DE3442145A1 - Process for the preparation of complex ether glycerolipids using plant cell cultures - Google Patents

Abstract

The invention relates to a process for preparing complex ether glycerolipids from 1-O-alkylglycerols, 1-O-(1'-alkenyl)glycerols or 2-O-alkylglycerols using plant cell cultures. Cell suspension cultures of rape (Brassica napus) and soya (Glycine max) synthesise neutral and ionic ether glycerolipids, mainly alkylacylglycerophosphocholines and 1-alkenylacylglycerophosphocholines, from exogenous alkylglycerols. The best yields of ether glycerolipids are obtained on incubation of the cells with optically active 1-O-alkyl-sn-glycerols. Racemic and symmetrical alkylglycerols are likewise metabolised by the cell cultures to optically active ionic ether lipids with 'natural' configuration. 1-O-Alkyl-2-acetyl-sn-glycero-3-phosphocholines (I) can be prepared in a simple way from the biosynthetic 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholines. The former compounds ('Platelet Activating Factor') are bioregulators with a plurality of physiological effects; they have been recognised as, inter alia, substances which lower blood pressure. <IMAGE>

Description

Process for the preparation of complex ether glycerolipids using plant cell cultures

Field of the invention

The invention relates to a process for the production of complex ether glycerolipids from exogenous 1-o-alkylglycerols, 1-0- (1'-alkeny glycerines. Or 2-o-alkylglycerols using plant cell cultures, for example from rapeseed (Brassica napüs) and soya {glycine Max).

State of the art

The biochemical production of compounds with biological medical effects using plant cell cultures has been recommended several times [W. Barz, E. Reinhard & M, H. Zenk (Eds) "Plant tissue culture and its bio-technological application ", Springer Verlag, pp. 153-171, Heidelberg-New York 1977; A. W. AIfermann & E. Reinhard (Eds)" Production of natural compounds by cell culture methods ", pp. 3-15, Gesellschaft für Strahlen- und Umweltforschung, Munich 1978; E. J. Staba (Ed.) "Plant tissue culture as a source of biochemicals", pp. 59-97, 167-190, CRC Press, Boca Raton, Fla. 1980].

The possibility of using canola or soy cell suspension cultures for the specific biotransformation of alkyl glycerols to complex neutral and ionic ether glycerolipids recently presented in the literature [N. Weber & H. K. Mangold, Planta JJ58_, 111-118 (1983); N. Weber & H. Benning, Chem. Phys. Lipids, 33_, 293-296 (1983)].

Ether glycerophospholipids are of great interest, as certain representatives of the class of the 1-O-alkyl ^ -acetyl-sn-glycero-S-phosphocholine as bioregulators with multiple physiological effects

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recognized as a "platelet activating factor" (PAF) are [J. Benveniste & B. B. Vargaftig in_ H; K. Mangold & F. Paltauf (Eds) "Ether lipids: Biochemical and biomedical aspects", pp. 355-376, Academic Press, New York 1983].

1-o-alkyl ~ sn-glycero-S-phosphocholine (Lyso-PAF) and 1-0-alkyl-Z-acetyl-sn-glycero-S-phosphocholine (PAF) have been obtained by total synthesis in 15-271 yield [ G. Hirth et al . HeIv. Chim. Acta ^, 1059-1084 (1982); ö £, 1210-1240 (1983); K., Fujita et al . Tetrahedron Lett. 2 ^, 3507-3510 (1982); ^ CA A / van Boeckel et al. Synthesis 1982 , 399-402].

DE-OS 3131524 describes the chemical synthesis of PAF starting from saturated and unsaturated 1-Q-alkyldiacylglycerols, which occur in the liver of certain fish, in particular the sea rat (Hydrolagus coliiei) .

Task and solution

Even after various synthetic routes leading to PAF and lyso-PAF have become known, there is still a need for simple processes for the production of these active ingredients and analogous compounds. A simpler production of PAF and Lyso-PAF could e.g. through the biotechnological production of suitable intermediate products can be achieved with the help of plant cell cultures. It has now been found that 1-O-alkyl ^ -acyl-sn-glycero-S-phosphocholine as well the corresponding 1-0- (1'-alkenyl) compounds of the formula I or 2-O-alkyl-1-acyl-sn-glycero-S-phosphocholine of the formula II, in which R is a saturated or unsaturated alkyl radical with 12-24 Carbon atoms, preferably 12-18 carbon atoms, and Ac is a saturated or unsaturated long-chain acyl radical having 12-24 carbon atoms, preferably 16 and 18 carbon atoms means can be obtained by using exogenous 1-o-alkylglycerols or 1-0- (1'-AlkenyDglycerine of the formula III and IV,

