EJEMPLOS DE OXIDORREDUCTASAS PDF
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The present invention relates to a process for the enantioselective enzymatic reduction of a keto compound to the corresponding chiral hydroxy compound in the ketonic ejem;los is reduced with an oxidoreductase. The invention further relates to novel oxidoreductases for use in the enantioselective reduction of keto compounds to give compounds of chiral hydroxyl.
The optically active compounds are valuable chiral hydroxyl modules with wide application for the synthesis of pharmacologically active compounds, aromatic substances, pheromones, agrochemicals and enzyme inhibitors. In this respect it is recorded particularly in djemplos pharmaceutical industry a growing need for chiral compounds and thus chiral synthesis technologies, since in the future and only be used as drugs racemic compounds.
Asymmetric reduction of prochiral ketone compounds is a stereoselective catalysis sector, wherein the biocatalysis technology represents an efficient competition with respect to chemical catalysis.
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The chemical asymmetric hydrogenation requires the use of heavy metal catalysts with highly toxic and contaminants of extreme reaction conditions and therefore energy-intensive and large amounts of organic solvents. Furthermore, these processes are characterized by side reactions often and insufficient enantiomeric excesses.
Reductions ketone compounds prochiral to give hydroxy compounds and conversely occur in nature in many biochemical pathways, both the primary metabolism and secondary metabolism, in any organism and are catalyzed by different types of secondary and oxidoreductases alcoholdeshidrogenasas.
Por regla general, estas enzimas dependen de cofactores. Generally, these enzymes dependent cofactors. The main feasibility of using biocatalysts for reduction of ketone compounds prochiral to give compounds of chiral hydroxyl demonstrated in the past repeatedly using model systems, and the conference both oxidoreductases isolated as different systems of biotransformation of whole cells. In this respect the biocatalytic approach ejemplox advantageous with respect to essentially mild reaction conditions, and absent byproducts obtainable with essentially enantiomeric excesses better oxidorrdductasas.
The use of isolated enzymes is advantageous in comparison to whole dr procedures regarding obtainable enantiomeric excess, the production of degradation products and side as compared to product isolation. It is also not possible using whole cell processes in any chemical company, because for that special equipment and expertise are needed. In the systems described in this connection the ketone compound to be reduced, in most cases poorly oxidkrreductasas in water forms together with the organic solvent the organic phase.
Partially can also dispensed organic solvent itself, then the ejemolos phase by the ketone compound to be reduced OxodorreductasasDE The coenzyme regeneration is made in this respect by the simultaneous oxidorreudctasas of secondary alcohols, being used in most cases the 2-propanol miscible with water, economic. Examples Ry oxidoreductases and dehydrogenase S-specific suitable high enantioselectivity: Sep de ; 62 4: Epub 26 de abril dey Rhodococcus ruber J Org Chem.
Epub 26 de junio deJ. Chem kxidorreductasas, 57, However, the known enzymes are currently not even remotely sufficient to realize the commercial potential of stereoselective reductions of keto compounds. This may be based on one hand in that the individual enzymes have very different properties with respect to the substrate spectrum, pH optima and stabilities temperature and solvents, often complement each other.
Therefore, even homologous enzymes proportionally similar may occur with respect to a particular substrate behavior completely different reaction.
Candida oxidoreductase nemodendra presents an equal activity against Rbutanol, 2-propanol ozidorreductasas 2-octanol-R, also has the enzyme also approximately equal activity against two-octanone. The pH optimum for the reduction reaction is for this oxidoreductase to 6. Candida nemodendra oxidoreductase has good pH stability and solvents shown in the pH range of 6.
Of the two enantiomers of 2-octanol preferentially oxidizes Soctanol.
Ubiquinol-citocromo-c reductasa – Wikipedia, la enciclopedia libre
It is also suitable for the reduction of 4-haloacetoacetato ester acid esters Rhalohydroxybutyric acid. Fusion proteins can be separated for example more easily from other proteins or can be recombinantly expressed in larger amounts. The preparation of these antibodies are performed according to known methods by immunizing suitable mammalian and subsequent production of antibodies.
Los anticuerpos pueden ser monoclonales o policlonales. The antibodies may be monoclonal or polyclonal. As scoring matrix for calculating analogy sequence was based on the PAM30 matrix. The invention further relates to a cloning vector containing one or more nucleic acid sequences encoding oxidorredudtasas according to SEQ ID NO: The invention further relates to an expression vector is a bacterial cell, insect, plant or mammalian and containing a nucleic acid sequence encoding the carbonilrreductasas re to SEQ ID NO: The invention further relates to a recombinant host cell is a bacterial, yeast, insect, plant or mammalian and transformed or transfected with an expression vector of this type and to a preparation process for obtaining a carbonyl reductase which is based on culturing a recombinant host cell of this type.
