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Phosphoric acid derivatives. XVI. Phosphatidylglycerols from diacylglycerol (o-acetoxyphenyl hydrogen phosphates)
Chemistry and Physics of Lipids, 46 (1988) 121--125 Elsevier Scientific Publishers Ireland Ltd.
121
Phosphoric acid derivatives. XVI. Phosphatidylglycerols from diacylglycerol (o-acetoxyphenyl hydrogen phosphates)* J. Calder6n and P. Yagiie Instituto de Qu~mica Org(~nica General del C.S.L C., Juan de la Cierva, 3, 28006 Madrid (Spain) The syntheses of saturated diacylglycero-3-phosphogylcerols are described. Diacylglycerol (o-acetoxyphenyl hydrogen phosphates) were coupled with 1,2-isopropylideneglycerol in the presence of 2,4,6-triisopylbenzenesulphonylchloride to phosphotriester intermediates, which were readily purified by short-column chromatography. Subsequently, the phosphateprotecting and the isopropylidene groups were removed by bydrogenolysis and acid hydrolysis, respectively.
Keywords: synthesis; saturated phospholipids; phosphatidylglycerols.
Introduction
Diacylphosphatidylglycerols were first detected by Maruo and Benson in higher plants and algae [2]. These phospholipids were also found to occur in bacterial membranes [3--5] and in minor amounts in animal tissues [6--8]. The structure of these naturally occurring lipids was determinated by Haverkate and van Deenen [9] to be 3-sn-phosphatidyl-l'- sn-glycerol. Phosphatidylglycerols isolated from extremely halophilic bacteria contain ether-linked alkyl groups rather than fatty acid ester groups, but their structures were established as l-sn-phosphatidyl-3'- sn-glycerol [10]. Chemical synthesis was carried out by one of the following general methods: (a) phosphorylation with phosphorus oxychloride of a 1,2-diacylglycerol and a protected glycerol; and (b) reaction of a 1,2-diacyl-3-iodo-l,2-propanediol with an appropriate silver phosphate. The first chemical synthesis by method (a) was published by Baer and Buchnea [11], but the stereochemical configuration of the non-acetylated moiety was opposite that of the natural compound. Saunders and Schwarz [12] used the same approach, but with 2,3-dibenzyl-sn-glycerol Correspondence to: J. Calder6n. *For Part XV, see Ref. 1.
instead of 1,2-isopropylidene-sn-glycerol as one of the starting materials. The product has the configuration of the naturally occurring phospholipids. Harlos and Eibl [13] prepared 1,2-tetradecyl-rac-glycero-3-phosphoglycerol by this approach using 1,2-isopropylidene-sn-glycerol. Method (b) was first performed by Bonsen et al. [14] from the silver salt of 2,3-isopropylidene-sn-glycerol-l-phosphoric acid benzylester and 1-oleyl-2-palmitoyl-sn-glycerol-3-iodohydrine. After removal of the protecting groups, the end product was obtained with sterochemical purity An alternative approach was described by Tocanne et al. [15]. They started from the silver salt of the 1,2-didecanoyl-sn-glycero-3-phosphoric acid benzylester and 2,3-dibenzylglycerolsn-l-iodohydrine. By applying the same route, Joe and Kates [10] prepared a dialkyl derivative of phosphatidylglycerol with the same configuration as the phospholipids isolated from halophilic bacteria. Finally, Lammers and van Boom [16] studied a new triester approach starting with a monofunctional reagent, R~R2POCI (R ~ and R 2 being phosphate protecting groups). In this way, 1,2distearoyl-sn-glycero-3-phospho-l'-glycerol was prepared. Method (a) is accompanied by the formation of many by-products due to the trifunctionality of phosphorus oxychloride. In method (b), the
0009-3084/88/$03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
122
mer and Lester reagent [18]. The crude products were purified by short column chromatography [19] with a column (30 cm x 9.6 cm 2) of 50 g of silica gel type 60 (Merck for thin-layer chromatography). Solvent systems were: (A) ether/ hexane (1 : 1); (13) chloroform/methanol (75:25); and (C) chloroform/methanol (80:20).
starting materials are not readily available and are light-sensitive. Furthermore a problem with this procedure is that many reaction steps are involved and the overall yield is low. The same considerations can be applied to the Lammers and van Boom procedure. In this report we describe the synthesis of the saturated phosphatidylglycerols by applying our recently published approach used for the preparation of the phosphatidylethanolamines and N, N-dimethylphosphatidylethanolamines. [11.
