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A facile procedure for the synthesis of saturated phosphatidylcholines
Chemistry and Physics of Lipids, 28 (1981 ) 111 - 115 © Elsevier/North-Holland Scientific Publishers Ltd.
Short Communication A FACILE PROCEDURE FOR THE SYNTHESIS OF SATURATED PHOSPHATIDYLCHOLINES
A. HERMETTER and F. PALTAUF Institut ffir Bioehemie und Lebensmittelchemie. Teehnische Universitdt Graz, Schlogelgasse 9, A-8010 Graz (Austria)
Received June 16th, 1980
accepted October 5th, 1980
1,2-Dipalmitoyl-, 1,2-distearoyl- and mixed chain 1,2-diacyl-sn-glycero-3-phosphocholines were synthesized by a modification of the method of Warner and Benson [7]. Glycerophosphocholine (GPC) cadmium chloride complex was acylated with imidazolides of the corresponding fatty acids in dimethylsulfoxide-tetrahydrofurane (DMSO-THF) using the methylsulfinylmethide anion as a catalyst. The imidazolides were used for the acylation step without prior isolation and purification. Phosphatidylcholines were obtained in yields over 70%, they were free of sn- l- and sn-2-isomers.
Introduction Partial syntheses o f phosphatidylcholines o f the 'natural' sn-3-configuration having well-defined acyl chains can be achieved by acylation of glycerophosphocholine (GPC). Acylation procedures involve the use of acyl chlorides, anhydrides [1,2] or imidazolides [3], most of them require the presence of a basic catalyst. Long reaction times, fairly high temperatures and strong bases can lead to side reactions such as isomerisation, racemisation and phosphodiester migration [3]. Particularly strong reaction conditions are required for the synthesis of long chain saturated lecithins because o f the rather lower reactivity and low solubility o f the saturated fatty acid derivatives in the solvent systems used [3,4]. More recent methods produce the desired lecithins in good yields based on GPC [4,5], but yields based on fatty acids are poor, because the methods require the isolation and purification o f the reactive fatty acid derivatives. It is known, that carboxylic acids can easily be esterified with alcohols in the presence of carbonyldiimidazole (CDI), which forms an imidazolide, and an alkoxide anion [6]. A similar principle underlies the acylation method of Warner and Benson [7], which is, however, restricted to the synthesis of unsaturated phosphatidylcholines [4,7]. In this procedure, GPC is acylated with the imidazolides of unsaturated fatty acids in dimethylsulfoxide (DMSO) in the presence of the DMSO anion. The reaction is complete within a few minutes at 17°C and thus avoids the above described shortcomings. The acyl imidazolides are prepared from fatty acids 111
112
A. Hermetter and F. Paltauf, Synthesis of saturated phosphatidyicholines
0! o
GPC.CdCt 2 *
2 R-
DMSO"
,sm,o
J,
R--C--O--CH
'0
,c.3,3
1. I!. lll Scheme I. Synthesis of saturated 1,2-diacyl-sn-glycero-3-phosphocholines.(I) R-CO = C16:o; (II) R-CO = Cls:o ; (III) R-CO = Cl,:o (10%), CI6:0 (69%), Cla:0 (16%), Cis:l (5%). GPC, snglycero-3-phosphocholine.
