Method for preparing (9S)-6, 7, 8, 9-tetrahydro-9-hydroxy-5H-cycloheptane [b] pyridine-5-ketone through catalysis of alcohol dehydrogenase

Abstract

The invention discloses a method for preparing (9S)-6, 7, 8, 9-tetrahydro-9-hydroxy-5H-cycloheptane [b] pyridine-5-ketone through catalysis of alcohol dehydrogenase, and cycloheptane [b] pyridine-5, 9-diketone is used as a raw material, alcohol dehydrogenase is used as a catalyst, and a target product is generated through reaction. The alcohol dehydrogenase is derived from Acetobacter aceti, the amino acid sequence of the alcohol dehydrogenase is as shown in SEQ ID NO. 1, and the nucleotide sequence for coding the alcohol dehydrogenase is as shown in SEQ ID NO. 2. According to the method, (9S)-6, 7, 8, 9-tetrahydro-9-hydroxy-5H-cycloheptane [b] pyridine-5-ketone is prepared under the optimized alcohol dehydrogenase catalytic reaction condition, the substrate concentration is 50 g / L, the reaction conversion rate is larger than 99%, the product chiral purity is larger than 99.5% ee, and good industrial application value is achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological enzyme catalysis, in particular to a method for preparing (9S)-6,7,8,9-tetrahydro-9-hydroxy-5H-cyclohepta[b]pyridin-5-one by alcohol dehydrogenase catalysis. BACKGROUND

[0002] Rimegepant is an effective, selective, competitive, orally active calcitonin gene-related peptide (CGRP) antagonist for the acute treatment of adult migraine. On February 27, 2020, it was approved for listing by the US Food and Drug Administration (FDA). (9R)-6,7,8,9-tetrahydro-9-hydroxy-5H-cyclohepta[b]pyridin-5-one is a key intermediate for synthesizing Rimegepant, and its enantiomer is (9S)-6,7,8,9-tetrahydro-9-hydroxy-5H-cyclohepta[b]pyridin-5-one. Both have similar physical and chemical properties, but there are significant differences in their biological activity, metabolic pathways and toxicity. Therefore, (9S)-6,7,8,9-tetrahydro-9-hydroxy-5H-cyclohepta[b]pyridin-5-one can be used as a reference standard for enantiomeric purity detection and impurity research to analyze the optical impurity content in the Rimegepant intermediate, and has important application value in the synthesis of Rimegepant (Rimegepant) drugs,

[0003] .

[0004] At present, the synthesis method of (9S)-6,7,8,9-tetrahydro-9-hydroxy-5H-cyclohepta[b]pyridin-5-one has not been reported in the literature. The traditional chiral synthesis method of chiral alcohol (such as chemical catalytic asymmetric reduction or resolution) often faces the problems of harsh reaction conditions, insufficient stereoselectivity, heavy environmental burden, high cost, etc., so it is particularly important to develop a green, environmentally friendly, simple, easy-to-obtain, low-cost, high-stereoselectivity synthesis method. In the present application, high-stereoselectivity alcohol dehydrogenase (ketoreductase) asymmetric catalytic reduction is used to realize the high-stereoselectivity synthesis of chiral alcohol (9S)-6,7,8,9-tetrahydro-9-hydroxy-5H-cyclohepta[b]pyridin-5-one, the product has extremely high ee value and high yield, the reaction conditions are mild, the by-products are few, the enzyme accessibility is good, and the operation is simple. It is a green technology suitable for industrial production. SUMMARY

[0005] In view of the deficiencies in the prior art, the application provides a method for synthesizing a drug intermediate (9S)-6,7,8,9-tetrahydro-9-hydroxy-5H-cyclohepta[b]pyridine-5-ketone by alcohol dehydrogenase catalysis, and the method for preparing (9S)-6,7,8,9-tetrahydro-9-hydroxy-5H-cyclohepta[b]pyridine-5-ketone by biological catalysis is simple in operation, green in environment protection, low in cost, high in efficiency and suitable for industrial application.

[0006] The application provides a method for preparing (9S)-6,7,8,9-tetrahydro-9-hydroxy-5H-cyclohepta[b]pyridine-5-ketone by alcohol dehydrogenase catalysis reaction of cyclohepta[b]pyridine-5,9-dione, and the alcohol dehydrogenase is derived from Acetobacter aceti.

