DE102005010804A1 - Process for the preparation of optically active alcohols - Google Patents

Abstract

A process for the preparation of optically active alkanols of the formula I, DOLLAR F1 in which DOLLAR A n is an integer from 0 to 5, DOLLAR A Cyc is an optionally substituted, mono- or polynuclear, saturated or unsaturated, carbocyclic or heterocyclic ring, and DOLLAR AR · 1 · is halogen, SH, OH, NO¶2¶, NR · 2 · R · 3 or NR · 2 · R · 3 · R · 4 + · X · - ·, where R · 2 R, R 3 and R 4 independently of one another are H or a lower alkyl or lower alkoxy radical and X is a counterion, DOLLAR A being embedded in an alkanone of the formula II, DOLLAR F 2 in which n, Cyc and R · 1 · have the meanings given above, DOLLAR A containing medium, an enzyme (E) selected from the classes of dehydrogenases, aldehyde reductases and carbonyl reductases, incubated in the presence of reducing equivalents, wherein the compound of formula II to the compound of formula I is reduced enzymatically, and in the course of Reacti On used Rduktionsäquivalente be regenerated by reacting a sacrificial alcohol to the corresponding sacrificial ketone with the aid of the enzyme (E) and the sacrificial ketone is at least partially removed from the reaction medium, and the product isolated (I).

Description

The The present invention relates to a process for the preparation of optically active alkanols by enzymatic reduction of the corresponding Ketones, in particular the preparation of (1S) -3-methylamino-1- (2-thienyl) -propan-1-ol and (1S) -3-chloro-1- (2-thienyl) -propan-1-ol.

(1S) -3-Methylamino-1- (2-thienyl) -propan-1-ol ("duloxetine alcohol") is a building block in the duloxetine synthesis. Duloxetine ^® is an active pharmaceutical ingredient that is currently being approved and is to be used in the indication area depression and incontinence.

EP-B-0273658 describes a method for producing the corresponding Base of duloxetine by reaction of 2-acetylthiophene in one Mannich reaction with formaldehyde and dimethylamine, reduction of Keto group of the resulting Mannich base to racemic (S) -3-N, N-dimethylamino-1- (thien-2-yl) propan-1-ol, Etherification of alcohol function with naphthyl fluoride and finally conversion of the Dimethylamino group in a methylamino function. The desired enantiomer of the naphthyl ether by using chiral starting materials or by racemate separation at the stage of the final product, for example via the salts with optically active acids or chromatography on a chiral stationary phase.

US 5,362,886 describes an analogous method, in which after reduction of the Keto group obtained racemic propanol with S-mandelic acid becomes. The resulting S-enantiomer of the alcohol is in the used subsequent reaction stages.

EP-A-0457559 also describes a method analogous to EP-B-0273658. in this connection becomes the keto group of Mannich base with the asymmetric reduction system LAH-Icb (lithium aluminum hydride - [(2R, 2S) - (-) - 4-dimethylamino-1,2-diphenyl-3-methyl-2-butanol]) to the alcohol reduced in the form of the S-enantiomer. The disadvantage here is next the cost the sensitivity of the reduction system LAH-Icb, the only a few minutes is stable.

umgesetzt, welches anschließend durch Behandlung mit Chlorwasserstoff in 3-Chlor-1-(thien-2-yl)-propan-1-on der Formel (VI)

überführt wird. Das so erhaltene Chlorpropanon wird anschließend unter Verwendung eines chiralen Oxazaborylidins und BH₃ zu (S)-3-Chlor-1-(thien-2-yl)-propan-1-ol der Formel (VII)

reduziert. Der so erhaltene Alkohol wird durch sukzessive Umsetzung mit Natriumiodid und anschließend mit Methylamin in (S)-3-Methylamino-1-(thien-2-yl)-propan-1-ol überführt. Durch nachfolgende sukzessive Umsetzung mit Natriumhydrid, 1-Fluornaphthalin und Chlorwasserstoff erhält man Duloxetine in Form des Hydrochlorids.

• Hal = Halogen

WJ Wheeler and F. Kuo describe in the Journal of Labeled Compounds and Radiopharmaceuticals, Vol. XXXVI, No. 3, pages 213 to 223, a process for the production of Duloxetine. For this purpose, thiophene-2-carboxylic acid chloride in a Stille coupling with vinyl tri-n-butylstannane in the presence of catalytic amounts of benzylchloro-bis (triphenylphosphine) palladium (II) in DMPU (dimethylpropyleneurea) to 1- (thien-2-yl) - propenone of the formula (V)

which is subsequently reacted by treatment with hydrogen chloride in 3-chloro-1- (thien-2-yl) -propan-1-one of the formula (VI)

is transferred. The chloropropanone thus obtained is then converted to (S) -3-chloro-1- (thien-2-yl) -propan-1-ol of the formula (VII) using a chiral oxazaborylidine and BH ₃ .

reduced. The alcohol thus obtained is obtained by successive reaction with sodium iodide and then with Methylamine in (S) -3-methylamino-1- (thien-2-yl) propan-1-ol transferred. Subsequent successive reaction with sodium hydride, 1-fluoronaphthalene and hydrogen chloride gives duloxetines in the form of the hydrochloride.

• Hal = halogen

T. Stillger et al. describe in chemistry engineer technique (74) pages 1035-1039, 2002, substrate-coupled cofactor regeneration methods for the enzymatic enantioselective reduction of 5-oxohexanoic acid ethyl ester to (S) -5-hydroxyhexanoic acid ethyl ester.

Short description of Invention:

Of the The invention therefore an object of the invention, a way to stereospecific Reduction of substituted alkanones, such as 3-methylamino-1- (2-thienyl) -propanone and 3-chloro-1- (2-thienyl) -propanone, the reaction procedure on inexpensive Way possible quantitatively lead to the product should.

These Task was by the surprising Findings resolved, that enzymes with dehydrogenase activity, especially those that can be produced from microorganisms of the genus Azoarcus, to the stereospecific Catalysis of the above reaction with simultaneous cofactor regeneration capable are.

worin
n für einen ganzzahligen Wert von 0 bis 5 steht;
Cyc für einen gegebenenfalls substituierten, ein- oder mehrkernigen, gesättigten oder ungesättigten, carbocyclischen oder heterocyclischen Ring steht, und
R¹ für Halogen, SH, OH, NO₂, NR²R³ oder NR²R³R⁴⁺X^– steht, wobei R², R³ und R⁴ unabhängig voneinander für H oder einen Niedrigalkyl- oder Niedrigalkoxy-Rest stehen und X^– für ein Gegenion steht,
wobei man in einem Alkanon der Formel II

worin n, Cyc und R¹ die oben angegebenen Bedeutungen besitzen,
enthaltenden Medium, ein Enzym (E), ausgewählt aus den Klassen der Dehydrogenasen, Aldehydreduktasen und Carbonylreduktasen, inkubiert in Gegenwart von Reduktionsäquivalenten, wobei die Verbindung der Formel II zur Verbindung der Formel I enzymatisch reduziert wird, und die im Laufe der Reaktion verbrauchten Reduktionsäquivalente durch Umsetzen eines Opfer-Alkohols zum entsprechenden Opfer-Keton mit Hilfe des Enzyms (E) wieder regeneriert werden und das Opfer-Keton zumindest partiell aus dem Reaktionsmedium entfernt wird, und man das gebildete Produkt (I) isoliert Erfindungsgemäß geeignete Enzyme (E) sind insbesondere die Enzyme der Familien der Aldo-Keto-Reduktasen der Aldo-Keto-Reduktase-Superfamily (K.M.Bohren, B.Bullock, B.Wermuth und K.H.Gabbay J.Biol. Chem. 1989, 264, 9547-9551) und der kurzkettigen Alkoholdehydrogenasen/Reduktasen (shortchain alcohol dehydrogenases/reductases [SDR]) zählen. Die letztere Enzymgruppe ist ausführlich beschrieben zum Beispiel bei H.Jörnvall, B.Persson, M.Krook, S.Atrian, R.Gonzalez-Duarte, J.Jeffery und D.Ghosh, Biochemistry, 1995, 34, S. 6003-6013 oder U.Oppermann, C.Filling, M.Hult, N.Shafqat, X.Q.Wu, M.Lindh, J.Shafqat, E.Nordling, Y.Kallberg, B.Persson und H.Jornvall, Chemico-Biological Interactions, 2003, 143, S. 247-253. Innerhalb dieser genannten Enzymklassen sind die kurzkettigen Alkoholdehydrogenasen besonders gut geeignet.A first subject of the invention relates to a process for the preparation of optically active alkanols of the formula I.

wherein
n is an integer from 0 to 5;
Cyc is an optionally substituted, mono- or polynuclear, saturated or unsaturated, carbocyclic or heterocyclic ring, and
R ¹ is halogen, SH, OH, NO _2, NR ² R ³ or NR ² R ³ R ⁴⁺ X ^-, where R ^2, R ³ and R ⁴ are independently H or a lower alkyl or lower alkoxy radical and X ^{- stands} for a counterion,
wherein in an alkanone of formula II

in which n, Cyc and R ^{1 have} the meanings given above,
containing medium, an enzyme (E) selected from the classes of dehydrogenases, aldehyde reductases and carbonyl reductases, incubated in the presence of reducing equivalents, wherein the compound of formula II is enzymatically reduced to the compound of formula I, and the reduction equivalents consumed in the course of the reaction Reacting a sacrificial alcohol to the corresponding sacrificial ketone with the aid of the enzyme (E) be regenerated and the sacrificial ketone is at least partially removed from the reaction medium, and the product (I) formed in accordance with the invention suitable enzymes (E) are in particular the enzymes of the families of aldo-keto-reductases of the aldo-keto-reductase superfamily (KMBohren, B.Bullock, B.Wermuth and KHGabbay J.Biol. Chem. 1989, 264, 9547-9551) and the short-chain alcohol dehydrogenases / reductases (shortchain alcohol dehydrogenases / reductases [SDR]). The latter enzyme group is described in detail, for example, in H.Jörnvall, B.Persson, M.Krook, S.Atrian, R. Gonzalez-Duarte, J.Jeffery and D.Ghosh, Biochemistry, 1995, 34, pp. 6003-6013 or U. Oppermann, C. Filling, M. Hult, N. Shafqat, XQWu, M. Lindh, J. Shafqat, E. Nordling, Y. Kallberg, B.Persson and H.Jornvall, Chemico-Biological Interactions, 2003, 143, pp. 247-253. Within these enzyme classes mentioned, the short-chain alcohol dehydrogenases are particularly well suited.

