434
METHODS Synthesis of Mixed-Acid Phosphatidylcholines and High Pressure Liquid Chromatographic Analysis of Isomeric Lysophosphatidylcholines ALLAN W. NICHOLAS, LILIANE G. KHOURI, JOE C. ELLINGTON, JR., and NED A. PORTER*, Paul M. Gross Chemical Laboratories, Duke University, Durham, NC 27706 ABSTRACT
A new method for the synthesis of mixed-chain phosphatidylcholines is reported. Silver ion catalyzed acylation of lysophosphatidylcholines by 2-thiopyridyl esters occurs rapidly (10 min) at room temperature in organic solvents. Yields of isomerically pure mixed-chain phosphatidylcholines (> 98% isomeric purity) are generally greater than 80%. The reaction proceeds with only 1.5- to 2-fold excess of thiopyridyl ester, thus offering some advantages over existing procedures when precious acylating agents are used. The major disadvantage of the procedure is its sensitivity to water. Phosphatidylcholines having hydroxy fatty acyl groups are prepared by protection of the hydroxyl as the levulinate ester, conversion to the 2-thiopyridyl ester, acylation, and removal of the levulinate with hydrazine. For purification of lysophosphatidylcholines, a reverse-phase high pressure liquid chromatographic method for separation of I-acylglycerophosphocholines from 2-acylglycerophosphocholines was developed. Lipids 18:434-438, 1983. Interest in membrane structure and function has stimulated an extensive search for synthetic methods leading to specific phospholipids. The synthesis of mixed-acyl phosphatidylcholines (PC) 2, in particular, has been the subject of several recent publications and partial syntheses utilizing I-acylsn-glycero-3-phosphocholine (lysoPC), 1, prepared by phospholipase A2 catalyzed hydrolysis of phosphatidylcholine have been reported (1-5). Our interest in phospholipid oxidation has led us to seek alternate methods of phospholipid synthesis. We report here a method that allows the efficient synthesis of mixed-acyl PC with high purity. Our method may be particularly useful when a lysoPC must be acylated with a precious fatty acid. Existing methods for mixed-acyl PC synthesis rely primarily on acylation of lysoPC with acid anhydrides. Either p-dimethylamino pyridine (2) or 4-pyrrolidinopyridine (I) may be used as catalyst. Yields are excellent and the reaction is convenient to carry out. The pyrrolidine catalyst is preferred, since less migration of acyl substituents Oi, CH2-O-C-R I
has been reported with this catalyst (1). On the other hand, the rate of acylation is relatively slow unless a large excess of anhydride is used and a 5fold excess of this reagent is typically utilized. Furthermore, one equivalent of the acyl substituent in the anhydride is wasted since fatty acyl carboxylate is the leaving group of the acylating agent. For this reason, and also because we have found the acid anhydride method less than ideal for the synthesis of phospholipids having hydroxy fatty acyl substituents, we searched for alternate synthetic approaches to mixed-acyl PC. The method reported here relies on a silver-ion catalyzed acylation of lysoPC with 2-pyridinethiol fatty acid esters (Scheme I) (6-9). A variety of diacyl PC have been prepared by this approach and the method would appear to be one of general utility. For example, phospholipids (2) containing the following substituents have been prepared: 3, R I = R2= 16:0;4, R ~ - 18:0, R2-- 16:0; 5, R~= 16:0, R2 = 18:2; 6, R~--- 16:0, R2= 18:1; 7, Ri = 16:0, R2 = 12-OH stearate; 8, R~ = 16:0, R~= O
Ag §
HO~C~H
C H , . ) - O - P I- O - C H ~ - C9 H ~ - NL- C Hi +~ ~
O.
R~C
CH3
/ " T o w h o m correspondence should be addressed.
