DE102004047774A1 - Process for the enzymatic hydroxylation of non-activated hydrocarbons - Google Patents
Process for the enzymatic hydroxylation of non-activated hydrocarbons Download PDFInfo
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- DE102004047774A1 DE102004047774A1 DE200410047774 DE102004047774A DE102004047774A1 DE 102004047774 A1 DE102004047774 A1 DE 102004047774A1 DE 200410047774 DE200410047774 DE 200410047774 DE 102004047774 A DE102004047774 A DE 102004047774A DE 102004047774 A1 DE102004047774 A1 DE 102004047774A1
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- 238000000034 method Methods 0.000 title claims abstract description 20
- 229930195733 hydrocarbon Natural products 0.000 title claims description 13
- 150000002430 hydrocarbons Chemical class 0.000 title claims description 11
- 238000005805 hydroxylation reaction Methods 0.000 title claims description 11
- 230000033444 hydroxylation Effects 0.000 title claims description 9
- 230000008569 process Effects 0.000 title claims description 7
- 230000002255 enzymatic effect Effects 0.000 title claims description 6
- 238000006243 chemical reaction Methods 0.000 claims abstract description 16
- 239000000126 substance Substances 0.000 claims abstract description 15
- 239000007800 oxidant agent Substances 0.000 claims abstract description 7
- 125000001931 aliphatic group Chemical group 0.000 claims abstract description 5
- 108010023506 peroxygenase Proteins 0.000 claims abstract description 4
- 239000000203 mixture Substances 0.000 claims abstract description 3
- 102000004190 Enzymes Human genes 0.000 claims description 15
- 108090000790 Enzymes Proteins 0.000 claims description 15
- 229940088598 enzyme Drugs 0.000 claims description 13
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 6
- 244000045069 Agrocybe aegerita Species 0.000 claims description 5
- 235000008121 Agrocybe aegerita Nutrition 0.000 claims description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 5
- 150000004945 aromatic hydrocarbons Chemical class 0.000 claims description 4
- 230000000694 effects Effects 0.000 claims description 4
- 241000222531 Bolbitiaceae Species 0.000 claims description 3
- 239000004366 Glucose oxidase Substances 0.000 claims description 2
- 108010015776 Glucose oxidase Proteins 0.000 claims description 2
- 230000015572 biosynthetic process Effects 0.000 claims description 2
- 229940116332 glucose oxidase Drugs 0.000 claims description 2
- 235000019420 glucose oxidase Nutrition 0.000 claims description 2
- 238000005580 one pot reaction Methods 0.000 claims description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims 3
- 241001660920 Agrocybe chaxingu Species 0.000 claims 1
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- 229910019142 PO4 Inorganic materials 0.000 claims 1
- 241000722337 Pholiota Species 0.000 claims 1
- 230000001133 acceleration Effects 0.000 claims 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical class [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims 1
- 235000019800 disodium phosphate Nutrition 0.000 claims 1
- 150000007524 organic acids Chemical class 0.000 claims 1
- 235000005985 organic acids Nutrition 0.000 claims 1
- 150000001451 organic peroxides Chemical class 0.000 claims 1
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- 150000003013 phosphoric acid derivatives Chemical class 0.000 claims 1
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- 125000003118 aryl group Chemical group 0.000 abstract description 3
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- 231100000719 pollutant Toxicity 0.000 abstract description 2
- 239000012736 aqueous medium Substances 0.000 abstract 1
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 19
- UFWIBTONFRDIAS-UHFFFAOYSA-N Naphthalene Chemical compound C1=CC=CC2=CC=CC=C21 UFWIBTONFRDIAS-UHFFFAOYSA-N 0.000 description 16
- 102000003992 Peroxidases Human genes 0.000 description 8
- 108040007629 peroxidase activity proteins Proteins 0.000 description 8
- KJCVRFUGPWSIIH-UHFFFAOYSA-N 1-naphthol Chemical compound C1=CC=C2C(O)=CC=CC2=C1 KJCVRFUGPWSIIH-UHFFFAOYSA-N 0.000 description 6
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- HUMNYLRZRPPJDN-UHFFFAOYSA-N benzaldehyde Chemical compound O=CC1=CC=CC=C1 HUMNYLRZRPPJDN-UHFFFAOYSA-N 0.000 description 6
- VTWDKFNVVLAELH-UHFFFAOYSA-N 2-methylcyclohexa-2,5-diene-1,4-dione Chemical compound CC1=CC(=O)C=CC1=O VTWDKFNVVLAELH-UHFFFAOYSA-N 0.000 description 4
- JWAZRIHNYRIHIV-UHFFFAOYSA-N 2-naphthol Chemical compound C1=CC=CC2=CC(O)=CC=C21 JWAZRIHNYRIHIV-UHFFFAOYSA-N 0.000 description 4
- 108010015742 Cytochrome P-450 Enzyme System Proteins 0.000 description 4
- 102000002004 Cytochrome P-450 Enzyme System Human genes 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 238000006911 enzymatic reaction Methods 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- QWVGKYWNOKOFNN-UHFFFAOYSA-N o-cresol Chemical compound CC1=CC=CC=C1O QWVGKYWNOKOFNN-UHFFFAOYSA-N 0.000 description 4