• (i) SEQ ID NO: 2 oder
• (ii) bei der bis zu 25% der Aminosäurereste gegenüber SEQ ID NO:2 durch Deletion, Insertion, Substitution oder einer Kombination davon verändert sind und die noch mindestens 50% der enzymatischen Aktivität von SEQ ID NO:2 besitzt, besitzt.

A particularly suitable embodiment of the invention is the use of enzymes (E) in the above-mentioned process, wherein E is a polypeptide sequence

• (i) SEQ ID NO: 2 or
• (ii) wherein up to 25% of the amino acid residues are altered from SEQ ID NO: 2 by deletion, insertion, substitution or a combination thereof and which still has at least 50% of the enzymatic activity of SEQ ID NO: 2.

wobei R¹ = Cl oder NHCH₃ bedeuten,
wobei man in einem ein Derivat des 1-(2-thienyl)-propanons der Formel IV

enthaltenden Medium diese Verbindung zur Verbindung der Formel III enzymatisch reduziert wird, und man das in im Wesentlichen enantiomerenreiner Form gebildete Produkt isoliert.In a particularly preferred embodiment, the process is used to prepare derivatives of 1- (2-thienyl) - (S) -propanol of the formula III

where R ¹ = Cl or NHCH ₃ ,
wherein one in a derivative of 1- (2-thienyl) -propanone of the formula IV

containing medium, this compound is enzymatically reduced to the compound of formula III, and isolated the product formed in substantially enantiomerically pure form.

Preferably In these methods, an enzyme having dehydrogenase activity derived from microorganisms is used genera Azoarcus, Azonexus, Azospira, Azovibrio, Dechloromonas, Ferribacterium, Petrobacter, Propionivibrio, Quadricoccus, Rhodocyclus, Sterolibacterium, Thauera and Zoogloea can be produced.

Especially Dehydrogenases from species of the genus Azoarcus are preferred.

by virtue of their amino acid sequence you can use the phenylethanol dehydrogenase from Azoarcus sp EbN1 too the short-chain alcohol dehydrogenases / reductases (shortchain alcohol dehydrogenases / reductases [SDR]). The enzyme group is described in detail for example with H.Jörnvall, B. Persson, M. Krook, S.Atrian, R. Gonzalez-Duarte, J.Jeffery and D.Ghosh, Biochemistry, 1995, 34, pp. 6003-6013 or U. Oppermann, C. Filling, M.Hult, N.Shafqat, X.Q.Wu, M.Lindh, J.Shafqat, E.Nordling, Y.Kallberg, B.Persson and H.Jornvall, Chemico-Biological Interactions, 2003, 143, pp. 247-253. Within the group of SDR, the amino acid sequences may be the individual representative differ greatly. Yet It is known that certain amino acids or amino acid regions within the group of SDR are highly conserved. In C. Filling, K.D.Berndt, J.Benach, S.Knapp, T.Prozorovski, E.Nordling, R.Ladenstein, H.Jornvall and U. Oppermann, Journal of Biological Chemistry, 2002, 277, pp. 25677-25684 are important conserved regions of SDR described.

Examples for Azoarcus species are Azoarcus anaerobius, Azoarcus buckelii, Azoarcus communis, Azoarcus evansii, Azoarcus indigens, Azoarcus toluclasticus, Azoarcus tolulyticus, Azoarcus toluvorans, Azoarcus sp., Azoarcus sp. 22Lin, Azoarcus sp. BH72, Azoarcus sp. CC-11, Azoarcus sp. CIB, Azoarcus sp. CR23, Azoarcus sp. EB1, Azoarcus sp. EbN1, Azoarcus sp. FL05, Azoarcus sp. HA, Azoarcus sp. HxN1, Azoarcus sp. mXyN1, azoarcus sp. PbN1, Azoarcus sp. PH002, Azoarcus sp. T and Azoarcus sp. TON1.

Especially Dehydrogenases from Azoarcus sp EbN1 are preferably used.

In a particularly preferred embodiment of the method, the enzyme with dehydrogenase activity is selected from enzymes which comprise an amino acid sequence according to SEQ ID NO: 2 or a sequence derived therefrom in which up to 25%, preferably up to 20%, particularly preferably up to in particular up to 10,9,8,7,6,5,4,3,2,1% of the amino acid residues by a deletion, a substitution one to 15% Insertion or a combination of deletion, substitution and insertion have been modified, wherein the SEQ ID NO: 2 modified polypeptide sequences nor at least 50%, preferably 65%, more preferably 80%, in particular more than 90% of the enzymatic activity of SEQ ID NO : 2 own. In this context, the enzymatic activity of SEQ ID NO: 2 is to be understood as the ability to reduce the ketones of the formula (IV) with R ¹ = Cl enantioselectively to the (S) -alcohol having the general formula (III). The exact conditions for determining the enzymatic activity are given in Examples 3 and 4.

The inventive method is added with the addition of reduction equivalents, especially from NADH or NADPH. For the regeneration of Reduction equivalents used in the reaction become a sacrificial alcohol, preferably 1-propanol, 2-butanol, 2-pentanol, 2-hexanol, 3-hexanol used under the reaction conditions by the enzyme (E) to the corresponding Sacrificial ketone is oxidized.

In a preferred embodiment In the invention, the added sacrificial alcohol is not only for regeneration the consumed reduction equivalents used, but also as a co-solvent. It works preferentially in a liquid 2-phase system, wherein the one phase of water or water-miscible solvent exists, and the other phase consists of the sacrificial alcohol. Prefers is used as sacrificial alcohol 2-pentanol.

The reducing equivalents are preferably in an amount of 0.001 to 100 mmol, especially preferably from 0.01 to 1 mmol reduction equivalents per mole of used Alkanone (II) used.

at the method according to the invention This will be done in the regeneration of the used reduction equivalents by sacrificial alcohol oxidation resulting sacrificial ketone from the Reaction mixture at least partially removed. Preferably removed completely remove the resulting sacrificial ketone from the reaction medium. This can be done in various ways, for example through selective membranes or through extraction or distillation processes. Preferably, the distillation is used to remove the ketone. Usually becomes in the distillative separation of the ketone also a part of the Sacrificial alcohol and some parts of the aqueous Phase with removed. These distillation losses are usually through After-dose the sacrificial alcohol and possibly the water balanced. The distillation rates are in the range of 0.02% / min to 2% / min, preferably from 0.05% / min to 1% / min based on the reaction volume. The jacket temperatures of the reactor are between 5-70 Kelvin, preferably between 10-40 Kelvin over the internal reactor temperature.

The Distillation is particularly good in a pressure range of 1-500 mbar, preferably 10-200 mbar performed.

A preferred embodiment it is the reaction of the compound of general formula II, such as e.g. of the formula IV, in the presence of a microorganism to let that selected is among bacteria of the families Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Lactobacillaceae, Streptomycetaceae, Rhodococcaceae and Nocardiaceae. The microorganism may be, in particular, a recombinant Microorganism which transforms with a nucleic acid construct is which for encodes an enzyme with dehydrogenase activity as defined above.

• – einen ein Enzym mit Dehydrogenase-Aktivität produzierenden Mikroorganismus aus einer natürlichen Quelle zu isolieren oder rekombinant herzustellen,
• – diesen Mikroorganismus zu vermehren,
• – aus dem Mikroorganismus das Enzym mit Dehydrogenase-Aktivität gegebenenfalls zu isolieren oder eine dieses Enzym enthaltende Proteinfraktion herzustellen, und
• – den Mikroorganismus gemäß Stufe b) oder das Enzym gemäß Stufe c) in ein Medium zu überführen, das eine Verbindung der Formel Ienthält.

The invention is particularly

• To isolate or recombinantly produce a microorganism producing an enzyme having dehydrogenase activity from a natural source;
• - to multiply this microorganism,
• Optionally isolating from the microorganism the enzyme having dehydrogenase activity or producing a protein fraction containing this enzyme, and
• - To convert the microorganism according to step b) or the enzyme according to step c) in a medium containing a compound of formula I.

object of the invention are also coding nucleic acid sequences, comprising the coding sequence for a polypeptide according to the above Definition.

