Chemistry and Physics of Lipids, 62 (1992) 263-268 Elsevier Scientific Publishers Ireland Ltd.
263
A facile synthesis of 1-O-alkyl-2-(R)-hydroxypropane-3phosphonocholine (lyso-phosphono-platelet activating factor) Jeffrey D. Schmitt, Andrew B. Nixon, Adalsteinn Emilsson, Larry W. Daniel and Robert L. Wykle Department of Biochemistry, Wake Forest University Medical Center, Bowman Gray School of Medicine, Medical Center Boulevard. Winston-Salem, NC 27157-1016 (USA) (Received April 7th, 1992; revision received August 6th, 1992; accepted August 7th, 1992~
The synthesis of 1-O-alkyl-2-(R)-hydroxypropane-3-phosphonocholine is described. An efficient alkylation procedure using (NaH/DMSO) catalysis is also described and applied to the synthetic scheme. The key intermediate l-O-alkyl-2-(R)-O-benzyl-3bromopropane was phosphonylated using tris(trimethylsilyl)phosphite; the resulting phosphonic acid was coupled to choline using trichloroacetonitrile/pyridine or triisopropylbenzenesulfonylchloride/pyridine followed by catalytic hydrogenation to yield 1-O-alkyl2(R)-hydroxypropane-3-phosphonocholine. Key words., phosphonate; phospholipase C; phospholipase D
Introduction
Phosphonate analogs of phospholipids promise to be valuable in assessing the presence of phospholipase C and D activities in human neutrophils and other cells. Upon activation, a number of cells convert choline-containing phosphoglycerides into diglyceride and phosphatidic acid [1]. These putative second messengers are thought to alter cellular metabolism through the activation of protein kinase C [2] and the mobilization of inCorrespondence to." Robert L. Wykle, Department of Biochemistry, Wake Forest University Medical Center, Bowman Gray School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157-1016, U.S.A. Abbreviations: BzBr, benzyl bromide; Call 2. calcium hydride; Chol Ph4B, choline tetraphenylborate; ChoTos, choline tosylate; DG, diglyceride; DMAP, 4-dimethylaminopyridine; DMF, dimethylformamide; DMSO, dimethylsulfoxide; Nail, sodium hydride; PMN, polymorphonuclear neutrophils; PA, phosphatidic acid; PC, choline-containing phosphoglycerides; PLC, phospholipase C; PLD, phospholipase D; TCAN, trichloroacetonitrile; TIPS, triisopropylbenzenesulfonyl chloride; TLC, thin-layer chromatography.
tercellular calcium [3], respectively. Choline containing phosphoglycerides can be converted to DG and PA via distinct pathways, as outlined in Fig. 1. The extent to which each of these pathways contributes to second messenger formation has not been clearly established. The interconversions of PA and DG as well as the interconversion of choline and phosphocholine make the elucidation of the relative contributions of PLC and PLD difficult. Since the phosphonate bond of phosphonolipids is resistant to hydrolysis by PLC and PLD, depending on the position of the C - - P bond, they provide promising tools for use in studies of DG and PA formation from PC. Phosphonolipids occur naturally in many organisms including Tetrahymenapyriformis [4l and since their first characterization a substantial effort has gone into developing synthetic strategies for these compounds. The synthesis of a number of phosphonolipids that contain a P - - C bond between the phosphorus and the head group has been described [5]. However, most of the definitive
0009-3084/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd, Printed and Published in Ireland
264 O-radyl r RCOO"~ I--QCho
PLD
fO-radyl
RCOO_~ O'radyl LOH Fig. 1. Conversion of phosphatidylcholine to phosphatidic acid and diglyceride.
phosphonate chemistry performed to date has focused on the synthesis and use of Wittig and Horner-Emmons reagents [7]. Engel et al. [6] recently described the total synthesis of an isosteric phosphono analog of diacyl-PC containing a C ~ P bond between the backbone and headgroup. As a part of our ongoing studies of platelet activating factor, ether lipid metabolism and the metabolic significance of PLC and PLD, we synthesized an analog of 1-O-alkyl-2-1yso-snglycero-3-phosphocholine containing a C--P bond between the backbone and the phosphocholine headgroup. This phosphono analog of lyso platelet activating factor is resistant to hydrolysis by phospholipase C. Materials and Methods
Materials 1-O-Hexadecyl-sn-glycerol ([a] = +4.45 deg) was obtained from Western Chemical Industries Ltd. (Vancover, Canada). Trityl chloride, 4-dimethylaminopyridine, sodium hydride (60% disp. in oil), bromine, trichloroacetonitrile and 20% palladium hydroxide on charcoal were 'purchased from Aldrich Chemical (Milwaukee, WI). Benzyl bromide, triphenylphosphine and dimethyl sulfoxide were purchased from MTM Lancaster Synthesis (Windham, N J). Tris(trimethylsilyl)phosphite was purchased from Fluka Chemical (Ronkonkoma, NY). Triisopropylbenzenesulfonyl
chloride was purchased from Alfa Chemical (Ward Hill, MA). Phospholipase C from Bacillus cereus was purchased from Boehringer Mannheim (Germany). Phospholipase D was prepared as described from cabbage [21] and from bacteria, Vibria cholera [22]. Choline tosylate was synthesized according to the method of Rosenthal [8] followed by two recrystalizations from acetone and vacuum dessication over phosphorus pentoxide. Pyridine was distilled over calcium hydride. All solvents were HPLC grade unless otherwise noted. TLC plates (Silica Gel-G, 250 t~m) were purchased from Analtech (Newark, DE). Proton NMR analysis was conducted using a Bruker 300 MHz spectrometer; chemical shift values are reported in PPM relative to tetramethylsilane. Mass spectrometry was conducted on a Ribermag quadrapole GC-MS equipped with Vector-2 data analysis software; spectra were acquired using ammonia or methane chemical ionization.
Chemistry The synthetic route to 1-O-alkyl-2-(R)-hydroxypropane-3-phosphonocholine (phosphono lysoplatelet activating factor) is outlined in Fig. 2.
