DE3442145A1 - Process for the preparation of complex ether glycerolipids using plant cell cultures - Google Patents
Process for the preparation of complex ether glycerolipids using plant cell culturesInfo
- Publication number
- DE3442145A1 DE3442145A1 DE19843442145 DE3442145A DE3442145A1 DE 3442145 A1 DE3442145 A1 DE 3442145A1 DE 19843442145 DE19843442145 DE 19843442145 DE 3442145 A DE3442145 A DE 3442145A DE 3442145 A1 DE3442145 A1 DE 3442145A1
- Authority
- DE
- Germany
- Prior art keywords
- alkyl
- formula
- compounds
- glycero
- phosphocholines
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 238000000034 method Methods 0.000 title claims description 19
- 238000002360 preparation method Methods 0.000 title claims description 6
- -1 ether glycerolipids Chemical class 0.000 title abstract description 22
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 title abstract description 14
- 238000004113 cell culture Methods 0.000 title abstract description 8
- 150000001875 compounds Chemical class 0.000 claims abstract description 23
- 240000002791 Brassica napus Species 0.000 claims abstract description 13
- 238000004114 suspension culture Methods 0.000 claims abstract description 12
- 238000004519 manufacturing process Methods 0.000 claims abstract description 8
- 235000004977 Brassica sinapistrum Nutrition 0.000 claims description 10
- 125000004432 carbon atom Chemical group C* 0.000 claims description 8
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 7
- 229920006395 saturated elastomer Polymers 0.000 claims description 7
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 claims description 6
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 5
- 239000002609 medium Substances 0.000 claims description 5
- 239000000741 silica gel Substances 0.000 claims description 5
- 229910002027 silica gel Inorganic materials 0.000 claims description 5
- 235000006008 Brassica napus var napus Nutrition 0.000 claims description 4
- 239000006870 ms-medium Substances 0.000 claims description 4
- 238000011097 chromatography purification Methods 0.000 claims description 3
- 229910014033 C-OH Inorganic materials 0.000 claims 4
- 229910014570 C—OH Inorganic materials 0.000 claims 4
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims 1
- TUCNEACPLKLKNU-UHFFFAOYSA-N acetyl Chemical compound C[C]=O TUCNEACPLKLKNU-UHFFFAOYSA-N 0.000 claims 1
- 125000003342 alkenyl group Chemical group 0.000 claims 1
- 125000002525 phosphocholine group Chemical class OP(=O)(OCC[N+](C)(C)C)O* 0.000 claims 1
- 235000010469 Glycine max Nutrition 0.000 abstract description 12
- 235000011187 glycerol Nutrition 0.000 abstract description 10
- 241000196324 Embryophyta Species 0.000 abstract description 7
- 244000068988 Glycine max Species 0.000 abstract description 5
- 230000007935 neutral effect Effects 0.000 abstract description 4
- HVAUUPRFYPCOCA-AREMUKBSSA-N 2-O-acetyl-1-O-hexadecyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCCOC[C@@H](OC(C)=O)COP([O-])(=O)OCC[N+](C)(C)C HVAUUPRFYPCOCA-AREMUKBSSA-N 0.000 abstract description 3
- 108010003541 Platelet Activating Factor Proteins 0.000 abstract description 3
- 238000011534 incubation Methods 0.000 abstract description 3
- 230000001766 physiological effect Effects 0.000 abstract description 3
- 150000002170 ethers Chemical class 0.000 abstract description 2
- 239000000126 substance Substances 0.000 abstract description 2
- 235000011293 Brassica napus Nutrition 0.000 abstract 1
- 230000001851 biosynthetic effect Effects 0.000 abstract 1
- 230000036772 blood pressure Effects 0.000 abstract 1
- 150000002314 glycerols Chemical class 0.000 abstract 1
- VLBPIWYTPAXCFJ-XMMPIXPASA-O Lyso-PAF C-16-d4 Chemical compound CCCCCCCCCCCCCCCCOC[C@@H](O)COP(O)(=O)OCC[N+](C)(C)C VLBPIWYTPAXCFJ-XMMPIXPASA-O 0.000 description 9
- 108700020675 O-deacetyl platelet activating factor Proteins 0.000 description 9
- 239000000203 mixture Substances 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N glycerol group Chemical class OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 5
- 150000002632 lipids Chemical class 0.000 description 5
- 210000004185 liver Anatomy 0.000 description 5
