New optically pure dimethylacetals of glyceraldehydes and their application for lipid and phospholipid synthesis

Chemistry and Physics of

ELSEVIER

Chemistry and Physics of Lipids 76 (1995) 211-224

LIPID$

New optic,ally pure dimethylacetals of glyceraldehydes and their application for lipid and phospholipid synthesis Ulrich Massing*, Hansj6rg Eibl Department 145, Membrane Biophysics, Max-Planck-lnstitute for Biophysical Chemistry, Am Fassberg 11, D-37077 G6ttingen, Germany

Received 25 January 1995; accepted 22 March 1995

Abstract

A convenient synthesis of new and enantiomerically pure 2-O-protected D-glyceraldehyde dimethylacetals as chiral C-3 building blocks for the synthesis of lipids and phospholipids is described. Benzyl- or allylethers are used as protecting groups in position 2 and 5 of D-mannitol. These intermediates are converted to 2-O-benzyl- or 2-O-allyl-D-glyceraldehyde dimethylacetals by cleavage with periodic acid in methanol. The two dimethylacetals are useful for the synthesis of mixed chain phospholipids with natural configuration of ester-ester, ester-ether or ether-ether composition. Also, triglycerides with three different alkyl chains, ester or ether, can be prepared. As an example of the versatility of the new intermediates, we describe the synthesis of 1-O-hexadecyl-2-O-acetyl-sn-glycero3-phosphocholine, the so-called 'platelet activating factor' (PAF), via 1-O-hexadecyl-2-O-benzyl-sn-glycerol. Keywords: Synthetic phospholipids; PAF; Phospholipid analogues; Synthesis; Chiral pool; Glyceraldehyde; C-3 building blocks; Optical purity

I. Introduction

Phospholipids and their metabolites such as lysophospholipids and diacylglycerols play an important role in signal transduction [1-3]. Therefore, synthetic chemists have improved the methodology for the preparation of signal molecules. This usually requires techniques for * Corresponding author, Clinical Research, Tumor Biology Center; Breisacher Str. 117, D-79106 Freiburg, Germany. Tel.: + 49/761/206 2177; Fax: + 49/761/206 1899. E-mail: umas@suu1"tum°rbi°'uni'freiburg'de

the preparation of lipid molecules well-defined in structure and configuration. In general, optically pure glycerol derivatives as C-3 building blocks are powerful starting materials for the synthesis of lipids [4-9]. Many new synthetic routes for chiral C-3 building blocks, including chiral-pool synthesis, asymmetric synthesis and enzymatic synthesis, have been developed in the last decade [10-16]. In this publication, we describe a new strategy based on protected D-glyceraldehyde dimethylacetals. Starting from D-mannitol, it leads to the well-known C-3 building block 1-O-allyl-2-

0009-3084/95/$09.50 © 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0009-3084(951102445-0

212

U. Massing, H. Eibl / Chemistry and Physics of Lipids 76 (1995) 211-224

benzaldehyde,

sulfuricacid, DMF ~ 39 % (94 %)

D-Mannit 1

62-15 or ~ OH O O allyJbromide benzylchloride, ~......~ .,,.J K-O-tert-butylate, THF, reflux F ~ 7 I~ O...]/O

OH

C6H5

,CBH5 O

O'l/O

OR1

2

IR1 Yield

C6H5 methanol,

3 Bn ~ % 9 All ~ %

periodic acid,

sulfuricacid, reflux

1.1 N HCI,70 *C q 2. sodiumborohydride

R20

8

OH R1 R2 Yield Bn Alll 8 6 %

12 A Bn 75%

OR1 I,,~OCH3

I R20

I OCH3

7

Nail, benzylchloride, THF, reflux or OR1 ~ K-O-tert-butylate, ~ OCH3 allylbromide,THF,reflux I I HO OCH3

R1 R2 Yield Bn All 99%

1t A Bn 72%

R1 Yield Bn 77% 10 A 72% 6

Scheme1. O-benzyl-sn-glycerol in a five-step synthesis, whereas earlier strategies required nine steps [1517]. Especially for the preparation of PAF and PAF-like compounds, 2-O-benzyl-D-glyceraldehyde dimethylacetal, obtained in three steps from D-mannitol, is useful and can be transformed to the products via 1-O-hexadecyl-2-O-benzyl-snglycerol. 2. Synthesis

2.1. 2-O-Benzyl- and 2-O-allyl-D-glyceraldehyde dimethylacetal The general educt for the synthesis of 2-O-allylor 2-O-benzyl-D-glyceraldehyde dimethylacetal is D-mannitol (1), which is converted to 1,3:4,6-diO-benzyliden-D-mannitol (2) by protection of the 1,3- and the 4,6-hydroxyl groups as benzylidenacetals using benzaldehyde and sulfuric acid catalysis [18]. The desired product 2 was isolated as white crystals after extracting the byproducts with boiling chloroform in yields of about 40%. The isomeric acetals extracted with chloroform can also be converted to product 2 by additional acid-catalyzed isomerisation and chloroform extraction steps.

The 2- and 5-hydroxyl groups in 2 are alkylated with benzyl chloride or allylbromide using potassium-tert-butylate as base to give the benzyl ether 3 (yield: 94%) or the allylether 9 (yield: 84%), respectively [19]. The 2-O-benzyl- or 2-O-allyl-glyceraldehyde dimethylacetals (6 and 10) were prepared from 3 or 9 in good yields (77% and 72%, respectively). The educts are dissolved in methanol containing catalytic amounts of sulfuric acid and reacted with 1.2 molequivalents of periodic acid. First, the cyclic acetal is removed by formation of 2,5-di-Obenzyl- or 2,5-di-O-allyl-D-mannitol. The D-mannitol derivatives containing vicinal 3,4-diol groups are immediately cleaved to the 2-O-benzyl- or the 2-O-allyl-glyceraldehyde in the presence of periodic acid and, directly after formation, converted to dimethylacetals. The three reaction steps of the above conversion can also be performed separately. This is demonstrated for the synthesis of the benzyl compound 6 (Scheme 2). The acetals of the D-mannitol compound 3 were cleaved by the treatment with aqueous acetic acid (45%) to give 2,5-di-Obenzyl-D-mannitol (4). The product 4 is quantitatively converted to 2-O-benzyl-D-glyeraldehyde

U. Massing, H. Eibl / Chemistry and Physics of Lipids 76 (1995) 211-224

benzaldehyde,

sulfuricacid, DMF ~

~

OBn OH OH I : I

86 %

O-,T/O

95 %

HO

OR1

THF/water1:1 NaBH4 ~-

OH OBn

213

OBn

&,

HO

0

~

C6H5 3

H+

OBn OBn HO

6

methanol, OCH3 ~ sulfuric acid, 94% OCH3

C@°" Bn~ ~

OH 5

S c h e m e 2.

OBn

OBn

OBn HC)

NO HObnoB n Bn~,.-OH S c h e m e 3.

by oxidative cleavage with sodium metaperiodate. The glyceraldehyde is unstable as a monomer and spontaniously forms the dioxolan-structure 5 by dimerisation. The dimer was identified by 1HNMR- and mass-spectroscopy. The dimerisation process, which is catalyzed by acidic conditions, takes place by intermolecular formation of an acetal and an hemiacetal (Scheme 3). A similar reaction of acetaldol is known, which forms paraldol [20]. The dinaer 5 can be isolated in good yield by precipitation from hexane and can be stored at 4°C. It can be converted to the dimethylacetal 6 in 94% yield by treatment with a catalytical amount of sulfuric acid in methanol.

with allylbromide and potassium-tert-butylate gives the l-O-allyl-2-O-benzyl-D-glyceraldehyde dimethylacetal (7) in 99% yield. The 1-O-benzyl2-O-allyl analogue 11 was prepared in 72% yield from 2-O-allyl-D-glyceraldehyde dimethylacetal (10) with benzyl chloride and sodium hydride under the catalysis of tetrabutylammonium iodide [21]. Compounds 7 and 11 can be easily converted into the respective well-known glycerol derivates 8 and 12 [16,17] by deacetalisation in dioxane/water/hydrochloric acid, followed by reduction with sodium borohydride. The products 8 and 12 were purified by distillation and obtained in 86% and 75% yield, respectively.