1-O-Hexadecyl-3-O-trityl-sn-glycerol (1) 1-O-Hexadecyl-sn-glycerol (3.0 g), (9.5 mmol) and 6.0 g (21.6 mmol) trityl chloride were combined in 70 ml of dry pyridine. A catalytic amount of 4-DMAP was added and the mixture was stirred overnight under nitrogen. After solvent removal by rotary evaporation, solids were resuspended in 200 ml hexane/ethyl ether (1:1 v/v) and extracted with an equal volume of water. The aqueous phase was washed with hexane/ethyl ether (1:1 v/v), the solvents were pooled, dried over Na2SO4, and rotary evaporated. The desired product eluted with hexane/ethyl ether (2:3 v/v) on silica gel flash chromatography. Yield 90%+. TLC Ry= 0.59 hexane/ether (2:3 v/v); ammonia CI-MS 559 (M + 1)+, 576 (MNH4) +. NMR (CDCI3) 6 1.25 (s(br), 26H, (CH2)n) , 6.6-7.5 (m, 15H, C6H5).
1-O-Hexadeeyl-2-O-benzyl-3- O-trityl-sn-glycerol (II) Sodium hydride (60% disp. in oil) 2.1 g, (52
265 Synthesis of phosphonate analog F OCH2R HO%o H
A
F OCH2R . HO-~ ~-OTr
B
Y BzO ~
OCH2R ~-OTr II
I
__•
OCH2R
BzO
C BzO
D
~--OH
OCH2R E
'--Br
Ill
FOCH2 R BzO "4k OH ~
v II "OH 0
IV FOCH2 R 8zO--~ o M_"
v,
6
+
G
FOCH2R . HO~ o-
u
÷
O
Fig. 2. Synthesis of phosphonate analog. A: Tritylchloride/Py; B: Benzyl bromide/sodium hydride/DMSO; C: HCl/ethanol/ diethyl ether; D:Triphenylphosphine dibromide/DMF; E: Tris(trimethylsilyl)phosphite; F: TCAN/ChoTos; G: H2/Palladium hydroxide.
mmol) was cautiously added over 5 min to 80 ml DMSO in a 3-neck flask equipped with a nitrogen inlet, condenser and dropper funnel. The suspension was stirred under nitrogen and heated to 60-70°C until the grey color was no longer visible (-- 10 min). The mixture should not be heated above 80°C due to the chance of violent decomposition, t After cooling to room temperature, 4 g (7.17 mmol) 1-O-hexadecyl-3-O-trityl-sn-glycerol and 2.2 g (12 mmol) benzyl bromide in 20 ml DMSO were added. The alkylation is usually complete within 30 min. An equal volume of water was carefully added, followed by two extractions with hexane/ethyl ether (2:1 v/v). The solvents were pooled, washed with an equal volume of 5% (w/v) aqueous NaHCO3 and dried over Na2SO4. The desired product eluted with hexane/ethyl ether (5:1) on silica gel flash chromatography. Yield 90%+. TLC Rf = 0.82 hexane/ethyl ether (2:3 v/v); ammonia CI-MS 647 (M + 1)÷, 664 (MNH4) +. NMR (CDCI3) 6 1.26 (s(br), 26H, (C_H2),), 4.63 (d, 2H, Ph - - C_H20), 6.4-7.5 (m, 2OH, C6_H5).
1-O-Hexadecyl-2-O-benzyl-sn-glycerol (III) 1-O-Hexadecyl-2-O-benzyl-3- O-trityl-sn-glycerol 13.8 g, (21.8 mmol) was dissolved in 100 ml ethanol:ethyl ether (1:1 v/v), 20 ml 6N HC1 (aq.) was then added. The mixture was stirred overnight
at room temperature. The reaction mixture was diluted to a final volume of 250 ml with H20 and extracted three times with 200 ml hexane:ethyl ether (1:1 v/v). The solvents were pooled and washed with 500 ml 5% w/v aqueous NaHCO3 and dried over Na2SO4. The deprotected alcohol eluted with hexane:ethyl ether (2:3 v/v) on silica gel flash chromatography. Yield 85-90%. TLC Rf = 0.45 hexane:ethyl ether (2:3 v/v); ammonia CI-MS 407 (M + 1)+, 424 (MNH4) +, NMR (CDCI3) 6 1.26 (s(br), 26H, (CH_2)n), 4.65 (d, 2H, Ph-CH_20).
1-O-Hexadecyl-2-( R )-O-benzyl-3-bromopropane (IV) Triphenylphosphine 6.3 g, (24 mmol) was dissolved in 80 ml dry acetonitrile followed by the addition of 1.25 ml Br2 (24 mmol) in an atomosphere of dry nitrogen. After 30 min the solution became clear or milky white (to react all free bromine the addition of extra triphenylphosphine may be required). Using a dropper funnel, 3.5 g (7.4 mmol) of 1-O-hexadecyl-2-O-benzyl-snglycerol in 30 ml acetonitrile:dimethylformamide (1:1 v/v) was slowly added. Reaction progress was followed by TLC and was complete within 1-2 h. The acetonitrile was then rotary evaporated followed by the addition of 100 ml water and the slurry was then extracted twice with hexane:ethyl
266
ether (3:1 v/v). The solvents were pooled, dried over Na2SO4 and rotary evaporated. Purification was accomplished using silica gel column chromatography and a stepwise gradient of ethyl ether in hexane (70 g silica; 3 column volumes each of: 100% hexane, 12.5% ether, 33% ether, 50"/,, ether and 100% ether). The product eluted with the 12.5% and 33% fractions. Yield 60-70%. TLC Rf = 0.79 hexane:ethyl ether (2:3 v/v); ammonia CIMS 470 (M + 1) +, 487 (MNH4) ÷. NMR (CDCI3) 6 0.88 (t, 3H, CH3), 1.26 (s(br), 26H, (CH2)n) , 1.54 (m, 2H, OCH2CH:R), 3.44 (t, 2H, OCH2), 3.56 (m, 2H, C_H20), 3.74 (septet, 1H,/3-C_H) 3.50 (d, 2H, C H2Br), 4.67 (d, 2H, Ph-C_H20), 7.31 (m, 5H, C5H5). 1- O-Hexadecyl-2- (R) - O-benzylpropane-3-phosphonic acid (V) 1-O-Hexadecyl-2-(R)-O-benzyl-3-bromopropane, 1.1 g (2.0 mmol) was placed in a screwcap vial fitted with a teflon liner, and 2 ml tris(trimethylsilyl)phosphite was added and the sample heated at 125-140°C under nitrogen for 24 h. The suspension was transferred to a microdistillation apparatus and the unreacted tris(trimethylsilyl)phosphite removed by cautious vacuum distillation (@50/z, 80-90°C). Overheating will cause substantial loss of product. The oily residue, consisting mostly of bisTMS phosphonolipid, was refluxed in 4.5 ml tetrahydrofuran and 0.5 ml water overnight to remove TMS groups. Solvents were removed by rotary evaporation and the product crystallized from hot acetonitrile as a free crystalline powder. Yield -70%. TLC RU = 0.1 CHC13:MeOH:NH4OH (65:35:8 v/v). TLC Rf = 0.6-0.7 CHC13:MeOH; acetic acid:H20 (50:25:8:3 v/v); ammonia CI-MS 471 (M + 1)+, 488 (MNH4) ÷, 453 - H 2 0 , 391 -PO3H2, 364 -PhCH20. NMR (CDCI3,CD3OD 2:1 v/v) 6 0.89 (t, 3H, C_H3), 1.26 (s(br), 26H, (CHz)n), 1.52 (t, 2H, OCHzC_H2R), 1.98 ([dxd], 2H, 2C_HzP), 3.43 (t, 2H, OCH2), 3.48 (m, 2H, OC_H2), 3.96 (Septet, 1H, 13-C_H), 4.65 (d, 2H, PhCH:O), 7.41 (m, 5H Cs_Hs).