- 229960001231 choline Drugs 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- JSSKAZULTFHXBH-UHFFFAOYSA-N 1-O-Tetradecylglycerol Chemical compound CCCCCCCCCCCCCCOCC(O)CO JSSKAZULTFHXBH-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 101001110286 Homo sapiens Ras-related C3 botulinum toxin substrate 1 Proteins 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- 102100022122 Ras-related C3 botulinum toxin substrate 1 Human genes 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 239000012737 fresh medium Substances 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 238000004161 plant tissue culture Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- OOWQBDFWEXAXPB-UHFFFAOYSA-N 1-O-palmitylglycerol Chemical compound CCCCCCCCCCCCCCCCOCC(O)CO OOWQBDFWEXAXPB-UHFFFAOYSA-N 0.000 description 1
- YBYIRNPNPLQARY-UHFFFAOYSA-N 1H-indene Chemical compound C1=CC=C2CC=CC2=C1 YBYIRNPNPLQARY-UHFFFAOYSA-N 0.000 description 1
- OVSKIKFHRZPJSS-DOMIDYPGSA-N 2-(2,4-dichlorophenoxy)acetic acid Chemical compound OC(=O)[14CH2]OC1=CC=C(Cl)C=C1Cl OVSKIKFHRZPJSS-DOMIDYPGSA-N 0.000 description 1
- VTDOEFXTVHCAAM-UHFFFAOYSA-N 4-methylpent-3-ene-1,2,3-triol Chemical compound CC(C)=C(O)C(O)CO VTDOEFXTVHCAAM-UHFFFAOYSA-N 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 241000219198 Brassica Species 0.000 description 1
- 235000011331 Brassica Nutrition 0.000 description 1
- 235000014698 Brassica juncea var multisecta Nutrition 0.000 description 1
- 240000000385 Brassica napus var. napus Species 0.000 description 1
- 235000006618 Brassica rapa subsp oleifera Nutrition 0.000 description 1
- 241000251472 Hydrolagus Species 0.000 description 1
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 description 1
- NJUISRMVIKYYCN-UHFFFAOYSA-N acetic acid;chloroform;methanol;hydrate Chemical compound O.OC.CC(O)=O.ClC(Cl)Cl NJUISRMVIKYYCN-UHFFFAOYSA-N 0.000 description 1
- 230000000397 acetylating effect Effects 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000001476 alcoholic effect Effects 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 238000005904 alkaline hydrolysis reaction Methods 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- OGBUMNBNEWYMNJ-UHFFFAOYSA-N batilol Chemical compound CCCCCCCCCCCCCCCCCCOCC(O)CO OGBUMNBNEWYMNJ-UHFFFAOYSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000013452 biotechnological production Methods 0.000 description 1
- 230000036983 biotransformation Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- SIHHLZPXQLFPMC-UHFFFAOYSA-N chloroform;methanol;hydrate Chemical compound O.OC.ClC(Cl)Cl SIHHLZPXQLFPMC-UHFFFAOYSA-N 0.000 description 1
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- IXORZMNAPKEEDV-OBDJNFEBSA-N gibberellin A3 Chemical compound C([C@@]1(O)C(=C)C[C@@]2(C1)[C@H]1C(O)=O)C[C@H]2[C@]2(C=C[C@@H]3O)[C@H]1[C@]3(C)C(=O)O2 IXORZMNAPKEEDV-OBDJNFEBSA-N 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000012280 lithium aluminium hydride Substances 0.000 description 1
- 230000007721 medicinal effect Effects 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 238000007127 saponification reaction Methods 0.000 description 1
- 238000006257 total synthesis reaction Methods 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/04—Plant cells or tissues
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/08—Esters of oxyacids of phosphorus
- C07F9/09—Esters of phosphoric acids
- C07F9/10—Phosphatides, e.g. lecithin
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Botany (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
Abstract
Description
Verfahren zur Herstellung komplexer Etherglycerolipide unter Verwendung von pflanzlichen ZellkulturenProcess for the preparation of complex ether glycerolipids using plant cell cultures
Gebiet der Erfindung Field of the invention
Die Erfindung betrifft ein Verfahren zur Herstellung komplexer Etherglycerolipide aus exogenen 1-o-Alkylglycerinen, 1-0-(1'-Alkeny glycerinen .oder 2-o-Alkylglycerinen unter Verwendung pflanzlicher Zellkulturen, beispielsweise von Raps (Brassica napüs) und Soja {Glycine max). 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).
