Chemistry and Physics of Lipids, 60 (1992) 253-261

253

Elsevier Scientific Publishers Ireland Ltd.

Synthesis of enantiomerically pure lysophosphatidylinositols and alkylphosphoinositols E c k h a r d F i l t h u t h a n d H a n s j S r g Eibl Max-Planck-lnstitut J~r biophysikalische Chemie, Am Faflberg, D-3400 G6ttingen (Germany) (Received August 29th, 1991; revision received November 25th, 1991; accepted November 29th, 1991) A convenient synthesis for enantiomeric pure 1-O-(1'-O.alkyl-sn-glycero-3'-phospho)-D (or L)-myo-inositol, 1-O-(I '-O-acyl-snglycero-3'-phospho)-D (or L)-myo-inositol and alkylphospho-I-D (or L)-myo-inositol has been described. Starting from myo-inositoi, penta-O-acetyl-myo-inositol was made in five steps, Then enantiomeric purification was done by a diastereomedc salts separation method, and the purity of each enantiomer was spectroscopically measured (tgF-NMR). The phosphodiester was made via phosphoramidites. The enantiomerie products (> 99% optical purity) of all compounds were easily obtained in large quantities (5-10 g). Synthetic phosphatidylinositoi analogues of precisely defined structure and configuration are interesting tools for studying signal transduction mechanism and cell activity modulation.

Key words: lysophosphatidylinositol; alkylphosphoinositol; phosphoinositol; optical purity; synthesis; phosphoramidite

Introduction The important role of inositol lipids in signal transduction was established in the early eighties [1,2]. The few synthetic methods that were published at that time, though pioneering, were difficult to reproduce and of low yield [3-5]. Therefore we developed a synthetic method that can be routinely used to synthesize derivatives of enantionmeric pure phosphatidylinositol (PI) in gram quantities. Three different molecular species were synthesized: two lysophosphatidyl inositols, 1-O(1 '-O-octadecyl-sn-glycero-3'-phospho)-o (or L)myo-inositol and 1-O-(1 "-O-palmitoyl-sn-glycero3'-phospho)-D ( o r L)-myo-inositol and one alkylphosphoinositol, hexadecyl-phospho-l-D (or Correspondence to: Eckhard Filthuth, Max-Planck-lnstitut f. biophysikalische Chemie, AG. 145, Am FaBberg 11, D-3400 G6ttingen, Germany. Abbreviations IX;C, dicyclohexyicarbodiimide; DMAP, 4-dimethylaminopyridine; DMSO, dimethylsulfoxide; EtOAc, ethylacetate; EtOH, ethanol; He, hexadecanol; MeOH, methanol; (R)-(+)-MTPA, (R)-(+)-methoxy-trifluoromethyi-phenylacetic acid; OcBG, l-O-octadecyl-2-O-benzyl-sn-glyoerol; OcG, I-O-octadecyl-sn-glycerol; PI, phosphatidylinositol.

L)-myo-inositol (Fig. 1). These lysophosphatidylinositols and analogues of defined stereochemistry are necessary and useful for studies on the substrate specificity of enzymes involved in PI metabolism. They may also be used for studies on the stimulation or inhibition of signal transduction mechanisms.

Synthesis Starting from myo-inositol, l(3),2,4(6),5,6(4)penta-O-acetyl-myo-inositol 5 is obtained in five steps (Fig. 2). This part of the synthetic route was developed by Angyal et al. [6-8]. It was used in this study with minor modifications. Resolution of the inositol pentaacetate in its enantiomers was achieved by largely modifying a method introduced by Molotkovskij and Bergelson [4] where oxalyl-pentaacetyl-myo-inositols were precipitated with optically active bases as diastereomeric salts. The esterification of the myo-inositol pentaacetate with oxalylchloride, the purification of the Lenantiomer by precipitating the phenylethylammonium oxalate and the oxidative decarboxylation resulted in low yields and hence were modified. In our procedure the yield was raised

0009-3084/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

254

OH

0

il



H3C- (CH2)13-CH 2 - C - O-CH 2

I

NO ""C ", H

0

HO

OH

H2C_ 0 _

OH

NH~O-

3

o

OH

H3C- (CH2)13-CH2- ~ - 0 - CH2

I

NO" C .,.H 0

H

OH o°

B z O ~ BzO-~,~l~i,,,~OBz BzO

14])

I co

HO

BzO-~li~,.,.,~OBz

Bz0

o

o

OA¢ A c O ~

0Ac Ac0 I AcO-~.~--~

.

NL

0

OAc

(CH2)14-CH3 ~O

Ac0

Fig. 2. Schemeof l(3),2,4(6),5,6(4)-penta-O-acetyl-myo-inositol synthesis.

OH H O ~ - ~ '

-

I

0

NH~

AcO

OCH3

AcO

~0

0

~

ED

9D

OH

O ~ o HO

~L

2 - - ~ ~ / 0 -- CH2 -- ( CH2)14- CH3

HO H

g

HO

O

H 1

J O- NH~

(/ntc

1.4' L

Fig. I. l-O-(l'-O-palmitoyl-sn-glycero-Y-phospho)-D-myoinositol, 14 D; 1-O-(1'-O-palrtfitoyl-sn-glycero-Y-phospho)-Lmyo-inositol, 14 L; Hexadecyl-phospho-i-O-D-myo-inositol, 14' D; Hexadecyl-phospho-l-O-L-myo-inositol 14' L. J

from 22% to 40% for the D-isomer a n d purity was achieved for the L-isomer with a 25% yield. Purity was assessed by 19F- or l H - N M R - s p e c t r o s c o p y after the esterification [9,10] of the D- or the Lisomer with R-(+)-a-methoxy-t~-trifiuoromethylphenylacetic acid ( R - ( + ) - M T P A ) [1 l]. The optical purity of the e n a n t i o m e r s was 99% or better, according to N M R spectroscopy (Fig. 3). The phosphodiester between the inositol a n d the glycerol (or alcohol) moiety was m a d e by first

.L.--

I

~-72 0 i

J ,~-I

-72.0

.

i

i

-72.4

t

-72.5 t

-72.8

i

-73.0 i

-73.2

i

I

ppm

Fig. 3. 19F-NMR spectra of 9 rse, 9 t) and 9 L. Spectrum of 9 L: signal intensity ratio 9 D/9 L = 0.005;noise to signal ratio of the spectra of 9 l) and 9 L were 0.03% and 0.05%, respectively.

