Acyl and phosphoryl migration in lysophospholipids: importance in phospholipid synthesis and phospholipase specificity.

Three isomeric lysophosphatidylcholines (1-palmitoyl-sn-glycero-3-phosphorylcholine, 2-palmitoyl-snglycero-3-phosphorylcholirte, and 3-palmitoyl-sn-glycero-2phosphorylcholine) have been prepared by the action of phospholipase A2 or lipase on I ,2-dipalmitoyl-sn-glycero-3phosphorylcholine or phospholipase A2 on 1 ,3-dipalmitoylsn-glycero-2-phosphorylcholine. The structures of the lyso compounds have been confirmed by a complete assignment of the polar head groups using 1H NMR spectroscopy. The product of phospholipase A2 action on phosphatidylcholine is 1-acyl-sn-glycero-3-phosphorylcholine. Acyl migration between this compound and the 2-acyl isomer and phosphoryl migration between this compound and the 2-phosphoryl isomer were followed by 31P NMR. T he acyl migration was found to be first order in both lysophospholipid and acid or base with a base-catalyzed second-order rate constant of about 4 X 10-4 he rearrangement of lysophospholipids via acyl migration has long been recognized as a serious problem that has to be taken into account in the determination of phospholipase specificity (de Haas eta!., 1960; de Haas & van Deenen, 1961; van Deenen eta!., 1963), synthesis of mixed acyl phospholipids (Gupta eta!., 1977; Warner & Benson, 1977) , and the elucidation of biosynthetic pathways that lead to mixed acyl phospholipids (van Golde & van den Bergh, 1977) involving lysophospholipid intermediates (Homma eta!., 1981) . The importance of phosphoryl migration is illustrated by the recent observation that trace amounts of 1 ,3-diacyl-sn-glycero-2phosphorylcholine produced by phosphoryl migration during the synthesis of 1 ,2-diacyl-sn-glycero-3-phosphorylcholine (Lammers et a!., 1978; Ponpipom & Bugianesi, 1980) can increase the stability of sonicated vesicles of the latter by 100-fold (Larrabee, 1979) . There are six different lysophosphatidylcholines consisting of three enantiomeric pairs of positional isomers as shown in Figure l. The possible acyl and phosphoryl migrations are indicated. The lack of a suitable analytical tool has previously prevented a detailed investigation of the reaction rates and equilibria involved in migration. We have now used 31P NMR to follow the kinetics and determine the equilibria of these reactions. 1 The phenomenon of acyl migration was first discovered by Fischer (1920) and has subsequently been found to occur frequently in chemical and biochemical systems [reviews: Akahori, 1965; Pavlova & Rachinskii , 1968] . It has been characterized quite extensively in the rearrangement between 1and 2-monoacylglycerides, because the vicinal diol in 1monoacylglycerides can be easily quantitated by using a periodate determination (Martin, 1953; van Lohuizen & t From the Department of Chemistry, University of California at San Diego, La Jolla, California 92093 . R eceived July 8, 1981. These studies were supported by National Institutes of Health Grant GM-20,501 and National Science Foundation Grant PCM 79-22839. I Recipient of a predoctoral fellowship from the Studienstiftung des Deutschen Volkes. M-1 s-1• At alkaline pHs, the equilibrium mixture contains about 90% of the l-acy! and about 10% of the 2-acyl isomer. A slow acyl migration also occurs in organic solvents, most notably in the presence of basic cata lysts used in common acylation procedures for the synthesis of phospholipids from lysophospholipids. At alkaline pHs, no phosphoryl migra tion was detected in the time scale of acyl migration and hydrolysis. 31 P NMR could also directly demonstrate the positional specificity of phospholipase A2 and lipase, which acts as a phospholipase AI> by the direct observation of the products formed under conditions where migration was slow. While it is well-known that phospholipase A2 is specific for the sn-2 position of phospholipids in micelles and bilayer membranes, it was demonstrated by this technique that this specificity also holds for the monomeric phospholipid dibu tyrylphosphatidylcholine. Verkade, 1960; Wolfenden et a!., 1964; Verkade, 1966; Serdarevich, 1967). However, such a reaction is not possible in lysophospholipids. A number of techniques have been used for studies on the rearrangement of diglycerides, such as a thin-layer chromatographic separation (Freeman & Morton, 1966), enzymatic breakdown (Mattson & Volpenhein, 1962), and 1 H NMR spectroscopy (Serdarevich, 1967) . In the case_ of lysophospholipids, however, it was found to not be possible to separate compounds Ia and Ila by any thin-layer or paper chromatography technique (Slotboom et a!. , 1963; Eibl & Lands, 1970) . Column chromatography on silicic acid or alumina causes rapid equilibration of lysophospholipids (Lands & Merkel, 1963; de Haas & van Deenen, 1965; Eibl & Lands, 1970; Gupta eta!., 1977). No chemical methods have appeared which can quantitate mixtures of isomers of lysophospholipids without themselves causing isomerization (Hanahan et al., 1960). The difference in activity of phospholipase A2 toward Ia and Ila was used in an attempt to qualitatively assess the stability of the isomers (Slotboom eta!., 1963; Albright eta!., 1973), but this depends on the correct assignment by another, independent method. Another enzymatic technique, which has been employed to distinguish Ia and Ila, used the degradation of lysophospholipids to monoglycerides by phospholipase C (van den Bosch & van Deenen, 1965; Eibl & Lands, 1970) . Although the resulting 1and 2-monoglycerides can be analyzed by a variety of techniques (Hofmann, 1962; Wolfenden et a!., 1964; Johnson & Holman, 1966; Satouchi & Saito, 1977; Sugatani eta!., 1980; Matsuzawa & Hostetler, 1980), this is rather cumbersome and involves the problem of further acyl migration during the workup procedure. Spectroscopic methods such as IR (Slotboom et a!., 1963, 1967; de Haas & van Deenen, 1965), 13C NMR (Schmidt eta!., 1977), or 1H NMR (discussed below) can all detect spectral differences between the 1 A preliminary report of some of these results was presented at the International Conference on Phosphorus Chemistry, Duke University, Durham, NC, June 1-5, 198 1. Reprinted from Biochemistry, 1982, 21, 1743. Copyright© 1982 by t he American Chemical Society and reprinted by permission of the copyright owner.