EP1805313A1

EP1805313A1 - Method for producing chiral alcohols

Info

Publication number: EP1805313A1
Application number: EP05800828A
Authority: EP
Inventors: Antje Gupta; Maria Bobkova; Anke Zimmer
Original assignee: IEP GmbH
Current assignee: Cambrex IEP GmbH
Priority date: 2004-10-27
Filing date: 2005-10-26
Publication date: 2007-07-11
Also published as: AT501928B1; KR20070085458A; CN101120094B; JP2008517612A; WO2006045598A1; AT501928A1; CN101120094A; US20090148917A1; CA2585411A1

Abstract

The invention relates to a method for producing an enantiopure alcohol of general formula (Ia) or (Ib), wherein R1, R2, R.3, R4, R5 and R6 each represent hydrogen, halogen, a C1-C6 alkyl or C1-C6 alkoxy group, with the proviso that at least one of the groups R1, R2, R3, R4, R5 and R6 is different from the remaining five groups and with the additional proviso that at least one of the groups R1, R2, R3, R4, R5 and R6 is a halogen. The invention is characterized in that a ketone of general formula (II), wherein R1, R2, R3, R4, R5 and R6 are defined as above, is enzymatically reduced in the presence of an S-specific or R-specific dehydrogenase/oxidoreductase using NADH or NADPH as the cofactor.

Description

Process for the preparation of chiral alcohols

The invention relates to a process for preparing enantiomerically pure alcohols of the general formula Ia or Ib

where R 1, R ₂ , R ₃ , R ₄ , R ₅ and R ₆ each represent hydrogen, halogen, a C 1 -C ₆ -alkyl or C ₁ -C ₆ -alkoxy group, with the proviso that at least one of the radicals Ri , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ is different from the other five, and the proviso that at least one of Ri, R ₂ , R ₃ , R ₄ , R ₅ and R _{6 is} a halogen ,

Furthermore, the invention relates to a process for the preparation of enantiomerically pure alcohols of the general formula HIa or IHb

wherein R ₇ , R ₈ and R _{9 represent} a Ci-C ₆ alkyl group.

Enantiomerically pure alcohols of the general formulas Ia or Ib and IHa or IHb are valuable chiral building blocks for the synthesis of a variety of chiral compounds, which are of interest for the preparation of pharmaceutically active substances. However, many of these enantiomerically pure alcohols are chemically not or only very expensive representable and are therefore not available in larger quantities.

The invention therefore has as its object to provide a process which enables the economical preparation of enantiomerically pure alcohols of general formula Ia or Ib and IHa or IHb in high yield and high enantiomeric purity. This object is achieved in relation to the alcohols of the general formula Ia or Ib according to the invention characterized in that a ketone of the general formula II

wherein Ri, R _2, R _3, R _4, R ₅ and R ₆ are each hydrogen, halogen, a Ci-C ₆ alkyl or Ci-C ₆ - represent alkoxy group, with the proviso that at least one of the radicals Ri, R ₂ , R ₃ , R ₄ , R ₅ and R _{6 are} different from the other five, and with the proviso that at least one of Ri, R ₂ , R ₃ , R ₄ , R ₅ and R _{6 is} a halogen, in the presence of an S- or R-specific dehydrogenase / oxidoreductase using NADH or NADPH as cofactor is enzymatically reduced.

A preferred embodiment of the method is characterized in that Ri = R ₂ = Cl and R ₃ = R ₄ = R ₅ = R ₆ = H.

Another preferred embodiment is characterized in that Ri = R ₂ = R ₄ = Cl and R ₃ = R ₅ = R ₆ = H.

A further preferred embodiment is characterized in that Ri = CH ₃ , R ₂ = Cl and R ₃ = R ₄ = R ₅ = R ₆ = H.

Yet another preferred embodiment is characterized in that Cl and R ₂ = R ₃ = R ₄ = R ₅ = R ₆ = H.

The problem underlying the invention with respect to the alcohols of general formula IHa or HIb is achieved in that a ketone of the general formula IV

O O (IV)

R ₉ wherein R ₇ , R ₈ and R _{9 represent} a Ci-C ₆ alkyl group, is enzymatically reduced in the presence of an S- or R-specific dehydrogenase / oxidoreductase using NADH or NADPH as a cofactor.