255 phosphorylating the free hydroxyl group of inositol pentaacetate with chloro-N,N-diisopropylaminomethoxyphosphine [12]. The fully protected phosphoramidite was then activated with a weak acid and the chosen alcohol added. After esterification, oxidation of the intermediate was done with t-butylhydroperoxide. Synthesis of the glycerol moiety is described elsewhere [ 13,14]. Removal of the protecting groups was achieved stepwise. Methyl ester was cleaved off by lithium bromide [14], acetyl esters by hydrazine hydrate [4,51 and benzyl ether by hydrogenolysis (Fig. 4).

OAc

A c O ~ AcO~ J " ~ ' m ~ " " d ~ 0 H AcO

5,8

OAc

~ n l

I 10

lH-NMR spectra were recorded with a Bruker MSL 300 (300 MHz), TMS = 0 ppm and 19FN M R with a Bruker MS 360 (360 MHz), C 6 F 6 added as internal standard, CFCI 3 = 0 ppm. IR spectra were recorded with a Perkin Elmer 1420 spectrometer. A Bfichi 520 apparatus was used to measure melting points. Elemental analysis data were from Mikroanalytisches Laboratorium Belier (G6ttingen, Germany). Chemicals, pro analyse grade, were purchased from Aldrich, Baker, Fluka and Merck and used without further purification. Exceptions were tetrazol (sublimated), oxalylchloride (distilled) and the solvents used for reactions under anhydrous conditions which were dried and distilled. Chloro-N,N-diisopropylaminomethoxyphosphine was synthesized from methyl-dichlorophosphite [ 12]. Removal of solvents, if not otherwise specified, was done in a rotatory evaporator under reduced pressure.

1 (3),2,4(6),5,6(4)-Penta-O-acetyl-myo-inositol, 5 l (3),2, 4( 6 ),5, 6(4 )-pen ta-O-acetyi-m yo-inosi tol was

AcOI ~ ~ A c O 2~ ~o ~ ~ N ' ( i p r )

AcO

Materials and Methods

H 2C- 0 - R1 J HC-O-R 2

synthesized according to Angyal [6-8] with the following modifications.

H2C-OH

l ( 3 ).'2-O-Cyclohexylidene-myo-inositol, 1

I

OMe

-

Only 0.4 g p-toluenesulfonic acid were used for 1O0 g myo-inositol. Benzene and petrolether were

OAc

AcO 11

H2C- O- R1

o

A c O ~ ' ~ O

~ njo-

AcO

H C,- 0 - R2

/,

CH2

I OMe

OH

HO

1_4

HO~'~.~/O~ HO

1 o

H2C - 0 - R1

HC-O-R2

I

IpI/ O- CH2, I oNH~

Fig. 4. Scheme of P1 analogue synthesis. Rm: alkyl, acyl; R2: benzyl, H; Ac: acetyl; Bz: benzyl; Me: methyl; ipr: isopropyl.

substituted by toluene and hexane respectively. Yield: up to 91%, (131 g); 74% [7]; Fp: 180.4°C; lit.: 181-183°C [7]. IH-NMR (DMSO/D20; 9.5:0.5): 6 = 1.30-1.70 ppm (m; 10 H; - C H 2- ketal), 2.98 ppm (dd; 1 H; 3 J 1 = 9.5 Hz, 3 J 2 = 9.5 Hz; Cs-H), 3.30-3.44 ppm (m; 2 H; 3J I = 9.2 Hz; 3J2 = 2.8 Hz; C4-H, C6-H), 3.55 ppm (dd; 1 H; 3J m= 9.5 Hz; 3J2 = 3.9 Hz; C~(3)-H), 3.83 ppm (dd; 1 H; 3 J 1 = 7.5 Hz; 3 J 2 = 5.1 Hz; C 3 ( I ) - H ) , 4.21 ppm (dd; 1 H; 3J I = 4.7 Hz; 3J2 = 4.3 Hz; C2-H). C12H2006 (260.29) calc. C 55.37%, H 7.75%; found C 55.31%, H 7.87%.

3(1),4(6),5,6(4)-Tetra-O-benzyl-1 hexylidene-myo-inositol, 2

(3) :2-O-cyclo-

During the exothermic phase of the reaction, cooling was provided with a water/ice bath to keep

