WO2007099051A1

WO2007099051A1 - Method for the production of ketones by oxygenating secondary alcohols with the aid of an alcohol dehydrogenase in the presence of a cofactor and an ketone as a cosubstrate

Info

Publication number: WO2007099051A1
Application number: PCT/EP2007/051666
Authority: WO
Inventors: Christian Peschko; Jürgen STOHRER
Original assignee: Wacker Chemie Ag
Priority date: 2006-03-02
Filing date: 2007-02-21
Publication date: 2007-09-07
Also published as: DE102006009743A1

Abstract

Disclosed is a method for producing a ketone, which is characterized in that a secondary alcohol is reacted to the ketone in a reaction mixture containing water, an activated ketone, an alcohol dehydrogenase, and a cofactor.

Description

METHOD FOR THE PRODUCTION OF KETONS BY OXIDATION OF SECONDARY ALCOHOLS USING AN ALCOHOL DEHYDROGENASE IN THE PRESENCE OF A COFECTOR AND A KETONE AS A COSUBSTRATE

The present invention relates to an enzymatic process for the oxidation of secondary alcohols to the corresponding keto compounds.

Keto compounds are valuable building blocks in the synthesis of important compounds with pharmacological activity and other valuable properties.

Ketones are selected from the corresponding hydroxy compounds by oxidation reactions, e.g. the Oppenauer oxidation, gas-phase oxidations with air or oxygen, liquid-phase oxidations with nitric acid or bleach. However, such processes are often unsuitable for compounds that contain other functional groups that are not stable under the oxidation conditions (e.g., Oppenauer oxidation in strongly basic). Therefore, enzyme-catalyzed processes are increasingly used for the oxidation of functionalized hydroxy compounds, wherein the oxidation reaction is carried out with living or inactivated microorganisms or with completely or partially purified, isolated enzymes.

The dehydrogenases suitable for this purpose, and in particular alcohol dehydrogenases (ADH) or oxidoreductases, are known as valuable catalysts in numerous processes for obtaining chiral products by stereoselective reduction of organic keto compounds to the corresponding chiral alcohols (Current Opinion in Chemical Biology 2004, 8, 120- 126). In these reactions, the reduction of the ketone is coupled with the simultaneous oxidation of a reducing agent (cosubstrate). The catalyzing enzymes are derived, for example, from yeasts, horse liver, Thermoanaerobium brockii, Rhodococcus erythropolis (EP 1 499 716 A1), Lactobacillus kefir (US 5200335 A), Lactobacillus brevis (US Pat. No. 6,221,099 B1), Lactobacillus reuteri (US 2005 00191735 A1) or Rhodococcus ruber (US 2004 0157305 A1 or WO 2005/026338 Al). Frequently, overexpressed recombinant enzymes are used.

Alcohol dehydrogenases can in principle catalyze both the reduction of ketones and the oxidation of alcohols. From the prior art, in contrast to the reduction of ketones, only a few processes for the enzymatic oxidation of hydroxy compounds to the corresponding keto compounds by alcohol dehydrogenases are known (W. Stampfer et al., Biotechnology and Bioengineering, 2003, 81, 866-869 ).

DE 10 2004 037 669 A1 (Julich Enzyme Products), WO 2004 111083 A3 (Julich Enzyme Products), as well as US 2004 0265978 A1 (Julich Enzyme Products), et al. Alcohol dehydrogenase-catalyzed oxidations of secondary alcohols with aliphatic ketones and in particular acetone claimed as the oxidant, the authors prove this, however, by no means.

As the oxidizing agent acetone is used regularly. The advantageous use of other ketones, in particular of ketones whose oxidation capacity is increased, for example by heteroatoms in the vicinity of the carbonyl group, in comparison to acetone (referred to below as "activated ketones") is not known, only in K. Nakamura et al. , Tetrahedron Lett. 1994, 35,

4375-4376 describes that in the enzyme-catalyzed oxidation of 1-aryl alcohols cyclohexanone is added as solvent for the purpose of increasing the stereoselectivity.

In all known processes, the oxidant acetone is used in large excess.

In W. Kroutil et al. , Angew. Chem., 2002, 114, 1056-1059; or US 2004 0157305 Al and WO 2005/026338 Al an enzyme from Rhodococcus ruber (DSM 44541) is described, which is characterized by a high stability to acetone and is therefore used for oxidations of secondary alcohols with acetone as the oxidant. The NADH / NAD ⁺ -dependent enzyme is typically used in the form of lyophilized cells. The proportion of acetone used is typically three to ten molar equivalents relative to the alcohol to be oxidized and is typically 20% by volume of the batch size. The substrate loadings attainable therewith are typically well below 1 mol / L, which leads to low space-time performances of typically less than 20 mol / m ³ h (W.Kroutil et al., Tetrahedron: Asymmetry, 2003, 14 , 275-280).