- 230000003647 oxidation Effects 0.000 description 4
- 238000007254 oxidation reaction Methods 0.000 description 4
- IWDCLRJOBJJRNH-UHFFFAOYSA-N p-cresol Chemical compound CC1=CC=C(O)C=C1 IWDCLRJOBJJRNH-UHFFFAOYSA-N 0.000 description 4
- 150000002978 peroxides Chemical class 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- WVDDGKGOMKODPV-UHFFFAOYSA-N Benzyl alcohol Chemical compound OCC1=CC=CC=C1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 description 3
- QGJOPFRUJISHPQ-UHFFFAOYSA-N Carbon disulfide Chemical compound S=C=S QGJOPFRUJISHPQ-UHFFFAOYSA-N 0.000 description 3
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 229950011260 betanaphthol Drugs 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- QNGNSVIICDLXHT-UHFFFAOYSA-N para-ethylbenzaldehyde Natural products CCC1=CC=C(C=O)C=C1 QNGNSVIICDLXHT-UHFFFAOYSA-N 0.000 description 3
- 150000002989 phenols Chemical class 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- FYKDNWHPKQOZOT-UHFFFAOYSA-M sodium;dihydrogen phosphate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].OP(O)([O-])=O.OC(=O)CC(O)(C(O)=O)CC(O)=O FYKDNWHPKQOZOT-UHFFFAOYSA-M 0.000 description 3
- 239000007858 starting material Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- OEGPRYNGFWGMMV-UHFFFAOYSA-N (3,4-dimethoxyphenyl)methanol Chemical compound COC1=CC=C(CO)C=C1OC OEGPRYNGFWGMMV-UHFFFAOYSA-N 0.000 description 2
- 241000682855 Aegerita Species 0.000 description 2
- 239000004215 Carbon black (E152) Substances 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 101000610640 Homo sapiens U4/U6 small nuclear ribonucleoprotein Prp3 Proteins 0.000 description 2
- 101001110823 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) 60S ribosomal protein L6-A Proteins 0.000 description 2
- 101000712176 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) 60S ribosomal protein L6-B Proteins 0.000 description 2
- 102100040374 U4/U6 small nuclear ribonucleoprotein Prp3 Human genes 0.000 description 2
- 150000001298 alcohols Chemical class 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical group [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 239000011942 biocatalyst Substances 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 239000007979 citrate buffer Substances 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- -1 hydrocarbons Hydrocarbon Chemical class 0.000 description 2
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 2
- 238000006213 oxygenation reaction Methods 0.000 description 2
- 239000003642 reactive oxygen metabolite Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 150000003431 steroids Chemical class 0.000 description 2
- 241000222532 Agrocybe Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000221198 Basidiomycota Species 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 108010074633 Mixed Function Oxygenases Proteins 0.000 description 1
- 102000008109 Mixed Function Oxygenases Human genes 0.000 description 1
- ACFIXJIJDZMPPO-NNYOXOHSSA-N NADPH Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](OP(O)(O)=O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 ACFIXJIJDZMPPO-NNYOXOHSSA-N 0.000 description 1
- CBENFWSGALASAD-UHFFFAOYSA-N Ozone Chemical compound [O-][O+]=O CBENFWSGALASAD-UHFFFAOYSA-N 0.000 description 1
- 101150053185 P450 gene Proteins 0.000 description 1
- 235000014676 Phragmites communis Nutrition 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 150000001338 aliphatic hydrocarbons Chemical class 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 150000001555 benzenes Chemical class 0.000 description 1
- 235000019445 benzyl alcohol Nutrition 0.000 description 1
- 230000036983 biotransformation Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- ZCDOYSPFYFSLEW-UHFFFAOYSA-N chromate(2-) Chemical class [O-][Cr]([O-])(=O)=O ZCDOYSPFYFSLEW-UHFFFAOYSA-N 0.000 description 1
- HPXRVTGHNJAIIH-UHFFFAOYSA-N cyclohexanol Chemical compound OC1CCCCC1 HPXRVTGHNJAIIH-UHFFFAOYSA-N 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000006193 diazotization reaction Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 229910001882 dioxygen Inorganic materials 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 238000000769 gas chromatography-flame ionisation detection Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 231100001261 hazardous Toxicity 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- TUJKJAMUKRIRHC-UHFFFAOYSA-N hydroxyl Chemical compound [OH] TUJKJAMUKRIRHC-UHFFFAOYSA-N 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 238000007040 multi-step synthesis reaction Methods 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- WVDDGKGOMKODPV-ZQBYOMGUSA-N phenyl(114C)methanol Chemical compound O[14CH2]C1=CC=CC=C1 WVDDGKGOMKODPV-ZQBYOMGUSA-N 0.000 description 1
- 239000007981 phosphate-citrate buffer Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 150000003505 terpenes Chemical class 0.000 description 1