LIPIDS, VOL. 18, NO. 6 (1983)
o
("H 2 - 0 - C - R 1 0 ~ii R2-C-O~C~H
J
o '
O_
SCHEME I
c,~3
CH2-O-PI-O-CH2-CH2-N-CH3 I §
CH 3
METHODS ricinoleate. During the course of these synthetic studies, we also developed reverse-phase high pressure liquid chromatographic (HPLC) methods for the analysis and isolation of l-acyl lysoPC (1) and 2-acyl lysoPC. This HPLC analysis is convenient and may be generally useful in the analysis and preparation of pure lysoPC isomers. EXPERIMENTAL Materials
Phosphatidylcholines were obtained from Avanti Biochemicals (Birmingham, AL) as were lysophosphatidylcholines. Palmitic acid (NuChek Prep, Elysian, MN), linoleic acid (Sigma Chemical Co., St, Louis, MO), 12-hydroxystearic acid (Pfattz and Bauer, Inc., Stamford, CT; Supelco Inc., Bellefonte, PA), and ricinoleic acid (Sigma) were used without further purification. Phosgene in benzene came from MC&B and 2pyridine thiol was obtained from Aldrich (Milwaukee, Wl). HPLC
Simple diacyl phosphatidylcholines were purified by reverse-phase HPLC on a Waters #Bondapak C-18 column with methanol/water (97:3, v/v). PC having acyl groups with hydroxy substituents (7 and 8) were purified on the same column using methanol/water (95:5) as solvent. LysoPC were analyzed by reverse-phase H PLC on a Waters u-Bondapak C-18 column with methanol/water (85: 15). Detection for PC and lysoPC was by ultraviolet (UV) at 214 nm. Synthesis of 2-Pyridinethiol Esters
The method of Corey and Clark (10) was used. Thus, 2-thiopyridyl chloroformate was prepared from 2-pyridinethiol and phosgene. Commercial phosgene in benzene was found suitable and the reaction was carried out as described (10), with the exception that toluene solvent was replaced by benzene. The 2-thiopyridyl chloroformate was converted to the thiopyridyl ester by reaction with free fatty acid in the presence of triethylamine to scavenge HCI. Fatty acyl pyridinethioesters were purified by flash chromatography on silica (Waters Prep 500, I in, od column with refractive index detection). Nuclear magnetic resonance spectra were consistent with assigned structures of all 2pyridinethiol esters prepared (10). A typical procedure for conversion of 2-thiopyridyl chloroformate to the 2-pyridinethiol palrnitic acid ester is described below. Palmitic acid (512 mg, 2.0 mmol) and triethylamine (0.35 ml, 2.2 mmol) were taken up in dry
435
diethylether at 0 C under argon. A solution (CH2C12) of 2-thiopyridyl chloroformate (0.49 g, 2.5 rnmol) was added, and the milky mixture was stirred for 30 min at 0 C, dilutcd with 50 ml ether, and 0.2 g anhydrous MgSO4 was Ihen added. The solution was filtered, and the pale yellow filtrate was evaporated. The yellow solid that resulted was flash chromatographed on silica (Prep 500, 10% ethyl acetate/hexane on 200-400 mesh silica). Typical yields were 55-80%. LysoPC Preparation
The procedure of Mason et al. was used with minor modifications (1). The lysoPC was dried exhaustively by pumping at high vacuum. PhosphatidyIcholine Syntheses
The synthesis of l-stearoyl-2-palmitoyl-sn-glycerophosphocholine is typical and is presented below. Silver perchlorate (5-9 equiv) was dried for 20 hr (dark, P205, high vacuum) at room temperature and an additional 2 hr at relluxing ethanol temperature. The 2-lhiopyridyl palmitic acid ester (1,55 equiv) was dried overnight at room temperature under high vacuum. Freshly prepared lysoPC (one equivalent) was dried for 4 hr at room temperature under high vacuum. Benzene (2.5 ml/mmol lysoPC) was distilled into an oven-dried (argon) flask. The lysoPC was quickly added followed by solid thioester and AgCIOd. All additions were made in less than one min. After 10 min stirring under argon, thin layer chromatography showed complete reaction. Solvent was removed under vacuum and the crude white solid was suspended in 2.5 ml CHCI3 and chromatographed on silica (Prep 500, CHCI3/ MeOH/H20, 6:2.5:.3). Yield for l-stearoyl-2palmitoyl-sn-glycerophosphocholine was greater than 95% if appropriate precautions to exclude water were taken. PC of highest purity may be obtained by reverse-phase HPLC (vide infra) of material obtained from silica chromatography. If the procedures outlined above were followed without drying the 2-thiopyridyl ester or the lysoPC, yields of PC product were low (20%). Synthesis of 1-Palmitoyl-2-(12-hydroxystearoyl)sn-glycerophosphocholine
Protection of hydroxy as levulinate ester. Levulinic anhydride (!.5 g, 7 mmol) was added to 0.9 g (3 mmol) 12-hydroxystearic acid in 6 ml pyridine under argon and the mixture stirred 16-20 hr. Pyridine was removed in vacuo, the residue was taken up in chloroform/water and acidified with 1 N HCI to pH 4. The aqueous phase was extracted 3 times with chloroform, the chloroform extracts
LIPIDS, VOL. 18, NO. 6 (1983)
METHODS
436
were combined, dried, and the solvent removed. Chromatography on silica gel (Prep 500, 20% ethyl acetate in hexane, 0.7% acetic acid) gave the pure 12-1evulinate, 9.
