Enzymatic method for the enantioselective reduction of keto compounds

Abstract

The inventon relates to an enzymatic method for the enantioselective reduction of organic keto compounds to the corresponding chiral hydroxy compounds, an alcohol dehydrogenase from Lactobacillus minor and a method for the enantioselective production of (S)-hydroxy compounds from a racemate.

1 . A method for the enantioselective reduction of a keto compound of the formula I  
       R 1 —C(O)—R 2    (I)  
       where R 1  and R 2  are, independently of one another, identical or different and are 
 1. hydrogen,  
 2. —(C 1 -C 20 )-alkyl in which alkyl is straight-chained or branched,  
 3. —(C 2 -C 20 )-alkenyl in which alkenyl is straight-chained or branched and, optionally, comprises one, two, three or four double bonds,  
 4. —(C 2 -C 20 )-alkynyl in which alkynyl is straight-chained or branched and, optionally, comprises one, two, three or four triple bonds,  
 5. —(C 6 -C 14 )-aryl,  
 6. —(C 1 -C 8 )-alkyl —(C 6 -C 14 )-aryl or  
 7. R 1  and R 2  form in combination with the —C(O) radical a —(C 6 -C 14 )-aryl or a —(C 5 -C 14 )-hetercycle, 
 where the radicals defined above under 1. to 7. are unsubstituted or, independently of one another, mono- to trisubstituted by  
 a) —OH,  
 b) halogen such as fluorine, chlorine, bromine or iodine,  
 c) —NO 2 ,  
 d) —C(O)—O—(C 1 -C 20 )-alkyl in which alkyl is linear or branched and unsubstituted or mono- to trisubstituted by halogen, hydroxyl, amino or nitro, or  
 e) —(C 5 -C 14 )-heterocycle which is unsubstituted or mono- to trisubstituted by halogen, hydroxyl, amino or nitro,  
 said method comprising  
 
 a) a compound of the formula I with a proportion of equal to/greater than 5% to 30%, based on the total volume of the reaction mixture, alcohol dehydrogenase, water, cofactor NADPH or NADH and an organic solvent immiscible with water and having a logP of from 0.5 to 4.0;  
 b) are incubated in a two-phase system of water and organic solvent immiscible with water;  
 c) the oxidized cofactor produced by said alcohol dehydrogenase is steadily regenerated, and  
 d) the chiral hydroxy compound is isolated.  
 
     
     
         2 . The method as claimed in  claim 1 , wherein a compound of the formula I of the series ethyl 4-chloro-3-oxobutanoate, acetophenone, methyl acetoacetate, ethyl 2-oxo-4-phenylbutyrate, 2,5-hexanedione, ethyl pyruvate or 2-octanone is used.  
     
     
         3 . The method as claimed in  claim 1 , wherein an organic solvent having a logP of from 0.6 to 3.0, is used.  
     
     
         4 . The method as claimed in  claim 3 , wherein an organic solvent having a logP of from 0.63 to 1.75 is used.  
     
     
         5 . The method as claimed in  claim 1 , wherein the organic solvent used is diethyl ether, tert-butyl methyl ether, diisopropyl ether or ethyl acetate.  
     
     
         6 . The method as claimed in  claim 1 , wherein an alcohol dehydrogenases from yeast, equine liver,  Thermoanaerobium brockii, Rhodococcus erythropolis, Lactobacillus kefir, Lactobacillus brevis, Lactobacillus minor  or an alcohol dehydrogenase having the amino acid sequence according to SEQ ID NO: 4 is used.  
     
     
         7 . The method as claimed in  claim 1 , wherein a buffer selected from potassium phosphate, Tris/HCl or triethanolamine buffer, having a pH of from 5 to 10, is added.  
     
     
         8 . The method as claimed in  claim 7 , wherein magnesium ions at a concentration of from 0.2 mM to 10 mM are added to the buffer.  
     
     
         9 . The method as claimed in  claim 1 , wherein the cofactor added is NADPH or NADH in an amount of from 0.01 mM to 0.25 mM, based on the aqueous phase.  
     
