Stereocontrolled syntheses of water-soluble inhibitors of phosphatidylinositol-specific phospholipase C: inhibition enhanced by an interface.

Three inositol 1,2-(cyclic)-phosphate analogs, inositol cyclic phosphonates with different stereochemistry at the C-2 position of the inositol ring, have been synthesized as water-soluble inhibitors of phosphatidylinositol-specific phospholipase C (PI-PLC). Their inhibition of both phosphotransferase and cyclic phosphodiesterase activities has been studied in the absence and presence of an interface. Key results include the following. (i) Only the analog with the same stereochemistry at the C-2 position of the inositol ring as the natural substrate, myo-inositol 1,2-(cyclic)-phosphate (cIP), exhibits effective inhibition of PI-PLC. (ii) The inhibition of the PI-PLC cyclic phosphodiesterase activity by this cIP analog is enhanced by the presence of an interface (Triton X-100 or diC7PC micelles). This is the first observation of detergent enhancing the effectiveness of a water-soluble inhibitor competing with a water-soluble substrate. (iii) For the cyclic phosphodiesterase activity measured in the presence of 8 mM of the best (e.g., most activating) interface, diC7PC, myo-inositol 1,2-(cyclic)-2-methylenephosphonate (cICH2P) was shown to be a competitive inhibitor with a Ki of 12.3 mM. (iv) The IC50 obtained for the same compound inhibiting the PI-PLC hydrolysis of PI dispersed in DiC7PC micelles was consistent with a Ki approximately 10 mM for the phosphotransferase activity. The similarity of Ki for both PI and cIP processing by PI-PLC suggests both reactions occur at the same site on the enzyme.