Synthesis of phospholipid and removal of protective groups. The levulinate ester of 12-hydroxystearic acid, 9, was converted to the 2-thiopyridyl ester and l-palmitoyl lysoPC was acylated by this reagent as described above. Removal of levulinate from the protected PC to give 1-palmitoyl-2-(12hydroxystearoyl)-sn-glycerophosphocholine was carried out as described by vanBoom and Burgers (11) and Hassner et al. (12). The reaction was worked up by dilution of the pyridine/acetic acid solvent mixture with cold 1 N aq HC1 and extraction with CHCI3/CH3OH (2:1). Removal of the CHC13 followed by chromatography gave the pure phospholipid.
RESULTS AND DISCUSSION HPLC of Lysophosphatidylcholines
The most efficient synthesis of mixed-acyl phosphatidylcholines available before this work was initiated, involved acylation of lysoPC by acid anhydrides. One of the problems associated with this synthetic approach is the rearrangement of 1acyl lysoPC to the 2-acyl lysoPC isomer during the course of acylation. Nitrogen bases used as catalysts for the acylation also assist in acyl migration. A recent publication reports on the use of 3~p N MR to monitor this rearrangement and the details of migration catalysis have been worked out in this elegant study (13). During the course of our studies, we sought to analyze the isomeric purity of lysoPC and we have developed HPLC methods for their analysis. A reverse-phase liquid chromatogram of a mixture of lysoPC is presented in Figure 1. This lysoPC mixture contains myristoyl, palmitoyl, and stearoyl lysoPC, and the fractions A-F have been assigned as follows: A, 8.3 ml, 2-myristoyl-sn-glycero3-phosphocholine; B, 8.9 ml, l-myristoyl-sn-glycero3-phosphocholine; C, 12.9 ml, 2-palmitoyl-snglycero-3-phosphocholine; D, 14.1 ml, l-palmitoylsn-glycero-3-phosphocholine;E, 21.6 ml, 2-stearoylsn-glycero-3-phosphocholine; and, F, 23.8 ml, lstearoyl-sn-gtycero-3-phosphocholine. Freshly prepared lysoPC (snake venom) contains only the lacyl lyso compounds (B, D, and F) and upon standing, the 2-acyl substituted isomers are formed (A, C, and El. Commercial samples of lysoPC are quite variable in purity. Some sampled contained no detectable 2acyl lysoPC, while others showed as much as 8% of this isomer. We find, in fact, a direct correlation of lysoPC purity and isomeric PC purity of products
LIPiDS, VOL. 18, NO. 6 (1983)
B
D
O21nnm
F
I
t
I
i
0
5
10
15
~-
20
L
25
_
I
30
m/
FIG. 1. HPLC trace for chromatography of lysoPC. Solvent was methanol/water (85: 15), flowrate I ml/min, column Waters/~-Bondapak C-18. Fractions A-F as identified in text. Separation was achieved only with this column. Other highly coated columns proved ineffective in the separation. Compounds separated were as follows: A, 8.3 ml, 2-myristoyl-sn-glycero-3-phosphocholine;B, 8.9 ml, 1-myristoyl-sn-glycero-3-phosphoeholine; C, 12.9 ml, 2-palmitoyl-sn-glycero-3-phosphocholine;D, 14.1 m|, l-palmitoyl-sn-glycero-3-phosphocholine;E, 21.6 ml, 2stearoyl-sn-glycero-3-phosphocholine;and, F, 23.8 ml, lstearoyl-sn-glycero-3-phosphocholine.