Die biochemische Herstellung von Verbindungen mit biologischmedizinischer Wirkung unter Verwendung von pflanzlichen Zellkulturen ist mehrfach empfohlen worden [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, Gesellscha für Strahlen- und Umweltforschung, München 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 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].
Die Möglichkeit der Verwendung von Raps- oder Soja-Zellsuspensionskulturen zur spezifischen Biotransformation von Alkylglycerinen zu komplexen neutralen und ionischen Etherglycerolipiden wurde vor kurzem in der Literatur vorgestellt [N. Weber & H. K.Mangold, Planta JJ58_, 111-118 (1983); N. Weber & H. Benning, Chem. Phys. Lipids 33_, 293-296 (1983)].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)].
Etherglycerophospholipide sind von großem Interesse, da bestimmte Vertreter der Klasse der 1-O-Alkyl^-acetyl-sn-glycero-S-phosphocholine als Bioregulatoren mit mehrfacher physiologischer WirkungEther 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
-1--1-
BAD ORIGINALBATH ORIGINAL
3442 H53442 H5
u.a. als "Platelet activating factor" (PAF) erkannt worden sind [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].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) und 1-0-Alkyl-Z-acetyl-sn-glycero-S-phosphocholine (PAF) sind durch Totalsynthese in 15-271 Ausbeute gewonnen worden [G. Hirth et al . HeIv. Chim. Acta ^, 1059-1084 (1982); ö£, 1210-1240 (1983); K., Fujita et al . Tetrahedron Lett. 2^, 3507-3510 (1982); ^ C. A. A/ van Boeckel et al . Synthesis 1982, 399-402].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].
In der DE-OS 3131524 wird die chemische Synthese von PAF ausgehend von gesättigten und ungesättigten 1-Q-Alkyldiacylglycerinen beschrieben, die in der Leber bestimmter Fische insbesondere der Seeratte (Hydrolagus coliiei) vorkommen.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) .
Auch nach Bekanntwerden verschiedener Synthesewege die zu PAF und Lyso-PAF führen, besteht ein Bedarf an einfachen Verfahren zur Herstellung dieser Wirkstoffe und analoger Verbindungen. Eine einfachere Herstellung von PAF und Lyso-PAF könnte z.B. durch die biotechnologische Produktion geeigneter Zwischenprodukte mit Hilfe pflanzlicher Zellkulturen erreicht werden. Es wurde nun gefunden, daß 1-O-Alkyl^-acyl-sn-glycero-S-phosphocholine sowie die entsprechenden 1-0-(1'-Alkenyl)-Verbindungen der Formel I oder 2-O-Alkyl-1-acyl-sn-glycero-S-phosphocholine der Formel II, worin R einen gesättigten oder ungesättigten Alkyl-Rest mit 12-24 Kohlenstoffatomen, vorzugsweise 12-18 Kohlenstoffatomen, und Ac einen gesättigten oder ungesättigten langkettigen Acyl-Rest mit 12-24 Kohlenstoffatomen vorzugsweise 16 und 18 Kohlenstoffatomen bedeutet, erhalten werden können, wenn man exogene 1-o-Alkylglycerine bzw. 1-0-(1'-AlkenyDglycerine der Formel III und IV,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,
— 2 —- 2 -
EPO COPYEPO COPY
οο
ττ
mmmmmmmm
••
"T" CM
"T"
COCO
-^ζ:—-ο- ^ ζ: —- ο
II.
