CN109957585B - Method for preparing (S) - (4-chlorophenyl) -2-pyridinemethanol by biological catalysis process - Google Patents

Abstract

The invention relates to an enzymatic method for preparing chiral alcohol, belonging to the field of preparation of medical intermediates by using genetic engineering technology. A method for preparing (S) - (4-chlorphenyl) -2-pyridinemethanol by a biological catalysis process, wherein (4-chlorphenyl) (2-pyridyl) ketone is converted into (S) - (4-chlorphenyl) -2-pyridinemethanol under the catalysis of ketoreductase. The method screens out ketoreductase with a specific amino acid sequence for catalyzing and preparing (S) - (4-chlorphenyl) -2-pyridinemethanol, realizes the conversion rate of a substrate of more than 99 percent, ensures that the ee value of the prepared product is not less than 99.5 percent, ensures that the concentration of the substrate of the reaction is maximally close to 330g/L, and has the value of industrial amplification production. The reaction can completely and efficiently convert the substrate into the target product, and the prepared product is simple to separate and purify, low in post-treatment cost, high in environmental friendliness of the whole process flow and high in atom utilization rate.

Description

Method for preparing (S) - (4-chlorphenyl) -2-pyridinemethanol by biological catalysis process

Technical Field

The invention relates to a preparation method of an intermediate, in particular to a method for preparing (S) - (4-chlorphenyl) -2-pyridinemethanol by a biocatalysis process.

Background

Bepotastine besilate is a histamine H1 receptor antagonist jointly developed by Tanabe Seiyaku and Nippon Japan (UBEIindustries), is a non-sedative high-selectivity histamine H1 receptor antagonist drug, has antiallergic and antipruritic effects, can inhibit eosinophilic granulocyte infiltration into peripheral tissues, and is effective in relieving inflammatory reaction of nasal mucosa, namely nasal obstruction compared with the existing drugs; moreover, the bepotastine besilate rarely enters the brain, no sedation is caused, the choline resistance effect is separated from the histamine resistance effect, other adverse reactions are extremely small, the bepotastine besilate has excellent pharmacodynamic effects and clinical effects, the action intensity is superior to that of ketotifen, terfenadine, cetirizine and epinastine, and the bepotastine besilate has wide clinical application prospects. Approved for urticaria/pruritus in 2002, sold in japan by Mitsubishi Tanabe Pharma under the trade name Talion (tanglion). The besilate tablet is imported in China and is used for treating allergic rhinitis, urticaria and pruritus caused by skin diseases (eczematous dermatitis, prurigo and cutaneous pruritus). The chemical name of the compound is (+) - (S) -4- [4- [ (4-chlorophenyl) (2-pyridyl) methoxy ] piperidino ] butyric acid monobenzenesulfonate, and the chemical structural formula of the effective component is as follows:

formula I

Bepotastine was first reported in japanese patent JP1990025465 as a racemate of a pair of enantiomers, but subsequent studies found that S-configuration bepotastine is less toxic than R-configuration, and thus S-configuration bepotastine gradually replaces the racemate to enter the market. Most of the currently published chiral bepotastine synthetic routes have low yield and high cost.

For example, JP1998237070 and JP2000198784 report the preparation route of bepotastine by first resolving racemic compound to obtain 4- [ (S) - (4-chlorophenyl) -2-pyridylmethoxy ] -1-piperidine, then carrying out condensation reaction with ethyl bromobutyrate to obtain bepotastine ethyl ester, and then hydrolyzing to obtain bepotastine compound. The preparation method is complex and high in cost because an intermediate compound with a complex structure needs to be prepared in the route, so that the application of the method is severely limited.

The process route disclosed by CN1242013A is that 2- [ (4-chlorophenyl) (4-piperidinyloxy) methyl ] pyridine is split by a splitting agent to obtain S-2- [ (4-chlorophenyl) (4-piperidinyloxy) methyl ] pyridine N-acetyl-L-phenylalanine salt, then S-4- [ (4-chlorophenyl) (2-pyridylmethoxy) piperidino ] ethyl butyrate is obtained by reacting with ethyl 4-bromobutyrate, and bepotastine besilate is obtained by hydrolysis and salt forming reaction, and finally, three continuous coupling reactions, hydrolysis reactions and salt forming reactions respectively adopt different solvents, and respectively need crystallization drying, so that the overall process time is long, the operation is tedious, and the yield is low.

