader insulin superfamily. These structurally similar peptides include the insulin-like growth factors and also the related peptides such as relaxin that signal through G-protein-coupled receptors. After a half-century of advances in insulin chemistry, we have reached a point where synthesis is no longer limiting structural and biological investigation within this family of peptide hormones. The future will increasingly focus on the refinement of structure to meet medicinal purposes that have long been pursued, such as the development of a glucose-sensitive insulin. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

The use of carboxylate side chains to induce peptide helicity upon binding to dirhodium centers is examined through experimental and computational approaches. Dirhodium binding efficiently stabilizes alpha helicity or induces alpha helicity in otherwise unstructured peptides for peptides that contain carboxylate side chains with i, i+4 spacing. Helix induction is furthermore possible for sequences with i, i+3 carboxylate spacing, though in this case the length of the side chains is crucial: ligating to longer glutamate side chains is strongly helix inducing, whereas ligating the shorter aspartate side chains destabilizes the helical structure. Further studies demonstrate that a dirhodium metallopeptide complex persists for hours in cellular media and exhibits low toxicity toward mammalian cells, enabling exploitation of these metallopeptides for biological applications.

Selective bond formation of biomolecule substrates requires synthetic chemistry in an aqueous, functional-grouprich environment to alter and control the structure, function, and aggregation state of biomolecules. Ideally, new methods would be non-denaturing, would utilize chemistry that is orthogonal to existing methods, [1] and would be reversible in response to external stimuli. We here report a reversible method for addressing and bridging glutamate and aspartate carboxylates under mild aqueous conditions.One powerful way to affect polypeptide structure is through methods that link, or bridge, two amino-acid side chains to form a cyclic product, but there exist few methods to selectively and reversibly bridge amino acid side chains. Cysteine is commonly used as a handle for selective bond formation through redox-mediated formation of disulfide linkages [2] or selective alkylation reactions, [3] but working with cysteine-containing peptides can be difficult. Other methods to address naturally occurring amino acids include activated esters of organic dicarboxylic acids for cross-linking lysine residues, but reversibility in this case is limited.Metal ions serve structural roles in metalloproteins, where side chains serve as ligands which are bridged by a metal ion to create a folded metal-binding pocket. Taking a cue from these biological examples, the effects of metal binding on peptide structures is an active area of study. [4][5][6] Peptidemetal interactions have been used to understand metalloprotein folding and energetics and to shed light on potential toxicity pathways. [7] Many transition metals can bind to peptides in aqueous solution, most commonly through cysteine or histidine residues. [5,8] Although metalloproteins can bind extremely tightly to metal ions, smaller designed polypeptides often bind metal ions dynamically in aqueous solution, and systems involving a small number of binding groups often require excesses of metal ion in solution. We set out to develop a robust metal-binding method that would selectively address side chain functional groups in a manner complementary to extant methods. Carboxylates are common, naturally occurring polypeptide side chains, and the dirhodium-carboxylate interaction is sufficiently stable that ligand exchange is not observed under a variety of biologically relevant conditions. Selective binding to polycarboxylate regions has been observed with lanthanides, [9] but well defined bridging of a small number of carboxylate side chains has not been demonstrated.We set out to explore the ability of dirhodium tetracarboxylates to bind chemoselectively and tightly to side chain carboxylates and hoped to develop a reversible metal ligation protocol to link two carboxylate side chains under nondenaturing conditions. In contrast to equilibrium binding exhibited in many peptide-metal binding interactions the robust carboxylate À dirhodium bond allows us to enforce structural changes of an isolable peptide À metal adduct.