Since many dehydrogenases often have only a low stability towards organic reactants in a higher concentration, the concentrations of organic compounds in the reaction mixture are kept as low as possible (W. Stampfer et al., Biotechnology and Bioengineering, 2003, 81, 866-869).

The use of excessively overstoichiometric amounts of acetone as oxidant thus only leads to low concentrations of the alcohol to be oxidized, which leads to a poor space-time yield and makes such enzymatic oxidation processes uneconomical. In addition, the high amounts of acetone used in a considerable effort in the workup and disposal and unokonomisch high consumption. Furthermore, the oxidation of secondary alcohols with non-activated ketones, such as acetone, even with high ketone excesses, does not generally lead to complete conversion of the alcohol used. However, the incomplete oxidation achievable in this way is problematic, since a separation of the ketone formed by oxidation from the remaining alcohol used is often extremely difficult and expensive. In addition, the incomplete conversion of the alcohol used naturally leads to poor yields, which is not very economical, especially with expensive alcohols.

The known enzymatic oxidation processes using non-activated ketones such as acetone are therefore uneconomical and impractical for industrial applications. The object of the invention is therefore to provide an economical enzymatic process for the preparation of ketones by oxidation of alcohols.

This object is achieved by a process for the preparation of ketones which is characterized in that a secondary alcohol in a reaction mixture containing water, an activated ketone as the oxidizing agent, an alcohol dehydrogenase and a cofactor is converted to the ketone.

The inventive method is characterized by high space-time performance at the same time low enzyme consumption and high chemical yields of up to> 95% based on the amount of secondary alcohol used, which allows a considerably easier isolation of the ketone. The use of large amounts of enzyme-damaging oxidizing agents, as is the case in the known processes with acetone as the oxidizing agent, is avoided. Furthermore, the method according to the invention is very simple in terms of apparatus and can therefore be implemented industrially.

As secondary alcohols it is generally possible to use chiral or achiral, racemic or enantiomerically enriched, secondary alcohols, preferably those having 3 to 40 C atoms.

In a preferred embodiment of the process according to the invention, secondary alcohols of the general formula (I)

R 1 is CH (OH) -R ² (I)

used, where

R ¹ and R ^{2 are the} same or different and are selected from the group Ci-C ₂ o-alkyl, C ₃ -C ₂ o-cycloalkyl, C ₅ -C ₂ o-aryl, C ₂ -C ₂₀ - heteroaryl, C C ₂ -C ₂₀ -alkenyl, C ₂ -C ₂₀ -alkynyl, C ₅ -C ₂₀ -aralkyl and C ₅ - C ₂ o-alkylaryl or R ¹ and R ² together with the group -CH (OH) - form a ring can consist of three to 40 ring members and R ¹ and R ² , or a ring formed by R ¹ and R ² with the group -CH (OH) -, may be optionally substituted with one or more radicals Z independently of one another, wherein

Z is selected from the group consisting of fluorine, chlorine, bromine, iodine, -CN, -NO ₂ , -NO, -NR ³ OR ³ , -C (O) R ³ , -SO ₃ R ³ , -C (O) OR ³ , -C (O) NR ³ R ³ , or -R ³ and

R ^{3 is} hydrogen or may have the meaning of R ¹ and in R ¹ and R ² optionally independently one or more methylene groups by the same or different groups

Y can be replaced, where

Y is selected from the group -CR ³ = CR ³ -, -C≡C-, -C (O) -, -C (O) O-, -OC (O) -, -C (O) OC (O ) -, -0-, -0-0-, -CR ³ = N-,

-C (O) -NR ³ -, -N = N-, -NR ³ -NR ³ -, -NR ³ -O-, -NR ³ -, -P (O) (OR ³ ) O-, -OP (O) (R ³ ) 0-, -P (R ³ ) -, -P (O) (R ³ ) -, -S-, -SS-, -S (O) -, -S (O) ₂ -, -S (O) NR ³ -, -S (O) (OR ³ ) 0-, -Si (R ³ ) ₂ -, -Si (R ³ ) ₂ O-, -Si (R ³ ) (OR ³ ) -,

-OSi (R ³ ) ₂ O-, -OSi (R ³ ) ₂ -or -Si (R ³ ) ₂ O Si (R ³ ) ₂ -.

Preferred C ₅ -C ₂ o-aryl or C ₂ -C ₂ o-heteroaryl radicals for R ¹ and R ² are in particular selected from the group consisting of phenyl, naphthyl, indolyl, benzofuranyl, thiophenyl, pyrrolyl, pyridinyl, imidazolyl, oxazolyl , Isoxazolyl, furanyl or thiazolyl.