- 235000007586 terpenes Nutrition 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
- C12P7/26—Ketones
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
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- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
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- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
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- Health & Medical Sciences (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
Aufgabe war es, die Ausgangsverbindungen aufwandgering, insbesondere mit geringem Energie- und Chemikalieneinsatz sowie preiswerten Kosubstraten, schadstoffarm, ohne erhöhte Anforderungen an sterile bzw. semisterile Reaktionsführungen und mit möglichst kurzen Inkubationszeiten in wässrigen Medien umzusetzen. DOLLAR A Erfindungsgemäß wird ein Einstufen-Reaktionsverfahren vorgeschlagen, bei dem die Substanzen bzw. Substanzgemische zumindest durch Zugabe und ggf. Zudosierung von neu gefundenen Haloperoxidasen mit Peroxygenaseaktivität und zumindest eines geeigneten Oxidationsmittels in einem wässrigen Milieu zur Reaktion gebracht werden, wobei der aromatische Ring oder die aliphatische Kette jeweils hydroxyliert werden. DOLLAR A Das Verfahren kann in verschiedensten Bereichen der Synthesechemie eingesetzt werden, u. a. zur Herstellung von Pharmazeutika oder Aromastoffen.The task was to reduce the initial compounds, in particular with low energy and chemical use and low-cost cosubstrates, low in pollutants, without increased demands on sterile or semisterile reaction routes and with the shortest possible incubation times in aqueous media. DOLLAR A According to the invention, a one-stage reaction process is proposed, in which the substances or mixtures of substances are brought at least by addition and optionally addition of newly found haloperoxidases with peroxygenase and at least one suitable oxidizing agent in an aqueous environment for reaction, wherein the aromatic ring or each aliphatic chain be hydroxylated. DOLLAR A The method can be used in various fields of synthetic chemistry, u. a. for the production of pharmaceuticals or flavorings.
Description
Die Erfindung betrifft ein Verfahren zur enzymatischen Hydroxylierung nicht-aktivierter Kohlenwasserstoffe, insbesondere nicht-aktivierte Kohlenwasserstoffmoleküle aromatischer Ringe (beispielsweise die selektive Umsetzung von Naphthalin zu 1-Naphthol).The The invention relates to a method for enzymatic hydroxylation non-activated hydrocarbons, especially non-activated hydrocarbons Hydrocarbon molecules aromatic rings (for example, the selective conversion of naphthalene to 1-naphthol).
Das Verfahren kann in verschiedensten Bereichen der Synthesechemie eingesetzt werden, u. a. zur Herstellung von Pharmazeutika, Terpenen, Steroiden oder Fettsäuren.The Process can be used in various fields of synthetic chemistry be, u. a. for the production of pharmaceuticals, terpenes, steroids or fatty acids.
Es ist allgemein bekannt, dass die direkte und selektive Einführung von Sauerstofffunktionen (Oxygenierung) in nicht-aktivierte aromatische oder aliphatische Kohlenwasserstoffe ein Problem für die chemische Synthese darstellt. Die meisten chemischen Hydroxylierungsreaktionen beruhen darauf, dass in Gegenwart eines Elektronendonators sowie molekularen Sauerstoffs (O2) durch einen Katalysator eine reaktive Sauerstoffspezies (z. B. das Hydroxylradikal) erzeugt wird, welches die C-H-Bindungen am nicht-aktivierten Kohlenwasserstoff angreift. Auf Grund der hohen Reaktivität und geringen Selektivität der reaktiven Sauerstoffspezies sind die Ausbeuten bei chemischen Hydroxylierungen, vor allem bei der Herstellung chiraler Produkte, gering. Andere Möglichkeiten der chemischen Hydroxylierung erfordern aufwendige, mehrstufige Syntheseschritte, wie z. B. das Cumolhydroperoxidverfahren oder die Diazotierung, die zur Herstellung von Phenolen aus Benzol und Benzolderivaten eingesetzt werden (Vollhardt und Schore 2000, Organische Chemie, Wiley-VCH).It is well known that the direct and selective introduction of oxygen functions (oxygenation) into unactivated aromatic or aliphatic hydrocarbons presents a problem for chemical synthesis. Most chemical hydroxylation reactions rely on the generation of a reactive oxygen species (eg, the hydroxyl radical) in the presence of an electron donor and molecular oxygen (O 2 ) by a catalyst, which attacks the CH bonds on the non-activated hydrocarbon. Due to the high reactivity and low selectivity of the reactive oxygen species, the yields in chemical hydroxylations, especially in the production of chiral products, are low. Other possibilities of chemical hydroxylation require complex, multi-step synthesis steps, such as. As the Cumolhydroperoxidverfahren or diazotization, which are used for the preparation of phenols from benzene and benzene derivatives (Vollhardt and Schore 2000, Organic Chemistry, Wiley-VCH).