2.2. 1-O-Benzyl-2-O-allyl-sn-glycerol and l-O-allyl-2-O-benzyl-sn-glycerol

2.3. Optical purity

Alkylation of the primary 1-hydroxyl group in 2-O-benzyl-D-glyceraldehyde dimethylacetal (6)

The synthesis of the C-3 building blocks described in this paper was developed in order to

214

u. Massing, H. Eibl / Chemistry and Physics oS Lipids 76 (1995) 211-224

synthesize natural compounds and analogues of natural compounds based on glycerol, which we need for biological experiments. Most importantly, for meaningful biological experiments, is the optical purity of these compounds. To prove the optical purity of the C-3 building blocks, 8 and 12 were converted to the respective 'Mosher esters' [22] 19 and 20 with the 'Mosher acid' R-( + )- 1-methoxy- 1-phenyl-trifluoromethyl-acetic acid (DCC/DMAP-method [23]). The lgF-NMR spectra of the 'Mosher esters' 19 and 20 were compared with the 19F-NMR spectra of the 'Mosher esters' of racemic 8 and 12 (rac-19 and rac-20). In the 19F-NMR-spectra of 19 and 20 no diastereomeric signal can be detected, the d-values of 8 and 12 were better than 99%. The high optical purity is demonstrated by the ]gF-NMRspectra of the 'Mosher esters' 19 and rac-19 in Fig. 1. It is experimental proof, that the high optical purity of the chiral pool compound, Dmannitol, was not decreased by the periodate cleavage reaction of the vicinal hydroxyl groups, by liberation of the aldehyde group by acidic treatment of the dimethylacetal and by reduction of the aldehyde with sodium borohydride. The compounds 6, 10, 7, 11, 8 and 12 can be stored at 4°C for several month without loss of optical purity. ..~-72.09

2.4. l-O-Hexadeyl-2-O-acetyl-sn-glycero-3phosphocholine (PAF)

The first step in the synthesis of PAF is the alkylation of 6 with hexadecylmesylate and potassium-tert-butylate to get 1-O-hexadeyl-2-O-benzyl-D-glyceraldehyde dimethylacetal (13) in 95% yield. The aldehyde group is deprotected in dioxane/water/hydrochloric acid and reducted with sodium borohydride to 1- O-hexadecyl-2- O-benzylsn-glycerol (14). The phosphorylation of the free primary hydroxyl group in 14 with phosphoroxychloride, phospholane ring formation with Nmethyl-aminoethanol and specific hydrolysis of the PN-bond generates the respective phospho-Nmethylethanolamine, which is converted to the respective phosphocholine by methylation to give 1-O-hexadecyl-2-O-benzyl-sn-glycero-3-phosphocholine (15) (for details see Section 3.16.) [24-26]. The catalytic cleavage of the benzyl ether 15 with H2Pd/C in methanol/THF led to 1-O-hexadecylsn-glycero-3-phosphocholine (Lyso-PAF) (16) in quantitative yield. The acylation of 16 with acetic anhydride in the presence of DMAP as catalyst and base results in the formation of PAF, 1 - Ohexadecyl-2- O-acetyl-sn-glycero- 3-phosphocholine (17), in 84% yield [23,26,27]. The completion of the acylation is supported by ultrasonication. (See Scheme 4.) -72.09~

-72.13 /

-72.0S o72.1 -72.t5 ppm

-72.0

-72.1 -72.2 ppm

Fig. 1. ]gF-NMR spectra of the 'Mosher esters' of 1-O-benzyl-2-O-allyl-sn-glycerol and 1-O-benzyl-2-O-allyl-rac-glycerol (19 and rac-19).

U. Massing, H. Eibl / Chemistry and Physics of Lipids 76 (1995) 211-224

B[~ HO

OCH3

OCH3

215

hexadecylmesyate /

triethylamine/

THF/ reflux 94 %

13 ~ O C H

~--

~(CH2)15 I O

3

OCH3

3

15

I

OBn

triethylamine

3. dimethylsulfate, ~1 potassiumcarbonate

O.. ,.O o~.P..o@N(CH3)3

~
ll. 1 N HCI 82 %;2. sodiumborohydride

1. POCI 2. " ~ N ~ OH

OBn

&

~(CH2)15/0

OH 14

/

90 % /Pd/C 5 %, H2 (from 14)~,methanol/ THF (1:1) OH

O...O

~(CH2) 15/O

o~,P,.O~

N(CH3)3

l

acetanhydride/

84 % DMAP,

)))))

O" )'0 17 (PAFI

~(CH2)15 j O

O...,O

o~.P..o@

N(CH3)3 Scheme 4.

3. Material and methods

~H-NMR spectra were recorded with a Bruker MSL 300 spectrometer (300 MHz, T M S = 0 ppm) and ~9F-NMR spectra with a Bruker WP 80 SY (75.398 MHz, CFC13 = 0 ppm, internal standard: C 6 F 6 == 162.28 ppm). Infrared spectra were measured using a Perkin-Elmer 1420 spectrophotometer. The mass-spectra was recorded with a Varian MAT 311 spectrometer. A Biichi 520 apparatus was used to measure melting points. Elemental analysis of the compounds was performed from Mikroanalytisches Laboratorium Belier (G6ttingen, Germany). Silica gel (Kieselgel

60, 35-70 mesh) and TLC-plates (art. 5721) were obtained from Merck (Darmstadt, FRG). Solvent mixtures for chromatography are given in volume ratios and ammonia stands for a 25% aqueous solution. Chemicals and solvents were of p.a. grade from Aldrich, Baker, Fluka and Merck, and were used without further purification. Removal of solvents, if not otherwise specified, was done in a rotory evaporator under reduced pressure.

3.1. 1,3:4,6-Di-O-benzylidene-D-mannitol (2) D-Mannitol (1) (300 g; 1.65 mol) and benzaldehyde (368 ml; 3.62 mol) were dispersed in 1 1

216

U. Massing, H. Eibl / Chemistry and Physics of Lipids 76 (1995) 211-224

of DMF. The mixture was cooled with ice and 60 ml of conc. sulfuric acid were added dropwise. The reaction mixture was stirred for 3 days at room temperature, then the mixture was poured into 12 1 of ice water containing 120 g of potassium carbonate and 1 1 of petrol ether under vigorous stirring. When the mixture reached 20°C, the pre-cipitate was filtered off and washed with 1 1 of petrol ether. The crude product was dispersed in 500 ml of chloroform and heated under reflux for 15 min under vigorous stirring. The undissolved precipitate was collected after 24 h at 4°C. The extracting procedure was repeated two times with 500 ml of chloroform. Yield: 230 g (0.64 mol; 39%) R / 0.44 (chloroform/methanol, 9:1) [7]D: --9.9 ° ( C = 1, acetone); lit.: [~]D: --9.1° (C = 1.06, acetone) [18] Fp: 158°C IH-NMR (CDC13): 6 = 3.55 (dd, 3Jlax,2(6ax.5) = 10.4 Hz, 2jgem" = 10.5 Hz, 2 H, lax-H and 6ax-H); 3.81 (dddd 3J2,3(5,4~ = 9.2 Hz, 3J2,1eq(5.6eq) = 5.2 Hz, 3J2,1~x,~s,6ax~ = 10.4 Hz, 3J2,o~5.oH) = 5.8 Hz, 2 H, 2-H and 5-H); 3.91 (d, 3J3.2~4.5) = 9.2 Hz, 2 H, 3-H and 4-H); 4.16 (dd, 2Jg~m" = 10.5 HZ, 3Jleq.2(6eq,5) = 5.2 Hz, 2 H, leq-H and 6eq-H); 4.36 (d, 3JoH.2(OH,5) = 5.8 HZ, 2 H, OH); 5.25 (s, 2 H, acetal-H); 7.30-7.55 (m, 10 H, arom. benzyliden-H) IR (KBr): [cm-l] 3430, 3090, 3070, 3040, 2980, 2960, 2950, 1970, 1910, 1830, 1605, 1495, 1460, 750, 740, 700 C20H2206 (358.39) calc. C 67.03% H 6.19% O 26.79%; found C 66.85% H 6.14% O 26.65%.

