An improved synthesis of 7-nitrobenz-2-oxa-1,3-diazole analogs of CDP-diacylglycerol and phosphatidylinositol.

A protocol utilizing chemical and enzymatic steps to synthesize several fluorescent (7-nitrobenz-2-oxa-1,3 diazole) analogs of cytidine diphosphate diacylglycerol and phosphatidylinositol in high yields is described. The fluorescent analogs were characterized by phospholipase C digestion, fast atom bombardment mass spectrometry, and HPLC analysis. Studies of the metabolism and intracellular distribution of the fluorescent phosphatidylinositol analogs in Swiss 3T3 cells further revealed that all the analogs were substrates for a previously described cell surface phosphatidylinositol-specific phospholipase C (A.E. Ting and R.E. Pagano (1990) J. Biol. Chem. 265, 5337-5340). These fluorescent lipids should serve as useful tools for studying phosphatidylinositol metabolism and transport in cells.

Inhibition of platelet phosphatidylinositol synthetase by an analog of CDP-diacylglycerol.

Unpublished portions of the synthesis of a phosphinate-phosphonate diether analog of CDPdiacylglycerol are reported. The liponucleotide analog was found to be a very powerful inhibitor of platelet PI synthetase; kinetic data suggest a competitive inhibition mechanism. The structural specificity of CDPdiacylglycerol for liponucleotide-mediated biosynthetic reactions is discussed.

Stereospecific synthesis and enzyme studies of CDP-diacylglycerols.

The fatty acid specificity of two enzymes that metabolize CDPdiacylglycerol, CDPdiacylglycerol hydrolase (EC 3.6.1.26) and CDPdiacylglycerol: inositol phosphatidyltransferase (EC 2.7.8.11), has been examined in guinea pig brain. Mixed CDPdiacylglycerols were stereospecifically synthesized by the following sequence: (i) hydrolysis of a homodiacyl lecithin to 1-acyl lysoPC by action of snake venom phospholipase A2, (ii) reacylation with the anhydride of the desired second fatty acid and dimethylaminopyridine, (iii) hydrolysis of the resultant heterodiacyl lecithin to phosphatidate with cabbage phospholipase D, and (iv) reaction of phosphatidate with CMPmorpholidate to give CDPdiacylglycerol. CDPdiacylglycerol: inositol phosphatidyltransferase showed the following rates of conversion of 40-microM suspensions of CDPdiacylglycerol in 0.15% Triton X-100 to phosphatidylinositol relative to the 1-stearoyl-2-oleoyl derivative (100%): dipalmitoyl, 70%; distearoyl, 38%; diarachidonoyl, 9%; 1-arachidonoyl-2-stearoyl, 6%; 1-stearoyl-2-arachidonoyl, 4%. These results indicate that the composition of isolated phosphatidylinositol and related lipids is not explained by the fatty acid specificity of the biosynthetic enzymes and supports the intervention of a deacylation-reacylation sequence. The rates of hydrolysis of the synthetic CDPdiacylglycerols at 76 microM, in 0.3% Triton X-100, by the CDPdiacylglycerol hydrolase relative to the 1-stearoyl-2-oleoyl derivative (100%) were: dipalmitoyl, 70%; distearoyl, 32%; 1-arachidonoyl-2-stearoyl, 30%; 1-stearoyl-2-arachidonoyl, 28%; diarachidonoyl, 22%. Inhibition of this enzyme by AMP was shown to be non-competitive, with a Ki of 40 microM. The lysosomal localization of the mammalian hydrolase was confirmed.

Cytotoxic liponucleotide analogs. I. Chemical synthesis of CDP-diacylglycerol analogs containing the cytosine arabinoside moiety.

Cytidine and deoxycytidine diphosphate diacylglycerol are metabolic liponucleotides which are substrates for the biosynthesis of several classes of cellular phosphoglycerides. In addition to their essential biochemical function, liponucleotides can be considered unique from the point of view of molecular structure (lipid, phosphorus, sugar, heterocyclic moieties) and biophysical properties. Liponucleotides, therefore, have been investigated as possible models for anticancer drug design and development. The chemical synthesis of several liponucleotide analogs of cytidine diphosphate diacylglycerol (CDPdiacylglycerol/dCDPdiacylglycerol) containing the 1-beta-D-arabinofuranosyl moiety was undertaken for the purpose of evaluation of the antitumor activity of these compounds. The analogs were synthesized by reaction of 1-beta-D-arabinofuranosylcytosine-5'-(hydrogen morpholinophosphonate):N,N'-dicyclohexyl-4-morpholine carboxamide (1 : 1) in pyridine with either egg lecithin-derived phosphatidic acid, synthetic phosphatidic acid, or synthetic analogs of phosphatidic acid. The yields of liponucleotide analogs after purification were approx. 25-40%. Although reaction yields were not optimized, the condensation of phosphatidic acids and nucleotides represents an expedient laboratory-scale synthetic approach to liponucleotides, especially when phosphatidic acids are obtained from natural sources or by semisynthetic methods, and when 5'-nucleotides can be synthesized directly (i.e., without use of protecting groups) from precursor nucleosides.

Synthesis of biologically active spin-labelled radioactive cytidine diphosphodiglyceride, a novel probe for biological membranes.

A versatile synthesis of spin-labelled radioactive cytidine diphospho-sn-1,2-diacylglycerol (CDP-diglyceride) has been developed based on the combination of the enzymatic acylation of radioactive sn-glycero-3-phosphate with 12-doxyl stearic acid and the chemical conversion of the thus obtained spin-labelled radioactive phosphatidic acid with cytidine monophosphomorpholi-date into spin-labelled radioactive CDP-diglyceride. The method for the isolation and purification of the latter compound was described. This obtained CDP-[2-3H]diglyceride contained 10% of fatty acids of paramagnetic nature, presumably present as a covalently bound 12-doxyl stearic acid esters. The biological activity was tested by using the synthesized compound as a substrate in the mitochondrial biosynthesis of phosphatidylglycerol. It was found that spin-labelled CDP-[2-3H]diglyceride prepared as described can be converted in the presence of sn-[2-14C]-glycero-3-phosphate into a spin-labelled [2-3H, 2'-14C]phosphatidylglycerol with isolated rat liver mitochondria, establishing therefore that the site of its utilization is identical with the site of phosphatidylglycerol synthesis in isolated mitochondria, i.e. inner mitochondrial membrane. Results described demonstrate that the synthesized spin-labelled CDP-diglyceride can be used as a specific probe for the spin- and radioactive covalent labelling of polyglycerophosphatides of mitochondrial membranes. Some implications and further possibilities in the study of biological membranes using the spin-labelled radioactive CDP-diglyceride are discussed.