Chemical Synthesis of Insulin Analogs through a Novel Precursor

Abstract

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.

Insulin—From its Discovery to the Industrial Synthesis of Modern Insulin Analogues

“…[62] Analysis of its X-ray structure [63] indicates that Lys B28 is located in proximity to the N-terminus of the A-chain. [64] Therelated 49 residue polypeptide named the DesDi precursor (1)w as compared with the single-chain mini-proinsulin (2) [31] and with the porcine insulin precursor PIP (3) [60] in its ability to fold into the correct insulin disulfide framework. [64] Therelated 49 residue polypeptide named the DesDi precursor (1)w as compared with the single-chain mini-proinsulin (2) [31] and with the porcine insulin precursor PIP (3) [60] in its ability to fold into the correct insulin disulfide framework.…”

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

“…These conditions were applicable to the native hormones and translated to a synthetic target that had previously required orthogonal stepwise synthesis, a four-disulfide containing insulin analog 22 . The successful syntheses of two individual penicillamine substituted insulin analogs, that we could not prepare by native folding using a bio-mimetically linked insulin precursor 15 , demonstrate a unique virtue to this synthetic approach. The analogs complete an otherwise full set of selective penicillamine substitutions for each of the native cysteines (Table 1).…”

“…The cleavage of the DKP-peg-bis linker was achieved in 9 h (pH 7.0, 56°C), to provide analogs 4 and 5 in total yields of 30% and 28% (Supplementary Figures 43 and 48 Supplementary Figure 11). The other four single-site, penicillamine insulin analogs (A6, A11, A20, and B7) were chemically synthesized using a linear desDI single-chain precursor without issue, (Supplementary Figures 55-59) 15 . The A7 and B19 proved to be synthetically accessible only by the Ntermini ligation approach we describe in this manuscript.…”

“…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.…”

“…The efficiency and versatility of the method are demonstrated in the synthetic yield of insulin and the direct translation to the synthesis of relaxin. The non-native N-N linkage enables the synthesis of two site-specific penicillamine-substituted analogs 4 and 5, that fail when using a high-efficiency N-C folding intermediate, named des-DI insulin 15 . This synthetic approach is based upon an orthogonal, non-native N-N linkage of individual peptide chains that is synthetically straightforward and of high efficiency in synthesis of insulin-like peptides.…”