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3 442145 _ <ρ ■ - ( χ O . (ο- - —- X X χ , ζ ■ ο- O - (J— O Ι χ ®ι χ I. | I. ο ο— ι
ο —Ο ο: ■ CNJ
τ (Z CM
mmmm CNJ _> L_ X CM X O T O D I Ο — Ol = O ι »-« υ I · - « < O
• O I. I. O ο- CM CM
"T" • JL ·

CO

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X CM: α: -J- X EPO COPY - O X ο - (J - O I. I. ι I. I. I. ο— I. —— (J · - « O O
I. CM ο CM U I.
-CL = O X χ X (X ■ < O I. I. U - -ο CM CM X X

R has the meaning given above, cell suspension cultures of photomixotrophic rapeseed cells in modified MS medium or heterotrophic soy cells in Br medium are added and these are left under culture conditions for a few days Cells isolated in a manner known per se and the compounds of the formulas I or II obtained therefrom in a manner known per se.

Oilseed rape and soy cell suspension cultures

The photomixotrophic cell suspension cultures of oilseed rape are in "Modified Medium according to Murashige and Skoog" [Physiol. Plant 1_5, 473-479 (1962)] which contains 1x10 " ⁶ M 2,4-dichlorophenoxyacetic acid, 1x10 M 6-benzylaminopurine and 1x10 ~ M gibberelic acid (hereinafter referred to as modified MS medium). The cultures are under continuous Exposure shaken at 25 ° C. and transferred to fresh medium every 10-14 days The heterotrophic cell suspension cultures of soy are in B _c medium [Gamborg and Shyluk, Plant Physiol. 45, 598-600 (1970)]

- f \
increased with the addition of 1x10 M 2 ^ -dichlorophenoxyacetic acid. The cells are shaken in the dark at 25 ^° C. and transferred to fresh medium every 10 days.

The dry weight of the cells is determined inden>. the cells are collected on a sintered glass suction filter, washed with distilled water and dried at 110 ° C. for three hours, 10 g of rapeseed cells result in 900 mg of dried cell material, 10 g of soy cells _for 700 mg of dried cell material.

Alkyl glycerols of the formulas III and IV

The alkyl glycerols of the formulas III and IV are best known per se can be prepared by processes known per se. Some of them are in stores. Examples include 1-o-hexadecyl-glycerine and 2-o-hexadecyl-sn-glycerin, which are commercially available

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are. Racemic 1-O-tetradecylglycerin, rac-1-O-hexadecylglycerin, rac-1-O-octadecylglycerin and rac-1 ~ 0 - [(z) ~ 9'-octadecenyl! Glycerin can be prepared from the corresponding long-chain alcohols and rac-isopropylidene glycerin according to the method of WJ Baumann & HKMangold [J. Org. Chem. 29 ^ ₅ 3055-3057 (1964)]. Mixtures of 1-o-alkyl-sn-glycerols and mixtures of 1-0- (1'-alkenyl) -sn-glycerols are made from 1-o-alkyldiacylglycerols or 1-o-alkenyldiacylglycerols, which are obtained, for example, from the liver oil of the sea rat (Hydroiagus coliiei) were isolated, produced by reduction with lithium aluminum hydride [WG Brown, Org. Reactions 6_ _P 469-509 (1951). If appropriate, radioactively labeled compounds of the formulas III and IV can also be used in the process.

Implementation of the procedure

a.) Incubation of alkyl glycerols with vegetable cell suspension cultures: 0.5 mg of the optically active compounds of the formula III or 1 mg of the racemates of the Formula III or 1 mg of the compounds of formula IV per 1 g of cell material (wet weight) are in alcoholic Solution (1 g compound of formula III or IV per 5 ml ethanol) to 50 g photomixotrophic rapeseed cells in 150 ml MS medium or 50 g heterotrophic soy cells in 150 ml B medium on the 7th, 8th and 9th day added to growth. On .10. Day the cells are collected on a sintered glass suction filter and distilled with Water washed. The cells are then dissolved in 200 ml of a 1: 2 (v / v) chloroform-methanol mixture suspended and using an Ultra-Turrax mixer (IKA-Werke, Staufen, Federal Republic of Germany) homogenized »The lipids are extracted in a manner known per se, for example according to the method of M, Kates & E. Eberhardt [Can. J. Bot. 35: 895-905 (1957)].

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b,) Isolation and analysis of the lipids:

The total lipids obtained according to a) - approx. 4 g / kg moist cell material - - will thin layer chromatography on silica gel H (E. Merck, Darmstadt, Federal Republic of Germany) fractionated in the solvent system chloroform-methanol-water (65: 25: 4, v / v). The choline glycerophospholipids of the formulas I or II with R ^ - value approx. 0.25 and the neutral lipids with R ^ - value 0.8 1.0 are so separated. The choline glycerophospholipids can by multiple chromatography according to ' The above description is further purified and by co-chromatography with a standard substance be identified.