and CDI in tetrahydrofurane (THF). After evaporation of the solvent, they can be used for the acylation step. The procedure is unsuitable for the acylation of GPC with imidazolides of long-chain saturated fatty acids, probably because of their poor solubility in DMSO. We have found, that the cadmium chloride complex of GPC can be readily acylated with an excess of imidazolides of long-chain saturated fatty acids in the presence of the DMSO anion within 15 min at 20°C, provided a mixed solvent system DMSO-THF is used and the temperature is slightly increased before adding the catalyst. Following this procedure (Scheme I), dipalmitoyl- (I), distearoyl- (II) and saturated mixed acid (III) lecithins were synthesized. The phosphatidylcholines were obtained in approx. 70% yield after ion exchange chromatography and further purification by chromatography on silica gel. Materials and Methods Materials
The cadmium chloride complex of GPC was purchased from Lipid Products, South Nutfield, U.K. Fatty acids (3>99% pure) were obtained from Nu Chek Prep, Elysian, Minnesota, U.S.A. CDI (98%) was purchased from EGA, Steinheim, F.R.G. Ion Exchange Resins, IRC-50 and IRA-68 were purchased from Serva, Heidelberg, F.R.G. Silica Gel 60 H and silica Gel 60 for column chromatography, 0.0400.063 mm, were purchased from Merck, Darmstadt, F.R.G. Snake venom phospholipase A2 (Crotalus) was a product of Boehringer, Mannheim, F.R.G. Solvents were purified by chromatography on basic alumina, activity I (Merck)immediately before use. Anhydrous conditions were maintained throughout all synthetic operations. Methods
The purity of phospholipids was checked by thin-layer chromatography (TLC) on silica gel H with the following solvent systems: (A) CHC1JMeOH/7 M-NH3, 50 : 25 : 6, v/v/v; (B) CHC1JMeOH/H20, 95 : 35 : 6, v/v/v. Solvent B has been described previously for the separation of sn-3- and sn-2-1ecithin isomers [3]. Lipids
A. Hermetter and F. Paltauf, Synthesis of saturated phosphatidylcholines
113
were detected by spraying with molybdic aicd and charring after spraying with 50% H2SO4. 'Medium pressure liquid chromatography' [8] was carried out on a column, 35 X 3 cm, packed with silica gel, 0.040-0.063 mm. A pump (RP-D/O-SSY) purchased from Fluid Metering Inc., New York, was used for solvent delivery at a rate of 50 ml/min. Samples dissolved in 20 ml chloroform were applied to the column and eluted with a gradient of chloroform/methanol starting with the ratio 7 : 3 (v/v). After elution with 1.8 1 of solvent the ratio 3 : 7 (v/v) was reached. Fractions (100-ml) were collected and analyzed by TLC. Pure lecithins were eluted with fractions 7-18. Lipid phosphorus was determined by the method of Bartlett [9]. Carboxyl esters were analyzed by the hydroxamate method [10]. Optical rotation was measured on a Perkin Elmer 141 polarimeter in a 10-cm microcell at the Na/D line. Lipid samples (c = 1.1 g/100 ml) were dissolved in CHCla]MeOH (1 : 1, v/v). Cleavage of the sn-3-1ecithins with phospholipase A2 was carried out as described [111. The fatty acid composition of lecithin III was determined by gas chromatography of the methyl esters obtained by interesterification with BF3-MeOH [12]. The methyl esters were separated on a column, 2 m × 1.8 turn, packed with 8% BDS on Chromosorb W using a Packard Chromatograph Model 419 equipped with flame ionisation detector; carrier gas N2, 50 ml/min; column temperature 170°C, detector and injector temperature 220°C. Preparation o f imidazolides o f saturated fatty acids. Fatty acid (8 mmol) and 10 mmol CDI in 10 ml tetrahydrofurane (20 ml in the case of stearic acid) are stirred for 45 min at room temperature. During the reaction imidazolide precipitates, while CO2 is being released. Acylation o f the GPC-CdCl2 complex. The GPC-CdC12 complex (878 rag, 2 mmol) is dissolved in 40 ml DMSO at 40°C. The imidazolide suspension is clarified by heating on a water bath at 40°(2 and the resultant solution is added to the GPCCdC12 solution in DMSO. After addition of 10 mmol metallic sodium, dissolved in 16 ml DMSO, the reaction starts arm the formed lecithin precipitates. The tightly closed reaction vessel is left at 20°C for 15 min and is slightly shaken in intervals of a few minutes. The reaction mixture is then cooled in an ice bath and rapidly neutralized with 100 ml 0.1 N acetic acid in water. The lecithin is extracted with 120 ml and then twice with 60 ml portions of CHCI3/MeOH (2 : 1, v/v). The combined chloroform phases are washed with two 60-ml portions of methanol]water (1 : 1 ,v/v) and evaporated under reduced pressure. Water is removed by evaporation in the presence of ethanol]benzene (3 : 2, v/v). Residual DMSO is removed under high vacuum at about 60°12. TLC of the residue in both solvent systems A and B showed, after spraying with molybdic acid solution, only one phosphorus-containing spot corresponding to lecithin. The residue is dissolved in 200 ml CHC13/MeOH/H20 (5 : 4 : 1, v/v/v) and passed through a mixed bed ion exchange column containing 20 g IRA-68 and 10 g IRC-50.
