Synthesis of human [9-leucine-B] Insulin

The synthesis and isolation in purified form of [Leu9-B]insulin, a biologically active analogue of human insulin, is described. This analogue differs from the parent molecule in that the polar residue, serine, occupying position 9 in the B chain and located on the outside of the insulin molecule, has been replaced with the hydrophobic leucine residue. For the synthesis of this analogue the [Leu9]B chain of human insulin was chemically synthesized by the fragment condensation approach and isolated in the S-sulfonated form. Combination of this compound with the sulfhydryl form of human A chain afforded [Leu9-B]insulin. Separation of this analogue from the combination mixture and isolation as the hydrochloride in purified form were accomplished by chromatography on a carboxymethylcellulose column with an acetate buffer (pH 3.3) and an exponential sodium chloride gradient. [Leu9-B]Insulin possesses a potency of 13-14 IU/mg when assayed by the mouse convulsion method and 11-12 IU/mg by the radioimmunoassay method as compared to 23-25 IU/mg possessed by the natural hormone.