WO2008028526A1 - Process for the preparation of optically active 1-arylalcohols using the haloperoxidase of agrocybe aegerita - Google Patents
Process for the preparation of optically active 1-arylalcohols using the haloperoxidase of agrocybe aegerita Download PDFInfo
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- the invention relates to a process for the enzymatic hydroxylation of alkylaromatics, to the corresponding aromatically substituted chiral alkanols.
- biocatalysts are the cytochrome P450-dependent monooxygenases, which are found in almost all organisms. You are u.a. involved in the detoxification of toxic compounds in the human liver or in the synthesis of secondary metabolites (e.g., steroids used as membrane constituents, vitamins, hormones, bile acids or cardiac active).
- secondary metabolites e.g., steroids used as membrane constituents, vitamins, hormones, bile acids or cardiac active
- Whole cell catalysis uses whole organisms (bacteria, yeasts, molds) instead of isolated hydroxylating enzymes. In this way it is possible, in the sense of biotransformations to a series of substrates hydroxylate. Examples are the nicotinic acid hydroxylation by Achromobacter xylosooxidans, the hydroxylation of biphenyl derivatives by E. coli or the steroid transformation with the aid of molds of the genus Rhizopus (Liese et al., 2000, Industrial Biotransformations, Wiley-VCH, Weinheim). Disadvantages of catalysis with whole cells exist in the time and cost intensive
- the object of the present invention is to carry out a process for the preparation of chiral alcohols from the corresponding precursors with the least possible procedural and equipment expense while simultaneously using inexpensive cosubstrates.
- the reaction of the starting compounds should take place in the shortest possible incubation times without increased requirements for sterile or semisterile reaction conditions.
- the reaction products are to be isolated with the least possible effort and a complex separation of enantiomers should be eliminated.
- the present process solves this problem and relates to a process for the enzymatic, stereoselective hydroxylation of alkylaromatics to chiral aromatic-substituted alkanols by reacting alkylaromatics with an agrocyte aegerita peroxidase (AaP) in the presence of at least one oxidation agent in a one-step reaction process.
- AaP agrocyte aegerita peroxidase
- the starting compounds are in this case reacted with the enzyme designated Agrocybe- aegerita peroxidase (AaP), which has a particularly high peroxygenase activity, and at least one oxidizing agent, wherein the stereoselective oxygenation of certain C-H bonds takes place.
- AaP Agrocybe- aegerita peroxidase
- the oxidizing agent according to the invention preferably H 2 O 2
- organic peroxides or hydroperoxides such as tert-butyl hydroperoxide, air or oxygen used.
- expensive electron donors, such as NAD (P) H can at dispensed with the present process (concentration of the oxidizing agent: from 0.01 to 10 mmol / L, preferably 0.1 to 2 mmol / LH 2 O 2 ).
- the reaction mixture can additionally accelerate the reaction of the starting compound with the AaP enzyme H 2 O 2 -generating enzymes, in particular oxidases such as glucose oxidase or aryl alcohol oxidase and their substrates, eg. As glucose or benzyl alcohol may be added.
- H 2 O 2 -generating enzymes in particular oxidases such as glucose oxidase or aryl alcohol oxidase and their substrates, eg. As glucose or benzyl alcohol may be added.
- Oxidizing agent e.g., peroxides
- Oxidizing agent especially in buffered aqueous solutions, oxidizes aliphatic side chains of aromatic compounds (e.g., ethylbenzene) to the corresponding chiral 1-phenylalkanols, thereby achieving high enantiomeric purity (> 95% ee).
- the enzyme used is a special haloperoxidase with peroxygenase function. It is produced by basidiomycetes of the families Bolbitiaceae (e.g., Agrocybe spp.) And Coprinaceae (e.g., Coprinus spp.) And is characterized by particular catalytic properties that clearly distinguish it from previously described peroxidases and cytochrome P450 enzymes. Enzyme production takes place in liquid culture, preferably in bioreactors and nitrogen-rich media (R. Ullrich, 2005, dissertation, IHI Zittau, M. Kluge, 2006, diploma thesis, IHI Zittau).
- the reactions catalyzed by the enzyme termed Agrocybe aegerita peroxidase (AaP) do not require highly concentrated aggressive and environmentally hazardous reagents, and product recovery can dispense with chemical and time-consuming purification steps to separate the racemic mixtures.
- the enzyme is used in a concentration of 0.02 U / mL to 10 U / mL AaP, in particular 0.09 to 8 U / mL AaP. This makes the reaction process shown particularly environmentally friendly.
