NBl-C16-Insulin: Site-Specific Synthesis,Purification, and Biological Activity

Purpose. To develop a simple and efficient method for the synthesis and purification of NB1-lipid-modified-insulin without the use of protecting agents.Methods. Bovine insulin was allowed to react with cis-9-hexadecenal in an aqueous-organic medium in the presence of NaBH₃CN at room temperature overnight. HPLC and ESI LC/MS coupled with dithiothreitol and trypsin treatment were employed for product identification and optimization. The product was purified by a differential C18 solid-phase extraction. The biological effects of the modified insulin were evaluated by receptor binding assay and hypoglycemic effect measurement.Results. NB1-cis-9-hexadecenyl insulin was synthesized by a one-step reductive alkylation in sodium salicylate and isopropanol solution in high yield (80%). The site selectivity and yield of the reaction were found to be affected by pH, medium, and insulin-to-aldehyde ratio. After solid phase extraction, the purity was found to be approximately 98%. This derivative showed a Kd to the insulin receptor of 5.72 X 10 ^{– 9} M and a significantly slower glucose lowering rate than insulin.Conclusions. NB1-hexadecenyl insulin was synthesized by reductive alkylation without the use of protective agents in high yield. NB1-hexadecenyl insulin retained significant binding affinity to insulin receptor and showed a pronounced hypoglycemic effect.