Biomimetic Synthesis of Lispro Insulin via a Chemically Synthesized "Mini-Proinsulin" Prepared by Oxime-Forming Ligation
Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA. Journal of the American Chemical Society
(Impact Factor: 12.11).
11/2009; 131(44):16313-8. DOI: 10.1021/ja9052398
Here we report a proof-of-principle study demonstrating the efficient folding, with concomitant formation of the correct disulfides, of an isolated polypeptide insulin precursor of defined covalent structure. We used oxime-forming chemical ligation to introduce a temporary "chemical tether" to link the N-terminal residue of the insulin A chain to the C-terminal residue of the insulin B chain; the tether enabled us to fold/form disulfides with high efficiency. Enzymatic removal of the temporary chemical tether gave mature, fully active insulin. This chemical tethering principle could form the basis of a practical, high yield total synthesis of insulin and analogues.
Available from: Ming Chen
- "These peptides are functionalized with groups that react chemoselectively with only one group of the acceptor peptide preserving the integrity of unprotected side chains, i.e., using these ligation methods unprotected peptide segments can be coupled efficiently to form long peptide chains and even large proteins, usually in good yield (42–95 % total yield) and of high purity (Böhm et al. 2012; Raibaut et al. 2013; Bodapati et al. 2013). The methods range from thioester ligation to imine ligation (Tam et al. 1999, 2001), and several peptides and proteins such as cytochrome b562 (106 AAs) (Low et al. 2001), His-tagged interleukin-2 (133 AAs) (Tolbert et al. 2005), and insulin lispro (51 AAs) (Sohma and Kent 2009) were produced in this way. "
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ABSTRACT: This review focuses on chemical ligation methods for the preparation of oligopeptides and proteins. Chemical ligation is a practical and convenient methodology in peptide and protein synthesis. Longer peptides and proteins can be obtained with high yield in aqueous buffer solutions by coupling unprotected peptide segments even without activation by enzymes or further chemical agents. Several methods and protocols were developed in the past. The potential of the most important approaches of the thioester- and imine-ligation techniques is demonstrated by a broad spectrum of applications. In addition, special features and protocols such as the template-directed ligation, ligation with novel additives or solvent media, microwave-assisted ligation, and the achievements obtained with those are also highlighted herein.
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ABSTRACT: (Figure Presented). Insulin folding: An ester-linked polypeptide proinsulin surrogate folded efficiently with concomitant disulfide bond formation, and saponification gave native insulin having full biological activity. This strategy overcomes the low yield combi- nation of individual insulin A and B chains, and provides a simple and effective approach to the total chemical synthesis of human insulin and its analogues.
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