Microscale synthesis of phosphatidyl-[3H]choline from 1,2-diacylglycerol. Assessment of isomerization by reversed-phase high-performance liquid chromatography.
The synthesis of rac-1-palmitoyl-2-oleoylglycero-3-phospho-[3H]choline of high specific activity was carried out on a microscale by making 7 mumol of rac-1-palmitoyl-2-oleoylglycerol react first with an equimolar amount of POCl3 and then of [3H]choline. After purification by thin-layer chromatography and normal-phase high-performance liquid chromatography and normal-phase high-performance liquid chromatography (HPLC), the yield of the synthesis of [3H]phosphatidylcholine (120 microCi/mumol) was 22%. rac-1-Palmitoyl-2-oleoylglycerol was purified before use by reversed-phase HPLC under conditions which were nonisomerizing and allowed the separation of 1,2- and 1,3-isomers of diacylglycerol. Ethanol, but not benzene, was shown to cause isomerization of long-chain diacylglycerol and, therefore, was not used for drying the substrate before reaction. A rapid and complete separation of 1,2- and 1,3-isomers of long-chain phosphatidylcholine was obtained by reversed-phase HPLC using 20 mM choline chloride in methanol/acetonitrile/water (50:50:1, by vol) isocratically as the mobile phase. Under these conditions, analysis of the synthesized rac-1-palmitoyl-2-oleoylglycero-3-phospho-[3H]choline showed a total absence of 1,3-isomer.
Bugaut
M
M
Unité de Nutrition Cellulaire et Métabolique, Faculté des Sciences Mirande, Université de Bourgogne, Dijon, France.
Gresti
J
J
Gagniarre
J
J
D002849:Chromatography, Gas; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D004075:Diglycerides / Q000737:chemistry*; D010713:Phosphatidylcholines / Q000138:chemical synthesis*; D012015:Reference Standards; D013237:Stereoisomerism
C011439:1,2-diacylglycerol; D004075:Diglycerides; D010713:Phosphatidylcholines; C028694:1-palmitoyl-2-oleoylphosphatidylcholine