Patent CN102675283A, CN103121966A disclose that a compound with optical activity of (S) - (4-chlorophenyl) - (pyridine-2-yl) -methanol is used to directly synthesize bepotastine, so that the synthesis yield of the bepotastine raw material drug is substantially improved. The synthetic route is as formula II:

formula II

According to formula II, (S) - (4-chlorophenyl) - (pyridin-2-yl) -methanol is a key intermediate for the preparation of optically active bepotastine. Several synthetic methods are currently co-searched for the preparation of the chiral compounds:

the Chinese patent with the application number of 201110369002.4 adopts BINAL-H to reduce 4-chlorphenyl- (pyridine-2-yl) -ketone to obtain (4-chlorphenyl) - (pyridine-2-yl) -methanol with R or S configuration, and the optical purity reaches 80-95.7%.

In the Chinese patent with the application number of 201110419992.8, Candida kluyveri JNU-KR1 (the strain preservation number: CCTCC NO: M2011385) is adopted to catalyze and reduce 4-chlorphenyl- (pyridine-2-yl) -methanone to obtain (4-chlorphenyl) - (pyridine-2-yl) -methanol with R or S configuration, the ee value can reach 86% to the maximum, and the concentration of a reaction substrate is 6 g/L.

In addition, international patent application No. PCT/IB2014/066031 screens 9 for different sources of microorganisms, and reduces 4-chlorophenyl- (pyridine-2-yl) -methanone to obtain R or S configuration (4-chlorophenyl) - (pyridine-2-yl) -methanol, wherein the ee value of the optimized R-configuration (4-chlorophenyl) - (pyridine-2-yl) -methanol is up to 94%, the ee value of the S-configuration (4-chlorophenyl) - (pyridine-2-yl) -methanol is up to 96%, and the concentration of the reaction substrate is not more than 5 wt%.

The Chinese patent with the application number of 201510346906.3 adopts bis (1, 5-cyclooctadiene) rhodium tetrafluoroborate to catalytically reduce 4-chlorophenyl- (pyridine-2-yl) -methanone to obtain (4-chlorophenyl) - (pyridine-2-yl) -methanol with S configuration, the highest ee value of 99% and the highest conversion rate of 99% can be achieved, but the rhodium noble metal catalyst causes the reaction cost to be increased, and the high-pressure hydrogenation catalytic reduction further improves the process difficulty and the production cost of the reaction, and is only suitable for small-batch gram-grade and milligram-grade production and preparation. Similar techniques for the preparation of chiral alcohols by metal-catalyzed reduction are also known from org. Lett. 2015, 17, 4144-.

The School of Pharmaceutical Sciences, University of Shizuoka,52-1 Yada, Shizuoka 422, japan. December 141995, discloses a process for yeast reduction of 4-chlorophenyl- (pyridin-2-yl) -methanone to give (4-chlorophenyl) - (pyridin-2-yl) -methanol in R or S configuration, but has low optical purity and no practical applicability.

In view of the prior art, the process for reducing 4-chlorophenyl- (pyridine-2-yl) -methanone to obtain (4-chlorophenyl) - (pyridine-2-yl) -methanol with R or S configuration mostly has the problem of low optical purity of the product. Even if some biological enzymes can achieve an ee value close to 95.7%, the gap is still quite short from the qualified line of ICH (international registration technical requirement for human medicine) for the optical purity of chiral drugs not less than 99% ee, and the prepared bulk drugs can meet the drug production specifications given by the ICH only through a chiral purification process.

Secondly, the prior art has the problems of low yield, expensive catalyst, environmental-friendly reaction system and the like, and the process of chiral (4-chlorphenyl) - (pyridine-2-yl) -methanol is prevented from being applied to industrial production.

Therefore, a new technology suitable for industrial application is urgently needed at present, the chiral (4-chlorophenyl) -2-pyridinemethanol medical intermediate with high optical purity can be prepared efficiently and at low cost, and the production requirements of bepotastine and medicines with similar structures are met.