The compounds of the general formula (I) can generally also be used as salts.

Particularly preferred compounds of the general formula (I) are 2-hydroxyalkanes, 3-hydroxyalkanes, 4-hydroxyalkanes, 2-hydroxyalkenes, 3-hydroxyalkene, 4-hydroxyalkene, 2-hydroxyalkynes, 3-hydroxyalkynes, 4-hydroxyalkynes, γ-hydroxyketones, δ-hydroxyketones, γ-hydroxyesters, δ-hydroxyesters, γ-hydroxyamides, δ-hydroxyamides, CC-hydroxy alcohols, β-

Hydroxy alcohols, γ-hydroxy alcohols or δ-hydroxy alcohols. Particularly suitable compounds of the general formula (I) are 2-butanol, 2-pentanol, 2-hexanol, 2-heptanol, 2-octanol, 2-nonanol, 2-decanol, 3-pentanol, 3-hexanol, 3-heptanol , 3-octanol, 3-nonanol, 3-decanol, 3-buten-2-ol, 4-penten-2-ol, 5-hexen-2-ol, 6-hepten-2-ol.

In the process according to the invention, the ketones formed from alcohols of the formula (I) are not identical to the ketones of the formula (II). Likewise, the alcohols of formula (I) are not the same as the alcohols formed from ketones of formula (II) when present in mixtures side by side.

The compounds of the general formula (I) are used in the process according to the invention in an amount of 1% by weight to 50% by weight, based on the total volume of the reaction mixture, preferably in an amount of 3% by weight to 25% by weight, in particular of 5% by weight to 15% by weight.

The reaction mixture should generally have a pH of from 5 to 11, preferably from 6 to 10.

The reaction mixture preferably contains a buffer, in particular a potassium phosphate / potassium hydrogenphosphate, tris (hydroxymethyl) aminomethane / HCl or triethanolamine / HCl buffer having a pH of from 5 to 11, preferably a pH of from 6 to 10. The buffer concentration is preferably from 5 mM to 150 mM.

In addition, the reaction mixture may also contain magnesium ions, for example in the form of added MgCl 2 in a concentration of 0.2 mM to 10 mM, preferably 0.5 mM to 2 mM, based on the amount of water used. In addition, the reaction mixture may contain other salts such as NaCl, and other additives such as dimethyl sulfoxide, glycerol, glycol, ethylene glycol, sorbitol, mannitol or sugar. The cofactor used is NADP, NADPH, NAD, NADH or salts thereof. The concentration of cofactor in the aqueous phase of the reaction mixture is preferably from 0.01 mM to 0.25 mM, more preferably from 0.02 mM to 0.1 mM.

As the oxidizing agent, the reaction mixture is preferably an activated ketone of the general formula (II)

R ⁴ -C (O) -X (II)

added, where

R ^{4 is} selected from the group Ci-C ₂ o-alkyl, C3-C2o-cycloalkyl, C ₅ -C ₂ o-aryl, C ₂ -C ₂ o-heteroaryl, C ₂ -C ₂ o-alkenyl, C ₂ -C ₂₀ alkynyl, C ₅ - C ₂ o-aralkyl and Cs-C ₂ o-alkylaryl wherein

R ^{4 may} optionally be substituted by one or more radicals Z, where

Z is selected from the group consisting of fluorine, chlorine, bromine, iodine I, --CCNN ,, --NOOO ₂₂ ,, --NOOO ,, --NNPR ³ OR ³ , -C (O) R ³ , -SO ₃ R ³ , -C (O) OR ³ , -C (O) NR ³ R ³ , or -R ³ and

R is hydrogen or may have the meaning of R and

in R ⁴ optionally one or more methylene groups may be replaced by identical or different groups Y, wherein

Y is selected from the group -C (O) -, -C (O) O-, -OC (O) -, -C (O) -NR ³ and

X is -C (O) R ⁵ , -C (O) OR ⁵ , -C (O) SR ⁵ , -C (O) NHR ⁵ , -C (O) NR ⁵ R ⁶ , -CN, or a group CH _n Q (3- _n) , where

R ⁴ and X together with the group -CO- can form a ring with three to 40 ring members and n = 0, 1 or 2 i st and

Q is selected from the group fluorine, chlorine, bromine, iodine, - OC (O) R ⁵ , -OC (O) OR ⁵ , -SO ₃ R ⁵ , -OR ⁵ , -SR ⁵ , -NO ₂ , -NO , -N ₃ , -NR ⁵ OR ⁶ , -CN, -C (O) R ⁵ , -C (O) OR ⁵ , -C (O) SR ⁵ , -C (O) NR ⁵ R ⁶ , wherein

R ⁵ and R ^{6 are} independently hydrogen, or may have the meaning of R ⁴ .