Materials
Experiment
Methods
Microanalyses of carbon, hydrogen and nitrogen were performed on the CHN-O Rapid, Heraeus. Phosphorus was determined according to Ma and McKinley [17]. Infrared spectra were recorded for solid films (triesters) and Nujol (diesters) on a Perkin-Elmer Model 687 spectrometer; for proton NMR (denterochloroform solutions), Varian EN 390 and XL 300 spectrometers were used. Reactions were monitored by thin-layer chromatography on silica gel 60 F 254 (Merck plates). The spots were visualised with the Ditto o
0
-. Lj.o.,..o.
u
v
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('mn')
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'- C~CHOHCHzOH
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Organic solvents and liquid reagents were dried and stored over molecular sieves 4A. The residual water was checked by the Karl Fischer method. 2,4,6-triisopropylbenzenesulphonyl chloride (TPS) was purchased from Fluka. 1,2-1sopropyiideneglycerol was prepared according to Ref 20. rac- l ,2-dipalmitoylglycerol 3- and rac- l ,2-distearoylglycerol 3-(o-hydroxyphenyl hydrogen phosphates) (III, R a = R t,~ and R ~ = R m.2 were synthesized as described previously [21]. Preparition of pkospbotriesters v m Procedure
The preparation of intermediate phosphotriesters (VIII) was carried out as in the case of phosphatidylethanolamines [1]. The rac-l,2-diacylglycerol 3-(o-hydroxyphenyl hydrogen phosphates) (Ill) (200---700) mg was rendered anhydrous by repeated evaporation with dry pyridine under reduced pressure. The residue was dissolved in anhydrous pyridine and acetic anhydride (6 ml and 1 ml, respectively per 400 mg). After 24 h, the concentrated solution was taken up in ether. The ethereal solution was washed several times with diluted sodium chloride solution and dried over sodium sulphate. After evaporating the ether, the acetylated compound (VII) was treated with pyridine in the same way as described above. To the glassy residue in anhydrous pyridine (5 ml), TPS and 1,2isopropylideneglycerol were added. The molar ratio of III/1,2-isopropylideneglycerol/TPS was 1 : 2 : 3. After stirring the mixture for 48 h at room temperature, ether was added. The solu-
123 tion was washed with sodium chloride solution, dried over sodium sulphate, and the solvent was removed with rotary evaporation. The residue was fractionated by short-column chromatography. rac- 1,2-Dipaimito ylg lycerol-3-ph osph oisopropylidene-glycerol o-acetoxyphenylester (VIII, R ~ = RI,9 The acetylated derivative (VII) (R ~ = R 1,~) was prepared from III (R ~ = R I,~) (433 mg) and coupled with 1,2-isopropylideneglycerol in the presence of TPS. The phosphotriester was isolated by short-column chromatography (eluent, system A) (451 mg, 87o70, R/0.30, system A. Calc. for C49HssO12P (897.2): C 65.60; H 9.55. Found: C 65.74; H 9.76. rac- 1, 2-Distearo ylglycerol-3-phsoph oisopropylideneglycerol o- acetoxyphenylester (VIII, R 1 =
RI,9
In the same way, starting from III (R ~ = R ~.2) (217 mg), the phosphotriester was also separated from the reaction mixture after shortcolumn chromatography (eluent, system A) (186 mg, 72.5°70; Rf0.30, system A). Calc. for C53H93OI2P (953.3): C 66.78; H 9.83. Found: 66.60; H 9.96. Hydrogenolysis of the triestes VIII Procedure A solution of VIII (200--700 mg) in 10--25 ml of ethanol and a mixture of platinic oxide (10 --15°70 of VIII) and palladium black (10--15070 of VIII) was shaken in an atmosphere of pure hydrogen at an initial pressure of 40--50 cm of water until the absorption of hydrogen ceased (about 1 h). The catalyst was removed by filtration and washed with ethanol. The reaction was quantitative (TLC). The filtrate was brought to dryness in vacuo and the residue was dissolved in a mixture of equal volumes of acetic acid (70070 solution in water) and ether. After 5 h, the solution showed one spot of phosphorus by TLC (system B). The solution was diluted with more ether and
