Methods for producing optically active alcohols

Abstract

A method for producing optically active alcohols is provided. Optically active alcohols are useful intermediates in pharmaceutical production. The method of the present invention enables simple and efficient production of optically active alcohols with a high optical purity. According to the production method disclosed, optically active alcohols are produced via asymmetric reduction of 3-quinuclidinone using tropinone reductase-I. For example, the use of tropinone reductase-I derived from plants like Datura stramonium and Hyoscyamus niger allows the production of high optical purity (R)-3-quinuclidinol as shown in Formula (1) below. Formula (1):

What is claimed is:  
     
         1 . A method for producing an optically active alcohol, the method comprising the steps of contacting a ketone with an enzymatic material having tropinone reductase-I activity in the presence of a reduced coenzyme to perform asymmetric reduction and recovering an optically active alcohol.  
     
     
         2 . The method according to  claim 1 , wherein the ketone is 3-quinuclidinone and the optically active alcohol is (R)-3-quinuclidinol.  
     
     
         3 . The method according to  claim 1 , wherein the enzymatic material has tropinone reductase-I activity to produce an optically active alcohol with 70% ee or higher optical purity.  
     
     
         4 . The method according to  claim 1 , wherein the enzymatic material is derived from a plant belonging to the genus Datura or the genus Hyoscyamus.  
     
     
         5 . The method according to  claim 4 , wherein the plant belonging to the genus Datura is  Datura stramonium.    
     
     
         6 . The method according to  claim 4 , wherein the plant belonging to the genus Hyoscyamus is  Hyoscyamus niger.    
     
     
         7 . The method according to  claim 1 , wherein the enzymatic material comprises a protein selected from the group consisting of (a) to (d): 
 (a) a protein comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4;    (b) a protein comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 wherein one or more amino acids have been substituted, deleted, inserted, and/or added, and having the activity of producing (R)-3-quinuclidinol from 3-quinuclidinone by asymmetric reduction;    (c) a protein comprising an amino acid sequence having 85% or higher identity to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, and having the activity of producing (R)-3-quinuclidinol from 3-quinuclidinone by asymmetric reduction; and    (d) a protein encoded by a polynucleotide capable of hybridizing to the polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 under a stringent condition, and having the activity of producing (R)-3-quinuclidinol from 3-quinuclidinone by asymmetric reduction.    
     
     
         8 . The method according to  claim 1 , wherein the enzymatic material is a transformant carrying a DNA encoding tropinone reductase-I or a vector comprising a DNA encoding tropinone reductase-I, or a processed product of the transformant.  
     
     
         9 . The method according to  claim 8 , wherein the vector comprising the DNA selected from the group consisting of (a) to (d): 
 (a) a DNA comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3;    (b) a DNA that encodes a protein comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 wherein one or more amino acids have been substituted, deleted, inserted, and/or added and having the activity of producing (R)-3-quinuclidinol from 3-quinuclidinone by asymmetric reduction;    (c) a DNA comprising a nucleotide sequence having 85% or higher identity to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3, and encoding a protein having the activity of producing (R)-3-quinuclidinol from 3-quinuclidinone by asymmetric reduction; and    (d) a DNA that is capable of hybridizing to a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 under a stringent condition, and encodes a protein having the activity of producing (R)-3-quinuclidinol from 3-quinuclidinone by asymmetric reduction.    
     
     
         10 . The method according to  claim 8 , wherein the vector further comprises a DNA encoding an enzyme of regenerating a reduced coenzyme from the oxidized form.  
     
     
         11 . The method according to  claim 8 , wherein the transformant further comprises a DNA encoding an enzyme of regenerating a reduced coenzyme from the oxidized form or a vector comprising a DNA encoding an enzyme of regenerating a reduced coenzyme from the oxidized form.  
     
     
         12 . The method according to  claim 1 , wherein the reduced coenzyme is NADPH or NADH.  
     
     
         13 . The method according to  claim 10 , wherein the enzyme of regenerating a reduced coenzyme from the oxidized form is selected from the group consisting of glucose dehydrogenase, glutamate dehydrogenase, formate dehydrogenase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, phosphogluconate dehydrogenase, alcohol dehydrogenase, and glycerol dehydrogenase.  
     
     
         14 . The method according to  claim 13 , wherein the enzyme of regenerating a reduced coenzyme from the oxidized form is a glucose dehydrogenase.  
     
     
         15 . The method according to  claim 14 , wherein the glucose dehydrogenase is derived from  Bacillus subtilis  or  Thermoplasma acidophilum.    
     
     
         16 . The method according to  claim 1 , wherein the asymmetric reduction is preformed at pH 6.5 to 8.5.  
     
