"Synthesis of 1,2-diacyl-sn-glycerophosphatidylserine from egg phosphatidylcholine by phosphoramidite methodology" Morillo, M., Sagristá, M.L., de Madariaga, M.A., Eritja, R. Lipids, 31(5), 541-546 (1996). doi: 10.1007/BF02522649

Synthesis of 1,2-Diacyl-sn -Glycerophosphatidylserine from Egg Phosphatidyicholine by Phosphoramidite Methodology

Margarita Morillo a, M. Luisa Sagristá a,* , M. Africa de Madariaga a, and Ramón Eritja b aDepartment of Biochemistry and Physiology, Faculty of Chemistry, University of Barcelona, O8028 Barcelona, Spain, and bDepartment of Molecular Genetics, CID-CSIC, 08034 Barcelona, Spain

*To whom correspondence should be addressed at the Department of Bio chemistry and Physiology, Faculty of Chemistry, University of Barcelona, Martí i Franqués, 1-11,08028 Barcelona, Spain.

Abbreviations: BE, N-t-BOC-L-serine benzhydril ; BEP, N-t-BOC-L- serine benzhydril ester phosphoramidite; DAG, diacylglycerol; DDM, diazodiphenylmethane; NMR, nuclear magnetic resonance; PC, phosphatidylcholine; PS, phosphatidylserine; P Sp, O-(1,2-diacylglycero-3- phospho-)-N-t-BOC-L-serine benzhydril ester; t-BOC, N-tert - butoxycarbonyl; TES, N-tris(hydroxymethyl)methyl-2-aminoethane- sulfonica cid; TFA, trifluoroacetic acid; TLC, thin-layer chromatography

ABSTRACT : A simple chemical method for the synthesis of 1,2-diacyl-sn - glycerophosphatidylserine (PS), with the same fatty acid composition in the sn -1 and sn -2 glycerol positions as egg phosphatidylcholine (PC), is described. PS synthesis was carried out by a phosphite-triester approach, using 2-cyanoethyI- N,N,N' ,N' -tetraisopropylphosphorodiamidite (phosphoramiditate) as the phosphorylating agent, for the formation of phosphate linkage between serine and diacylglycerol. 1,2- Diacylglycerol, obtained from PC hydrolysis by phospholipase C, was coupled with N-t-BOC- L-serine benzhydryl ester phosphoramidite with tetrazole as catalyst. Phosphite-triester was oxidized to the corresponding phosphate-triester with

30% H202 in CH 2Cl 2. The cyanoethyl group was removed by addition of an Et 3N/CH 3CN/pyridine mixture, and trifluoroacetic acid was used to eliminate the protecting groups of O-(1,2-diacylglycero- 3-phospho)-N-t-BOC-serine benzhydril ester. Purified PS was identified by thin-layer chromatography, infrared, and 1H nuclear magnetic resonance.