Preferably used compounds of general formula (II) are the group 3-0xo-carboxylic acid ester, 3-0xo-carboxylic acid amide, 2-0xo-carboxylic acid ester, 2-0xo-carboxylic acid amide, 2-oxo alcohols, 2- 0xoalkohol-ester, 2-0xoether, 2-0xoalkyl- halide, 2-Oxoalkyldihalogenid, 2-0xoalkyltrihalogenid, ß-diketone associated.

Particularly preferred compounds of general formula (II) are methyl acetoacetate, ethyl acetoacetate, t-butyl acetoacetate, ethylene glycol bisacetoacetate, methyl benzoylacetate, ethyl benzoylacetate, methyl benzoylformate, ethyl benzoylformate, methyl pyruvate, pyruvic acid ethyl ester, methoxyacetone, 2-methoxy-cyclohexanone, 1,3-dimethoxyacetone, chloroacetone, 2,4-pentanedione.

Very particular preference is given to methyl acetoacetate and ethyl acetoacetate.

The amount of activated ketone is 1 to 10 molar equivalents based on the alcohol to be oxidized in the reaction mixture, preferably 1 to 5 molar equivalents, more preferably 1.1 to 3 molar equivalents.

Suitable alcohol dehydrogenases are derived, for example, from hay fever liver or Rhodococcus erythropolis, which enzymes require NADH as cofactor, or from Thermoanaerobium spec, Lactobacillus kefir or Lactobacillus brevis, these enzymes requiring NADPH as cofactor. Particularly suitable alcohol dehydrogenases are the alcohol dehydrogenases ADH-LB (from Lactobacillus brevis) and ADH-T (from Thermoanaerobium spec), both commercially available from Jeutelh, Jülich, Germany.

The alcohol dehydrogenases can be used in the process of the invention either completely purified or partially purified or used in cells containing. The cells used can be native, permeabilized or lysed.

The volume activity of the alcohol dehydrogenases used is preferably 100 units / ml (U / ml) to 5,000 U / ml, more preferably about 1,000 U / ml.

In the reaction mixtures, preferably 20,000 to 700,000 U of alcohol dehydrogenase (ADH) are available for the reaction of each 1 kg of compound of the general formula (I). Particularly preferably, the ADH are used in the reaction mixtures with more than 5 U / ml.

The alcohol dehydrogenases can be selected in the process according to the invention so that the reaction of the alcohol to be oxidized takes place partially or completely stereoselectively (W. Hummel et al., Ann. N.Y. Acad. Sci. 1996, 755, 713-716). The use of stereospecific enzymes in the oxidation of a partially or completely racemic alcohol mixture in a manner known to those skilled in the preferred oxidation of a handedness of the alcohol, the isomeric form can not or only partially converted to the ketone and separated from the product mixture. The process according to the invention thus also makes it possible to prepare chiral enantiomerically pure or enriched alcohols by oxidative racemate resolution.

The simultaneous implementation of both handfulnesses of a partially or fully racemic alcohol mixture can be achieved by the use of enzymes with little or no stereospecificity, or the simultaneous or sequential use of enzymes with complementary stereospecificity.

The stepwise implementation of the complementary hand of an alcohol mixture can be carried out with the respective stereospecific enzymes in the manner described above.

The temperature of the reaction mixture is preferably from 0 ⁰ C to 60 ⁰ C, more preferably from 20 ⁰ C to 40 ° C.

In the course of the reaction, further activated ketone, in particular in the form of ketones of the general formula (II), or further secondary alcohol, in particular in the form of alcohols of the general formula (I), may be added.

The reaction is preferably carried out at a pressure of 10 mbar to 5 bar, more preferably at a pressure of 30 mbar to 1 bar.

Depending on the nature and amount of the alcohol dehydrogenases used and the compound of the general formula (I) used, the reaction time is between 30 minutes and 120 hours, preferably 1 hour to 50 hours.

The reaction mixture can be a homogeneous liquid phase, consist of two or more liquid phases, contain organic solvents and contain solid components. Two or more liquid phases may, for example, occur in the presence of limited or immiscible substances or of poorly or not water-miscible solvents in the reaction mixture. Solid constituents may, for example, be carrier materials for the enzymes used, such as eupergite, celite, kieselguhr or other materials known for this purpose.

The separation of the ketone formed during the reaction can be carried out by phase separation of the reaction mixture, or in a manner known to the skilled worker by extraction of the entire reaction mixture. reaction mixture or parts of the reaction mixture, carried out with an organic extraction phase.

