# Patent

## Title
Process for the enantioselective enzymatic reduction of hydroxy keto compounds

## Abstract

In a process for the enantioselective enzymatic reduction of a hydroxy ketone of general formula I




wherein R1═C1-C6 alkyl and R2═—Cl, —CN, —OH, —H or C1-C6 alkyl, into a chiral diol of general formula II





wherein R1 and R2 have the same meaning as in formula I, the hydroxy ketone is reduced with an oxidoreductase in the presence of NADH or NADPH as a cofactor, wherein

    a) the hydroxy ketone is provided in the reaction at a concentration of ≧50 g/l,
    b) the oxidized cofactor NAD or NADP having formed is regenerated continuously by oxidation of a secondary alcohol of general formula RXRYCHOH, wherein RX, RY independently represent hydrogen, branched or unbranched C1-C8-alkyl and Ctotal≧3, and
    c) the reduction of the hydroxy ketone and the oxidation of the secondary alcohol are catalyzed by the same oxidoreductase.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a Divisional Application of co-pending U.S. application Ser. No. 12/086,704 filed Nov. 10, 2008, now U.S. Pat. No. 8,980,592 which is the National Phase of PCT/EP2006/012037 filed on Dec. 14, 2006, which claims priority to Patent Application No. 2027/2005 filed in Austria on Dec. 19, 2005, all of which are hereby expressly incorporated by reference into the present application.


The invention relates to a process for the enantioselective enzymatic reduction of a hydroxy ketone of general formula I




wherein R1═C1-C6 alkyl and R2═—Cl, —CN, —OH, —H or C1-C6 alkyl, into a chiral diol of general formula II





wherein R1 and R2 have the same meaning as in formula I, with the hydroxy ketone being reduced with an oxidoreductase in the presence of a cofactor.