Farther The invention relates to expression cassettes comprising in operative shortcut having at least one regulatory nucleic acid sequence encoding one nucleic acid sequence according to the above Definition.

One Another object of the invention are recombinant vectors comprising at least one such expression cassette.

The Invention also relates to prokaryotic or eukaryotic hosts, which are transformed with at least one vector according to the invention.

A final object of the invention relates to the use of an enzyme having dehydrogenase activity as defined above or a microorganism producing this enzyme for the preparation of compounds of the formulas I or III, and their further processing, for example for the preparation of duloxetines (formula VIII)

Detailed description the invention:

A. General Terms and definitions

• „Halogen" steht für Fluor, Chlor, Brom oder Jod, insbesondere Fluor oder Chlor.
• „Niedrigalkyl" steht für geradkettige oder verzweigte Alkylreste 1 bis 6 C-Atomen, wie Methyl, Ethyl, i- oder n-Propyl, n-, i-, sec.- oder tert.-Butyl, n-Pentyl oder 2-Methyl-Butyl, n-Hexyl, 2-Methyl-pentyl, 3-Methyl-pently, 2-Ethyl-butyl.
• „Niedrigalkenyl" steht für die ein- oder mehrfach, vorzugsweise einfach oder zweifach ungesättigten Analoga oben genannter Alkylreste mit 2 bis 6 Kohlenstoffatomen, wobei die Doppelbindung in beliebiger Position der Kohlenstoffkette liegen kann.
• „Niedrigalkoxy" steht für die Sauerstoff-terminierten Analoga obiger Alkylreste.
• „Aryl" steht für einen ein- oder mehrkernigen, vorzugsweise ein- oder zweikernigen, gegebenenfalls substituierten aromatischen Rest, insbesondere für Phenyl oder für ein über eine beliebige Ringposition gebundenes Naphthyl, wie 1- oder 2-Naphthyl. Diese Arylreste können gegebenenfalls 1 oder 2 gleiche oder verschiedene Substituenten tragen, ausgewählt unter Halogen, Niedrigalkyl, Niedrigalkoxy gemäß obiger Definition oder Trifluormethyl.

If no other information is given, the following general meanings apply:

• "Halogen" is fluorine, chlorine, bromine or iodine, in particular fluorine or chlorine.
• "Lower alkyl" represents straight-chain or branched alkyl radicals of 1 to 6 C atoms, such as methyl, ethyl, isopropyl or n-propyl, n-, i-, sec- or tert-butyl, n-pentyl or 2-methyl Butyl, n-hexyl, 2-methylpentyl, 3-methylpentane, 2-ethyl-butyl.
• "Lower alkenyl" represents the mono- or polysubstituted, preferably mono- or diunsaturated, analogs of the abovementioned alkyl radicals having 2 to 6 carbon atoms, it being possible for the double bond to be in any position of the carbon chain.
• "Lower alkoxy" refers to the oxygen-terminated analogs of the above alkyl groups.
• "Aryl" is a mononuclear or polynuclear, preferably mono- or binuclear, optionally substituted aromatic radical, in particular phenyl or a naphthyl bound via an arbitrary ring position, such as 1- or 2-naphthyl These aryl radicals may optionally be 1 or 2 bear identical or different substituents selected from halogen, lower alkyl, lower alkoxy as defined above or trifluoromethyl.

Substituted alkanones, (S) -alkanols and derivatives thereof Alkanols obtainable by enzymatic catalysis according to the invention are those of the above formula (I) embedded image wherein
n is an integer from 0 to 5;
Cyc is an optionally substituted, mono- or polynuclear, saturated or unsaturated, carbocyclic or heterocyclic ring, and
R ¹ is halogen, SH, OH, NO _2, NR ² R ³ or NR ² R ³ R ⁴⁺ X ^-, where R ^2, R ³ and R ⁴ are independently H or a lower alkyl or lower alkoxy radical and X ^{- stands} for a counterion.

The used for enzymatic synthesis alkanones the above formula II are compounds known in the art and generally used of known organic synthesis methods (see, for example, EP-A-0 273 658).

Preferably n in the above compounds stands for 0, 1 or 2, in particular for 1.

Examples of carbo- and heterocyclic groups Cyc are, in particular, mono- or binuclear, preferably mononuclear, groups having up to 4, preferably 1 or 2, identical or different ring heteroatoms selected from among O, N and S:
These carbo- or heterocyclic rings comprise in particular 3 to 12, preferably 4, 5 or 6 ring carbon atoms. As examples may be mentioned cyclopropyl, cyclobutyl, cyclopenty, cyclohe xyl, cycloheptyl, the mono- or polyunsaturated analogs thereof, such as cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclohexadienyl, cycloheptadienyl; and 5- to 7-membered saturated or mono- or polyunsaturated heterocyclic radicals having 1 to 4 heteroatoms, which are selected from O, N and S, wherein the heterocycle may optionally be condensed with another heterocycle or carbocycle. Particular mention may be made of heterocyclic radicals derived from pyrrolidine, tetrahydrofuran, piperidine, morpholine, pyrrole, furan, thiophene, pyrazole, imidazole, oxazole, thiazole, pyridine, pyran, pyrimidine, pyridazine, pyrazine, cumarone, indole and quinoline.

The Radicals Cyc can over it any ring position, preferably via a ring carbon atom, be bound to the alkanone or the alkanol.

Examples suitable for Cyc radicals are 2-thienyl, 3-thienyl; 2-furanyl, 3-furanyl; 2-pyridyl, 3-pyridyl or 4-pyridyl; 2-thiazolyl, 4-thiazolyl or 5-thiazolyl; 4-methyl-2-thienyl, 3-ethyl-2-thienyl, 2-methyl-3-thienyl, 4-propyl-3-thienyl, 5-n-butyl-2-thienyl, 4-methyl-3-thienyl, 3-methyl-2-thienyl; 3-chloro-2-thienyl, 4-bromo-3-thienyl, 2-iodo-3-thienyl, 5-iodo-3-thienyl, 4-fluoro-2-thienyl, 2-bromo-3-thienyl, and 4-chloro-2-thienyl.

The radicals Cyc can furthermore be monosubstituted or polysubstituted, for example monosubstituted or disubstituted. Preferably, the substituents are on a ring carbon atom. Examples of suitable substituents are halogen, lower alkyl, lower alkenyl, lower alkoxy, -OH, -SH, -NO ₂ or NR ² R ³ , wherein R ² and R ^{3 have the} above meanings, preferably halogen or lower alkyl.

R ¹ represents in particular halogen, NR ² R ³ or NR ² R ³ R ⁴⁺ X ^-, where R ^2, R ³ or R ^2, R ³ and R ⁴ independently represent H or a lower alkyl or lower alkoxy radical and X ^{- is} a counterion, wherein preferably one of the radicals R ² , R ³ and R ^{4 is} H. Suitable counterions are, for example, acid anions, as obtained, for example, in the preparation of an acid addition salt. Examples of this are mentioned, for example, in EP-A-0 273 658, to which reference is hereby made. Preferred examples of radicals R ¹ are in particular fluorine or chlorine, and also NR ² R ³ in which R ² and R ^{3 are} identical or different and are H or methyl, ethyl or n-propyl; R ¹ particularly preferably represents chlorine or -NHMethyl.

C. Suitable enzymes with Dehydrogenase activity

preferred Enzymes with dehydrogenase activity comprise an amino acid sequence according to SEQ ID NO: 2.

Includes according to the invention are also "functional equivalents" of the specifically disclosed Enzymes with dehydrogenase activity and the use of these in the methods of the invention.

"Functional equivalents" or analogues of Specifically disclosed enzymes are within the scope of the present invention different polypeptides, which furthermore the desired biological Activity, such as. Substrate specificity, have. For example, "functional equivalents" are understood to mean enzymes derived from 3-chloro-1- (thien-2-yl) -propan-1-one reduce to the corresponding S-alcohol and that at least 50%, preferably 60%, more preferably 75%, most preferably 90% of the activity an enzyme having the amino acid sequence listed in SEQ ID NO: 2 having. Functional equivalents are also preferred stable between pH 4 to 10 and advantageously have a pH optimum between pH 5 and 8 and a temperature optimum in the range of 20 ° C to 80 ° C.

"Functional equivalents" are understood according to the invention in particular also mutants which in at least one sequence position of the above mentioned amino acid sequences but have a different than the specified amino acid but still possess one of the above biological activities. "Functional equivalents" thus include the by one or more amino acid additions, Substitutions, deletions and / or inversions available Mutants, said changes in any sequence position may occur, as long as they become a mutant with the property profile according to the invention to lead. Functional equivalence is especially given when the patterns of reactivity between mutant and unchanged Polypeptide match qualitatively, i.e. for example, the same substrates with different speeds be implemented.

Examples of suitable amino acid substitutions are shown in the following table: Original rest Examples of substitution Ala Ser bad Lys Asn Gln; His Asp Glu Cys Ser Gln Asn Glu Asp Gly Per His Asn; Gln Ile Leu; Val Leu Ile; Val Lys Arg; Gln; Glu mead Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu

"Functional equivalents" are understood according to the invention in particular also mutants which in at least one sequence position of the above mentioned amino acid sequences but have a different than the specified amino acid but still possess one of the above biological activities. "Functional equivalents" thus include the by one or more amino acid additions, Substitutions, deletions and / or inversions available Mutants, said changes in any sequence position may occur, as long as they become a mutant with the property profile according to the invention to lead. Functional equivalence is especially given when the patterns of reactivity between mutant and unchanged Polypeptide match qualitatively, i.e. for example, the same substrates with different speeds be implemented.

