Synthesis of fluorescent and radiolabeled analogues of phosphatidic acid

Abstract

Procedures for the synthesis of fluorescent and radiolabeled analogues of phosphatidic acid are described. The fluorophore 7-nitrobenzo-2-oxa-1,3-diazole (NBD) was coupled to 6-aminocaproic acid and 12-aminododecanoic acid by reaction of NBD-chloride with the amino acids under mild alkaline conditions at room temperature. 1,2-Dioleoyl- sn[U- 14C]glycerol 3-phosphate was prepared by acylation of sn-[U- 14C]glycerol 3-phosphate with oleic acid anhydride using dimethylaminopyridine as the catalyst. This compound was converted to 1-oleoyl- sn-[U- 14C]glycerol 3-phosphate by hydrolysis with phospholipase A 2. The lysophosphatidic acid was reacylated with NBD-aminocaproyl imidazole or NBD-aminododecanoyl imidazole to form the fluorescent, radiolabeled analogue of phosphatidic acid. Fluorescent, non-radiolabeled analogues of phosphatidic acid were prepared by phospholipase D hydrolysis of fluorescent phosphatidylcholine.