PHOSPHONATE ANALOGS OF TRIACYLGLYCEROLS ARE POTENT INHIBITORS OF LIPASE

1,2-Dioctylcarbamoylglycero-3-O-p-nitrophenyl alkylphosphonates, with alkyl being methyl or octyl, were synthesised and tested as irreversib le inhibitors of cutinase from Fusarium solani pisi and Staphylococcus hyicus lipase. Rapid inactivation of these enzymes occurred with a co ncomitant release of one mole of p-nitrophenol per mole of enzyme. Wit h both lipases a higher reactivity was observed when the alkyl substit uent on the phosphonate is a methyl rather than an octyl chain, Both l ipases are highly selective for the chirality of these compounds at gl ycerol and at phosphorus. Rapid inactivation at an inhibitor concentra tion of 0.1 mol% in 100 mM NaTDOC (t(1/2) < 60 min.) occurred when the glycerol moiety had the (R) configuration, while inhibitors of the (S ) configuration react 4-10-fold more slowly. The isomer with the p-nit rophenyl octylphosphonate attached to the secondary hydroxyl group of glycerol hardly inhibited (t(1/2) > 1 day) the lipases, These results reflect the known positional- and stereopreference of these enzymes wh ich preferentially release the fatty acid at sn-3 of natural triacylgl ycerols. The enzymes appeared to be even more selective for the chiral ity at phosphorus, the differences in reactivity of the faster and slo wer reacting isomers being as high as about 250-fold for the methylpho sphonates and about 60-fold for the octylphosphonates. These phosphona tes can be regarded as true active site-directed inhibitors. The inhib ited enzymes can be considered as analogues of the tetrahedral interme diate in the acylation step that occurs during triacylglycerol hydroly sis.