Disclosure of Invention

The invention aims to provide a method for preparing (S) - (4-chlorophenyl) -2-pyridinemethanol by a biocatalytic process, which can prepare (S) - (4-chlorophenyl) -2-pyridinemethanol by an industrialized process with high conversion rate and high optical purity.

Technical scheme

A method for preparing (S) - (4-chlorphenyl) -2-pyridinemethanol by a biological catalysis process is shown in a formula III, wherein (4-chlorphenyl) (2-pyridyl) ketone is converted into (S) - (4-chlorphenyl) -2-pyridinemethanol under the catalysis of ketoreductase:

formula III

The Ketoreductase (KRED) employed in formula III has the amino acid sequence as described in (a) or (b):

(a) the ketoreductase has an amino acid sequence as shown in a table SEQ ID No. 2;

(b) the ketoreductase is a ketoreductase which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in an amino acid sequence shown in SEQ ID No.2 and can catalyze the conversion of (4-chlorophenyl) (2-pyridyl) methanone into (S) - (4-chlorophenyl) -2-pyridinemethanol.

Furthermore, the amino acid sequence of the ketoreductase in (b) has more than 85 percent of homology with the amino acid sequence of the ketoreductase in (a), and the ketoreductase coded by the amino acid sequence can catalyze the conversion of (4-chlorphenyl) (2-pyridyl) ketone into (S) - (4-chlorphenyl) -2-pyridinemethanol.

Further, the ketoreductase of (b) has an amino acid sequence which is 90% or more, preferably 93% or more, more preferably 95% or more, and most preferably 97% or more homologous to the amino acid sequence of the ketoreductase of (a).

Further, the ketoreductase has the following genes (c) or (d):

(c) the nucleotide sequence of the gene is shown in a sequence table SEQ ID No. 1;

(d) and (c) has N% or more homology (N is selected from 85, 90, 95 and 99), and can encode a gene that catalyzes a ketoreductase for converting (4-chlorophenyl) (2-pyridyl) methanone into (S) - (4-chlorophenyl) -2-pyridinemethanol.

A recombinant expression vector, a recombinant bacterium or a transgenic cell line containing the gene.

Furthermore, the ketoreductase takes part in catalytic reaction in the forms of ketoreductase enzyme powder, ketoreductase enzyme liquid, ketoreductase-containing cells and the like, and is used for catalyzing the conversion of (4-chlorophenyl) (2-pyridyl) methanone into (S) - (4-chlorophenyl) -2-pyridinemethanol.

Further, the process steps of preparing (S) - (4-chlorphenyl) -2-pyridinemethanol by catalyzing (4-chlorphenyl) (2-pyridyl) methanone by the ketoreductase comprise: preparing (4-chlorphenyl) (2-pyridyl) ketone, ketoreductase enzyme powder or a cell containing ketoreductase (the 'ketoreductase-containing cell' in the technical scheme refers to engineering bacteria commonly used in the technical field for fermentation production, such as saccharomycetes, escherichia coli and the like), coenzyme and a buffering agent into a water/isopropanol mixed solution, and reacting to obtain a product.

Further, the concentration of the (4-chlorphenyl) (2-pyridyl) ketone is 1-330 g/L, preferably 180-320 g/L, and more preferably 200-300 g/L;

further, the ratio of the using mass of the ketoreductase enzyme powder to the using mass of the ketoreductase-containing cells is 1: 4-6. Namely, the catalytic efficiency of 1 part by mass of ketoreductase enzyme powder is equivalent to that of 4-6 parts by mass of ketoreductase cells.

Furthermore, the cell concentration of the ketoreductase is 20-90 g/L, preferably 30-70 g/L. If the ketoreductase enzyme powder is adopted to replace ketoreductase cells, the using concentration of the ketoreductase enzyme powder is 4-20 g/L;

further, in the water/isopropanol solution, the volume ratio of isopropanol to water is 1: 1-3, preferably 1: 1.5-2.5;

furthermore, coenzyme can be added into the reaction system to promote the reaction, when the ketoreductase-containing cell is adopted, a small amount of coenzyme is contained in the cell, and the coenzyme can also not be added at the time; in some cases, the ketoreductase enzyme powder prepared also contains a small amount of coenzyme, in which case no coenzyme may be added. But coenzyme can be added into the reaction system to further promote the reaction, and when the coenzyme is added into the reaction system to promote the reaction, the coenzyme is selected from NAD ⁺ 、NADH、NADP ⁺ And NADPH or a combination thereof, preferably NADP; the concentration of the coenzyme is 0.02-0.5 g/L, preferably 0.1-0.4 g/L.

