METHODS
Simple Synthesis of Diastereomerically Pure Phosphatidylglycerols by Phospholipase D-Catalyzed Transphosphatidylation Rina Satoa, Yutaka Itabashia,*, Hironori Fujishimaa, Hidetoshi Okuyamab, and Arnis Kuksisc a
Graduate School of Fisheries Sciences, Hokkaido University, Hakodate 041-8611, Japan, bGraduate School of Environmental Earth Science, Hokkaido University, Sapporo 060-0810, Japan, and cBanting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6, Canada
ABSTRACT: A simple method for synthesizing diastereomerically pure phosphatidylglycerols (PtdGro), namely, 1,2-diacyl-sn-glycero-3-phospho-3’-sn-glycerol (R,R configuration) and 1,2-diacylsn-glycero-3-phospho-1’-sn-glycerol (R,S configuration), was established. For this purpose, diastereomeric 1,2-O-isopropylidene PtdGro were prepared from 1,2-diacyl-sn-glycero-3-phosphocholine (PtdCho) and enantiomeric 1,2-O-isopropylideneglycerols by transphosphatidylation with phospholipase D (PLD) from Actinomadura sp. This species was selected because of its higher transphosphatidylation activity and lower phosphatidic acid (PtdOH) formation than PLD from some Streptomyces species tested. The reaction proceeded well, giving almost no hydrolysis of PtdCho to PtdOH in a biphasic system consisting of diethyl ether and acetate buffer at 30°C. The isopropylidene protective group was removed by heating the diastereomeric isopropylidene PtdGro at 100°C in trimethyl borate in the presence of boric acid to obtain the desired PtdGro diastereomers. The purities of the products, which were determined by chiral-phase HPLC, were exclusively dependent on the optical purities of the original isopropylideneglycerols used. The present method is simple and can be utilized for the synthesis of pure PtdGro diastereomers having saturated and unsaturated acyl chains. Paper no. L9482 in Lipids 39, 1025–1030 (October 2004).
Phosphatidylglycerol (PtdGro) is known to be widely distributed in animals, plants, and microorganisms (1), where it plays important roles as one of the acidic polyglycerophospholipids (2–4). The structure of naturally occurring PtdGro may be 1,2diacyl-sn-glycero-3-phospho-1′-sn-glycerol (sn-3,sn-1′, R,S configuration) (1,5–7). However, Itabashi and Kuksis (8) and Fujishima et al. (9) have recently found that PtdGro from some bacteria, including Escherichia coli, contain a nonnegligible amount of 1,2-diacyl-sn-glycero-3-phospho-3′-sn-glycerol (sn-3,sn-3′, R,R configuration). Y. Itabashi, H. Fujishima, and A. Kuksis (unpublished results) also have found that the content of the R,R isomer in E. coli PtdGro increases with increasing growth temperature. This observation suggests that the bac*To whom correspondence should be addressed at Graduate School of Fisheries Sciences, Hokkaido University, 3-1-1 Minato-cho, Hakodate, Hokkaido 041-8611, Japan. E-mail:
[email protected] Abbreviations: DNPU, 3,5-dinitrophenylurethane; ESI-MS, electrospray ionization mass spectrometry; PLD, phospholipase D; PtdCho, phosphatidylcholine; PtdGro, phosphatidylglycerol.