Weiterhin ist bekannt, dass einzelne Hydroxylgruppen enzymatisch mit Hilfe von Monooxygenasen (EC 1.14.13., EC 1.14.14., EC 1.14.99) in nicht-aktivierte Kohlenwasserstoffe eingeführt werden können. Man kennt heute über 100 verschiedene Enzyme, die solche Reaktionen katalysieren. Sie kommen ausschließlich intrazellulär vor und benötigen NAD(P)H oder andere komplexe Elektronendonatoren sowie molekularen Sauerstoff als Kofaktoren. Besonders vielseitig sind die Cytochrom P450-abhängigen Monooxygenasen, die in fast allen Organismen vorkommen und u. a. an der Synthese von Wirkstoffen (z. B. Steroide), der Metabolisierung von Fremdstoffen (z. B. Mono- und Polyaromaten) sowie an der Aktivierung von Mineralölkohlenwasserstoffen (z. B. n-Alkane) beteiligt sind (Rehm and Reed 2000, Biotechnology Vol. 8b: biotransformations II, Wiley-VCH). Die Nutzbarkeit von P450-Enzymen in der Synthesechemie ist allerdings stark eingeschränkt, da sie schwierig zu isolieren, wenig stabil und ihre Kosubstrate – NADH oder NADPH – extrem teuer sind.Farther It is known that single hydroxyl groups are enzymatically using of monooxygenases (EC 1.14.13., EC 1.14.14., EC 1.14.99) in unactivated Hydrocarbons introduced can be. One knows about today 100 different enzymes that catalyze such reactions. they are coming exclusively intracellularly before and need NAD (P) H or other complex electron donors as well as molecular ones Oxygen as cofactors. Particularly versatile are the cytochrome P450-dependent monooxygenases, which occur in almost all organisms and u. a. at the synthesis of Active substances (eg steroids), the metabolisation of foreign substances (For example, mono- and polyaromatics) as well as the activation of mineral oil hydrocarbons (eg. N-alkanes) (Rehm and Reed 2000, Biotechnology Vol. 8b: biotransformations II, Wiley-VCH). The usability of P450 enzymes in synthetic chemistry, however, is severely limited since they are difficult to isolate, little stable and their cosubstrates - NADH or NADPH - extremely expensive are.
Es gibt Versuche, durch gentechnische Manipulation von herkömmlichen P450-Enzymen neue Biokatalysatoren zu entwickeln, die an Stelle von NAD(P)H preiswerte Peroxide als Kosubstrate nutzen (Roberts 1999, The power of evolution: accessing the synthetic potential of P450s. Chemistry & Biology 6: R269-R272). Die so erzeugten Enzyme verfügen jedoch noch nicht über die, für chemische Synthesen notwendige Effizienz und Stabilität.It There are attempts by genetic manipulation of conventional P450 enzymes to develop new biocatalysts in place use inexpensive peroxides as co-substrates of NAD (P) H (Roberts 1999, The power of evolution: accessing the synthetic potential of P450s. Chemistry & Biology 6: R269-R272). However, the enzymes produced in this way do not yet have the for chemical Syntheses necessary efficiency and stability.
Aufgabe der vorliegenden Erfindung ist es, die Prozesse zur enzymatischen Darstellung von hydroxylierten Produkten aus entsprechenden nicht-aktivierten Kohlenwasserstoffen mit möglichst geringem Aufwand verfahrenstechnischer und apparativer Art sowie unter Verwendung kostengünstiger Kosubstrate durchzuführen.task The present invention is the processes for enzymatic Preparation of hydroxylated products from corresponding non-activated Hydrocarbons with as possible little effort procedural and apparatus type and using cheaper To perform cosubstrates.