phonic acid, 0.20 g (0.43 mmol) was placed in a flame-dried screwcap tube with 0.85 g (2.13 mmol) choline tosylate and vacuum dessicated overnight (at 0.050 Torr, over phosphorous pentoxide); 5 ml of dry pyridine and 2 ml of freshly distilled trichloroacetonitrile were then added, using flame dried pipettes. After 24 h at 55-60°C the reaction was essentially complete. Solvents were removed under a stream of nitrogen and the lipid was extracted according to the method of Bligh and Dyer [9]. Yield 90%+. TLC Rj= 0.47 CHCI3:MeOH: NH4OH, (65:35:8 v/v). CI-MS 556 (M + 1)+, 571 (MNH4) +. NMR (CDCI3, CD3OD 2:1 v/v) 6 0.88 (t, 3H, C_H3), 1.27 (s(br), 26H, (CH2)13), 1.57 (p, 2H, OCHzC_H2R), 1.93 (dx[dx[dxt]], Jn - p = 18.8 Hz, Jn -- ~n = 3.2 Hz, Jn -- ocn2 = 1-2 Hz, 2H, C_H2P), 3.07 (s, 9H, N(C_H3)3), 3.37 and 3.70 (d, 2H, C_H20), 3.48 (m, 2H, OC_H2), 4.06 (septet, 1H, H-CH), 4.13 (m(br), 2H, POC_H2), 4.67 (d, 2H, PhC_H:O), 7.33 (m, 5H, Pb-_H). 1-O-Hexadecyl-2- ( R )-hydroxypropane-3-phosphonocholine (VII) A mixture of 0.5 g (1.07 mmol) 1-O-hexadecyl(R)-benzylpropane-3-phosphonocholine and 0.1 g palladium hydroxide (20% on carbon) were combined in 10 ml absolute ethanol and hydrogenated at 40 ATM on a Parr hydrogenator for 2 h. Ten milliliters of chloroform were added to kill the catalyst, then the mixture was filtered through a pad of celite and rinsed with ethanol. Solvents were removed yielding a white solid. [od = +4.60 deg CH2C12:MeOH (1:2 v/v) Yield 98%+. TLC Rf= 0.19 CHC13/MeOH/acetic acid/H20 (50:25: 8:4 v/v); methane CI-MS 466 (M + 1) +, 452 -CH 3, 407-N(CH3)3 +, 395 -OCH2CHzN(CH3)3 +, NMR (CDCI3, CD3OD 1:1 v/v) 6 0.86 (t, 3H, C_H3), 1.27 (s(br), 26H, (C_H2)I3), 1.58 (p, 2H, OCH2C_H:R), 1.80 (dx[dxt], Jn--p = 18.8 Hz, Jn ~n = 3.2 Hz, 2H, C_H2PO), 3.23 (s, 9H, N(CH3)3), 3.41 and 3.72 (d, 2H, C_H20), 3.47 (t, 2H, OC_H 2), 3.59 (t, 2H, C_H2N), 4.12 (septet, 1H,/3-C_H), 4.27 (m(br), 2H, POC_H2). Results and Discussion
1- O-Hexadecyl-2- ( R)- O-benzylpropane-3-phosphonocholine (VI)
l-O-Hexadecyl-2-(R)-O-benzylpropane-3-phos-
We have developed a facile method for synthesizing 1-O-alkyl-2-(R)-hydroxypropane-3-phos-
267
phonocholine (1-O-alkyl-2-1yso-GnPC), which employs some novel techniques which to our knowledge are new to the field of lipid synthesis*. The synthesis starts with commercially available, optically active 1-O-alkyl-sn-glycerol. Tritylation of the sn-3-hydroxyl group was accomplished with tritylchloride/pyridine [10]; we found the reaction rate to be greatly enhanced by the addition of a catalytic amount of 4-dimethylaminopyridine [11]. Purification of the tritylated lipid by rapid column chromatography resulted in a higher purity product compared to products obtained by precipitation from acetone or other solvents. The methods commonly used to make benzyl ethers are often harsh (e.g., potassium metal, benzyl bromide, xylene-reflux) and often result in variable yields. With l-O-alkyl-3-O-tritylglycerol we found that high yields of 1-O-alkyl-2-O-benzyl3-O-trityl glycerol were obtained using sodium methylsulfonylmethidecatalyzed alkylation conditions (DMSO, Nail, BzBr). This observation was extended to include other lipophilic starting materials (data not shown); the use of up to 20% THF to promote solubility is acceptible. Detritylation of 1-O-alkyl-2-O-benzyl-3-O-tritylglycerol proceeded most smoothly by acid hydrolysis using hydrochloric acid in ethanol/ethyl ether [12]. Bromination (i.e., R - - CH2OH -- R - - CH2Br) of 1-O-alkyl-2-O-benzyl-sn-glycerol was accomplished in nearly quantitative yield using triphenylphosphine dibromide/DMF [13]. We found this reagent to be far superior to PBr3 [14] or LiBr/acetone [15], as both of these reagents gave poor yields and many side products.