of the acylation procedures we have developed. The quality of commercial samples depends dramatically on history in transit and storage. The HPLC analysis reported here should allow for easy and direct analysis of commercial or laboratory samples. The 2-acyl lysoPC isomers isolated by HPLC have JH NMR spectra consistent with their proposed structures. For example, in CDCI 3 for 2palmitoyl-sn-glycero-3-phosphocholine (Fraction C), the sn-2-methine proton appears at 4.95 6, characteristic of an acyl substituted methine, and inconsistent with an acyl substituted methylene (14). In DMSO-d6, the sn-2-methine proton appears at 4.75 6 as a quintet and the primary -OH is observed at 5.7 6 as the expected triplet. We should note that other reverse-phase HPLC columns were not found suitable for lysoPC separations. The utility of the column appears to relate directly to the amount of C-18 bonded phase present. The 10 ~ column successfully used in these studies does not contain a highly coated silica and it was only with this column material that a successful separation was achieved. Even 5 /~ or 3 # spherical head columns that are highly coated are not suitable for the separation of lysoPC isomers. The differences in solubility of the isomeric lysoPC is also noteworthy. Whereas the 2-acyl lysoPC were soluble in benzene, DMSO, chloroform, and methanol, the l-acyl lysoPC were readily dissolved only in methanol.
METHODS
437
TABLE 1 Synthetic Phosphatidylcholinesfrom Acylationof Lysophosphatidylcholinr with 2-ThiopyridylEsters Phosphatidylcholines
Isomericimpurity~ (%)
F]b (%)
F~ (%)
1,2-Palmitoyl-snglycerophosphocholine
Yieldc (%) 98
1 -Stearoyl-2-palmitoyl-sn~
1.5
49.3
50.7
95
l-Palmitoyl-2-1inoleoyl-snglycerophosphocholine
1.8
49.0
51.0
93
glycerophosphoeh01ine
~Amountof mixed-chainimpurity,analysisas describedin ref. 1. bFractionof fatty acids at the 1 and 2 positions;see ref. 1. CYieldbased on lysoPC.
Phosphatidylcholine Synthesis
The yield and isomeric purity of 3 PC prepared by 2-thiopyridyl ester acylation of lysophosphatidylcholines are presented in Table 1. Yields are 90% or greater and the isomeric impurity present was comparable to that obtained by the method of Mason et al. (1). In fact, we have oreoared both of the mixed-chain PC reported in Table 1 by acylation of lysoPC with acid anhydride in the presence of 4-pyrrolidine-pyridinecatalyst, and the isomeric purity obtained by the 2-thiopyridyl ester method was slightly better in each case than that obtained by the acid anhydride method. Yields reported here are for material purified by normalphase HPLC. If PC of highest purity are required, we recommend additional reverse-phase HPLC chromatography as described in the Experimental section. 1-Palmitoyl-2-(12-hydroxystearoyl)glycerophosphocholine, 10
Several approaches to the synthesis of PC having acyl groups containing hydroxyl functions were attempted. Silyl protecting groups for the hydroxyl (t-butyldimethylsilyl) were utilized, but the best protecting group found for the free' hydroxyl was the levulinate ester (11,12). While silyl protected fiydroxy fatty acid derivatives could be used to acylate lysoPC, we found removal of the tbutyldimethyl silyl group to lead to impurities that could not be removed even by reverse-phase HPLC. The preferred synthetic approach to hydroxy fatty acyl phosphatidylcholines is illustrated in Scheme 2. Protection and removal of the protecting group are easy and convenient, and yields for the overall sequence of acylation with