Die Ausgangsverbindungen sollen insbesondere mit geringem Energie- und Chemikalieneinsatz, schadstoffarm, ohne erhöhte Anforderungen an sterile bzw. semisterile Reaktionsführungen und mit möglichst kurzen Inkubationszeiten umgesetzt werden.The Starting compounds are intended in particular with low energy and Chemical use, low in pollutants, without increased demands on sterile or semisterile reaction guides and with as possible short incubation times are implemented.
Erfindungsgemäß wird ein Verfahren zur enzymatischen Darstellung hydroxylierter Kohlenwasserstoffe aus entsprechenden nicht-aktivierten Ausgangsverbindungen in einem Einstufen-Reaktionsverfahren vorgeschlagen, bei dem die Substanzen bzw. Substanzgemische zumindest durch Zugabe und ggf. Zudosierung von speziellen Haloperoxidasen mit Peroxygenaseaktivität und zumindest eines Oxidationsmittels, wie beispielsweise Wasserstoffperoxid, in einem wässrigen Milieu zur Reaktion gebracht werden, wobei die Oxygenierung nicht-aktivierter C-H-Bindungen erfolgt.According to the invention is a Process for the enzymatic preparation of hydroxylated hydrocarbons from corresponding non-activated starting compounds in one One-stage reaction method proposed in which the substances or Mixtures of substances, at least by addition and, if necessary, addition of special haloperoxidases with peroxygenase activity and at least an oxidizing agent, such as hydrogen peroxide, in an aqueous Be reacted to the environment, wherein the oxygenation is not activated C-H bonds occur.
Das zellfreie, enzymatische Verfahren beruht dabei auf einem neu gefundenen extrazellulären Pilzenzym, wobei diese Haloperoxidase (EC 1.11.1.10) in Gegenwart des Oxidationsmittels in vorzugsweise gepufferten wässrigen Lösungen aromatisch Kohlenwasserstoffe (wie Naphthalin oder Toluol) zu entsprechenden Phenolen unmittelbar, d. h. in dem besagten Einstufen-Reaktionsverfahren, umsetzt. Prinzipiell werden auf die gleiche Weise auch andere Verbindungen (beispielsweise Aliphaten und aliphatische Seitenketten von Aromaten sowie Cycloaliphaten) hydroxyliert.The Cell-free, enzymatic method is based on a newly found extracellular Fungal enzyme, said haloperoxidase (EC 1.11.1.10) in the presence of the oxidizing agent in preferably buffered aqueous solutions aromatic hydrocarbons (such as naphthalene or toluene) to corresponding Phenols directly, d. H. in said one-step reaction process, implements. In principle, in the same way, other compounds (For example, aliphatic and aliphatic side chains of aromatics and cycloaliphatic) hydroxylated.
Das neu gefundene und zunächst als Arylalkohol-Arylaldehyd-Peroxidase (AAP) bezeichnete Enzym, das inzwischen jedoch als eine besondere Haloperoxidase mit Peroxygenasefunktion erkannt wurde, wird vorzugsweise von Basidiomyceten aus der Familie Bolbitiaceae (z. B. Agrocybe sp.) gebildet und zeichnet sich durch besondere katalytische Eigenschaften aus, die keine der bisher bekannten Peroxidasen oder P450-Enzyme besitzt.The newly discovered enzyme, initially known as aryl alcohol-aryl aldehyde peroxidase (AAP), which has since been recognized as a particular peroxygenase-functional haloperoxidase, is preferably produced and characterized by basidiomycetes of the family Bolbitiaceae (eg Agrocybe sp.) by special catalytic properties, none of the previously known Pe has roxidases or P450 enzymes.
Die Reaktionen mit dieser Agrocybe-aegerita-Peroxidase (AaP) sind umweltfreundlich (d. h. sie erfordern keine aggressiven und umweltbelastenden Chemikalien). So sind Oxidationsmittel nur in katalytischen Mengen zur Gewährleistung der Peroxidaseaktivität, nicht aber zur direkten Umsetzung der Ausgangsverbindungen erforderlich. Die chemische Oxidation von Alkoholen und Aldehyden erfordert hingegen äquimolare Mengen an umweltgefährdenden Oxidationsmitteln (Peroxide, Ozon, Permanganate, Chromate).The Reactions with this agrocyte aegerita peroxidase (AaP) are environmentally friendly (ie they do not require aggressive and polluting chemicals). Thus, oxidizing agents are only in catalytic amounts to ensure the peroxidase activity, but not required for the direct conversion of the starting compounds. The chemical oxidation of alcohols and aldehydes, however, requires equimolar Amounts of environmentally hazardous oxidants (Peroxides, ozone, permanganate, chromates).
