NB1-C16-insulin: Site-specific synthesis, purification, and biological activity

Purpose. To develop a simple and efficient method for the synthesis and pur ification of NB1-lipid-modified-insulin without the use of protecting agent s. Methods. Bovine insulin was allowed to react with cis-9-hexadecenal in an a queous-organic medium in the presence of NaBH3CN at room temperature overni ght. HPLC and ESI LC/MS coupled with dithiothreitol and trypsin treatment w ere employed for product identification and optimization. The product was p urified by a differential C18 solid-phase extraction. The biological effect s of the modified insulin were evaluated by receptor binding assay and hypo glycemic effect measurement. Results. NE 1-cis-9-hexadecenyl insulin was synthesized by a one-step reduc tive alkylation in sodium salicylate and isopropanol solution in high yield (80%). The site selectivity and yield of the reaction were found to be aff ected by pH, medium, and insulin-to-aldehyde ratio. After solid phase extra ction, 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 alkylatio n without the use of protective agents in high yield. NB1-hexadecenyl insul in retained significant binding affinity to insulin receptor and showed a p ronounced hypoglycemic effect.