Phospholipids are major components of cell membranes, have many functions in living organisms, and are involved in a myriad of industrial processes (1). Phospholipids, particularly phosphatidylcholine (PC) and phosphatidylserine (PS), are used extensively in the formation of liposomes that have an enormous potential as drug delivery systems (2,3). Liposomes have also been used as model membrane systems to study cell-cell or liposome-cell interactions, as well as the molecular mechanisms of biological membrane fusion (4). The isolation of phospholipids in pure and homogeneous form from natural sources or phospholipids mixtures, as well as the total synthesis of phospholipids by chemical reactions, is a complicated, expensive, and time- consuming task (5). Preparation of pure phospholipids, including PS, has been an area of interest because of their use in biochemical and membrane research. The conversion of phospholipids using synthetic methodology can be achieved both chemically and enzymatically. The economic efficiency of the chemical or biochemical process depends mainly upon high rates of production of desired products and minimizing the formation of undesirable by-products (6). Although PS formation by enzymatic methods, using cabbage phospholipase D, has been reported by many scientists (7-9), the yields obtained were low and formation of the byproduct phosphatidic acid was very high. Nevertheless, conversion of PC to PS by streptomyces phospholipase D was achieved in high yield (10). PS with saturated or unsaturated acyl moieties also have been synthesized by a variety of chemical methods, most of which involve condensation of phosphatidic acid with serine protected at the amino and carboxyl functions or the coupling of diacylglyceroliodohydrines with suitable protected O-phosphoserines (5,11-14). In some cases, the amino and carboxyl protecting groups have been removed by two successive reactions leading to lower yield of the end product (12). Hermetter et al. (13) used the N-tert -butoxycarbonyl (t-BOC) residue and the benzhydryl group to protect serine at the amino and carboxyl groups, respectively. These two protective groups can easily be coupled to serine and removed in a single step under mild conditions, but the condensation yield of N-t-BOC- serine benzhydryl ester with defined molecular species of phosphatidic acids depends on the acyl chain lengths and their unsaturation degree (13). We have studied the synthesis of PS starting from PC, the main constituent of natural phospholipids, using the phosphite- triester approach for the formation of a phosphate linkage between the serine and the diacylglycerol (DAG). Although phosphite coupling reactions have been well established and applied for oligonucleotides synthesis (15-19), the utilization of these tactics for the construction of phosphoiipids and their derivatives has received limited attention (20-25). Traditional phosphate coupling procedures and modifications thereof have generally provided a suitable mean for the preparation of phosphatidylethanolamine, PC, and derivatives bearing different fatty acid side chains and/or polar head groups, in general, with good to excellent yields. However, the application of these standard techniques to achieve an efficient synthesis of PS may be problematic; in fact, only single molecular species of PS have been synthesized by this technology. The aim of this work was the synthesis of a PS with the same fatty acid composition in the sn- 1 and sn-2 glycerol positions as egg PC used as the starting material. This will enable us to prepare liposomes with different PC/PS ratios but identical fatty acyl composition of each component, thus allowing us to correlate the changes in behavior of these model systems with the head polar group modification.

MATERIALS AND METHODS

Egg lecithin (65%) for synthesis was purchased from Merck A.G. (Darmstadt, Germany) and purified by column and thinlayer chromatography (TLC) before use. CH 3CN [Solvants Documentation Syntheses (SDS), Peypin, France; water content <50 ppm] was stored over

CaH2 (Merck A.G.). All other reagents were of analytical grade. Water was twice-distilled (Millipore Systems, Molsheim, France), and all organic solvents were redistilled before use. DAG was obtained by PC hydrolysis with phospholipase C (26) (Lecithinase C; E.C. 3.1.4.3.) Type I of CIostridium perfringens ( C. welchii , 10-20 units per mg protein; Sigma Chemical Co., St. Louis, MO). Phospholipase C (33 mg) dissolved in 60 mL of 0.1 M Tris-HCl buffer (pH

7.2) and 75 mL of a 5 mM CaCl 2 solution were added to a solution of PC (300 mg, 0.4 mmol) in peroxide free diethyl (75 mL). The mixture was stirred under N2 at room temperature for 2 h and 45 min. 1,2-DAG~ were then extracted with (4 x 75 mL). The combined organic extracts were evaporated under reduced pressure, without heating, to reduce the initial volume four times. The resulting suspension was dried over anhydrous Na 2SO 4, the solvent was evaporated under reduced pressure to give a crude product, which was dissolved in dry CH 2Cl 2, and the drying and evaporation processes were repeated. This compound was used without further purification in the next step. The extent of the phosphocholine group elimination was checked by TLC, 1H nuclear magnetic resonance (NMR), and infrared. DAG was used immediately in order to prevent isomerization from 1,2-DAG to 1,3-DAG. Diazodiphenylmethane (DDM), often used as a carboxyl protecting group, was prepared from hydrazine (>98%; Merck) by oxidation with metachloroperbenzoic acid (55%; Merck) in the presence of iodine as catalyst and N,N,N',N' -tetramethylguanidine as base (Merck A.G.)

(27). N,N,N',N' -tetramethylguanidine (1.6 mL) and a solution of I 2 (1%, p/v) in 0.32 mL of distilled CHCl 3 were added to a solution of benzophenone (1638.1 mg, 8.36 mmol) in 8 mL of distilled CHCl 3. After stirring for 5 min at 0 ºC solid 55% metachloroperbenzoic acid (3409.8 mg, 10.87 mmol) was added slowly over 1 h at 0ºC. The mixture was stirred for an additional 15 min, then washed with water until the washings were at pH 6.