*Phosphonolipid nomenclature." We have adopted the following abbreviation system for these phosphoglyceride analogs, wherein 'n' is placed either before or after the 'P' in the phospholipid abbreviations to denote the location of the PC bond. The diradyl phosphono-PC analog with a PC bond between the phosphorus and the choline moiety will be abbreviated PnC, whereas the corresponding analog with a PC bond between the glycerol backbone and the phosphorus will be abbreviated nPC. Similarly, the system can be used to name these as sn-glycero-3-phosphocholine (-GPC) analogs, 1-Oalkyl-2-acyl-GPnC and -GnPC to denote a P--C bond between the phosphorus and the choline, or between the backbone and the phosphorus, respectively.
1-O-Alkyl-2-O-benzylpropane-3-phosphonic acid was prepared using tris(trimethylsilyl)-phosphite [16]; this reagent was deemed superior to trimethyl phosphite because of the ease with which the intermediate diester hydrolizes. We tested a number of choline coupling conditions to ascertain the one best suited to generate 1O-alkyl-2-O-benzylpropane-3-phosphonocholine from the phosphonic acid. Trichloroacetonitrile [17] and triisopropylbenzenesulfonyl chloride [18] were used as coupling reagents; choline tosylate and choline tetraphenylborate [19] were chosen as the choline salt candidates. Of the four possible combinations (TIPS/CholTos, TIPS/Chol Ph4B, TCAN/CholTos, TCAN/Chol Ph~B) the cleanest reaction and highest yields were obtained with the TCAN/CholTos combination. Choline tetraphenylborate has been reported to offer certain advantages over choline tosylate in phosphatidylcholine synthesis [19]. Unfortunately, this salt, even when twice-recrystallized, gave brown-black reaction mixtures with many side products. Finally 1- O-alkyl-2-(R)-hydroxypropane-3-phosphonocholine was obtained by catalytic hydrogenation of the 2-O-benzyl material over palladium hydroxide [20]. Proton NMR peak assignment for compounds IV-VII was aided by gated homonuclear decoupling. The alkyl chain, benzyl protecting group, sn-I and sn-2 glycerol protons as well as the choline moiety had absorptions and multiplicities that were in agreement with published data [21]. The sn-3 glycerol protons in compound VI (phosphonocholine) were split through a long range interaction with the benzilic CH 2 protons. This interaction was not observed in compound V (phosphonic acid). We attribute this difference to rotational hindrance caused by the introduction of the choline moiety. The sn-3 glycerol protons in compound VI were shifted upfield relative to the same protons in compound V; this observation is consistant with published data on phosphate mono- and diesters [22]. To test susceptibility of the phosphonate to phospholipase D hydrolysis, we first acylated the phosphono-lyso PAF with palmitoyl chloride/ pyridine. The isolated product was then treated with phospholipase D from cabbage [23] and from
268
bacteria [24]. The cabbage enzyme failed to hydrolize the 1-O-alkyl-2-palmitoyl-GnPC to 1-Oalkyl-2-palmitoyl-GnPA although it hydrolyzed egg PC, while the bacterial enzyme hydrolyzed the phosphonolipid at a slower rate as compared to egg phosphatidylcholine (data not shown). In experiments where equimolar amounts of egg PC and 1-O-alkyl-2-palmitoyl-GnPC were combined there was no apparent inhibition of egg PC hydrolysis using the cabbage enzyme. The nature of the 2-acyl residue may influence the susceptibility to hydrolysis by PLD. Further experiments are needed to determine if differing rates of PLD hydrolysis with enzymes from different sources can be correlated to differences in enzyme mechanism. Finally, as expected, no reaction occurred upon treatment of 1-O-alkyl-2-palmitoylGnPC with phospholipase C from Bacillus cereus. Using this synthetic scheme we have synthesized
1-O-octadec-9-enyl-2-(R)-benzylpropane-3-phosphonocholine from 1-O-octadec-9-enyl-sn-glycerol in high yield. With this intermediate we hope to obtain 1-O-[9,10- 3H]octadecyl-2(R)-hydroxypropane-3-phosphonocholine by sequential catalytic tritiation of the A9 double bond followed by hydrogenation under pressure to remove the benzyl group. This material will then be used to label cellular phospholipid pools. These procedures provide a simple means to generate phosphonate ether lipid analogs as well as providing a contribution to existing lipid chemistry.
Acknowledgements We would like to thank Mike Samuel for the mass spectral analysis, Dr. Michael Thomas for help in interpretation of NMR data, Dr. Craig Miller for advice on coupling reactions and Susan I. Britt for preparing the manuscript. This work was supported by grants CA43297, CA48995 and HL26818 from the National Institutes of Health. We are grateful for the use of the Membrane Lipid and Mass Spectrometry core facilities of the Corn-
prehensive Cancer Center of Wake Forest University (Supported by CA 12197).
References 1 J.H, Exton (1990) J. Biol. Chem. 265(1), 1-4. 2 R.M. Bell and D.J, Burns (1991) J. Biol. Chem. 266(8), 4661-4664. 3 S.B. Bocckino, P.F. Blackmore and J.H. Exton (1985) J. Biol. Chem. 260(26), 14201-14207. 4 H. Rosenberg (1964) Nature 203, 299. 5a A.F. Rosenthal (1966) J. Lipid Res. 7, 779-785. 5b E. Baer and N.Z. Stanacev (1965) J. Biol. Chem. 240(10), 3754-3759. 6 P. Waters-Schwartz, B.E. Tropp and R. Engel (1988) Chem. Phys. Lipids, 48, 1-7. 7 Organophosphorus Reagents in Organic Synthesis, (1980) in: J.I.G. Cadogan (Ed.), Academic Press. 8 A.F. Rosenthal (1966) J. Lipid Res. 7, 779-785. 9 E.G. Bligh and W.L. Dyer (1959) Can J. Biochem. Physiol. 37, 911-918. 10 B. Helferich, P.E. Speidel and W. Toeldte (1925) Ber. Bonn. Univ. Poliklin. Mund. Zahn. Kieferkr. 58, 872. 11 W. Steglich and G. H6fle (1969) Agnew. Chem. Int. Ed. 8, 981. 12 G.A. Wiley, R.L. Hershkowitz, B.M, Rein and B.C. Chung (1964) J. Am. Chem. Soc. 86, 964. 13 C.R. Noller and R. Dinsmore (1943) Org. Syn. Coll. 2, 358. 14 M.C. Moschidis (1986) Chem. Phys. Lipids 39, 265-269. 15 M. Sekine and T. Hata (1978) Chem. Commun. 285, 125. 16 F. Cramer and H.J. Baldauf (1959) Ber. Bonn. Univ. Poliklin. Mund. Zahn. Kieferkr. 92, 370. 17 I.L. Doerr, J.C. Tang, A.F. Rosenthal, R. Engel and B.E. Tropp (1977) Chem. Phys. Lipids 19, 185. 18 R. Lohrmann and H.G. Khorana (1966) J. Am. Chem. Soc. 88, 4. 19 G.S. Harbison and R.G. Griffin (1984) J. Lipid Res. 25, 1140-1142.
