Synthesis of azide-modified glycerophospholipid precursor analogs for detection of enzymatic reactions

Glycerophospholipids (GPLs) are major cell membrane components. Although various phosphorylated molecules are attached to lipid moieties as their headgroups, GPLs are biosynthesized from phosphatidic acid (PA) via its derivatives, diacylglycerol (DAG) or cytidine diphosphate diacylglycerol (CDP-DAG). A variety of molecular probes capable of introducing detection tags have been developed to investigate biological events involved in GPLs. In this study, we report the design, synthesis, and evaluation of novel analytical tools suitable to monitor the activity of GPL biosynthetic enzymes in vitro. Our synthetic targets, namely, azide-modified PA, azide-modified DAG, and azide-modified CDP-DAG, were successfully obtained from solketal as their common starting material. Moreover, using CDP-diacylglycerol-inositol 3-phosphatidyltransferase (CDIPT), an enzyme that catalyzed the final reaction step in synthesizing phosphatidylinositol, we demonstrated that azide-modified CDP-DAG worked as a substrate for CDIPT. Azide-modified glycerophospholipid precursor analogs were successfully synthesized. One of the analogs, azide-modified cytidine diphosphate diacylglycerol, functioned as the substrate for the well-known enzyme CDP-diacylglycerol-inositol 3-phosphatidyltransferase (CDIPT) and the reaction product was easily detected using the fluorescent labeling technique via strain-promoted azide-alkyne cycloaddition.image