washed three times with water. The ether extract was dried over sodium sulphate, then evaporated under reduced pressure, then kept under vacuum (oil pump) for 1 h. The residue was dissolved in methanol and neutralized with methanolic NaOH (0.75 g/100 ml, phenolphthalein end point) and brought to dryness. A chloroform solution of the product was diluted with a mixture of equal volumes of water and methanol. The chloroform extract was dried and evaporated. A 1 ml chloroform solution of sodium salt was diluted with 10 ml of acetone and cooled. The precipitate was centrifuged and washed with cold acetone. The operation, including the neutralization step, must be performed in a working day to avoid a redistribution of the phosphate molecule within the glycerol moiety which leads to the formation of a significant amount of 2phosphatidylglycerol isomer to the acidic conditions. rac-l,2-Dipalmitoylglycero-3-phosphoglycerol (IX, R 1 = RI.9 VIII (R ~ = R 1,~ (274.40 mg) was hydrogenated in 10 ml of ethanol. The final product (157.33 mg, 68070) showed a single spot (TLC, system B). Calc. for C3sH740~0PNa • H20 (762.6): C 59.79; H 10.04; P 4.06. Found: C 59.99; H 10.05; P 3.85. The synthesized phosphatidylglycerol was found to be unstable in the free acid form. When the product in acid solution was kept for several hours, a partial formation of the 2phosphatidylglycerol was detected (TLC, system B). After neutralization, the isomer can be separated by a short-column using solvent system B. rac-l,2-Distearoylglycerol-3-phosphoglycerol (IX, R 1 = R1,9 In the same way starting from VIII (R t = R ~,2) (346.82 mg) the product IX (R ~ =" R~,2) was prepared by hydrogenolysis and neutralization (203.02 mg, 68°70). Its solution showed a single spot of phoshorus (TLC, system B).
124
The infrared spectra of all the synthesized compounds were as follows (the tentative assignments of the bands were made according to Ref. 1. For triesters VIII (measured as solid film), the bands appears at the same positions: 3075 (v C--H, aryl), 1780, 1750 (v C = O, ester), 1600, 1500 (v C = C, aryl), 1260 (v P = O) 1180 (v,~ C--OMC), 1105--1030 (v CMO--(P)), vs C--O --C), 830 (vs O--P--O), 760, 740 (v O - - P - - O , doop C--H, aryl), 720 cm -l 0'r (CH2)n)" In the case of the diesters IX including the 2phosphatidylglycerol isomer (sodium salts, in KBr), the assignments were: 3500 (v "O--H), 1745 (v C = O , ester), 1240---1220 ( v Pq), 1110 --1050 (v PO~ v C--O--(P), v C--O--C), 720 cm- 1 (Yr (CH2))"
corresponding to the AB part of an ABX system and these protons can be assigned to the CH2OCO protons. When the CHOCO protons was decoupled, the AB part of the system ABX of the C_H2OCO protons could be clearly seen. The assignments for the C_H2OCO protons are then ¢JA = 4.39 ppm and d a = 4.17 ppm (JAa = 11.05 HZ). The centered broad absorption at 3.94 ppm must include both the C_H2OH and the C HOH signals. The C H2OH protons can be assigned to the signal at 3.74 ppm in accordance with that reported for the sngiycero-3-phosphorylcholine [22]. This signal does not appear in the spectrum of the possible 2-phosphatidylglycerol. In this case, both equivalent C_H2OH protons absorb as a doublet. Spin decoupling experiments indicate that these four protons overlap the signal of the H~ protons of the C H2OCO group (multiplet centered at 4.10 ppm). In this isomer, the other signals agreed with the structure.
