Semisynthesis of A6-A11 lactam insulin

Abstract

Insulin replacement therapy is essential for the management of diabetes. However, despite the relative success of this therapeutic strategy, there is still a need to improve glycaemic control and the overall quality of life of patients. This need has driven research into orally available, glucose-responsive and rapid-acting insulins. A key consideration during analogue development is formulation stability, which can be improved via the replacement of insulin's A6-A11 disulfide bond with stable mimetics. Unfortunately, analogues such as these require extensive chemical synthesis to incorporate the nonnative cross-links, which is not a scalable synthetic approach. To address this issue, we demonstrate proof of principle for the semisynthesis of insulin analogues bearing nonnative A6-A11 cystine isosteres. The key feature of our synthetic strategy involves the use of several biosynthetically derived peptide precursors which can be produced at scale cost-effectively and a small, chemically synthesised A6-A11 macrocyclic lactam fragment. Although the assembled A6-A11 lactam insulin possesses poor biological activity in vitro, our synthetic strategy can be applied to other disulfide mimetics that have been shown to improve thermal stability without significantly affecting activity and structure. Moreover, we envisage that this new semisynthetic approach will underpin a new generation of hyperstable proteomimetics.

Keywords: cystine mimetic; diabetes; disulfide bond; insulin; peptide synthesis; semisynthesis.

FIGURE 1

Structure of native human insulin, with its two peptide chains and three disulfide bonds.

SCHEME 1

Synthesis of the A6–A11 lactam insulin. (A) 1 eq. COMU, 10 equation N‐methylmorpholine, DMF, 25°C, 18 h (1 pre‐activated). (B) 30 eq. piperidine, 10 min. (C) Preparative RP‐HPLC. (D) 10 equation N‐methylmorpholine, DMF, 25°C, 2 h. (E) Preparative RP‐HPLC. (F) 10 eq. DPDS, 2% H₂O, 2% thioanisole, 96% TFA, 25°C, 1 h. (G) Preparative RP‐HPLC. (H) Aqueous 6 M guanidinium hydrochloride, 0.2 M Na₂HPO₄, pH 7, 25°C, 30 min. (I) Preparative RP‐HPLC.

SCHEME 2

Synthesis of the protected A6–A11 lactam fragment. (A) 3% TFA, CH₂Cl₂, 25°C, 18 h. (B) 2 eq. COMU, 5 eq. DIEA, CH₂Cl₂, 25°C, 2 h. (C) Preparative RP‐HPLC. (D) 5 eq. SnMe₃OH, dichloroethane, 55°C, 24 h. (E) Preparative RP‐HPLC.

FIGURE 2

Analytical data of the protected ZR‐A6–A11 lactam fragment synthesis. (A) RP‐HPLC of crude 10 (linear material). (B) RP‐HPLC of the crude 11 (macrocyclic product). (C) RP‐HPLC of purified 11. (D) RP‐HPLC of crude 1 (hydrolysed product). (E) RP‐HPLC of purified 10. (F) ESI‐MS of purified 1 ([M+H⁺]⁺).

FIGURE 3

Analytical data of A6–A11 lactam insulin assembly. (A) RP‐HPLC of Fmoc‐protected 3*. (B) RP‐HPLC of crude 3 (Fmoc deprotected). (C) RP‐HPLC of purified 3. (D) RP‐HPLC of protected 5. (E) RP‐HPLC of crude 6 (bis‐pyridylsulfenyl A‐chain). (F) RP‐HPLC of purified 6. (G) RP‐HPLC of crude 8 (two isomers). Zoomed inset: A = A6‐A11 lactam insulin isomer A (8A), B = A6 A11 lactam insulin isomer B (8B), C = reduced B‐chain, D = oxidised B‐chain, E = bicyclic A‐chain. 1. (H) RP‐HPLC of purified 8A. (I) RP‐HPLC of purified 8B. J ESI‐MS of purified 8B ([M+4H⁺]⁴⁺, [M+5H⁺]⁵⁺, [M+6H⁺]⁶⁺).

FIGURE 4

Analysis of the insulin lactam isomers and native insulin. (A) CD spectra. (B) Thermal stability assay (PBS at pH 7.4, 70°C). (C) IR phosphorylation.

FIGURE 5

Chromatographic analysis of the ZR lactam insulin isomers co‐injected with the analogue formed through regioselective disulfide bond formation. (A) Isomer ‘A’. (B) Isomer ‘B’.