3.2. 1,3:4,6-Di- 0 -benzylidene-2, 5-di- O-benzylD-mannitol (3) The benzylidene acetal 2 (383 g; 1.07 tool) and

potassium-tert-butylate (288 g; 2.57 mol) in 1.5 1 T H F were heated under reflux for 3 h. Benzyl chloride (298 g; 2.35 mol) in 700 ml T H F was added dropwise within 1 h. Heating under reflux was continued for 60 min. The mixture was cooled to room temperature and extracted with 700 ml water. Extraction of the organic phase was repeated twice with sodium chloride solution (100 g/l). The combined organic phases were freed

from solvents. The crude product was filtered over silica gel (column: length (1)= 15 cm, diameter (130 = 8 cm) with 1.5 1 of chloroform. The product was purified by crystallisation in methanol at 4°C for 24 h. Yield: 541 g (1.01 mol; 94%) Rs: 0.64 (chloroform); Rs: 0.52 (diisopropylether); Rs: 0.52 (diisopropylether/hexane, 1:9) [~]D: --39.1° (C = 1, chloroform) Fp: 106°C IH-NMR (CDC13): 6 = 3.61 (dd, 3J~x.2~,x.5~ = 10.0 Hz, 2Jgem" = 10.6 HZ, 2 H, lax-H and 6ax-H); 3.99 (ddd, 3J2.3(5,4) = 10.0 nz, 3J2,1eq(5,6eq) = 4.9, 3J2.1ax,(5,6ax ) = 10.0 Hz, 2 H, 2-H and 5-H); 4.08 (d, 3J3.2(4.5~ = 10.0 Hz, 2 H, 3-H and 4-H); 4.35 (dd, 2jgem" = 10.6 Hz, 3Jleq,2(6eq,5) = 4.9 Hz, 2 H, leq-H and 6eq-H); 4.55 (d, 2Jgem" = 11.7 HZ, 2 H, benzyl-CH2); 4.61 (d, ZJgem" = 11.7 HZ, 2 H, benzyl-CH2); 5.40 (s, 2 H, acetal-H); 7.29-7.30 (m, 10 H, arom. benzyl-H); 7.31-7.48 (m, 10 H, arom. benzyliden-H) IR (KBr): [cm 1] 3080, 3060, 3040, 2970, 2960, 2920, 2870, 2850, 1980, 1955, 1885, 1605, 1495, 1455, 750, 700 C34H340 6 (538.64) calc. C 75.82% H 6.36% O 17.82%; found C 75.72% H 6.29% O 17.87%.

3.3. 2.5-Di-O-benzyl-D-mannitol (4) The benzylated diacetal 3 (202 g; 0.37 mol) was heated under reflux in 2 1 acetic acid (45%) for 2 h. After cooling with ice, the pH of the solution was adjusted to 6.5 by the addition of 870 g of solid potassium hydroxide. The solution was kept at 4°C over night. The precipitation was filtered off and washed first with petrol ether then with water. The precipitate was dried by aceotropic distillation with toluene. The residue was dissolved in 1 1 of methanol, 50 g of sodium carbonate was added and the mixture was heated under reflux for 20 h. Saturated NaC1 (1 1) was added and the crude product was extracted with 600 ml chloroform. The solvents of the organic phase were removed and the product was recystallized twice from diisopropylether. Yield: 106 g (0.29 mol; 86%) RF: 0.42 (chloroform/methanol, 9:1); R / 0.42 (ether); R~ 0.30 (chloroform/acetone, 1:1)

U. Massing, H. Eibl / Chemistry and Physics of Lipids 76 (1995) 211-224

[~]D: - 7 . 7 ° ( C = 1, ethanol); lit.: [C~]D: --7.7 ° (C = 1.4, ethanol) [28] Fp: 120°C 1H-NMR (CDC10: ~ = 3.61 (ddd, 3.12,3(5,4) = 7.3 H Z , 3J2,1,(5,6, ) = ,4.4 H z , 3J2,1,(5,6 ) = 4.3 Hz, 2 H, 2-H and 5-H); 3.78 (dd, 3Ji.2(6.5) = 4.3 Hz, 2j gem. = 11.8 Hz, 2 H, 1-H and 6-H); 3.90 (dd, 2jg~m" = 11.8 Hz, 3J1,2(6,5 ) = 4.4 Hz, 2 H, I'-H and 6'-H); 3.98 (d, 3J3.2(4.5) = 7.3 Hz, 2 H, 3-H and 4-H); 4.62 (d, 2Jgem' = 11.3 HZ, 2 H, benzyl-CH2); 4.73 (d, 2j gem. = 11.7 Hz, 2 H, benzyl-CH2); 7.20-7.40 (m, 10 H, arom. benzyl-H) IR (KBr): [cm-l] 3480, 3380, 3270, 3090, 3070, 3050, 2970, 2940, 2920, 2910, 2890, 1975, 1960, 1885, 1825, 1605, 1585, 1495, 750, 740, 700 C 2 0 H 2 6 0 6 (362.42) calc. C 66.28% H 7.23% O 26.49%; found C 66.08% H 7.10% O 26.52%.

3.4. (2RS,4R,5R)-5-Benzyloxy-2-((l-R)-lbenzyloxy-2-hydroxyethyl)-4-hydroxy- 1,3dioxane (5) The product 4. (60 g; 170 mmol), was dissolved in 900 ml THF/water (1:1, v/v) at room temperature and a solution of sodium periodate (42.7 g; 187 mmol) in 300 ml water was added. The solution was stirred for 4 h. The precipitate was filtered off. The organic solvent was removed and the aqueous residue was extracted twice with 300 ml of dichlorornethane. The combined organic phases were reextracted with sodium chloride solution (100 g/l) and filtered over silica gel (column: 1 = 5 crn; O = 4 cm). The organic solvent was removed and the oily product was dried by aceotropic distilllation with toluene. Yield: 58.3 g (160 mmol; 95°,4,). Crystallisation: 20 g of the oily product was dissolved in 50 rnl toluene at 40°C and 4 drops of hydrochloric acid (1 N) was added. The solution was kept at 4°C for 3 days, the precipitate was filtered off and washed with hexane. Yield: 16 g (80%) R:: 0.07 (diisopropylether); R:: 0.55 (chloroform/ acetone, 1:1) [~]D: + 36. 2o (C = 1, chloroform) Fp: 94.0-94.5°C ~H-NMR (CDCI3): ~ = 2.28 (s, br, 1 H, 8-OH); 3.35 (ddd, 3J5,6a x = 10.4 Hz, 3Js,6e q = 4.6 Hz,