[0007] Further, the amino acid sequence of the alcohol dehydrogenase is shown as SEQ ID NO. 1.

[0008] Further, the alcohol dehydrogenase is prepared by the following method:

[0009] (1) synthesizing a nucleotide sequence encoding the alcohol dehydrogenase to construct an engineering bacterium;

[0010] (2) inducing the engineering bacterium to culture, centrifuging to collect the bacterial body, and then preparing the required enzyme preparation;

[0011] The nucleotide sequence of the alcohol dehydrogenase is shown as SEQ ID NO. 2.

[0012] Further, in the preparation reaction, a water buffer solution is used as a reaction medium, a co-substrate, a coenzyme, a coenzyme regenerating enzyme and a substrate cyclohepta[b]pyridine-5,9-dione are added, and then the alcohol dehydrogenase is added to react to obtain (9S)-6,7,8,9-tetrahydro-9-hydroxy-5H-cyclohepta[b]pyridine-5-ketone, and the reaction formula is as follows:

[0013] ,

[0014] Further, the concentration of the substrate is 10-200 g / L, and preferably 50 g / L.

[0015] Further, the reaction medium is a triethanolamine-hydrochloric acid buffer solution, a Tris-HCl buffer solution, a phosphate buffer solution or a boric acid-borax buffer solution, and preferably a triethanolamine-hydrochloric acid buffer solution.

[0016] Further, the catalytic reaction temperature is 5-50 DEG C, and preferably the reaction temperature is 20 DEG C.

[0017] Further, the initial reaction pH value is 6-10, preferably the initial reaction pH is 7.5.

[0018] Further, the auxiliary substrate is glucose, and the addition amount is glucose: substrate = 1:1-2.5:1, preferably glucose: substrate = 2:1 (W / W).

[0019] Further, the auxiliary substrate is glucose, and the addition amount is glucose: substrate = 1:1-2.5:1, preferably glucose: substrate = 2:1 (W / W).

[0020] Further, the auxiliary substrate is glucose, and the addition amount is glucose: substrate = 1:1-2.5:1, preferably glucose: substrate = 2:1 (W / W). + The addition amount is 0.1-1.0 g / L, preferably 0.3 g / L.

[0021] Further, the auxiliary substrate is glucose, and the addition amount is glucose: substrate = 1:1-2.5:1, preferably glucose: substrate = 2:1 (W / W).

[0022] The technical principle of the present application is: to develop and screen alcohol dehydrogenase from Acetobacter aceti as a catalyst to catalyze the asymmetric reduction of the substrate cycloheptane and [b] pyridine-5,9-dione to generate the product (9S)-6,7,8,9-tetrahydro-9-hydroxy-5H-cycloheptano[b]pyridine-5-ketone. The alcohol dehydrogenase can catalyze the reaction of the substrate with a concentration of 50 g / L, and the conversion rate is greater than 99%, and it has high selectivity, which indicates that the enzyme has strong catalytic activity on the ketone substrate, and can carry out the reaction of high-concentration substrate. This may be related to the strong affinity between the enzyme and the substrate, thereby providing the possibility for realizing the large-scale industrial production of enzyme-catalyzed preparation of (9S)-6,7,8,9-tetrahydro-9-hydroxy-5H-cycloheptano[b]pyridine-5-ketone.

[0023] Compared with the prior art, the present application has the following beneficial effects:

[0024] (1) The present application firstly develops and screens the genetically engineered Escherichia coli containing the alcohol dehydrogenase gene of Acetobacter aceti, then cultures and induces the expression of alcohol dehydrogenase to prepare enzyme liquid, which can be applied to catalyze the reaction of cycloheptane and [b] pyridine-5,9-dione to synthesize the drug intermediate (9S)-6,7,8,9-tetrahydro-9-hydroxy-5H-cycloheptano[b]pyridine-5-ketone. The alcohol dehydrogenase catalyzes the reaction of raw material with a concentration of 50 g / L, the enzyme substrate ratio is 1 / 1 (enzyme liquid / raw material), the conversion rate of raw material is >99%, and the optical purity of the product is >99.5%.