Chiral diols of general formula II are important intermediates in the production of pharmaceutical products, in particular in the production of HMG-CoA reductase inhibitors. Such chiral diols are, for example, tert. butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate, tert. butyl (3R,5R)-6-cyano-3,5-dihydroxyhexanoate or tert. butyl (5S,3R)-3,5,6-trihydroxy-hexanoate.
Diols of this kind are produced in particular by enantioselective reduction of the corresponding hydroxy ketones of formula I such as, e.g., tert. butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate, tert. butyl (5S)-6-chloro-5-hydroxy-3-oxohexanoate or tert. butyl (5S)-5,6-dihydroxy-3-oxohexanoate. In doing so, the chemically catalyzed reduction has the disadvantage that, on the one hand, it may result in byproducts due to the harsh reaction conditions and, on the other hand, yields unsatisfactory enantiomeric and diastereomeric excesses, respectively, and is technically feasible only with very large efforts.
For this reason, there have, for quite some time, been endeavors to develop biocatalytic processes which allow for the enantioselective reduction of the above-mentioned hydroxy ketones. Biocatalytic processes usually operate under mild conditions, which is why they can be expected to enable the reduction of 5-hydroxy-3-oxohexanoate derivatives, which are rather unstable anyway, without the formation of further byproducts. So far, however, it has not been possible to find any suitable biocatalysts by means of which the enzymatic reduction of the above-mentioned 5-hydroxy-3-oxohexanoate derivatives is feasible in an effective manner and with isolated enzymes.
For instance, the U.S. Pat. No. 6,001,615 and the international patent application WO 01/85975 A1 describe the reduction of tert. butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate and of tert. butyl (5S)-5,6-dihydroxy-3-oxohexanoate, respectively, with various yeasts of the genus Beauvaria, Pichia, Candida, Kluyveromyces and Torulaspora. Thereby, however, the conversions occur only with whole cells of wild strains and therefore can only be carried out at very low concentrations of far below 5%. So far, it has not yet been possible to identify the enzymes and DNA sequences responsible for the conversions.
Furthermore, microbial conversions of structurally similar compounds are described in EP 0 569 998 B1. Therein, it has even been possible to purify an NADH-dependent enzyme from Acinetobacter calcoaceticus ATCC 33305, which is also used in an isolated state together with glucose dehydrogenase for coenzyme regeneration. In the process described, the substrate is used at concentrations of 1% and a “total turn over number” of the NADH of merely 10 is achieved. An industrially applicable process has not been presented.
In the U.S. Pat. No. 6,645,746 B1, an amino acid sequence from Candida magnoliae is disclosed, which may be used for reducing tert. butyl (5S)-6-chloro-5-hydroxy-3-oxohexanoate to tert. butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate with the aid of NADPH. In the specification of said document, the enzyme is preferably used in a state in which it is coexpressed with glucose dehydrogenase from Bacillus megaterium, wherein the regeneration of cofactor NADPH occurs with the aid of the glucose dehydrogenase and with glucose as a cosubstrate.
It is the object of the invention to provide a process which enables the economical production of enantiomerically pure diols of general formula II in high yields and with high enantiomeric purity without any byproducts.
According to the invention, this object is achieved by a process of the initially mentioned kind, wherein the oxidoreductase is reduced in the presence of NADH or NADPH as a cofactor and which is characterized in that
a) the hydroxy ketone is provided in the reaction at a concentration of ≧50 g/l,
b) the oxidized cofactor NAD or NADP having formed is regenerated continuously by oxidation of a secondary alcohol of general formula RXRYCHOH, wherein RX, RY independently represent hydrogen, branched or unbranched C1-C8-alkyl and Ctotal≧3, and
c) the reduction of the hydroxy ketone and the oxidation of the secondary alcohol are catalyzed by the same oxidoreductase.
A preferred embodiment of the process is characterized in that the oxidoreductase
a) comprises an amino acid sequence in which at least 50% of the amino acids are identical to those of amino acid sequence SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:11 or SEQ ID NO:14,
b) is encoded by the nucleic acid sequence SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:15, or
c) is encoded by a nucleic acid sequence which hybridizes to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:15 under stringent conditions.
According to the invention, the above-mentioned object is also achieved by a process of the initially mentioned kind, wherein the oxidoreductase
a) comprises an amino acid sequence in which at least 50% of the amino acids are identical to those of amino acid sequence SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:11 or SEQ ID NO:14,
b) is encoded by the nucleic acid sequence SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:15, or
c) is encoded by a nucleic acid sequence which hybridizes to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:15 under stringent conditions.
It has been found that the polypeptides comprising amino acid sequences SEQ ID NO:1, SEQ ID NO:8 and SEQ ID NO:11 show oxidoreductase activity and can be used for reducing tert. butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate with a diastereomeric excess of >99% to tert. butyl (3R,5R)-6-cyano-3,5-dihydroxyhexanoate. Other hydroxy ketones of general formula I can be reduced in the same manner by means of amino acid sequences SEQ ID NO:1, SEQ ID NO:8 and SEQ ID NO:11.
In addition, the aforementioned oxidoreductases have the advantage of being able to regenerate the oxidized cofactor that is formed during the reduction by reducing a secondary alcohol. Thus, a particular economic advantage of said oxidoreductases also consists in that, in contrast to processes of the prior art (U.S. Pat. No. 6,645,746 B and EP 0 569 998 B), no further enzyme is required for cofactor regeneration.
A DNA sequence SEQ ID NO:2, which codes for a polypeptide comprising SEQ ID NO:1, is obtainable, for example, from the genome of the organism Rubrobacter xylanophilus DSM 9941. In addition, it has been found that the DNA sequence SEQ ID NO:3 can be used for expressing the polypeptide of SEQ ID NO:1 in Escherichia. 
A DNA sequence SEQ ID NO:9, which codes for a polypeptide comprising SEQ ID NO:8, is obtainable, for example, from the genome of the organism Geobacillus thermodenitrificans DSM 465. In addition, it has been found that the DNA sequence SEQ ID NO:10 can be used for expressing the polypeptide of SEQ ID NO:8 in Escherichia. 
A DNA sequence SEQ ID NO:12, which codes for a polypeptide comprising SEQ ID NO:11, is obtainable, for example, from the genome of the organism Chloroflexus aurantiacus DSM 635.
A DNA sequence SEQ ID NO:15, which codes for a polypeptide comprising SEQ ID NO:14, is obtainable, for example, from the organism Candida magnoliae CBS 6396.
Thus, the present invention also relates to a process for the reduction of hydroxy ketones of general formula I into diols of general formula II, using a polypeptide comprising amino acid sequence SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:11 or SEQ ID NO:14, or a polypeptide which comprises an amino acid sequence which is identical by at least 50% to the amino acid sequence SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:11 or SEQ ID NO:14, i.e., a polypeptide which can be derived by substitution, insertion, deletion or addition from at least one amino acid of SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:11 or SEQ ID NO:14, or using a polypeptide which is encoded by the nucleic acid sequence SEQ ID NO:2, SEQ ID NO:9, SEQ ID NO:12 or SEQ ID NO:15 or by a nucleic acid sequence which hybridizes to SEQ ID NO:2, SEQ ID NO:9, SEQ ID NO:12 or SEQ ID NO:15 under stringent conditions.
By a nucleic acid sequence which hybridizes, for example, to SEQ ID NO:2 under stringent conditions, a polynucleotide is understood which can be identified via the colony hybridization method, the plaque hybridization method, the Southern hybridization method or comparable methods, using SEQ ID NO:2 as a DNA probe.
For this purpose, the polynucleotide immobilized on a filter is hybridized, for example, to SEQ ID NO:2 in a 0.7-1 M NaCl solution at 60° C. Hybridization is carried out as described, for instance, in Molecular Cloning, A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, 1989) or in similar publications. Subsequently, the filter is washed with a 0.1 to 2-fold SSC solution at 65° C., wherein a 1-fold SSC solution is understood to be a mixture consisting of 150 mM NaCl and 15 mM sodium citrate.
In the process according to the invention, the polypeptide comprising the sequence SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:11 or SEQ ID NO:14 and polypeptides derivable from said polypeptides, respectively, can be used either in a completely purified state, in a partially purified state or as cells containing the polypeptide SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:11 or SEQ ID NO:14. Thereby, the cells used can be provided in a native, permeabilized or lysed state. Preferably, the polypeptide comprising the sequence SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:11 or SEQ ID NO:14 and derivatives derivable therefrom, respectively, are overexpressed in a suitable host organism such as, for example, Escherichia coli, and the recombinant polypeptide is used for reducing the hydroxy ketone of general formula I.
5.000 to 10 Mio U of oxidoreductase SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:11 or SEQ ID NO:14 or derivatives thereof, respectively, are used per kg of the compound of Formula I to be reacted (upwardly open). Herein, the enzyme unit 1 U corresponds to the enzyme amount which is required for reacting 1 μmol of the hydroxy ketone of formula I per minute (min).
The enzymatic reduction according to the invention proceeds under mild conditions so that the degradation of the frequently unstable hydroxy ketone and thus the formation of undesired byproducts can be largely avoided. The process according to the invention has a high service life and a diastereomeric purity of normally >99% of the chiral diols produced.
A preferred embodiment of the invention is characterized in that the cofactor used in the process is continuously reduced with a cosubstrate. Preferably, NAD(P)H is used as the cofactor, with the NAD(P) formed in the reduction again being reduced to NAD(P)H by means of the cosubstrate.
Secondary alcohols such as 2-propanol, 2-butanol, 2-pentanol, 3-pentanol, 4-methyl-2-pentanol, 2-heptanol, 2-octanol or cyclohexanol are thereby preferably used as cosubstrates. According to a particularly preferred embodiment, 2-propanol is used for coenzyme regeneration. The amount of cosubstrate for the regeneration can range from 5 to 95% by volume, based on the total volume.
Coenzyme regeneration is, for example, likewise effected via the polypeptide comprising SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:11 or SEQ ID NO:14.
In the process according to the invention, the hydroxy ketone of general formula I is preferably used in an amount of from 5 to 50% by weight (50 g/1 to 50 g/1), based on the total volume, preferably from 8 to 40% by weight, in particular from 10 to 25% by weight.
A particularly preferred embodiment is characterized in that tert. butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate, tert. butyl (5S)-6-chloro-5-hydroxy-3-oxohexanoate or tert. butyl (5S)-5,6-dihydroxy-3-oxohexanoate is used as the hydroxy ketone of general formula (I).
Preferably, the process according to the invention is carried out in an aqueous organic two-phase system.
The aqueous portion of the reaction mixture in which the enzymatic reduction proceeds preferably contains a buffer, e.g., a potassium phosphate, tris/HCl or triethanolamine buffer, having a pH value of from 5 to 10, preferably a pH of from 6 to 9. In addition, the buffer can contain ions for stabilizing or activating the enzymes such as, for example, zinc ions or magnesium ions.
While carrying out the process according to the invention, the temperature suitably ranges from about 10° C. to 70° C., preferably from 20° C. to 45° C.
In a further preferred embodiment of the process according to the invention, the enzymatic conversion is carried out in the presence of an organic solvent which is not miscible with water or is miscible with water only to a limited degree. Said solvent is, for example, a symmetric or unsymmetric di(C1-C6)alkyl ether, a linear-chain or branched alkane or cycloalkane or a water-insoluble secondary alcohol which, at the same time, represents the cosubstrate. The preferred organic solvents are diethyl ether, tertiary butyl methyl ether, diisopropyl ether, dibutyl ether, butyl acetate, heptane, hexane, 2-octanol, 2-heptanol, 4-methyl-2-pentanol and cyclohexanol. In case of the last-mentioned secondary alcohols, the solvent can simultaneously also serve as a cosubstrate for cofactor regeneration.
If water-insoluble solvents and cosubstrates, respectively, are used, the reaction batch consists of an aqueous phase and an organic phase. According to its solubility, the hydroxy ketone is distributed between the organic phase and the aqueous phase. In general, the organic phase has a proportion of from 5 to 95%, preferably from 10 to 90%, based on the total reaction volume. The two liquid phases are preferably mixed mechanically so that, between them, a large surface area is generated. Also in this embodiment, the NAD formed in the enzymatic reduction, for example, can again be reduced to NADH with a cosubstrate, such as described above.
The concentration of the cofactor, in particular of NADH or NADPH, respectively, in the aqueous phase generally ranges from 0.001 mM to 10 mM, in particular from 0.01 mM to 1 mM.
In the process according to the invention, a stabilizer of oxidoreductase/dehydrogenase can also be used. 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, e.g., nitrogen or air.
According to another possible embodiment of the invention, the oxidized cosubstrate (e.g. acetone) can be removed continuously and/or the cosubstrate (e.g. 2-propanol) can be newly added in a continuous manner in order to shift the reaction equilibrium towards the reaction product (diol of general formula II).
In a further embodiment, the addition of the oxidoreductase according to SEQ ID NO:1, SEQ ID NO:8, SEQ ID NO:11 or SEQ ID NO:14 and/or of the cosubstrate can also be effected gradually in the course of the process.
After completion of the reduction, the reaction mixture is processed. For this purpose, the aqueous phase is optionally separated from the organic phase and the organic phase containing the product is filtered. Optionally, the aqueous phase can also be extracted and processed further like the organic phase. Thereupon, the solvent is evaporated from the organic phase and the diol of general formula II is obtained as a crude product. The crude product can then be purified further or used directly for the synthesis of a follow-up product.
In the following, the invention is illustrated further by way of examples.