- Another advantage over purely chemical syntheses is the process control due to the AaP-catalyzed reaction according to the invention at room temperature and normal atmospheric pressure.
- the process is carried out in aqueous, buffered solutions.
- buffers based on organic acids preferably citric acid, and phosphates, preferably potassium hydrogen phosphates, may be added to the reaction mixture (buffer concentration 5 mmol / L to 500 mmol / L, preferably 20-100 mmol / L).
- phosphates preferably potassium hydrogen phosphates
- organic solvents may be added to the reaction mixture.
- Solvents which can be used according to the invention are, for example, protic solvents, for example ethers (inter alia diisopropyl ether), methanol, acetone, acetonitrile, DMF (N, N-dimethylformamide) or DMSO (dimethyl sulfoxide).
- the reaction is carried out in a range of 5 to 60 0 C, preferably at 20 to 30 0 C.
- the reaction times are usually in the range of 1 to 90 minutes, in particular in the range of 5 to 30 minutes.
- the achieved ee values are in the range of 80 to 99.9%, preferably greater than 95% ee; the yields are between 30 and 90%.
- the advantages of the AaP-catalyzed reactions to catalysis with conventional cytochrome P450 enzymes (monooxygenases) are: i) the high enantiomeric purity of the reaction products, ii) the use of inexpensive peroxides instead of expensive electron donors [NAD (P) H], iii) in the independence of the hydroxylating enzyme of
- AaP over conventional peroxidases (including chloroperoxidase, horseradish peroxidase, lignin peroxidase) are: i) a broader substrate spectrum, ii) a more pronounced peroxygenase activity, iii) an overall higher stability.
- Fig. 1 General scheme
- Fig. 2 Schematic diagram according to the embodiment
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Abstract
Description
Beschreibung description
VERFAHREN ZUR HERSTELLUNG VON OPTISCH AKTIVEN 1-ARYLALKOHOLEN UNTER VERWENDUNG DER HALOPEROXIDASE VON AGROCYBE AEGERITAPROCESS FOR THE PREPARATION OF OPTICALLY ACTIVE 1-ARYL ALCOHOLS USING THE HALOPEROXIDASE OF AGROCYBE AEGERITA
Die Erfindung betrifft ein Verfahren zur enzymatischen Hydroxylierung von Alkylaromaten, zu den entsprechenden aromatisch substituierten chiralen Alkanolen.The invention relates to a process for the enzymatic hydroxylation of alkylaromatics, to the corresponding aromatically substituted chiral alkanols.
Es ist allgemein bekannt, dass eine direkte stereoselektive Einführung von Sauerstoff-Funktionen (Oxygenierung) in organische Moleküle ein Problem in der chemischen Synthese darstellt. Chemische Hydroxylierungsreaktionen beruhen zumeist darauf, dass in Gegenwart von Elektronendonatoren und molekularem Sauerstoff (O2) durch einen Katalysator eine reaktive Sauerstoffspezies generiert wird, welche die C-H-Bindung am α-Kohlenstoffatom angreift. Diese hoch reaktiven Sauerstoffspezies sind nur wenig regio- und nicht stereoselektiv. Aus diesem Grund sind die Ausbeuten bei chemischen Hydroxylierungen, insbesondere bei der Synthese chiraler, d.h. enantiomerenreiner Produkte relativ gering (Campestrini & Tonellato, 2005, Selective Catalytic Oxidations in Supercritical Carbon Dioxide. Current Organic Chemistry 9: 31-47; Warner et al., 2004, Green Chemistry. Environmental Impact Assessment Review 24: 775-799; Krause, N., 1995, Metallorganische Chemie. Spektrum Akademischer Verlag Heidelberg, Berlin Oxford).It is well known that a direct stereoselective introduction of oxygen functions (oxygenation) into organic molecules is a problem in chemical synthesis. Chemical hydroxylation reactions are usually based on the fact that in the presence of electron donors and molecular oxygen (O 2 ) by a catalyst, a reactive oxygen species is generated, which attacks the CH bond at the α-carbon atom. These highly reactive oxygen species are only slightly regio- and not stereoselective. For this reason, yields in chemical hydroxylations, especially in the synthesis of chiral, ie, enantiomerically pure products, are relatively low (Campestrini & Tonellato, 2005, Selective Catalytic Oxidations in Supercritical Carbon Dioxide, Current Organic Chemistry 9: 31-47, Warner et al. 2004, Green Chemistry, Environmental Impact Assessment Review 24: 775-799, Krause, N., 1995, Organometallic Chemistry, Spectrum Academic Publishing Heidelberg, Berlin Oxford).