114
A. Hermetter and F. Paltauf, Synthesis of saturated phosphatidylcholines
The column is washed with 200 ml CHC1JMeOH/H20 (5 : 4 : 1, v/v/v) and the combined eluents are evaporated under reduced pressure. Residual water is removed by evaporation with ethanol/benzene (3 : 2, v/v) and with benzene. The phospholipid is purified by 'medium pressure liquid chromatography' as described above. Yields of lecithin amount to 1.1 g (72%). The sn-3-1ecithins synthesized are free of sn-2-phosphatidylcholines. Phosphorus and carboxyl ester analyses of the phospholipids agree well with the calculated values. Optical rotation of lecithin I, [aD] + 6.5 °, corresponds to the values known from the literature [4] indicating that racemisation does not occur. Phospholipase A2 cleaved the phosphatidylcholine III completely to lysolecithin and fatty acid.
Discussion GPC is readily acylated by imidazolides of unsaturated fatty acids in the presence of the DMSO anion in DMSO, but not by saturated fatty acid derivatives because of their low solubility in this solvent [4,7]. We have found, that the imidazolides of saturated fatty acids are sufficiently soluble in a mixed THF-DMSO system at 40°C. The GPC-CdCI: complex is also readily soluble in the solvent mixture and reacts rapidly with the imidazolides after addition of the DMSO- catalyst at room temperature within 15 min (Scheme I). Under these mild conditions pure phosphatidylcholines are obtained in good yields, free of positional and steric isomers. Racemisation induced by the basic catalyst is not observed; this can be explained by the action of DMSO- as a sterically hindered base [7]. Under the experimental conditions described, imidazolides with saturated chains of 14, 16 and 18 carbon atoms react with GPC to the same extent. This was shown by quantitative fatty acid analysis of an 1-acyl-GPC derived from lecithin III by phospholipase A2 cleavage; the fatty acid composition of the lysolecithin corresponded well to the mixture used for acylation. Since the reactive acyl derivatives prepared from the acids and CDI need not be isolated and purified for the acylation step, good yields of lecithins, based on fatty acids, are obtained. Thus the procedure described offers a simple and attractive way for the synthesis of phosphatidylcholines with saturated photo-, spin-, deuteriumor radioactively labelled acyl chains.
Acknowledgements We thank Mr. H. Stfitz for expert technical assistance. This work was supported in part by the 'Fonds zur FiSrderung der wissenschaftlichen Forschung in Osterreich' (Projekt Nr. 3982).
A. Hermetter and F. Paltauf, Syn thesis of saturated phosphatidylcholines
115
References 1 A.J. Slotboom, H.M. Verkeij and G.H. de Haas, Chem. Phys. Lipids, 11 (1973) 295. 2 M. Kates, in E.D. Korn (Ed.), Methods in Membrane Biology, Vol. 8, Plenum Press, New York, 1977, p. 219. 3 J.G. Lammers, Th. J. Liefkens, J. Bus and J. van der Meer, Chem. Phys. Lipids, 22 (1978) 293. 4 K.M. Patel, J.D. Morrisett and J.T. Sparrow, J. Lipid Res., 20 (1979) 674. 5 C.M. Gupta, R. Radhakrishnan and H.G. Khorana Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 4315. 6 R.G. Hiskey and J.B. Adams, J. Am. Chem. Soc., 87 (1965) 3969. 7 T.G. Warner and A.A. Benson, J. Lipid Res., 18 (1977) 548. 8 H. Loibner and G. Seidl, Chromatographia, 12 (1979) 600. 9 G.R. Bartlett, J. Biol. Chem., 234 (1959) 466. 10 J. Stern and B. Shapiro, J. Clin. Pathol., 6 (1953) 158. 11 C. Long and I.F. Penny, Biochem. J., 65 (1957) 382. 12 W.R. Morrison and I.M. Smith, J. Lipid Res., 5 (1964) 600.
