Semisynthesis and biological activity of porcine [LeuB24]insulin and [LeuB25]insulin.

Abstract

Two analogs of porcine insulin with substitutions of Leu for Phe in the COOH-terminal region of the insulin B chain were prepared by a combination of solid-phase synthesis and semisynthesis. Solid-phase synthesis of the substituted octapeptides B23-B30 bearing the trifluoroacetyl group on Lys-B29, enzymatic coupling of the octapeptides to bis(tert-butyloxycarbonyl)desoctapeptide insulin by trypsin, and deprotection of the corresponding addults in formic acid and piperidine resulted in 2 insulin derivatives, one with Leu at position B24 and the other with Leu at position B25. These analogs had only about 10% and 1%, respectively, of the activity of porcine insulin in competing for the binding of [125I]iodoinsulin to both rat adipocytes and human IM-9 lymphocytes. The relative potencies of the analogs in stimulating glucose oxidation by rat adipocytes decreased in the order porcine insulin > [LeuB24]insulin > [LeuB25]insulin. A high concentrations both analogs had full agonist activity. Experiments in which the semisynthetic insulins were mixed with the native hormone showed that [LeuB24]insulin, but not [LeuB25]insulin, was an active antagonist of insulin action. The antagonistic activity of a human insulin variant having Leu at position B24 or B25 can be assigned to the molecule with the sequence Gly-Leu-Phe-Tyr (residues B23-B26) in its active site.