Copyright © 2004 by AOCS Press
terium adapts to temperature by alternating not only the degree of unsaturation but also the stereoisomer composition of PtdGro. To clarify the differences in biochemical properties and physiological functions between the R,S and R,R diastereomers in bacteria, we concerned ourselves with the synthesis of the pure PtdGro diastereomers, which are not commercially available. Although the enantiomeric and diastereomeric PtdGro can be synthesized chemically via several steps involving phosphorylation of enantiomeric DAG (10,11), in this study we investigated a simpler enzymatic method using phospholipase D (PLD). PLD catalyzes transphosphatidylation in which the phosphatidyl moiety of substrate phospholipids is transferred to acceptor alcohols to produce phosphatidyl alcohols (12,13). Thus, 1,2-diacyl-sn-glycero-3-phosphocholine (R configuration) in the presence of glycerol is readily converted into PtdGro, which is a mixture of the R,R and R,S diastereomers (8,14–16). In this study, to obtain diastereomerically pure R,R and R,S PtdGro, we used enantiomerically pure 1,2and 2,3-O-isopropylidene-sn-glycerols instead of glycerol, which are the general intermediates for the synthesis of chiral glycerolipids (17,18). The sequence of reactions used is shown in Figure 1. D’Arrigo et al. (16) reported a similar enzymatic method for obtaining the diastereomeric PtdGro from phosphatidylcholine (PtdCho) and isopropylideneglycerols by transphosphatidylation using PLD from Streptomyces species; the diastereomeric nature of the products was implied from the starting materials (1,2- and 2,3-O-isopropylidenesn-glycerols), which were not enantiomerically characterized or their source indicated. The previous paper (16) claims that the two PtdGro diastereomers could be easily differentiated from their 1H NMR spectra. However, there is no indication of which particular feature of the 1H NMR spectra of the PtdGro diastereomers permits the degree of purity of either diastereomer to be established, chiral-shift reagents were not used. In the present study, we established the enantiomeric purities of the starting materials (1,2- and 2,3-O-isopropylidene-sn-glycerols) and the diastereomeric purities of the products (R,R and R,S PtdGro) using chiral-phase HPLC (8,19). In addition, we selected PLD from Actinomadura sp. because of its higher transphophatidylation activity and lower phosphatidic acid formation when compared with the Streptomyces species tested (20).
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FIG. 1. Synthesis of a diastereomerically pure phosphatidylglycerol using phospholipase D (PLD)-catalyzed transphosphatidylation. PtdCho, phosphatidylcholine (1,2-diacyl-sn-glycero-3-phosphocholine, R configuration); IPG, isopropylideneglycerol (1,2-O-isopropylidene-sn-glycerol, S configuration); IPPG, isopropylidene phosphatidylglycerol (1,2-diacyl-sn-glycero-3-phospho-1′,2′-O-isopropylidene-sn-glycerol, R,R, configuration); PtdGro, phosphatidylglycerol (1,2-diacyl-sn-glycero-3-phospho-3′-sn-glycerol, R,R, configuration).
MATERIALS AND METHODS Materials. PLD from Actinomadura sp. (Seikagaku Kogyo, Tokyo, Japan) was used for transphosphatidylation. (+)-1,2O-Isopropylidene-sn-glycerol (S configuration) and (−)-2,3O-isopropylidene-sn-glycerol (R configuration) were obtained from Sigma (St. Louis, MO) and Tokyo Kasei Kogyo (Tokyo, Japan), respectively. The optical purities of these enantiomers were determined as 98.6% (enantiomeric excess, e.e.) and 86.2% (e.e.), respectively, by chiral-phase HPLC (19). 1,2-O-Isopropylidene-rac-glycerol was a product of Tokyo Kasei Kogyo. Pure 1,2-dipalmitoyl-sn-glycero-3phosphocholine (16:0–16:0) and 1,2-dioleoyl-sn-glycero-3phosphocholine (18:1–18:1) were obtained from Sigma and Avanti Polar Lipids (Alabaster, AL), respectively. Fresh salmon (Oncorhynchus keta) roe was purchased at a fish market in Hakodate, and the PtdCho fraction was isolated from the total lipids by TLC as described previously (21). The major molecular species of the roe PtdCho were as follows: 16:0–22:6 (23%), 16:0–20:5 (19%), 16:0–22:5 (6%), 18:0–22:6 (11%), 18:0–20:5 (7%), and 18:1–20:5 (5%). Trimethyl borate and boric acid were obtained from Aldrich (Milwaukee, WI) and Kanto Kagaku (Tokyo, Japan), respectively. HPLC-grade solvents (hexane, dichloromethane, methanol, and trifluoroacetic acid) were obtained from Wako Pure Chemicals (Osaka, Japan). All other chemicals and solvents were of analytical grade or better from commercial sources. Preparation of diastereomeric isopropylidene PtdGro. 1,2Diacyl-sn-glycero-3-phospho-1′,2′-O-isopropylidene-sn-glycLipids, Vol. 39, no. 10 (2004)