256 the solution from reaching the temperature of boiling benzylchloride ( < 180°C). The temperature of the reaction solution then was held around 95°C. After 19 h the solution was cooled to 20°C and water was added (e.g. 1 I water for the conversion of 100 g 1). The product was extracted with diisopropylether (1 1). The solvent of the organic phase was removed and the product was subjected to acidolysis (next step) without isolation. For analytical purposes the solvent was removed by high vacuum distillation (0.05 mbar) and the product was crystallized twice in ethanol. Yield: 75% (179.6 g from 100 g 1); 74% [6]; Fp: 83.6-84.2°C; lit.: 80-84°C [6]. IH-NMR (CDC13): 8 = 1.25-1.80 ppm (m; 10 H; - C H 2- ketal), 3.42 ppm (dd; 1H; 3J 1 = 9.5 Hz, 3J2 = 8.6 Hz; Cs-H), 3.69 ppm (dd; 1 H; 3J1 = 8.5 Hz, 3 J 2 - 3.8 HZ; E l - H ) , 3.81 ppm (dd; 1 H; 3J l = 9.7 Hz, 3J2 = 7.0 Hz; C6-H), 3.93 ppm (dd; 1 H; 3J 1 = 8.5 Hz, 3J2 = 8.5 Hz; C4-H), 4.10 ppm (dd; 1 H; 3J 1 = 6.9 Hz, 3J2 = 5.7 Hz; C3-H), 4.27 ppm (dd; 1 H; 3J l = 5.7 Hz, 3J2 = 3.8 Hz; C2-H), 4.72-4.92 ppm (m; 8 H; - C H 2 - benzyl), 7.28-7.40 ppm (m; 20 H; H aryl). C40H4406 (620.79) calc. C 77.40%, H 7.14%; found C 77.40%, H 6.90%.

3 (1), 4 ( 6),5,6 ( 4 )- Tetra-O-benz yl-myo-inositol, 3 The product was extracted with chloroform. The solvent of the organic phase was removed and the product was precipitated with diisopropylether (1 1). Yield: - 9 5 % (153.6 g from 100 g 1); 84% [61. Fp: 113.9°C; lit.: 114-115°C [6]. 1H-NMR: (CDCI3): 6 = 3.48 ppm (dd; 3 H; 3J I = 9.3 Hz, 3J2 = 8.2 Hz; C - H ) , 3.84 ppm (dd; 1 H; 3J 1 = 9.5 Hz, 3J 2 = 9.5 Hz; C-H), 3.97 ppm (dd; 1 H; 3J 1 = 9.5 Hz, 3J2 = 9.5 Hz; C - H ) , 4.21 ppm (dd; 1 H; 3J l = 2.5 Hz, 3J2 = 2.5 Hz; Cs-H) 4.70-4.97 ppm (m; 8 H; - C H 2 - benzyl), 7.20-7.40 ppm (m; 20 H; H aryl). C34H360 6 (540.66) calc. C 75.35%, H 6.71%; found C 75.63%, H 6.71%. l ( 3 ),2, 4 ( 6 ),5,6 ( 4 )-Penta-O-acetyi-3 (1)-O-benzyl-myo-inositol, 4 To a stirred solution of 475 ml acetic anhydride

(4.30 mol) and 2.4 ml 70% perchloric acid were added 92 g of 3 (0.172 mol) at 0°C. The reaction solution was stirred 31 h at 0°C. Then 450 ml of an ice-cold NaOH solution (0.4 mol) was added and the mixture was stirred for another 10 min. The pH of the solution remained acidic. The product was extracted with chloroform against water, the organic phase was washed with 1 N HCI and water and the solvent removed. The residue was dissolved in 450 ml boiling ethanol and the product crystallized overnight at 4°C. Recrystallization from boiling ethanol gave fine colourless needles. Yield: 80% (65 g from 92 g 3); 83% [8]. Fp: 165.5°C; lit.: 167-168°C [8]. IH-NMR (CDCI3): ~ = 1.99 ppm (s; 3 H; -CH3), 2.00 ppm (s; 3 H; -CH3), 2.02 ppm (s; 3 H; -CH3), 2.17 ppm (s; 3 H; -CH3), 2.20 ppm (s; 3 H; -CH3), 3.61 ppm (dd; 1H; 3j~ = 10.0 Hz, 3J2 = 2.8 Hz; C3-H), 4.39 and 4.66 ppm (AB; 2 H; 2j = 12.1 Hz; -CH2-benzyl), 4.94 ppm (dd; 1 H; 3J 1 = 10.5 Hz, 3J2 = 2.8 Hz; CI-H), 5.08 ppm (dd; 1 H; 3J1 = 9.9 Hz; 3J2 = 9.9 Hz; Cs-H), 5.43 ppm (dd; 1 H; 3J I = 9.8 Hz, 3J2 = 9.8 nz; C4(6)-H ) 5.49 ppm (dd; 1 H; 3J l = 10.1 Hz, 3J2 = 10.1 Hz; C6(4)-H), 5.76 ppm (dd; 1 H; 3J 1 2.8 Hz, 3J2 = 2.8 Hz; C2-H), 7.20-7.36 ppm (m; 5 H; H aryl). C23H28011 (480.47) calc. C 57.50%, H 5.87%; found C 57.51%, H 6.22%.

l ( 3),2, 4 ( 6 ),5,6 ( 4 )-Penta-O-acetyl-myo-inositol, 5 In 500 ml glacial acetic acid, 500 ml methanol, 50 ml water and 15 ml formic acid were dissolved 20 g of 4 (41.6 mmol). In the presence of 10 g palladium (10%) on activated charcoal, hydrogenolysis was completed after 5-12 h in a closed system at atmospheric pressure. Catalyst was removed by membrane filtration and the filtrate extracted with chloroform. The same catalyst was always reused for this synthetic step. The solvent of the organic phase was removed. The residue was dissolved in dichloromethane and the product was precipitated with diethylether. Yield: 79% (12.8 g); 76% [8]. Fp: 166-168°C; lit.: 161-164°C [8]. 1H-NMR (CDCI3): ~ = 1.99 ppm (s; 3 H; -CH3), 2.01 ppm (s; 3 H; -CH3), 2.02 ppm (s; 3 H; -CH3), 2.09 ppm (s; 3 H; -CHa), 2.21 ppm (s;