Example 1
Cloning of oxidoreductase from Rubrobacter xylanophilus DSM 9941
A) Cultivation of Rubrobacter xylanophilus DSM 9941
Cells of Rubrobacter xylanophilus DSM 9941 were cultivated in the following medium at 50° C. (pH 7.2) and 140 rpm in a bacterial shaker: 0.1% yeast extract, 0.1% tryptone, 0.004% CaSO4×2 H2O, 0.02% MgCl2×6 H2O, 0.01% nitrilotriacetic acid, 100 ml phosphate buffer [5.44 g/l KH2PO4, 43 g/l Na2HPO4×12 H2O], 500 μl/10.01 M Fe citrate, 500 μl/l trace element [500 μl/l H2SO4, 2.28 g/l MnSO4×H2O, 500 mg/l ZnSO4×7 H2O, 500 mg H3BO3, 25 mg/l CuSO4×5 H2O, 25 mg/l Na2MoO4×2 H2O, 45 mg/l CoCl2×6 H2O]. On day 6 of the cultivation, cells were separated from the culture medium by centrifugation and stored at −80° C.
B) Amplification of the Gene Coding for Selective Oxidoreductase
Genomic DNA was extracted according to the method described in “Molecular Cloning” by Manniatis & Sambrook. The resulting nucleic acid served as a template for the polymerase chain reaction (PCR) involving specific primers which were derived from the gene sequence published under number 46106817 in the NCBI database. In doing so, the primers were provided in a 5′-terminal position with restriction sites for the endonucleases Nde I and Hind III or Sph I, respectively (SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7), for subsequent cloning into an expression vector.
Amplification was carried out in a PCR buffer [10 mM tris-HCl, (pH 8.0); 50 mM KCl; 10 mM MgSO4; 1 mM dNTP Mix; per 20 pMol of primer and 2.5 U of Platinum Pfx DNA polymerase (Invitrogen)] with 500 ng of genomic DNA and the following temperature cycles:
Cycle 1: 94° C., 2 min
Cycle 2×30: 94° C., 15 sec