The term "NADH" is understood to mean reduced jSficotinarmd-adenine dmucleotide and by the term "NAD" nicotinamide adenine dinucleotide. By the term "NADPH" is meant reduced nicotinamide adenine dinucleotide phosphate and by the term "NADP" nicotinamide adenine dinucleotide phosphate.

A preferred embodiment of this process is characterized in that R ₇ = R ₈ = R ₉ = CH ₃ .

Another preferred embodiment is characterized in that R ₇ = CH ₃ and R ₈ = R ₉ = C ₂ H ₅ .

The ketones of the general formula II or IV which are used according to the invention as starting material are generally available in a simple and inexpensive manner.

The dehydrogenase used for the enzymatic reduction is obtained according to a preferred embodiment of microbial starting material. Which configuration of the products is formed predominantly or exclusively depends on the type of dehydrogenase / oxidoreductase as well as the type of cofactor.

Preferably, in the process for the preparation of enantiomerically pure alcohols of the general formulas Ia or Ib and IHa or HIb as R-specific dehydrogenase a secondary alcohol dehydrogenase from Lactobacilli of the genus Lactobacilliales, in particular Lactobacillus kefir, Lactobacillus brevis or Lactobacillus minor, or from Pseudomonas used ,

In this context, R-specific secondary alcohol dehydrogenases are understood as meaning those which reduce the keto group in a group H ₃ CC (C =O) -CH ₂ -C to the corresponding (R) -configured alcohol. Such R-specific secondary alcohol dehydrogenases are described, for example, in US Pat. No. 5,200,335, DE 196 10 984 A1, DE 101 19 274 or US Pat. No. 5,385,833.

As S-specific dehydrogenase is preferably a secondary alcohol dehydrogenase from the genus Pichia or Candida, in particular Candida boidinii ADH, Candida parapsilosis or Pichia capsulata. Such S-specific dehydrogenases are described, for example, in US Pat. No. 5,523,223 or DE 103 27 454.

The enzyme does not have to be used in pure form. Likewise, it is also possible to use enzyme-containing microorganisms or more or less purified lysates thereof. If the reaction is to be carried out continuously, immobilized enzymes can also be used. The immobilization can be carried out, for example, by including the enzymes - in particular in polymeric networks or in semipermeable membranes - or by binding to a support, for example by absorption or by ionic or covalent bonds. Preferably, however, the dehydrogenases are used in free form.

The enzymatic reduction itself proceeds under mild conditions, so that the alcohols produced do not continue to react. The inventive method have a long service life, an enantiomeric purity of more than 95% of the prepared chiral alcohols of formulas Ia and Ib and purple or Erb and a high yield based on the amount of keto compounds of formula II or IV.

The oxidoreductases can either be completely purified or partially purified in the inventive process, used in the form of cell lysates or in the form of whole cells. The cells used may be native or permeabilized. Cloned and overexpressed oxidoreductases (for example known from US Pat. No. 5,523,223, DE 103 27 454 or DE 101 19 274) are preferably used.

According to a preferred embodiment of the method, the volume activity of the oxidoreductase used is 10 U / ml to 5000 U / ml, preferably 100 U / ml to 1000 U / ml.

Per kg of ketone to be reduced, 5000 to 10,000,000 U, preferably 10,000 to 1,000,000 U, of oxidoreductase are used in the process. The enzyme unit 1 U corresponds to the amount of enzyme required to react 1 μmol of the keto compound of the formula II or IV per minute.

A preferred embodiment of the invention is further characterized in that the NAD or NADP formed during the reduction is continuously reduced with a cosubstrate to NADH or NADPH. Preferred cosubstrates are primary and secondary alcohols, such as ethanol, 2-propanol, 2-butanol, 2-pentanol, 4-methyl-2-pentanol, 2-octanol or cyclohexanol.