Andere Wege der chemischen Hydroxylierung erfordern aufwändige, mehrstufige Reaktionsschritte. Für die Gewinnung enantiomerenreiner Produkte ((R)- oder (S)-) sind zudem aufwändige Isolierungs- und Reinigungsschritte nach der eigentlichen Reaktion notwendig. Die Reinheit von Enantiomeren ist vor allem in der Pharmazeutischen Industrie eine Grundvoraussetzung für die Wirksamkeit von Medikamenten und die Vermeidung von Nebenwirkungen (Mitsudome et al., 2005, Liquid-phase Epoxidation of Alkenes Using Molecular Oxygen Catalyzed byOther routes of chemical hydroxylation require elaborate, multi-step reaction steps. For the recovery of enantiomerically pure products ((R) - or (S) -) also elaborate isolation and purification steps after the actual reaction are necessary. The purity of enantiomers is a prerequisite, especially in the pharmaceutical industry, for drug efficacy and avoidance of side effects (Mitsudome et al., 2005, Liquid-phase Epoxidation of Alkenes Using Molecular Oxygen Catalyzed by
Vanadium Cation-exchanged Montmorillonite. Chemistry Letters 34: 1626-1631 ; Festel et al., 2004, Einfluss der Biotechnologieauf Produktionsverfahren in der Chemieindustrie. Chemie Ingenieur Technik 76: 307-312; Breuer et al., 2004, Industrielle Verfahren zur Herstellung optisch aktiver Zwischenprodukte. Angewandte Chemie 116: 806-843).Vanadium cation-exchanged montmorillonite. Chemistry Letters 34: 1626-1631; Festel et al., 2004, Biotechnological influence on production processes in the chemical industry. Chemical Engineer Technology 76: 307-312; Breuer et al., 2004, Industrial processes for the preparation of optically active intermediates. Angewandte Chemie 116: 806-843).
Außerdem ist bekannt, dass einzelne Hydroxylgruppen enzymatisch und stereoselektiv mit Hilfe von Monooxygenasen (E. C. 1.14.) in Kohlenwasserstoffe eingeführt werden können. Aktuell sind mehr als 100 solcher Enzyme bekannt, die jedoch nur zu einem geringen Teil stereoselektiv agieren. Es handelt sich dabei ausschließlich um intrazelluläre Enzyme, die NAD(P)H oder andere komplexe Elektronendonatoren, Hilfsproteine (Flavinreduktasen, Fer-ridoxine) sowie molekularen Sauerstoff als Cofaktoren benötigen (Buchholz et al., 2005,In addition, it is known that individual hydroxyl groups can be introduced into hydrocarbons enzymatically and stereoselectively with the aid of monooxygenases (E.C. 1.14.). Currently, more than 100 such enzymes are known, but only a small part of them act stereoselectively. These are exclusively intracellular enzymes which require NAD (P) H or other complex electron donors, auxiliary proteins (flavin reductases, ferridoxins) as well as molecular oxygen as cofactors (Buchholz et al., 2005,
Biocatalysts and Enzyme Technology. Wiley-VCH, Weinheim; Aehle (Ed.), 2004, Enzymes in Industry. Wiley-VCH, Weinheim; Holland, 1998, Hydroxylation and Dihydroxylation. In: Biotechnology. Vol. 8a: Biotransformations I, Wiley-VCH, Weinheim).Biocatalysts and Enzyme Technology. Wiley-VCH, Weinheim; Aehle (Ed.), 2004, Enzymes in Industry. Wiley-VCH, Weinheim; Holland, 1998, Hydroxylation and Dihydroxylation. In: Biotechnology. Vol. 8a: Biotransformations I, Wiley-VCH, Weinheim).