    
    
        54° C., 30 sec
        68° C., 60 sec

Cycle 3: 68° C., 7 min

        4° C., ∞
    
    


The resulting PCR product having a size of about 750 bp was restricted after purification over a 1% agarose gel with the aid of the endonucleases Nde I and Hind III or Sph I and Hind III, respectively, and was ligated into the backbone of the pET21a vector (Novagen) or of the pQE70 vector (Qiagen), respectively, which backbone had been treated with the same endonucleases. After transforming 2 μl of the ligation batch into E. coli Top 10 F′ cells (Invitrogen), plasmid DNA of ampicillin-resistant colonies was tested for the presence of an insert having a size of 750 bp by means of a restriction analysis with the endonucleases Nde I and Hind III or Sph I and Hind III, respectively. Plasmid preparations from the clones which were positive for the fragment were subjected to a sequence analysis and subsequently transformed into Escherichia coli BL21 Star (Invitrogen) and E. coli RB791 (genetic stock, Yale), respectively.
Example 2
Efficient Expression of Polypeptide SEQ ID NO:1 in Escherichia coli Cells
In order to obtain efficient expression of the polypeptide SEQ ID NO:1 in Escherichia coli cells, for cloning into an expression vector coding DNA SEQ ID NO:3 was used as a template in a PCR reaction. In the region of the first 160 base pairs, this DNA sequence differed in 51 bases from the previously known DNA sequence (SEQ ID NO:2). This modification was conservative and did not result in a change in the amino acid sequence.
Amplification was carried out in a PCR buffer [10 mM tris-HCl, (pH 8.0); 50 mM KCl; 10 mM MgSO4; 1 mM dNTP Mix; per 20 pMol of primer (SEQ ID NO:6, SEQ ID NO:5) and 2.5 U of Platinum Pfx DNA polymerase (Invitrogen)] with 50 ng of DNA SEQ ID NO:3 as a template and the following temperature cycles:
Cycle 1: 94° C., 2 min
Cycle 2×30: 94° C., 40 sec


    
    
        56° C., 30 sec
        68° C., 60 sec

Cycle 3: 68° C., 7 min

        4° C., ∞
    
    