257

3 H; -CH3), 3.87 ppm (dd; 1 H; 3Jl = 10.0 Hz, 3J2 = 2.8 Hz; C30) -H), 3.88 ppm (t; 1 H; 3j = 2.8 Hz; -OH), 4.97 ppm (dd; 1 H; 3J l = 10.5 Hz, 3J2 = 2.8 Hz; CI¢3)-H), 5.14 ppm (dd; 1 H; 3J l = 9.8 Hz, 3J2 = 9.8 Hz; Cs-H), 5.30 ppm (dd; 1 H; 3J l = 10.0 Hz, 3J2 = 10.0 nz; C4(6)-H), 5.45 ppm (dd; 1 H; 3J 1 = 10.2 Hz, 3Jz = 10.0 Hz; C6(4)-H ) 5.59 ppm (dd; 1 H; 3J 1 = 2.9 Hz, 3J2 = 2.9 Hz; Cz-H). C16H22011 (390.35) calc. C 49.23%, H 5.68%; found C 49.63%, H 5.79%.

Resolution of diastereomeric salts 3 (1)-O-Oxalyl-1 ( 3 ),2, 4 ( 6 ),5,6 ( 4 )-penta-O-acetylmyo-inositol, 6 Pyridine (9.04 ml; 113 mmol) was added dropwise at room temperature to 40 g of 5 (105 mmol) stirred in 450 ml anhydrous dichloromethane and 44 ml freshly distilled oxalylchloride (512 mmol). Synthesis was complete immediately after the addition of pyridine. Water (20 ml) was carefully added dropwise. Product was extracted with chloroform against water. The solvent of the organic phase was removed. The residue was dried by azeotropic destillation with toluene and used directly for the next step. For analysis, it was dissolved in dichloromethane and the product was precipitated with diethylether. Yield: 90% (42.6 g); 81% [41. Fp: 191.8192.1°C; lit.: 191-193°C [41. IH-NMR (CDCI3): 6 = 2.02 ppm (s; 6 H; -CH3), 2.03 ppm (s; 6 H; -CH3), 2.24 ppm (s; 3 H; -CH3), 5.12 ppm (dd; 1 H; 3J l = 10.5 Hz, 3J2 = 2.8 Hz; C3~I)-H), 5.18 ppm (dd; 1 H; 3J 1 = 10.5 Hz, 3J2 = 2.9 Hz; CI(3)-H ) 5.21 ppm (dd; 1 H; 3J 1 = 9.8 Hz, 3J2 = 9.8 Hz; Cs-H), 5.54 ppm (dd; 1 H; 3J l = 10.2 Hz, 3J2 = 10.1 Hz; C4(6)-H), 5.60 ppm (dd; 1 H; 3J l = 10.2 Hz, 3,/2 = 10.1 Hz; C6~4)-H), 5.70 ppm (dd; 1 H; 3 J 1 = 2.8 Hz, 3 J 2 = 2.8 Hz; C 2 - H ) . C18H22014 (462.36) calc. C 46.76%, H 4.80%; found C 46.71%, H 4.87%. l-O-Oxalyl-2,3, 4,5,6.penta- O-acetyl-D-myo-inositol, 7D

In 300 ml of anhydrous dichloromethane/methanol 2/1 were dissolved 40 g of 6 (86.7 retool) and

24 g chinidine (66.6 mmol) dissolved in 220 ml dry dichloromethane were added with 390 ml dry methanol. Dichloromethane was removed and the volume of the solution reduced to 240 ml in a rotatory evaporator. This solution was kept overnight at 4°C. The precipitate was filtered, washed with cold methanol and then dissolved in dichloromethane/5% HCI. The organic phase was washed three times with 5% HCI. The solvent of the organic phase was removed. The residue was dissolved in dichloromethane and the product was precipitated with diethylether. Yield: 40% (16 g); 22% [41. Analytical data (see data of 6).

1- O- Oxalyl-2, 3, 4, 5,6-pen ta- O-ace tyl-L-myoinositol 7 L In 360 ml of anhydrous dichloromethane/ ethanol (2/1) were dissolved 40 g of 6 (86.9 mmol). Then 8 ml (S)-(-)-ot-phenylethylamine (62.7 mmol) and 240 ml dry ethanol were added. The dichloromethane was removed and the volume of the solution reduced to 240 ml in a rotatory evaporator. This solution was kept overnight at 4°C. The precipitate was filtered, washed with cold ethanol and then dissolved in dichloromethane/5% HCI. The organic phase was washed three times with 5% HCI. The solvent of the organic phase was removed and the residue dried by azeotropic distillation with toluene. The optical purity of the residue was 40% diastereomeric excess (de). In order to achieve enantiomeric purity, it was necessary to do three complete cycles of the above steps, while keeping the molar ratio of (S)-(-)-ot-phenylethylamine to 8 L respectively 1.5 to 1. The pure product was then precipitated as described before (for 7 D). Yield: 25% (10 g); 26% [4]. Analytical data (see data of 6). 2,3,4,5,6-Penta-O-acetyl-D-myo-inositol, 8 D In 150 ml dioxane were dissolved 10 g of 7 D (21.6 mmol). Oxygen was kept away from the reaction with argon. Successively 0.6 g cupric diacetate (4.5 mmol), 20 g lead tetraacetate (45 mmol) and 60 ml pyridine were added. The remainder of the procedure is exactly as described by Molotkovskij and Bergelson [4]. Crystallization was done with dichloromethane/diethylether as described for 5.

258

Yield: 80% (6.75 g); 88% [4]. IH-NMR data (see data of 5). C16H22OI1 (390.35) calc. C 49.23%, H 5.68%; found C 49.13%, H 5.74%.

Phosphorylation 2,3,4,5,6-Penta-O-acetyl-l-O- (N,N-diisopropylaminomethoxyphosphoramidite)-o-myo-inositol, 10

1,2, 4,5,6-Penta-O-acetyl-o-myo-inositol, 8 t Same procedure as for 8 V.