Short Communication A FACILE PROCEDURE FOR THE SYNTHESIS OF SATURATED PHOSPHATIDYLCHOLINES
A. HERMETTER and F. PALTAUF Institut ffir Bioehemie und Lebensmittelchemie. Teehnische Universitdt Graz, Schlogelgasse 9, A-8010 Graz (Austria)
Received June 16th, 1980
accepted October 5th, 1980
1,2-Dipalmitoyl-, 1,2-distearoyl- and mixed chain 1,2-diacyl-sn-glycero-3-phosphocholines were synthesized by a modification of the method of Warner and Benson [7]. Glycerophosphocholine (GPC) cadmium chloride complex was acylated with imidazolides of the corresponding fatty acids in dimethylsulfoxide-tetrahydrofurane (DMSO-THF) using the methylsulfinylmethide anion as a catalyst. The imidazolides were used for the acylation step without prior isolation and purification. Phosphatidylcholines were obtained in yields over 70%, they were free of sn- l- and sn-2-isomers.
Introduction Partial syntheses o f phosphatidylcholines o f the 'natural' sn-3-configuration having well-defined acyl chains can be achieved by acylation of glycerophosphocholine (GPC). Acylation procedures involve the use of acyl chlorides, anhydrides [1,2] or imidazolides [3], most of them require the presence of a basic catalyst. Long reaction times, fairly high temperatures and strong bases can lead to side reactions such as isomerisation, racemisation and phosphodiester migration [3]. Particularly strong reaction conditions are required for the synthesis of long chain saturated lecithins because o f the rather lower reactivity and low solubility o f the saturated fatty acid derivatives in the solvent systems used [3,4]. More recent methods produce the desired lecithins in good yields based on GPC [4,5], but yields based on fatty acids are poor, because the methods require the isolation and purification o f the reactive fatty acid derivatives. It is known, that carboxylic acids can easily be esterified with alcohols in the presence of carbonyldiimidazole (CDI), which forms an imidazolide, and an alkoxide anion [6]. A similar principle underlies the acylation method of Warner and Benson [7], which is, however, restricted to the synthesis of unsaturated phosphatidylcholines [4,7]. In this procedure, GPC is acylated with the imidazolides of unsaturated fatty acids in dimethylsulfoxide (DMSO) in the presence of the DMSO anion. The reaction is complete within a few minutes at 17°C and thus avoids the above described shortcomings. The acyl imidazolides are prepared from fatty acids 111
112
A. Hermetter and F. Paltauf, Synthesis of saturated phosphatidyicholines
0! o
GPC.CdCt 2 *
2 R-
DMSO"
,sm,o
J,
R--C--O--CH
'0
,c.3,3
1. I!. lll Scheme I. Synthesis of saturated 1,2-diacyl-sn-glycero-3-phosphocholines.(I) R-CO = C16:o; (II) R-CO = Cls:o ; (III) R-CO = Cl,:o (10%), CI6:0 (69%), Cla:0 (16%), Cis:l (5%). GPC, snglycero-3-phosphocholine.