Als besonders vielseitige Biokatalysatoren sind die Cytochrom-P450-abhängigen Monooxygenasen bekannt, die in nahezu allen Organismen vorkommen. Sie sind u.a. an der Entgiftung von toxischen Verbindungen in der menschlichen Leber oder an der Synthese von Sekundärmetaboliten beteiligt (z.B. Steroide, die als Membranbestandteile, Vitamine, Hormone, Gallensäuren oder herzaktiveParticularly versatile biocatalysts are the cytochrome P450-dependent monooxygenases, which are found in almost all organisms. You are u.a. involved in the detoxification of toxic compounds in the human liver or in the synthesis of secondary metabolites (e.g., steroids used as membrane constituents, vitamins, hormones, bile acids or cardiac active
Alkaloide fungieren). Derzeit ist die Nutzung dieser Enzyme jedoch noch stark eingeschränkt, da sie schwierig zu isolieren, wenig stabil und die benötigten Kosubstrate z.T. teuer sind. Aus diesem Grund versucht man mittels gezielter gerichteter Evolution bekannte P450 Enzyme (z.B. P450cam) so zu verändern, dass sie anstatt der komplexen Kosubstrate einfache Peroxide nutzen (Roberts, 1999, The power of evolution: accessing the synthetic potential of P450s. Chemistry & Biology 6: R269-R272). Die auf diesem Weg erzeugten Enzyme weisen allerdings noch nicht die gewünschte Effizienz und Stabilität für chemische Synthesen auf.Alkaloids act). Currently, however, the use of these enzymes is still severely limited because they are difficult to isolate, less stable and the required Kosubstrate are partly expensive. For this reason, it is attempted to modify known P450 enzymes (eg P450 ca m) by targeted directed evolution in such a way that they use simple peroxides instead of the complex cosubstrates (Roberts, 1999, The power of evolution: accessing the synthetic potential of P450s. Biology 6: R269-R272). However, the enzymes generated in this way do not yet have the desired efficiency and stability for chemical syntheses.
Die Ganzzellkatalyse verwendet anstelle der isolierten hydroxylierenden Enzyme ganze Organismen (Bakterien, Hefen, Schimmelpilze). Auf diese Weise ist es möglich, im Sinne von Biotransformationen eine Reihe von Substraten zu hydroxylieren. Beispiele sind die Nikotinsäurehydroxylierung durch Achromobacter xylosooxidans, die Hydroxylierung von Biphenylderivaten durch E. coli oder die Steroidtransformation mit Hilfe von Schimmelpilzen der Gattung Rhizopus (Liese et al., 2000, Industrial Biotransformations, Wiley-VCH, Weinheim). Nachteile der Katalyse mit ganzen Zellen bestehen in den zeit- und kostenintensivenWhole cell catalysis uses whole organisms (bacteria, yeasts, molds) instead of isolated hydroxylating enzymes. In this way it is possible, in the sense of biotransformations to a series of substrates hydroxylate. Examples are the nicotinic acid hydroxylation by Achromobacter xylosooxidans, the hydroxylation of biphenyl derivatives by E. coli or the steroid transformation with the aid of molds of the genus Rhizopus (Liese et al., 2000, Industrial Biotransformations, Wiley-VCH, Weinheim). Disadvantages of catalysis with whole cells exist in the time and cost intensive
Fermentationen, der möglichen Degeneration der Produktionsstämme und der komplizierten Aufarbeitung der komplexen Fermentationsbrühen.Fermentations, the possible degeneration of the production strains and the complicated processing of the complex fermentation broths.
Die Aufgabe der vorliegenden Erfindung ist es, einen Prozess zur Darstellung von chiralen Alkoholen aus den entsprechenden Vorstufen mit möglichst geringem verfahrenstechnischen und apparativem Aufwand bei gleichzeitigem Einsatz kostengünstiger Kosubstrate durchzuführen. Die Umsetzung der Ausgangsverbindungen soll in möglichst kurzen Inkubationszeiten ohne erhöhte Anforderungen an sterile bzw. semisterile Reaktionsbedingungen erfolgen. Die Reaktionsprodukte sind dabei mit möglichst geringem Aufwand zu isolieren und eine aufwändige Enantiomerentrennung soll entfallen.The object of the present invention is to carry out a process for the preparation of chiral alcohols from the corresponding precursors with the least possible procedural and equipment expense while simultaneously using inexpensive cosubstrates. The reaction of the starting compounds should take place in the shortest possible incubation times without increased requirements for sterile or semisterile reaction conditions. The reaction products are to be isolated with the least possible effort and a complex separation of enantiomers should be eliminated.