The resulting suspension was dried over anhydrous Na 2SO4, the solvent was evaporated under reduced pressure and the residue suspended in MeOH. An 526 -1 -1 assay at 526 nm ( ε DDM (MeOH) = 100 M cm ) of a suitable diluted sample corresponded to a 90% yield of DDM. Tetrazole (99%) and 2-cyanoethyl N,N,N',N' - tetraisopropylphosphorodiamidite (phosphorodiamidate) were from Aldrich Chemical Co. Ltd. (Milwaukee, WI). The phosphorodiamidate was checked 31 by P-NMR ( δp 123.5) and TLC (Table 1) before use. Impurities observed were 5.1% (ipr) 2N-P(OCH 2CH 2CN) 2 (ISe 149.06), 1% ((ipr) 2N) 2PO(CH 2CH 2CN) (δp 31.4), and phosphoramiditate hydrolysis products with low δp values. N-t-BOC-L-serine benzhydril ester phosphoramidite (BEP) was obtained from N-t-BOC-L-serine benzhydril ester (BE). First, BE was prepared from N-t-BOC-L-serine (Sigma chemical Co.) and an excess of DDM (13). DDM (938 mg, 4.83 mmol) was added to a solution of N-t-BOC-L -serine (717.7 mg, 3.5 mmol) in dry diethyl ether (20 mL), and additional diethyl ether was added to give a total volume of 30 mL. The reaction mixture was stirred overnight at room temperature. The excess of DDM was destroyed by the addition of glacial acetic acid and followed by the color change from red to yellow. The solvent was removed by evaporation under reduced pressure. The crude product was dissolved in CHCl 3 containing 1% per volume of aqueous NH 3 and purified afterwards by column chromatography on silica gel (27 g, 0.063-0.1 mm; Macherey Nagel, Düren,

Germany). Elution was performed with chloroform containing 0.5% NH 3 to prevent decomposition of the product. Then 10-mL fractions were collected at a flow rate of 0.8-1.1 mL/mim. BE was contained in fractions 8-24. The combined fractions were filtered, dried, and evaporated under reduced pressure. BE (149 mg, 0.401 mmol) was dried by its dissolution in dry

CH 3CN, and the solvent was evaporated under reduced pressure. Dry BE was redissolved under N 2 in dry CH 3CN (1.46 mL) by continuous stirring. Tetrazole (26 mg, 0.37 mmol) was added with stirring at room temperature, followed by phosphoramiditate (120.4 mg, 0.4 mmol). The molar ratio of reagents was BE/phosphine/tetrazole (1:0.99:0.93). After stirring for 40 min, the reaction was stopped by the addition of 5% Et 3N in CH 2Cl 2. The diisopropylammonium tetrazolide formed was removed by washings with saturated aqueous NaCl and filtration. The solution was evaporated to dryness, and the residue was redissolved in CH 2Cl 2 and dried 31 again with Na 2SO 4. The product, BEP, was identified by P NMR and TLC on silica gel.

TABLE 1

aNBS, N-t-BOC-L-serine; BE, N-t-BOC-L-serine benzhydri] ester; BEP, N-t-

BOC- L -serine benzhydril ester phosphoramidite; DAG, diacylglycerol; P Sp, O-(1,2-diacylglycero-3-phospho-)-N-t-BOC -serine benzhydril ester; PS, phosphatidylserine. b Solvent systems: (1) CHCl 3/MeOH/NH 3 (28%) (65:35:5, by vol); (2) hexane/diethyl ether/Et 3N (30:60:1, by vol); (3) hexane/diethyl ether (30:70, vol/vol). Adsorbent: Silica Gel G (Merck, Darmstadt, Germany). Data represent the mean ± SD of at least five determinations.

DAG (156.58 mg, 0.26 mmol) and BEP (265.9 mg crude BEP, 0.34 mmol pure BEP) were coevaporated in dry CH 3CN. The residue was dissolved in 1.65 mL of CH 3CN, and tetrazole (90.92 mg, 1.3 mmol) was then added. The reaction vessel was deaerated with N 2, and the mixture was stirred for 15 min (reagents molar ratio was DAG/BEP/tetrazole,