and CDI in tetrahydrofurane (THF). After evaporation of the solvent, they can be used for the acylation step. The procedure is unsuitable for the acylation of GPC with imidazolides of long-chain saturated fatty acids, probably because of their poor solubility in DMSO. We have found, that the cadmium chloride complex of GPC can be readily acylated with an excess of imidazolides of long-chain saturated fatty acids in the presence of the DMSO anion within 15 min at 20°C, provided a mixed solvent system DMSO-THF is used and the temperature is slightly increased before adding the catalyst. Following this procedure (Scheme I), dipalmitoyl- (I), distearoyl- (II) and saturated mixed acid (III) lecithins were synthesized. The phosphatidylcholines were obtained in approx. 70% yield after ion exchange chromatography and further purification by chromatography on silica gel. Materials and Methods Materials
The cadmium chloride complex of GPC was purchased from Lipid Products, South Nutfield, U.K. Fatty acids (3>99% pure) were obtained from Nu Chek Prep, Elysian, Minnesota, U.S.A. CDI (98%) was purchased from EGA, Steinheim, F.R.G. Ion Exchange Resins, IRC-50 and IRA-68 were purchased from Serva, Heidelberg, F.R.G. Silica Gel 60 H and silica Gel 60 for column chromatography, 0.0400.063 mm, were purchased from Merck, Darmstadt, F.R.G. Snake venom phospholipase A2 (Crotalus) was a product of Boehringer, Mannheim, F.R.G. Solvents were purified by chromatography on basic alumina, activity I (Merck)immediately before use. Anhydrous conditions were maintained throughout all synthetic operations. Methods
The purity of phospholipids was checked by thin-layer chromatography (TLC) on silica gel H with the following solvent systems: (A) CHC1JMeOH/7 M-NH3, 50 : 25 : 6, v/v/v; (B) CHC1JMeOH/H20, 95 : 35 : 6, v/v/v. Solvent B has been described previously for the separation of sn-3- and sn-2-1ecithin isomers [3]. Lipids
A. Hermetter and F. Paltauf, Synthesis of saturated phosphatidylcholines
113
were detected by spraying with molybdic aicd and charring after spraying with 50% H2SO4. 'Medium pressure liquid chromatography' [8] was carried out on a column, 35 X 3 cm, packed with silica gel, 0.040-0.063 mm. A pump (RP-D/O-SSY) purchased from Fluid Metering Inc., New York, was used for solvent delivery at a rate of 50 ml/min. Samples dissolved in 20 ml chloroform were applied to the column and eluted with a gradient of chloroform/methanol starting with the ratio 7 : 3 (v/v). After elution with 1.8 1 of solvent the ratio 3 : 7 (v/v) was reached. Fractions (100-ml) were collected and analyzed by TLC. Pure lecithins were eluted with fractions 7-18. Lipid phosphorus was determined by the method of Bartlett [9]. Carboxyl esters were analyzed by the hydroxamate method [10]. Optical rotation was measured on a Perkin Elmer 141 polarimeter in a 10-cm microcell at the Na/D line. Lipid samples (c = 1.1 g/100 ml) were dissolved in CHCla]MeOH (1 : 1, v/v). Cleavage of the sn-3-1ecithins with phospholipase A2 was carried out as described [111. The fatty acid composition of lecithin III was determined by gas chromatography of the methyl esters obtained by interesterification with BF3-MeOH [12]. The methyl esters were separated on a column, 2 m × 1.8 turn, packed with 8% BDS on Chromosorb W using a Packard Chromatograph Model 419 equipped with flame ionisation detector; carrier gas N2, 50 ml/min; column temperature 170°C, detector and injector temperature 220°C. Preparation o f imidazolides o f saturated fatty acids. Fatty acid (8 mmol) and 10 mmol CDI in 10 ml tetrahydrofurane (20 ml in the case of stearic acid) are stirred for 45 min at room temperature. During the reaction imidazolide precipitates, while CO2 is being released. Acylation o f the GPC-CdCl2 complex. The GPC-CdC12 complex (878 rag, 2 mmol) is dissolved in 40 ml DMSO at 40°C. The imidazolide suspension is clarified by heating on a water bath at 40°(2 and the resultant solution is added to the GPCCdC12 solution in DMSO. After addition of 10 mmol metallic sodium, dissolved in 16 ml DMSO, the reaction starts arm the formed lecithin precipitates. The tightly closed reaction vessel is left at 20°C for 15 min and is slightly shaken in intervals of a few minutes. The reaction mixture is then cooled in an ice bath and rapidly neutralized with 100 ml 0.1 N acetic acid in water. The lecithin is extracted with 120 ml and then twice with 60 ml portions of CHCI3/MeOH (2 : 1, v/v). The combined chloroform phases are washed with two 60-ml portions of methanol]water (1 : 1 ,v/v) and evaporated under reduced pressure. Water is removed by evaporation in the presence of ethanol]benzene (3 : 2, v/v). Residual DMSO is removed under high vacuum at about 60°12. TLC of the residue in both solvent systems A and B showed, after spraying with molybdic acid solution, only one phosphorus-containing spot corresponding to lecithin. The residue is dissolved in 200 ml CHC13/MeOH/H20 (5 : 4 : 1, v/v/v) and passed through a mixed bed ion exchange column containing 20 g IRA-68 and 10 g IRC-50.