20 21
22
23 24
W.M. Pearlman (1987) Tetrahedron Lett. 4, 1663-1664. F. Heymans, E. Michel, M-C. Borrel, B. Wichrowski, J-J. Godfroid, O. Convert, E. Coeffier, M. Tence and J. Benveniste (1981) Biochim. Biophys. Acta 666, 230-237. J.R. Surles, R.L. Wykle, J.T. O'Flaherty, W.L. Salzer, M.J. Thomas, F. Snyder and C. Piantadosi (1985) J. Med. Chem. 28, 73-78. F.M. Davidson and C. Long (1958) Biochem. J. 69, 458-466. A.S. Kreger and M.H. Kothany (1985) Infect. Imrnun. 49, 25.
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A facile synthesis of 1-O-alkyl-2-(R)-hydroxypropane-3phosphonocholine (lyso-phosphono-platelet activating factor) Jeffrey D. Schmitt, Andrew B. Nixon, Adalsteinn Emilsson, Larry W. Daniel and Robert L. Wykle Department of Biochemistry, Wake Forest University Medical Center, Bowman Gray School of Medicine, Medical Center Boulevard. Winston-Salem, NC 27157-1016 (USA) (Received April 7th, 1992; revision received August 6th, 1992; accepted August 7th, 1992~
The synthesis of 1-O-alkyl-2-(R)-hydroxypropane-3-phosphonocholine is described. An efficient alkylation procedure using (NaH/DMSO) catalysis is also described and applied to the synthetic scheme. The key intermediate l-O-alkyl-2-(R)-O-benzyl-3bromopropane was phosphonylated using tris(trimethylsilyl)phosphite; the resulting phosphonic acid was coupled to choline using trichloroacetonitrile/pyridine or triisopropylbenzenesulfonylchloride/pyridine followed by catalytic hydrogenation to yield 1-O-alkyl2(R)-hydroxypropane-3-phosphonocholine. Key words., phosphonate; phospholipase C; phospholipase D
Introduction
Phosphonate analogs of phospholipids promise to be valuable in assessing the presence of phospholipase C and D activities in human neutrophils and other cells. Upon activation, a number of cells convert choline-containing phosphoglycerides into diglyceride and phosphatidic acid [1]. These putative second messengers are thought to alter cellular metabolism through the activation of protein kinase C [2] and the mobilization of inCorrespondence to." Robert L. Wykle, Department of Biochemistry, Wake Forest University Medical Center, Bowman Gray School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157-1016, U.S.A. Abbreviations: BzBr, benzyl bromide; Call 2. calcium hydride; Chol Ph4B, choline tetraphenylborate; ChoTos, choline tosylate; DG, diglyceride; DMAP, 4-dimethylaminopyridine; DMF, dimethylformamide; DMSO, dimethylsulfoxide; Nail, sodium hydride; PMN, polymorphonuclear neutrophils; PA, phosphatidic acid; PC, choline-containing phosphoglycerides; PLC, phospholipase C; PLD, phospholipase D; TCAN, trichloroacetonitrile; TIPS, triisopropylbenzenesulfonyl chloride; TLC, thin-layer chromatography.
tercellular calcium [3], respectively. Choline containing phosphoglycerides can be converted to DG and PA via distinct pathways, as outlined in Fig. 1. The extent to which each of these pathways contributes to second messenger formation has not been clearly established. The interconversions of PA and DG as well as the interconversion of choline and phosphocholine make the elucidation of the relative contributions of PLC and PLD difficult. Since the phosphonate bond of phosphonolipids is resistant to hydrolysis by PLC and PLD, depending on the position of the C - - P bond, they provide promising tools for use in studies of DG and PA formation from PC. Phosphonolipids occur naturally in many organisms including Tetrahymenapyriformis [4l and since their first characterization a substantial effort has gone into developing synthetic strategies for these compounds. The synthesis of a number of phosphonolipids that contain a P - - C bond between the phosphorus and the head group has been described [5]. However, most of the definitive
0009-3084/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd, Printed and Published in Ireland
264 O-radyl r RCOO"~ I--QCho
PLD
fO-radyl
RCOO_~ O'radyl LOH Fig. 1. Conversion of phosphatidylcholine to phosphatidic acid and diglyceride.
phosphonate chemistry performed to date has focused on the synthesis and use of Wittig and Horner-Emmons reagents [7]. Engel et al. [6] recently described the total synthesis of an isosteric phosphono analog of diacyl-PC containing a C ~ P bond between the backbone and headgroup. As a part of our ongoing studies of platelet activating factor, ether lipid metabolism and the metabolic significance of PLC and PLD, we synthesized an analog of 1-O-alkyl-2-1yso-snglycero-3-phosphocholine containing a C--P bond between the backbone and the phosphocholine headgroup. This phosphono analog of lyso platelet activating factor is resistant to hydrolysis by phospholipase C. Materials and Methods
Materials 1-O-Hexadecyl-sn-glycerol ([a] = +4.45 deg) was obtained from Western Chemical Industries Ltd. (Vancover, Canada). Trityl chloride, 4-dimethylaminopyridine, sodium hydride (60% disp. in oil), bromine, trichloroacetonitrile and 20% palladium hydroxide on charcoal were 'purchased from Aldrich Chemical (Milwaukee, WI). Benzyl bromide, triphenylphosphine and dimethyl sulfoxide were purchased from MTM Lancaster Synthesis (Windham, N J). Tris(trimethylsilyl)phosphite was purchased from Fluka Chemical (Ronkonkoma, NY). Triisopropylbenzenesulfonyl
chloride was purchased from Alfa Chemical (Ward Hill, MA). Phospholipase C from Bacillus cereus was purchased from Boehringer Mannheim (Germany). Phospholipase D was prepared as described from cabbage [21] and from bacteria, Vibria cholera [22]. Choline tosylate was synthesized according to the method of Rosenthal [8] followed by two recrystalizations from acetone and vacuum dessication over phosphorus pentoxide. Pyridine was distilled over calcium hydride. All solvents were HPLC grade unless otherwise noted. TLC plates (Silica Gel-G, 250 t~m) were purchased from Analtech (Newark, DE). Proton NMR analysis was conducted using a Bruker 300 MHz spectrometer; chemical shift values are reported in PPM relative to tetramethylsilane. Mass spectrometry was conducted on a Ribermag quadrapole GC-MS equipped with Vector-2 data analysis software; spectra were acquired using ammonia or methane chemical ionization.