1H-NMR spectra
Results and discussion
The ~H-NMR spectra also agree with the expected structures of the synthesized compounds and were obtained from solutions in CDCI 3 using the Varian FM 390 spectrometer at 90 MHz for the triester VIII and the Varian XL 300 spectrometer at 300 MHz for the diesters IX. The chemical shifts (d) are given in ppm downfield from internal TMS. The triesters gave the following NMR d values: 0.85 (t, J = 6.6 Hz, C H3CH2) , 1.25 (c, (CH2)n), 1.28 (s, C__Ha C), 1.35 (s, CH3C), 1.57 (c, CH2CH2CO), 2.10--2.40 (c, CH2CO), 2.28 (s, CH3CO), 3.60--4.40 (c, CH_~OCO, CH_2OP, CH2OC, CH_OC), 5.25 (c, CH_OCO), 7.00--7.30 (c, H, aryl). In the case of the diesters IX (sodium salts) the following assignments were made: 0.88 (t, J = 6.6 Hz, C H3CH2), 1.25 (c, (CH2)n), 1.58 (c, C_H2CH2CO), 2.28 (c, C_H2CO). The signals within the range 5.23--3.74 ppm correspond to both glycerol backbones, the low-field single proton (CHOCO, 5.23 ppm) is coupled to four CH 2 protons in the region 3.9--4.6 ppm. One CH 2 group gives a simple eight-line multiplet
In this report we described the application of the new synthesis of saturated phosphatidylethanolamines and N,N-dimethylphosphatidylethanolamines for the preparation of the phosphatidylglycerols based on the same synthetic route [1]. We prepared first the rac-l,2diacylglycerol 3-(o-hydroxyphenyl hydrogen phosphates) (III) from o-phenylene phosphorochloridate (I) via the o-phenylenecyclophosphate intermediate (II) [21], which was not isolated. The acetylation of the phenolic hydroxyl group of III affords the corresponding acetylated compound (VII), which was not isolated but was condensed with 1,2-isopropylidene glycerol in the presence of TPS to give the triesters VIII in high yields. The molar ratio of III/isopropylidenegiycerol/TPS was 1:2: 3. The rac1,2-dipalmitoyl and rac-l,2-disteroylglycerol-3phosphoisopropylideneglycerol oaeetoxyphenylestexs (VIII, R ~ = R ~,~ and R ~ = W ,2) were obtained. The catalytic hydrogenolysis of the triesters VIII quantitively removed the o-acetoxyphenyl
Calc. for C42H82OIoPNa H20 (818.6): C 61.56; H 10.34. Found: C 61.71; H 10.13.
Infrared spectra
125
group. Afterwards the isopropylidene group was removed by aqueous acetic acid under very strict conditions to avoid a redistribution of the phosphate moiety within the glycerol molecule, and so a partial formation of 2-phosphatidylglycerol isomer was detected. Thus rac-l,2-dipalmitoyl and rac- 1,2-distearoylglycerol-3-phosphoglycerols (IX, R 1 = R I,~ and R 1 = R ~,2) were synthesized as their sodium salts. The washing of the chloroform solutions (sodium salts) with the water and methanol mixture is necessary to eliminate the sodium acetate excess, since its removal is difficult to achieve by any other procedure. The structure of all the synthesized compounds was confirmed by elemental analysis as well as IR and tH-NMR spectra. Acknowledgement
5 6 7 8 9 10 11 12 13 14 15
16
The work was supported by the Comisi6n Asesora de Investigaci6n Cientifica y T6cnica (Madrid).
18
References
19
! 2 3 4
J. Calder6n and P. Yagiie, Chem. Phys. Lipids, 41 (1986) 147--157. B. Maruo and A.A. Benson, J. Am. Chem. Soc., 79 (1957) 4564--4565. M.G. Macfarlane, Biochem. J., 80 (1961) 44--51. M. Kates, D.J. Kushner and A.T. James, Can. J. Biochem. Physiol., 40 (1962) 83--94.