Furthermore, the coenzymes used in the technical scheme are all from Shangke biological medicine (Shanghai) Co., Ltd.

Further, the buffer is a phosphate-phosphate buffer, a citrate-citrate buffer, preferably a phosphate-phosphate buffer; the pH value of the buffering agent is 6.0-9.0, preferably 6.4-8.7; the buffer has a molar concentration of 0.01 to 0.5mol/L, preferably 0.05 to 0.2 mol/L.

Further, the reaction temperature for catalyzing (4-chlorophenyl) (2-pyridyl) methanone to prepare (S) - (4-chlorophenyl) -2-pyridinemethanol is 22-45 ℃, and the reaction time is 5-36 h. According to the examples in the description, the reaction time can be shortened to 5h by controlling the reaction temperature, the material concentration and the mixture ratio. However, the reaction operation behavior that the reaction can be completed within 5h and the reaction time is still intentionally prolonged is also considered to be within the scope of the present invention. However, too long a reaction time leads to increased side reactions and reduced analytical and optical purity of the product, and therefore, it is generally not preferable to exceed 36 hours.

Further, the ketoreductase is involved in the reaction in the form of an enzyme powder, a cell disruption solution containing the ketoreductase or whole cells, preferably in the form of an enzyme powder.

Furthermore, in the present embodiment, the ketoreductase is derived fromNovosphingobium aromaticivoransBy the pairNovosphingobium aromaticivoransScreening a mutant library of the ketoreductase of (1).From Novosphingobium aromaticivoransThe wild-type ketoreductase of (a) is at NCBI accession number WP _ 011906790.1. And all amino acid sequences and their possible corresponding gene sequences in this patent can be made by commercial whole gene synthesis services.

Further, the ketoreductase is obtained by fermenting genetically engineered bacteria, wherein the genetically engineered bacteria are selected from saccharomycetes and escherichia coli, and are preferably saccharomycetes.

Furthermore, as will be appreciated by those skilled in the art, the last three bases of the DNA sequence encoding the ketoreductase enzyme are stop codons, and the stop codons can have various base sequences, and after the stop codons of different base sequences are replaced, the essence of the technical scheme is consistent with the technical scheme applied by the inventor, and the protection scope of the present invention is also considered.

Advantageous effects

The invention provides a method for preparing (S) - (4-chlorphenyl) -2-pyridinemethanol by a biocatalytic process, which is used for catalyzing (4-chlorphenyl) (2-pyridyl) ketone to be converted into (S) - (4-chlorphenyl) -2-pyridinemethanol by screening out ketoreductase with a specific amino acid sequence, so that the production process is efficiently carried out in a water phase, the conversion rate of raw materials of the reaction is over 99 percent, the ee value of the prepared product (S) - (4-chlorphenyl) -2-pyridinemethanol is not less than 99.5 percent, the concentration of a substrate is close to 330g/L, the substrate can be completely and efficiently converted into a target product, the prepared product is simple to separate and purify, the post-treatment cost is low, the environment-friendly degree of the whole process flow is high, the atom utilization rate is high.

Compared with the prior art, the technical scheme provides a ketoreductase, an amino acid sequence and a gene sequence thereof, the ketoreductase coded by the gene sequence can realize a catalytic result with the conversion rate not lower than 99% under the environment-friendly and pollution-free reaction process condition, and the (S) - (4-chlorophenyl) -2-pyridinemethanol with the ee value of more than 99.5% is prepared, so that the defects that in the enzyme catalytic preparation process of the prior art, the substrate concentration is low, and the ee value of a product is lower than the value required by ICH for optically active drugs are overcome, and the process can be practically applied to the industrial preparation of (S) - (4-chlorophenyl) -2-pyridinemethanol.