217

6.8 Hz, 1 H, 5-H); 3.42 (dd, 3j 6ax,5 = 10.4 Hz, 2Jgem" = 10.2 HZ, 1 H, 6ax-H); 3.54 (ddd, 3j 7,2 = 4.8 Hz, 3j 7,8 = 4.2 Hz, 3 j 7,8' = 4 . 5 H z , 1 H, 7-H); 3.73 (dd, 2Jgem" = 11.5 HZ, 3,]8, 7 ~--- 4.2 Hz, 1 H, 8-H); 3.76 (dd, 2Jgem" = 11.5 HZ, 3J8,,7 = 4.5 Hz, 1 H, 8'-H); 3.93 (s, br, 1 H, 4-OH); 4.12 (dd, 3j6~q.s = 4.6 Hz, 2jgem" = 10.3 Hz, 1 H, 6eq-H); 4.70 (d, 3j 2,7 = 4.8 Hz, 1 H, 2-H); 4.83 (d, 3J4.5 = 6.8 Hz, 1 H, 4-H); 4.60-4.90 (m, 4 H, benzyl-CH2) ~ = 7.26-7.40 (m, 10 H, arom. benzyl-H) IR (KBr): [cm-l] 3400, 3290, 3090, 3060, 2970, 2920, 2890, 2760, 1970, 1950, 1890, 1875, 1820, 1610, 1585, 1495, 1470, 1455, 735, 725, 695 C 2 o H 2 4 0 6 (360.41) calc. C 66.65% H 6.71% O 26.64%; found C 66.54% H 6.69% O 26.65%. MS (DCI-NH3, 200 eV): m/z = 378 (4.5%, (C10H1203)2+*NH4) m/z = 198 (100%, (CIoH1203)+*NH4) 3 j 5,4 =

3.5. 2-O-Benzyl-D-glyceraldehyde dimethylacetal

(6) The oily product 5 (25.0 g; 690 mmol) was dissolved in 600 ml of methanol and 10 ml of conc. sulfuric acid was added carefully. The solution was heated under reflux for 16 h. A solution of 11 g potassium hydroxide in 300 ml of water was added and the methanol was removed by vacuum distillation. The residue was extracted three times with ether. The combined ether phases were dried with potassium carbonate. The crude product was purified by distillation (10 cm vigreux-column); bpl0 3mbar 115-116°C. Yield: 31.2 g (131 mmol; 94%) R:: 0.19 (diisopropylether); Rf: 0.60 (chloroform/ acetone, 1:1) [~]i): + 25-97° (without solvent) 1H-NMR (CDCI3): ~ = 2.24 (s, br, 1 H, --OH); 3.46 (s, 6 H, --OCH3); 3.52 (ddd, 3Jz,3 = 4.8 Hz, 3J2,1 = 4.8 Hz, 3J2,v = 4.8 Hz, 1 H, 2-H); 3.67 (dd, 3J3,2 = 4.8 Hz, 2Jger,. = 11.8 Hz, 1 H, 3'-H); 3.76(dd, 2Jgem" = 11.8 Hz, 3J3.2 = 4.8 Hz, 1 H, 3-H) 6 = 4.39 (d, 3J1.z = 5.9 Hz, 1 H, acetal-H); 4.66 (d, 2Jg~m" = 11.6 Hz, 1 H, benzyl-CH2) = 4.77 (d, 2Jgem. = 11.6 HZ, 1 H, benzyl-CHz); 7.29-7.44 (m, 5 H, arom. benzyl-H) IR (KBr): [cm-1] 3460, 3080, 3060, 3030, 2980-

u. Massing, H. Eibl / Chemistry and Physics of Lipids 76 (1995) 211-224

218

2850, 2830, 1960, 1880, 1815, 1605; 1585, 1500, 1455, 740, 700 C12H1804 (226.27) calc. C 63.69% H 8.02% O 28.28%;found C 63.83%H8.02%O28.19%.

3. 6. 3-O-Allyl-2-O-benzyl-D-glyceraldehyde dimethylacetal (7) The reaction product 6 (100 g; 420 mmol) and potassium-tert-butylate (65 g; 580 mmol) in 1 1 THF were heated under reflux for 3 h. Allylbromide (70 g; 580 mmol) in 250 ml THF was added dropwise within 30 min. Heating under reflux was continued for 30 min. The THF phase was first extracted with 600 ml of water, then twice with 300 ml of sodium chloride (100 g/l). The solvent of the organic phase was removed and the residue was dried by aceotropic distillation with toluene. The product was purified by distillation (10 cm vigreux-column); bpl0- 4mbar 113-- 115°C. Yield: 113 g (410 mmol; 99%) Ry: 0.56 (diisopropylether); RF: 0.73 (chloroform/ acetone, 1:1); R / 0.21 (hexane/diisopropylether, 3:1) [~]D: + 23.56° (without solvent) IH-NMR (CDCI3): ~ = 3.42 (s, 3 H, --OCH3); 3.44 (s, 3 H, --OCH3); 3.54-3.70 (m, 3 H, 2-H, 3-H and 3'-H); 4.03 (dddd, 2j,CH2__CH~(gem.) --- 12.8 H Z , 3j,CH2--CH~,CH-= 5.5 Hz, 4j,CH2--CH~ , t. . . . . . c/~2 = 1.4 Hz, 4fC H 2 - - C H = , c i s _ = C H 2 = 1.7 Hz, 1 H, CH2--CH=CH); 4.06 (dddd, 2JCH2__CH=(gem.) -~- 12.8 Hz, 3.1,,CH2__CH=,CH~ = 5.5 Hz, 4Jc~2_CH= t. . . . . . c~2 = 1.4 Hz, 4j,CH2--CH=, c i s - = C H 2 = 1.7 Hz, 1 H, CH2--CH=CH); 4.39 (d, 3J1,2 = 5.1 nz, 1 H, acetal-H); 4.55 (s, 2 H, benzyl-CH2); 5.17 (dddd, 2J~i~=cn2 (gem.) = 1.7 nz,3J~is=fH2.CH= = 10.5 Hz, 4 j c i s = C H 2 , C H 2 _ _ C H ~ = 1.5 Hz, 4 j c i s = C H 2 , - - C H 2 - - C H = = 1.4 Hz, 1 H, cis=CH2); 5.28 (dddd, 2j,. . . . . cH2 (gem.) = 1.7 Hz, 3Jt. . . . . CH2. CH= = 17.1 Hz, 4 J t . . . . . CH2,CH2--CH= -1.7 Hz, 4 j t r a n s ~ C H 2 , --CH2--CH= = 1.7 Hz, 1 H, trans=CH2); 5.91(dddd, 3j.C H = t r a n s ~ C H 2 --- 17.1 Hz, 3JcH=" cis:CH 2

=

10.5 HZ, 3JcH=,CH2__CH=

3jCH=,CH2--CH=

=

5.5 H Z ,

Hz, 1 H, CH2--CH=CH2); 7.22-7.41 (m, 5 H, arom. benzyl-H) IR (KBr): [cm-~] 3080, 3060, 3030, 2980, 29702850, 2830, 1960, 1870, 1810, 1645, 1605, 1585, 1500, 1455, 740, 700 = 5.5

C15H220 4 (266.34) calc. C 67.65% H 8.33% O

24.03%; found C 67.61% H 7.91% O 24.00%

3. 7. 1-O-Allyl-2-O-benzyl-sn-glycerol (8) The dimethylacetal 7 (50 g; 190 mmol) was dispersed in 1 N hydrochloric acid. The dispersion was stirred vigorously for 5 h at 70°C. The reaction mixture was extracted three times with 200 ml of dichloromethane. The combined organic phases were reextracted with NaHCO3 solution (conc.) and the solvent was removed by distillation. The residue (crude 1-O-allyl-2-O-benzyl-Dglyceraldehyde) was dispersed in 400 ml of THF/water by vigorous stirring, which was continued during the addition of sodium borohydride (7 g; 4 eq.). After 30 min, the pH was adjusted with formic acid (10%) to 7.0. The solution was extracted three times with ether and the combined ether phases were dried with sodium carbonate. The solvents were removed and the product was purified by distillation (10 cm vigreux-column); b p l 0 - 4mbar 100-105°C. Yield: 35.8 g (160 mmol; 86%) Rs: 0.28 (diisopropylether) [~]o: --5 .470 (without solvent) 1H-NMR (CDCI3): ~ = 2.11 (dd, 3j.OH,3 -- 5.8 HZ, 3JoH,3, = 5.8 n z , 1 H, OH); 3.54-3.62 (m, 5 H, I-H, I'-H, 2-H, 3-H and 3'-H); 3.99-4.03 (m, 2 H, --CH2--CH=CH2); 4.62 (d, 2Jgcm" = 11.8 Hz, 1 H, benzyl-CH2); 4.73 (d, 2jgem = 11.8 HZ, 1 H, benzyl-CH2); 5.20 (dddd, 2Jcis=Cn2(gem.) = 1.6 HZ, 3Jcis=CH2,CH= = 10.9 Hz, 4Jcis=CH2. CH2__CH= =-