[0025] (2) The alcohol dehydrogenase in the application has significant catalytic activity, substrate affinity and regioselectivity on cycloheptano[b]pyridine-5,9-dione, high substrate and product tolerance, high yield, less waste and low cost, and shows good industrialization potential.

[0026] (3) The method for preparing (9S)-6,7,8,9-tetrahydro-9-hydroxy-5H-cycloheptano[b]pyridine-5-one by enzyme catalysis developed in the application has no related literature reports at present. DETAILED DESCRIPTION

[0027] The following further illustrates a method for preparing (9S)-6,7,8,9-tetrahydro-9-hydroxy-5H-cycloheptano[b]pyridine-5-one by alcohol dehydrogenase catalysis, which aims to better understand the content of the application, but these examples do not constitute a limitation on the application.

[0028] The analysis method is as follows: 1) chiral HPLC analysis method: chromatographic column: CHIRALPAK IC 250 mm x 4.6 mm x 5 µm, mobile phase A: 10 mM aqueous ammonium acetate (pH 5.0), mobile phase B: methanol-acetonitrile (9:1, v / v), mobile phase ratio: mobile phase A (% V / V): mobile phase B (% V / V) = 60:40, time: 30 min, flow rate: 1 mL / min, detection wavelength: UV 235 nm.

[0029] Example 1 Preparation of alcohol dehydrogenase

[0030] 1. Construction of alcohol dehydrogenase-producing recombinant engineering bacteria

[0031] Through enzyme information mining and screening, alcohol dehydrogenase from Acetobacter aceti is obtained, the amino acid sequence of which is shown as SEQ ID NO. 1, and the corresponding gene sequence thereof is shown as SEQ ID NO. 2, NdeI and EcoRI sites are introduced at both ends of the gene, and the gene is cloned into a pET21a vector. The constructed plasmid is transformed into E. coli BL21 (DE3) competent cells by a chemical transformation method, spread on an LB plate containing 50 μg / ml ampicillin resistance, and cultured at 37°C for 8-12 h. Single colony cells are picked to obtain a genetically engineered strain that can induce expression of alcohol dehydrogenase.

[0032] 2. Inducing expression of alcohol dehydrogenase

[0033] The above-mentioned strain expressing alcohol dehydrogenase was inoculated into LB medium containing Amp (0.1 mL of an antibiotic aqueous solution with a concentration of 50 g / L was added to 100 mL of medium) and cultured overnight at 37°C, 200 rpm. The culture was inoculated into fermentation medium containing Amp (0.1 mL of an antibiotic aqueous solution with a concentration of 50 g / L was added to 100 mL of medium) at a volume fraction of 1% (1 mL of seed liquid was added to 100 mL of medium). The culture was further incubated at 37°C, 200 rpm for 3 h, and then IPTG was added to a final concentration of 50 mg / L. The culture was induced at 25°C, 200 rpm for 20 h.

[0034] 3. Preparation of alcohol dehydrogenase enzyme solution

[0035] The alcohol dehydrogenase cell obtained after centrifugation of the fermentation broth was uniformly suspended in 0.1 M pH 7.0 triethanolamine-hydrochloric acid buffer (20% cell concentration), and then broken in an ultrasonic disrupter to obtain an enzyme solution.

[0036] Example 2. Synthesis of (9S)-6,7,8,9-tetrahydro-9-hydroxy-5H-cyclohepta[b]pyridin-5-one catalyzed by alcohol dehydrogenase under different substrate concentrations

[0037] 10 mL reaction system, different substrate concentrations of raw materials, glucose: substrate = 2:1 (W / W), coenzyme regeneration enzyme solution (glucose dehydrogenase enzyme solution): substrate = 1 / 2 (W / W), alcohol dehydrogenase enzyme solution: substrate = 1 / 2 (W / W), 0.5 g / L NADP+, pH 7.0 Tris-HCl buffer solution to 10 mL, 30°C, 800 rpm for 24 h, intermittent adjustment to pH 7.0 during the reaction, and sampling analysis at the end of the reaction.

