Related Experiment Videos
NB1-C16-insulin: site-specific synthesis, purification, and biological activity
1Division of Pharmaceutics, College of Pharmacy, Comprehensive Cancer Center, Ohio State University, Columbus 43210, USA.
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 5.72x10(-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.