These cosubstrates are converted by means of an oxidoreductase and NAD or NADP to the corresponding aldehydes or ketones and NADH or NADPH. This leads to the regeneration of NADH or NADPH. The proportion of the cosubstrate for the regeneration is from 5 to 95% by volume, based on the total volume.

For the regeneration of the cofactor, an alcohol dehydrogenase may additionally be added. Suitable NADH-dependent alcohol hydro genases are obtainable, for example, from baker's yeast, from Candida boidinii, Candida parapsilosis or Pichia capsulata. Suitable NADPH-dependent alcohol dehydrogenases are also found in Lactobacillus brevis (DE 196 10 984 A1), Lactobacillus minor (DE 101 19 274), Pseudomonas (US 5,385,833) or in Thermoanaerobium brockii. Suitable cosubstrates for these alcohol dehydrogenases are the abovementioned secondary alcohols, such as ethanol, 2-propanol (isopropanol), 2-butanol, 2-pentanol, 4-methyl-2-pentanol, 2-octanol or cyclohexanol.

Further, cofactor regeneration may also be performed, for example, with NAD- or NADP-dependent formate dehydrogenase (Tishkov et al., J. Biotechnol., Bioeng., 1999, 64, 187-193, Pilot-scale production and isolation of recombinant NAD and NADP specific Formats dehydrogenase) are performed. Suitable cosubstrates of formate dehydrogenase are, for example, salts of formic acid, such as ammonium formate, sodium formate or calcium formate. Preferably, however, the processes according to the invention are carried out without such an additional dehydrogenase, i. a substrate-coupled coenzyme regeneration takes place.

The aqueous portion of the reaction mixture in which the enzymatic reduction takes place preferably contains a buffer, for example potassium phosphate, tris / HCl or triethanolamine buffer, having a pH of from 5 to 10, preferably a pH of from 6 to 9. The buffer may additionally contain ions for stabilizing or activating the enzymes, for example zinc ions or magnesium ions.

The temperature during the execution of the inventive method expediently from about 10 ° C to 7O ⁰ C, preferably from 20 ° C to 40 ° C. In a further preferred embodiment of the process according to the invention, the enzymatic reaction is carried out in the presence of an organic solvent which is immiscible or only slightly miscible with water. This solvent is, for example, a symmetrical or unsymmetrical di (C ₁ -C ₆ ) alkyl ether, a straight-chain or branched alkane or cycloalkane or a water-insoluble secondary alcohol which simultaneously represents the cosubstrate. The preferred organic solvents are, for example, diethyl ether, tert-butyl methyl ether, diisopropyl ether, dibutyl ether, butyl acetate, heptane, hexane, 2-octanol, 2-heptanol, 4-methyl-2-pentanol or cyclohexane.

The reaction mixture consists of the use of water-insoluble solvents or cosubstrates of an aqueous and an organic phase. The substrate is distributed according to its solubility between organic and aqueous phase. The organic phase generally has a content of from 5 to 95%, preferably from 20 to 90%, based on the total reaction volume. The two liquid phases are preferably mechanically mixed so that a large surface is created between them. In this embodiment too, the NAD or NADP formed during the enzymatic reduction can be reduced again to NADH or NADPH with a cosubstrate as described.

The concentration of the cofactor NADH or NADPH in the aqueous phase is generally 0.001 mM to ImM, in particular 0.01 mM to 0.1 mM.

In the process according to the invention, it is also possible to use a stabilizer of the oxidoreductase / dehydrogenase. Suitable stabilizers are, for example, glycerol, sorbitol, 1,4-DL-dithiothreitol (DTT) or dimethyl sulfoxide (DMSO).

The process according to the invention is carried out, for example, in a closed reaction vessel made of glass or metal. For this purpose, the components are transferred individually into the reaction vessel and stirred under an atmosphere of, for example, nitrogen or air. The reaction time is from 1 hour to 48 hours, especially from 2

Hours to 24 hours.

Subsequently, the reaction mixture is worked up. For this purpose, the aqueous phase is separated, the organic phase is filtered. If appropriate, the aqueous phase can be extracted once more and, like the organic phase, worked up further. Thereafter, if necessary, the solvent is evaporated from the filtered organic phase. The invention will be explained in more detail by way of examples.