1:1.3:5). The reaction was stopped by the addition of 1.6 mL of 5% Et 3N in CH 2Cl 2, and the solution was washed in the same way as indicated for BEE The amount of tetrazole necessary to activate the phosphoramidite was higher than that required to activate the phosphorodiamidite. Subsequent oxidation of the intermediate phosphite delivered the corresponding phosphate-triester. The oxidation was carried out by the addition of 1.2-1.5 equivalent of an aqueous solution of H202 (30%) to the residue containing the phosphite-triester dissolved in 1.6 mL of CH 2Cl 2. The mixture was stirred vigorously at room temperature for two hours. The reaction mixture was washed with a saturated aqueous NaCl solution (2 • 1.6 mL), the layers were separated, and the combined organic phases were dried with anhydrous Na 2SO 4 and evaporated under reduced pressure. The phosphate- triester was deprotected without further purification. Egg PC, and most of the naturally-occurring and biologically- active phospholipids, contain unsaturated sn -2-acyl chains possessing from one to four double bonds which can easily undergo oxidation. Thus, before the development of the synthetic method presented in this paper, we tested the

H202 effect on fatty acid double bonds. We prepared an incubate containing a mixture of unsaturated fatty acids in CH 2Cl 2 and twice the theoretical amount of H 202 necessary for the oxidation of the phosphite to the phosphate-triester. The mixture was stirred vigorously for two hours at room temperature and, after standard work up, fatty acids analysis by gas- liquid chromatography showed the same composition as the starting mixture (results not shown). Thus, no evidence of oxidation of fatty acyl double bonds in the presence of hydrogen peroxide used for phosphite oxidation was detected. The cyanoethyl group was removed by addition of 1 mL of an

Et 3N/CH 3CN/pyridine (1:1:1, by vol) mixture to the dry residue. After 90 min, the solvent mixture was eliminated by vacuum. The O-(1,2- diacylglycero-3-phospho)-N-t-BOC-serine benzhydril ester (P Sp) obtained was purified by column chromatography on silica gel (27 g, 0.1-0.2 mm;

Macherey Nagel) saturated with CHCl 3 containing 0.5% NH 3 and eluting with CHCl 3 and CHCl 3/MeOH mixtures containing 0.1% NH 3. Then 5-mL fractions were collected at a flow rate of 0.7-1.0 mL/min. Pure PSp was eluted when the CHCl 3/MeOH ratio was 90:10 (vol/vol). The combined fractions were filtered, dried, and evaporated under reduced pressure. trifluoroacetic acid

(TFA) (1 mL) was added to the product dissolved in 1 mL of CHCl 3 to eliminate protecting groups of PSp and was then removed by vacuum. Et 3N (0.4 mL), dissolved in CHCl 3, was added to the residue to neutralize any remaining TFA. The organic layer was washed with the following cold mixtures--MeOH/H 20/NaHCO 3sat (2:1:1), MeOH/100 mM NaHCOO pH 3.0 (4:5), and MeOH/100 mM N tris(hydroxymethyl)methyl-2-aminoethane- sulfonic acid pH 7.4--to eliminate remaining Et 3N. The solvent was evaporated under reduced pressure and the product was dried over P205. The crude PS was purified by CM-Cellulose (CM52; Whatman, Kent,

England) column chromatography using CHCl 3/MeOH mixtures as eluent, at a flow rate of 0.48 mL/min. Pure PS was eluted with CHCl 3/MeOH, 65:35 (vol/vol). The combined fractions were filtered, dried, and evaporated under reduced pressure. Infrared spectra were obtained from a film of lipid solutions in chloroform (approximately 5 mg/mL) on a KCI pellet, using a Perkin-Elmer 687 infrared spectrophotometer (Norwalk, CT). All spectra were recorded at room temperature against a reference KCl pellet. 1H NMR (200 MHz) and 31 P NMR (81 MHz) spectra were obtained, in

CDCl 3 (15-25 mg lipid/mL), on Geminis-200 (Palo Alto, CA) and Varian XL- 200 (Palo Alto, CA) spectrometers, respectively, operating in the Fourier transform mode, at room temperature. Typically 200-300 scans were averaged. Chemical shifts values are expressed in units of δ relative to tetramethylsilane set at 0 ppm or trimethyl phosphite set at 140-141 ppm, respectively, for 1H and 31 P NMR spectra. Methyl of the fatty acids were analyzed by gasliquid chromatography on a Perkin-Elmer 990 gas chromatograph equipped with flame-ionization detector. Data were obtained with a 2 m x 2 mm i.d. column packed with 15% polymeric ethyleneglycol succinate as stationary phase on Chromosorb W (100-120 mesh; Johns-Manville, Denver, CO). Helium at 30 mL/min was used as carrier gas. Injector, column, and detector temperatures were 230, 188, and 250 ºC respectively. Samples were applied as hexane solutions.