Chemistry The synthetic route to 1-O-alkyl-2-(R)-hydroxypropane-3-phosphonocholine (phosphono lysoplatelet activating factor) is outlined in Fig. 2.
1-O-Hexadecyl-3-O-trityl-sn-glycerol (1) 1-O-Hexadecyl-sn-glycerol (3.0 g), (9.5 mmol) and 6.0 g (21.6 mmol) trityl chloride were combined in 70 ml of dry pyridine. A catalytic amount of 4-DMAP was added and the mixture was stirred overnight under nitrogen. After solvent removal by rotary evaporation, solids were resuspended in 200 ml hexane/ethyl ether (1:1 v/v) and extracted with an equal volume of water. The aqueous phase was washed with hexane/ethyl ether (1:1 v/v), the solvents were pooled, dried over Na2SO4, and rotary evaporated. The desired product eluted with hexane/ethyl ether (2:3 v/v) on silica gel flash chromatography. Yield 90%+. TLC Ry= 0.59 hexane/ether (2:3 v/v); ammonia CI-MS 559 (M + 1)+, 576 (MNH4) +. NMR (CDCI3) 6 1.25 (s(br), 26H, (CH2)n) , 6.6-7.5 (m, 15H, C6H5).
1-O-Hexadeeyl-2-O-benzyl-3- O-trityl-sn-glycerol (II) Sodium hydride (60% disp. in oil) 2.1 g, (52
265 Synthesis of phosphonate analog F OCH2R HO%o H
A
F OCH2R . HO-~ ~-OTr
B
Y BzO ~
OCH2R ~-OTr II
I
__•
OCH2R
BzO
C BzO
D
~--OH
OCH2R E
'--Br
Ill
FOCH2 R BzO "4k OH ~
v II "OH 0
IV FOCH2 R 8zO--~ o M_"
v,
6
+
G
FOCH2R . HO~ o-
u
÷
O
Fig. 2. Synthesis of phosphonate analog. A: Tritylchloride/Py; B: Benzyl bromide/sodium hydride/DMSO; C: HCl/ethanol/ diethyl ether; D:Triphenylphosphine dibromide/DMF; E: Tris(trimethylsilyl)phosphite; F: TCAN/ChoTos; G: H2/Palladium hydroxide.
mmol) was cautiously added over 5 min to 80 ml DMSO in a 3-neck flask equipped with a nitrogen inlet, condenser and dropper funnel. The suspension was stirred under nitrogen and heated to 60-70°C until the grey color was no longer visible (-- 10 min). The mixture should not be heated above 80°C due to the chance of violent decomposition, t After cooling to room temperature, 4 g (7.17 mmol) 1-O-hexadecyl-3-O-trityl-sn-glycerol and 2.2 g (12 mmol) benzyl bromide in 20 ml DMSO were added. The alkylation is usually complete within 30 min. An equal volume of water was carefully added, followed by two extractions with hexane/ethyl ether (2:1 v/v). The solvents were pooled, washed with an equal volume of 5% (w/v) aqueous NaHCO3 and dried over Na2SO4. The desired product eluted with hexane/ethyl ether (5:1) on silica gel flash chromatography. Yield 90%+. TLC Rf = 0.82 hexane/ethyl ether (2:3 v/v); ammonia CI-MS 647 (M + 1)÷, 664 (MNH4) +. NMR (CDCI3) 6 1.26 (s(br), 26H, (C_H2),), 4.63 (d, 2H, Ph - - C_H20), 6.4-7.5 (m, 2OH, C6_H5).
1-O-Hexadecyl-2-O-benzyl-sn-glycerol (III) 1-O-Hexadecyl-2-O-benzyl-3- O-trityl-sn-glycerol 13.8 g, (21.8 mmol) was dissolved in 100 ml ethanol:ethyl ether (1:1 v/v), 20 ml 6N HC1 (aq.) was then added. The mixture was stirred overnight
at room temperature. The reaction mixture was diluted to a final volume of 250 ml with H20 and extracted three times with 200 ml hexane:ethyl ether (1:1 v/v). The solvents were pooled and washed with 500 ml 5% w/v aqueous NaHCO3 and dried over Na2SO4. The deprotected alcohol eluted with hexane:ethyl ether (2:3 v/v) on silica gel flash chromatography. Yield 85-90%. TLC Rf = 0.45 hexane:ethyl ether (2:3 v/v); ammonia CI-MS 407 (M + 1)+, 424 (MNH4) +, NMR (CDCI3) 6 1.26 (s(br), 26H, (CH_2)n), 4.65 (d, 2H, Ph-CH_20).
