A novel approach to structure proof of glyceryl ether-containing glycerophospholipids. Base- catalyzed rnethanolysis of platelet-activating factor (AGEPC) at 6OoC

A novel reaction was explored in which synthetic platelet-activating factor, l-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC), upon treatment with 1 N NaOH in methanol at 6OoC for 20 min, sequentially released the acetyl group, then the choline moiety with concomitant formation of the monomethyl ester of 1-0-alkyl-glycero-phosphoric acid. A mechanism is proposed in which a transient cyclic phosphate intermediate is formed and then attacked by a CHsO moiety to yield a mixture of the sn-2 and sn-3 methyl esters. Proof of structure of the monomethyl ester derivative was achieved through the use of thin-layer chromatography, aluminum oxide chrornatography, and examination of the trimethylsilyl derivative of the monomethyl ester by gas-liquid chromatography-mass spectrometry. Replacement of the acyl group on the 2 position with an ethyl or methyl residue completely prevented any attack by 1 N NaOH in methanol at 6OOC. Sphingomyelin was not attacked and only acetate removal was noted with l-O-alkyl-2acetyl-sn-glycero-3-phosphoethanolamine under similar conditions. The significance of these findings as they relate to the influence of substituents on the chemical and biological reactivity of AGEPC is discussed. "anahan, D. J., S. T. Weintraub, S. J. Friedberg, A. Tokumura, and D. E. Ayer. A novel approach to structure proof of glyceryl ether-containing glycerophospholipids. Base-catalyzed methanolysis of platelet-activating factor (AGEPC) at 6OOC. J Lipid Res. 1985. 26: 1345-1355. Supplementary key words methyl phosphate ester formation * AGEPC analogs GLC mass spectrometry high potency at levels in the range of 1 X 10"' M to 1 x lo-" M toward washed rabbit platelets in initiating aggregation and in causing serotonin release (1-3). It also has been shown to have hypotensive activity (4). The high biological activity together with the observation that very low amounts of this factor are generated in stimulated phagocytic cells (5) has emphasized the need for more refined and definitive assay and structure proof procedures than are available at present. Recently, Kumar, Weintraub, and Hanahan (6) reported that a diether glycerophospholipid, i.e., 1-0-alkyl-2-0-methyl-sn-glycero3-phosphocholine was resistant to acetolysis whereas an analogous compound, the 2 acetyl derivative (AGEPC), was easily attacked. This observation intimated that current structure proof techniques might not be adequate for identification of these types of naturally occurring compounds. In the course of this research study designed to provide a more efficient route to structural analysis of naturally produced platelet-activating factor, a novel reaction was observed in which base-catalyzed methanolysis of 1-0alkyl-2-acetyl-sn-glycero-3-phosphocholine l d to the rapid release of choline and also produced a unique acidic phosphorus-containing product. The various aspects of this reaction are described in detail. The biochemical importance of glyceryl ether-containing lipids in metabolic processes in mammalian cells has assumed a new dimension with the discovery of a potent Abbreviations: TMS, trimethylsilyl; DMS, dimethylsilyl; TLC, thinfactor (PAF). This substance has been identified as 1-0spectrometry; AGEPC, acetyl glyceryl ether phosphocholine; GEPC, glycerophospholipid chemical mediator, platelet-activating layer chromatography; GLC-MS, gas-liquid chromatography-mass alkyl-2~acetyl-sn-glycero-3-phosphocholine (AGEPC), in glyceryl ether phosphocholine. which the alkyl group is primarily composed of long chain 'To whom inquiries should be addressed. 'Current address: Facultv of Pharmaceutical Sciences. Tokushima hydrocarbon (16:0, 18:O) residues (1-3). This lipid exhibits University, Shomachi, Tokushima 770, Japan Journal of Lipid Research Volume 26, 1985 1345 by gest, on N ovem er 6, 2017 w w w .j.org D ow nladed fom