17
20 21 22
F. Haverkate, U.M.T. Houtsmuller and L.L.M. van Deenen, Biochim. Biophys. Acta, 63 (1962) 547--549. E.H. Strickland and A.A. Benson, Arch. Biochem. Biophys., 88 (1960) 344--348. J.Y. Kiyasu, R.A. Pieringer, H. Paulus and E.P. Kennedy, J. Biol. Chem., 238 (1963) 2293--2298. G.M. Gray, Biochim. Biophys. Acta, 84 (1964) 35--40. F. Haverhate and L.L.M. van Deenen, Biochim. Biophys. Acta, 84 (1964) 106--108. C.N. Joo and M. Kates, Biochim. Biophys. Acta, 176 (1969) 278--297. E. Baer and D. Buchnea, J. Biol. Chem., 232 (1958) 895--901. M. Saunders and H.P. Schwarz, J. Am. Chem. Soc., 88 (1966) 3844--3847. K. Harlos and M. Eibl, Biochemistry, 19 (1980) 895-899. P.P.M. Bonsen, G.H. de Haas and L.L.M. van Deenen, Chem. Phys. Lipids, 1 (1966) 33--40. J.F. Tocanne, H.M. Verheij, J.A.F. Op den Karnp and L.L.M. van Deenen, Chem. Phys. Lipids, 13 (1974) 389--403. J.G. Lammers and J.H. van Boom, Recl. Tray. Chem. Pays-Bas, 98 (1979) 243--250. T.S. Ma and J.D. MacKinley, Jr., Mikrochim. Acta (1953) 4--13. J.L. Dittmer and R.L. Lester, J. Lipid Res., 5 (1964) 126--127. B.J. Hunt and W. Rigby, Chem. Ind. (London), 0967) 1868--1869. Organic Synthesis, Vol. 28, John Wiley and Sons, London, 1946, p. 73. J. Calder6n and P. Yagiie, An. Quire., 81C (1985) 97-101. N.J.M. Birdsall, J. Feeney, A.G. Lee, Y.K. Levine and J.C. Metcalfe, J. Chem. Soc., Perkin 11, (1972) 1441-1445.
121
Phosphoric acid derivatives. XVI. Phosphatidylglycerols from diacylglycerol (o-acetoxyphenyl hydrogen phosphates)* J. Calder6n and P. Yagiie Instituto de Qu~mica Org(~nica General del C.S.L C., Juan de la Cierva, 3, 28006 Madrid (Spain) The syntheses of saturated diacylglycero-3-phosphogylcerols are described. Diacylglycerol (o-acetoxyphenyl hydrogen phosphates) were coupled with 1,2-isopropylideneglycerol in the presence of 2,4,6-triisopylbenzenesulphonylchloride to phosphotriester intermediates, which were readily purified by short-column chromatography. Subsequently, the phosphateprotecting and the isopropylidene groups were removed by bydrogenolysis and acid hydrolysis, respectively.
Keywords: synthesis; saturated phospholipids; phosphatidylglycerols.
Introduction
Diacylphosphatidylglycerols were first detected by Maruo and Benson in higher plants and algae [2]. These phospholipids were also found to occur in bacterial membranes [3--5] and in minor amounts in animal tissues [6--8]. The structure of these naturally occurring lipids was determinated by Haverkate and van Deenen [9] to be 3-sn-phosphatidyl-l'- sn-glycerol. Phosphatidylglycerols isolated from extremely halophilic bacteria contain ether-linked alkyl groups rather than fatty acid ester groups, but their structures were established as l-sn-phosphatidyl-3'- sn-glycerol [10]. Chemical synthesis was carried out by one of the following general methods: (a) phosphorylation with phosphorus oxychloride of a 1,2-diacylglycerol and a protected glycerol; and (b) reaction of a 1,2-diacyl-3-iodo-l,2-propanediol with an appropriate silver phosphate. The first chemical synthesis by method (a) was published by Baer and Buchnea [11], but the stereochemical configuration of the non-acetylated moiety was opposite that of the natural compound. Saunders and Schwarz [12] used the same approach, but with 2,3-dibenzyl-sn-glycerol Correspondence to: J. Calder6n. *For Part XV, see Ref. 1.