erol (R,R configuration) was prepared from PtdCho and 1,2-Oisopropylidene-sn-glycerol by PLD-catalyzed transphosphatidylation (Fig. 1), whereas 1,2-diacyl-sn-glycero-3-phospho-2′,3′O-isopropylidene-sn-glycerol (R,S configuration) was prepared by using enantiomeric 2,3-O-isopropylidene-sn-glycerol instead of 1,2-O-isopropylidene-sn-glycerol. The transphosphatidylation reaction was carried out according to the procedures described previously (20). Briefly, a mixture containing 10 mg of PtdCho, 1.9 mL of 0.4 M acetate buffer (pH 5.6), 1 mL of 0.2 M CaCl2, 0.1 mL of 1,2- or 2,3-O-isopropylidene-sn-glycerol/0.4 M acetate buffer (1:4, vol/vol), and 2 mL of water was preincubated at 30°C for 10 min, and the buffered PLD preparation (0.4 M acetate buffer, pH 5.6) from Actinomadura sp. (1.25 U) was then added. The reaction was initiated by the addition of 2.5 mL of diethyl ether and performed at 30°C for 1.5 h with stirring. The progress of the reaction was monitored by TLC on Silica gel 60F254 (Merck, Darmstadt, Germany) using chloroform/ methanol/water (65:25:4, by vol) as the developing solvent. The reaction was stopped by the addition of 10 mL of chloroform/methanol (2:1, vol/vol). After centrifugation for 5 min, the lower phase was collected and dehydrated under anhydrous sodium sulfate. The lipids were obtained by evaporating the solvent under a stream of nitrogen. Preparation of diastereomeric PtdGro. To the isopropylidene PtdGro diastereomers (ca. 10 mg each ) dissolved in 1.5 mL of trimethyl borate was added 80 mg of boric acid, and the mixture was heated at 100°C for 2.5 h. After the solvent was evaporated under a stream of nitrogen, the reaction mixture was dissolved in 6 mL of chloroform/methanol (2:1,
METHODS
vol/vol), then washed with 1 mL of distilled water. The suspension formed was centrifuged at 1500 × g for 5 min, and the resulting chloroform layer was recovered and dried over anhydrous sodium sulfate. After the solvent was evaporated, the crude PtdGro were purified by preparative TLC on a Silica gel 60F254 plate (Merck) using chloroform/methanol/ water (65:25:4, by vol) as the developing solvent. The PtdGro band was detected by spraying with 0.2% 2′,7′-dichlorofluorescein ethanol and extracted from the adsorbent with three portions of chloroform/methanol/acetic acid (20:10:1, by vol). The purified PtdGro showed single spots on analytical TLC using the same solvent system as described above and the Dittmer–Lester reagent for detection (22). MS. Aliquots of the transphosphatidylation products were analyzed by electrospray ionization MS (ESI-MS) in the negative ion mode on a LCQ mass spectrometer to confirm product identities. Samples dissolved in chloroform/methanol (2:1, vol/vol, ca. 10 µg/mL) were introduced directly into the ESI probe by flow injection (3 µL/min). The heated capillary temperature was 200°C, and the spray voltage was 4.2 kV. The nitrogen sheath gas was set at 30 arb (arbitrary units) by the software. Mass spectra were taken in the mass range of 150–2000 m/z. Chiral-phase HPLC. To determine the purities of the R,R and R,S diastereomers, the resulting PtdGro were converted to bis(3,5-dinitrophenylurethane) (bis-DNPU) derivatives and analyzed by chiral-phase HPLC on an (R)-1-(1-naphthyl)ethylamine column (YMC A-K03, 25 cm × 4.6 mm i.d.; YMC, Kyoto, Japan) using hexane/dichloromethane/methanol/trifluoroacetic acid (60:20:20:0.2, by vol) as the mobile phase at a flow rate of 1 mL/min (8,20). RESULTS AND DISCUSSION Synthesis of diastereomeric isopropylidene PtdGro. PLD-catalyzed transphosphatidylation has usually been carried out in a biphasic system containing water and an organic solvent such as diethyl ether or ethyl acetate (12–16,23), although the reaction also has been tried in anhydrous organic solvents (24,25). Organic solvents play an important role by dissolving the PtdCho and preserving the modified phospholipid. A serious drawback of the biphasic reaction system containing water is the possibility of undesirable PLD-catalyzed hydrolysis of PtdCho, which would lower the yield of PtdGro. However, in this study, almost no PtdOH was detected in the reaction products by TLC and MS. Figure 2 shows the negative ESI-MS spectra of the 1,2-dipalmitoyl-sn-glycero-3-phospho-1′,2′-O-isopropylidene-sn-glycerol generated from 1,2dipalmitoyl-sn-glycero-3-phosphocholine (R configuration) and 1,2-O-isopropylidene-sn-glycerol by PLD-catalyzed transphosphatidylation. In the negative ion mode, isopropylidene PtdGro (R,R configuration) gave a prominent singly charged [M − H]− ion (m/z 763), but no fragment ions showing PtdOH were seen in the spectrum. A similar simple spectrum was also observed for 1,2-dipalmitoyl-sn-glycero-3phospho-2′,3′-O-isopropylidene-sn-glycerol, which was generated using 2,3-O-isopropylidene-sn-glycerol instead of