After completion of the reaction of a secondary alcohol with the ketone and separation of the ketone, the enzyme-containing aqueous phase can again be admixed with alcohol and / or activated ketone as the oxidizing agent and used for a further reaction.

By purifying the reaction mixture or the organic extraction solution containing the crude product mixture, for example by distillation or other cleaning methods known in the art, such as recrystallization or chromatography, the desired ketone is obtained.

The products thus obtained are typically characterized by yields> 95% and chemical purities> 95%.

The process according to the invention for the oxidation of secondary alcohols enables the preparation of ketones from alcohols under conditions which also allow the presence of further chemically sensitive functions in the educts or products which under classical chemical reaction conditions (eg Oppenauer oxidation, oxidation with air or Oxygen or oxidation processes with nitric acid or bleach).

By using activated ketones of the general formula (II) as enzyme-compatible, organic oxidizing agents, the process according to the invention makes it possible to use these activated ketones in enzyme-compatible concentrations. The high reactivity and the enzyme compatibility of the activated ketones make possible, with low consumption of enzyme and oxidant, more complete reactions of the alcohol to be oxidized, and permit the reuse of the aqueous, enzyme-containing reaction mixture. The resulting high space-time performances thus allow the cost-effective conversion of alcohols to ketones using enzymes.

The inventive method allows the production of functionalized ketones, which were difficult to access from corresponding alcohols by oxidation, such as. 4-pentene-2-one.

The processes known from the prior art for the enzyme-catalyzed production of ketones from secondary alcohols give the expert no indication that the use of activated keto compounds for the reaction of alcohols under enzyme-contractual conditions, without high excesses of oxidizing agent and with high space Time achievements is possible.

The invention is further illustrated by the following examples:

Example Ia:

Reaction of 4-penten-2-ol with methyl acetoacetate and ADH-LB

Carrying out 100 mL of a mixture of water, phosphate buffer (50 mM), methyl acetoacetate (0.6 M), 25 U / mL ADH-LB (crude extract), NADP disodium salt (0.0415 mM) and racemic 4-penten-2-ol ( 0.5 M) was stirred vigorously at pH 8.0 in a 250 mL round bottom flask with magnetic stirrer and reflux condenser at 30 ⁰ C. After 24 hours, the solution was extracted with 4 × 50 ml of methyl tert-butyl ether (MTBE) and the product mixture was isolated by evaporation of the organic phases. By GC and NMR spectroscopy, the conversion to ketone and the amount of 4-penten-2-ol was determined in the balance.

Result

Conversion to ketone: 24 mmol (96% of theory) • 4-pentene-2-ol: 26 mmo1

• Space-time performance: 10 mol / m ³ h ketone

Example Ib:

Reaction of 4-penten-2-ol with methyl acetoacetate and ADH-T

execution

100 mL of a mixture of water, phosphate buffer (50 mM), methyl acetoacetate (0.6 M), 10 U / mL ADH-T (crude extract), NADP disodium salt (0.0415 mM) and 4-penten-2-ol (0.5 M ) of pH 8.0 was stirred round bottom flask with magnetic stirrer and reflux condenser vigorously at 30 ⁰ C in a 250 mL. After 24 hours, the solution was extracted with 4 × 50 ml of methyl tert-butyl ether (MTBE) and the product mixture was isolated by evaporation of the organic phases. By GC and NMR spectroscopy, the conversion to ketone and the amount of 4-penten-2-ol was determined in the balance.

Result

• conversion to ketone: 24 mmol (96% d.Th

• 4-penten-2-ol: 26 mmo1 • room-time: 10 mol / m ³ h ketone

Example Ic:

Reaction of 4-penten-2-ol with methyl acetoacetate, ADH-LB and ADH-T

execution

100 mL of a mixture of water, phosphate buffer (50 mM), methyl acetoacetate (0.6 M), 10 U / mL ADH-T (crude extract), 25 U / mL ADH-LB (crude extract) NADP disodium salt (0.083 mM ) and 4-penten-2-ol (0.5 M) of pH 8.0 were vigorously stirred at 30 ^° C. in a 250 ml round bottom flask with magnetic stirrer and reflux condenser. stir. After 24 hours, the solution was extracted with 4 × 50 ml of methyl tert-butyl ether (MTBE) and the product mixture was isolated by evaporation of the organic phases. By GC and NMR spectroscopy, the conversion to ketone in the weight was determined.