instead of 1,2-isopropylidene-sn-glycerol as one of the starting materials. The product has the configuration of the naturally occurring phospholipids. Harlos and Eibl [13] prepared 1,2-tetradecyl-rac-glycero-3-phosphoglycerol by this approach using 1,2-isopropylidene-sn-glycerol. Method (b) was first performed by Bonsen et al. [14] from the silver salt of 2,3-isopropylidene-sn-glycerol-l-phosphoric acid benzylester and 1-oleyl-2-palmitoyl-sn-glycerol-3-iodohydrine. After removal of the protecting groups, the end product was obtained with sterochemical purity An alternative approach was described by Tocanne et al. [15]. They started from the silver salt of the 1,2-didecanoyl-sn-glycero-3-phosphoric acid benzylester and 2,3-dibenzylglycerolsn-l-iodohydrine. By applying the same route, Joe and Kates [10] prepared a dialkyl derivative of phosphatidylglycerol with the same configuration as the phospholipids isolated from halophilic bacteria. Finally, Lammers and van Boom [16] studied a new triester approach starting with a monofunctional reagent, R~R2POCI (R ~ and R 2 being phosphate protecting groups). In this way, 1,2distearoyl-sn-glycero-3-phospho-l'-glycerol was prepared. Method (a) is accompanied by the formation of many by-products due to the trifunctionality of phosphorus oxychloride. In method (b), the
0009-3084/88/$03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
122
mer and Lester reagent [18]. The crude products were purified by short column chromatography [19] with a column (30 cm x 9.6 cm 2) of 50 g of silica gel type 60 (Merck for thin-layer chromatography). Solvent systems were: (A) ether/ hexane (1 : 1); (13) chloroform/methanol (75:25); and (C) chloroform/methanol (80:20).
starting materials are not readily available and are light-sensitive. Furthermore a problem with this procedure is that many reaction steps are involved and the overall yield is low. The same considerations can be applied to the Lammers and van Boom procedure. In this report we describe the synthesis of the saturated phosphatidylglycerols by applying our recently published approach used for the preparation of the phosphatidylethanolamines and N, N-dimethylphosphatidylethanolamines. [11.
Materials
Experiment
Methods
Microanalyses of carbon, hydrogen and nitrogen were performed on the CHN-O Rapid, Heraeus. Phosphorus was determined according to Ma and McKinley [17]. Infrared spectra were recorded for solid films (triesters) and Nujol (diesters) on a Perkin-Elmer Model 687 spectrometer; for proton NMR (denterochloroform solutions), Varian EN 390 and XL 300 spectrometers were used. Reactions were monitored by thin-layer chromatography on silica gel 60 F 254 (Merck plates). The spots were visualised with the Ditto o
0
-. Lj.o.,..o.
u
v
(I)
(II}
('mn')
0 \ 101 / O R
" ~
~.,~
0 0~....0 RI
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(zz)
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l
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I
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'- C~CHOHCHzOH
I
Organic solvents and liquid reagents were dried and stored over molecular sieves 4A. The residual water was checked by the Karl Fischer method. 2,4,6-triisopropylbenzenesulphonyl chloride (TPS) was purchased from Fluka. 1,2-1sopropyiideneglycerol was prepared according to Ref 20. rac- l ,2-dipalmitoylglycerol 3- and rac- l ,2-distearoylglycerol 3-(o-hydroxyphenyl hydrogen phosphates) (III, R a = R t,~ and R ~ = R m.2 were synthesized as described previously [21]. Preparition of pkospbotriesters v m Procedure
The preparation of intermediate phosphotriesters (VIII) was carried out as in the case of phosphatidylethanolamines [1]. The rac-l,2-diacylglycerol 3-(o-hydroxyphenyl hydrogen phosphates) (Ill) (200---700) mg was rendered anhydrous by repeated evaporation with dry pyridine under reduced pressure. The residue was dissolved in anhydrous pyridine and acetic anhydride (6 ml and 1 ml, respectively per 400 mg). After 24 h, the concentrated solution was taken up in ether. The ethereal solution was washed several times with diluted sodium chloride solution and dried over sodium sulphate. After evaporating the ether, the acetylated compound (VII) was treated with pyridine in the same way as described above. To the glassy residue in anhydrous pyridine (5 ml), TPS and 1,2isopropylideneglycerol were added. The molar ratio of III/1,2-isopropylideneglycerol/TPS was 1 : 2 : 3. After stirring the mixture for 48 h at room temperature, ether was added. The solu-
