Intramolecularly Quenched BODIPY-Labeled Phospholipid Analogs in Phospholipase A2 and Platelet-Activating Factor Acetylhydrolase Assays and in Vivo Fluorescence Imaging

Abstract

Phospholipase substrate analogs containing both a fluorescent BODIPY group and a quenching 2,4-dinitrophenyl (DNP) group were synthesized. They showed little fluorescence, but upon hydrolysis became fluorescent as the quenching group was removed. Two substrates were phosphatidylethanolamine analogs with a BODIPY-pentanoyl group at the sn-2 position and DNP linked to the amino head group. The third was a phosphatidylcholine analog with a BODIPY-labeled alkyl ether at the sn-1 position and a N-(DNP)-8-amino-octanoyl group at the sn-2 position. These compounds were evaluated as substrates for cytosolic (85 kDa) phospholipase A2 (cPLA2) and plasma platelet-activating factor acetylhydrolase (rPAF-AH). Two were good substrates for cPLA2 (specific activities: 18 and 5 nmol min−1 mg−1) and all were good for rPAF-AH (specific activities: 17, 11, and 6 μmol min−1 mg−1). The minimal amount of enzyme detectable was 50 ng for cPLA2 and 0.1 ng for rPAF-AH. These substrates were active in assays of PLA2 in zebrafish embryo extracts and one was well suited for imaging of PLA2 activity in living zebrafish embryos. Embryos were injected with substrate at the one- to four-cell stage and allowed to develop until early somitogenesis when endogenous PLA2 activity increases dramatically; substrate persisted (12 h) and specifically labeled cells of the developing notochord.