Examples of choline glycerophospholipids, which according to the invention Process can be produced can be found in the following table (Table I),

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3U2U5

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Table I. Yield of complex ether glycerolipids obtained from rapeseed or soy cell suspension cultures after incubation were obtained over 3 days with different substrates.

Substrates of the formulas
III and IV

Ether glycerolipids isolated from rapeseed cells * (mg / kg wet weight)

iO-alkyl-2-acylglycerols or
2-0-alkyl-1-acylglycerols

1-o-alkyl-3-acylglycerols

1-0-Alkyl-2-ac sn-glycero-3-p phocholine or 2-0-Alkyl-1-ac sn -glycero-3-p phocholine

rac-i-O-tetradecyl glycerol 41

rac HO-hexadecylglycerol 36

r-ic -IO-octadecylglycerin 21

rac -1-0 - [(z) -9'-octadecenyl]

<glycerin 32

i-O-alkyl-stf-glycerine (mixture

jaus sea rat liver oil) 49

JJ-C-hexadecyl-sn-glycerin 73

41
50
22nd

32

51
0

190
96
80

233

288
95

The investigation of the specific optical rotations of the neutral and ionic ether glycerolipids obtained from rapeseed or soybean cells, which were then subjected to alkaline hydrolysis and / or acetylation, showed: Optically active ether glycerolipids with the sn -1 configuration are both starting from rac -1 -θ - alkyl glycerols. as well as 2-0 -alkyl glycerols or from 1-o-alkyl-sn-glycerols.

The process is recommended for the preparation of 1-o-alkyl ~ and 1-o ~ (1'-alkenyl) -2-acyl-sn-glycero-3-phosphocholines (so-called "Choline plasmalogens") of the formula I from 1-o-alkyl-sn-glycerols,

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"" rac-1-O-alkylglycerols or 1-o- (1. ^f -alkenyl) -sn-glycerols of the formula III and for the preparation of 2-o-alkyl-1 ~ acyl-sn ~ glycero-S-phosphocholines of the Formula II from 2-0-alkylglycerols of the formula IV, radioactive compounds of the formulas I and II are also easily accessible by this process.

Production and characterization of the biologically active compounds V-VIII ether glycerophospholipids with physiological effects such as the 1-o-alkyl-2-acetyl-s2r-glycero-3-phosphocholine (PAF) and 1-o ~ alkyl-sn-glycero-S- Phosphocholines (Lyso-PAF) can be prepared in a simple manner from the 1 -o- alkyl-2-acylsn-glycero-3-phosphocholines obtained by the process according to the invention. For example, alkaline saponification of 1-0-alkyl-2- acyl-si7-glycero-3-phosphocholines of the formula I with dilute methanolic potassium hydroxide solution (20 g potassium hydroxide / l methanol) and subsequent thin-layer chromatographic purification on silica gel H in the solvent system chloroform-methanol-conc. Ammonia (70: 35: 7, v / v) the corresponding lyso compounds of the formula V (Lyso-PAF). By acetylating these lyso compounds with acetic anhydride in methylene chloride with the addition of 1 g of 4- (Ν, Ν-dimethyl- 'amino) pyridine and subsequent * ¹ purification by thin layer chromatography on silica gel H in the solvent system chloroform-methanol-acetic acid-water (70:35 : 5: 5, v / v) the corresponding compounds of the formula VI can be prepared. The 2-0-alkyl analogs of Lyso-PAF of the formula VII and the 2-0-alkyl analogs of PAF of the formula VIII are obtained in the same way from compounds of the formula II.

Examples of compounds of the formulas V (Lyso-PAF) and VI (PAF) and their 2-0-alkyl analogues of the formulas VII and VIII, which are based on can be prepared by the process according to the invention can be found in Table II below.

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34421

/ a

' χ
ο

co ι

cn

X "O

co O

co Ο—

co O

CM CM

X O

O = O

I ο

he

I o

CM

CO

CO

Ο — Ζ — O

CM

•O
I.

O-CL =

I O

CM

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Table II. Specific optical rotations of 1-o-alkyl-sn-

'glycero-S-phosphocholines (Lyso-PAF) and 1-o-

"Alkyl-Z-acetyl-sn-glycero-S-phosphocholinen (PAF)

. as well as their 2-o-alkyl analogues /

i-O-Alkylglycero-S-phosphocholine CLyso-PAF) or 2-o-alkylglycero-3-phosphocholines

Alkyl radicals ^ ^a ^ l °

in chloroform-methanol (1: 1, v / v)

1-0-tetradecyl -6.0

1-O-hexadecyl -5.8

Τ-0-octadecyl -5.3 '