-CL=O I.
-CL = O
R die oben bezeichnete Bedeutung besitzt, Zellsuspensionskulturen von photomixotrophen Raps-Zellen in modifiziertem MS-Medium oder heterotrophen Soja-Zellen in Br-Medium zugibt, diese über einige Tage bei Kulturbedingungen beläßt, die Zellen in an sich bekannter Weise isoliert und daraus in an sich bekannter Weise die Verbindungen der Formeln I oder II gewinnt.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.
Die photomixotrophen Zeilsuspensionskulturen von Raps werden in "Modifiziertem Medium nach Murashige und Skoog" [Physiol. Plant 1_5, 473-479 (1962)] fortgepflanzt, das 1x10"6M 2,4-Dichlorphenoxyessigsäure, 1x10 M 6-Benzylaminopurin und 1x10~ M Gibberelinsäure enthält (im folgenden als modifiziertes MS-Medium bezeichnet). Die Kulturen werden unter kontinuierlicher Belichtung bei 25°C geschüttelt und alle 10-14 Tage auf frisches Medium übertragen. Die heterotrophen Zeilsuspensionskulturen von Soja werden in Bc-Medium [Gamborg und Shyluk, Plant Physiol. 45, 598-600 (1970)]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\
unter Zusatz von 1x10 M 2^-Dichlorophenoxyessigsäure vermehrt.
Die Zellen werden im Dunkeln bei 250C geschüttelt und alle 10 Tage
auf frisches Medium übertragen.- 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.
Das Trockengewicht der Zellen wird bestimmt, inden>. man die Zellen auf einer Sinterglasnutsche sammelt, mit destilliertem Wasser wäscht und bei 110°C drei Stunden lang trocknet, 10 g Rapszellen ergeben 900 mg getrocknetes Zellmaterial, 10 g Sojazellenf 700 mg getrocknetes Zellmaterial.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.
Die Alkylglycerine der Formeln III und IV sind an sich bekanntste können nach an sich bekannten Verfahren hergestellt werden. Zum Teil sind sie im Handel. Genannt seien z.B. 1-o-Hexadecyl-snglycerin und 2-o-Hexadecyl-sn-glycerin, die käuflich erhältlichThe 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
-4--4-
EPO COPY JEPO COPY J
sind. Racemisches 1-O-Tetradecylglycerin, rac-1-O-Hexadecylglycerin, rac-1-O-Octadecylglycerin und rac-1~0-[(z)~9'-Octadecenyl!glycerin können aus den entsprechenden langkettigen Alkoholen und rac-Isopropylidenglycerin nach der Methode von W. J. Baumann & H. K.Mangold [J. Org. Chem. 29^5 3055-3057 (1964)] gewonnen werden. Gemische von 1-o-Alkyl-sn-glycerinen und Gemische von 1-0-(1'-Alkenyl)-sn-glycerinen werden aus 1-o-Alkyldiacylglycerinen bzw. 1-o-Alkenyldiacylglycerinen, die beispielsweise aus dem Leberöl der Seeratte (Hydroiagus coliiei) isoliert wurden, dui Reduktion mit Lithiumaluminiumhydrid [W. G. Brown, Org. Reactions 6_P 469-509 (1951) hergestellt. Gegebenenfalls können auch radioaktiv markierte Verbindungen der Formeln III und IV in das Verfahren eingesetzt werden.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.
