NB1-C16-insulin: site-specific synthesis, purification, and biological activity.
OBJECTIVE
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.
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Division of Pharmaceutics, College of Pharmacy, Comprehensive Cancer Center, Ohio State University, Columbus 43210, USA.
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D000478:Alkylation; D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003921:Diabetes Mellitus, Experimental / Q000188:drug therapy*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007004:Hypoglycemic Agents / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D066298:In Vitro Techniques; D007328:Insulin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D008055:Lipids / Q000737:chemistry*; D008297:Male; D013058:Mass Spectrometry; D011869:Radioligand Assay; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism; D014407:Tumor Cells, Cultured
D007004:Hypoglycemic Agents; D007328:Insulin; D008055:Lipids; D011972:Receptor, Insulin