Des weiteren laufen die rein chemischen Hydroxylierungen Oxidationen nur in Gegenwart geeigneter Lösungsmittel (Methanol, Dimethylsulfoxid, Aceton) ab und erfolgen in wässrigen, lediglich gepufferten Reaktionslösungen nicht mit befriedigender Ausbeute. Während chemische Umsetzungen in der Regel eine Prozessführung bei höheren Temperaturen (Heizquelle) und/oder höheren Drücken benötigen, zeigt sich, dass die erfindungsgemäße AaP-katalysierte Umsetzung jeweils bei Raumtemperatur realisiert werden kann und keine speziellen Apparaturen (wie Druckreaktoren o. ä.) oder apparative Aufwände erfordert.Of further, the purely chemical hydroxylations undergo oxidation only in the presence of suitable solvents (Methanol, dimethyl sulfoxide, acetone) and take place in aqueous, only buffered reaction solutions not with satisfactory yield. During chemical reactions usually a litigation at higher Temperatures (heat source) and / or higher pressures, it turns out that the AaP-catalyzed according to the invention Implementation can be implemented in each case at room temperature and no special equipment (like pressure reactors or similar) or equipment expenses requires.
Die Vorteile der zellfreien, enzymatischen AaP-Umsetzungen gegenüber einer ebenfalls möglichen Hydroxylierung von nicht-aktivierten Kohlenwasserstoffen durch ganze Zellen (Bakterien, Hefen, Schimmelpilze, tierische oder pflanzliche Zellkulturen) bestehen in den relativ kurzen Reaktionszeiten, die keine sterile oder semisterile Reaktionsführung erforderlich machen. Im Vergleich zu P450-abhängigen Enzymen bestehen Vorteile bezüglich der Kosubstrate (preiswerte und stabile Peroxide anstelle von NAD(P)H) sowie bezüglich der Enzymgewinnung und -stabilität (extrazelluläre Enzyme anstelle von intrazellulären, z. T. membrangebundenen Enzymen).The Advantages of cell-free, enzymatic AaP reactions over one also possible Hydroxylation of non-activated hydrocarbons through whole Cells (bacteria, yeasts, molds, animal or vegetable Cell cultures) exist in the relatively short reaction times, the do not require sterile or semi-sterile reaction. Compared to P450-dependent Enzymes have advantages regarding of the cosubstrates (inexpensive and stable peroxides instead of NAD (P) H) as well as regarding Enzyme production and stability (Extracellular Enzymes instead of intracellular, z. T. membrane-bound enzymes).
Mit den AaP-katalysierten Reaktionen ist erstmals möglich, nicht-aktivierte Kohlenwasserstoffe mit Hilfe eines einzelnen, extrazellulären Biokatalysators, der lediglich ein Peroxid als Kofaktor benötigt, in einem einstufigen Prozess zu den entsprechenden Phenolen oder Alkoholen zu oxidieren.With The AaP-catalyzed reactions is possible for the first time, non-activated hydrocarbons with the help of a single, extracellular biocatalyst that only requires a peroxide as a cofactor, in a one-step process to the corresponding phenols or To oxidize alcohols.
Die Erfindung soll nachstehend anhand von in der Zeichnung dargestellten Ausführungsbeispielen näher erläutert werden, wobei die Erfindung nicht auf die behandelten Aromaten an sich beschränkt sein soll.The Invention will be described below with reference to the drawing embodiments be explained in more detail, the invention is not limited to the aromatics treated per se should.
Es zeigen:It demonstrate:
Ausführungsbeispiel 1:Embodiment 1
200
nmol Naphtalin wurden in Natriumphosphat-Citrat-Puffer (50 mM, pH
7) zusammen mit 2 mM Wasserstoffperoxid und 0,1 U Agrocybe-aegerita-Peroxidase
(0,1 Unit bezüglich
der Oxidation von Veratrylalkohol) in einem Gesamtvolumen von 1
ml bei 24 °C
in einem offenem Glasgefäß kurz gerührt und
acht Minuten stehen gelassen. Dann wurden 20 μl des Versuchsansatzes entnommen
und mittels High Performance Liquid Chromatography (HPLC) vermessen
[Säule:
LiChrospher® RP18
5 μm 125/4 (Firma
Merck Darmstadt), Trennbedingungen: Gradient 20–80 % Acetonitril (0 bis 5
min, 20 %; 20 min 80 %, 20-25 min 80 %) in 0.05 % Phosphorsäure, konstante
Flußrate
1 ml/min] (vgl.