Das vorliegende Verfahren löst diese Aufgabe und betrifft ein Verfahren zur enzymatischen, stereoselektiven Hydroxylierung von Alkylaromaten zu chiralen aromatisch substituierten Alkanolen durch Umsetzung von Alkylaromaten mit einer Agrocybe-aegerita-Peroxidase (AaP) in Gegenwart mindestens eines Oxidations- mittels in einem Einstufen-Reaktionsverfahren.The present process solves this problem and relates to a process for the enzymatic, stereoselective hydroxylation of alkylaromatics to chiral aromatic-substituted alkanols by reacting alkylaromatics with an agrocyte aegerita peroxidase (AaP) in the presence of at least one oxidation agent in a one-step reaction process.
Die Ausgangsverbindungen (Alkylaromaten) werden dabei mit dem Agrocybe- aegerita-Peroxidase (AaP) bezeichneten Enzym, das eine besonders hohe Peroxygenaseaktivität besitzt, und zumindest eines Oxidationsmittels, zur Reaktion gebracht, wobei die stereoselektive Oxygenierung bestimmter C-H- Bindungen erfolgt.The starting compounds (alkylaromatics) are in this case reacted with the enzyme designated Agrocybe- aegerita peroxidase (AaP), which has a particularly high peroxygenase activity, and at least one oxidizing agent, wherein the stereoselective oxygenation of certain C-H bonds takes place.
Als Oxidationsmittel werden erfindungsgemäß vorzugsweise H2O2, organische Peroxide oder Hydroperoxide, wie z.B. tert.-Butylhydroperoxid, Luft oder Sauerstoff verwendet. Auf teure Elektronendonatoren, wie z.B. NAD(P)H kann bei dem vorliegenden Verfahren verzichtet werden (Konzentration des Oxidationsmittel: von 0,01 bis 10 mmol/L, bevorzugt 0,1 bis 2 mmol/L H2O2).As the oxidizing agent according to the invention preferably H 2 O 2 , organic peroxides or hydroperoxides, such as tert-butyl hydroperoxide, air or oxygen used. On expensive electron donors, such as NAD (P) H can at dispensed with the present process (concentration of the oxidizing agent: from 0.01 to 10 mmol / L, preferably 0.1 to 2 mmol / LH 2 O 2 ).
Dem Reaktionsgemisch können zur weiteren Beschleunigung der Umsetzung der Ausgangsverbindung mit dem AaP-Enzym zusätzlich H2O2-generierende Enzyme, insbesondere Oxidasen, wie z.B. Glucose-Oxidase oder Arylalkohol-Oxidase sowie deren Substrate, z. B. Glucose oder Benzylalkohol, zugesetzt werden. Die Grundlage des erfindungsgemäßen enzymatischen, zellfreien Verfahrens ist eine neuartige extrazelluläre Haloperoxidase, die über P450-ähnliche Katalyseeigenschaften verfügt und in Gegenwart eines geeignetenThe reaction mixture can additionally accelerate the reaction of the starting compound with the AaP enzyme H 2 O 2 -generating enzymes, in particular oxidases such as glucose oxidase or aryl alcohol oxidase and their substrates, eg. As glucose or benzyl alcohol may be added. The basis of the enzymatic, cell-free method according to the invention is a novel extracellular haloperoxidase which has P450-like catalytic properties and in the presence of a suitable
Oxidationsmittels (z.B. Peroxiden), insbesondere in gepufferten wässrigen Lösungen aliphatische Seitenketten von aromatischen Verbindungen (z.B. Ethylbenzol) zu den entsprechenden chiralen 1-Phenylalkanolen oxidiert, und dabei eine hohe Enantiomerenreinheit erreicht (> 95% ee).Oxidizing agent (e.g., peroxides), especially in buffered aqueous solutions, oxidizes aliphatic side chains of aromatic compounds (e.g., ethylbenzene) to the corresponding chiral 1-phenylalkanols, thereby achieving high enantiomeric purity (> 95% ee).
Bei dem eingesetzten Enzym handelt es sich um eine besondere Haloperoxidase mit Peroxygenasefunktion. Es wird von Basidiomyceten der Familien Bolbitiaceae (z.B. Agrocybe spp.) und Coprinaceae (z.B. Coprinus spp.) gebildet und zeichnet sich durch besondere katalytische Eigenschaften aus, die es von bisher beschriebenen Peroxidasen und Cytochrom-P450-Enzymen deutlich unterscheidet. Die Enzymherstellung erfolgt in Flüssigkultur, vorzugsweise in Bioreaktoren und stickstoffreichen Medien (R. Ullrich, 2005, Dissertation, IHI Zittau; M. Kluge, 2006, Diplomarbeit, IHI Zittau).The enzyme used is a special haloperoxidase with peroxygenase function. It is produced by basidiomycetes of the families Bolbitiaceae (e.g., Agrocybe spp.) And Coprinaceae (e.g., Coprinus spp.) And is characterized by particular catalytic properties that clearly distinguish it from previously described peroxidases and cytochrome P450 enzymes. Enzyme production takes place in liquid culture, preferably in bioreactors and nitrogen-rich media (R. Ullrich, 2005, dissertation, IHI Zittau, M. Kluge, 2006, diploma thesis, IHI Zittau).