123 tion was washed with sodium chloride solution, dried over sodium sulphate, and the solvent was removed with rotary evaporation. The residue was fractionated by short-column chromatography. rac- 1,2-Dipaimito ylg lycerol-3-ph osph oisopropylidene-glycerol o-acetoxyphenylester (VIII, R ~ = RI,9 The acetylated derivative (VII) (R ~ = R 1,~) was prepared from III (R ~ = R I,~) (433 mg) and coupled with 1,2-isopropylideneglycerol in the presence of TPS. The phosphotriester was isolated by short-column chromatography (eluent, system A) (451 mg, 87o70, R/0.30, system A. Calc. for C49HssO12P (897.2): C 65.60; H 9.55. Found: C 65.74; H 9.76. rac- 1, 2-Distearo ylglycerol-3-phsoph oisopropylideneglycerol o- acetoxyphenylester (VIII, R 1 =
RI,9
In the same way, starting from III (R ~ = R ~.2) (217 mg), the phosphotriester was also separated from the reaction mixture after shortcolumn chromatography (eluent, system A) (186 mg, 72.5°70; Rf0.30, system A). Calc. for C53H93OI2P (953.3): C 66.78; H 9.83. Found: 66.60; H 9.96. Hydrogenolysis of the triestes VIII Procedure A solution of VIII (200--700 mg) in 10--25 ml of ethanol and a mixture of platinic oxide (10 --15°70 of VIII) and palladium black (10--15070 of VIII) was shaken in an atmosphere of pure hydrogen at an initial pressure of 40--50 cm of water until the absorption of hydrogen ceased (about 1 h). The catalyst was removed by filtration and washed with ethanol. The reaction was quantitative (TLC). The filtrate was brought to dryness in vacuo and the residue was dissolved in a mixture of equal volumes of acetic acid (70070 solution in water) and ether. After 5 h, the solution showed one spot of phosphorus by TLC (system B). The solution was diluted with more ether and
washed three times with water. The ether extract was dried over sodium sulphate, then evaporated under reduced pressure, then kept under vacuum (oil pump) for 1 h. The residue was dissolved in methanol and neutralized with methanolic NaOH (0.75 g/100 ml, phenolphthalein end point) and brought to dryness. A chloroform solution of the product was diluted with a mixture of equal volumes of water and methanol. The chloroform extract was dried and evaporated. A 1 ml chloroform solution of sodium salt was diluted with 10 ml of acetone and cooled. The precipitate was centrifuged and washed with cold acetone. The operation, including the neutralization step, must be performed in a working day to avoid a redistribution of the phosphate molecule within the glycerol moiety which leads to the formation of a significant amount of 2phosphatidylglycerol isomer to the acidic conditions. rac-l,2-Dipalmitoylglycero-3-phosphoglycerol (IX, R 1 = RI.9 VIII (R ~ = R 1,~ (274.40 mg) was hydrogenated in 10 ml of ethanol. The final product (157.33 mg, 68070) showed a single spot (TLC, system B). Calc. for C3sH740~0PNa • H20 (762.6): C 59.79; H 10.04; P 4.06. Found: C 59.99; H 10.05; P 3.85. The synthesized phosphatidylglycerol was found to be unstable in the free acid form. When the product in acid solution was kept for several hours, a partial formation of the 2phosphatidylglycerol was detected (TLC, system B). After neutralization, the isomer can be separated by a short-column using solvent system B. rac-l,2-Distearoylglycerol-3-phosphoglycerol (IX, R 1 = R1,9 In the same way starting from VIII (R t = R ~,2) (346.82 mg) the product IX (R ~ =" R~,2) was prepared by hydrogenolysis and neutralization (203.02 mg, 68°70). Its solution showed a single spot of phoshorus (TLC, system B).
124
The infrared spectra of all the synthesized compounds were as follows (the tentative assignments of the bands were made according to Ref. 1. For triesters VIII (measured as solid film), the bands appears at the same positions: 3075 (v C--H, aryl), 1780, 1750 (v C = O, ester), 1600, 1500 (v C = C, aryl), 1260 (v P = O) 1180 (v,~ C--OMC), 1105--1030 (v CMO--(P)), vs C--O --C), 830 (vs O--P--O), 760, 740 (v O - - P - - O , doop C--H, aryl), 720 cm -l 0'r (CH2)n)" In the case of the diesters IX including the 2phosphatidylglycerol isomer (sodium salts, in KBr), the assignments were: 3500 (v "O--H), 1745 (v C = O , ester), 1240---1220 ( v Pq), 1110 --1050 (v PO~ v C--O--(P), v C--O--C), 720 cm- 1 (Yr (CH2))"
corresponding to the AB part of an ABX system and these protons can be assigned to the CH2OCO protons. When the CHOCO protons was decoupled, the AB part of the system ABX of the C_H2OCO protons could be clearly seen. The assignments for the C_H2OCO protons are then ¢JA = 4.39 ppm and d a = 4.17 ppm (JAa = 11.05 HZ). The centered broad absorption at 3.94 ppm must include both the C_H2OH and the C HOH signals. The C H2OH protons can be assigned to the signal at 3.74 ppm in accordance with that reported for the sngiycero-3-phosphorylcholine [22]. This signal does not appear in the spectrum of the possible 2-phosphatidylglycerol. In this case, both equivalent C_H2OH protons absorb as a doublet. Spin decoupling experiments indicate that these four protons overlap the signal of the H~ protons of the C H2OCO group (multiplet centered at 4.10 ppm). In this isomer, the other signals agreed with the structure.
