Synthesis of hydrophobic insulin-based peptides using a helping hand strategy

Abstract

The introduction of solid-phase peptide synthesis in the 1960s improved the chemical synthesis of both the A- and B-chains of insulin and insulin analogs. However, the subsequent elaboration of the synthetic peptides to generate active hormones continues to be difficult and complex due in part to the hydrophobicity of the A-chain. Over the past decade, several groups have developed different methods to enhance A-chain solubility. Two of the most popular methods are use of isoacyl dipeptides, and the attachment of an A-chain C-terminal pentalysine tag with a base-labile 4-hydroxymethylbenzoic acid linker. These methods have proven effective but can be limited in scope depending on the peptide sequence of a specific insulin. Herein we describe an auxiliary approach to enhance the solubility of insulin-based peptides by incorporating a tri-lysine tag attached to a cleavable Fmoc-Ddae-OH linker. Incorporation of this linker, or "helping hand", on the N-terminus greatly improved the solubility of chicken insulin A-chain, which is analogous to human insulin, and allowed for coupling of the insulin A- and B-chain via directed disulfide bond formation. After formation of the insulin heterodimer, the linker and tag could be easily removed using a hydrazine buffer (pH 7.5) to obtain an overall 12.6% yield based on A-chain. This strategy offers an efficient method to enhance the solubility of hydrophobic insulin-based peptides as well as other traditionally difficult peptides.

Fig. 1

Current methods to increase solubility of insulin-based peptides. (A) Liu et al. Isoacyl peptide method using a threonine and serine to perform O-to-N acyl shift. (B) Hossain et al. a C-terminus 4-hydroxymethoxybenzoic linker with pentalysine tag. (C) Our method using a N-terminus Fmoc-Ddae-OH linker with trilysine tag.

Fig. 2

Human insulin sequence versus chicken insulin sequence. A-chain has an amino acid sequence homology of 85% and B-chain has an 87% sequence identity.

Fig. 3

pAKT cell-based assay measuring the bioactivity of chicken insulin versus human insulin in NIH 3T3 cells overexpressing the human insulin receptor. (Native Insulin EC₅₀ = 3.27 nM, Chicken Insulin EC₅₀ = 3.33 nM).

Fig. 4

Phospho-IR beta cell-based assay measuring the endogenous levels of insulin receptor beta only when phosphorylated at Tyr 1150/1151 when stimulated by human insulin versus chicken insulin in NIH 3T3 cells overexpressing the human insulin receptor. (Native Insulin EC₅₀ = 22.20 nM, Chicken Insulin EC₅₀ = 10.54 nM).

Scheme 1

Synthesis of Fmoc-Ddae-OH linker. (A) Reaction conditions previously reported in the literature. (b) Reaction conditions using DCC produced the highest yield, however N, N’ dicyclohexylurea byproduct formation made purification difficult. (c) Reaction conditions with little byproduct formation and high yield consisted of N-Fmoc-amido dPEG-acid, 1.1 eq. dimedone, 1.1 eq. EDC-HCl, 1.1 eq. DIEA and 0.1 eq. DMAP in DCM.

Scheme 2

Synthesis of chicken insulin and coupling of Fmoc-Ddae-OH to the N-terminus of the A-chain. (i) Standard tBu/Fmoc SPPS protocol. (ii) 0.2 mmol Fmoc-Ddae-OH (1), 25uM resin scale in NMP, 4h RT. (iii) 20% piperidine in DMF. (iv) Standard tBu/Fmoc SPPS protocol.

Scheme 3

Orthogonal deprotection and cleavage of chicken insulin A-chain with Fmoc-Ddae-OH linker and trilysine tag.

Scheme 4

Orthogonal deprotection and cleavage of chicken insulin B-chain

Scheme 5

Three-step combination protocol for removal of solubility tag and linker after formation of the first intermolecular bridge (12) and removal of solubility tag and linker after formation of the second intermolecular bridge (13).