Synthesis and Evaluation of Fluorogenic Substrates for Phospholipase D and Phospholipase C

Abstract

Fluorogenic analogues of phosphatidylcholine and lysophosphatidylcholine, DDPB and lysoDDPB, were synthesized by an enzyme-assisted strategy. The analogues were evaluated as substrates for phospholipases C and D, and lysophospholipase D. DDPB was cleaved by bacterial and plant phospholipase D (PLD) enzymes and represents the first direct fluorogenic substrate for real-time measurement of PLD activity. Both fluorogenic substrates have potential in screening for PLD and PC-PLC inhibitors, and for monitoring spatiotemporal changes in PLD activity in cells.PLC, PLD, and lysoPLD are three phospholipases that catalyze hydrolysis of phospholipid (PL) head groups. PLC catalyzes the hydrolysis of the PL phosphodiester glyceryl P-O bond to give diacylglycerol and a phosphomonoester. PLD cleaves the head group P-O bond of the phosphodiester linkage, to produce phosphatidic acid (PA) and an alcohol. LysoPLD catalyzes the same reaction as PLD, but is selective for lysophospholipids. Most PLDs also catalyze transphosphatidylation, 1 in which a primary alcohol replaces water as the cleaving nucleophile.PLC isozymes selectively hydrolyze PLs with either inositol or choline headgroups. There are at least eleven different phosphoinositide-selective PLCs (PI-PLC), 2 two putative phosphatidylcholine-selective PLCs (PC-PLCs) 3 in mammals, and one PC-PLC in plants. 4 PLD isozymes found in mammals are involved in a diversity of normal and disease-related biological processes. 5-7 The PLD enzymes found in mammals, plants, and some bacteria are called "HKD PLD" because they share a consensus amino acid sequence (HxKx 4 Dx 6 GG/S), and employ a common catalytic mechanism involving a covalent histidine intermediate. 8 The "non-HKD PLD" enzymes lack an HKD consensus sequence, and have a catalytic mechanism in which metal ions are required to position the PL substrate and activate the attacking nucleophile. 9 S. chromofuscus PLD (scPLD) is an archetypical non-HKD PLD. Similar to the Email: Glenn.Prestwich@hsc.utah.edu. We report here the synthesis of fluorogenic PC and lysoPC analogues that contain a fluorescence quencher (dabcyl, a.k.a p-methyl red) at each acyl chain terminus, and a fluorophore appended to the PL head group through a choline-mimetic linker. These PL analogues, denoted DDPB and lysoDDPB, were evaluated in microtiter plate assays as substrates for lysoPLD, scPLD, PLC, and phospholipase A 2 (PLA 2 ), as well as several commercially-available HKD PLD enzymes. NIH Public AccessDDPB and lysoDDPB were synthesized efficiently as illustrated in Schemes 1 and 2. The lysophosphatidylcholine intermediate 2 containing the shorter dabcyl acyl chain was obtained a selective monoacylation of the commercially-available glycerol phosphocholine, 1, followed by installation of the longer-chain dabcyl quencher at the sn-2 position (4, Scheme 1). While the longer, dodecanoyl acyl chain at sn-2 improved the lipophilicity of the analogue over compounds with two hexanoyl chains, adding a second C 12 linker at sn-1 afforded a less soluble ...

Phospholipase C families: Common themes and versatility in physiology and pathology

“…For in vitro assays, the main focus has been on the development of inositol lipid based chemical probes that could be used as a substrate in screens. Despite the initial difficulties, some promising, fluorogenic substrate analogues were reported [448][449][450][451][452]. One of the more recent examples is a soluble PI(4,5)P 2 analog with a cleavable tag (compound WH-15) that is hydrolyzed by PLCs to produce a blue fluorescent molecule [453][454][455]; a related compound (XY-69), that can be incorporated into lipid vesicles, has also been synthesised and used in vitro [83,456].…”

Synthesis of Lysophospholipids

“…Afterwards the PL is submitted to the action of the right phospholipase A 1 or A 2 or a lipase in order to get the desired 1-LPL or 2-LPC (see Figure 13 ). With a similar procedure and starting from commercially available GPC and PLD, fluorogenic analogues of lysophosphatidylcholine were synthesized and evaluated as substrates for enzymatic assays [ 103 ].…”

Practical Labeling Methodology for Choline‐Derived Lipids and Applications in Live Cell Fluorescence Imaging

“…Jao et al used this method to examine phospholipid distribution by fluorescence microscopy in fixed cells. While this method has the advantage of facile synthesis, it is not able to specifically interrogate a single class of phospholipids, as can be done with synthesis (9,16). Additional examples of synthetic alkyne-labeled lipid probes have been used to detect lipase activity in cells (17), and have been used as affinity tags (18).…”