a.) Inkubation von Alkylglycerinen mit pflanzlichen ZeIlsuspensionskulturen: 0.5 mg der optisch aktiven Verbindungen der Formel III bzw. 1 mg der Racemate der Formel III oder 1 mg der Verbindungen der Formel IV pro 1 g Zellmaterial (Feuchtgewicht) werden in alkoholischer Lösung (1 g Verbindung der Formeln III oder IV pro 5 ml Ethanol) zu 50 g photomixotrophen Rapszellen in 150 ml MS-Medium oder 50 g heterotrophen Sojazellen in 150 ml B,--Medium jeweils am 7., 8. und 9 Tag des Wachstums zugesetzt. Am .10. Tag werden die Zellen auf einer Sinterglasnutsche gesammelt und mit destilliertem Wasser gewaschen. Die Zellen werden dann in 200 ml eines 1:2 (v/v) Chloroform-Methanol-Gemisches suspendiert und mittels eines Ultra-Turrax-Mixers (IKA-Werke, Staufen, Bundesrepublik Deutschland) homogenisiert» Die Lipide werden in an sich bekannter Weise extrahiert, beispielsweise nach dem Verfahren von M, Kates & E. Eberhardt [Can. J. Bot. 35, 895 -905 (1957)].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)].
-5--5-
EPO COPYEPO COPY
b,) Isolierung und Analyse der Lipide:b,) Isolation and analysis of the lipids:
Die gesamten Lipide, die nach a) erhalten wurden - ungefähr 4 g / kg feuchtes Zellmaterial - - werden dünnschichtchromatographisch an Silikagel H (E. Merck, Darmstadt, Bundesrepublik Deutschland) im Laufmittelsystem Chloroform-Methanol-Wasser (65:25:4, v/v) fraktioniert. Die Cholinglycerophospholipide der Formeln I oder II mit R^ - Wert ca. 0.25 und die neutralen Lipide mit R^ - Wert 0.8 1.0 werden so aufgetrennt. Die Cholinglycerophospholipide können durch mehrfache Chromatographie gemäß ' obiger Beschreibung weiter gereinigt und durch Ko-Chromatographie mit einer Standardsubstanz identifiziert werden.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.
Beispiele für Cholinglycerophospholipide, die nach dem erfindungsgemäßen Verfahren hergestellt werden können, lassen sich der folgenden Tabelle entnehmen (Tabelle I),Examples of choline glycerophospholipids, which according to the invention Process can be produced can be found in the following table (Table I),
-6--6-
EPO COPYEPO COPY
3U2U53U2U5
./O./O
Tabelle I. Ausbeute an komplexen Etherglycerolipiden, die aus Raps« oder Soja-Zellsuspensionskulturen nach Inkubation während 3 Tagen mit verschiedenen Substraten gewonnen wurden.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.
Substrate der Formeln
III und IVSubstrates of the formulas
III and IV
Etherglycerolipide isoliert aus Rapszellen* (mg/kg Feuchtgewicht)Ether glycerolipids isolated from rapeseed cells * (mg / kg wet weight)
i-O-Alkyl-2-acylglycerine
oder
2-0-Alkyl-1-acylglycerine
iO-alkyl-2-acylglycerols or
2-0-alkyl-1-acylglycerols
1-o-Alkyl-3-acylglycerine 1-o-alkyl-3-acylglycerols
1-0-Alkyl-2-ac sn-glycero-3-p phocholine ode 2-0-Alkyl-1-ac sn -glycero-3-p phocholine1-0-Alkyl-2-ac sn-glycero-3-p phocholine or 2-0-Alkyl-1-ac sn -glycero-3-p phocholine
rac-i-O-Tetradecylglycerin 41rac-i-O-tetradecyl glycerol 41
rac H-O-Hexadecylglycerin 36 rac HO-hexadecylglycerol 36
r-ic -I-O-Octadecylglycerin 21 r-ic -IO-octadecylglycerin 21
rac -1-0-[(z)-9'-0ctadecenyl] rac -1-0 - [(z) -9'-octadecenyl]
< glycerin 32<glycerin 32
i-O-Alkyl-stf-glycerine (Gemischi-O-alkyl-stf-glycerine (mixture
jaus Seeratte-Leberöl) 49jaus sea rat liver oil) 49
JJ-C-Hexadecyl-sn-glycerin 73JJ-C-hexadecyl-sn-glycerin 73
41
50
2241
50
22nd
3232
51
051
0
190
96
80190
96
80
233233
288
95288
95
Die Untersuchung der spezifischen optischen Drehungen der aus Raps- oder Sojazellen gewonnenen neutralen und ionischen Etherglycerolipide j die anschließend der alkalischen Hydrolyse und/ oder Acetylierung unterworfen wurden, ergab: Optisch aktive Etherglycerolipide mit sn -1-Konfiguration werden sowohl ausgehend von rac -1 -θ—Alkylglycerinen. als auch 2-0 -Alkylglycerinen oder von 1-o-Alkyl-sn-glycerinen erhalten.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.