RESULTS AND DISCUSSION

Scheme 1 shows the scheme used for PS synthesis via phosphite coupling. 31 P NMR was used to check the reaction course. DAG (98%) obtained by PC hydrolysis gave single spots on silica gel G plates using different solvent systems (Table 1), showing that the isomerization of 1,2 diglyceride to 1,3 diglyceride (side product) was not done during the reaction time course. Moreover, the extent of acyl migration was low (<4%) during the condensation step between DAG and + BEE. The signal at δH 3.35 corresponding to (CH 3)3N - from PC disappears - for DAG, and the signals at δH 3.8-4.6 due to -(CH 20) 2P(O)O are not shown in DAG spectrum. On the other hand, the absorption bands at 970, 1090, and 1240 cm -1 related with the phosphate group are missing in DAG infrared spectrum.

SCHEME 1

BE was identified by TLC (Table 1), ultraviolet-spectrum (CHCl 3) ( λmax -1 -1 1 = 258 nm, ε = 450 M cm ) and H NMR (signals at δH 1.44 and δH 7.32- 7.34 ppm corresponding to -C(CH 3)3 and -C6H5 groups, respectively) (13). The homogeneity of BEP, obtained from N-t-BOC-L-serine, was checked by TLC (Table 1). BEP 31 P NMR spectrum showed two main signals at δP 148.1 and 147.4 ppm. The two signals at similar δP obtained for BEP with different values showed the stereoselectivity of the reaction. The only impurities observed were little amounts of dimers (δP 137.8 ppm) and hydrolysis products ( δP 2.1-18.5 ppm). The process was carried out with an excellent yield (95.5%), and BEP purity was ca. 71.7%. The appearance in the 31 P NMR spectra of two new signals, after DAG and BEP condensation, at δP 136.37 and 135.85 ppm and the disappearance of the characteristic peaks of BEP isomers showed the formation of the desired phosphite-triester (93%). The oxidation of the phosphite to phosphate-triester was performed using a two-phase system with aqueous

H202 and dichloromethane (21). This milder treatment was preferred over iodine oxidation (16) to avoid possible addition of iodine to fatty acid double bonds and because the oxidation solution is easy to remove from the phospholipid solution. The oxidation reaction was completed after two hours (94%) as confirmed by the substitution of phosphite-triester signals by two 31 signals at δP -4.54 and -4.93 ppm in the P NMR spectra. The elimination of the cyanoethyl group and the purification of the obtained P Sp by column chromatography on silica gel was achieved by a 50% yield. BEP, phosphite-triester, and phosphate-triester were not extensively purified since the side products, largely consisting of hydrolysis products, did not interfere with the coupling, oxidation, and cyanoethyl group elimination processes. PS obtained after deprotection with TFA was purified by CM-Cellulose (51%) column chromatography because the utilization of silica gel always gave impure PS. CMCellulose was used in older literature to separate PS and phosphatidic acid (8), although the basis for lipid separation on CM- Cellulose is not completely understood. It seems likely that in chloroform, which has a low dielectric constant, phospholipids are bound to CM- Cellulose by nonspecific adsorption and possibly by hydrogen bonding. The addition of methanol will increase the dielectric constant and the repulsion of gradually dissociating negative charges, resulting in elution of lipids. The analysis by gas-liquid chromatography of PS, obtained after the elimination of all protecting groups and purified by CM-Cellulose column chromatography, showed that PS and PC fatty acids compositions were the same (Table 2). This result seems to indicate that the coupling of particular molecular species is not preferred and that the double bonds were not affected under the mild oxidation conditions used. PS was identified by infrared and 1H NMR (Fig. 1), being the obtained spectra identical to those reported in the bibliography (13, 28). PC and PS 31 P NMR spectra showed a sole signal at δP -1.244 and -0.265 ppm, respectively. The relative upfield shift of phosphate group of PC vs. PS can be correlated with the quaternary of choline (29).

