Synthesis of fluorescent phospholipid analogs for kinetic and mechanistic studies of phospholipase A2 enzymes

Includes bibliographical references (pages 58-61)The synthesis and characterization of two fluorogenic phospholipase A2 enzyme substrates (PC-1, PC-2) have been accomplished. (R)-3-amino-1,2-propanediol was employed as the chiral precursor for synthesizing the optically active phospholipid molecules. 7-mercapto-4-methyl coumarin was employed as the fluorophore which acts as a donor while 2,4-dinitroaniline was used as the energy acceptor acting as a quencher. PC-1 contains a long chain quencher at the sn-1 position of the glycerol and a long chain-terminal fluorescent reporter group at the sn-2 position of the glycerol. Phosphocholine is incorporated into the sn-3 position of the glycerol. Enzymatic hydrolysis studies of PC-1 using bee-venom phospholipase A2 clearly confirmed that the molecule is biologically active. The specific activity of the enzyme in the long chain-carrying quencher phosphocholine (PC-1) has been found to be 238 ??mol/mg/min which is about two fold less than the natural phospholipid dipalmitoylphosphatidylcholine (DPPC). PC-2 contains a short chain-carrying quencher at the sn-2 position of the glycerol and a chain-terminal fluorescent reporter group at the sn-1 position of the glycerol. Phosphocholine is incorporated into the sn-3 position of the glycerol. PC-2 showed very little biological activity upon the addition of bee-venom phospholipase A2 enzyme. The specific activity of the enzyme in the short chain-linked quencher phosphocholine (PC-2) has been found to be 45 ??mol/mg/min. Further studies need to be done as to how the length of carbon chain affects the hydrolysis rate. The preparation of the synthetic compounds (PC-1 and PC-2) opens the way to study the fluorescent resonance energy transfer (FRET) between the donor-acceptor pair of the phospholipid analogs and to develop FRET-based assays of the enzyme