"Functional equivalents" in the above sense are also "precursors" of the described Polypeptides and "functional Derivatives "and" salts "of the polypeptides.

"Precursors" are natural or synthetic precursors of the polypeptides with or without the desired biological activity.

Under The term "salts" is understood both Salts of carboxyl groups as well as acid addition salts of amino groups the protein molecules of the invention. salts of carboxyl groups are prepared in a conventional manner and include inorganic Salts such as sodium, calcium, ammonium, iron and Zinc salts, and salts with organic bases, such as amines, such as Triethanolamine, arginine, lysine, piperidine and the like. Acid addition salts, such as salts with mineral acids, such as hydrochloric acid or sulfuric acid and salts with organic acids, like acetic acid and oxalic acid are also the subject of the invention.

"Functional Derivatives of polypeptides according to the invention can on functional amino acid side groups or at their N- or C-terminal end by known techniques also be prepared. Such derivatives include, for example aliphatic esters of carboxylic acid groups, Amides of carboxylic acid groups, available by reaction with ammonia or with a primary or secondary amine; N-acyl derivatives free amino groups, prepared by reaction with acyl groups; or O-acyl derivatives of free hydroxy groups prepared by reaction with acyl groups.

Of course, "functional equivalents" also include Polypeptides which are accessible from other organisms, as well as naturally occurring Variants. For example, areas can be identified by sequence comparison homologous sequence regions and based on the concrete Specifications of the invention equivalent Determine enzymes.

"Functional equivalents" also include fragments, preferably single domains or sequence motifs, of the polypeptides of the invention which, for example, have the desired biological function sen.

"Functional equivalents" are also fusion proteins, which is one of the above-mentioned polypeptide sequences or derived therefrom functional equivalents and at least one other, functionally distinct, heterologous Sequence in functional N- or C-terminal linkage (i.e. without mutual essential functional impairment the fusion protein parts). Nonlimiting examples for such heterologous sequences are e.g. Signal peptides or enzymes.

According to the invention, "functional equivalents" encompassed are homologs to the specifically disclosed proteins. These have at least 60 %, preferably at least 75%, in particular at least 85%, such as e.g. 90%, 95% or 99%, homology to one of the specifically disclosed Amino acid sequences, calculated according to the algorithm of Pearson and Lipman, Proc. Natl. Acad, Sci. (USA) 85 (8), 1988, 2444-2448. A percentage homology a homologous invention Polypeptide means in particular percentage identity of the amino acid residues based on the total length one of the amino acid sequences specifically described herein.

in the Trap of a possible Protein glycosylation includes "functional equivalents" of the type defined above according to the invention in deglycosylated or glycosylated form and by changing the Glycosylation pattern available modified forms.

homologous the proteins of the invention or polypeptides produced by mutagenesis, e.g. by point mutation or shortening of the Protein.

homologous of the proteins according to the invention can by screening combinatorial libraries of mutants, e.g. Truncation mutants, be identified. For example, a variegated bank of protein variants by combinatorial mutagenesis at the nucleic acid level be generated, such. by enzymatic ligation of a mixture synthetic oligonucleotides. There are a variety of procedures that for making banks of potential homologs from a degenerate Oligonucleotide sequence can be used. The chemical synthesis a degenerate gene sequence can be in a DNA synthesizer carried out and the synthetic gene can then be transformed into a suitable expression vector be ligated. The use of a degenerate set of sentences allows the Provision of all sequences in a mixture that the desired Encoding a set of potential protein sequences. Process for synthesis degenerate oligonucleotides are known to those skilled in the art (e.g., Narang, S.A. (1983) Tetrahedron 39: 3; Itakura et al. (1984) Annu. Rev. Biochem. 53: 323; Itakura et al., (1984) Science 198: 1056; Ike et al. (1983) Nucleic Acids Res. 11: 477).

in the The prior art is several techniques for screening gene products combinatorial banks produced by point mutations or truncation and to screen cDNA libraries for gene products a selected one Property known. These techniques can be applied to the fast Screening of gene libraries by combinatorial mutagenesis homologue according to the invention have been generated. The most common used techniques for screening large libraries that an analysis at high throughput include cloning the library into replicable expression vectors, transform the appropriate Cells with the resulting vector library and expressing the combinatorial Genes under conditions under which the detection of the desired activity the isolation of the vector encoding the gene whose product has been detected was relieved. Recursive ensemble mutagenesis (REM), a technique the frequency functional mutants magnified in the banks, can be combined with used in screening tests to identify homologs (Arkin and Yourvan (1992) PNAS 89: 7811-7815; Delgrave et al. (1993) Protein Engineering 6 (3): 327-331).

D. Coding for the dehydrogenases nucleic acid sequences

object In particular, nucleic acid sequences (single and double-stranded DNA and RNA sequences, e.g. cDNA and mRNA) necessary for an enzyme encode with inventive dehydrogenase activity. Preference is given to nucleic acid sequences, which e.g. For amino acid sequences according to SEQ ID NO: 2 or encode characteristic partial sequences thereof, or nucleic acid sequences according to SEQ ID NO: 1 or characteristic partial sequences thereof.

All nucleic acid sequences mentioned herein can be prepared in a manner known per se by chemical synthesis from the nucleotide units, for example by fragment condensation of individual overlapping, complementary nucleic acid units of the double helix. The chemical synthesis of oligo nucleotides can be carried out, for example, in a known manner by the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897). The attachment of synthetic oligonucleotides and filling gaps with the aid of the Klenow fragment of the DNA polymerase and ligation reactions and general cloning methods are described in Sambrook et al. (1989), Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press.

object The invention also includes nucleic acid sequences (single and double stranded DNA and RNA sequences, e.g. cDNA and mRNA) encoding a the above polypeptides and their functional equivalents, e.g. under Use artificial Nucleotide analogs accessible are.

The The invention relates both to isolated nucleic acid molecules which are suitable for polypeptides according to the invention or encode proteins or biologically active portions thereof, as well as nucleic acid fragments, the e.g. for use as hybridization probes or primers for Identification or amplification of coding according to the invention nucleic acids can be used.

The Nucleic acid molecules according to the invention can additionally untranslated sequences from the 3 'and / or 5 'end of the coding Gene range included

The The invention further encompasses the nucleotide sequences specifically described complementary Nucleic acid molecules or a section of it.

The nucleotide sequences according to the invention enable the generation of probes and primers used for identification and / or Cloning of homologous sequences in other cell types and organisms usable are. Such probes or primers usually comprise a nucleotide sequence region, under "stringent" conditions (see below) at least about 12, preferably at least about 25, such as e.g. about 40, 50 or 75 consecutive nucleotides of a sense strand a nucleic acid sequence according to the invention or a corresponding antisense strand hybridizes.

An "isolated" nucleic acid molecule is used by separated from other nucleic acid molecules, in the natural Source of nucleic acid are present and can also essentially free of other cellular material or culture medium when produced by recombinant techniques, or be free from chemical precursors or other chemicals, though it is chemically synthesized.

One Nucleic acid molecule according to the invention can by means of Standard molecular biology techniques and the invention provided Sequence information can be isolated. For example, cDNA from a suitable cDNA library can be isolated by one of the specifically disclosed complete Sequences or a portion thereof as a hybridization probe and Standard hybridization techniques (such as described in Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). moreover let yourself a nucleic acid molecule comprising one of the disclosed sequences, or a portion thereof, by polymerase chain reaction isolate using the oligonucleotide primers based on this Sequence were created to be used. The so amplified nucleic acid can be cloned into a suitable vector and characterized by DNA sequence analysis become. The oligonucleotides according to the invention can further by standard synthetic methods, e.g. with an automatic DNA synthesizer, manufactured become.

The Nucleic acid sequences of the invention can be identified in principle from all organisms and isolate. Advantageously, the nucleic acid sequences of the invention can be or the homologues thereof, from fungi, yeasts, arkens or bacteria isolate. As bacteria are gram-negative and gram-positive bacteria called. The nucleic acids according to the invention are preferred Gram-negative bacteria advantageous from α-proteobacteria, β-proteobacteria or γ-proteobacteria, most preferably bacteria of the orders of Burkholderiales, Hydrogenophilales, Methylophilales, Neisseriales, Nitrosomonadales, Procabacteriales or Rhodocyclales. Very particularly preferred Bacteria of the Rhodocyclaceae family. Especially preferred from the genus Azoarcus. Especially preferred from species Azoarcus anaerobius, Azoarcus buckelii, Azoarcus communis, Azoarcus evansii, Azoarcus indigens, Azoarcus toluclasticus, Azoarcus tolulyticus, Azoarcus toluvorans, Azoarcus sp., Azoarcus sp. 22Lin, Azoarcus sp. BH72, Azoarcus sp. CC-11, Azoarcus sp. CIB, Azoarcus sp. CR23, Azoarcus sp. EB1, Azoarcus sp. EbN1, Azoarcus sp. FL05, Azoarcus sp. HA, Azoarcus sp. HxN1, Azoarcus sp. mXyN1, Azoarcus sp. PBN1, Azoarcus sp. PH002, Azoarcus sp. T and Azoarcus sp. TON1.