Examples:

analytics:

a) Ami de:

The determination of the ee (enantiomeric excess) was carried out by means of chiral gas chromatography.

To this was added a gas chromatograph GC-17 A from Shimadzu with a chiral separation column

CP-Chirasil-DEX CB (Varian Chrompack, Darmstadt, Germany), Flame Ionization

Detector and helium used as carrier gas.

The separation of N, N-dimethyl-3-hydroxybutanamide was carried out at 0.86 bar and 10 min

120 ° C, 2 ° C / min-> 125 ° C.

Retention times were: (3R) 10.42 min and (3S) 10.09 min.

The separation of N, N-diethyl-3-hydroxybutanamide was carried out at 0.75 bar and 10 min

130 ° C, 2 ° C / min- 135 ° C.

Retention times were: (3R) 11.6 min and (3S) 11.3 min.

b) Chlorine compounds:

FS-Hydrodex β-6-TBDM (Machery-Nagel, Düren, Germany), Flame Ionization

Detector and helium used as carrier gas.

The separation of 1-chloropropan-2-ol was carried out at 0.94 bar and 15 min at 40 ⁰ C, l ° C / min →> 50 ° C.

Retention times were: (2R) 20.3 min and (2S) 20.9 min.

The separation of l, l-dichloropropan-2-ol was carried out at 0.69 bar and 15 min at 80 ⁰ C,

2 ° C / min-> 95 ° C.

Retention times were: (2R) 20.8 min and (2S) 21.4 min.

The separation of l, l, 3-trichloropropan-2-ol was carried out at 0.69 bar and 30 min at 120 ⁰ C isothermal.

Retention times were: (2R) 25.0 min and (2S) 24.5 min.

The separation of 3-chlorobutan-2-ol was isothermic at 0.98 bar and 25 min at 50 ° C.

The retention times were:

(3R) -3-chlorobutan-2-one: 6.0 min

(3S) -3-Chlorobutan-2-one: 6.2 min

(3R, 2R) -3-chlorobutan-2-ol: 17.5 min (3R, 2S) -3-chlorobutan-2-ol: 18.1 min (3S, 2R) -3-chlorobutan-2-ol: 20.7 min (3S, 2S) -3-chlorobutan-2-ol: 22.1 min

1. Synthesis of (S) -3-hydroxy-N, N-diethylbutanamide from N, N-diethylacetoacetamide

For the synthesis of (R) -3-hydroxy-N, N-diethylbutanamide, a mixture of 172 ml buffer (100 mM triethanolamine, pH = 7, 0.5 mM DTT, 20% glycerol), 18 ml 2-propanol (0 , 23 mol), 10 ml of N, N-Diethylecetoacetamid (63 mmol), 200 mg NAD and 30000 units of recombinant alcohol sehydrated from Candida parapsilosis incubated for 24 h at room temperature with constant mixing. After 24 h, 97% of the N, N-diethylacetoacetamide used had been reduced to (S) -3-hydroxy-N, N-diethylbutanamide. The reaction mixture was then extracted with ethyl acetate and the solvent removed on a rotary evaporator. The crude product thus obtained was purified by means of vacuum distillation. It was possible to obtain 2.5 g of (S) -3-hydroxy-N, N-diethylbutanamide having a purity of> 98% and an enantiomeric excess of> 99.9%.

Analysis results:

Elemental analysis% found (calculated):. C ₈ Hi ₇ NO ₂ C: 59.8 (60.4) H: 10.7 (10.8) N: 8.9 (8.8)

¹ H-NMR CDCl ₃ m:

¹³ C-NMR in CDCl ₃

Specific rotations:

The specific rotation [OC] _D ^{20 of} the enantiomers was measured with a precision polarimeter

POL-S2 measured at a layer thickness of 1 dm. For the study, 0.5 g

Sample dissolved in 25 ml of EtOH.