The resulting PCR product having a size of about 750 bp was ligated after purification over a 1% agarose gel with the aid of the endonucleases Nhe I and Hind III into the backbone of the pET21a vector (Novagen), which backbone had been treated with the same endonucleases. After transforming 2 μl of the ligation batch into E. coli Top 10 F′ cells (Invitrogen), plasmid DNA of ampicillin-resistant colonies was tested for the presence of an insert having a size of 750 bp by means of a restriction analysis with the endonucleases Nhe I and Hind III. Plasmid preparations from the clones which were positive for the fragment were subjected to a sequence analysis and subsequently transformed into Escherichia coli BL21 Star (Invitrogen).
Example 3
Preparation of Oxidoreductase from Rubrobacter xylanophilus DSM 9941
The Escherichia coli strains BL21 Star (Invitrogen, Karlsruhe, Germany) and RB791 (E. coli genetic stock, Yale, USA), respectively, which had been transformed with the expression construct, were cultivated in a medium (1% tryptone, 0.5% yeast extract, 1% NaCl) with ampicillin (50 μg/ml) until an optical density of 0.5, measured at 550 nm, was reached. The expression of recombinant protein was induced by adding isopropylthiogalactoside (IPTG) at a concentration of 0.1 mM. 16 hours after the induction at 25° C. and 220 rpm, the cells were harvested and frozen at −20° C.
For enzyme recovery, 30 g cells were suspended in 150 ml of triethanolamine buffer (100 mM, pH=7, 2 mM MgCl2, 10% glycerol) and broken down by means of a high-pressure homogenizer. Subsequently, the enzyme solution was mixed with 150 ml of glycerol and stored at −20° C.
The enzyme solution thus obtained was used for the synthesis of tert. butyl (3R,5R)-6-cyano-3,5-dihydroxyhexanoate.
In the diluted state, the enzyme solution obtained was also used for the corresponding enzymatic measurements. Thereby, the activity test was made up of the following: 870 μl of 100 mM TEA buffer, pH 7.0, 160 μg NAD(P)H, 10 μl diluted cell lysate. The reaction was initiated by adding 100 μl of the 100 mM substrate solution tert. butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate to the reaction mixture.
Example 4
Conversion of tert. butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate into tert. butyl (3R,5R)-6-cyano-3,5-dihydroxyhexanoate by means of oxidoreductase SEQ ID NO:1
For the conversion of tert. butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate into tert. butyl (3R,5R)-6-cyano-3,5-dihydroxyhexanoate, a mixture of 900 μl buffer (100 mM TEA, pH=7, 1 mM MgCl2), 100 μl 2-propanol, 10 μl tert. butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate crude product (enantiomeric purity>99%), 0.1 mg NAD and 100 μl enzyme suspension (see Example 3) was incubated in an Eppendorf reaction vessel for 24 h at room temperature, under continual mixing. After 24 h, 96% of the tert. butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate used had been reduced to tert. butyl (3R,5R)-6-cyano-3,5-dihydroxyhexanoate. The diastereomeric excess amounted to >99%.
The determination of the conversion as well as of the diastereomeric excess was performed by chiral gas chromatography. For this purpose, a gas chromatograph GC-17A from Shimadzu comprising a chiral separating column CP-Chirasil-DEX CB (Varian Chrompack, Darmstadt, Germany), a flame ionization detector and helium as a carrier gas was used.
The separation of tert. butyl-6-cyano-3,5-dihydroxyhexanoate occurred at 0.72 bar and for 10 min at 50° C., 5° C./min→200° C. for 10 min.
The retention times were: (R—BCH) 4.4 min; (R,R—BCH) 47.1 min and (R,S—BCH) 48.2 min.
Example 5
Synthesis of tert. butyl (3R,5R)-6-cyano-3,5-dihydroxyhexanoate from tert. butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate by means of oxidoreductase SEQ ID NO:1
For a further conversion of tert. butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate into tert. butyl (3R,5R)-6-cyano-3,5-dihydroxyhexanoate, a mixture of 550 μl buffer (100 mM TEA, pH=7, 1 mM MgCl2), 150 μl 2-propanol, 200 μl tert. butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate crude product (enantiomeric purity>99%), 0.1 mg NAD and 200 μl enzyme suspension (see Example 3) was incubated in an Eppendorf reaction vessel. In the course of the reaction, the acetone/2-propanol mixture formed was evaporated repeatedly by the introduction of nitrogen, and fresh 2-propanol and 50 μl of enzyme were added. After 2 to 3 repeats at 24-hour intervals, >90% of the tert. butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate used had been reduced to tert. butyl (3R,5R)-6-cyano-3,5-dihydroxyhexanoate. The diastereomeric excess amounted to >99%.
Example 6
Synthesis of tert. butyl (3R,5R)-6-cyano-3,5-dihydroxyhexanoate from tert. butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate by means of oxidoreductase SEQ ID NO:1
For a further conversion of tert. butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate into tert. butyl (3R,5R)-6-cyano-3,5-dihydroxyhexanoate, a mixture of 6.7 ml buffer (100 mM TEA, pH=9), 1.7 ml 2-propanol, 2 ml tert. butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate crude product (enantiomeric purity>99%), 1.0 mg NAD and 150 mg frozen cells E. coli BL21 Star, containing oxidoreductase SEQ ID NO:1, (see Example 3) was incubated in a reaction vessel at 45° C. After 24 h, >90% of the tert. butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate used had been reduced to tert. butyl (3R,5R)-6-cyano-3,5-dihydroxyhexanoate. The diastereomeric excess amounted to >99%.
Example 7
Synthesis of tert. butyl (3R,5R)-6-cyano-3,5-dihydroxyhexanoate from tert. butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate by means of oxidoreductase SEQ ID NO:8
For a further conversion of tert. butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate into tert. butyl (3R,5R)-6-cyano-3,5-dihydroxyhexanoate, a mixture of 5.0 ml buffer (100 mM TEA, pH=7.5), 2.0 ml 2-propanol, 4.0 ml tert. butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate crude product (enantiomeric purity>99%), 1.0 mg NAD and 250 mg frozen cells E. coli RB 791, containing oxidoreductase SEQ ID NO:8, (corresponding to Examples 2 and 3) was incubated in a reaction vessel at 40° C. After 24 h, >90% of the tert. butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate used had been reduced to tert. butyl (3R,5R)-6-cyano-3,5-dihydroxyhexanoate. The diastereomeric excess amounted to >99%.
Example 8 
Synthesis of tert. butyl (3R,5R)-6-cyano-3,5-dihydroxyhexanoate from tert. butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate by means of oxidoreductase SEQ ID NO:11
For the conversion of tert. butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate into tert. butyl (3R,5R)-6-cyano-3,5-dihydroxyhexanoate, a mixture of 350 μl buffer (100 mM potassium phosphate, pH=7), 150 μl 2-propanol, 50 μl tert. butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate crude product (enantiomeric purity>99%), 0.025 mg NAD and 15 μl enzyme suspension SEQ ID NO:11 (see Example 3) was incubated in an Eppendorf reaction vessel for 48 h at room temperature, under continual mixing. After 48 h, >80% of the tert. butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate used had been reduced to tert. butyl (3R,5R)-6-cyano-3,5-dihydroxyhexanoate. The diastereomeric excess amounted to >99%.