2,3, 4,5,6-Penta-O-acetyl-l-( ( R )-( +)-ot-methoxyot-trifluoromethyl-phenylacetic-acid)-D-( or L)-myoinositol, 9 D or 9 L To 131 mg (R)-(+)-MTPA (0.56 mmol) in 1 ml anhydrous dimethylformamide were added 10 mg D M A P (0.08 mmol) and 141 mg 8 D or 8 L (0.48 mmol). The temperature of the solution was decreased to 0°C. 160 mg DCC (0.77 mmol) in 1.6 ml anhydrous dichloromethane were added dropwise. The solution was stirred 5 min at 0°C and 3 h at room temperature. The precipitated urea was filtered off, washed with dichloromethane and the solvent of the filtrate was removed. The residue was dissolved in dichloromethane, washed twice with 1 N HC1 and once with 1 N NaHCO3. The organic phase was dried over Na2SO4, filtered and the solvent was removed. The product was purified chromatographically on a 12 g silicagel column with dichloromethane. Yield: 85%. Rf (CH2C12): 0.10, (CHCI3/EtOAc/EtOH/ HCOOH, 10:10:2:1): 0.78. IH-NMR (CDCI3) 9 D: 5 = 1.93 ppm (s; 3 H; - C H 3 acetyl), 1.98 ppm (s; 3 H; - C H 3 acetyl) 2.00 ppm (s; 3 H; - C H 3 acetyl), 2.01 ppm (s; 3 H; - C H 3 acetyl), 2.02 ppm (s; 3 H; - C H 3 acetyl), 3.45 ppm (s; 3 H; - O - C H 3 ) (9 L: 3.40 ppm), 5.08 ppm (dd; 1 H; 3J 1 = 10.5 Hz, 3J 2 = 2.8 Hz; C 3 - H ), 5.17 ppm (dd; 1 H; 3J 1 = 9.8 Hz, 3J2 = 9.8 Hz; Cs-H), 5.26 ppm (dd; 1 H; 3J 1 = 10.6 Hz, 3J2 = 2.9 Hz; CL-H), 5.48 ppm (dd; 1 H; 3J 1 = 10.2 Hz, 3,/2 = 10.1 Hz; Ca-H), 5.60 ppm (dd; 1 H; 3J1 = 10.5 n z , 3J2 = 9.9 Hz; C 6 - H ), 5.72 ppm (dd; 1 H; 3j! = 2.9 Hz, 3Jz = 2.8 Hz; C2-H), 7.30-7.48 ppm (m; 5 H; H aryl). 19F-NMR (CDCI3): 9 D -72.0 ppm; 9 L -71.86 ppm; 9 rac -71.85 ppm, -72.0 ppm.

D

Phosphorylation was done under anhydrous conditions with 10 g of 8 I~ (25.65 mmol) dissolved in 60 ml dichloromethane and 5.6 ml triethylamine (40.2 mmol). Chloro-N,N-diisopropylaminomethoxyphosphine (7.5 ml, 38.6 mmol) was added dropwise at room temperature. After completion of the reaction the solution was poured into 400 ml NaHCO3-washed ethylacetate. The organic phase was washed with saturated NaCI solution. The solvent was removed. The residue was dissolved in dichloromethane and the product was precipitated with diethylether. Yield 87% (12.3 g). Rf ( d i i s o p r o p y l e t h e r / d i e t h y l e t h e r / d i c h l o r o methane, 5:4:1): 0.50. IH-NMR (CDC13): 5 = 1.17 ppm (d; 6 H; 3j = 6.8 Hz; - C H 3 isopropyl), 1.27 ppm (d; 6 H; 3j = 6.8 Hz; - C H 3 isopropyl), 1.99 ppm (s; 3 H; - C H 3 acetyl), 2.00 ppm (s; 3 H; -CH3 acetyl), 2.01 ppm (s; 3 H; - C H 3 acetyl), 2.04 ppm (s; 3 H; -CH3 acetyl), 2.05 ppm (s; 3 H; - C H 3 acetyl), 3.35 ppm (d; 3 H; 3j = 13 Hz; - O - C H 3 ) , 3.60-3.75 ppm (m; 2 H; H isopropyl), 4.49 and 4.52 ppm (dd; 1 H; 3J 1 = 2.5 Hz, 3J2 = 2.5 Hz; C2-H), 4.81 ppm (dd; 1 H; 3J 1 = 10.4 Hz, 3J I = 2.5 Hz; C3-H), 4.90-4.97 ppm (m; 1 H; C I - H ) , 5.13 ppm (dd; 1 H; 3J 1 = 9.8 Hz, 3J2 = 9.8 Hz; Cs-H), 5.55 ppm (dd; 1 H; 3Jz = 10.2 Hz, 3J2 = 10.2 Hz; C 4 ( 6 ) - H ) , 5.58 ppm (dd; 1 H; 3J 1 = 10.2 Hz, 3J2 = 10.2 Hz; C6(4)-H) C23H38012PN (551.53) calc. C 50.09%, H 6.95%, P 5.62%; found C 48.75%, H 7.10%, P 5.64%.

1,2, 4,5,6-Penta-O-acetyl-l-O- ( N,N-diisopropylaminomethoxyphosphoramidite)-o-myo-inositol, 10 L Same procedure as for 10 V. The procedure for the last part of the synthesis is the same using either hexadecanol, 1-O-octadecyl-2-O-benzyl-sn-glycerol or 1-O-palmitoyl-2O-benzyl-sn-glycerol. Description of the method is done exemplarily with 1-O-palmitoyl-2-O-benzyl-

259 sn-glycerol and corresponding analytical data are given.

