Total Chemical Synthesis of Palmitoyl-Conjugated Insulin

[Image: see text] Commercially available insulins are manufactured by recombinant methods for the treatment of diabetes. Long-acting insulin drugs (e.g., detemir and degludec) are obtained by fatty acid conjugation at LysB29 ε-amine of insulin via acid–amide coupling. There are three amine groups in insulin, and they all react with fatty acids in alkaline conditions. Due to the lack of selectivity, such conjugation reactions produce non-desired byproducts. We designed and chemically synthesized a novel thiol-insulin scaffold (CysB(29)-insulin II), by replacing the Lys(B29) residue in insulin with the Cys(B29) residue. Then, we conjugated a fatty acid moiety (palmitic acid, C16) to CysB(29)-insulin II by a highly efficient and selective thiol–maleimide conjugation reaction. We obtained the target peptide (palmitoyl-insulin) rapidly within 5 min without significant byproducts. The palmitoyl-insulin is shown to be structurally similar to insulin and biologically active both in vitro and in vivo. Importantly, unlike native insulin, palmitoyl-insulin is slow and long-acting.