(S) -3-hydroxy-N, N-diethylbutanamide (100%) [α] _D ²⁰ = + 19.92 +1 ° x 1 / g.dm

2. Synthesis of (R) -3-hydroxy-N, N-diethylbutanamide from N ₅ N-diethyl acetoacetamide

For the synthesis of (R) -3-hydroxy-N, N-diethylbutanamide, a mixture of 290 ml buffer (100 mM triethanolamine, pH = 7, 1 mM MgCl ₂ , 10% glycerol), 100 ml 2-propanol (1, 3 mol), 10 ml of N, N-Diethylecetoacetamid (63 mmol), 20 mg NADP and 60,000 units of recombinant alcohol dehydrogenase from Lactobacillus minor (DE-A 101 19 274) for 24 h at room temperature with constant mixing. After 24 h, 60% of the N, N-diethylacetoacetamide used was reduced to (R) -3-hydroxy-N, N-diethylbutanamide. The reaction mixture was then extracted with ethyl acetate and the solvent removed on a rotary evaporator. The crude product thus obtained was purified by means of vacuum distillation. It was possible to obtain 2.5 g of (R) -3-hydroxy-N, N-diethylbutanamide having a purity of> 9δ% and an enantiomeric excess of> 99%.

Analysis results:

Elemental Analysis% F (over): C ₈ Hi ₇ NO ₂ C: 59.8 (60.4) H: 10.7 (10.8) N: 8.9 (8.8)

¹ H-NMR in CDCl ₃ : Results analogous to Example 1

¹³ C-NMR in CDCl ₃ : Results analogous to Example 1

Specific rotations:

(R) -3-hydroxy-N, N-diethylbutanamide (100%) [α] _D ²⁰ = -19.7 ± 1 ° x 1 / g.dm

The structure of the alcohols (S) - and (R) -3-hydroxy-N, N-diethylbutanamide was confirmed by ¹ H-NMR, ¹³ C-NMR and elemental analysis. The exact determination of the enantiomeric excess was carried out by means of chiral GC and determination of the rotational value [αjα ^{20 '} ] ^.

3. Synthesis of (R) -3-hydroxy-N, N-dimethylbutanamide from N, N-dimethylacetoacetamide

For the synthesis of (R) -3-hydroxy-N, N-dimethylbutanamide, a mixture of 525 ml buffer (100 mM triethanolamine, pH = 7, 1 mM MgCl ₂ , 10% glycerol), 90 ml 2-propanol (1, 18 mol), 15 ml of N, N-dimethylacetoacetamide (120 mmol), 30 mg NADP and 50,000 units of recombinant Alkoholdehydro genäse from Lactobacillus minor (DE-A 101 19 274) incubated for 24 h at room temperature with constant mixing. After 24 h, 95% of the N, N-dimethylacetoacetamide used was reduced to (R) -3-hydroxy-N, N-diethylbutanarnide. The reaction mixture was then extracted with ethyl acetate and the solvent removed on a rotary evaporator. The crude product thus obtained was purified by means of vacuum distillation. O 2.3 g of (R) -3-hydroxy-N, N-dimethylbutanamide having a purity of> 98% and an enantiomeric excess of> 99.0% could be obtained.

Analysis results:

Elemental Analysis% F (over): C ₆ Hi ₃ NO ₂ C: 54.2 (54.9) H: 9.7 (10.0) N: 10.4 (10.7) ¹ H-NMR in CDCl ₃ :

Specific rotations:

(R) -3-hydroxy-N, N-dimethylbutanamide (100%) [α] _D ²⁰ = - 29.1 + 1 ° x 1 / g.dm

4. Synthesis of (S) -3-hydroxy-N, N-dimethylbutanamide from N, N-dimethylacetoacetamide

For the synthesis of (S) -3-hydroxy-N, N-dimethylbutanamide, a mixture of 89 ml buffer (100 mM triethanolamine, pH = 7, 1 mM ZnCl ₂ , 10% glycerol), 9 ml 2-propanol (0, 12 mol), 2.5 ml of N, N-dimethylacetoacetamide (19 mmol), 10 mg NAD and 16,000 units of recombinant alcohol dehydrogenase from Candida parapsilosis incubated for 24 h at room temperature with constant mixing. After 24 h, 95% of the N ₅ N-dimethylacetoacetamide used had been reduced to (S) -3-hydroxy-N, N-diethylbutanamide. The reaction mixture was then extracted with ethyl acetate and the solvent removed on a rotary evaporator. The crude product thus obtained was purified by means of vacuum distillation. It was thus possible to obtain (S) -3-hydroxy-N, N-dimethylbutanamide having a purity of> 98% and an enantiomeric excess of> 99.0%. Specific rotations:

(S) -3-hydroxy-N, N-dimethylbutanamide (100%) [α] _D ²⁰ = + 29.1 ± 1 ° x 1 / g.dm

5. Synthesis of (S) -I, l-dichloro-2-propanol from 1,1-dichloroacetone

For the synthesis of (S) -l, l-dichloro-2-propanol, a mixture of 640 ml buffer (100 mM triethanolamine, pH = 7, 1 mM ZnCl ₂ , 20% glycerol), 80 ml 2-propanol (1, 05 mol), 20 ml of 1,1-dichloroacetone (0.2 mol), 40 mg of NAD and 13,000 units of recombinant alcohol dehydrogenase from Pichia capsulata (DE-A 103 27 454) for 24 h at room temperature with constant mixing. After 24 h, 100% of the 1,1-dichloroacetone used was reduced to (S) -1,1-dichloro-2-propanol. The reaction mixture was then extracted with ethyl acetate and the solvent removed on a rotary evaporator. The crude product thus obtained was purified by means of vacuum distillation. It was possible to obtain 6.5 g of (S) -I, 1-dichloro-2-propanol with a purity of> 99% and an enantiomeric excess of> 99.9%.

Analysis results:

Elemental analysis and chlorine determination% F (over): C ₃ H ₆ Cl ₂ OC: 26.7 (27.9) H: 4.7 (4.7) O: 14.8 (12.4) Cl: 53, 4 (55)

¹ H-NMR in CDCl

13 C-NMR in CDCl ₃

Specific rotations:

(S) -I, l-dichloro-2-propanol (100%) [α] _D ²⁰ = - 19.1 + 1 ° x 1 / g.dm

6. Synthesis of (R) -I, 1-dichloro-2-propanol from 1,1-dichloroacetone

For the synthesis of (R) -I, l-dichloro-2-propanol, a mixture of 320 ml buffer (100 mM triethanolamine, pH = 7, 1 mM MgCl ₂ , 10% glycerol), 60 ml 2-propanol (0, 78 mol), 20 ml of 1,1-dichloroacetone (0.2 mol) dissolved in 40 ml of ethyl acetate, 40 mg of NADP and 8000 units of recombinant alcohol dehydrogenase from Lactobacillus minor (DE-A 101 19 274) for 24 h at room temperature with constant Mixing incubated. After 24 h, 100% of the 1,1-dichloroacetone used was reduced to (R) -I, l-dichloro-2-propanol. The reaction mixture was then extracted with ethyl acetate and the solvent removed on a rotary evaporator. The crude product thus obtained was purified by means of vacuum distillation. It was 4.8 g of (R) -I, l-dichloro-2-propanol with a purity of> 98% and an enantiomeric excess of> 95% are obtained.

Analysis results:

¹ H-NMR in CDCl ₃ : analogously to Example 5

¹³ C-NMR in CDCl ₃ : analogously to Example 5

Specific rotations:

(R) -I, l-dichloro-2-propanol (100%) [α] _D ²⁰ = + 19.56 ± 1 ° x 1 / g.dm

7. Synthesis of (R) -I, 1,3-trichloro-2-propanol from 1,1,3-trichloroacetone

For the synthesis of (R) -I, l, 3-trichloro-2-propanol, a mixture of 110 ml buffer (100 mM triethanolamine, pH 7, 1 mM MgCl ₂ , 10% glycerol), 40 ml 2-propanol ( 0.52 mol), 10 ml of 1,1,3-trichloroacetone (93 mmol) dissolved in 40 ml of ethyl acetate, 20 mg NADP and 12,000 units of recombinant alcohol dehydrogenase from Lactobacillus minor (DE-A 101 19 274) incubated for 24 h at room temperature with constant mixing. After 24 h, 100% of the 1,1,3-trichloroacetone used was reduced to (R) -I, l, 3-trichloro-2-propanol. The reaction mixture was then extracted with ethyl acetate and the solvent removed on a rotary evaporator. The crude product thus obtained was purified by means of vacuum distillation. It was possible to obtain 8.9 g of (R) -1,3,1-trichloro-2-propanol with a purity of> 99% and an enantiomeric excess of> 97%.