2,3,4,5,6-Penta-O-acetyl-l-O-( l "-O-palmitoyl-2'O-benzyl-sn-glycero-3 '-methoxyphosphoryl)-omyo-inositol, 11 D To 2.76 g of 10 O (5 mmol) in 20 ml acetonitrile 0.7 g tetrazole (10 retool) dissolved in 20 ml acetonitrile were added at room temperature. After activation had proceeded 2.35 g 1-Opalmitoyl-2-O-benzyl-sn-glycerol (5.6 mmol) were added. Oxidation was done about 2 h later with tbutyl-hydroperoxide (6.9 mmol). The solvent was removed. The residue was dissolved in chloroform/methanol and extracted against water. The solvent of the organic phase was removed and the product was purified chromatographically on a silicagel column (200 g silicagel, solvent gradient dichloromethane/diethylether 99/1 to 79/30) Yield: 53%; with 1-O-octadecyl-2-O-benzyl-snglycerol (OcBG): 80%; with hexadecanol (He): 68%. Rf (diethylether): 0.35. IR (film): (cm -l) 3060-3020 (o C - H , aryl), 2920 (Vasym -CH2-), 2850 (I)sy m -CH2-), 1755 (v C=O, acetyl), 1450 and 1430 (6asym -CH2-), 1365 (6sym C - H , acetyl), 1220 (v P=0), l l l 5 (Vasym C - O - C acyl), 1040 (v P - O - C ) , 740 and 700 (O -CH2). ~H-NMR (CDC13): 6 = 0.88 ppm (t; 3 H 3j = 6.3 Hz; - C H 3 alkyl), 1.25 ppm (s; 24 H; -CH2-), 1.57 ppm (s; 3 H; - C H 3 acetyl), 1.52~-1.65 ppm (m; 2 H; C O - C H 2 - C H ? - ) , 1.98-2.21 ppm (m; 12 H; - C H 3 acetyl), 2.31 ppm (t; 2 H; 3j = 7.5 Hz; C O - C H ~ - C H 2 - ) , 3.65-3.73 ppm (m; 3 H; -O-CH3), 3.75-3.83 ppm (m; 1 H; CH), 4.05-4.28 ppm (m; 4 H; CH2OP, C H 2 - O - C O - ) , 4.56-4.62 ppm (m; 1 H; CI-H), 4.64-4.67 ppm (m; 2 H; C6Hs-_CHH2-), 4.82-5.04 ppm (m; 1 H; C3-H), 4.96-5.16 ppm (m; 1 H; Cs-H), 5.43 ppm (dd; 1 H; 3J l = 10.6 Hz, 3J2 = 10.2 Hz; C4t62-H), 5.46 ppm (dd; 1 H; 3j! 10.6 Hz, ~J2 = 10.2 Hz; C6(4)-H), 5.66-5.75 ppm (m; 1 H; C2-H), 7.27-7.39 ppm (m; 5 H; aryl). C 4 3 H 6 7 0 1 7 P (886.97) calc. C 58.23%, H 7.61%, P 3.49%; found C 58.70%, H 8.02%, P 3.65%.

Deprotection 2,3,4,5,6-Penta-O-acetyl-l-O-( l '-O-palmitoyl-2'O-benzyl-sn-glycero-3 ' -phospho)-D-myo-inositol, Li +, 12 D Lithiumbromide (0.6 g; 6.91 mmol) was added to 3 g 11 D (3.38 mmol) dissolved in 70 ml acetone. The solution was heated to reflux for 3-5 h. The solvent was removed and the residue was used for the following step without purification. The reaction is quantitative. Rf(dichloromethane/acetone; 9:1): 0.0. IH-NMR (CDCI3/CD3OD; 1:1) t5 = 0.89 ppm (t; 3 H; J = 6.1 Hz; - C H 3 alkyl), 1.27 ppm (s; 24 H; -CH2-), 1.53-1.67 ppm (m; 2 H; CO-CHE-CHT-), 1.97 ppm (s; 3 H; - C H 3 acetyl), 2.01 ppm (s; 3 H; - C H 3 acetyl), 2.02 ppm (s; 3 H; -CH3 acetyl), 2.08 ppm (s; 3 H; -CH3 acetyl), 2.18 ppm (s; 3 H; -CH3 acetyl), 2.31 ppm (t; 2 H; 3j = 7.4 Hz; CO-CHT-CH2-), 3.82-3.96 ppm (m; 3 H; CH, CH2OP), 4.10-4.19 ppm (m; 1 H; C H ~ I a - O - C O ) , 4.30-4.39 ppm (m; 1 H; CIZI_AHB-O-CO), 4.46-4.55 ppm (m; 1 H; CI-H), 4.62-4.77 ppm (m; 2 H; C6Hs-CHg-), 5.09 ppm (dd; 1 H; 3 J t = 10.6 Hz, 3J 2 = 2.8 Hz; C3-H), 5.15-5.25 ppm (m; 1 H; C5-H), 5.44 ppm (dd; 1 H; 3J l = 9.8 Hz, 3J2 = 9.4 Hz; C4(6)-H), 5.47 ppm (dd; 1 H;3J1 = 9.8 Hz, 3J2 = 9.4 Hz; C6(4)-H), 5.80 ppm (dd; 1 H; 3 J 1 = 2.8 Hz, 3J2 = 2.8 Hz; C2-H), 7.26-7.39 ppm (m; 5 H; aryl).

