DESIGN, SYNTHESIS, AND EVALUATION OF PHOSPHOLIPID ANALOGS AS INHIBITORS OF THE BACTERIAL PHOSPHOLIPASE-C FROM BACILLUS-CEREUS

Enzymes belonging to the phospholipase C (PLC) family hydrolyze the ph osphodiester bond of phospholipids to give a diacylglycerol and a phos phorylated head group. The bacterial phospholipase C from Bacillus cer eus (PLC(Bc)) has been studied extensively, and there is a wealth of i nformation regarding those structural features that are important for substrate activity. In contrast, there is virtually no data available regarding structure-activity relationships for inhibitors of this enzy me. To address this shortcoming, a series of optically pure analogues of 1,2-dihexanoyl-sn-glycero-3-phosphocholine (2) containing different replacements of the phosphate group were first synthesized including the phosphoramidates 4 and 8, the phosphonate 5, the (difluoromethylen e)phosphonate 6, the thiophosphate 7, the diastereomeric phosphorothio ates 9 and 10, and the phosphorodithioate 11. Each of these phosphatid ylcholine derivatives was tested for inhibitor or substrate activity w ith PLC(Bc) using the water-soluble phosphatidylcholine 2 as the monom eric substrate. The measurements were conducted below the critical mic ellar concentrations of both 2 and the inhibitor. Of the analogues, on ly 7 and 9 underwent observable enzymatic hydrolysis under the assay c onditions used. The k(cat) of the (Sp)-phosphorothioate 9 was approxim ately one-fifth that of 2, and when compared to 2, 7 was hydrolyzed on ly very slowly by the enzyme. Kinetic studies indicated that the phosp holipid analogues tested were competitive inhibitors with increasing K -i's follows: 7 approximate to 11 approximate to 10 < 4 approximate to 8 < 5 approximate to 6.