Enzymatic semisynthesis of [Leu B30] insulin

Experimental conditions for the preparation of [Leu^B30] insulin by coupling of des-Ala^B30 insulin with Leu-OBu^t were determined using Achromobacter protease I and trypsin as catalysts. Successful coupling required a large excess of the amine component (0.8 M), a high concentration of organic cosolvent (35–50%) and neutral pH of the reaction mixture. The coupling yield of Achromobacter protease I after 24 h at 37°C was almost the same or a little higher than that at 25°C. With trypsin, the coupling yield at 37°C after 24 h was considerably lower than at 25°C. This was partly ascribed to the difference in concentration of organic cosolvent at 37°C and 25°C; 35% and 50%, respectively, or possibly of enzyme stability at these temperatures. The maximum product yield was about 90% with both enzymes under optimal conditions. A preparative scale experiment was performed with Achromobacter protease I; the yield of [Leu^B30] insulin was 51% using porcine insulin as the starting material. This semisynthetic insulin was identified by HPLC and amino acid analysis. No difference was observed in CD spectra between [Leu^B30] insulin and human insulin.