1.5 H Z , 4 j c i s = C H 2 , --CH2--CH= =

1.5

Hz, 1 H, cis=CH2); 5.28 (dddd, 2Jr. . . . . cn2~gem.) = 1.6 HZ, 3j t r a n s ~ C H 2 , C H = = 17.4 HZ, 4J t r a n s = C H 2, CH2--CH

=

=

1.5 H z , 4 J t . . . . .

CH2,--CH2--CH= =

1.5 Hz, 1 H, trans=CH2); 5 . 9 0 (dddd, 3j.C H ~ , t r a n s = C H 2 = 17.4 Hz, 3j,c n = , c i s ~ C H 2 = 10.9 Hz, 3jCH~-,CH2--CH= = 5.5 Hz, 3jCH=,CH2--CH= = 5.5 Hz, 1 H, CH2--CH=CH2); 7.27-7.39 (m, 5 H, arom. benzyl-H) IR (KBr): [cm-1] 3430, 3080, 3060, 3030, 29802800, 1950, 1870, 1820, 1645, 1610, 1585, 1495, 1450, 1170-1010, 740, 700 C13H1803 (222.28) calc. C 70.25% H 8.16% O 21.59%; found C 70.53% H 8.13% O 21.54%

U. Massing, H. Eibl / Chemistry and Physics of Lipids 76 (1995) 2 1 1 - 2 2 4

3.8. 2-O-Benzyl-D-glyceraldehyde dimethylacetal (6) (from 3) The D-mannitol derivative 3 (100 g; 190 mmol) was dissolved in 1.2 1 methanol at 20°C. Periodic acid (50.7 g; 2221mmol) in 200 ml methanol and 0.5 ml conc. sulfuric acid in 20 ml methanol were added and the solution was heated under reflux for 3 h. Then, 18 g potassium hydroxide was added and the precipitate was filtered off. After the addition of 400 ml water to the reaction mixture the organic solvent was removed by distillation. The aqueous residue was extracted four times with 100 ml of ether and the combined organic phases were dried with potassium carbonate. The solvents were removed and the product was purified by distillation; bpm 3mbar 119°C. Yield: 67.7 g (290 mmol; 77%) (Physical data, see Section 3.5.)

3.9. 1,3:4,6-Di-O-benzylidene-2,5-di-O-allyl-Dmannitol (9) The product 2 (101 g; 280 mmol) and potas-

sium-tert-butylate (82.4 g; 730 mmol) in 1 1 T H F were heated under reflux for 3 h. Allylbromide (82.0 g; 680 mmol) in 200 ml T H F was added dropwise. Heating under reflux was continued for 60 min. The reaction mixture was first extracted with 500 ml water, then three times with 300 ml sodium chloride solution (100 g/l). The combined organic phases were removed and the residue was dried by azeotropic distillation with toluene. The residue was dissolved in 1.5 1 of chloroform and filtered through silicagel (column: 1 = 10 cm, O = 8 cm). The solvent was removed and the product was recrystallized from 400 ml methanol. Yield: 104 g (2~0 mmol; 84%) Rf: 0.43 (hexane/diisopropylether, 1:1); Rf: 0.52 (chloroform) [~]i~: - 4 4 . 2 ° ( C = I , chloroform); lit: [~]D: --40.9 ° (C = 4.'7, chloroform) [28] Fp: 59.9°C; lit: Fp: 59-60°C [28] ~H-NMR (CDC13): c$ = 3.67 (dd, 2jgem = 10.5 H z , 3Jlax,2(6ax,5) = 10.0 Hz, 2 H, lax-H and 6ax-H); 3.91 (ddd, 3J2,3(5.4) = 9.1 Hz, 3J2,1eq(5.6eq) = 5.0 Hz, 3J2,~x(s,6~x) = 10.0 Hz, 2 H, 2-H and 5-H); 4.01 (d, 3J3.2(4,5) = 9.1 Hz, 2 H, 3-H and 4-H); 4.06 (dddd, 2JcH~__CH=(gem.) = 12.0 Hz, 3JCH2--CH=. CH=: = 5.8 Hz, 4j CH2--CH~, trans-~CH 2

219

= 1.5 Hz, 4JcH2__CH=, cis-=CH'~ ~" 1.5 Hz, 2 H, C H 2 - - C H = C H ) ; 4.10 (dddd, 2jCH2__CH=(gem.) = 12.0 Hz, 3Jc/_/2_cn= ' c u = = 5.8 Hz, 4Jc/42_cu=" ,-....... c/42 = 1.5 Hz, 4Jc/_,2_cn=" ,.i.,-=cH2 = 1.6 Hz, 2 H, C H 2 - - C H ~ - C H ) ; 4.41 (dd, 2Jgem. = 10.5 Hz, 3Jleq,2(6eq,5) = 5.0 Hz, 2 H, leq-H and 6eq-H); 5.50 (s, 2 H, acetal-H); 5.13 (dddd, 2Jcis=CH~- (gem.) -- -- 1.5 Hz, 3j cis=CH2,CH= = 10.3 Hz, 4 j cis~CH 2, CH2--CH~ = 1.5 Hz, 4 j cis~CH 2. --CH,--CH= = 1.5 Hz, 2 H, cis=CH2); 5.22 (dddd, 2Jt,. . . . . . CH,(gem.) = 1.5 Hz, 3 j trans=CH 2, C H ~ = 17.2 Hz, 4j,,....... CH2, CZ/2--C.----- = 1.5 Hz, 4 j trans~CH2, - - C H 2 - - C H = 1.5 HZ, 2 H, trans~CH2); 5.87 (dddd, 3Jcn= ' ,,....... cu2 = 17.2 Hz, 3j CH~,cis=CH 2 = 10.3 Hz, 3J C H = . CH2--CH~ = 5.8 Hz, 3JcH= ' Cn2--CH= = 5.8 Hz, 2 H, C H 2 - - C H = C H 2 ) ; 7.30-7.55 (m, 10 H, arom. benzyliden-H) IR (KBr): [cm-L] 3090, 3060, 3030, 3000, 2980, 2920, 2900, 2860, 1950, 1810, 1645, 1500, 1465, 1450, 740, 700, 695 C26H300 6 (438.52) calc. C 71.21% H 6.90% O 21.89%; found C 71.20% H 6.91% O 22.03%.