Die von dem als Agrocybe-aegerita-Peroxidase (AaP) bezeichneten Enzym katalysierten Reaktionen benötigen im Gegensatz zu chemischen Synthesen keine hochkonzentrierten aggressiven und umweltgefährdenden Reagenzien, und bei der Produktgewinnung kann auf Chemikalien- und zeitintensive Reinigungsschritte zur Trennung der racemischen Gemische verzichtet werden. Üblicherweise wird das Enzym in einer Konzentration von 0,02 U/mL bis 10 U/mL AaP, insbesondere von 0,09 bis 8 U/mL AaP eingesetzt. Dies macht das dargestellte Reaktionsverfahren besonders umweltfreundlich. Ein weiterer Vorteil gegenüber rein chemischen Synthesen besteht in der Prozessführung auf Grund der erfindungsgemäßen AaP-katalysierten Umsetzung bei Raumtemperatur und normalem Luftdruck.In contrast to chemical syntheses, the reactions catalyzed by the enzyme termed Agrocybe aegerita peroxidase (AaP) do not require highly concentrated aggressive and environmentally hazardous reagents, and product recovery can dispense with chemical and time-consuming purification steps to separate the racemic mixtures. Usually, the enzyme is used in a concentration of 0.02 U / mL to 10 U / mL AaP, in particular 0.09 to 8 U / mL AaP. This makes the reaction process shown particularly environmentally friendly. Another advantage over purely chemical syntheses is the process control due to the AaP-catalyzed reaction according to the invention at room temperature and normal atmospheric pressure.
In einer bevorzugten Ausführungsform wird das Verfahren in wässrigen, gepufferten Lösungen durchgeführt. Dem Reaktionsgemisch können hierbei zur Stabilisierung der Reaktion im wässrigen Medium Puffer auf Basis organischer Säuren, vorzugsweise Zitronensäure, sowie Phosphate, vorzugsweise Kaliumhydrogenphosphate, zugesetzt werden (Pufferkonzentration 5 mmol/L bis 500 mmol/L, bevorzugt 20-100 mmol/L). Darüber hinaus ist es möglich, die Reaktion im pH-Staten ohne Puffer unter kontinuierlicher Zudosierung von Säuren/Basen durchzuführen.In a preferred embodiment, the process is carried out in aqueous, buffered solutions. To stabilize the reaction in the aqueous medium, buffers based on organic acids, preferably citric acid, and phosphates, preferably potassium hydrogen phosphates, may be added to the reaction mixture (buffer concentration 5 mmol / L to 500 mmol / L, preferably 20-100 mmol / L). In addition, it is possible to carry out the reaction in the pH state without buffer under continuous addition of acids / bases.
Zur Verbesserung der Löslichkeit können dem Reaktionsgemisch organische Lösungsmittel zugesetzt werden. Erfindungsgemäß einsetzbare Lösungsmittel sind z.B. protische Lösungsmittel, wie z.B. Ether (u.a. Diisopropylether), Methanol, Aceton, Acetonitril, DMF (N.N-Dimethylformamid) oder DMSO (Dimethylsulfoxid). Als Ausgangsverbindungen der Formel (I) werden insbesondere Verbindungen aus folgenden Gruppen eingesetzt: Alkylbenzole und am Aromaten (Ar) substituierte Alkylbenzole (RrAr-R2; R1 = X, NO2, NH2, OH; R2 = Alkyl-, insbesondere Ethyl-Substituent).To improve the solubility, organic solvents may be added to the reaction mixture. Solvents which can be used according to the invention are, for example, protic solvents, for example ethers (inter alia diisopropyl ether), methanol, acetone, acetonitrile, DMF (N, N-dimethylformamide) or DMSO (dimethyl sulfoxide). The starting compounds of the formula (I) used are in particular compounds from the following groups: alkylbenzenes and aromatic (Ar) -substituted alkylbenzenes (RrAr-R 2 ; R 1 = X, NO 2 , NH 2 , OH; R 2 = alkyl, especially ethyl substituent).