TABLE 2

aPC, Phosphathidylcholine; other abbreviations as in Table 1. bData represent the mean ± SD of at least three determinations

The methods previously described for obtaining PS by the phosphoramidite approach (20-23,25) have been applied to molecular species with the same fatty acid composition at the sn- 1 and sn-2 positions of glycerol. This paper describes a new method, and with excellent yield, for the synthesis of PS with the same fatty acid composition at the sn-1 and sn- 2 positions of glycerol as egg PC, using a phosphite-triester approach. Phosphodiester (14) and phosphate-triester (14) methods have been described for the preparation of PS. Low yields, formation of undesirable products, and long reaction times have been some of the problems associated with these methodologies. The methods described here are faster, simpler, and more selective. It is based on the phosphite-triester method that has been proven to be more efficient in solid phase oligonucleotide synthesis (16,19). The separation of the unreacted materials also is easier due to the large differences on their polarity, and it could be performed using materials readily available. Moreover, phosphorothioate and other phosphate derivatives of PS can be prepared from the intermediate phosphite-triester. On the other hand, PS also could be prepared in higher yields by the transphosphatidylation method using Streptomyces phospholipase D (10). Nevertheless, the differences in yields obtained with the method we propose and the transphosphatidylation viewed in the context of the costs of reagents indicate that the synthetic method described in this paper may be less expensive, depending upon the relative costs of phospholipase D from streptomyces species and additional labor needed for our method. The protective groups would be easily cleaved in two steps. The cyanoethyl group was eliminated by addition of Et 3N/CH 3CN/pyridine mixture, and TFA was used to simultaneously eliminate the protecting groups of P Sp. It was not possible to use other functional protective groups which could be uniformly removed since phosphorous protecting groups cleavable in acid media are not available. A β-elimination mechanism for cyanoethyl group removal could yield phosphatidic acid as a by-product, but that secondary reaction did not take place since phosphatidic acid was not detected by TLC analysis.

FIG. 1. (A) Infrared and (B) 1H nuclear magnetic resonance spectra of phosphatidylcholine (PC) and phosphatidylserine (PS).

ACKNOWLEDGMENT This work was supported by Grant No. PB86-0042 from C.I.C.Y.T. (Spain).