3. I0. 2-O-Allyl-D-glyceraldehyde dimethylacetal

(lO) The D-mannitol derivative 9 (85.0 g; 190 mmol) was dissolved in 1 1 of methanol at 20°C. Periodic acid (53 g; 230 mmol) in 250 ml of methanol and 0.6 ml conc. sulfuric acid in 20 ml of methanol were added and the solution was heated under reflux for 6 h. Then, 20 g potassium hydroxide was added and the precipitate was filtered off. After the addition of 400 ml of water to the reaction mixture, the organic solvent was removed by distillation. The aqueous residue was extracted seven times with 200 ml of ether. The combined organic phases were dried with potassium carbonate. The solvents were removed and the product was purified by distillation; bpl0-2mbar 56-57°C. Yield: 49.2 g (280 mmol; 72%) R~ 0.52 (chloroform/acetone, 1:1) [CqD: + 24.98 ° (without solvent) 1H-NMR (CDC13): J = 2.25 (dd, 3JoH,3 = 6.3 HZ, 3JoH,3, 3.3 Hz, 1 H, OH); 3.45 (ddd, 3j 2,3 ---= 4.8 HZ, 3 j 2.3' = 4 . 9 Hz, 3J2.1 = 5.9 Hz, 1 H, 2-H); 3.45 (s, 3 H, --OCH3); 3.48 (s, 3 H, --OCH3); 3.65 (ddd, 2jgem = 11.7 HZ, 3,/3.2 ----

220

U. Massing, H. Eibl / Chemistry and Physics of Lipids 76 (1995) 211 224

4.9 Hz, 3 J 3 , o H ~--- 6.3 Hz, 1 H, 3'-H); 3.74 (ddd, 2jgem" = 11.7 Hz, 3J3.2 = 4.8 Hz, 3 J 3 , o H = 6.3 Hz, 1 H, 3-H); 4.17 (dddd, 2JcH2__CH=(g~m.) = 12.7 Hz, 3j C H 2 - - C H = , C H ~ = 5.8 Hz, 4 j C H 2 - - C H ~ , ,........ cu2 = 1.4 Hz, 4JcH2--CH=" c i s . = C H 2 = 1.4 Hz, 1 H, CH2--CH---CH); 4.22 (dddd, 2JcH2__CH=(gem.) = 12.7 Hz, 3JcH2_CH="cn= = 5.6 Hz, 4JcH2__CH=" t~,m..=Ci12 = 1.4 Hz, 4JcH2__CH=" c i s . = C H 2 = 1.4 HZ, 1 H, C H z - - C H = C H ) ; 4.36 (d, 3J~,2= 5.9 Hz, 1 H, acetal-H) ~ = 5.20 (dddd, 2J,.~s=CH2(g~m) = 1.8 HZ, 3j c i s = C H 2, C H = = 10.3 Hz, 4j c i s = C H 2. C H 2 - - C H = = 1.4Hz, 4Jc i s ~ C H 2, - - C H 2 - - C H = = 1.4 Hz, 1 H , cis--~-CH2)", 5.30 (dddd, 2Jt....... CH2(g~m.) = 1.8 HZ, 3Jt . . . . . C H 2, CH= = 17.1 Hz, 4j t r a n s = C H 2, 2 - - C H = - 1.4 H z , 4 j t r a n s ~ C H 2. - - C H 2 - - C H ~ = 1.4 Hz, 1 H, trans=CH2); 5.93 (dddd, 3j.C H = , t r a n s = C H 2 - 17.1 Hz, 3j C H = , c i s ~ C H 2 = 10.3 Hz, 3Jcn=" CH2--CH= = 5.8 Hz, 3JcH=" CH2--CH= = 5.6 Hz, 1 H, C H 2 - - C H = C H 2 ) ; IR (KBr): [cm-~] 3470, 3080, 2980-2850, 2830, 1645, 1455 C 8 H 1 6 0 4 (176.21) calc. C 54.53% H 9.15% O 36.32%; found C 54.52% H 9.00% O 36.46%.

3.1 I. 2-O-Allyl-3-O-benzyl-D-glyceraldehyde dimethylacetal (11) A solution of 10 (30.0 g; 170 mmol) in 750 rnl T H F was cooled to 0°C, sodium hydride (5.4 g; 80% in oil) and tetrabutylammonium iodide (1.25 g; 1/50 eq.) was added. The dispersion was warmed to room temperature. After 15 rain, benzyl chloride (21.2 ml; 179 mmol) in 200 ml T H F was added dropwise. Stirring was continued for 20 h. The organic phase was extracted with 250 ml of water and the solvents of the organic phase were removed. The residue was solved in 700 ml of ether and was extracted four times with 75 ml water. The organic phase was dried with potassium carbonate and the product was purified by distillation (vigreux column; bpl0 3mb~r92-96°C). Yield: 35.2 g (132 mmol; 72%) Rj: 0.56 (chloroform); Rj: 0.45 (diisopropylether) [c~]t): + 17.85 ° (without solvent) ~H-NMR (CDC13): 6 = 3.40 (s, 3 H, ~OCH3); 3.46 (s, 3 H, --OCH3); 3.54-3.70 (m, 3 H, 2-H, 3'-H and 3-H); 4.17 (dddd, 2Jc~/2__CH=(g~m.) = 12.8 Hz, 3JcH2__CH= ' CH-- = 5.7 Hz, 4JcH2__CH= '

CH 2 1.4 Hz, 4 j C H 2 _ _ C H ~ _ . " c i s - ~ C H Hz, 1 H, C H 2 - - C H = C H ) ; 4.22 2JcH2_CH=(gem.) = 12.8 Hz, 3j C H 2 - - C H = , t.......

5.7

Hz,

4JcH2__CH=t

.......

CH 2

= 1.4 (dddd, = CH= = 1.4 Hz, Hz, 1 H, 2

1.4 C H 2 - - C H = C H ) ; 4.38 (d, 3 j 1,2 = 5.8 H z , 1 H , acetal-H); 4.50 (d, 2jgem" = 12.4 Hz, 1 H, benzylCH2); 4.59 (d, 2jgem" = 12.4 HZ, 1 H, benzyl-CH2); 5.16 (dddd, 2J, i.~=CH2(gem.) = 1.4 H z , 3 j c i s = C H 2 , C H ~ = 10.3 Hz, 4j ,~'i.';=CH2, C H 2 - - C H = 1.4 Hz, = 4j c i s ~ C H 2, - - C H 2 - - C H = -- - 1.4 Hz, 1 H, cis=CH2); 5.28 (dddd, 2Jt. . . . . . CH2(gem.) = 1.4 Hz, 3j, ...... CH2, CH= = 17.1 Hz, 4j t r a n s ~ C H 2, 2 - - C H = -1.4 Hz, -4j t r a n a ~ C H 2. - - C H 2 - - C H = -1.4 Hz, 1 H, -trans=CH2); 5.94 (dddd, 3JcH=t ...... CH2 = 17.1 HZ, 3j.C H = , c i s = C H 2 = 10.3 Hz, 3j.C H = , C H 2 - - C H = = 5.7 Hz, 3j C H = , C H 2 - - C H = = 5.7 Hz, 1 H, CH 2 C H = C H 2 ) ; 7.23-7.38 (m, 5 H, benzyl-arom.-H) IR (KBr): [cm ~] 3470, 3080, 2980-2850, 2830, 1645, 1455 C 1 5 H 2 2 0 4 (266.34) calc. C 67.65% H 8.33% O 14.03%; found C 68.46% H 8.43% O 23.53% 4 j CH2--CH=,

cis-=CH 2

--

--

3.12. 2-O-Allyl- 1-O-benzyl-sn-glycerol (12) The dimethylacetal 11 (22.3 g; 84 mmol) was dispersed in 600 ml of 1 N hydrochloric acid. The dispersion was stirred vigorously for 5 h at 70°C in a beaker. The reaction mixture was extracted three times with 200 ml dichloromethane. The combined organic phases were reextracted with conc. NaHCO3 solution and the solvent was removed by distillation. The residue (crude 2-O-allyl-3-O-benzyl-D-glyceraldehyde) was dispersed in 400 ml of THF/water. Sodium borohydride (3.5 g; 4 eq.) was added and the reaction mixture was stirred vigorously for 30 min. The pH was adjusted with formic acid (10%) to 7.0. The solution was extracted three times with ether and the combined ether phases were dried with sodium carbonate. The solvents were removed and the product was purified by column chromatography using 500 g silica gel. First, the apolar byproducts were eluted with hexane, then the product was eluted with hexane/diisopropylether (1:1, v/v) and finally with diisopropylether. Yield: 13.8 g (62 retool; 75%) R / 0.62 (diisopropylether); Rj: 0.0 (hexane); Rr: 0.24 (hexane/diisopropylether, 1:1)