The extract obtained cells can be used either directly or be purified further. For this, for example, the cell extract and the supernatant obtained is subjected to ion exchange chromatography, for example by ion exchange chromatography on Q-Sepharose Fast Flow Pharmacia and oxidoreductasas.
By the oxidorreductazas aryl residues of aromatic hydrocarbon it is meant having 6 to 14 carbon atoms in ekemplos ring.
Los restos arilo C6-C14 son por ejemplo fenilo, naftilo, por ejemplo 1-naftilo, 2-naftilo, bifenililo, por ejemplo 2-bifenililo, 3bifenililo y 4-bifenililo, antrilo o fluorenilo. The aryl C6-C14 are for example phenyl, naphthyl, for example 1-naphthyl, 2-naphthyl, biphenylyl, for example 2-biphenylyl, 4-biphenylyl 3bifenililo and, anthryl or fluorenyl.
Los restos bifenililo, oxidorreduftasas naftilo y en particular restos fenilo son restos arilo preferentes. Biphenylyl residues, naphthyl residues and, in particular phenyl radicals are preferred aryl radicals.
By the oxidorreductasas “halogen” means an element of the series fluorine, chlorine, bromine or iodine. By the term “C0” means a covalent bond.
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By the term ” C3-C7 ” cyclic hydrocarbon residues such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl means. The term “heterocycle C5-C14 ” represents a heterocyclic ring of 5-membered to membered bicyclic or monocyclic which is partially or completely saturated. Examples of heteroatoms N, O and S. Oxidorreductassa particular residues 2-or 3pirrolilo are preferred, fenilpirrolilo as 4- or ejfmplos, 2-furyl, 2-thienyl, 4-imidazolyl, methyl-imidazolyl, 1-methyl eg, -4 -o imidazolyl, 1,3-thiazolylpyridyl, 3-pyridyl, 4-pyridyl, N-oxide of 2- 3- or 4-pyridyl, 2-pyrazinyl, 2- 4- or 5-pyrimidinyl group, 2- 3- or 5-indolylindolyl substituted, for oxidorreduchasas 1-methyl- 5-methyl- 5-methoxy- 5-benzyloxy- 5-chloro- or 4,5 -dimethylindolyl, 1-benzyl or indolyl, 4,5,6,7-tetrahydroindolyl, cyclohepta [b] pyrrolyl, 2- 3- ejempls 4quinolilo, 1- 3- or 4-isoquinolyl, 1-oxo-1,2-dihydroisoquinolyl, 2-quinoxalinyl, 2-benzofuranyl, 2-benzo-thienyl, benzothiazolyl or dihydropyridinyl 2benzoxazolilo or, pyrrolidinyl, for example 2- or 3- N-methylpyrrolidinylpiperazinyl, morpholinyl, thiomorpholinyl, tetrahydrothienyl or benzodioxolanyl.
Oxidoreductases may be used in the process according to the invention or fully purified form or partially purified or they may be performed with cells containing oxidoreductases according to the invention. Sjemplos cells used in this respect may be native, permeabilized or lysed way. Per kg of compound of formula I to be reacted are used from to 10 million U of oxidoreductase. Enzymatic reduction itself takes place in mild conditions, so that the alcohols do not react subsequently generated.
Como cosustrato se usan a este respecto preferentemente alcoholes primarios y secundarios, tales como etanol, 2propanol, 2-butanol, 2-pentanol, 3-pentanol, 4-metilpentanol, 2-heptanol, 2-octanol o ciclohexanol.
Cosubstrate are used in this respect preferably primary and secondary, such as ethanol, 2-propanol, 2-butanol, 2-pentanol, 3-pentanol, 4-methylpentanol, 2-heptanol, 2-octanol or cyclohexanol alcohols. For cofactor regeneration can be further oxidorreductasa one alcohol dehydrogenase. NADH dependent alcoholdeshidrogenasas suitable obtainable, for example bakers yeast, Candida boidinii, Candida parapsilosis oxidorrdductasas Pichia capsulata.
Los cosustratos adecuados para estas alcoholdeshidrogenasas son los alcoholes secundarios ya mencionados tales como etanol, 2propanol isopropanol2-butanol, 2-pentanol4-metilpentanol, 2-octanol o ciclohexanol. Suitable cosubstrates for these alcoholdeshidrogenasas are the already mentioned secondary alcohols such as ethanol, 2-propanol isopropanol2-butanol, 2-pentanol, 4-methylpentanol, 2-octanol or cyclohexanol.