Result

Conversion to ketone: 48 mmol (96% of theory)

• Space-time performance: 20 mol / m ³ h ketone

Example Id:

Reuse of the extracted aqueous phase from Ia with ADH-LB

execution

The remaining after extraction with MTBE water phase

Example Ia was filled into a 250 mL round bottom flask with magnetic stirrer and reflux condenser, with methyl acetoacetate

(0.06 mol) and 4-penten-2-ol (0.05 mol) and stirred vigorously after adjustment to pH 8.0 at 30 ⁰ C.

After 24 hours, the solution was extracted with 4 × 50 ml MTBE and the product mixture was isolated by evaporation of the organic phases. By GC and NMR spectroscopy, the conversion to ketone and the amount of 4-penten-2-ol was determined in the balance.

Result

• conversion to ketone: 23 mmol (92% of theory) • 4-penten-2-ol: 27 mmo1

• Space-time performance: 9.6 mol / m ³ h ketone

Example Ie:

Reuse of the extracted aqueous phase from Ib with ADH-T

execution The remaining after extraction with MTBE water phase from Example Ib was placed in a 250 mL round bottom flask with magnetic stirrer and reflux condenser, with methyl acetoacetate (0.06 mol) and 4-penten-2-ol (0.05 mol) and after adjustment to pH 8, 0 vigorously stirred at 30 ⁰ C.

Result

• conversion to ketone: 23 mmo1 '92 d.Th.

• 4-pentene-2-ol: 27 mmo1

• Space-time performance: 9.6 mol / m ³ h ketone

Example 2a:

Reaction of hexan-2-ol with methyl acetoacetate and ADH-LB

execution

100 mL of a mixture of water, phosphate buffer (50 mM), methyl acetoacetate (0.54 M), 25 U / mL ADH-LB (crude extract), NADP disodium salt (0.0415 mM) and hexan-2-ol (0.49 M) of pH

6.5 was vigorously stirred at 30 ^° C. in a 250 ml round bottom flask with magnetic stirrer and reflux condenser. After 6 hours, the solution was extracted with 4 × 50 ml of methyl tert-butyl ether (MTBE) and the product mixture was isolated by evaporation of the organic phases. By GC and NMR spectroscopy, the conversion and yield of hexane-2-one was determined in the balance.

Result

Hexane-2-one yield: 21 mmo1 of theory

Hexane-2-ol: 28 mmol

Space-time performance: 35 mol / m ³ h hexane-2-one In game 2b:

Reaction of hexan-2-ol with methyl acetoacetate and ADH-T

execution

100 mL of a mixture of water, phosphate buffer (50 mM), methyl acetoacetate (0.54 M), 10 U / mL ADH-T (crude extract), NADP disodium salt (0.0415 mM) and hexan-2-ol (0.49 M) of pH 6.5 was stirred vigorously in a 250 ml round bottom flask with magnetic stirrer and reflux condenser at 30 ^° C. After 6 hours, the solution was extracted with 4 × 50 ml of methyl tert-butyl ether (MTBE) and the product mixture was isolated by evaporation of the organic phases. By GC and NMR spectroscopy, the conversion and yield of hexane-2-one was determined in the balance.

Result

• hexane-2-ye yield: 21 mmol (86% of theory) • hexane-2-ol: 28 mmol

Space-time performance: 35 mol / m ³ h hexane-2-one

Example 2c:

Reaction of hexan-2-ol with methyl acetoacetate, ADH-LB and ADH-T

execution

100 mL of a mixture of water, phosphate buffer (50 mM), acetic acid methyl ester (0.54 M), 10 U / mL ADH-T (crude extract), 25 U / mL ADH-LB (crude extract) NADP disodium salt (0.083 mM ) and hexane-2-ol (0.49 M) of pH 6.5 was filled in a 250 mL round bottom flask with magnetic stirrer and reflux condenser and stirred vigorously at 30 ^° C. After 6 hours, the solution was extracted with 4 × 50 ml of methyl tert-butyl ether (MTBE) and the product mixture was isolated by evaporation of the organic phases. By GC and NMR spectroscopy, the conversion and yield of hexan-2-one was determined in the weight. Result

Hexane-2-one yield: 43 mmol (87% of theory)

• space-time power: 72 mol / m ^J h hexane-2-one

Example 2d:

Reaction of hexan-2-ol with 2 equivalents of methyl acetoacetate, ADH-LB and ADH-T

execution

100 mL of a mixture of water, phosphate buffer (50 mM), acetic acid methyl ester (1.00 M), 10 U / mL ADH-T (crude extract), 25 U / mL ADH-LB (crude extract) NADP disodium salt (0.083 mM ) and hexane-2-ol (0.49 M) of pH 6.5 was filled in a 250 mL round bottom flask with magnetic stirrer and reflux condenser and stirred vigorously at 30 ^° C. After 6 hours, the solution was extracted with 4 × 50 ml of methyl tert-butyl ether (MTBE) and the product mixture was isolated by evaporation of the organic phases. By GC and NMR spectroscopy, the conversion and yield of hexan-2-one was determined in the weight.