Ausführungsbeispiel 2:Embodiment 2:
200
nmol Toluol wurden in Natriumphosphat-Citrat-Puffer (50 mM, pH 7)
zusammen mit 2 mM H2O2 und
0,1 Unit Agrocybe-aegerita-Peroxidase in einem Gesamtvolumen von
1 ml bei 24 °C
in einem offenem Glasgefäß kurz gerührt und
acht min stehen gelassen. Dann wurden 20 μl des Versuchsansatzes entnommen
und mittels High Performance Liquid Chromatography (HPLC) vermessen
[Säule:
LiChrospher® RP18
5 μm 125/4
(Firma Merck Darmstadt), Trennbedingungen: Gradient 20–80 % Acetonitril
(0 bis 5 min, 20 %; 20 min 80 %, 20–25 min 80 %) in 0.05 % Phosphorsäure, konstante
Flußrate
1 ml/min] (vgl.
Ausführungsbeispiel 3:Embodiment 3
200 nmol Cyclohexan wurden in Natriumphosphat-Citrat-Puffer (50 mM, pH 7) und 10 vol.% Ethanol zusammen mit 10 mM Glukose, 0,1 Unit Glukose-Oxidase (Sigma) und 0,1 Unit Agrocybe-aegerita-Peroxidase in einem Gesamtvolumen von 1 ml bei 24 °C in einem offenem Glasgefäß 30 min gerührt. Dann wurde der gesamte Versuchsansatz mit 3 ml Schwefelkohlenstoff (CS2) extrahiert und der Extrakt mittels Gaschromatographie (GC/FID) vermessen [Säule: 60 m × 0.32 mm DB-1 capillary column, 1 m df]. Im Verlauf der enzymatischen Reaktion nahm die Cyclohexankonzentration um 96 nmol ab und Cyclohexanol (47,2 nmol) konnte als Produkt identifiziert werden.200 nmol of cyclohexane were dissolved in sodium phosphate-citrate buffer (50 mM, pH 7) and 10 vol.% Ethanol together with 10 mM glucose, 0.1 unit glucose oxidase (Sigma) and 0.1 unit agrocybe aegerita peroxidase a total volume of 1 ml at 24 ° C in an open glass vessel for 30 min. Then, the entire assay was extracted with 3 ml of carbon disulfide (CS 2 ) and the extract was measured by gas chromatography (GC / FID) [column: 60 m × 0.32 mm DB-1 capillary column, 1 m df]. In the course of the enzymatic reaction, the cyclohexane concentration decreased by 96 nmol and cyclohexanol (47.2 nmol) could be identified as a product.
Claims (10)
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE200410047774 DE102004047774A1 (en) | 2004-09-28 | 2004-09-28 | Process for the enzymatic hydroxylation of non-activated hydrocarbons |
| DE112005003060T DE112005003060A5 (en) | 2004-09-28 | 2005-09-27 | Process for the enzymatic hydroxylation of non-activated hydrocarbons |
| PCT/DE2005/001734 WO2006034702A1 (en) | 2004-09-28 | 2005-09-27 | Method for the enzymatic hydroxylation of non-activated hydrocarbons |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE200410047774 DE102004047774A1 (en) | 2004-09-28 | 2004-09-28 | Process for the enzymatic hydroxylation of non-activated hydrocarbons |
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| DE102004047774A1 true DE102004047774A1 (en) | 2006-03-30 |
Family
ID=35569499
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| DE200410047774 Withdrawn DE102004047774A1 (en) | 2004-09-28 | 2004-09-28 | Process for the enzymatic hydroxylation of non-activated hydrocarbons |
| DE112005003060T Withdrawn DE112005003060A5 (en) | 2004-09-28 | 2005-09-27 | Process for the enzymatic hydroxylation of non-activated hydrocarbons |
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| DE112005003060T Withdrawn DE112005003060A5 (en) | 2004-09-28 | 2005-09-27 | Process for the enzymatic hydroxylation of non-activated hydrocarbons |
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| DE (2) | DE102004047774A1 (en) |
| WO (1) | WO2006034702A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE102006010910B3 (en) * | 2006-03-09 | 2007-10-18 | Dechema Gesellschaft Für Chemische Technik Und Biotechnologie E.V. | Process for the conversion of terpenes |
| WO2022171606A2 (en) | 2021-02-09 | 2022-08-18 | F. Hoffmann-La Roche Ag | Methods for base-level detection of methylation in nucleic acids |
Families Citing this family (25)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE102006041493A1 (en) * | 2006-09-05 | 2008-03-06 | Clariant Specialty Fine Chemicals (Deutschland) Gmbh | Process for the stereoselective hydroxylation of alkylaromatics |