1-0- ( l '-O-Palmitoyl-2'-O-benzyl-sn-glycero-3"phospho)-D-myo-inositol, NH4 +, 13 D H2NNH2. H20 (1.6 ml; 32.7 mmol) was added to 2.28 g 12 D (2.9 mmol) dissolved in 40 ml 80% ethanol. The sohition was stirred at room temperature. After completion of the reaction (2-5 h, depending on the substrate) the solution was neutralised with acetic acid. The solvent was removed and the residue was purified chromatographically on a silicagel column (CHC13/MeOH/ NHaOH 25%; 90:10:0.5; 85:15:0.5; 14:5:1). The product was lyophilized. Yield relative to 11 D: 60%; (OcBG): 92%; (He): 45%. Rf (CHC13/CH3OH/NH4OH 25%, 16:15:1): 12 D: 0.5.

260 IR (KBr): (cm -1) 3400-3200 (u O-H), 3010 (u H aryl), 2905 (Oasyrn-CH2-), 2840 (u sym -CH2-), 1730 (u asym C=O, ester), 1460 (8 asym -CH2-) 1400 (u N - H , NH4+), 1205 (u P=O), 1110 (o C - O - C , ester), 1035 (o P - O - C ) , 730 and 695 (p -CH2- ). !H-NMR (CDCI3/CD3OD; 1/1): 8 = 0.89 ppm (t; 3 H; 3j = 6.5 Hz; -CH3), 1.27 ppm (s; 24 H; -CH2-), 1.57-1.63 ppm (m; 2 H; C O - C H 2CH~-), 2.31 ppm (t; 2 H; 3j = 7.5 Hz; CO-C__H_2HCH2-), 3.21 ppm (dd; 1 H; 3Jl = 9.3 Hz, 3J2 = 9.3 Hz; Cs-H), 3.37 ppm (dd; 1 H; 3j! = 10.1 Hz, 3J2 = 2.9 Hz; C3-H), 3.61-3.69 ppm (m; 1 H; 3J1 = 9.8 Hz, 3J2 = 9.7 Hz; C4-H), 3.75-3.82 ppm (m; 1 H; 3Jl = 9.6 Hz, 3J2 = 9.3 Hz; C6-H), 3.86-3.96 ppm (m; 2 H; CH, C!-H), 4.02-4.09 ppm (m; 2 H; CH2OP), 4.13-4.22 ppm (m; 2 H CHA_.HB-O-CO, C2-H), 4.28-4.38 ppm (m; 1 H (~_AHB-O-CO), 4.74 ppm (m; 2 H; C6Hs-CH~-), 7.27-7.38 ppm (m; 5 H; aryl).

1-O-( l '-O-Palmitoyl-sn-glycero-3 '-phospho )-Dmyo-inositol, NH4 +, 14 D To 1.39 g 13 D (2.05 mmol) dissolved in 50 ml CHCl¢CH3OH/H20/HCOOH, 40:50:15:0.5, were added 0.3 g 10% palladium on activated charcoal. The hydrogenolysis was done at room temperature and atmospheric pressure. The catalyst was then filtered off and the solvent removed. The product was purified chromatographically on a silicagel column (CHC13/CH3OH/NH4OH 25%, 80/20/0.5; 65/25/8 and 60/30/10). The product was lyophilized. Yield: 77%. (OcG): 73%. Rf (CHCI3/MeOH/NH4OH 25%; 6/3/1): 0.23. IR (KBr): (cm -1) 3400-3200 (v O-H), 2910 (Oasyrn - c a 2 - ) , 2840 (Usym-CH2-), 1735 (v C=O, ester), 1460 and 1440 (8 asym - c a 2 - ) , 1400 (v N-H, NH4+), 1205 (v P=O), 1105 (Oasym C - O - C , ester), 1035 (v P-O-C), 720 (O -CH2-). IH-NMR (CDC13/CD3OD; 1:1): 8 = 0.89 ppm (t; 3 H; 3j = 6.5 Hz;-CH3), 1.27 ppm (s; 24 H; -CH2-), 1.57-1.68 ppm (m; 2 H; CO-CH2CH~-), 2.36 ppm (t; 2 H; 3j = 7.6 Hz; CO-CH~CH2-), 3.23 ppm (dd; 1 H; 3Jt = 9.2 Hz, 3J2 = 9.2 Hz; Cs-H), 3.40 ppm (dd; 1 H; 3j! = 10.2 Hz, 3J2 = 2.8 Hz; C3-H), 3.56-3.61 ppm (m; 1 H; CH2OP), 3.66 ppm (dd; 1 H;

3J1 = 9.6 Hz, 3J2 = 9.6 Hz; C4-H), 3.67-3.74 ppm (m; 1 H; CH2OP), 3.79 ppm (dd; 1 H; 3Jl = 9.7 Hz, 3J2 = 9.3 Hz; C6-H), 3.88-3.96 ppm (m; 1 H; CI-H), 3.96-4.07 ppm (m; 2 H; CH, CH~HB-O-CO), 4.12-4.18 ppm (m; 1 H; CI&-I_AHB-O-CO), 4.21 ppm (m; 1 H; C2-H). Results and Discussion

We developed a versatile procedure for the synthesis of large quantities (5-10 g) enantiomerically pure (1-alkyl-) or (1-acyl-)glycerophosphoinositols. Alkylphosphoinositols were also prepared. These latter compounds have similar physical properties as lysophosphatidylinositols but may differ strongly in their biological properties. Two steps of this synthesis were of fundamental importance. First the separation of the pentaacetyl-myo-inositol enantiomers which was achieved through diastereomeric salts precipitation. According to our experience with the method of Molotkovskij and Bergelson the yield of the esterification of inositol pentaacetates 5 with oxalyl chloride dropped dramatically on a larger scale (e.g. 40 g 5). Good yields (90%) were possible after modifying the procedure of this reaction step. The precipitated a-phenylethylamonium oxalate 7 L had only a low optical purity. Multiple recrystallizations, as proposed [4], did not lead to any improvement. Excellent results were obtained with our purification procedure (see methods). After the first precipitation cycle, NMR analysis showed a purity of 400 de, after the second cycle a purity of 95% de. Finally, after the third precipitation cycle, the optical purity of the Lisomer was 99% or more. The following step, oxidative decarboxylation, was also modified because the original procedure led to coloured byproducts that were very difficult to separate from the wanted product. The formation of these byproducts was avoided when pyridine was added last. The second fundamental step was the building of the phosphodiester between the inositol and the glycerol moiety. The chosen phosphorylation method through phosphoramidites was developed for oligonucleotide synthesis; it lacks the usual