Die Reaktion wird in einem Bereich von 5 bis 600C, vorzugsweise bei 20 bis 300C durchgeführt. Die Reaktionszeiten liegen üblicherweise im Bereich von 1 bis 90 Minuten, insbesondere im Bereich von 5 bis 30 Minuten.The reaction is carried out in a range of 5 to 60 0 C, preferably at 20 to 30 0 C. The reaction times are usually in the range of 1 to 90 minutes, in particular in the range of 5 to 30 minutes.
Die erzielten ee-Werte liegen im Bereich von 80 bis 99,9 %, vorzugsweise größer als 95% ee; die Ausbeuten liegen zwischen 30 und 90 %. Die Vorteile der AaP-katalysierten Umsetzungen gegenüber Katalysen mit herkömmlichen Cytochrom-P450-Enzymen (Monooxygenasen) bestehen: i) in der hohen erreichbaren Enantiomerenreinheit der Reaktionsprodukte, ii) im Einsatz preiswerter Peroxide anstelle teurer Elektronendonatoren [NAD(P)H], iii) in der Unabhängigkeit des hydroxylierenden Enzyms vonThe achieved ee values are in the range of 80 to 99.9%, preferably greater than 95% ee; the yields are between 30 and 90%. The advantages of the AaP-catalyzed reactions to catalysis with conventional cytochrome P450 enzymes (monooxygenases) are: i) the high enantiomeric purity of the reaction products, ii) the use of inexpensive peroxides instead of expensive electron donors [NAD (P) H], iii) in the independence of the hydroxylating enzyme of
Flavin-Reduktasen und Elektronentransportproteinen (Ferridoxine), iv) in der einfachen Enzymgewinnung und - v) in der hohen Stabilität der extrazellulärer Enzyme im Vergleich zu den instabilen intrazellulärer und oftmals membrangebundenenIv) in simple enzyme production and - v) in the high stability of extracellular enzymes compared to the unstable intracellular and often membrane bound proteins
P450-Enzyme.P450 enzymes.
Vorteile der AaP gegenüber herkömmlichen Peroxidasen (u.a. Chlorperoxidase, Meerrettich-Peroxidase, Lignin-Peroxidase) sind: i) ein breiteres Substratspektrum, ii) eine stärker ausgeprägte Peroxygenase-Aktivität, iii) eine insgesamt höhere Stabilität.Advantages of AaP over conventional peroxidases (including chloroperoxidase, horseradish peroxidase, lignin peroxidase) are: i) a broader substrate spectrum, ii) a more pronounced peroxygenase activity, iii) an overall higher stability.
Mit den AaP-katalysierten Reaktionen ist es erstmals möglich, Alkylaromaten mit Hilfe eines einzelnen extrazellulären Biokatalysators, welcher lediglich ein Peroxid als Cosubstrat benötigt in einem einstufigen Prozess stereoselektiv zu chiralen aromatisch substituierten Alkanolen, wie z.B. (R)-I-Phenylethanol) zu oxidieren. Das Verfahren kann in verschiedensten Bereichen der Synthesechemie eingesetzt werden, u.a. zur Herstellung von chiralen Pharmazeutika und deren Vorstufen, Aromastoffen und Ausgangsstoffen für nachfolgende chemische Synthesen. Die Erfindung soll nachstehend anhand von dem in der Zeichnung dargestellten Ausführungsbeispiel näher erläutert werden, wobei die Erfindung nicht auf die Beispiele beschränkt ist. With the AaP-catalyzed reactions, it is now possible to oxidize alkylaromatics stereoselectively to chiral aromatically substituted alkanols, such as (R) -I-phenylethanol, with the aid of a single extracellular biocatalyst, which only requires a peroxide as cosubstrate. The process can be used in various fields of synthetic chemistry, including the preparation of chiral pharmaceuticals and their precursors, flavorings and precursors for subsequent chemical syntheses. The invention will be explained in more detail below with reference to the embodiment shown in the drawing, wherein the invention is not limited to the examples.