• Result hexane-2 - one Yield: 48 mmol ^'98 d. Th. )

• Space-time performance: 80 mol / m ³ h hexane-2-one

Example 3:

Oxidation of 1-phenylethanol with ethyl acetoacetate, ADH-LB and ADH-T

Procedure 100 mL of a mixture of water, phosphate buffer (50 mM), ethyl acetoacetate (0.54 M), 10 U / mL ADH-T (crude extract), 25 U / mL ADH-LB (crude extract) NADP disodium salt (0.083 mM) and 1-phenyl-ethanol (0.49 M) of pH 6.5 was dissolved in a 250 mL round bottom filled with magnetic stirrer and reflux condenser and stirred vigorously at 30 ^° C. After 6 hours, the solution was extracted with 4 × 50 ml of methyl tert-butyl ether (MTBE) and the product mixture was isolated by evaporation of the organic phases. By GC and NMR spectroscopy, the conversion and yield of acetophenone in the balance was determined.

Result

• acetophenone yield: 46 mmol (94% of theory) • space-time performance: 77 mol / m ³ h acetophenone

Claims

Claims:

A process for producing a ketone which comprises reacting a secondary alcohol in a reaction mixture containing water, an activated ketone, an alcohol dehydrogenase and a cofactor to which ketone is added.

2. The method according to claim 1, characterized in that the secondary alcohols are a compound of the general

Formula (I)

R 1 is CH (OH) -R ² (I) or a salt of this compound, wherein

R ¹ and R ² are identical or different and are selected from the group Ci-C2o-alkyl, C3-C2o-cycloalkyl, C ₅ -C2o-aryl, _{C2-C2o-heteroaryl,} C ₂ -C ₂ o-alkenyl , C ₂ -C ₂ o-alkynyl, C ₅ -C ₂ O-aralkyl and C ₅ -C ₂ o-alkylaryl or R ¹ and R ² together with the group -CH (OH) - can form a ring consisting of consists of three to 40 ring members and R ¹ and R ² , or one by R ¹ and R ² with the group -

CH (OH) - formed ring, independently of one another may be substituted by one or more radicals Z, wherein Z is selected from the group consisting of fluorine, chlorine, bromine, iodine, -CN, -NO ₂ , -NO, -NR ³ OR ³ , -CHO, -SO ₃ H, -COOH or -R ³ and

R ^{3 is} hydrogen or may have the meaning of R ¹ and in R ¹ and R ^{2 may} optionally be replaced independently of one another one or more methylene groups by identical or different groups Y, wherein

Y is selected from the group -CR ³ = CR ³ -, -C≡C-, -C (O) -,

-C (O) O-, -OC (O) -, -C (O) OC (O) -, -O-, -O-O-, -CR ³ = N-, -C (O) -NR ³ -, - N = N-, -NR ³ -NR ³ -, -NR ³ -O-, -NR ³ -, -P (O) (OR ³ ) 0 -, -OP (O) (R ³ ) 0-, -P (R ³⁾ -, -P (O) (R ³⁾ -, -S-, -SS-, -S (O) -, - S (O) ₂ -, -S (O) NR ³ -, -S (0) (0R ³⁾ 0-, -Si (R ³⁾ ₂ -, -Si (R ³⁾ ₂ O-, -

Si (R ³⁾ (OR ³⁾ -, -OSi (R ³⁾ ₂ O-, -OSi (R ³⁾ ₂ -or -Si (R ³⁾ ₂ 0Si (R ³⁾ ₂ -.

3. The method according to claim 1 or 2, characterized in that the secondary alcohol is selected from the group 2-hydroxyalkanes, 3-hydroxyalkanes, 4-hydroxyalkanes, 2-hydroxyalkenes, 3-hydroxyalkenes, 4-hydroxyalkenes, 2-hydroxyalkynes, 3 Hydroxyalkynes, 4-hydroxyalkynes, γ-hydroxyketones, δ-hydroxyketones, γ-hydroxyesters, δ-hydroxyesters, γ-hydroxyamides, δ-hydroxyamides, OC-hydroxy alcohols, β-hydroxy alcohols, γ-hydroxy alcohols and δ-hydroxy alcohols.

4. The method according to any one of claims 1 to 3, characterized in that the cofactor is selected from compounds of the group NAD, NADP, NADH, NADPH and salts thereof.

