Fluorogenic phospholipid and metabolically stabilized inositol analogues as signal transduction probes

PLases (phospholipases) participate in a wide variety of cellular signals for healthy and diseased processes. PLA (phospholipase A), PLC (phospholipase C), and PLD (phospholipase D) enzymes cleave PLs (phospholipids) to give distinct, bioactive products. Fluorogenic substrate analogues offer the possibility of detecting PLase activity in vitro and in living cells and tissues in real-time and with high sensitivity.; Here, fluorogenic analogues of the PLs PA (phosphatidic acid), PC (phosphatidylcholine), PE (phosphatidylethanolamine), PG (phosphatidylglycerol), and PS (phosphatidylserine) were synthesized as PLA substrates for determining the influence of PL head group modifications on cell signaling in vitro and in cells. The initial synthetic route to a fluorogenic analogue of PA used exclusively chemical transformations. Later, an enzyme-assisted synthetic route was employed, which included remodeling of the sn-2 position of the diacylglyceryl moiety with cobra venom PLA2 and transphosphatidylation with a particular PLD. This enzyme-assisted synthesis allowed the PA analogue to be synthesized more efficiently than by purely chemical methods and also provided ready access to a variety of different head groups. The resulting fluorogenic Dabcyl- and BODIPY-containing PL analogues---DBPA, DBPC, DBPE, DBPG, and DBPS---were used to determine PLA 2 kinetics in mixed micelle assays. DBPC was then used to determine the Xi(50) value of a common PLA2 inhibitor. Finally, the head group selectivity of a series of commercially available PLA2 enzymes was established using the DBPL substrates.; For assaying PLD activity in vitro and in cells, a series of fluorogenic analogues of PC and LPC (lysophosphatidylcholine), including DDPB and lysoDDPB, were synthesized, again by an enzyme-assisted strategy. The analogues were evaluated as substrates for PLC, PLD, and lysoPLD (lysophospholipase D). DDPB was cleaved by PC-PLC and by bacterial, plant, and human PLD and represents the first direct fluorogenic substrate for mammalian-type enzymes.; Inositol polyphosphates, products of PL hydrolysis by PLC, also mediate cell signaling. In the concluding chapter, metabolically stabilized inositol polyphosphate analogues are proposed that are designed to be long-lived agonists/antagonists at intracellular inositol polyphosphate binding sites. Synthetic studies toward these analogues are detailed, culminating in a new synthetic route to the stabilized inositol analogue inositol(1,4,5)tris(methylphosphonate).