1-o - [(Z) -9'-Octadecenyl] -'5.3

1-o-alkyl (mixture of Seerate liver oil) -5.5

2-o-hexadecyl +6.6

i-o-Alkyl-Z-acetyl-glycero-S-phosphocholine (PAF) or 2-o-alkyl-1-acetyl-glycero-3-phosphocholine Alkyl radicals Ca] ₀ - in chloroform 1-o-tetradecyl ~ 1 .1 1-0-hexadecyl -1.1 1-O-octadecyl -1.5 1-0 - [(Z) -9'-octadecenyl] -1.1 1-o-alkyl (mixture of Seerate liver oil) -1.1 2-0-hexadecyl + 13.5

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The compounds of the formulas V (Lyso-PAF) and VI (PAF) are characterized by high biological activities that exceed those of fully synthetic products. As an example, the biological activity of 1-o-hexadecyl-Z-acetyl-sn-glycero-S-phosphocholine is given

_ 1 1 -

2. 2 χ ¹ i 0 M (determined according to M. Tence et.al., in J. Benveni: and B. Arnoux (Eds) "Platelet-activating factor and structurally related ether lipids", pp. 41-48, -Elsevier , Amsterdam 1983). The compounds of the formulas VII and VIII (2-0-alkyl analogs) can be used as antagonists of the first-mentioned compounds.

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Claims (3)

Claim 1.) Process for the production of 1-o-J phosphocholines and the I-O- (1'-alkenyl) compounds of the formula I. or 2-0-alkyl-1-acyl-sn-glycero-S-phosphocholines of the formula IT .. H ₂ C-OR H ₂ C-OAc 3 ~ CH RO-CH γ f ^H 3 I Φ® CH _? H ₂ COPO- (CH ₂ ) TN ^ -CH ₃ H ₂ COPO- (CH ₇ ) ON ^ -CH, Il I -—- Ii \ ⁰ CH ₃ O CH ₃ I II where R is a saturated or unsaturated alkyl radical having 12 to 24 carbon atoms and Ac is a saturated or unsaturated one Acyl radical with 12-24 carbon atoms, characterized in that one exogenous I-O-alkylglycerols or I-0- (1'alkenyl) -glycerols of the formula III or 2-o-alkylglycerols of the formula IV, H ₂ C-OR H ₂ C-OH HO-CH RO-CH H ₂ C-OH. H ₂ C-OH ¹¹¹ IV wherein R has the meaning given above, photomixotrophic cell suspension cultures of oilseed rape in modified MS medium or heterotrophic cell suspension cultures of soy in Bj. medium adds, leaves this under culture conditions for a few days, "isolates the rapeseed cells in a manner known per se and from them per se BATH ORIGINAL 3442Η5 known way the compounds of formula I or II wins. 2.) Process for the preparation of 1-p -alkyl-sn -glycero-3-phosphocholines and the 1 -o- (1'-alkenyl) compounds of the formula V or 2 ~ o ~ alkyl-sn-glycero-3- phosphocholines of formula VII ₈ H ₂ C-OR H ₂ C-OH HO-CH RO-CH T f ^H 3 - I H ₂ COPO- (CH ₂ J ₂ -N ^ -CH ₃ H ₂ COPO- (CH ₂ J ₂ -N ^ C ⁰ CH ₃ 0 CH ₃ ^Y YII wherein R is a saturated or unsaturated alkyl radical with 12-24 Means carbon atoms, characterized in that compounds of the formulas I or II are obtained from photomixotrophic cell suspension cultures of rapeseed or heterotrophic cell suspension cultures of soy according to method 1.) were obtained with dilute methanolic Potassium hydroxide is hydrolyzed and the compounds of the formulas V or VII are obtained by chromatographic purification on silica gel. 3.) Process for the preparation of 1-o-alkyl-2-acetyl-sn-glycero-3-phosphocholines and the 1-0- (1'-alkenyl) compounds of the formula VI or 2-o-alkyl-1-acetyl-sn -glycero-3-phosphocholines of the formula VIII 0 Ii H ₂ COC-CH ₃ H ₂ C-OR 0 I. -0 — CH 0 ^Θ Cl RO-CH CH ₃ -CO-CH f CH H ₂ COPO- (CH ₂ J ₂ -N-CH ₃ H ₂ C-0-PO- (CH ₂ J ₂ -N Ii- I ^ Il I 0 CH ₃ 0 Cf VIII YI -13- BATH ORIGINAL where R is a partially saturated or unsaturated alkyl radical with 12-24 carbon atoms and CH - CO- is an acetyl radical ^ "characterized in that compounds of the formula V or VII ₅ which were obtained by process 2.) were obtained with acetic anhydride acetylated in methylene chloride with the addition of 4- (N, N-dimethylamino) pyridine and the compounds of the formulas VI and VIII are obtained by chromatographic purification on silica gel, -14- BATH ORIGINAL