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1,2-O-isopropylidene-sn-glycerol (spectrum not shown). No PtdOH was observed in the reaction products on TLC. These results demonstrate that the transphosphatidylation reaction with PLD from Actinomadura sp. proceeds without the hydrolysis of PtdCho to produce isopropylidene PtdGro. This is probably due to the high transphosphatidylation activity of Actinomadura PLD and the physicochemical properties of isopropylideneglycerol as an accepter in the reaction. The high transphosphatidylation activity of Actinomadura PLD also has been observed for the synthesis of PtdGro from PtdCho and glycerol (20). Preparation of diastereomeric PtdGro. The isopropylidene group was easily removed by heating at 100°C in the presence of boric acid and trimethyl borate as established previously (17,18). Like the R,R isomer of isopropylidene PtdGro, the R,R isomer of PtdGro also gave a simple ESI-MS spectrum, showing a prominent singly charged [M − H]− ion (m/z 722) and a weak [M + K]− ion (m/z 763) for 16:0–16:0 (Fig. 2B). A similar spectrum also was observed for the R,S isomer (spectrum not shown). Figures 3 and 4 show the chiral-phase HPLC profiles of the bis-DNPU derivatives of PtdGro synthesized from PtdCho and enantiomeric and racemic 1,2-Oisopropylideneglycerols. Under the conditions employed (8), PtdGro produced from PtdCho and racemic 1,2-O-isopropylideneglycerol were clearly resolved into the R,R and R,S diastereomers and had almost the same peak areas (Figs. 3C, 4C). These chromatograms showed that no racemization occurred during the removal of the protective isopropylidene group and that the purity of each R,R and R,S isomer could be measured accurately. The partially resolved and broad peaks seen in the chromatograms of salmon roe PtdGro (Fig. 4) are due to the shifts of retention times of different molecular species including polyunsaturated ones. Itabashi and Kuksis (8) previously showed that the bis-DNPU derivatives of synthetic PtdGro were eluted in the order of increasing double bonds and decreasing carbon numbers on chiral-phase HPLC under the same conditions as those used in this study. The small peaks appearing ahead of or behind the major ones (Figs. 3A, 3B; Figs. 4A, 4B) arise from small amounts of enantiomers contained in the original 1,2- and 2,3-O-isopropylideneglycerols, the optical purities of which were 99% (e.e.) and 86% (e.e.), respectively (19). Accordingly, the purities (diastereomer excess) of the R,S and R,R diastereomers of saturated and unsaturated PtdGro obtained in this study were 85–91% and 96–98%, respectively (Table 1). The overall yield of the reaction was about 65%. Losses were mainly due to incomplete recoveries of the isopropylidene PtdGro and PtdGro from the adsorbent on silicic acid TLC. Comparison of the methods. This study emphasizes the diastereomeric purity of the products produced by PLD-catalyzed transphosphatidylation. Only recently has it become possible to establish the diastereomeric nature of the transphosphatidylation products of various PLD enzymes and to characterize their relaxed substrate specificity (20,26). In an earlier study (16), the diastereomeric nature of the products was implied from the starting
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FIG. 2. Negative electrospray ionization (ESI)-MS spectra of (A) 1,2-dipalmitoyl-sn-glycero-3phospho-1′,2′-O-isopropylidene-sn-glycerol (R,R configuration) and (B) 1,2-dipalmitoyl-snglycero-3-phospho-3′-sn-glycerol (R,R, configuration) generated from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (R configuration) and 1,2-O-isopropylidene-sn-glycerol (S configuration) by PLD-catalyzed transphosphatidylation. ESI-MS conditions are as given in the text.