Das Verfahren empfiehlt sich zur Herstellung von 1-o-Alkyl~ und 1-o~(1'-Alkenyl)-2-acyl-sn-glycero-3-phosphocholinen (sogenannten "Cholinplasmalogenen") der Formel I aus 1-o-Alkyl-sn-glycerinen,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,
EPO COPYEPO COPY
""rac-1- O-Alkylglycerinen oder 1-o-(1.f-Alkenyl)-sn-glycerinen der Formel III sowie zur Herstellung von 2-o-Alkyl-1~acyl-sn~ glycero-S-phosphocholinen der Formel II aus 2-0-Alkylglycerinen der Formel IV, Radioaktive Verbindungen der Formeln I und II sind nach diesem Verfahren ebenfalls leicht zugänglich."" 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.
Herstellung und Charakterisierung der biolog, aktiven Verbindungen V-VIII Etherglycerophospholipide mit physiologischer Wirkung wie die 1-o-Alkyl-2-acetyl-s2r-glycero-3-phosphocholine (PAF) und 1-o~ Alkyl-sn-glycero-S-phosphocholine (Lyso-PAF) können aus den nach dem erfindungsgemäßen Verfahren gewonnenen 1 -o -Alkyl-2-acylsn-glycero-3-phosphocholinen auf einfache Weise hergestellt werden.Beispielsweise erhält man durch alkalische Verseifung von 1-0-Alkyl-2-acyl-si7-glycero-3-phosphocholinen der Formel I mit verdünnter methanolischer Kalilauge (20 g Kaliumhydroxid/l Methanol) und anschließender dünnschichtchromatographischer Reinigung an Silikagel H im Laufmittelsystem Chloroform-Methanol-konz. Ammoniak (70:35:7, v/v) die entsprechenden Lyso-Verbindungen der Formel V (Lyso-PAF). Durch Acetylierung dieser Lyso-Verbindungen mit Acetanhydrid in Methylenchlorid unter Zusatz von 1 g 4-(Ν,Ν-Dimethyl- ' amino)pyridin und anschließender*1 dünnschichtchromatographischer Reinigung an Silikagel H im Laufmittelsystem Chloroform-Methanol-Essigsäure-Wasser (70:35:5:5, v/v) kann man die entsprechenden Verbindungen der Formel VI herstellen. Aus Verbindungen der Formel II erhält man in gleicher Weise die 2-0-Alkyl-Analogen von Lyso-PAF der Formel VII und die 2-0-Alkyl-Analogen von PAF der Formel VIII.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.
Beispiele für Verbindungen der Formel V (Lyso-PAF) und VI (PAF) sowie deren 2-0-Alkyl-Analogen der Formeln VII und VIII, die nach dem erfindungsgemäßen Verfahren hergestellt werden können, lassen sich der folgenden Tabelle II entnehmen.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.
EPO COPYEPO COPY
3442134421
/a·/ a
' χ
ο ' χ
ο
co ιco ι
cncn
X" OX "O
co Oco O
co Ο—co Ο—
co Oco O
CM CMCM CM
X OX O
O=OO = O
I οI ο
erhe
I o I o
CMCM
COCO
COCO
Ο—Ζ—OΟ — Ζ — O
CMCM
•o
I •O
I.