Drawings

FIG. 1 is a chiral HPLC analysis spectrum of a reaction substrate (4-chlorophenyl) (2-pyridyl) methanone;

FIG. 2 is a chiral HPLC analysis spectrum of the reaction solution of example 2;

FIG. 3 is a chiral HPLC analysis spectrum of the reaction solution of example 3;

FIG. 4 is a chiral HPLC analysis spectrum of the reaction solution of example 4;

FIG. 5 is a chiral HPLC analysis spectrum of the reaction solution of example 5;

FIG. 6 is a chiral HPLC analysis spectrum of racemate;

FIG. 7 is a conventional HPLC analysis spectrum of the reaction solution of example 2;

FIG. 8 is a conventional HPLC analysis chart of the reaction solution of example 3;

FIG. 9 is a conventional HPLC analysis chart of the reaction solution of example 4;

FIG. 10 is a conventional HPLC analysis chart of the reaction solution of example 5;

FIG. 11 is a photograph showing the results of detection of 225nm light by TLC plate in the course of the reaction of example 2;

wherein: 1-raw material point, 2-mixing point of reaction liquid and raw material, and 3-reaction liquid point.

Detailed Description

The invention is further illustrated below with reference to specific embodiments and the accompanying drawings.

EXAMPLE 1 preparation of ketoreductase

The gene engineering bacterium (carrier pET21a, host cell) containing the coding gene (SEQ ID No. 1) of the ketoreductaseE.ColiBL21(DE 3)) was inoculated into 5mL of ampicillin-containing LB test tube medium for activated culture (37 ℃ for 12 hours), the activated culture was transferred to 400mL of ampicillin-containing LB liquid medium at 1% inoculum size, OD was adjusted to 0.6-0.8 at 37 ℃ and IPTG (final concentration of 0.1 mM) was added to induce culture at 25 ℃ for 16 hours. Centrifugally collecting thalli to obtain ketoreductase cells, re-suspending the thalli by 40mL of phosphate buffer solution (10 mM, pH 7.5), ultrasonically crushing for 15min in an ice water bath, centrifugally collecting supernate, pre-freezing at-20 ℃, carrying out vacuum freeze drying for 48h, and grinding to obtain the recombinant ketoreductase enzyme powder.

EXAMPLE 2 preparation of (S) - (4-chlorophenyl) -2-pyridinemethanol

Adding isopropanol (2.4 mL) and substrate (4-chlorophenyl) (2-pyridyl) methanone (1.5 g) into a reaction vessel, stirring uniformly, adding enzyme powder 0.1g and coenzyme NADP 1mg, diluting to 10mL by using phosphate buffer solution with pH of 8.5, magnetically stirring at 28 ℃ for reaction, and simultaneously detecting the reaction progress by TLC (thin layer chromatography), wherein the attached figure 11 shows that the concentration of the enzyme powder is high. And after the reaction is finished for 8 hours, filtering the diatomite, extracting the liquid phase for three times by adopting an organic phase, combining the organic phases, drying the organic phases by using anhydrous sodium sulfate, and performing reduced pressure spin drying to obtain the product. The HPLC chromatogram before the reaction of the reaction solution is shown in FIG. 1; chiral HPLC of racemic 2-chloro-1- (3-hydroxyphenyl) ethanol is shown in figure 6; the chiral HPLC of the reaction solution is shown in figure 2, figure 7 is the conventional HPLC purity detection of the reaction solution, and the ee value and the conversion rate of the detection product of figures 2 and 7 can be known: substrate conversion =99.7%, and ee =99.7% for the S-form product. The obtained product is prepared into a sample of 5g/L and dissolved in chloroform, and after three detections and an average value is taken, the optical rotation value is +61.0 degrees, and the product can be determined as an S-type product.