1-O-Hexadecyl-2-( R )-O-benzyl-3-bromopropane (IV) Triphenylphosphine 6.3 g, (24 mmol) was dissolved in 80 ml dry acetonitrile followed by the addition of 1.25 ml Br2 (24 mmol) in an atomosphere of dry nitrogen. After 30 min the solution became clear or milky white (to react all free bromine the addition of extra triphenylphosphine may be required). Using a dropper funnel, 3.5 g (7.4 mmol) of 1-O-hexadecyl-2-O-benzyl-snglycerol in 30 ml acetonitrile:dimethylformamide (1:1 v/v) was slowly added. Reaction progress was followed by TLC and was complete within 1-2 h. The acetonitrile was then rotary evaporated followed by the addition of 100 ml water and the slurry was then extracted twice with hexane:ethyl
266
ether (3:1 v/v). The solvents were pooled, dried over Na2SO4 and rotary evaporated. Purification was accomplished using silica gel column chromatography and a stepwise gradient of ethyl ether in hexane (70 g silica; 3 column volumes each of: 100% hexane, 12.5% ether, 33% ether, 50"/,, ether and 100% ether). The product eluted with the 12.5% and 33% fractions. Yield 60-70%. TLC Rf = 0.79 hexane:ethyl ether (2:3 v/v); ammonia CIMS 470 (M + 1) +, 487 (MNH4) ÷. NMR (CDCI3) 6 0.88 (t, 3H, CH3), 1.26 (s(br), 26H, (CH2)n) , 1.54 (m, 2H, OCH2CH:R), 3.44 (t, 2H, OCH2), 3.56 (m, 2H, C_H20), 3.74 (septet, 1H,/3-C_H) 3.50 (d, 2H, C H2Br), 4.67 (d, 2H, Ph-C_H20), 7.31 (m, 5H, C5H5). 1- O-Hexadecyl-2- (R) - O-benzylpropane-3-phosphonic acid (V) 1-O-Hexadecyl-2-(R)-O-benzyl-3-bromopropane, 1.1 g (2.0 mmol) was placed in a screwcap vial fitted with a teflon liner, and 2 ml tris(trimethylsilyl)phosphite was added and the sample heated at 125-140°C under nitrogen for 24 h. The suspension was transferred to a microdistillation apparatus and the unreacted tris(trimethylsilyl)phosphite removed by cautious vacuum distillation (@50/z, 80-90°C). Overheating will cause substantial loss of product. The oily residue, consisting mostly of bisTMS phosphonolipid, was refluxed in 4.5 ml tetrahydrofuran and 0.5 ml water overnight to remove TMS groups. Solvents were removed by rotary evaporation and the product crystallized from hot acetonitrile as a free crystalline powder. Yield -70%. TLC RU = 0.1 CHC13:MeOH:NH4OH (65:35:8 v/v). TLC Rf = 0.6-0.7 CHC13:MeOH; acetic acid:H20 (50:25:8:3 v/v); ammonia CI-MS 471 (M + 1)+, 488 (MNH4) ÷, 453 - H 2 0 , 391 -PO3H2, 364 -PhCH20. NMR (CDCI3,CD3OD 2:1 v/v) 6 0.89 (t, 3H, C_H3), 1.26 (s(br), 26H, (CHz)n), 1.52 (t, 2H, OCHzC_H2R), 1.98 ([dxd], 2H, 2C_HzP), 3.43 (t, 2H, OCH2), 3.48 (m, 2H, OC_H2), 3.96 (Septet, 1H, 13-C_H), 4.65 (d, 2H, PhCH:O), 7.41 (m, 5H Cs_Hs).
phonic acid, 0.20 g (0.43 mmol) was placed in a flame-dried screwcap tube with 0.85 g (2.13 mmol) choline tosylate and vacuum dessicated overnight (at 0.050 Torr, over phosphorous pentoxide); 5 ml of dry pyridine and 2 ml of freshly distilled trichloroacetonitrile were then added, using flame dried pipettes. After 24 h at 55-60°C the reaction was essentially complete. Solvents were removed under a stream of nitrogen and the lipid was extracted according to the method of Bligh and Dyer [9]. Yield 90%+. TLC Rj= 0.47 CHCI3:MeOH: NH4OH, (65:35:8 v/v). CI-MS 556 (M + 1)+, 571 (MNH4) +. NMR (CDCI3, CD3OD 2:1 v/v) 6 0.88 (t, 3H, C_H3), 1.27 (s(br), 26H, (CH2)13), 1.57 (p, 2H, OCHzC_H2R), 1.93 (dx[dx[dxt]], Jn - p = 18.8 Hz, Jn -- ~n = 3.2 Hz, Jn -- ocn2 = 1-2 Hz, 2H, C_H2P), 3.07 (s, 9H, N(C_H3)3), 3.37 and 3.70 (d, 2H, C_H20), 3.48 (m, 2H, OC_H2), 4.06 (septet, 1H, H-CH), 4.13 (m(br), 2H, POC_H2), 4.67 (d, 2H, PhC_H:O), 7.33 (m, 5H, Pb-_H). 1-O-Hexadecyl-2- ( R )-hydroxypropane-3-phosphonocholine (VII) A mixture of 0.5 g (1.07 mmol) 1-O-hexadecyl(R)-benzylpropane-3-phosphonocholine and 0.1 g palladium hydroxide (20% on carbon) were combined in 10 ml absolute ethanol and hydrogenated at 40 ATM on a Parr hydrogenator for 2 h. Ten milliliters of chloroform were added to kill the catalyst, then the mixture was filtered through a pad of celite and rinsed with ethanol. Solvents were removed yielding a white solid. [od = +4.60 deg CH2C12:MeOH (1:2 v/v) Yield 98%+. TLC Rf= 0.19 CHC13/MeOH/acetic acid/H20 (50:25: 8:4 v/v); methane CI-MS 466 (M + 1) +, 452 -CH 3, 407-N(CH3)3 +, 395 -OCH2CHzN(CH3)3 +, NMR (CDCI3, CD3OD 1:1 v/v) 6 0.86 (t, 3H, C_H3), 1.27 (s(br), 26H, (C_H2)I3), 1.58 (p, 2H, OCH2C_H:R), 1.80 (dx[dxt], Jn--p = 18.8 Hz, Jn ~n = 3.2 Hz, 2H, C_H2PO), 3.23 (s, 9H, N(CH3)3), 3.41 and 3.72 (d, 2H, C_H20), 3.47 (t, 2H, OC_H 2), 3.59 (t, 2H, C_H2N), 4.12 (septet, 1H,/3-C_H), 4.27 (m(br), 2H, POC_H2). Results and Discussion
1- O-Hexadecyl-2- ( R)- O-benzylpropane-3-phosphonocholine (VI)
l-O-Hexadecyl-2-(R)-O-benzylpropane-3-phos-
We have developed a facile method for synthesizing 1-O-alkyl-2-(R)-hydroxypropane-3-phos-
267
phonocholine (1-O-alkyl-2-1yso-GnPC), which employs some novel techniques which to our knowledge are new to the field of lipid synthesis*. The synthesis starts with commercially available, optically active 1-O-alkyl-sn-glycerol. Tritylation of the sn-3-hydroxyl group was accomplished with tritylchloride/pyridine [10]; we found the reaction rate to be greatly enhanced by the addition of a catalytic amount of 4-dimethylaminopyridine [11]. Purification of the tritylated lipid by rapid column chromatography resulted in a higher purity product compared to products obtained by precipitation from acetone or other solvents. The methods commonly used to make benzyl ethers are often harsh (e.g., potassium metal, benzyl bromide, xylene-reflux) and often result in variable yields. With l-O-alkyl-3-O-tritylglycerol we found that high yields of 1-O-alkyl-2-O-benzyl3-O-trityl glycerol were obtained using sodium methylsulfonylmethidecatalyzed alkylation conditions (DMSO, Nail, BzBr). This observation was extended to include other lipophilic starting materials (data not shown); the use of up to 20% THF to promote solubility is acceptible. Detritylation of 1-O-alkyl-2-O-benzyl-3-O-tritylglycerol proceeded most smoothly by acid hydrolysis using hydrochloric acid in ethanol/ethyl ether [12]. Bromination (i.e., R - - CH2OH -- R - - CH2Br) of 1-O-alkyl-2-O-benzyl-sn-glycerol was accomplished in nearly quantitative yield using triphenylphosphine dibromide/DMF [13]. We found this reagent to be far superior to PBr3 [14] or LiBr/acetone [15], as both of these reagents gave poor yields and many side products.