Especially Dehydrogenases from Azoarcus sp EbN1 are preferably used.

Nucleic acid sequences according to the invention can be, for example, with usual Hybridization method or PCR technique from other organisms, e.g. above genomic or cDNA libraries, isolate. These DNA sequences hybridize under standard conditions with the sequences according to the invention. For hybridization become advantageous short oligonucleotides of the conserved areas for example, from the active center, via comparisons with a dehydrogenase according to the invention can be determined in a manner known to those skilled in the art used. It can but also longer Fragments of the nucleic acids or the complete Sequences for the hybridization can be used. Depending on the nucleic acid used (oligonucleotide, longer Fragment or complete Sequence) or whichever nucleic acid type DNA or RNA for hybridization used, these standard conditions vary. So lie for example, the melting temperatures for DNA: DNA hybrids about 10 ° C lower as that of DNA: RNA hybrids of equal length.

Under Standard conditions are, for example, temperatures depending on the nucleic acid between 42 and 58 ° C in an aqueous buffer solution a concentration between 0.1 to 5 x SSC (1 x SSC = 0.15 M NaCl, 15 mM Sodium citrate, pH 7.2) or in addition in the presence of 50% formamide such as 42 ° C in 5x SSC, 50% To understand formamide. Advantageously, the hybridization conditions are for DNA: DNA hybrids at 0.1x SSC and temperatures between about 20 ° C to 45 ° C, preferably between about 30 ° C to 45 ° C. For DNA: RNA hybrids the hybridization conditions are advantageously 0.1 × SSC and temperatures between about 30 ° C up to 55 ° C, preferably between about 45 ° C up to 55 ° C. These indicated temperatures for hybridization are exemplary calculated melting temperature values for one nucleic acid with a length of about 100 nucleotides and a G + C content of 50% in the absence of formamide. The experimental conditions for DNA hybridization are described in relevant textbooks Genetics, such as Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory, 1989; described and can be after the expert known formulas for example, dependent of the length the nucleic acids, calculate the type of hybrid or G + C content. additional Information for hybridization, one skilled in the art can refer to the following textbooks: Ausubel et al. (eds), 1985, Current Protocols in Molecular Biology, John Wiley & Sons, New York; Hames and Higgins (eds), 1985, Nucleic Acids Hybridization: A Practical Approach, IRL Press at Oxford University Press, Oxford; Brown (ed), 1991, Essential Molecular Biology: A Practical Approach, IRL Press at Oxford University Press, Oxford.

object The invention also relates to derivatives of the specifically disclosed or derivable Nucleic acid sequences.

So can further nucleic acid sequences according to the invention derived from SEQ ID NO: 1 and differing therefrom by addition, substitution, Distinguish insertion or deletion of single or multiple nucleotides, but continue for Polypeptides with the desired Encode property profile.

According to the invention are also such nucleic acid sequences, include the so-called silent mutations or according to the Codon usage of a particular source or host organism, in comparison are changed to a specific sequence, as well as naturally occurring Variants, such as splice variants or allelic variants, of which.

object are also protected by conservative nucleotide substitutions (i.e. Amino acid is by an amino acid same charge, size, polarity and / or solubility replaced) available Sequences.

object of the invention are also those by sequence polymorphisms of the specifically disclosed nucleic acids derived molecules. These genetic polymorphisms can occur between individuals of a population due to natural variation exist. This natural Variations usually work a variance of 1 to 5% in the nucleotide sequence of a gene.

Under Derivatives of a nucleic acid sequence according to the invention For example, allelic variants are to be understood that are at least 40% homology at the deduced amino acid level, preferably at least 60% homology, most preferably at least 80, 85, 90, 93, 95 or 98% homology over have the entire sequence region (with respect to homology at the amino acid level be to the above comments refer to the polypeptides). About subregions of the sequences can the homologies are advantageously higher lie.

Farther Derivatives are also homologs of the nucleic acid sequences according to the invention for example fungal or bacterial homologs, shortened sequences, Single-stranded DNA or RNA of the coding and non-coding DNA sequence, to understand. For example, at the DNA level a homology of at least 40%, preferably at least 60%, more preferably of at least 70%, more preferably of at least 80 % above the entire specified DNA range.

Besides, they are Derivatives for example, to understand mergers with promoters. The promoters preceding the indicated nucleotide sequences are, can by one or more nucleotide exchanges, insertions, inversions and / or deletions changed be, but without the functionality or effectiveness of the promoters impaired are. Furthermore, you can the promoters by change their effectiveness increased or completely by more effective Promoters of alien organisms are exchanged.

Under Derivatives are also variants to understand their nucleotide sequence in the range of -1 to -1000 Bases upstream before the start codon or 0 to 1000 bases downstream changed the stop codon so have been altered that alter gene expression and / or protein expression is increased.

Farther The invention also encompasses nucleic acid sequences which have been described above These coding sequences hybridize under "stringent conditions" Polynucleotides can be used in the screening of genomic or cDNA libraries find and, where appropriate, with suitable primers by means of Multiply PCR and then for example, with suitable probes isolate. Furthermore can Polynucleotides according to the invention also be synthesized by chemical means. Under this property one understands the ability a poly or oligonucleotide under stringent conditions to a almost complementary To bind sequence while under these conditions non-specific bonds between non-complementary partners remain under. For this, the sequences should be 70-100%, preferably to 90-100%, complementary be. The property complementary For example, you can make sequences that bind to each other specifically in the Northern or Southern Blot technique or in the primer binding use in PCR or RT-PCR. Usually For this purpose, oligonucleotides from a length of 30 base pairs are used. Under stringent conditions one understands for example in the Northern blotting technique, the use of a 50-70 ° C, preferably 60-65 ° C warm Wash solution, For example 0.1x SSC buffer with 0.1% SDS (20x SSC: 3M NaCl, 0.3M Na citrate, pH 7.0) for the elution of nonspecifically hybridized cDNA probes or oligonucleotides. Remain, as mentioned above, only to a high degree complementary nucleic acids tied together. The setting of stringent conditions is known in the art and is z: B. in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. described.

E. Embodiments of inventive constructs

object of the invention are also Expression constructs containing under genetic control regulatory nucleic acid sequences one for a polypeptide according to the invention coding nucleic acid sequence; and vectors comprising at least one of these expression constructs.

Preferably include such constructs of the invention 5'-upstream of the respective coding sequence a promoter and 3'-downstream one Terminator sequence and optionally other common regulatory elements, each operatively linked with the coding sequence.

Under an "operational Linking "one understands the Sequential arrangement of promoter, coding sequence, terminator and optionally other regulatory elements such that each the regulatory elements have their function in the expression of the coding Sequence can meet as intended. examples for operatively linkable Sequences are targeting sequences as well as enhancers, polyadenylation signals and the same. Other regulative elements include selectable ones Markers, amplification signals, origins of replication, and the like. suitable regulatory sequences are e.g. described in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).

Under a nucleic acid construct according to the invention In particular, those are to be understood in which the gene for a dehydrogenase according to the invention with one or more regulatory signals for control, e.g. Increase, gene expression was linked operatively or functionally.

In addition to These regulatory sequences may be the natural regulation of these sequences be present before the actual structural genes and optionally genetically modified have been so natural Regulation switched off and the expression of genes was increased. The nucleic acid construct but can also be simpler, that is, there were no additional Regulation signals inserted before the coding sequence and the natural Promoter with its regulation was not removed. Instead becomes the natural one Regulatory sequence mutated so that no regulation takes place and gene expression is increased.

One preferred nucleic acid construct contains advantageously also one or more of the already mentioned "enhancer" sequences, functional connected with the promoter, which increased one Expression of the nucleic acid sequence enable. Also at the 3'-end the DNA sequences can additional advantageous sequences are inserted, such as further regulatory Elements or terminators. The nucleic acids according to the invention can be used in one or more copies in the construct. In the construct can even more markers, such as antibiotic resistance or auxotrophs complementing genes, optionally for selection on the construct be included.

Advantageous regulatory sequences for the novel process are for example in promoters such as cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacI ^q-, T7 T5, T3 , gal, trc, ara, rhaP (rhaP _BAD ) SP6, lambda P _R - or contained in the lambda P _L promoter, which are advantageously used in gram-negative bacteria application. Further advantageous regulatory sequences are contained, for example, in the gram-positive promoters amy and SPO2, in the yeast or fungal promoters ADC1, MFalpha, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH. In this context, the promoters of pyruvate decarboxylase and methanol oxidase, for example from Hansenula, are also advantageous. It is also possible to use artificial promoters for regulation.

The nucleic acid construct is advantageously inserted into a host organism for expression in a vector, such as a plasmid or a phage, which enables optimal expression of the genes in the host. Vectors other than plasmids and phages are also all other vectors known to those skilled in the art, ie viruses such as SV40, CMV, baculovirus and adenovirus, transposons, IS elements, phasmids, cosmids, and linear or circular DNA. These vectors can be autonomously replicated in the host organism or replicated chromosomally. These vectors represent a further embodiment of the invention. Suitable plasmids are described, for example, in E. coli pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pKK223-3, pDHE19.2, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III ¹¹³ -B1, IgT11 or pBdCl, in Streptomyces pIJ101, pIJ364, pIJ702 or pIJ361, in Bacillus pUB110, pC194 or pBD214, in Corynebacterium pSA77 or pAJ667, in fungi pALS1, pIL2 or pBB116, in yeasts 2alphaM, pAG -1, YEp6, YEp13 or pEMBLYe23 or in plants pLGV23, pGHlac ⁺ , pBIN19, pAK2004 or pDH51. The plasmids mentioned represent a small selection of the possible plasmids. Further plasmids are well known to the person skilled in the art and can be found, for example, in the book Cloning Vectors (Eds. Pouwels PH et al., Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018 ).

advantageously, contains the nucleic acid construct For expression of the other genes contained additionally 3'- and / or 5'-terminal regulatory Sequences to increase expression, depending on the selected host organism and gene or genes for optimal expression selected become.