Sequence listing



SEQ ID NO: 1



  1 mlegkvavit gagsgigrat alrfaregar vvvaelderr



 41 geevvreile sggeavfvrt dvsefeqvea averaveeyg


 81 tldvmfnnag ighyaplleh dpehydrvvr vnqygvyygi


121 laagrkmael enpgviinta svyaflaspg vigyhaskga


161 vkmmtqaaal elaphgirvv aiapggvdtp iiqgykdmgl


201 gerlargqmr rrlqtpeqia gavvllatee adaingsvvm


241 tddgyaefk


 


SEQ ID NO: 2



  1 atgctcgaggggaaggtcgcggtcatcacgggggccggca



 41 gcggcataggccgggccaccgcgctcaggttcgcccgcga


 81 aggggcccgggtggtcgtggcggagctcgacgagcggagg


121 ggggaggaggtcgtccgggagatcctcgagtccggcgggg


161 aggccgtcttcgtgaggacggacgtctcggagttcgagca


201 ggttgaggccgccgtcgagcgcgccgtcgaggagtacggg


241 acgctggacgtcatgttcaacaacgccggcatcgggcact


281 acgcccccctgctggagcacgacccggagcactacgaccg


321 ggtggtccgggtgaaccagtacggcgtctactacgggata


361 ctcgccgccggcaggaagatggccgagctggagaaccccg


401 gcgtgatcatcaacaccgcctcggtctacgctttcctggc


441 ctcccccggtgtgatcggctatcacgcttccaagggggcg


481 gtgaagatgatgacccaggccgcagccctggagctcgccc


521 cccacggcatacgggtcgtcgccatcgccccgggcggggt


561 ggacaccccgatcatccagggctacaaggacatgggcctc


601 ggtgagcggctggcccgcggccagatgcgtcgcaggctcc


641 agacccccgagcagatcgccggcgccgtcgtcctgctcgc


681 caccgaggaggcagacgccataaacggctcggtggtgatg


721 accgacgacggctacgcggagttcaagtaa


 


SEQ ID NO: 3



  1 atgctggaag gtaaagtggc agtcatcacc ggtgcaggca



 41 gcggcattgg gcgtgccact gcgctgcgtt ttgcgcgtga


 81 aggcgctcgc gtcgttgtgg ccgagctgga tgaacgtcgc


121 ggtgaggaag ttgtacgtga gattctggaa tctggcgggg


161 gggaggccgt cttcgtgagg acggacgtct cggagttcga


201 gcaggttgag gccgccgtcg agcgcgccgt cgaggagtac


241 gggacgctgg acgtcatgtt caacaacgcc ggcatcgggc


281 actacgcccc cctgctggag cacgacccgg agcactacga


321 ccgggtggtc cgggtgaacc agtacggcgt ctactacggg


361 atactcgccg ccggcaggaa gatggccgag ctggagaacc


401 ccggcgtgat catcaacacc gcctcggtct acgctttcct


441 ggcctccccc ggtgtgatcg gctatcacgc ttccaagggg


481 gcggtgaaga tgatgaccca ggccgcagcc ctggagctcg


521 ccccccacgg catacgggtc gtcgccatcg ccccgggcgg


561 ggtggacacc ccgatcatcc agggctacaa ggacatgggc


601 ctcggtgagc ggctggcccg cggccagatg cgtcgcaggc


641 tccagacccc cgagcagatc gccggcgccg tcgtcctgct


681 cgccaccgag gaggcagacg ccataaacgg ctcggtggtg


721 atgaccgacg acggctacgc ggagttcaag taa


 


SEQ ID NO: 4



GGGAATTCCATATGATGCTCGAGGGGAAGGTCG



 


SEQ ID NO: 5



CCCAAGCTTATTACTTGAACTCCGCGTAGCCGTC



 


SEQ ID NO: 6



CCTAGCTAGCATGCTGGAAGGTAAAGTGGC



 


SEQ ID NO: 7



CACATGCATGCGAATGCTCGAGGGGAAGGTC



 


Geobacillus thermodenitrificans DSM 465 protein sequence carbonyl reductase


SEQ ID NO: 8



  1 mrlkgkaaiv tggasgigra tairfaeega kvaysdinee ggeetvrlir ekggeaifvq



 61 tdvadskqvs rlvqtavdaf gglhilfnna gighsevrst dlseeewdry invnlkgvfl


121 gikyavpvmk qcgggaivnt ssllgikgkk yesaynaska gvilltknaa leygkfniry


181 naiapgvidt niitpwkqde rkwpiiskan algrigtpee vanavlflas deasfitgat


241 lsvdgggltf


 


Geobacillus thermodenitrificans DSM 465 nucleic acid sequence carbonyl reductase


SEQ ID NO: 9



  1 atgaggctaa aaggaaaagc ggcgattgtc accggcggcg cgagcggcat cggccgggcg



 61 acggcgattc gctttgcgga agaaggcgcc aaagtggcgg tgagcgacat caatgaggaa


121 ggaggggaag aaacggtccg cctgattcgg gaaaaaggag gggaggcgat ttttgtccaa


181 acggacgtag ccgattccaa gcaagtgagc cgccttgtcc aaacggcggt tgatgccttt


241 ggcggcctac atattctctt taacaatgcc ggcatcggcc attcggaagt gcggagcacc


301 gacttgtctg aagaagagtg ggaccgggtc atcaacgtta atttgaaagg agtgttcctt


361 ggcatcaaat acgcggtgcc cgtgatgaag caatgcggtg gcggggccat tgtcaacaca


421 tcgagcctgc ttggaatcaa agggaaaaag tacgaatcgg cctacaacgc ctcgaaggcc


481 ggggtgattt tgttgacgaa aaatgcagca ttggaatatg ggaagtttaa cattcgcgtc


541 aatgccattg caccgggggt cattgatacg aacatcatca cgccgtggaa acaagatgag


601 cgcaaarggc cgatcatttc gaaagcgaac gccctcggcc gcatcgggac gccagaggaa


661 gtggcgaacg cggtgttgtt tttggcgtcc gatgaagcgt cgtttatcac cggcgcgaca


721 ttgtcggtcg acggcggcgg gctgacgttt tag


 