| DE102007016139A1 (en) | 2007-03-30 | 2008-10-02 | Jenabios Gmbh | Method for regioselective oxygenation of N-heterocycles |
| US9222109B2 (en) | 2010-03-28 | 2015-12-29 | Novozymes A/S | Enzymatic hydroxylation of aliphatic hydrocarbon |
| WO2013004639A2 (en) | 2011-07-07 | 2013-01-10 | Novozymes A/S | Enzymatic preparation of diols |
| CN103732740B (en) | 2011-08-10 | 2018-07-31 | 诺维信公司 | Polypeptide with peroxidase activity and polynucleotide encoding the polypeptide |
| WO2013021060A1 (en) | 2011-08-10 | 2013-02-14 | Novozymes A/S | Polypeptides having peroxygenase activity and polynucleotides encoding same |
| WO2013021065A1 (en) | 2011-08-10 | 2013-02-14 | Novozymes A/S | Polypeptides having peroxygenase activity and polynucleotides encoding same |
| WO2013021064A1 (en) | 2011-08-10 | 2013-02-14 | Novozymes A/S | Polypeptides having peroxygenase activity and polynucleotides encoding same |
| WO2013021062A1 (en) | 2011-08-10 | 2013-02-14 | Novozymes A/S | Polypeptides having peroxygenase activity and polynucleotides encoding same |
| WO2013021061A1 (en) | 2011-08-10 | 2013-02-14 | Novozymes A/S | Polypeptides having peroxygenase activity and polynucleotides encoding same |
| WO2013021059A1 (en) | 2011-08-10 | 2013-02-14 | Novozymes A/S | Polypeptides having peroxygenase activity and polynucleotides encoding same |
| WO2013079533A1 (en) | 2011-12-02 | 2013-06-06 | Novozymes A/S | Polypeptides having peroxygenase activity and polynucleotides encoding same |
| WO2013079531A2 (en) | 2011-12-02 | 2013-06-06 | Novozymes A/S | Polypeptides having peroxygenase activity and polynucleotides encoding same |
| WO2013144105A1 (en) | 2012-03-31 | 2013-10-03 | Novozymes A/S | Epoxidation using peroxygenase |
| US9534208B2 (en) | 2012-10-12 | 2017-01-03 | Novozymes A/S | Polypeptides having peroxygenase activity |
| US9458435B2 (en) | 2012-10-12 | 2016-10-04 | Novozymes A/S | Polypeptides having peroxygenase activity |
| EP2906688B1 (en) | 2012-10-12 | 2018-08-29 | Novozymes A/S | Polypeptides having peroxygenase activity |
| CN104704114A (en) | 2012-10-12 | 2015-06-10 | 诺维信公司 | Polypeptides having peroxygenase activity |
| US9453207B2 (en) | 2012-10-12 | 2016-09-27 | Novozymes A/S | Polypeptides having peroxygenase activity |
| WO2014056927A2 (en) | 2012-10-12 | 2014-04-17 | Novozymes A/S | Polypeptides having peroxygenase activity |
| CN104704116B (en) | 2012-10-12 | 2018-09-28 | 诺维信公司 | Polypeptides with peroxygenase activity |
| CN104718288B (en) | 2012-10-12 | 2018-11-16 | 诺维信公司 | Polypeptides with peroxygenase activity |
| WO2015079064A2 (en) | 2013-11-29 | 2015-06-04 | Novozymes A/S | Peroxygenase variants |
| US20240010996A1 (en) | 2020-09-23 | 2024-01-11 | Gecco Biotech B.V. | Bacterial unspecific peroxygenases (BUPO's) and methods and uses thereof |
| CN113999879B (en) * | 2022-01-04 | 2022-04-08 | 中国科学院天津工业生物技术研究所 | A kind of method for peroxidase catalyzed oxidation of aromatic hydrocarbons and derivatives thereof |
-
2004
- 2004-09-28 DE DE200410047774 patent/DE102004047774A1/en not_active Withdrawn
-
2005
- 2005-09-27 WO PCT/DE2005/001734 patent/WO2006034702A1/en not_active Ceased
- 2005-09-27 DE DE112005003060T patent/DE112005003060A5/en not_active Withdrawn
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE102006010910B3 (en) * | 2006-03-09 | 2007-10-18 | Dechema Gesellschaft Für Chemische Technik Und Biotechnologie E.V. | Process for the conversion of terpenes |
| WO2022171606A2 (en) | 2021-02-09 | 2022-08-18 | F. Hoffmann-La Roche Ag | Methods for base-level detection of methylation in nucleic acids |
Also Published As
| Publication number | Publication date |
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| DE112005003060A5 (en) | 2007-09-13 |
| WO2006034702A1 (en) | 2006-04-06 |
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