SYNTHESIS AND PURIFICATION OF NB1-PALMITOYL INSULIN

A procedure for synthesizing NB1-palmitoyl insulin for incorporation i nto liposomes for targeting to hepatocytes was developed. The amino gr oup of the first amino acid phenylalanine on the B chain (B1) of insul in was selected for conjugation with palmitic acid in anticipation tha t its binding to the insulin receptor would be preserved. Two other fr ee amino groups present in insulin, the first amino acid glycine on th e A chain (Al) and the 29th amino acid lysine on;the B chain (B29), we re first protected with a t-butoxycarbonyloxy (t-BoC) group to yield N A1, B29-di-(t-Boc) insulin. The identity of this di-(t-Boc) insulin wa s confirmed by amino acid analysis as well as by enzyme hydrolysis cou pled with matrix-assisted laser-desorption time of flight mass spectro metry (MALDI-TOF MS). NA1,B29-Di-(t-Boc) insulin was then reacted with the N-hydroxysuccinimide ester of palmitic acid, followed by deblocki ng the t-Boc groups, to yield NB1-palmitoyl insulin, the structure of which was further confirmed by MALDI-TOF MS analysis. NB1-palmitoyl in sulin was found to interact with the insulin receptor on fat cells, th ereby catalyzing the conversion of [C-14]glucose into lipids, at reduc ed efficiency (30-40%].