Geobacillus thermodenitrificans DSM 465 nucleic acid sequence synthetic gene


SEQ ID NO: 10



  1 atgcgcctga aagggaaagc ggcaattgtg acgggtggcg ccagcggcat cgggcgcgcg



 61 actgcgatcc gttttgcaga agagggtgcg aaagttgccg ttagcgacat taacgaggaa


121 ggcggtgagg aaaccgttcg cctgatccgt gaaaaaggcg gtgaggcaat cttcgtgcag


181 acggatgtgg ccgactcaaa acaggtatct cgtctggttc agaccgcggt cgacgcgttt


241 ggtggcctgc acatcctgtt caataacgcc ggcattggcc atagcgaagt gcgtagtact


301 gatctgagcg aggaagagtg ggatcgcgtg attaacgtga acctgaaagg tgtgtttctg


361 ggtattaagt atgcagtccc tgttatgaaa cagtgtggcg gtggtgcgat tgtgaatacc


421 tctagtctgt tgggaatcaa agggaaaaag tatgaatcgg cctacaacgc atcgaaagcc


481 ggcgtcatcc tgctgaccaa aaatgcggcc ctggagtatg gcaagttcaa tattcgtgtc


541 aacgcgatcg ctccaggcgt tatcgatacc aacatcatta caccgtggaa gcaagatgaa


601 cgcaagtggc cgattatctc caaagctaat gcgctgggcc gtatcggtac gccggaagaa


661 gtggctaatg cggttctgtt tctggcaagc gacgaagcga gctttattac gggtgcaacc


721 ctctccgtag atgcgggcgg gttaaccttc taa


 


Chloroflexus auratiacus DSMZ 635 protein sequence carbonyl reductase


SEQ ID NO: 11



  1 meppfigkva lvtgaaagig rasalafare gakvvvadvn veggeetial cralntdamf



 61 vrcdvsqrde verlialavd tfgridfahn nagiegvqam ladypeevwd rvieinlkgv


121 wlcmkyeirh mlkqgggaiv ntssvaglag srgvsayvas khgivgitka aaleyarngi


181 rvnaicpgti htamidrftq gdpqllaqfa egepigrlgs peevanaviw lcsdkasfvt


241 gatlavdggr la


 


 


Chloroflexus auratiacus DSMZ 635 nucleic acid sequence carbonyl reductase


SEQ ID NO: 12



  1 atggagccac ctttcattgg gaaggttgcg ctggtcaccg gcgcagcagc cggtattggt



 61 cgtgcttcag cactggcgtt tgcccgtgag ggtgccaagg ttgtcgttgc tgatgtgaat


121 gtcgagggcg gggaagagac gattgcgctg tgtcgggctt tgaataccga tgcaatgttc


181 gtgcgttgtg atgtttcgca acgcgatgaa gtggagcgat taattgctct ggcagttgac


241 acgttcggtc ggatcgactt tgcgcacaac aacgccggga ttgaaggcgt gcaggcaatg


301 ctggccgatt atcccgaaga ggtctgggat cgggtgatcg agatcaacct caaaggggtc


361 tggttgtgta tgaagtacga aatccggcac atgctcaagc agggtggcgg tgcgattgtg


421 aatacctcat cggtcgccgg tctggccgga tcacgtggcg tttcggcgta tgtagccagc


481 aagcacggta ttgttggtat taccaaagcg gcagcccttg agtatgcgcg taacggtatt


541 cgtgtcaacg caatctgtcc aggtacgatt catactgcga tgatcgaccg ctttacccag


601 ggtgatcccc aactgcttgc ccagttcgct gagggtgaac cgattggtcg gctcggctcg


661 cctgaagagg tcgccaatgc ggtgatctgg ctctgctcag ataaggcttc gtttgtgacc


721 ggagcgacac tggcggttga tggtggccgc ctggcgtaa


 


Chloroflexus auratiacus DSMZ 635 nucleic acid sequence synthetic gene


SEQ ID NO: 13



  1 atggagcccc catttatcgg gaaagttgcg ttagttacgg gggcagcggc agggatcggc



 61 agggcgagtg ccctggcgtt tgctagagaa ggggccaagg tcgttgtggc agacgttaac


121 gtagagggtg gcgaagagac aattgcttta tgcagagctc tcaacactga tgccatgttc


181 gtccgctgtg atgtgtcaca gcgagacgaa gtcgaaaggc taatcgccct agcggtagac


241 acattcggcc gtattgactt tgctcataat aacgcgggca tagagggagt acaagcaatg


301 ttggctgact atcctgagga agtatgggat cgagtaattg aaatcaatct caagggggtt


361 tggctgtgta tgaagtacga aataaggcac atgctcaagc aaggtggcgg agcgatcgta


421 aacactagct ctgtcgccgg tctagcagga tctcgggggg tttccgcata cgtcgcctcg


481 aaacacggca ttgtagggat taccaaagct gcagcccttg agtatgcccg aaatggaata


541 agagtgaatg ctatctgccc aggcacaata catactgcaa tgatagatcg gtttacgcag


601 ggtgatccgc aacttttggc gcagttcgcc gaaggtgagc ctataggtcg ccttggtagc


661 ccggaagagg tcgctaatgc ggtgatttgg ttgtgttcag acaaagcaag tttcgtgacg


721 ggagctaccc tggcagtgga tggaggacgt ttagct


 


 