U. Massing, H. Eibl / Chemistry and Physics of Lipids 76 (1995) 211-224

[~]D: -- 6.16 ° (without solvent) tH-NMR (CDCI3): 6 = 2.12 (dd, 3JoH.3 = 5.9 HZ, 3JoH,3, = 5.9 H z , 1 H, OH); 3.52-3.81 (m, 5 H, I-H, I'-H, 2-H, 3-H and 3'-H); 4.09 (dddd, 2JcH2__CH=(gem.) = 12.7 Hz, 3JcH2__CH=" C H = = 5.8 Hz, 4• C H 2 - - - C H ~ , trans-~fH 2 = 1.4 Hz, 4JcH 2__CH=' cis_==CH 2 = 1.4 Hz, 1 H, C H z - - C H = C H ) ; 4.18 (dddd, 2JcH2__CH=(gem.) = 12.7 HZ, 3Jc~2__CH=' c/4= = 5.5 HZ, 4Jcn2__cn=" t....... CH2 = 1.4 Hz, 4JCH2__CH= ' c i s - = C H 2 = 1.4 Hz, 1 H, C H 2 - - C H = C H ) 6 = 4.50 (d, 2jgem = 12.4 Hz, 1 H, benzyl-CH2); 4.59 (d, 2jg,m = 12.4 HZ, 1 H, benzyl-CH2) ~5 = 5.19 (dddd, 2Jcis=CH2(gem.) = 1 . 2 H Z , 3Jcis=CH2,CH= = 10.2 Hz, 4 j cis~CH2, CH2--CH-~- =

1.4

Hz, 4 j cis~CH2, - - C H 2 - - C H =

1.4 Hz, 1 H, cis=CH2); 5.28 (dddd, 2 J t . . . . . CH2(gem.) = 1.2 Hz, 3Jtrans_.~_~CH2,C H = = 17.2 H Z , 4 J t . . . . CH2, C H 2 _ _ C H = = 1.4 H Z , 4 j lrai~CH2, ~CH2__CH= = ][.4 Hz, 1 H, trans=CH2); 5.93 (dddd, 3 J c H = t. . . . . . CH2 = 17.2 Hz, 3JCH=cis=CH2 = 10.2 Hz, 3 J c , = ' CH2--CH= = 5.5 Hz, 3j C H ~ . CH2--CH= = 5.8 Hz, 1 H, C H 2 - - C H = C H 2 ) ; 7.26-7.41 (m, 5 H, arom. benzyl-H) IR (KBr): [cm-1] 3430, 3070, 3050, 3020, 29702850, 1950, 1870, 1820, 1645, 1600, 1580, 1490, 1450, 1180-1010, 730, 690 C I 3 H 1 8 0 3 (222.28) calc. C 70.25% H 8.16% O 21.59%; found C 70.27% H 8.22% O 21.51%. =

3.13. 'Mosher ester' (R)-( + )-~-methoxy-~trifluoromethyl-phenylacetic ester of the alcohols 8, rac-8, 12 and rac-12 The respective 1,2-protected glycerol (55.6 mg; 250/~mol); DCC (79.6 mg; 368 /zmol), ( R ) - ( + ) Table 1 No.

Alcohol

X1 (mg)

Xt (mmol)

X4 (%)

1 2 3 4

8 12 rac-8 rac-12

135.7 110.1 129.3 118.1

0.213 0.173 0.203 0.185

85 69 81 74

19F-NMR (CDCI3) .5: 'Mosher ester' of

8

rac-8

12

rac-12

-72.09 --

-72.09 -72.13

-72.14 --

-72.14 -72.16

221

-methoxy-ct-trifluoromethyl-phenylacetic acid (82.0 mg; 350 /~mol) and DMAP (2 mg) were stirred for 3 h in 1.5 ml choroform (ethanol free). The solvent was removed and the products were purified by column chromatography (25 g silica gel). First, the apolar byproducts were eluted with petrolether, then the 'Mosher esters' were eluted with ether/petrolether (1:2, v/v) Yield: X 1 mg (X2 mmol; X3 %)

3.14. 3-O-Hexadecyl-2-O-benzyl-Dglyceraldehyde dimethylacetal (13) The 2-O-benzyl-D-glyceraldehyde dimethylacetal 6 (12.0 g; 53.2 mmol) and triethylamine (11.1 ml; 80 mmol) in 120 ml T H F was heated under reflux. Hexadecylmesylate (20.4 g; 64 mmol) in 100 ml T H F was added dropwise and the reaction mixture was heated under reflux for 12 h. The organic phase was extracted with 120 ml water and the organic solvent was removed. The residue was dissolved in 300 ml ether and was extracted three times with 100 ml water. The product was purified by chomatography with hexane/diisopropylether (2:1, v/v). Yield: 22.5 g (50.0 mmol; 94%) Rj: 0.60 (diisopropylether); Rs: 0.21 (hexane/diisopropylether, 2:1) ~H-NMR (CDC13): 6 = 0.88 (t, 3j = 6.5 Hz, 3 H, --CH3); 1.26 (s (b), 28 H, --CH2--); 1.54 (t, 3j = 6.5 Hz, 2 H, 1--CH2--); 3.42 (s, 3 H, --OCH3); 3.44 (s, 3 H, --OCH3); 3.54-3.70 (m, 3 H, 2-H, 3-H and 3'-H); 4.39 (d, 3J1. 2 = 5.1 H z , 1 H, acetal-H); 4.55 (s, 2 H, benzyl-CH2); 7.22-7.41 (m, 5 H, arom. benzyl-H) IR (KBr): [cm-1] 3080, 3060, 3030, 2980, 29702850, 2830, 1960, 1870, 1810, 1605, 1585, 1500, 1455, 740, 700 C 2 8 H 5 0 0 4 (450.71) calc. C 74.61% H 11.18% O 14.19%; found C 74.40% H 11.21% O 14.32%. 3.15. 1-O-Hexadecyl-2-O-benzyl-sn-glycerol (14) The dimethylacetal 13 (20 g; 44.4 mmol) was dispersed in 250 ml 1 N hydrochloric acid. The dispersion was stirred vigorously for 5 h at 70°C. The reaction mixture was extracted three times with 70 ml dichloromethane, the combined organic phases were reextracted with conc. NaHCO3 solution and the solvent was removed by distilla-

222

U. Massing, H. Eibl ./Chemistry and Physics of Lipid~ 76 (1995) 211 224

tion. The residue (crude 1-O-hexadeyl-2-O-benzyl-D-glyceraldehyde) was dispersed in 150 ml THF/water by vigorous stirring, which was combined during the addition of sodium borohydride (1.5 g; 4 eq.). After 30 rain, the pH was adjusted with formic acid (10%) to 7.0. The solution was extracted three times with ether and the combined ether phases were dried with sodium carbonate. The solvents were removed and the product was finally purified by column chromatography on 300 g silica gel with hexane/diisopropylether (1:1, v/v). Yield: 14.8 g (36.4 mmol; 82%) Rf: 0.35 (diisopropylether) ~H--NMR (CDC13):/~ = 0.88 (t, 3j = 6.4 Hz, 3 H, --CH3); 1.28 (s (b), 28 H, --CH2--); 1.54 (t, 3j --- 6.5 Hz, 2 H, 1 - - C H 2 - - ) ; 2.11 (dd, 3j OH,3 = 5.8 H Z , 3JoH.3, = 5.8 H z , 1 H, OH); 3 . 5 4 - 3 . 6 2 (m, 5 H, I-H, I'-H, 2-H, 3-H and 3'-H); 4.62 (d, 2jgem" = 11.8 Hz, 1 H, benzyl-CH2); 4.73 (d, 2jgem" = 11.8 HZ, 1 H, benzyl-CH2); 7.22-7.41 (m, 5 H, arom. benzyl-H) IR (KBr): [cm-1] 3430, 3080, 3060, 3030, 29802800, 1950, 1870, 1820, 1645, 1610, 1585, 1495, 1450, 1170-1010, 740, 700 C26H4603 (406.65) calc. C 76.79% H 11.40% O 11.80%; found C 76.58% H 11.52% O 11.84%.

