Facile folding of insulin variants bearing a prosthetic C-peptide prepared by α-ketoacid-hydroxylamine (KAHA) ligation

Abstract

The chemical synthesis of insulin is an enduring challenge due to the hydrophobic peptide chains and construction of the correct intermolecular disulfide pattern. We report a new approach to the chemical synthesis of insulin using a short, traceless, prosthetic C-peptide that facilitates the formation of the correct disulfide pattern during folding and its removal by basic treatment. The linear precursor is assembled by an ester forming α-ketoacid-hydroxylamine (KAHA) ligation that provides access to the linear insulin precursors in good yield from two readily prepared segments. This convergent and flexible route provides access to various human, mouse, and guinea pig insulins containing a single homoserine mutation that shows no detrimental effect on the biological activities.

Scheme 1. Folding of insulin variants directed by a prosthetic C-peptide.

Scheme 2. (a) Synthesis of building blocks with incorporated cleavable linkers. N-methylmorpholine (NMM) (b) synthesis of peptide segment 6 and incorporation of linker building block 5a by standard Fmoc-SPPS.

Scheme 3. (a) Sequences of synthetically prepared insulin variants. (b) Chemical synthesis of M2 insulin variant (11). (c) Course of KAHA ligation monitored by analytical RP HPLC. (d) Course of the peptide folding and O → N shift. Bottom HPLC trace of purified folding precursor (9). Middle HPLC trace after 12 hours folding and 4 hours basic incubation. Top HPLC trace of purified folded peptide (10). (e) HPLC of purified M2 insulin variant (11). (f) HR MS trace of purified M2 insulin variant (11). (g) CD spectra of 11.

Scheme 4. (a) Sequences of synthetically prepared human insulin variant. (b) Chemical synthesis of human insulin variant. (c) Course of KAHA ligation monitored by analytical RP-HPLC. (d) Course of the peptide folding and O → N shift. Bottom RP-HPLC trace of purified folding precursor (14). Middle RP-HPLC trace after 12 hours folding and 4 hours basic incubation. Top RP-HPLC trace of purified folded peptide (15). (e) RP-HPLC of purified human insulin variant (16). (f) HR MS trace of purified human insulin variant (16). (g) CD spectra of 16.

Fig. 1. (a) Insulin receptor phosphorylation of recombinant insulin (gray, EC₅₀ = 7.43 nM) synthetic human insulin (16) variant (black, EC₅₀ = 3.01 nM). (b) Insulin receptor phosphorylation of recombinant insulin (gray, EC₅₀ = 6.34 nM) synthetic M2 (11) variant (black, EC₅₀ = 3.41 nM). (c) Insulin receptor phosphorylation of recombinant insulin (gray, EC₅₀ = 7.43 nM) synthetic mouse insulin (33) variant (black, EC₅₀ = 23.67 nM). (d) Insulin receptor phosphorylation of recombinant insulin (gray, EC₅₀ = 12.36 nM) synthetic guinea pig insulin (39) variant (black, EC₅₀ > 100 nM).