Analysis results:

Elemental analysis and chlorine determination% F (over): C ₃ H ₅ Cl ₃ OC: 22.1 (22.1) H: 2.8 (3.1) O: 11.1 (9.8) Cl: 63, 9 (65,1)

¹ H-NMR in CDCl ₃

Specific rotations:

(R) -1, 1, 3-trichloro-2-propanol (100%) [α] _D ² ° = + 10.1 ± 1 ° x 1 / g.dm

8. Synthesis of (S) -l, l _? 3-trichloro-2-propanol from 1,1,3-trichloroacetone For the synthesis of (S) -1,1,3-trichloro-2-propanol, a mixture of 9 ml buffer (100 mM triethanolamine, pH = 7, 1 mM ZnCl ₂ , 20% glycerol), 0.8 ml 2- Propanol (10 mmol), 0.25 ml of 1,1,3-trichloroacetone (0.2 mol), 10 mg NAD and 2000 units of recombinant alcohol dehydrogenase from Pichia capsulata (DE-A 103 27 454) for 24 h at room temperature with constant Mixing incubated. After 24 h, 97% of the 1,1,3-trichloroacetone used was reduced to (S) -1,1,3-trichloro-2-propanol with an enantiomeric excess of> 60%.

Specific rotations:

(S) -I, l, 3-trichloro-2-propanol (100%) [α] _D ²⁰ = -10.1 ± 1 ° x 1 / g.dm

9. Synthesis of S-chloro-2-propanol from chloroacetone

For the synthesis of S-chloro-2-propanol, a mixture of 0.6 ml buffer (100 mM triethanolamine, pH = 7, 10% glycerol, 1 mM ZnCl ₂ ), 400 ul 4-methyl-2-propanol, 100 ul Chloraceton, lmg NAD and 60 units recombinant alcohol dehydrogenase from Pichia capsulata (DE-A 103 27 454) or Candida parapsilosis incubated for 24 h at room temperature with constant mixing. After 24 h, 100% of the chloroacetone used was reduced to S-chloro-2-propanol with an enantiomeric excess of> 97%.

10. Synthesis of R-chloro-2-propanol from chloroacetone

For the synthesis of R-chloro-2-propanol, a mixture of 0.4 ml buffer (100 mM triethanolamine, pH = 7, 10% glycerol), 300 ul 2-propanol, 100 ul chloroacetone dissolved in 200 ul ethyl acetate, 1 mg NADP and 30 units of recombinant alcohol dehydrogenase from Lactobacillus minor (DE-A 101 19 274) for 24 h at room temperature with constant mixing. After 24 h, 100% of the chloroacetone used was reduced to R-chloro-2-propanol with an enantiomeric excess of> 95%.

11. Synthesis of (2S) -3-chloro-2-butanol from 3-chloro-2-butanone

For the synthesis of (2S) -3-chloro-2-butanol, a mixture of 0.45 ml buffer (100 mM triethanolamine, pH = 7, 10% glycerol, 1 mM ZnCl ₂ ), 450 μl 4-methyl-2- propanol, 100 ul of 3-chloro-2-butanone (1 mmol), 0.1 mg NAD (0.15 .mu.mol) and 60 units of recombinant alcohol dehydrogenase from Pichia capsulata (DE-A 103 27 454) or Candida parapsilosis for 24 h at Room temperature incubated with constant mixing. After 24 h were 100% of the 3-chloro-2-butanone used reduced to (2S) -3-chloro-2-butanol with an enantiomeric excess of> 98%.