3.16. l -O-Hexadecyl-2-O-benzyl-sn-glycero- 3phosphoeholine (15) A solution of 14 (17.9 g; 44.1 mmol) and triethylamine (10.8 ml; 77.2 mmol) in 60 ml THF was added dropwise to phosphoroxychloride (4.63 ml; 50.7 mmol). The temperature of the vigorously stirred reaction mixture was controlled and should not exceed 10°C. Stirring was continued for 10 min. Then, a solution of N-methylethanolamine (4.7 ml; 58.7 mmol) and triethylamine (10.8 ml; 77.2 mmol) in 32 ml THF was added dropwise. The temperature of the reaction mixture should not exceed 40°C. The precipitate was filtered off and the filtrate was added to 10 ml 6 N HC1. After 10 min, the pH value was adjusted to 7.0 with ammonia. The organic solvent was removed and the aqueous residue dissolved in 120 ml chloroform and 150 ml methanol. After extraction with 100 ml water, the solvents of the organic phase were removed. The residue was dissolved in 120 ml dichloromethane/2-propanol (1:3, v/v) and

dimethylsulfate (10.2 ml; 106 mmol) was added. The mixture was warmed up to 40°C and a solution of potassium carbonate (15.4 g; 110 mmol) in 50 ml water was added in 1 rain, while the mixture was intensively stirred. Stirring was continued for 30 min at 40°C and the reaction mixture was cooled to 20°C. After phase separation, the solvents of the organic phase were removed and the residue was dissolved in 300 ml methanol and 250 ml chloroform. The organic phase was extracted with 250 ml water, the organic solvents were removed and the residue was dried by aceotropic distillation with toluene. The residue was dissolved in 25 ml dichloromethane and precipitated with 250 ml acetone. After 12 h, the precipitated crude product was used in the next step without further purification. For analytical purposes, a small amount of the crude product was purified by column chromatography with chloroform/ methanol/ammonia (6%) (60:40:6, v/v). R/: 0.17 (chloroform/methanol/ammonia (6%) 60:40:6) ~H-NMR (CDCI3/CD3OD 3:1): ~ = 0.88 (t, 3j = 6.5 Hz, 3 H, --CH3); 1.28 (s (b), 28 H, --CH2); 1.57 (s (b), 2 H, 1--CH2--); 3.22 (s, 9 H, N(CH3)3); 3.32-3.52 (m, 2 H, 1-H and I'-H); 3.60 (m, 2 H, XX'-part of the XX'MM'-system (PO--CH2--CH2--N)); 3.90-4.10 (m, 3 H, 3-H, 3'-H and 2-H); 4.18-4.30 (m, 2 H, MM'-part of the MM'XX'-system (PO--CH2--CH2--N)); 4.60 (d, 2j = 11.3 Hz, 1 H, benzyl-CH2); 4.69 (d, 2 j = 11.3 Hz, 1 H, benzyl-CH2); 7.25-7.24 (m, 5 H, arom. benzyl-H) IR (KBr): [cm-q 3060, 3020, 2910, 2840, 1470, 1240, 1080, 960 C31HssO6NP (571.78) calc. C 65.11% H 10.22%; found C 64.98% H 10.41%.

3.17. 1-O-Hexadecyl-sn-glycero-3phosphocholine(Lyso-PAF) (16) The crude product 15 (about 44.1 mmol) was dissolved in 200 ml methanol/THF (1:1). Then, 5 g palladium on activated charocoal (5%) in 20 ml water and 20 ml 1 N HCI were added. Hydrogenolysis was performed under intensive stirring at room temperature and atmospheric pressure until hydrogen uptake was completed, The catalyst was filtered off and the pH value of

U. Massing, H. Eibl / Chemistry and Physics of Lipids 76 (1995) 211-224

the mixture was adjusted to 7.0 with ammonia. The solvents were removed. The residue was dissolved in 250 ml methanol and 200 ml chloroform and extracted twice with 200 ml NaCI solution (150 g/l). The solvents of the organic phase were removed and the residue was dried by aceotropic distillation with toluene. The residue was dispersed under reflux in 70 ml chloroform and precipitated with 700 ml acetone. After 48 h at 4°C, the precipitate was collected by filtration. Yield: 18.0 g (37.5 mmol; 85% from 14) Rf: 0.11 (chloroform/methanol/ammonia, 60:40:7) ~H-NMR ( C D C ] . 3 / C D 3 O D 3:1): d = 0.88 (t, 3j = 6.5 Hz, 3 H, - - C H 3 ) ; 1.28 (s (b), 28 H, --CH2--); 1.57 (s (b), 2 H, 1--CH2--); 3.22 (s, 9 H , N(CH3)3); 3.34-3.46 (m, 2 H, 1-H and I'-H); 3.60 (m, 2 H, XX'-part of the XX'MM'-system (PO--CH2--CH:,--N)); 3.82-4.10 (m, 3 H, 3-H, 3'-H and 2-H); 4.18-4.30 (m, 2 H, MM'-part of the MM'XX'-system) (PO--CH2--CH2--N)) IR (KBr): [cm -1] 3406, 2920, 2850, 1653, 1470, 1250, 1055, 972, 873 C24Hs206NP (479.64) calc. C 60.10% H 10.50%; found C 59.84% H 10.52%.

223

system was increased. The product was finally eluted with chloroform/methanol/ammonia (50:50:9, v/v) and dried by aceotropic distillation with toluene. Yield: 1.97 g (3.75 mmol; 84%) Rs: 0.23 (chloroform/methanol/ammonia, 60:40:7) IH-NMR (CDCI3/CD3OD 3:1): d = 0.88 (t, 3j = 6.5 Hz, 3 H, --CH3); 1.28 (s (b), 28 H, --CH2--); 1.57 (s (b), 2 H, 1--CH2--); 2.10 (s, 3 H, acetyl--CH3); 3.22 (s, 9 H, N(CH3)3); 3.323.52 (m, 2 H, 1-H and I'-H); 3.60 (m, 2 H, XX'-part of the XX'MM'-system (PO--CH 2 CH2--N)); 3.90-4.10 (m, 2 H, 3-H- and 3'-H); 4.18-4.30 (m, 2 H, MM'-part of the MM'XX'system (PO--CH2--CH2--N)); 5.14 (quint, 3j = 5.2 Hz, 1 H, 2-H) IR (KBr): [cm -1] 3418, 2917, 2850, 2360, 1736, 1468, 1376, 1244, 1092, 968, 820 C26H5407NP (523.69) calc. C 59.63% H 10.39%; found C 59.29% H 10.40%.

Acknowledgement We thank B. Seeger, Max-Planck-Institute fiir Experimentelle Medizin, G6ttingen, for recording the 19F-NMR spectra.

3.18. 1-O-Hexadecyl-2-O-acetyl-sn-glycero-3phosphocholine (PAF) (17)

References

Lyso-PAF (16) (2.00 g; 4.44 mmol), DMAP (1.14 g; 9.32 mmol) and acetic anhydride (0.85 ml; 8.88 mmol) were dissolved in alcohol-free chloroform. The acylation was performed in a stoppered flask at 30°C and supported by ultrasonication. After 16 h, 1 ml of methanol was added and the solvents were removed. The residue were dissolved in 4 ml chloroform and the crude product was precipitated at - 25°C by the addition of 250 ml acetone. The precipitate was collected by filtration and then dissolved in 70 ml chloroform and 80 ml methanol. The organic phase was extracted with conc. NaCI solution and the upper aqueous phase was reextracted with 90 ml chloroform/ methanol (2:1, v/v). The solvents of the organic phase were removed and the residue was dried by aceotropic distillation with toluene. The product was finally purified by column chromatography with 200 g silica gel. First, apolar byproducts were eluted with chloroform/methanol/ammonia (60:40:2, v/v), then the polarity of the elution

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