O—CL=O-CL =
I OI O
CMCM
-9--9-
Tabelle II. Spezifische optische Drehungen von 1-o-Alkyl-sn-Table II. Specific optical rotations of 1-o-alkyl-sn-
' glycero-S-phosphocholinen (Lyso-PAF) und 1-o-'glycero-S-phosphocholines (Lyso-PAF) and 1-o-
" Alkyl-Z-acetyl-sn-glycero-S-phosphocholinen (PAF)"Alkyl-Z-acetyl-sn-glycero-S-phosphocholinen (PAF)
. sowie deren 2-o-Alkyl-Analogen/. as well as their 2-o-alkyl analogues /
i-O-Alkylglycero-S-phosphocholine CLyso-PAF) oder 2-o-Alkylglycero-3-phosphocholinei-O-Alkylglycero-S-phosphocholine CLyso-PAF) or 2-o-alkylglycero-3-phosphocholines
Alkyl-Reste ^a^l° Alkyl radicals ^ a ^ l °
in Chloroform-Methanol (1:1, v/v)in chloroform-methanol (1: 1, v / v)
1-0-Tetradecyl -6.01-0-tetradecyl -6.0
1-O-Hexadecyl -5.81-O-hexadecyl -5.8
Τ-0-Octadecyl -5.3 'Τ-0-octadecyl -5.3 '
1-o-[(Z)-9'-Octadecenyl] -'5.31-o - [(Z) -9'-Octadecenyl] -'5.3
1-o-Alkyl (Gemisch aus Seerate-Leberöl) -5.51-o-alkyl (mixture of Seerate liver oil) -5.5
2-o-Hexadecyl +6.62-o-hexadecyl +6.6
-10--10-
3U2U53U2U5
Die Verbindungen der Formeln V (Lyso-PAF) und VI (PAF) zeichnen siel durch hohe biologische Aktivitäten aus ,welche die vollsynthetischer Produkte übertreffen. Als Beispiel wird die biologische Aktivtät von 1-o-Hexadecyl-Z-acetyl-sn-glycero-S-phosphocholin gegeben, dieThe 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 -_ 1 1 -
2 . 2 χ 1i 0 M beträgt (bestimmt nach M. Tence et.al., in J. Benveni: und B. Arnoux (Eds) "Platelet-activating factor and structurally related ether lipids", pp. 41 - 48, -Elsevier, Amsterdam 1983). Die Verbindungen der Formeln VII und VIII (2-0-Alkyl-Analoge) können als Antagonisten der erstgenannten Verbindungen verwendet werden..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.
-11--11-
BADBATH
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WO2004022530A1 (en) * | 2002-09-05 | 2004-03-18 | Dbl Australia Pty Ltd | Urea-, glycerate- and, hydroxyamide-headed hydrocarbon chain lyotropic phases forming surfactants |
WO2009133351A3 (en) * | 2008-04-28 | 2010-06-17 | Naturally Scientific Energy Limited | Production of biofuel from plant tissue culture sources |
AU2013201607B2 (en) * | 2008-04-28 | 2015-03-12 | Naturally Scientific Technologies Limited | Production of biofuel from tissue culture sources |
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WO2004022530A1 (en) * | 2002-09-05 | 2004-03-18 | Dbl Australia Pty Ltd | Urea-, glycerate- and, hydroxyamide-headed hydrocarbon chain lyotropic phases forming surfactants |
WO2009133351A3 (en) * | 2008-04-28 | 2010-06-17 | Naturally Scientific Energy Limited | Production of biofuel from plant tissue culture sources |
AU2009241909B2 (en) * | 2008-04-28 | 2015-02-12 | Naturally Scientific Technologies Limited | Production of biofuel from plant tissue culture sources |
AU2013201607B2 (en) * | 2008-04-28 | 2015-03-12 | Naturally Scientific Technologies Limited | Production of biofuel from tissue culture sources |
US9447442B2 (en) | 2008-04-28 | 2016-09-20 | Naturally Scientific Technologies Limited | Production of biofuel from tissue culture sources |
US10465215B2 (en) | 2008-04-28 | 2019-11-05 | Naturally Scientific Technologies Limited | Production of biofuel from tissue culture sources |
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