261

drawbacks of phosphorylating reagents in PI synthesis (e.g. long reaction time and appearance of byproducts as reported for POCI3). This phosphorylation method offers the advantage of a monofunctional reagent used under mild conditions. The overall yield (three steps) is very good for this kind of phosphorylation (60%). The phosphorylated D- or t-inositol isomers 8 O and 8 L, which can be produced in large quantities and stored, are used as starting compounds for the synthesis of inositolphospholipids with variations in the apolar region. Recently, Ishaq et al. [15] described the synthesis of ether-linked derivatives of PI. The compounds were racemic in the glycerol and also in the inositol moiety. Ward and Young [16] published an interesting synthetic route for 1-O-(1 ',2'-di-Opalmitoyl-sn-glycero-3 '-phospho)-D-myo-inositol. However, one of the main steps of the synthesis, the stereoselective monosilylation, was not described. Reaction conditions and quantities of reactants were missing. As reported by these authors [17] the chromatographic separation of the diastereomers (silyl ether derivatives esterified with camphanic acid chloride) was difficult and resulted in low yields. Therefore they replaced the camphanic acid chloride by a (R)-proline derivative. In their last publication, where the synthesis of the four stereoisomers of dipalmitoylphosphatidylinositol was reported [181, the first separation method was reused, without giving the yield. We have already used our synthetic P1 analogues for experiments in biological systems (e.g. substrate specificity for O-alkylglycerol monooxygenase EC 1.14.16.5), cytotoxicity towards cultured Raji and HL 60 cells). For example l-O-( l ' -O-octadecyl-sn-glycero- 3 ' -phospho )-myoinositol is toxic for Raji cells at concentrations below 80 /~M whereas l-O-(l'-O-palmitoyl-snglycero-3'-phospho)-myo-inositol is not. The ether-lysophosphatidylinositol with the inositol in L configuration is more toxic than the one with the D-configurated inositol (LDs0 = 35 #M and 50 /zM respectively) [19]. These results, together with those from Young et al. [18] who showed that the isomers with L-myo-inositol configuration were

not phosphorylated by a phosphatidylinositol 4-kinase, emphasize the importance of synthetic PI analogues of high optical purity as tools for studying the PI metabolism and the associated signal transduction mechanism.

Acknowledgement We thank B. Seeger, Max-Planck-Institut f/ir experimentelle Medizin, G6ttingen, for recording the 19F-NMR spectra.

References 1 M.J. Berridge (1983) Biochem. J. 212, 849-858. 2 M.J. Berridge (1985) Biol. Chem. Hoppe-Seyler 367, 447-456. 3 B.A. Klyashchitskij, E.G. Zhelvakova, V.V. Pimenova, V.I. Shvets, R.P. Evstigneeva and N.A. Preobrazhenskij (1971) J. Gen. Chem. (USSR) 41, 1391-1397. 4 Ju.G. Molotkovskij and L.D. Bergelson (1973) Chem. Phys. Lipids II, 135-147. 5 A.I. Lyutik, V.I. Sukhanov, V.I. Shvets and R.P. Evstigneeva (1974) J. Gen. Chem. (USSR) 44, 2559. 6 S. Angyal and M.E. Tate (1965) J. Chem. Soc. 6949-6955. 7 S. Angyal, G.C. Irving, D. Rutherford and M.E. Tate (1965) J. Chem. Soc. 6662-6664. 8 S. Angyal, M.H. Randall and M.E. Tate (1967) J. Chem. Soc. C 919-922. 9 B. Neises and W. Steglich (1978) Angew. Chem. 90, 556-557. 10 G. H6fle, W. Steglich and H. Vorbriiggen (1978) Angew. Chem. 90, 602-615. 11 J.A. Dale, D.L. Hull and H.S. Mosher (1969) J. Org. Chem. 34, 2543-2548. 12 L.J. MacBride and M.H. Caruthers (1983) Tetrahedron Lett. 24, 245-248. 13 H. Eibl and P. Woolley (1988) Chem. Phys. Lipids 47, 47-53. 14 P. Woolley and H. Eibl (1988) Chem. Phys. Lipids 47, 55-62. 15 K.S. lshaq, M. Capobianco, C. Piantadosi, A. Noseda, L.W. Daniel and E.J. Modest (1989) Pharm. Res. 6, 216-224. 16 J.G. Ward and R.C. Young (1988) Tetrahedron Lett. 29, 6013-6016 17 M. Jones, K.K. Rana, J.G. Ward and R.C. Young (1989) Tetrahedron Lett. 30, 5353-5356. 18 Young R.C., C.P. Downes, D.S. Eggleston, M. Jones, C.H. Macphee, K.K. Rana and J.G. Ward (1990)J. Med. Chem. 33, 641-646. 19 E. Filthuth, Ph.D. Thesis (1991) Technische Universit~it Braunschweig, Germany, pp. 44-50.

Synthesis of enantiomerically pure lysophosphatidylinositols and alkylphosphoinositols.

A convenient synthesis for enantiomeric pure 1-O-(1'-O-alkyl-sn-glycero-3'-phospho)-D (or L)-myo-inositol, 1-O-(1'-O-acyl-sn-glycero-3'-phospho)-D (or...
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