Synthesis of mixed-chain phosphatidylcholines including coumarin fluorophores for FRET-based kinetic studies of phospholipase A2 enzymes

Abstract

Phospholipase A2 (PLA2) enzymes catalyze the hydrolysis of the sn-2 ester linkage of glycerophospholipids to produce fatty acids and lysophospholipids. A significant number of mammalian phospholipases comprise a family of secreted PLA2 enzymes, found in specific tissues and cellular locations, exhibiting unique enzymatic properties and distinct biological functions. Development of new real-time spectrofluorimetric PLA2 assays should facilitate the kinetic characterization and mechanistic elucidation of the isozymes in vitro, with the potential applicability to detect and measure catalytic PLA2 activity in tissues and cellular locations. Here we report a new synthesis of double-labeled phosphatidylcholine analogues with chain-terminal reporter groups including coumarin fluorophores for fluorescence resonance energy transfer (FRET)-based kinetic studies of PLA2 enzymes. The use of coumarin derivatives as fluorescent labels provides reporter groups with substantially decreased size compared to the first generation of donor-acceptor pairs of fluorescent phospholipids. The key advantage of the design is to interfere less with the physicochemical properties of the acyl chains, thereby improving the substrate quality of the synthetic probes. In order to assess the impact of the fluorophore substituents on the catalytic hydrolysis and on the phospholipid packing in the lipid-water interface of the assay, we used the experimentally determined specific activity of bee-venom phospholipase A2 as a model for the secretory PLA2 enzymes. Specifically, the rate of PLA2 hydrolysis of the coumarin labeled phosphatidylcholine analogues was less than three times slower than natural substrate dipalmitoyl phosphatidylcholine (DPPC) under the same experimental conditions. Furthermore, variation of the mole fraction of the synthetic phosphatidylcholine vs. that of the natural DPPC substrate showed nearly ideal mixing behavior in the phospholipid-surfactant aggregates of the assay. The synthesis provides a rapid and efficient method for preparation of new synthetic phosphatidylcholines with the desired target structures for enzymatic and physicochemical studies.

Synthesis of phospholipids on a glyceric acid scaffold: design and preparation of phospholipase A2 specific substrates

“…These results indicate that introduction of chain-terminal coumarin labels maintain good substrate quality, in good agreement with what we observed with coumarin-labeled fluorescent glycerophospholipids. 14 …”

“…Specifically, we have shown recently, that phospholipid probes with coumarin labeled hydrocarbon side-chains could readily be incorporated into micellar interfaces of natural phospholipids, showing near ideal mixing behavior with the unlabeled phospholipid components in micellar aggregates. 14 …”

Activity-based targeting of secretory phospholipase A2 enzymes: A fatty-acid-binding-protein assisted approach

“…Primary alcohol of the amide 7 was protected by treating it with para-methoxytriylchloride and DMAP for 90 minutes to afford 8 . Formation of a sulfide linkage by base catalyze alkylation of 7-mercapto-4-methylcoumarin using 11-bromoundecanoic acid in acetone gave chemically stable thioether 9 (Wang, et al 2013). sn -2 position of the glycerol 8 was acylated using DMAP as a catalyst to give 10 in quantitative yield.…”

“…(Wang, et al, 2013) While the fluorescence data demonstrate the intended use of the flouorescence/quencher labeled phospholipid, the impact of attaching reporter groups on the rate of substrate hydrolysis can be assessed only by comparing the rates of hydrolysis of the labeled lipids with that of unmodified natural phospholipids.…”

“…We have been engaged in developing new fluorogenic probes and assays for the detection and kinetic characterization of secretory phospholipase A 2 enzymatic activity (Wang, et al, 2013). Small reporter groups employed in the present design should help minimize any impact on the structure of the lipid membrane system (Wang, et al, 2013). The FABP-assisted fluorescence assay described below enables a quantitative assessment of these considerations.…”