These Regulatory sequences are designed to target the expression of genes and enable protein expression. For example, depending on the host organism, this may mean that the gene is expressed or overexpressed after induction, or that it expresses immediately and / or overexpressed becomes.

The Regulatory sequences or factors can preferably the Gene expression of the introduced Positively influence genes and thereby increase them. So can a reinforcement of the regulatory elements advantageously at the transcriptional level by using strong transcription signals such as promoters and / or enhancers. But there is also a reinforcement Translation possible, for example, by improving the stability of the mRNA.

In In a further embodiment of the vector, the nucleic acid construct of the invention may be or the nucleic acid containing the invention Vector also advantageously in the form of a linear DNA in the Introduced microorganisms be and over heterologous or homologous recombination into the genome of the host organism to get integrated. This linear DNA can be linearized Vector such as a plasmid or only from the nucleic acid construct or the nucleic acid according to the invention.

For an optimal Expression of heterologous genes in organisms, it is advantageous the nucleic acid sequences according to the specific "codon usage" used in the organism. The "codon usage" can be on the basis of computer evaluations of other, known genes of the concerned Easily detect organism.

The Production of an Expression Cassette According to the Invention takes place by fusion of a suitable promoter with a suitable one coding nucleotide sequence and a terminator or polyadenylation signal. For this one uses common ones Recombination and cloning techniques, as described for example in T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and in T.J. Silhavy, M.L. Berman and L.W. Enquist, experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987) are described.

The recombinant nucleic acid construct or gene construct becomes expression in a suitable host organism advantageously inserted into a host-specific vector, which allows optimal expression of the genes in the host. Vectors are well known to those skilled in the art and may be obtained, for example, from "Cloning Vectors" (Pouwels P.H. et al., eds, Elsevier, Amsterdam-New York-Oxford, 1985) become.

F. Usable according to the invention host organisms

With Help the vectors of the invention or constructs are recombinant microorganisms to produce, which for example with at least one vector according to the invention are transformed and used to produce the polypeptides of the invention can be. Advantageously, the recombinant invention described above Constructs are introduced and expressed in a suitable host system. In this case, preference is given to familiar cloning methods known to the person skilled in the art. and transfection methods, such as co-precipitation, Protoplast fusion, electroporation, retroviral transfection and the like, used to bind said nucleic acids in respective expression system for expression. suitable Systems are used, for example, in Current Protocols in Molecular Biology, F. Ausubel et al., Eds., Wiley Interscience, New York 1997, or Sambrook et al. Molecular Cloning: A Laboratory Manual. 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

Also according to the invention homologously recombined microorganisms can be produced. This will be a Vector prepared containing at least a portion of a gene of the invention or a coding sequence, wherein optionally at least an amino acid deletion, Addition or substitution has been introduced to the sequence of the invention to change, e.g. Functionally to disrupts ("knockout" vector). The introduced sequence may e.g. also a homologue from one be a related microorganism or from a mammalian, yeast or insect source be derived. The vector used for homologous recombination may alternatively be designed so that the endogenous gene in homologous Recombination mutated or otherwise altered, but still the functional protein (e.g., upstream regulatory area be changed in such a way that thereby the Expression of the endogenous protein is changed). The modified section of the gene according to the invention is in the homologous recombination vector. The construction of suitable Vectors for homologous recombination are e.g. described in Thomas, K. R. and Capecchi, M.R. (1987) Cell 51: 503.

When recombinant host organisms for the nucleic acid according to the invention or the nucleic acid construct In principle, all prokaryotic or eukaryotic organisms come in question. Advantageously, microorganisms are used as host organisms like bacteria, fungi or yeasts used. Beneficial are gram-positive or gram-negative bacteria, preferably bacteria of the families Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Streptomycetaceae or Nocardiaceae, particularly preferred Bacteria of the genera Escherichia, Pseudomonas, Streptomyces, Nocardia, Burkholderia, Salmonella, Agrobacterium or Rhodococcus used.

All particularly preferred is the genus and species Escherichia coli. Further beneficial bacteria are about it addition, in the group of alpha-proteobacteria, beta-proteobacteria or to find gamma proteobacteria.

Of the Host organism or the host organisms according to the invention contain it preferably at least one of those described in this invention Nucleic acid sequences, nucleic acid constructs or vectors for encode an enzyme with dehydrogenase activity according to the invention.

The in the process according to the invention used organisms are depending on the host organism in the expert well-known or cultivated. Become microorganisms usually in a liquid Medium containing a carbon source mostly in the form of sugars, a Nitrogen source mostly in the form of organic nitrogen sources like yeast extract or salts like ammonium sulfate, trace elements like Iron, manganese, magnesium salts and optionally vitamins Temperatures between 0 ° C and 100 ° C, preferably between 10 ° C up to 60 ° C attracted under oxygen fumigation. In this case, the pH of the nutrient fluid be kept at a fixed value, that is regulated during the cultivation be or not. The cultivation can be batchwise, semi-batchwise or continuous respectively. nutrient can submitted at the beginning of the fermentation or semicontinuously or continuously refilled become. The ketone can be added directly to the culture or advantageous after cultivation. The enzymes can according to the method described in the examples from the organisms be isolated or used as crude extract for the reaction.

G. Recombinant production the polypeptides of the invention

object The invention furthermore relates to processes for recombinant production polypeptides of the invention or functional, biologically active fragments thereof, wherein a polypeptide-producing microorganism is cultured, optionally the expression of the polypeptides induced and these from the culture isolated. The polypeptides can so also in large-scale scale produced if desired.

Of the Recombinant microorganism can be cultured by known methods and fermented. Bacteria can be found in TB or LB medium and at a temperature of 20 to 40 ° C and a pH be increased from 6 to 9. Specifically, suitable cultivation conditions for example in T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989).

The Cells then become, if the polypeptides are not in the culture medium be secreted, open-minded and the product according to known Protein isolation method recovered from the lysate. The cells can optionally by high frequency ultrasound, by high pressure, e.g. in a French pressure cell, by osmolysis, by the action of detergents, lytic enzymes or organic solvents, by homogenizers or digested by combining several of the listed methods become.

A Purification of the polypeptides can be carried out by known, chromatographic Be achieved, such as molecular sieve chromatography (gel filtration), like Q-Sepharose chromatography, Ion exchange chromatography and hydrophobic chromatography, as well with other usual ones Methods such as ultrafiltration, crystallization, salting out, dialysis and native gel electrophoresis. Suitable methods are, for example in Cooper, F.G., Biochemical Working Methods, Verlag Walter de Gruyter, Berlin, New York or in Scopes, R., Protein Purification, Springer Verlag, New York, Heidelberg, Berlin.

Advantageous it may be to isolate the recombinant protein vector systems or oligonucleotides that encode the cDNA for particular nucleotide sequences extend and for that changed Encode polypeptides or fusion proteins, e.g. a simpler one Serve cleaning. Such suitable modifications are, for example anchors acting as anchors, such as. as a hexa-histidine anchor known modification or epitopes recognized as antigens of antibodies can be (described, for example, in Harlow, E. and Lane, D., 1988, Antibodies: A Laboratory Manual. Cold Spring Harbor (N.Y.) Press). These anchors can for attaching the proteins to a solid support, e.g. a polymer matrix, serve, for example, be filled in a chromatography column can be used, or on a microtiter plate or on another carrier can be.

simultaneously can these anchors can also be used to recognize the proteins. to Detection of proteins can also usual markers, like fluorescent dyes, enzyme markers after reaction with a Substrate form a detectable reaction product, or radioactive Marker, alone or in combination with the anchors for derivatization the proteins are used.

H. Implementation of the inventive method for the preparation of (S) -alkanols

The inventively used enzymes with dehydrogenase activity can in the process according to the invention be used as a free or immobilized enzyme.

The inventive method is advantageous at a temperature between 0 ° C to 95 ° C, preferably between 10 ° C up to 85 ° C, more preferably between 15 ° C up to 75 ° C carried out.

Of the pH value in the process according to the invention is advantageously between pH 4 and 12, preferably between pH 4.5 and 9, more preferably between pH 5 and 8.

Under enantiomerically pure or chiral products or optically active Alcohols are in the process of the invention To understand enantiomers that show an enantiomeric enrichment. Preferably in the process enantiomeric purities of at least 70% ee, preferably from min. 80% ee, more preferably from min. 90% ee, most preferably min. 98% ee achieved.

For the inventive method can growing cells are used, the nucleic acids according to the invention, nucleic acid constructs or vectors. Also dormant or open-minded cells can be used. Under-opened cells are for example Understand cells that are over made a treatment with, for example, solvents permeable or cells are over an enzyme treatment, about a mechanical treatment (e.g. French Press or ultrasound) or over another method was broken up. The crude extracts thus obtained are for the inventive method advantageous suitable. Also purified or purified enzymes may be used for the procedure be used. Also suitable are immobilized microorganisms or enzymes that can be used to advantage in the reaction.