REFERENCES

1. Hanin, I., and Pepeu, G. (eds.) (1990) Phospholipids: Biochemical, Pharmaceutical and Analytical Considerations , Plenum Press, New York. 2. Cuypers, P.A., Corsel, J.W., Jansen, M.P., Kop, J.M.M., Hermens, W.T., and Hemker, H.C. (1983) The Adsorption of Prothrombin to Phosphatidylserine Multilayers Quantitated by EIlipsometry, J. Biol. Chem . 258; 2426-2431. 3. Scherphof, G.L. (1986) Liposomes in Biology and Medicine (a biased review), in Lipids and Membranes: Past, Present and Future (Op den Kamp, J.A.F., Roelofsen, B., and Wirtz, K.W.A., eds.) pp. 113-136, Elsevier Science Publishers, Amsterdam. 4. Wilschut, J., and Hoekstra, D. (1986) Membrane Fusion: Lipid Vesicles as a Model System, Chem. Phys. Lipids 40, 145-166. 5. Eibl, H. (1980) Synthesis of Glycerophospholipids, Chem. Phys. Lipids 26, 405-429. 6. Shield, J.W., Ferguson, H.D., Bammarius, A.S., and Hatton, T.A. (1986) Enzymes in Reversed Micelles as Catalysts for Organic- phase Synthesis Reactions , Ind. Eng. Chem. Fundam 25, 603-612. 7. Yang, S.F., Freer, S., and Benson, A.A. (1967) Transphosphatidylation by Phospholipase D, J. Biol. Chem 242, 477-484. 8. Comfurius, P., and Zwaal, R.F.A. (1977) The Enzymatic Synthesis of Phosphatidylserine and Purification by CM-Cellulose Column Chromatography, Biochim. Biophys. Acta 488, 36-42. 9. Shuto, S., Imamura, S., Fukukawa, K., Sakakibara, H., and Murase, J. (1987) A Facile One-Step Synthesis of Phosphatidylhomoserines by Phospholipase D-Catalyzed Transphosphatidylation, Chem. Pharm. Bull . 35, 447-449. 10. Juneja, L.R., Kazuoka, T., Goto, N., Yamane, T, and Shimizu, S. (1989) Conversion of Phosphatidylcholine to Phosphatidylserine by Various Phospholipases D in the Presence of L- or D-Serine, Biochim. Biophys. Acta 1003, 277-283. 11. Slotboom, A.J., and Bonsen, P.P. (1970) Recent Developments in the Chemistry of Phospholipids, Chem. Phys. Lipids 5, 301-397. 12. Browning, J., and Seelig, J. (1979) Synthesis of Specifically Deuterated Saturated and Unsaturated Phosphatidylserines, Chem. Phys. Lipids 24, 103-118. 13. Hermetter, A., Paltauf, F., and Hauser, H. (1982) Synthesis of Diacyl and Alkylacyl Glycerophosphoserines, Chem. Phys. Lipids 30, 35-45. 14. Paltauf, F., and Hermetter, A. (1994) Strategies for the Synthesis of Glycerophospholipids, Progr. Lipid Research 33, 240-328. 15. Nielsen, J., Taagard, M., Marugg, J.E., van Boom, J.H., and Dahl, O. (1986) Application of 2-Cyanoethyl N,N,N',N' -Tetraisopropyl- phosphorodiamidite for in Situ Preparation of Deoxyribonucleoside Phosphoramidites and Their Use in Polymer- Supported Synthesis of Oligodeoxyribonucleotides, Nucleic Acid Research 14, 7391-7401. 16. Caruthers, M.H., Barone, A.D., Beaucage, S.L., Dodds, D.R., Fisher, E.F., McBride, I.J., Matteucci, M., Stabinsky, Z., and Tang, J.Y. (1987) Chemical Synthesis of Deoxyoligonucleotides by the Phosphoramidite Method, in Methods in Enzymology (Wu, R., and Grossman, L., eds.) Vol. 154, pp. 287-313, Academic Press Inc., London. 17. Marugg, J.E., Tromp, M., Kuyl-Yeheskiely, E., van der Marel, G.A., and van Boom, J.H. (1986) A Convenient and General Approach to the Synthesis of Properly Protected d-Nucleoside- 3'-Hydrogenphosphonates via Phosphite Intermediates, Tetrahedron Lett . 27, 2661-2664. 18. Beaucage, S.L., and Iyer, R.P. (1992) Advances in the Synthesis of Oligonucleotides by the Phosphoramidite Approach, Tetrahedron 48, 2223- 2311. 19. Beaucage, S.L., and Iyer, R.P. (1993) The Synthesis of Modified Oligonucleotides by the Phosphoramidite Approach and Their Applications, Tetrahedron 49, 6123-6194. 20. Lemmen, P., Buchweitz, K.M., and Stumpf, R. (1990) A Synthesis of Phospholipids Using a Phosphite-Triester Approach and Uniformly Cleavable β-Halogenated Protecting Groups, Chem. Phys. Lipids 53, 65-75. 21. Martin, S.F., and Josey, J.A. (1988) A General Protocol for the Preparation of Phospholipids via Phosphite Coupling, Tetrahedron Lett . 29, 3631-3634. 22. Lindh, I., and Stawinski, J. (1989) A General Method for the Synthesis of Glycerophospholipids and Their Analogues via H-Phosphonate Intermediates, J. Org. Chem . 54, 1338-1342. 23. Wooley, P., and Eibl, H. (1988) Synthesis of Enantiomerically Pure Phospholipids Including Phosphatidylserine and Phosphatidylglycerol, Chem. Phys. Lipids 47, 55-62. 24. McGuinan, C., and Swords, B. (1990) Synthesis of Phospholipids by Phosphoramidite Methodology, J. Chem. Soc. Perkin Trans . I, 783-787. 25. Bittman, R. (1993) Chemical Preparation of Glycerolipids: A Review of Recent Synthesis, in Phospholipids Handbook (Cevc, G., ed.) pp. 141-232, Marcel Decker Inc., New York. 26. Renkonen, O. (1965) Individual Molecular Species of Different Phospholipid Classes. II. A Method of Analysis, J. Am. Oil Chem. Soc . 42, 298-304. 27. Adamson, J.R., Bywood, R., Eastlick, D.T., Gallagher, G., Walker, D., and Wilson, E.M. (1975) Amino-Acids and Peptides. Part II. A New Method for Preparing Diazodiphenylmethane and Related Compounds, J. Chem. Soc. Perkin Trans . I Vol. III, 2030-2033. 28. Nelson, G.J. (1970) Lipids of Sheep Red Blood Cells. IV Identification of a New Phospholipid, N-Acyl Phosphatidyl Serine, Biochem. Biophys. Res. Commun . 38, 261-265. 29. Henderson, T.O., Glonek, T., and Myers, T.C. (1974) Phosphorus-31 Nuclear Magnetic Resonance Spectroscopy of Phospholipids, Biochemistry 13, 623-628.