     
         10 . The method as claimed in  claim 1 , wherein glycerol, sorbitol or dimethyl sulfoxide is added as stabilizer for alcohol dehydrogenase.  
     
     
         11 . The method as claimed in  claim 1 , wherein isopropanol is added.  
     
     
         12 . The method as claimed in  claim 1 , wherein the compounds of the formula I are used in an amount of from 5% to 30% based on the total volume.  
     
     
         13 . The method as claimed in  claim 1 , wherein the reaction is carried out at a temperature of from about 10° C. to 70° C.  
     
     
         14 . The method as claimed in  claim 1 , wherein the organic solvent are used in an amount of from 1% to 90%, based on the total volume of the reaction mixture.  
     
     
         15 . The method as claimed in  claim 1 , wherein the ratio of organic solvent to water is from 9 to 1 to 1 to 9.  
     
     
         16 . The method as claimed in  claim 10 , wherein the stabilizer is used in an amount of from 5% to 30%, based on the volume of the total reaction mixture.  
     
     
         17 . The method as claimed in  claim 11 , wherein isopropanol is used in an amount of from 5% to 30%, based on the volume of the total reaction mixture.  
     
     
         18 . The method as claimed in  claim 6 , wherein the alcohol dehydrogenase is used in an amount of from 20 000 U to 200 000 U per kg of compound of the formula I to be reacted.  
     
     
         19 . The method according to  claim 18 , comprising a  Lactobacillus minor  alcohol dehydrogenase having the amino acid sequence according to SEQ ID NO: 4.  
     
     
         20 . The method according to  claim 18 , comprising a  Lactobacillus minor  alcohol dehydrogenase having the amino acid sequence according to SEQ ID NO: 3.  
     
     
         21 . A method for obtaining the alcohol dehydrogenase from  Lactobacillus minor  as claimed in  claim 19 , wherein the DNA coding for  Lactobacillus minor  alcohol dehydrogenase is expressed in a suitable prokaryotic or eukaryotic microorganism, in particular in cells of  Escherichia coli  cell deposited under DSM 14196, and, optionally, said alcohol dehydrogenase is purified.  
     
     
         22 . A method for obtaining an enantioselective (S)-hydroxy compound of the formula II  
       R 1 —C(OH)—R 2    (II)  
       where R 1  and R 2  are, independently of one another, identical or different and are 
 1. hydrogen,  
 2. —(C 1 -C 20 )-alkyl in which alkyl is straight-chained or branched,  
 3. —(C 2 -C 20 )-alkenyl in which alkenyl is straight-chained or branched and, optionally, comprises one, two, three or four double bonds,  
 4. —(C 2 -C 20 )-alkynyl in which alkynyl is straight-chained or branched and, optionally, comprises one, two, three or four triple bonds,  
 5. —(C 6 -C 14 )-aryl,  
 6. —(C 6 -C 8 )-alkyl—(C 6 -C 14 )-aryl or  
 7. R 1  and R 2  form in combination with the —C(O) radical a —(C 6 -C 14 )-aryl or a —(C 6 -C 14 )-heterocycle, 
 where the radicals defined above under 1. to 7. are unsubstituted or, independently of one another, mono- to trisubstituted by  
 a) —OH,  
 b) halogen such as fluorine, chlorine, bromine or iodine,  
 c) —NO 2 ,  
 d) —C(O)—O—(C 1 -C 20 )-alkyl in which alkyl is linear or branched and unsubstituted or mono- to trisubstituted by halogen, hydroxyl, amino or nitro, or  
 e) —(C 6 -C 14 )-heterocycle which is unsubstituted or mono- to trisubstituted by halogen, hydroxyl, amino or nitro,  
 said method comprising  
 
 a) a racemic mixture comprising the compound of the formula II, alcohol dehydrogenase, water, cofactor NADP or NAD and an organic solvent having a logP of from 0.6 to 1.9, from the series diethyl ether, tert-butyl methyl ether, diisopropyl ether, ethyl acetate,  
 b) is incubated in a two-phase system of water and organic solvent immiscible with water and  
 C) the enantiomerically pure (S)-hydroxy compound is isolated.  
 
     
     
         23 . The method as claimed in  claim 22 , wherein acetone is added.