Become for the inventive method free organisms or enzymes used, these are the ones before Extraction conveniently separated over, for example a filtration or centrifugation.

The in the process according to the invention prepared product, for example (1S) -3-methylamino-1- (2-thienyl) -propan-1-ol, let yourself advantageous from the aqueous Reaction solution via extraction or win distillation. The extraction can increase the Yield be repeated several times. Examples of suitable extraction agents are solvents, such as toluene, methylene chloride, butyl acetate, diisopropyl ether, benzene, MTBE or ethyl acetate, without being limited thereto.

alternative this can be done in the process according to the invention prepared product, for example (1S) -3-methylamino-1- (2-thienyl) -propan-1-ol, advantageous from the organic phase of the reaction solution via extraction or win distillation or / and crystallization. The extraction can increase the yield can be repeated several times. Examples of suitable Extractants are solvents, such as toluene, methylene chloride, butyl acetate, diisopropyl ether, benzene, MTBE or ethyl acetate, without being limited thereto.

To Concentration of the organic phase can the products usually in good chemical purities, that is greater than 80% chemical purity, be recovered. After extraction, the organic phase with the product but only partially be narrowed and the Product be crystallized. For this, the solution is advantageous to a temperature of 0 ° C up to 10 ° C cooled. The crystallization can also be directly from the organic solution or from an aqueous solution respectively. The crystallized product can again in the same or in another solvent be re-crystallized and recrystallized again become. By the subsequent advantageous at least once performed crystallization, the Enantiomeric purity of the product further increased if necessary.

at The aforementioned work-up methods can be the product of the method according to the invention in yields of 60 to 100%, preferably from 80 to 100%, especially preferably from 90 to 100%, based on the used for the reaction Substrate, e.g. of 3-methylamino-1- (2-thienyl) -propan-1-one, isolate. The isolated product is characterized by a high chemical Purity of> 90 %, preferably> 95 % particularly preferred of> 98 % out. Furthermore, the products have a high enantiomeric purity, the advantageous if necessary by the crystallization on can be increased.

The inventive method can be operated batchwise, semi-batchwise or continuously.

The execution of the method can advantageously take place in bioreactors, such as. described in Biotechnology, Volume 3, 2nd edition, Rehm et al. Eds., (1993) especially Chapter II.

The The above description and the following examples are for the purposes of the Clarification of the invention. The obvious to the expert, numerous possible Modifications are also according to the invention includes.

Of the Advantage of the method according to the invention lies in the particularly high yield of the optically active alkanol of the formula (I) or the almost quantitative conversion of alkanone (II).

Experimental part:

Example 1: Cloning the phenylethanol dehydrogenase from Azoarcus sp EbN1 by means of synthetic Oligonucleotides.

The sequence of the phenylethanol dehydrogenase gene from Azoarcus sp EbN1 is stored in databases (Genbank ID 25956124, region: 25073 to 25822). From the nucleic acid sequence of the phenylethanol dehydrogenase gene oligonucleotides were derived from which the gene was synthesized by known methods. The DNA sequence of the oligonucleotides is summarized in Table 1. 3 shows the position of each oligonucleotide to the entire phenylethanol dehydrogenase gene from Azoarcus sp EbN1. Methods for preparing synthetic genes are described, for example, in YP Shi, P. Das, B. Holloway, V. Udhayakumar, JETongren, F. Candal, S. Biswas, R. Ahmad, SE Hasnain and AA Lal, Vac-cine 2000, 18 , Pp. 2902-2914. The sequence obtained is identical to the published sequence.

The PCR product was digested with the restriction endonucleases Ndel and BamHI and in appropriately digested pDHE19.2 vector ( DE19848129 ) cloned. The ligation mixtures were transformed into E. coli XL1 Blue (Stratagene). The plasmid was pDHEEbN1 into strain E.coli TG10 transformed pAgro4 pHSG575 (TG10: an RhaA ^- derivative of E.coli TG1 (Stratagene); pAgro4: Takeshita, S; Sato, M; Toba, M; Masahashi, W; Hashimoto -Gotoh, T (1987) Gene 61, 63-74; pHSG575: T. Tomoyasu et al (2001), Mol. Microbiol. 40 (2), 397-413).

The Recombinants E. coli are designated E. coli LU 11558.

Table 1 Oligonucleotides for the preparation of the synthetic phenylethanol dehydrogenase gene from Azoarcus sp EbN1

Example 2: Cloning of phenylethanol dehydrogenase from Azoarcus sp EbN1 by PCR

The sequence of the phenylethanol dehydrogenase gene from Azoarcus sp EbN1 is available in databases (Genbank ID 25956124, region 25073 to 25822). From the nucleic acid sequence of the phenylethanol dehydrogenase gene, oligonucleotides were derived (primers MKe0387 and MKe0388) which served to clone the dehydrogenase gene by PCR amplification as follows.

PCR:

primer:

ever 130 ng of primers MKe0387 and MKe0388 were mixed equimolarly. The PCR was performed according to Stratagene standard protocol with PfuTurbo polymerase (Roche Applied Science) and the following temperature gradient program carried out: 95 ° C for 5 min; 30 cycles at 95 ° C for 45 sec, 55 ° C for 45 sec, and 72 ° C for 1 min; 72 ° C for 10 min .; 10 ° C to for use. The PCR product (~ 0.75 kb) was purified by agarose gel electrophoresis (1.2% E-gel, Invitrogen) and column chromatography (GFX kit, Amersham Pharmacia) and then sequenced (Sequencing primer: MKe0387 and MKe0388). The sequence obtained is with identical to the published sequence.

The PCR product was digested with the restriction endonucleases Ndel and BamHI and in appropriately digested pDHE19.2 vector ( DE19848129 ) cloned. The ligation mixtures were transformed into E. coli XL1 Blue (Stratagene). The sequencing of corresponding clones gave as an insert in the plasmid pDHEEbN1 thus obtained the nucleic acid sequence shown in SEQ ID NO: 1,

The plasmid was pDHEEbN1 into strain E.coli TG10 transformed pAgro4 pHSG575 (TG10: an RhaA ^- derivative of E.coli TG1 (Stratagene); pAgro4: Takeshita, S; Sato, M; Toba, M; Masahashi, W; Hashimoto -Gotoh, T (1987) Gene 61, 63-74; pHSG575: T. Tomoyasu et al (2001), Mol. Microbiol. 40 (2), 397-413).

The Recombinants E. coli are designated E. coli LU 11558.

Example 3: Provision recombinant phenylethanol dehydrogenase from E. coli LU 11558

E.coli LU 11558 were amplified in 20mL LB-Amp / Spec / Cm (100μg / L ampicillin; 100μg / L spectinomycin; 20 μg / l chloramphenicol), 0.1mM IPTG, 0.5g / L rhamnose in 100mL Erlenmeyer flasks (baffles) 18 h at 37 ° C attracted, at 5000 x g / 10 min centrifuged, washed once with 10mM TRIS * HCl, pH 7.0 and in 2 mL of the same buffer resuspended.

cell-free Protein crude extract was prepared by cell paste of E. coli LU 11558 with 0.7ml glass balls (d = 0.5mm) in a vibrating mill (3 × 5min with intercooling on ice) was unlocked.

Example 4: Activity determination recombinant dehydrogenase from E. coli LU 11558

Six transformants each were grown in 100mL LBAmp / Spec / Cm (100μg / l amp; 100mg / l Spec; 20μg / lcm) 0.1mM IPTG 0.5g / L rhamnose in 100mL Erlenmeyer flasks (baffles) for 18h at 37 ° C Centrifuged, washed once with 10 mM Tris / HCl pH 7.0 and resuspended in 2 mL of the same buffer. 100 ml of cell suspension were incubated for 20 min in 900 μl of 50 mM MES pH6 with 50 μl / ml glucose-DH, 100 mM glucose, 100 mM NaCl, 1 mM NADH, 1 mM NADPH and with 10 mM 3-chloro-1- (thien-2-yl) -propane-1-on shaking incubated. The batches were analyzed analogously to Example 4. On average, 0.13 mM of 3-chloro-1- (thien-2-yl) -propan-1-ol was formed, which corresponds to an activity of 6.6 U / L of culture suspension. In ana Logen mixtures with crude extract, which was obtained by cell disruption with 0.7 ml glass beads (d = 0.5 mm) in a vibrating mill (3 × 5 min with intercooling on ice), 0.21 mM 3-chloro-1- (thien-2 -yl) -propan-1-ol, corresponding to an activity of 10.7 U / L.

In Control experiments without the addition of rhamnose at the time of cultivation was not 3-chloro-1- (thien-2-yl) -propan-1-ol detectable.