1H-NMR spectra
Results and discussion
The ~H-NMR spectra also agree with the expected structures of the synthesized compounds and were obtained from solutions in CDCI 3 using the Varian FM 390 spectrometer at 90 MHz for the triester VIII and the Varian XL 300 spectrometer at 300 MHz for the diesters IX. The chemical shifts (d) are given in ppm downfield from internal TMS. The triesters gave the following NMR d values: 0.85 (t, J = 6.6 Hz, C H3CH2) , 1.25 (c, (CH2)n), 1.28 (s, C__Ha C), 1.35 (s, CH3C), 1.57 (c, CH2CH2CO), 2.10--2.40 (c, CH2CO), 2.28 (s, CH3CO), 3.60--4.40 (c, CH_~OCO, CH_2OP, CH2OC, CH_OC), 5.25 (c, CH_OCO), 7.00--7.30 (c, H, aryl). In the case of the diesters IX (sodium salts) the following assignments were made: 0.88 (t, J = 6.6 Hz, C H3CH2), 1.25 (c, (CH2)n), 1.58 (c, C_H2CH2CO), 2.28 (c, C_H2CO). The signals within the range 5.23--3.74 ppm correspond to both glycerol backbones, the low-field single proton (CHOCO, 5.23 ppm) is coupled to four CH 2 protons in the region 3.9--4.6 ppm. One CH 2 group gives a simple eight-line multiplet
In this report we described the application of the new synthesis of saturated phosphatidylethanolamines and N,N-dimethylphosphatidylethanolamines for the preparation of the phosphatidylglycerols based on the same synthetic route [1]. We prepared first the rac-l,2diacylglycerol 3-(o-hydroxyphenyl hydrogen phosphates) (III) from o-phenylene phosphorochloridate (I) via the o-phenylenecyclophosphate intermediate (II) [21], which was not isolated. The acetylation of the phenolic hydroxyl group of III affords the corresponding acetylated compound (VII), which was not isolated but was condensed with 1,2-isopropylidene glycerol in the presence of TPS to give the triesters VIII in high yields. The molar ratio of III/isopropylidenegiycerol/TPS was 1:2: 3. The rac1,2-dipalmitoyl and rac-l,2-disteroylglycerol-3phosphoisopropylideneglycerol oaeetoxyphenylestexs (VIII, R ~ = R ~,~ and R ~ = W ,2) were obtained. The catalytic hydrogenolysis of the triesters VIII quantitively removed the o-acetoxyphenyl
Calc. for C42H82OIoPNa H20 (818.6): C 61.56; H 10.34. Found: C 61.71; H 10.13.
Infrared spectra
125
group. Afterwards the isopropylidene group was removed by aqueous acetic acid under very strict conditions to avoid a redistribution of the phosphate moiety within the glycerol molecule, and so a partial formation of 2-phosphatidylglycerol isomer was detected. Thus rac-l,2-dipalmitoyl and rac- 1,2-distearoylglycerol-3-phosphoglycerols (IX, R 1 = R I,~ and R 1 = R ~,2) were synthesized as their sodium salts. The washing of the chloroform solutions (sodium salts) with the water and methanol mixture is necessary to eliminate the sodium acetate excess, since its removal is difficult to achieve by any other procedure. The structure of all the synthesized compounds was confirmed by elemental analysis as well as IR and tH-NMR spectra. Acknowledgement
5 6 7 8 9 10 11 12 13 14 15
16
The work was supported by the Comisi6n Asesora de Investigaci6n Cientifica y T6cnica (Madrid).
18
References
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