Los cosustratos adecuados de la formiato Suitable ejemmplos of formate. Preferably procedures are performed in accordance with the invention however without additional dehydrogenase of this type, ie does not occur coenzyme regeneration coupled to the substrate.
In another oxidorreductawas embodiment of the methods according to the invention the enzymatic reaction in the presence of a water-immiscible or water-miscible organic solvent only limited performed.
This solvent is for example a dialkyl C1-C6 symmetrical or asymmetrical ether, a straight or branched alkane or cycloalkane chain or secondary alcohol insoluble in water, which in turn represents the cosubstrate.
Preferred organic solvents are for example diethyl ether, tert-butyl methyl ether, diisopropyl oxidorreductaaas, dibutyl ether, butyl acetate, heptane, hexane, 2-octanol, 2-heptanol, 4-methylpentanol or cyclohexane. The solvent may also serve in this respect as cosubstrate simultaneously cofactor regeneration. The reaction mixture, in case of using water-insoluble solvents or co-substrates, is composed of an aqueous phase and an organic phase.
The substrate is distributed correspondingly to its solubility between the organic and aqueous phase. The two liquid phases are preferably mixed mechanically so generated therebetween a large area.
The process according to the invention is performed for example in a closed reaction vessel made of glass or metal. For this, the components are transferred individually into the reaction vessel and stirred under an atmosphere of for example nitrogen or air.
The reaction time amounted to 1 hour to 48 hours, in particular from 2 hours to 24 hours. Then the reaction mixture is processed. For this, the aqueous oxiidorreductasas is separated, the organic phase is filtered.
The aqueous phase can be extracted again optionally as the organic phase can be further processed. After evaporating the solvent optionally the filtered organic phase.
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The reaction conditions are essentially the same as in the aforementioned method for the enantiospecific reduction of the ketone compound of formula I.
However, oxivorreductasas of an enantioselective reduction of the ketone compound of formula I, the oxivorreductasas mixture of the compound of formula II It oxidizes only one enantiomer of the hydroxy compound of formula II enantioselectively to give the corresponding ketone compound. Because it remains the opposite enantiomer of the hydroxy compound of formula II and can be isolated. Furthermore, the method is used instead of the alcohols used as cosubstrates such as ethanol, 2-propanol isopropanol2-butanol, 2-pentanol or 2-octanol, the corresponding ketones such as acetone for the regeneration of NAD.
The preferred embodiments of the invention is oxidorrwductasas explained in more detail by the following examples. The supernatant lysate obtained after centrifugation of 2 min at rpm was used in the next activity selection and for determining the enantiomeric excess ee value. As different substrates were used ketones such as 2-butanones, 2-octanone, 4-chloroacetoacetate, acetophenone or acid ethyloxophenylbutyric. Reaction mixture for the selection of activity: The reaction was continued for 1 min at nm.
Reaction mixture for the determination of ee value: The reaction mixtures for ee determination were extracted after 24 hours h for example with chloroform and by gas chromatography GC the enantiomeric excess was determined. The enantiomeric excess is calculated as follows: Tabla 1 Table 1. Definition of enzyme units: Para el aislamiento de las oxidorreductasas microbianas dependientes de NAD P H se cultivaron los microorganismos tal como se ha descrito en el ejemplo 1.
For the isolation of microbial dependent oxidoreductases NAD P H microorganisms cultured as described in example 1. After reaching the stationary phase cells were harvested by centrifugation and separated from the medium. Enzyme release was performed by wet grinding by means of glass beads, but may also be achieved by other methods disintegration. To this were suspended for example g of wet mass of cells with ml of disruption buffer mM triethanolamine, 1 mM MgCl2, pH 7.
The crude extract obtained after centrifugation rpm then it was further purified by FPLC fast protein liquid chromatography, fast liquid chromatography oxidorreductassas proteins. All oxidoreductases according to the invention could be purified by various combinations of ion exchange chromatography, for example on Q-Sepharose Fast Flow Pharmacia or One Q Biorad, Munich, Germany of hydrophobic interaction chromatography, for example in Octylsepharose Fast Flow or Butylsepharose Fast Flow Pharmaciahydroxyapatite chromatography and ceramic-gel permeation.
The resulting nucleic acid served as a matrix for the polymerase chain reaction PCR with degenerate primers.
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