Beispiele:Examples:
Es zeigen:Show it:
Abb. 1 : Allgemeines Schema Abb. 2: Formelschema gemäß AusführungsbeispielFig. 1: General scheme Fig. 2: Schematic diagram according to the embodiment
Ausführungsbeispiel:Embodiment:
50 μmol Ethylbenzen wurden in wässriger Kaliumphosphat-Pufferlösung (20 nriM, pH = 7.0) suspendiert und zusammen mit 250 μmol H2O2 (5 x je 50 μM im Abstand von je 5 min zugegeben) und 5 U Agrocybe-aegerita-Peroxidase (Unit bezogen auf die Oxidation von Veratrylalkohol zu Veratrylaldehyd; Ullrich et al., 2004, Appl. Environ. Microbiol. 70: 4575-81 ) in einem Gesamtvolumen von 50 ml bei 24 0C in einem verschlossenen Glasgefäß gerührt. Die Reaktionszeit betrug insgesamt 30 min. Das Enantiomerenverhältnis wurde anschließend mittels GC unter Verwendung einer chiralen Kapillarsäule zu 98,3 : 1 ,7 von R zu S (ee = 96,6%) bei einer Ausbeute von 82 % bestimmt. 50 .mu.mol of ethylbenzene were suspended in aqueous potassium phosphate buffer solution (20 .mu.m, pH = 7.0) and together with 250 .mu.mol H 2 O 2 (5 × 50 μM each 5 min added) and 5 U Agrocybe aegerita peroxidase ( Unit based on the oxidation of veratryl alcohol to veratryl aldehyde, Ullrich et al., 2004, Appl. Environ.Microbiol.70: 4575-81) in a total volume of 50 ml at 24 0 C in a sealed glass vessel. The reaction time was a total of 30 min. The enantiomeric ratio was then determined by GC using a chiral capillary column to be 98.3: 1, 7 from R to S (ee = 96.6%) with a yield of 82%.
Claims
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| DE102006041493.4 | 2006-09-05 | ||
| DE102006041493A DE102006041493A1 (en) | 2006-09-05 | 2006-09-05 | Process for the stereoselective hydroxylation of alkylaromatics |
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| PCT/EP2007/005230 Ceased WO2008028526A1 (en) | 2006-09-05 | 2007-06-14 | Process for the preparation of optically active 1-arylalcohols using the haloperoxidase of agrocybe aegerita |
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| WO2011120938A3 (en) * | 2010-03-28 | 2011-11-24 | Novozymes A/S | Regioselective hydroxylation of aliphatic hydrocarbons employing a fungal peroxygenase |
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| 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 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2006034702A1 (en) * | 2004-09-28 | 2006-04-06 | Jenabios Gmbh | Method for the enzymatic hydroxylation of non-activated hydrocarbons |
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2006
- 2006-09-05 DE DE102006041493A patent/DE102006041493A1/en not_active Withdrawn
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| WO2006034702A1 (en) * | 2004-09-28 | 2006-04-06 | Jenabios Gmbh | Method for the enzymatic hydroxylation of non-activated hydrocarbons |
Non-Patent Citations (3)
| Title |
|---|
| DATABASE CABA [online] CHEMICAL ABSTRACTS SERVICE; 2001, DANG, J. ET AL.: "Study on isoenzyme in Agrocybe chaxingu at different growth stages", XP002450062, Database accession no. 2002:138095 * |
| DATABASE MEDLINE [online] US NATIONAL LIBRARY OF MEDICINE (NLM); September 2007 (2007-09-01), DAU, H.A. ET AL.: "The Coprophilous Mushroom Coprinus radians Secretes a Haloperoxidase That Catalyzes Aromatic Peroxygenation", XP002450157, Database accession no. 2007505300 * |
| ULLRICH, R. ET AL.: "Novel Haloperoxidase from the Agaric Basidiomycete Agrocybe aegerita Oxidizes Aryl Alcohols and Aldehydes", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 70, no. 8, August 2004 (2004-08-01), pages 4575 - 4581, XP002364075 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011120938A3 (en) * | 2010-03-28 | 2011-11-24 | Novozymes A/S | Regioselective hydroxylation of aliphatic hydrocarbons employing a fungal peroxygenase |
| US9222109B2 (en) | 2010-03-28 | 2015-12-29 | Novozymes A/S | Enzymatic hydroxylation of aliphatic hydrocarbon |
| US9534238B2 (en) | 2010-03-28 | 2017-01-03 | Novozymes A/S | Enzymatic hydroxylation of aliphatic hydrocarbon |
| US9909147B2 (en) | 2010-03-28 | 2018-03-06 | Novozymes A/S | Enzymatic hydroxylation of aliphatic hydrocarbon |
| US10174342B2 (en) | 2010-03-28 | 2019-01-08 | Novozymes A/S | Enzymatic hydroxylation of aliphatic hydrocarbon |
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