Candida magnoliae CBS 6396 protein sequence carbonyl reductase


SEQ ID NO: 14



  1 msatsnalit gasrgmgeat aiklalegys vtlasrgieq lnaikeklpi vkkgqqhyvw



 61 qldlsdieaa stfkgaplpa ssydvffsna gvvdfapfad qsetaqkdlf tvnllspval


121 tktivkaiad kpretpahii ftssivgirg vpnvavysat kgaidsfars larefgpkni


181 hvncvnpgtt rtemtkgvdl aafgdvpikg wievdaiada vlflikskni tgqslvvdng


241 fgv


 


 


Candida magnoliae CBS 6396 nucleic acid sequence synthetic gene


SEQ ID NO: 15



  1 atgtctgcta cttcgaacgc tcttatcact ggtgccagcc gcggaatggg cgaggccaca



 61 gctattaagc ttgcccttga ggggtacagc gtcacccttg catcacgcgg tattgagcag


121 ctcaatgcca tcaaggaaaa actacccatc gtgaagaagg gccagcagca ctacgtttgg


181 cagctcgatc ttagtgacat cgaggcggct tccaccttca agggggctcc tctgcctgcc


241 agcagctacg acgtgttctt cagcaacgcc ggtgtggtgg actttgctcc gttcgcagac


301 caaagcgaga ctgcgcaaaa ggacctgttc acggttaacc tgctgtcgcc tgttgcgttg


361 accaagacca ttgttaaggc catcgccgac aagccccgcg agacgcctgc tcacattatc


421 ttcacctcgt ccattgtcgg aattcgcggt gttcccaacg tggcggtcta cagcgccacc


481 aagggcgcga ttgacagctt tgcgcgctcg cttgctcgtg agttcggtcc caagaacatc


541 cacgttaact gcgtgaaccc gggcacgacg cgcaccgaga tgacaaaggg cgttgatctc


601 gcggctttcg gcgatgttcc tatcaagggc tggatcgagg tcgatgcgat tgccgacgct


661 gtgctgtttt tgatcaagtc caagaacatc actggccagt cgctcgttgt tgacaacgga


721 ttcggtgttt aa

## Claims

1. A process for the enantioselective enzymatic reduction of a hydroxy ketone of general formula I



wherein R1═C1-C6 alkyl and R2═—Cl, —CN, —OH, —H or C1-C6 alkyl, to a chiral diol of general formula II




wherein R1 and R2 have the same meaning as in formula I,
wherein the hydroxy ketone is reduced with an oxidoreductase in the presence of a cofactor, wherein the oxidoreductase
a) comprises the amino acid sequence of SEQ ID NO:8,
b) is encoded by the nucleic acid sequence SEQ ID NO:9 or SEQ ID NO:10, or
c) is encoded by a nucleic acid sequence which hybridizes to SEQ ID NO:9 or SEQ ID NO:10 under stringent conditions comprising washing with 0.1-2.0×SSC solution at 65° C.

2. The process according to claim 1, wherein the cofactor is continuously reduced with a co-substrate.

3. The process according to claim 1, wherein NAD(P)H is used as a cofactor.

4. The process according to claim 2, wherein at least one member selected from the group consisting of 2-propanol, 2-butanol, 2-pentanol, 4-methyl-2-pentanol, 2-heptanol and 2-octanol is used as the co-substrate.

5. The process according to claim 1, wherein the hydroxy ketone is used in an amount of from 5 to 50% by weight based on the total reaction volume.

6. The process according to claim 1, wherein at least one member selected from the group consisting of tert. butyl (5R)-6-cyano-5-hydroxy-3-oxohexanoate, tert. butyl (5S)-6-chloro-5-hydroxy-3-oxohexanoate and tert. butyl (5S)-5,6-dihydroxy-3-oxohexanoate is used as the hydroxy ketone of general formula (I).

7. The process according to claim 1, wherein the TTN (total turn over number=mol of reduced hydroxy ketone/mol of cofactor used) is ≧103.

8. The process according to claim 1, wherein the process is carried out in an aqueous organic two-phase system.

9. The process according to claim 1, wherein, in addition, at least one organic solvent selected from the group consisting of diethyl ether, tertiary butyl methyl ether, diisopropyl ether, dibutyl ether, ethyl acetate, butyl acetate, heptane, hexane and cyclohexane is used.

10. A process for the enantioselective enzymatic reduction of a hydroxy ketone of general formula I



wherein R1═C1-C6 alkyl and R2═—Cl, —CN, —OH, —H or C1-C6 alkyl, to a chiral diol of general formula II




wherein R1 and R2 have the same meaning as in formula I,
wherein the hydroxy ketone is reduced with an oxidoreductase in the presence of a cofactor,
wherein the oxidoreductase comprises the amino acid sequence SEQ ID NO:8.

11. The process according to claim 1, wherein the hydroxy ketone is used in an amount of from 8-40% by weight, based on the total reaction volume.

12. The process according to claim 1, wherein the hydroxy ketone is used in an amount of from 10-25% by weight, based on the total reaction volume.

13. A process for the enantioselective enzymatic reduction of a hydroxy ketone of general formula I



wherein R1═C1-C6 alkyl and R2═—Cl, —CN, —OH, —H or C1-C6 alkyl, to a chiral diol of general formula II




wherein R1 and R2 have the same meaning as in formula I,
wherein the hydroxy ketone is reduced with an oxidoreductase in the presence of cofactor,
wherein said oxidoreductase is encoded by a nucleic acid sequence which hybridizes to SEQ ID NO:9 or SEQ ID NO:10 under stringent conditions comprising washing with 0.1-2.0×SSC solution at 65° C.