Example 5 Analysis of 3-Chloro-1- (thien-2-yl) -propan-1-one and 3-chloro-1 (thien-2-yl) -propan-1-ol

The Concentration of 3-chloro-1- (thien-2-yl) -propan-1-one and 3-chloro-1- (thien-2-yl) -propan-1-ol let determine by HPLC. Depending on the choice of stationary and Mobile phase can be determined in addition to the concentration and ee value.

a) achiral analytics

The quantification of the reaction was carried out with the following system: stationary phase: Chromoltih SpeedROD RP18, 50 · 4, 6μm, Merck (Darmstadt) tempered to 45 ° C mobile phase: Mobile phase A: 10 mM KH ₂ PO ₄ , pH 2.5 mobile phase B: acetonitrile gradient: 0-0.5 min, 35% B; 0.5-1.0 min 35 to 80% B; 1.0-1.2 min 80% B; 1.2-1.3 min 80% - 35% B; 1.3-2.0 min 35% B; Flow rate: 1.5 ml / min detection: UV detection at 230 and 260nm Retention times: 3-Chloro-1- (thien-2-yl) -propan-1-one: about 1.6 min 3-chloro-1- (thien-2-yl) -propan-1-ol: about 1.3 min

Authentic material is used to create a calibration series that can be used to determine the concentration of unknown samples. b) chiral analytics stationary phase: Chiracel OD-H, 250 x 4, 6μm, Daicel, tempered to 40 ° C mobile phase: Eluent A: n-hexane Eluent B: iso-propanol isocratic with 2.5% B Flow rate: 1.0 ml / min detection: UV detection at 230 and 260nm Retention times: 3-chloro-1- (thien-2-yl) -propan-1-one: about 9.5 min (1S) -3-chloro-1- (thien-2-yl) -propan-1-ol: ca. 16.6 min (1R) -3-chloro-1- (thien-2-yl) -propan-1-ol: ca. 18.3 min

With authentic material, a set of standards is prepared on the basis of which the concentration of unknown samples can be determined.

Example 6: Activity Determination recombinant dehydrogenase from E. coli LU 11558

Six transformants each of E.coli LU 11558 were added in 20mL LBAmp / Spec / Cm (100μg / l amp; 50mg / lpec; 10μg / lcm) 0.1mM IPTG 0.5g / L rhamnose in 100mL Erlenmeyer flasks (baffles) for 18h 37 ° C, centrifuged at 5000 x g / 10 min, washed once with 10 mM Tris / HCl pH 7.0 and resuspended in 2 mL of the same buffer. 100 ml of cell suspension were incubated for 20 min in 900 μl of 50 mM MES pH6 with 50 μl / ml glucose-DH (example...), 100 mM glucose, 100 mM NaCl, 1 mM NADH, 1 mM NADPH and 10 mM 3-chloro -1- (thien-2-yl) -propan-1-one shaking incubated. The mixtures were analyzed analogously to Example 4. On average, 0.13 mM of 3-chloro-1- (thien-2-yl) -propan-1-ol was formed, which corresponds to an activity of 6.6 U / L of culture suspension. In analogous runs with crude extract obtained by cell digestion with 0.7 ml glass beads (d = 0.5 mm) in a vibration mill (3 × 5 min with intercooling on ice), 0.21 mM 3-chloro-1- (thien-2-yl) -propan-1-ol, corresponding to an activity of 10.7 U / L. In control experiments without the addition of rhamnose at the time of cultivation, no 3-chloro-1- (thien-2-yl) -propan-1-ol was detectable.

Example 7: Preparation of (S) -3-chloro-1- (thien-2-yl) -propan-1-ol) with the recombinant Dehydrogenase from Azoarcus sp EbN1 with 2-pentanol

Resting cells of E. coli LU 11558 (1-20 g / L biomass) were incubated with 0.2 mM NAD ⁺ and 185 mM 3-chloro-1- (thien-2-yl) -propan-1-one (FIG. 5 ml) in 221 ml of 50 mM KH ₂ PO ₄ , and 250 ml of 2-pentanol at 40 ° C in a 0.75 L reactor (250 rpm). The reaction was maintained at pH 5.5 by titration with 5 M NaOH. The reaction was followed by HPLC analysis to a concentration of 50-350 mM TACA in the organic phase.

Example 8: Preparation of (S) -3-chloro-1- (thien-2-yl) -propan-1-ol) with the recombinant Dehydrogenase from Azoarcus sp EbN1 with 2-pentanol

Resting cells of E. coli LU 11558 (1-20 g / L biomass) were incubated with 0.2 mM NAD ⁺ and 370 mM 3-chloro-1- (thien-2-yl) -propan-1-one (200 mL ) in 1432 ml 50mM KH ₂ PO ₄ , and 2000 ml of 2-pentanol at 40 ° C in a 4 L reactor (250 rpm). The reaction was maintained at pH 5.5 by titration with 5 M NaOH. The reaction was followed by HPLC analysis to a concentration of 100-650 mM TACA in the organic phase.

Example 9: Production of (S) -3-chloro-1- (thien-2-yl) -propan-1-ol) with the recombinant Dehydrogenase from Azoarcus sp EbN1 with 2-pentanol

Resting cells of E. coli LU 11558 (1-20 g / L biomass) were incubated with 0.2 mM NAD ⁺ and 370 mM 3-chloro-1- (thien-2-yl) -propan-1-one (200 mL ) in 1432 ml 50mM KH ₂ PO ₄ , and 2000 ml of 2-pentanol at 40 ° C in a 4 L reactor (250 rpm). The reaction was maintained at pH 5.5 by titration with 5 M NaOH. A vacuum of about 85 mbar and a jacket temperature of 50-70 ° C was applied. The amounts of distillate of water and 2-pentanol obtained at distillation rates of 2-10 ml / min were added continuously during the reaction. The reaction was followed by HPLC analysis to a concentration of 300-700 mM TACA in the organic phase. The 3-chloro-1- (thien-2-yl) -propan-1-one was reacted to 0-30% residue.

Example 10: Production of (S) -3-chloro-1- (thien-2-yl) -propan-1-ol) with the recombinant Dehydrogenase from Azoarcus sp EbN1 with 2-pentanol

Resting cells of E. coli LU 11558 (1-20 g / L biomass) were incubated with 0.2 mM NAD ⁺ and 200 mM 3-chloro-1- (thien-2-yl) -propan-1-one (FIG. 5 ml) in 221 ml of 50 mM KH ₂ PO ₄ , and 250 ml of 2-pentanol at 40 ° C in a 0.75 L reactor (250 rpm). The reaction was maintained at pH 5.5 by titration with 5 M NaOH. The reaction was followed by HPLC analysis to a concentration of 50-320 mM TACA in the organic phase.

Claims (13)

Process for the preparation of optically active alkanols of the formula I

wherein n is an integer value of 0 to 5; Cyc is an optionally substituted, mono- or polynuclear, saturated or unsaturated, carbocyclic or heterocyclic ring, and R ^{1 is} halogen, SH, OH, NO ₂ , NR ² R ³ or NR ² R ³ R ^{4 +} X ^- , wherein R ² , R ³ and R ^{4 are} independently H or a lower alkyl or lower alkoxy radical and X ^{- is} a counterion, wherein in an alkanone of formula II

wherein n, Cyc and R ^{1 have} the meanings given above, an enzyme (E) selected from the classes of dehydrogenases, aldehyde reductases and carbonyl reductases, incubated in the presence of reducing equivalents, wherein the compound of formula II to the compound of formula I is reduced enzymatically, and the reduction equivalents used in the course of the reaction are regenerated by reacting a sacrificial alcohol to the corresponding sacrificial ketone with the aid of the enzyme (E) and the sacrificial ketone is at least partially removed from the reaction medium, and the resulting Product (I) isolated. Method according to claim 1, characterized in that the enzyme (E) is an alcohol dehydrogenase. Method according to claim 1, characterized in that the enzyme (E) is a polypeptide sequence (i) SEQ ID NO: 2 or (Ii) in up to 25% of the amino acid residues across from SEQ ID NO: 2 by deletion, insertion, substitution or a combination have changed and still at least 50% of the enzymatic activity of SEQ ID NO: 2 has. Process according to Claim 1, for the preparation of derivatives of 1- (2-thienyl) - (S) -propanol of the formula III

where R ¹ = Cl or NHCH ₃ , which is in a derivative of 1- (2-thienyl) -propanone of the formula IV

containing medium, this compound is enzymatically reduced to the compound of formula III, and isolated the product formed in substantially enantiomerically pure form. Method according to one of the preceding claims, wherein the enzyme is selected is among enzymes from microorganisms of the genus Azoarcus. Method according to one of the preceding claims, wherein the enzyme from a nucleic acid sequence according to SEQ ID NO: 1 or a functional equivalent of which is coded. Method according to one of the preceding claims, wherein as a victim alcohol 2-pentanol is used. Method according to one of the preceding claims, wherein one while the victim from the sacrificial alcohol reaction from the victim Reaction medium removed by distillation at least partially. Method according to claim 8, characterized in that that the removal of the sacrificial ketone from the enzyme reactor during the Reaction is carried out by distillation. Method according to one of the preceding claims, wherein the reaction of the compound of the formula II in the presence of a microorganism done, the selected is among bacteria of the families Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Lactobacillaceae, Streptomycetaceae, Rhodococcaceae and Nocardiaceae. Method according to one of the preceding claims, wherein the microorganism is a recombinant A microorganism transformed with a nucleic acid construct encoding an enzyme as defined in any one of claims 1 to 3. Use of an enzyme (E) with a polypeptide sequence i. SEQ ID NO: 2 or ii. in up to 25% of the amino acid residues across from SEQ ID NO: 2 by deletion, insertion, substitution or a combination changed are and still at least 50% of the enzymatic activity of SEQ ID NO: 2 owns, for the preparation of compounds of the formulas I or III. Use according to claim 12 in a method for Preparation of Duloxetine.