materials (isopropylideneglycerols), which were not enantiomerically characterized or their source indicated. In the present study, the enantiomeric purities of the commercially available 1,2- and 2,3-O-isopropylidene-sn-glycerols were established as 99 and 86%, respectively, by chiral-phase HPLC (19). Until now, adequate methods did not exist for establishing the purity of enantiomeric isopropylideneglycerols or for their resolution. In the earlier study (16), the PtdGro were hydrolyzed in aqueous methanol in the presence of toluene-p-sulfonic acid. It has been well established that removal of the protective isopropylidene groups by acid hydrolysis involves some acyl migration, which is difficult to control. A much more effective method of removing the isopropylidene groups involves refluxing with trimethyl-
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borate in the presence of boric acid, which results in replacing the isopropylidine groups with trimethylborate groups. The latter are readily removed by hydrolysis in water at room temperature without altering the structure (17,18,27,28), as shown in this study. In conclusion, this study establishes a method for preparing diastereomerically pure PtdGro by PLD-catalyzed transphosphatidylation. The reaction of PtdCho with enantiomeric 1,2-Oisopropylideneglycerols using Actinomadura PLD was shown to proceed efficiently, giving isopropylidene PtdGro under mild conditions and high yield. The isopropylidene protective group was decomposed chemically using boric acid and trimethylborate to produce the desired PtdGro diastereomers without racemiza-
METHODS
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FIG. 3. Chiral-phase HPLC profiles of the bis(3,5-dinitrophenylurethane) derivatives of the PtdGro generated from 1,2-dioleoyl-sn-glycero-3phosphocholine (R configuration) and 1,2-O-isopropylideneglycerols by PLD-catalyzed transphosphatidylation. (A) PtdGro from 1,2-O-isopropylidene-sn-glycerol; (B) PtdGro from 2,3-O-isopropylidene-sn-glycerol; (C) 1,2-O-isopropylidene-rac-glycerol. Column temperature, 10°C; detection, 254 nm UV. Other HPLC conditions are as given in the text. For abbreviations see Figure 1.
FIG. 4. Chiral-phase HPLC profiles of the bis(3,5-dinitrophenylurethane) derivatives of the PtdGro generated from salmon roe PtdCho and 1,2O-isopropylideneglycerols by PLD-catalyzed transphosphatidylation. (A) PtdGro from 1,2-isopropylidene-sn-glycerol; (B) PtdGro from 2,3-Oisopropylidene-sn-glycerol; (C) 1,2-O-isopropylidene-rac-glycerol. HPLC conditions and abbreviations are as given in the text. For abbreviations see Figure 1.
tion. The present method is simple and can be utilized for the synthesis of diastereomerically pure saturated and unsaturated PtdGro.
REFERENCES
ACKNOWLEDGMENT This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Sciences, Sports and Culture of Japan (Scientific Research B 13460088).
