Total Synthesis of desB30 Insulin Analogues by Biomimetic Folding of Single‐Chain Precursors

Abstract

Insulin is a peptide hormone consisting of 51 amino acids in two chains with three disulfide bridges. Human insulin and various analogues are used for the treatment of diabetes and are produced recombinantly at ton scale. Herein, we report the chemical synthesis of insulin by the step-wise, Fmoc-based, solid-phase synthesis of single-chain precursors with solubilising extensions, which under redox conditions, spontaneously fold with the correct pairing of the three disulfide bridges. The folded, single-chain, insulin precursors can be transformed into bioactive two-chain desB30 insulin by the simultaneous removal of the solubilising extension (4-5 residues) and the chain-bridging C-peptide (3-5 residues) by employing Achromobacter lyticus protease--a process well-known from the yeast-based recombinant production of insulin. The overall yields of synthetic insulins were as much as 6 %, and the synthetic process was straightforward and not labour intensive.

Discovery of High Potency, Single-Chain Insulin Analogs with a Shortened B-Chain and Nonpeptide Linker

“…This single chain insulin analog that was based entirely on the insulin sequence proved more difficult to prepare and was only obtained with the aid of an N-terminal polyglutamic acid leader sequence (peptide 20) that was removed proteolytically. 29 This demonstrates the biophysical virtue of IGF-1 based analogs. The additional amino acids at the N-terminal end of peptide 20 did not change activation at the insulin and IGF-1 receptors.…”

Recombinant expression, refolding, purification and characterization of Pseudomonas aeruginosa protease IV in Escherichia coli

“…These proteases robustly and specifically recognize and cleave C-terminal to the basic amino acid lysine, even following with proline or glutamic acid, making them useful enzymes for various applications, including proteomic sample preparation and human insulin production [2,5,6]. Current proteomic grade protease IV products of high purity and activity supplied by vendors such as Wako, and others, are extracted from the native bacteria L. enzymogenes using an inefficient process [2,7].…”

Synthesis of disulfide-rich heterodimeric peptides through an auxiliary N, N-crosslink

“…They share the native linear order of proinsulin where the N-terminus of the A-chain is indirectly connected to the C-terminus of the B-chain. Conversion to the two-chain form initially employed enzymatic conversion and was restricted by the requirement for a unique proteolytic site [11][12][13][14][15] . The more recent reports employing chemically labile linkers represent a leap forward by eliminating the need for a proteolytic site, and the enzyme itself.…”