EXAMPLE 3 preparation of (S) - (4-chlorophenyl) -2-pyridinemethanol

Adding isopropanol (3.6 mL) and a substrate (4-chlorophenyl) (2-pyridyl) ketone (2.8 g) into a reaction vessel, stirring uniformly, adding ketoreductase cells 0.3g, finally diluting to 10mL by using a phosphate buffer solution with the pH of 8.2, performing magnetic stirring reaction at 30 ℃, and detecting the reaction progress by TLC. Filtering the diatomite after the reaction is finished for 5 hours, extracting the liquid phase for three times by adopting an organic phase, combining the organic phases, drying by using anhydrous sodium sulfate, and performing reduced pressure spin drying to obtain the product. Chiral HPLC of the reaction solution is shown in figure 3, conventional HPLC spectrogram detection of the reaction solution is shown in figure 8, ee values and conversion rates of detection products in figures 3 and 8 can be known: substrate conversion =99.6%, and ee value of S-form product = 99.5%.

EXAMPLE 4 preparation of hectogram (S) - (4-chlorophenyl) -2-pyridinemethanol

Adding isopropanol (400 mL) and a substrate (4-chlorophenyl) (2-pyridyl) ketone (330 g) into a reaction vessel, uniformly stirring, adding 50g of ketoreductase cells, finally diluting to 1.0L by adopting a phosphate buffer solution with pH7.9, carrying out magnetic stirring reaction at 38 ℃, and simultaneously detecting the reaction progress by TLC. And after the reaction is finished for 15 hours, filtering the diatomite, extracting the liquid phase for three times by adopting an organic phase, combining the organic phases, drying the organic phases by using anhydrous sodium sulfate, and performing reduced pressure spin drying to obtain the product. Chiral HPLC of the reaction solution is shown in FIG. 4, a conventional HPLC purity spectrum of the reaction solution is shown in FIG. 9, and ee values and conversion rates of the detection products in FIGS. 4 and 9 can be known: substrate conversion =99.5%, and ee value of S-form product = 99.6%.

EXAMPLE 5 preparation of a kilogram grade of (S) - (4-chlorophenyl) -2-pyridinemethanol

Adding isopropanol (10L) and substrate (4-chlorophenyl) (2-pyridyl) methanone (6.0 Kg) into a reaction vessel, stirring well, adding ketoreductase enzyme powder 0.75Kg and coenzyme NADP ⁺ (5.5 g), finally, the volume is adjusted to 20L by adopting phosphate buffer solution with the pH value of 7.5, the reaction is magnetically stirred at the temperature of 45 ℃, and the progress of the reaction is detected by TLC. Filtering with diatomite after the reaction is finished for 30h, extracting the liquid phase for three times by adopting an organic phase, combining the organic phases, drying with anhydrous sodium sulfate, and carrying out reduced pressure spin drying to obtain the product. Chiral HPLC of the reaction solution is shown in figure 5, conventional HPLC spectrogram of the reaction solution is shown in figure 10, ee value and conversion rate of the detection product in figures 5 and 10 can be known: substrate conversion =99.7%, and ee =99.8% for the S-form product.

Sequence listing

<110> Shang Ke biomedical (Shanghai) Co., Ltd

<120> method for preparing (S) - (4-chlorophenyl) -2-pyridinemethanol by biocatalysis process

<160> 2

<170> SIPOSequenceListing 1.0

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atgaccgacc gtctgaaaca caaagttgct atcgttaccg gtggtaccat gggtatcggt 60

ctggctatcg ctgacaaata cgttgaagaa ggtgctaaag ttgttatcac cggtcgtcac 120

gctgacgttg gtgaaaaagc tgctaaatct atcggtggta ccgacgttat ccgtttcgtt 180

cagcacgacg tttctgacga agctggttgg accaaactgt tcgacaccac cgaagaaacc 240

ttcggtccgg ttaccaccgt tgttaacaac gctggttctt gggttaacaa atctgttaaa 300

gacaccacca ccgaagaatg gcgtaaactg ctgtctgtta acctggacgg tgttttcttc 360

ggtacccgtc tgggtatcca gcgtatgaaa aacaaaggtc tgggtgcttc tatcatcaac 420

atgtcttcta tcgccggtct ggttggtgac ccgtctctgg gtgcttacaa cgcttctaaa 480

ggtgctgttc gtatcatgtc taaatctgct gctctggact gcgctctgaa agactacgac 540

gttcgtgtta acaccgttca cccgggtccg atcaaaaccc agcgtctcga ccaggtgccg 600

ggttgggaag aaatgatgtc tcagcgtacc gttaccccga tgggtcgtat cggtgaaccg 660

aacgacatcg cttggatctg cgtttacctg gcttctgacg aatctaaatt cgctaccggt 720

gctgaattcg ttgttgacgg tggttacacc gctcagtaa 759

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<213> Artificial Sequence (Artificial Sequence)