1. Hostetler, K.Y. (1982) Polyglycerophospholipids: Phosphatidylglycerol, Diphosphatidylglycerol and Bis(monoacylglycero)phosphate, in Phospholipids (Hawthorne, J.N., and Ansell, G.B., eds.), pp. 215–261, Elsevier Biochemical Press, Amsterdam. 2. Shibuya, I. (1992) Metabolic Regulation and Biological Functions of Phospholipids in Escherichia coli, Prog. Lipid Res. 31, 245–299. 3. Gombos, Z., Várkonyi, Z., Hagio, M., Iwaki, M., Kovács, L.,
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TABLE 1 Diastereomer Composition of Phosphatidylglycerols Synthesized from Phosphatidylcholine and 1,2-O-Isopropylideneglycerols by Transphosphatidylation with Phospholipase D from Actinomadura sp. Phosphatidylcholinea
1,2-O-Isopropylideneglycerol
16:0–16:0 16:0–16:0 16:0–16:0 18:1–18:1 18:1–18:1 18:1–18:1 Salmon roe Salmon roe Salmon roe
rac-1,2sn-1,2- (S) sn-2,3- (R) rac-1,2sn-1,2- (S) sn-2,3- (R) rac-1,2sn-1,2- (S) sn-2,3- (R)
Diastereomer (mol%) sn-3,sn-3’ (R,R)b sn-3,sn-1’ (R,S)c 48.3 98.1 7.6 51.8 98.3 5.3 49.5 98.8 4.5
51.7 1.9 92.4 48.2 1.7 94.7 50.5 1.2 95.5
Diastereomer excess (%)d 3.4 (R,S) 96.2 (R,R) 84.8 (R,S) 3.6 (R,R) 96.6 (R,R) 89.4 (R,S) 1.0 (R,S) 97.6 (R,R) 91.0 (R,S)
a
1,2-Diacyl-sn-glycero-3-phosphocholine (R configuration). 1,2-Diacyl-sn-glycero-3-phospho-3’-sn-glycerol (R,R configuration). c 1,2-Diacyl-sn-glycero-3-phospho-1’-sn-glycerol (R,S configuration). d Diastereomer excess (%) = {([R,S] − [R,R])/([R,S] + [R,R])} × 100, where [R,S] and [R,R] denote the composition of the R,S and R,R diastereomers, respectively. b
4. 5. 6. 7.
8.
9. 10. 11. 12. 13.
14. 15.
16.
Masamoto, K., Itoh, S., and Wada, H. (2002) Phosphatidylglycerol Requirement for the Function of Electron Acceptor Plastoquinone QB in the Photosystem II Reaction Center, Biochemistry 41, 3796–3802. Veldhuizen, R., Nag, K., Orgeig, S., and Possmayer, F. (1998) The Role of Lipids in Pulmonary Surfactant, Biochim. Biophys. Acta 1408, 90–108. Hanahan, D.J. (1997) A Guide to Phospholipid Chemistry, pp. 165–194, Oxford University Press, New York. Haverkate, F., and Van Deenen, L.L.M. (1963) The Stereochemical Configuration of Phosphatidyl Glycerol, Biochem. Biophys. Acta 84, 106–108. Ruettinger, R.T., and Brewer, G.J. (1978) Strereoconfiguration of Phosphatidylglycerol in the Membrane of Bacteriophage PM2 and in Its Host, Pseudomonas BAL-31, Biochim. Biophys. Acta 529, 181–185. Itabashi, Y., and Kuksis, A. (1997) Reassessment of Stereochemical Configuration of Natural Phosphatidylglycerols by Chiral-Phase High-Performance Liquid Chromatography and Electrospray Mass Spectrometry, Anal. Biochem. 254, 49–56. Fujishima, H., Gamano, T., Taoka, Y., Sawabe, T., and Itabashi, Y. (2004) Stereoisomers of Marine Bacterial Phosphatidylglycerols, Nippon Suisan Gakkaishi (in Japanese) 70, 200–202. Baer, E., and Buchnea, D. (1958) Synthesis of Saturated and Unsaturated Phosphatidyl Glycerols. III. Cardiolipin Substitutes, J. Biol. Chem. 232, 895–901. Woolley, P., and Eibl, H. (1988) Synthesis of Enantiomerically Pure Phospholipids Including Phosphatidylserine and Phosphatidylglycerol, Chem. Phys. Lipids 47, 55–62. D’Arrigo, P., and Servi, S. (1997). Using Phospholipases for Phospholipid Modification, Trends Biotechnol. 