12. Synthesis of (2R) -3-chloro-2-butanol from 3-chloro-2-butanone

For the synthesis of (2R) -3-chloro-2-butanol, a mixture of 0.45 ml buffer (100 mM triethanolamine, pH = 7, 10% glycerol, 1 mM MgCl ₂ ), 450 μl 4-methyl-2- propanol, 100 ul of 3-chloro-2-butanone (1 mmol), 0.1 mg NADP (0.13 mol) and 60 units of recombinant alcohol dehydrogenase from Lactobacillus minor (DE-A 101 19 274) for 24 h at room temperature with constant mixing incubated. After 24 h, 100% of the 3-chloro-2-butanone used was reduced to (2R) -3-chloro-2-butanol with an enantiomeric excess of> 98%.

Claims

claims:

1. A process for preparing an enantiomerically pure alcohol of the general formula Ia or Ib

wherein Ri, R _2, R _3, R _4, R ₅ and R ₆ are each hydrogen, halogen, a Ci-C ₆ alkyl or Ci-C ₆ - represent alkoxy group, with the proviso that at least one of the radicals Ri, R ₂ , R ₃ , R ₄ , R ₅ and R _{6 are} different from the other five, and with the proviso that at least one of Ri, R ₂ , R ₃ , R ₄ , R ₅ and R _{6 is} a halogen, characterized in that a ketone of the general formula II

wherein Ri, R ₂ , R ₃ , R ₄ , R ₅ and R _{6 have} the abovementioned meaning, is enzymatically reduced in the presence of an S- or R-specific dehydrogenase / oxidoreductase using NADH or NADPH as cofactor.

2. The method according to claim 1, characterized in that Ri = R ₂ = Cl and R ₃ = R ₄ = R ₅ = R ₆ = H.

3. The method according to claim 1, characterized in that Cl and R ₃ = R ₅ = R ₆ = H.

4. The method according to claim 1, characterized in that Ri = CH ₃ , R ₂ = Cl and R ₃ = R ₄ = R ₅ = R ₆ = H.

5. The method according to claim 1, characterized in that Ri = Cl and R ₂ = R ₃ = R ₄ = R ₅ = R ₆ = H.

6. A process for the preparation of an enantiomerically pure alcohol of the general formula IHa or IHb

where R ₇ , R ₈ and Rg represent a C ₁ -C ₆ -alkyl group, characterized in that a ketone of the general formula IV

wherein R ₇ , R ₈ and R _{9 have} the abovementioned meaning, is enzymatically reduced in the presence of an S- or R-specific dehydrogenase / oxidoreductase using NADH or NADPH as cofactor.

7. The method according to claim 6, characterized in that R ₇ = R ₈ = R ₉ = CH ₃ .

8. The method according to claim 6, characterized in that R ₇ = CH ₃ and R ₈ = R ₉ = C ₂ H ₅ .

9. The method according to any one of claims 1 to 8, characterized in that as R-specific dehydrogenase, a secondary alcohol dehydrogenase from Lactobacilli of the genus Lactobacilliales, in particular Lactobacillus kefir, Lactobacillus brevis or Lactobacillus minor, or from Pseudomonas is used.

10. The method according to any one of claims 1 to 8, characterized in that as S-specific dehydrogenase a secondary alcohol dehydrogenase from the genus Pichia or Candida, in particular Candida boidinii ADH, Candida parapsilosis or Pichia capsulata.

1 1. The method according to any one of claims 1 to 10, characterized in that the volume activity of the oxidoreductase used is 10 U / ml to 5000 U / ml, preferably 100 U / ml to 1000 U / ml.

12. The method according to any one of claims 1 to 11, characterized in that per kg to be reduced ketone 5000 to 10,000,000 U, preferably 10,000 to 1,000,000 U, oxidoreductase be used.

13. The method according to any one of claims 1 to 12, characterized in that the formed during the reduction NAD or NADP is continuously reduced with a cosubstrate to NADH or NADPH.

14. The method according to claim 13, characterized in that as cosubstrate primary and secondary alcohols, such as ethanol, 2-propanol, 2-butanol, 2-pentanol, 4-methyl-2-pentanol, 2-octanol or cyclohexanol, are used.