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Met Thr Asp Arg Leu Lys His Lys Val Ala Ile Val Thr Gly Gly Thr

1 5 10 15

Met Gly Ile Gly Leu Ala Ile Ala Asp Lys Tyr Val Glu Glu Gly Ala

20 25 30

Lys Val Val Ile Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala

35 40 45

Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Val

50 55 60

Ser Asp Glu Ala Gly Trp Thr Lys Leu Phe Asp Thr Thr Glu Glu Thr

65 70 75 80

Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ser Trp Val Asn

85 90 95

Lys Ser Val Lys Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser

100 105 110

Val Asn Leu Asp Gly Val Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg

115 120 125

Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile

130 135 140

Ala Gly Leu Val Gly Asp Pro Ser Leu Gly Ala Tyr Asn Ala Ser Lys

145 150 155 160

Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu

165 170 175

Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys

180 185 190

Thr Gln Arg Leu Asp Gln Val Pro Gly Trp Glu Glu Met Met Ser Gln

195 200 205

Arg Thr Val Thr Pro Met Gly Arg Ile Gly Glu Pro Asn Asp Ile Ala

210 215 220

Trp Ile Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly

225 230 235 240

Ala Glu Phe Val Val Asp Gly Gly Tyr Thr Ala Gln

245 250

Claims (9)

1. A method for preparing (S) - (4-chlorphenyl) -2-pyridine methanol by a biological catalysis process is characterized by comprising the following steps: (4-chlorphenyl) (2-pyridyl) ketone is converted into (S) - (4-chlorphenyl) -2-pyridinemethanol under the catalysis of ketoreductase, and the amino acid sequence of the ketoreductase is shown as SEQ ID No. 2.

2. The biocatalytic process for preparing (S) - (4-chlorophenyl) -2-pyridinemethanol according to claim 1, characterized in that: the gene sequence of the ketoreductase is shown as SEQ ID No. 1.

3. The biocatalytic process for preparing (S) - (4-chlorophenyl) -2-pyridinemethanol according to claim 1, characterized in that: (4-chlorophenyl) (2-pyridyl) methanone is converted to (S) - (4-chlorophenyl) -2-pyridinemethanol by ketoreductase catalysis, comprising the steps of: preparing (4-chlorphenyl) (2-pyridyl) ketone, ketoreductase enzyme powder or ketoreductase-containing cells, coenzyme and a buffering agent into a water/isopropanol mixed solution, and reacting to obtain a product.

4. The method for preparing (S) - (4-chlorophenyl) -2-pyridinemethanol according to claim 3, comprising: the concentration of the (4-chlorphenyl) (2-pyridyl) ketone is 1-330 g/L; the concentration of ketoreductase enzyme powder is 2-20 g/L or the concentration of ketoreductase-containing cells is 20-90 g/L.

5. The biocatalytic process for preparing (S) - (4-chlorophenyl) -2-pyridinemethanol according to claim 3, characterized in that: the volume ratio of the isopropanol to the water in the water/isopropanol solution is 1: 1-3.

6. The biocatalytic process for preparing (S) - (4-chlorophenyl) -2-pyridinemethanol according to claim 3, characterized in that: the reaction temperature is 24-45 ℃.

7. The biocatalytic process for preparing (S) - (4-chlorophenyl) -2-pyridinemethanol according to claim 3, characterized in that: the reaction time is 5-36 h.

8. The method for preparing (S) - (4-chlorophenyl) -2-pyridinemethanol according to claim 4, comprising: adding coenzyme selected from NAD into the reaction solution ⁺ 、NADH、NADP ⁺ And NADPH or a combination thereof; the concentration of the coenzyme is 0.1-0.4 g/L.

9. The method for preparing (S) - (4-chlorophenyl) -2-pyridinemethanol according to claim 3, comprising: the ketoreductase-containing cell is selected from genetically engineered yeast or escherichia coli.

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