15, 90–96. D’Arrigo, P., de Ferra, L., Piergianni, V., Selva, A., Servi, S., and Strini, A. (1996) Preparative Transformation of Natural Phospholipids Catalysed by Phospholipase D from Streptomyces, J. Chem. Soc. Perkin Trans. 1, 2651–2656. Yang, S.F., Freer, S., and Benson, A.A. (1967) Transphosphatidylation by Phospholipase D, J. Biol. Chem. 242, 477–484. Joutti, A., and Renkonen, O. (1976) The Structure of Phosphatidyl Glycerol Prepared by Phospholipase D-Catalyzed Transphosphatidylation from Egg Lecithin and Glycerol, Chem. Phys. Lipids 17, 264–266. D’Arrigo, P., de Ferra, L., Giuseppe, P.-F., Scarcelli, D., Servi, S., and Strini, A. (1996) Enzyme-Mediated Synthesis of Two Diastereoisomeric Forms of Phosphatidylglycerol and of
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Diphosphatidylglycerol (cardiolipin), J. Chem. Soc. Perkin Trans. 1, 2657–2660. 17. Buchnea, D. (1978) Stereospecific Synthesis of Enantiomeric Acylglycerols, in Fatty Acids and Glycerides (Kuksis, A., ed.), Handbook of Lipid Research 1, pp. 233–287, Plenum Press, New York. 18. Eibl, H. (1980) Synthesis of Glycerophospholipids, Chem. Phys. Lipids 26, 405–429. 19. Itabashi, Y., Fujishima, H., and Sato, R. (2004) Chiral Phase High-Performance Liquid Chromatographic Separation of Enantiomeric 1,2- and 2,3-O-Isopropylidene-sn-glycerols as 3,5Dinitrophenylurethanes, J. Oleo Sci. 53, 405–412. 20. Sato, R., Itabashi, Y., Hatanaka, T., and Kuksis, A. (2004) Asymmetric in vitro Synthesis of Diastereomeric Phosphatidylglycerols from Phosphatidylcholine and Glycerol by Bacterial Phospholipase D, Lipids 39, 1013–1018. 21. Okabe, H., Itabashi, Y., and Ota, T. (1999) Determination of Molecular Species of Phosphatidylcholines as 2-Anthrylurethanes by Reversed-Phase HPLC with Fluorescence Detection and On-line Electrospray Ionization Mass Spectrometry, J. Jpn. Oil Chem. Soc. 48, 559–567. 22. Dittmer, J.C., and Lester, R.L. (1964) A Simple, Specific Spray for the Detection of Phospholipids on Thin-Layer Chromatograms. J. Lipid Res. 5, 126–127. 23. Juneja, L.R., Kazuoka, T., Goto, N., Yamane, T., and Shimizu, S. (1989) Conversion of Phosphatidylcholine to Phosphatidylserine by Various Phospholipase D in the Presence of L- or D-Serine, Biochim. Biophys. Acta 1003, 277–283. 24. Simpson, T.D. (1991) Phospholipase D Activity in Hexane, J. Am. Oil Chem. Soc. 68, 176–178. 25. Rich, J.O., and Khmelnitzky, Y.L. (2001) Phospholipase D-Catalyzed Transphosphatidylation in Anhydrous Organic Solvents, Biotechnol. Bioeng. 32, 374–377. 26. Sato, R., Itabashi, Y., Suzuki, A., Hatanaka, T., and Kuksis, A. (2004) Effect of Temperature on the Stereoselectivity of Phospholipase D Toward Glycerol in the Transphosphatidylation of Phosphatidylcholine to Phosphatidylglycerol, Lipids 39, 1019–1023. 27. Hartman, L. (1959) Hydrolysis of Isopropylidene Esters of Fatty Acids, J. Chem. Soc., 4134–4135. 28. Mattson, F.H., and Volpenhein, R.A. (1962) Synthesis and Properties of Glycerides, J. Lipid Res. 3, 281–296. [Received April 2, 2004; accepted November 11, 2004]