114
A. Hermetter and F. Paltauf, Synthesis of saturated phosphatidylcholines
The column is washed with 200 ml CHC1JMeOH/H20 (5 : 4 : 1, v/v/v) and the combined eluents are evaporated under reduced pressure. Residual water is removed by evaporation with ethanol/benzene (3 : 2, v/v) and with benzene. The phospholipid is purified by 'medium pressure liquid chromatography' as described above. Yields of lecithin amount to 1.1 g (72%). The sn-3-1ecithins synthesized are free of sn-2-phosphatidylcholines. Phosphorus and carboxyl ester analyses of the phospholipids agree well with the calculated values. Optical rotation of lecithin I, [aD] + 6.5 °, corresponds to the values known from the literature [4] indicating that racemisation does not occur. Phospholipase A2 cleaved the phosphatidylcholine III completely to lysolecithin and fatty acid.
Discussion GPC is readily acylated by imidazolides of unsaturated fatty acids in the presence of the DMSO anion in DMSO, but not by saturated fatty acid derivatives because of their low solubility in this solvent [4,7]. We have found, that the imidazolides of saturated fatty acids are sufficiently soluble in a mixed THF-DMSO system at 40°C. The GPC-CdCI: complex is also readily soluble in the solvent mixture and reacts rapidly with the imidazolides after addition of the DMSO- catalyst at room temperature within 15 min (Scheme I). Under these mild conditions pure phosphatidylcholines are obtained in good yields, free of positional and steric isomers. Racemisation induced by the basic catalyst is not observed; this can be explained by the action of DMSO- as a sterically hindered base [7]. Under the experimental conditions described, imidazolides with saturated chains of 14, 16 and 18 carbon atoms react with GPC to the same extent. This was shown by quantitative fatty acid analysis of an 1-acyl-GPC derived from lecithin III by phospholipase A2 cleavage; the fatty acid composition of the lysolecithin corresponded well to the mixture used for acylation. Since the reactive acyl derivatives prepared from the acids and CDI need not be isolated and purified for the acylation step, good yields of lecithins, based on fatty acids, are obtained. Thus the procedure described offers a simple and attractive way for the synthesis of phosphatidylcholines with saturated photo-, spin-, deuteriumor radioactively labelled acyl chains.
Acknowledgements We thank Mr. H. Stfitz for expert technical assistance. This work was supported in part by the 'Fonds zur FiSrderung der wissenschaftlichen Forschung in Osterreich' (Projekt Nr. 3982).
A. Hermetter and F. Paltauf, Syn thesis of saturated phosphatidylcholines
115
References 1 A.J. Slotboom, H.M. Verkeij and G.H. de Haas, Chem. Phys. Lipids, 11 (1973) 295. 2 M. Kates, in E.D. Korn (Ed.), Methods in Membrane Biology, Vol. 8, Plenum Press, New York, 1977, p. 219. 3 J.G. Lammers, Th. J. Liefkens, J. Bus and J. van der Meer, Chem. Phys. Lipids, 22 (1978) 293. 4 K.M. Patel, J.D. Morrisett and J.T. Sparrow, J. Lipid Res., 20 (1979) 674. 5 C.M. Gupta, R. Radhakrishnan and H.G. Khorana Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 4315. 6 R.G. Hiskey and J.B. Adams, J. Am. Chem. Soc., 87 (1965) 3969. 7 T.G. Warner and A.A. Benson, J. Lipid Res., 18 (1977) 548. 8 H. Loibner and G. Seidl, Chromatographia, 12 (1979) 600. 9 G.R. Bartlett, J. Biol. Chem., 234 (1959) 466. 10 J. Stern and B. Shapiro, J. Clin. Pathol., 6 (1953) 158. 11 C. Long and I.F. Penny, Biochem. J., 65 (1957) 382. 12 W.R. Morrison and I.M. Smith, J. Lipid Res., 5 (1964) 600.