protected hydroxy fatty acid and deprotection with hydrazine were on the order of 50%. Isomeric impurity in lecithin synthesized by the route in Scheme 2 was 1.3%. The levulinate protecting group could also be used with the acid anhydride method of PC synthesis. That is, the levulinate ester of 12-hydroxystearic acid could be converted to its anhydride and used to acylate lysoPC. This acylation appeared to be unusually slow, however, and overall yields for the acylation-deprotection sequence were on the order of 30%. Hydroxy fatty acid phosphatidylcholines have been isolated from natural sources (15,16), but the chemistry and biochemistry of these novel species have not been extensively explored. One might anticipate that these compounds would have unusual effects related to membrane structure and ion transport. The methods reported here for synthesis of PC and hydroxy-PC thus provide compounds of unique and novel structure for further study. A comparison of the acyl anhydride and 2thiopyridyl ester methods of PC synthesis should be made at this point. T h e 2-thiopyridyl ester method is fast and efficient with product isomeric purity equivalent to the acid anhydride method. In the case of precious or slow reacting acid anhydride acylating groups, the 2-t hiopyridyl method appears to have some advantages. A great excess of thiopyridyl ester is not required and valuable fatty acid is not used as the leaving group. Yields of the levulinate derivatives described here were better than the anhydride method. The one major disadvantage noted thus far for the 2-thiopyridyl ester method is the great sensitivity to moisture. This detracts from the convenience of the method. Nevertheless, this new method is significantly
LIP1DS, VOL. 18, NO. 6(1983)
438
METHODS
,o CH3_(CH2)5_CH.(CH2IIoCOOH OH
9
~- (CH3C-JCH2)2-C-]20 ~
CH3-(CH2)5-CH-(CH2)Io-COOH OI C=O t
c-o CH3
Ag§
,o CH3- (CH2)5-CH -(CH2)~0-~ \ S / ~ OI CJ (CH2)2
OPC, CHg-CH-CH ~ o o. O=C i
-
i
~
i
(c.2),,
c=o CH3
c.~
CH3
~fN2H4
OPC i CH~--CH-CH9 o
o.{
J
o
ii
0
H-C- O-C-(CH2)2-C-CH3 (CH2)5 CH3
o
o<, 0
(cn2)14 (CH2)10 CH3
OPC =- CH2 - - CH-CH 2 O O o oc o=~, (CH2)14 1CH2110
HC-OH I
(~H2)5 CH3 10 SCHEME 2
d i f f e r e n t f r o m c u r r e n t m e t h o d o l o g y a n d it t h u s provides a potentially useful alternative to existing procedures. ACKNOWI.EDGMENTS These studies were supported by grants from the National Institutes of Health (HL17921) and the National Science Foundation (CHE 80-26541). We thank Dr. J. T. Mason, The University of Virginia, for helpful discussions and for help in the analysis of mixed-chain phosphatidylcholines. REFERENCt~S I. Mason, J.l., Brocolli, A.V., and Huang, C. (1981) Anal. Biochem 113, 96-101. 2. Gupta, C.M., Radhakrishran, R , and Khorana, HG. (1977) Proc. Natl. Aead. Sci. USA 74, 4315-4319. 3. t.ammers, J.G., l.eifkens, J., Bus, J., and van dcr Moor, .1. (1978) Chem. Phys. Lipids 22, 293-305. 4. SIotboom, A.J., Verheij, H.M., and De Haas, G . H (1973) Chem. Phys. |.ipids I 1,295-317. 5. Eibl, H. (1980) Chem. Phys. l.ipids 26, 405-429. 6. Gerlach, H., and Thalmann, A. (1974) Heir. ['him. Acta 57, 2661-2663.
L I P I D S , VOL. 18, NO. 6 (1983)
7. Corey, EJ., and Nicolaou, K.C. (1974) J. Am. ('hem. Soc. 96, 5614-5616. 8. Nicolaou, KC. {1977)l'etrahedron 33, 683-710. 9. Masamune, S., Kamata, S., and Schilhng, W. {1975) J. Am. (_'hem. Soc. 97, 3515-3516. 10. Corey, EJ.. and Clark, I).A. (1979) ietrahcdron Left. 2875-2878. II. ,~anBoom, J.H., and Burgers, P.M.J. (1978) Recl. "lrav. Chim. Pays-Bas 97, 73-80. 12. Hassner, A., Strand, G., Ruhinstein, M., and Patchornik, A. (1975) J. Am. Chem. Soe. 97, 1641-1615. 13. Pliiekthun, A., and Dennis, E.A (1982) Biochemistry 21, 1743-1750. 14. Hauser, H., Guyer. W., Levine, B., Skrabal, P., and Williams, RJ.P. (1978) Biochlm. Biophys. Acta 508, 450-463. 15. Bonser, R.W., Siegel, M.I., Chung. S.M., McConnell, R.T.. and Cuatrecasas, P. 11981) Biochemistry 20. 5297-5301. 16. Stenson, W., and Parker, C. (1979) I'rostaglandins 18, 285-292.
[Received December 17, 1982]