*Phosphonolipid nomenclature." We have adopted the following abbreviation system for these phosphoglyceride analogs, wherein 'n' is placed either before or after the 'P' in the phospholipid abbreviations to denote the location of the PC bond. The diradyl phosphono-PC analog with a PC bond between the phosphorus and the choline moiety will be abbreviated PnC, whereas the corresponding analog with a PC bond between the glycerol backbone and the phosphorus will be abbreviated nPC. Similarly, the system can be used to name these as sn-glycero-3-phosphocholine (-GPC) analogs, 1-Oalkyl-2-acyl-GPnC and -GnPC to denote a P--C bond between the phosphorus and the choline, or between the backbone and the phosphorus, respectively.
1-O-Alkyl-2-O-benzylpropane-3-phosphonic acid was prepared using tris(trimethylsilyl)-phosphite [16]; this reagent was deemed superior to trimethyl phosphite because of the ease with which the intermediate diester hydrolizes. We tested a number of choline coupling conditions to ascertain the one best suited to generate 1O-alkyl-2-O-benzylpropane-3-phosphonocholine from the phosphonic acid. Trichloroacetonitrile [17] and triisopropylbenzenesulfonyl chloride [18] were used as coupling reagents; choline tosylate and choline tetraphenylborate [19] were chosen as the choline salt candidates. Of the four possible combinations (TIPS/CholTos, TIPS/Chol Ph4B, TCAN/CholTos, TCAN/Chol Ph~B) the cleanest reaction and highest yields were obtained with the TCAN/CholTos combination. Choline tetraphenylborate has been reported to offer certain advantages over choline tosylate in phosphatidylcholine synthesis [19]. Unfortunately, this salt, even when twice-recrystallized, gave brown-black reaction mixtures with many side products. Finally 1- O-alkyl-2-(R)-hydroxypropane-3-phosphonocholine was obtained by catalytic hydrogenation of the 2-O-benzyl material over palladium hydroxide [20]. Proton NMR peak assignment for compounds IV-VII was aided by gated homonuclear decoupling. The alkyl chain, benzyl protecting group, sn-I and sn-2 glycerol protons as well as the choline moiety had absorptions and multiplicities that were in agreement with published data [21]. The sn-3 glycerol protons in compound VI (phosphonocholine) were split through a long range interaction with the benzilic CH 2 protons. This interaction was not observed in compound V (phosphonic acid). We attribute this difference to rotational hindrance caused by the introduction of the choline moiety. The sn-3 glycerol protons in compound VI were shifted upfield relative to the same protons in compound V; this observation is consistant with published data on phosphate mono- and diesters [22]. To test susceptibility of the phosphonate to phospholipase D hydrolysis, we first acylated the phosphono-lyso PAF with palmitoyl chloride/ pyridine. The isolated product was then treated with phospholipase D from cabbage [23] and from
268
bacteria [24]. The cabbage enzyme failed to hydrolize the 1-O-alkyl-2-palmitoyl-GnPC to 1-Oalkyl-2-palmitoyl-GnPA although it hydrolyzed egg PC, while the bacterial enzyme hydrolyzed the phosphonolipid at a slower rate as compared to egg phosphatidylcholine (data not shown). In experiments where equimolar amounts of egg PC and 1-O-alkyl-2-palmitoyl-GnPC were combined there was no apparent inhibition of egg PC hydrolysis using the cabbage enzyme. The nature of the 2-acyl residue may influence the susceptibility to hydrolysis by PLD. Further experiments are needed to determine if differing rates of PLD hydrolysis with enzymes from different sources can be correlated to differences in enzyme mechanism. Finally, as expected, no reaction occurred upon treatment of 1-O-alkyl-2-palmitoylGnPC with phospholipase C from Bacillus cereus. Using this synthetic scheme we have synthesized
1-O-octadec-9-enyl-2-(R)-benzylpropane-3-phosphonocholine from 1-O-octadec-9-enyl-sn-glycerol in high yield. With this intermediate we hope to obtain 1-O-[9,10- 3H]octadecyl-2(R)-hydroxypropane-3-phosphonocholine by sequential catalytic tritiation of the A9 double bond followed by hydrogenation under pressure to remove the benzyl group. This material will then be used to label cellular phospholipid pools. These procedures provide a simple means to generate phosphonate ether lipid analogs as well as providing a contribution to existing lipid chemistry.
Acknowledgements We would like to thank Mike Samuel for the mass spectral analysis, Dr. Michael Thomas for help in interpretation of NMR data, Dr. Craig Miller for advice on coupling reactions and Susan I. Britt for preparing the manuscript. This work was supported by grants CA43297, CA48995 and HL26818 from the National Institutes of Health. We are grateful for the use of the Membrane Lipid and Mass Spectrometry core facilities of the Corn-
prehensive Cancer Center of Wake Forest University (Supported by CA 12197).
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