     
         17 . A method for producing (R)-3-quinuclidinol hydrochloride, the method comprising the steps of: 
 (a) making the pH of (R)-3-quinuclidinol solution alkaline to form free (R)-3-quinuclidinol;    (b) extracting free (R)-3-quinuclidinol with n-butanol;    (c) adding hydrochloric acid to the extract;    (d) removing moisture from the extract; and    (e) crystallizing (R)-3-quinuclidinol hydrochloride in the solution obtained in (d).    
     
     
         18 . A method for producing (R)-3-quinuclidinol, the method comprising the steps of: 
 (a) dissolving (R)-3-quinuclidinol hydrochloride in a first solvent, wherein the first solvent is capable of dissolving (R)-3-quinuclidinol hydrochloride at a concentration of 1% or higher;    (b) making the pH of the solution obtained in (a) alkaline to form free (R)-3-quinuclidinol;    (c) adding a second solvent to the (R)-3-quinuclidinol solution, wherein the second solvent can be substituted for the first solvent by distilling off the first solvent from a mixture of the first and second solvents, dissolves free (R)-3-quinuclidinol with lower solubility than the first solvent, and allows to crystallize free (R)-3-quinuclidinol from itself;    (d) distilling off the first solvent; and    (e) crystallizing (R)-3-quinuclidinol in the solution obtained in (d).    
     
     
         19 . The method according to  claim 18 , wherein the second solvent is selected from the group consisting of toluene, hexane, 4-methyl-2-pentanone, and butyl acetate.  
     
     
         20 . The method according to  claim 18 , wherein the first solvent is water, and the second solvent is toluene.  
     
     
         21 . A method for producing (R)-3-quinuclidinol, the method comprising the steps of: 
 (a) making the pH of (R)-3-quinuclidinol solution alkaline to form free (R)-3-quinuclidinol;    (b) extracting free (R)-3-quinuclidinol with n-butanol; and    (c) adding an organic solvent to the extract, wherein the organic solvent can be substituted for n-butanol by distilling off n-butanol from a mixture of n-butanol and the organic solvent, dissolves free (R)-3-quinuclidinol with lower solubility than n-butanol, and allows to crystallize free (R)-3-quinuclidinol from itself;    (d) distilling off n-butanol; and    (e) crystallizing (R)-3-quinuclidinol in the solution obtained in (d).    
     
     
         22 . The method according to  claim 21 , wherein the organic solvent is selected from the group consisting of toluene, 4-methyl-2-pentanone, and butyl acetate.  
     
     
         23 . A vector comprising and capable of expressing a DNA encoding tropinone reductase-I and a DNA encoding an enzyme of regenerating a reduced coenzyme from the oxidized form.  
     
     
         24 . The vector according to  claim 23 , wherein the DNA encoding tropinone reductase-I is a DNA selected from the group consisting of (a) to (d): 
 (a) a DNA comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3;    (b) a DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 wherein one or more amino acids have been substituted, deleted, inserted, and/or added and having an activity of producing (R)-3-quinuclidinol from 3-quinuclidinone by asymmetric reduction;    (c) a DNA comprising a nucleotide sequence having 85% or higher identity to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3, and encoding a protein having an activity of producing (R)-3-quinuclidinol from 3-quinuclidinone by asymmetric reduction; and    (d) a DNA capable of hybridizing to a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 under a stringent condition, and encoding a protein having the activity of producing (R)-3-quinuclidinol from 3-quinuclidinone by asymmetric reduction.    
     
     
         25 . The vector according to  claim 23 , wherein the reduced coenzyme is NADPH or NADH.  
     
     
         26 . The vector according to  claim 23 , wherein the enzyme of regenerating a reduced coenzyme from the oxidized form is selected from the group consisting of glucose dehydrogenase, glutamate dehydrogenase, formate dehydrogenase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, phosphogluconate dehydrogenase, alcohol dehydrogenase, and glycerol dehydrogenase.  
     
     
         27 . The vector according to  claim 23 , wherein the enzyme of regenerating a reduced coenzyme from the oxidized form is a glucose dehydrogenase.  
     
     
         28 . The vector according to  claim 27 , wherein the glucose dehydrogenase is derived from  Bacillus subtilis  or  Thermoplasma acidophilum.    
     
     
         29 . A transformant carrying and capable of expressing (1) a DNA encoding tropinone reductase-I or a vector comprising a DNA encoding tropinone reductase-I and (2) a DNA encoding an enzyme of regenerating a reduced coenzyme from the oxidized form or a vector comprising a DNA encoding an enzyme of regenerating a reduced coenzyme from the oxidized form.  
     
     
         30 . The transformant according to  claim 29 , wherein the transformant carries and is capable of expressing a vector comprising a DNA encoding tropinone reductase-I and a DNA encoding an enzyme of regenerating a reduced coenzyme from the oxidized form.  
     
     
         31 . The transformant according to  claim 29 , wherein a host is  Escherichia coli.