5. The method according to any one of claims 1 to 4, characterized in that the activated ketone is a compound of general formula (II)

R ^{4 is} -C (O) -X (II) wherein

R ^{4 is} selected from the group Ci-C2o-alkyl, C3-C20-

Cycloalkyl, C ₅ -C 20 -aryl, C ₂ -C 20 -heteroaryl, C ₂ -C 20 -alkenyl,

C ₂ -C ₂ o-alkynyl, C ₅ -C ₂ o-aralkyl and C ₅ -C ₂ o-alkylaryl where

R ^{4 may} optionally be substituted by one or more radicals Z, where

Z is selected from the group fluorine, chlorine, bromine, iodine, -

CN, -NO ₂ , -NO, -NR ³ OR ³ , -C (O) R ³ , -SO ₃ R ³ , -C (O) OR ³ ,

-C (O) NR ³ R ³ , and -R ³ and

R ^{3 is} hydrogen or has the meaning of R ⁴ and in R ⁴ optionally one or more methylene groups may be replaced by identical or different groups Y, wherein

Y is selected from the group -C (O) -, -C (O) O-, -OC (O) -,

-C (O) -NR ³ and X is -C (O) R ⁵ , -C (O) OR ⁵ , -C (O) SR ⁵ , -C (O) NHR ⁵ , -C (O) NR ⁵ R ⁶ , -

CN, or a group CH _n Q ( ₃ - _n) stands, or RR ⁴⁴ and XX zzuussaammmmeenn mmiitt ddeerr GGrruuppppee --CCOO-- can form a ring with three to 40 ring members and n = 0, 1 or 2 and

Q is selected from the group fluorine, chlorine, bromine, iodine, - OC (O) R ⁵ , -OC (O) OR ⁵ , -SO ₃ R ⁵ , -OR ⁵ , -SR ⁵ , -NO ₂ , -NO , -N ₃ , - NR ⁵ OR ⁶ , -CN, -C (O) R ⁵ , -C (O) OR ⁵ , -C (O) SR ⁵ , -C (O) NR ⁵ R ⁶ , wherein R ⁵ and R ^{6 are} independently hydrogen or have the meaning of R ⁴ .

6. The method according to claim 5, characterized in that the compounds of general formula (II) are selected from the group 3-0xo-carboxylic acid ester, 3-0xo- carboxylic acid amide, 2-0xo-carboxylic acid ester, 2- 0xo-carboxylic acid amide, 2-0xoalkohole, 2-0xoalkohol-ester, 2-oxoether, 2-0xoalkylhalogenid, 2-Oxoalkyldihalogenid, 2-Oxoalkyltrihalogenid, ß-diketone.

7. The method according to claim 5, characterized in that the compounds of general formula (II) are selected from the group of methyl acetoacetate, ethyl acetoacetate, t-acetoacetate, ethylene glycol bisacetoacetate, methyl benzoylacetate, ethyl benzoylacetate, benzoylformic acid methyl ester, benzoylformic acid ethyl ester, pyruvic acid methyl ester, pyruvic acid ethyl ester, methoxyacetone, 2-methoxycyclohexanone, 1, 3-dimethoxyacetone, chloroacetone, 2,4-pentanedione.

8. The method according to any one of claims 1 to 7, characterized in that the secondary alcohol in an amount of 1 wt.% To 50 wt.% Based on the total volume of the reaction mixture is used.

9. The method according to any one of claims 1 to 8, characterized in that activated ketone is used in an amount of 1 to 10 molar equivalents based on the alcohol to be oxidized in the reaction mixture.

10. The method according to any one of claims 1 to 9, characterized in that the ADH is present in the reaction mixture in an activity of more than 5 U / ml.

11. The method according to any one of claims 2 to 10, characterized in that per kg of the compound of general formula (I) 20 000 to 700 000 U alcohol dehydrogenase (ADH) are available.

12. The method according to any one of claims 1 to 11, characterized in that the cofactor is present in the aqueous phase in a concentration of 0.01 mM to 0.25 mM.

13. The method according to any one of claims 1 to 12, characterized in that it is carried out in a pH range of 5 to 11.

14. The method according to any one of claims 1 to 13, characterized in that it is carried out at a temperature of 0 ⁰ C to 60 ⁰ C.

15. The method according to any one of claims 1 to 14, characterized in that the ketone is extracted tion tion by means of a water-immiscible organic solvent from the reaction, wherein in addition to the ketone-containing organic solvent, an enzyme-containing aqueous phase is obtained.

16. The method according to any one of claims 1 to 15, characterized in that the enzyme-containing aqueous phase

Claim 15 is added with a secondary alcohol and / or activated ketone, the secondary alcohol is converted to the desired ketone.

17. Use of an activated ketone of the general formula (II) as oxidizing agent in an enzymatic catalyzed oxidation of a secondary alcohol.