[0038] Substrate concentration (g / L) Conversion rate Product ee% 10 80.1% 99.6% 30 70.2% 99.5% 50 65.3% 99.6% 100 20.7% 99.8% 200 10.5% 99.9%

[0039] Example 3. Synthesis of (9S)-6,7,8,9-tetrahydro-9-hydroxy-5H-cyclohepta[b]pyridin-5-one catalyzed by alcohol dehydrogenase in different reaction media

[0040] 10 mL reaction system, 50 g / L raw material, 100 g / L glucose, 25 g / L glucose dehydrogenase enzyme solution, 25 g / L ketoreductase enzyme solution, 0.5 g / L NADP+, pH 7.0 different buffer solutions to 10 mL, 30°C, 800 rpm for 24 h, intermittent adjustment to pH 7.0 during the reaction, and sampling analysis at the end of the reaction.

[0041] Reaction medium Conversion rate Product ee% value Triethanolamine-HCl buffer 76.3% 99.7% Tris-HCl buffer 66.5% 99.8% Phosphate buffer 40.4% 99.8% Boric acid-borax buffer 50.5% 99.7%

[0042] Example 4 Synthesis of (9S)-6,7,8,9-tetrahydro-9-hydroxy-5H-cyclohepta[b]pyridine-5- one catalyzed by alcohol dehydrogenase at different temperatures

[0043] 10 mL reaction system, 50 g / L raw material, 100 g / L glucose, 25 g / L glucose dehydrogenase enzyme solution, 25 g / L alcohol dehydrogenase enzyme solution, 0.5 g / L NADP+, use pH 7.0 triethanolamine-hydrochloric acid buffer solution to 10 mL, different reaction temperatures 5-50℃ reaction 24 h, intermittent process adjustment to pH 7.0, reaction end sampling analysis.

[0044] Reaction temperature (°C) Conversion rate Product ee% value 5 37.2% 99.77% 10 67.5% 99.71% 20 81.0% 99.73% 30 77.1% 99.83% 40 65.3% 99.5% 50 50.4% 99.8%

[0045] Example 5 Synthesis of (9S)-6,7,8,9-tetrahydro-9-hydroxy-5H-cyclohepta[b]pyridine-5- one catalyzed by alcohol dehydrogenase at different initial pH

[0046] 10 mL reaction system, 50 g / L raw material, 100 g / L glucose, 25 g / L glucose dehydrogenase enzyme solution, 25 g / L alcohol dehydrogenase enzyme solution, 0.5 g / L NADP+, use different pH triethanolamine hydrochloric acid buffer solution to 10 mL, 20℃, 800 rpm reaction 24 h, intermittent process adjustment to set pH, reaction end sampling analysis.

[0047] pH Conversion rate Product ee% value 6.0 50.2% 99.6% 6.5 70.2% 99.8% 7.0 80.7% 99.5% 7.5 89.2% 99.8% 8.0 66.3% 99.8% 8.5 54.5% 99.7% 9.0 30.2% 99.6% 9.5 5.6% 99.8% 10.0 3.3% 99.8%

[0048] Example 6 Synthesis of (9S)-6,7,8,9-tetrahydro-9-hydroxy-5H-cyclohepta[b]pyridine-5- one catalyzed by alcohol dehydrogenase at different glucose addition amounts

[0049] 10 mL reaction system, 50 g / L raw material, different glucose concentrations, 25 g / L glucose dehydrogenase enzyme solution, 25 g / L alcohol dehydrogenase enzyme solution, 0.5 g / L NADP+, use pH 7.5 triethanolamine hydrochloric acid buffer solution to 10 mL, 20℃, 800 rpm reaction 24 h, intermittent process adjustment to pH 7.5, reaction end sampling analysis.

[0050] Glucose: substrate (glucose addition amount) Conversion rate Product ee% value 1: 1 (50 g / L) 80.3% 99.8% 1.5: 1 (75 g / L) 85.5% 99.7% 2: 1 (100 g / L) 89.6% 99% 2.5: 1 (125 g / L) 90.7% 99%

[0051] Example 7 Synthesis of (9S)-6,7,8,9-tetrahydro-9-hydroxy-5H-cyclohepta[b]pyridine-5- one catalyzed by alcohol dehydrogenase at different coenzyme regeneration enzyme addition amounts

[0052] 10 mL reaction system, 50 g / L raw material, 100 g / L glucose, 10 g / L glucose dehydrogenase enzyme solution, 25 g / L alcohol dehydrogenase enzyme solution, 0.3 g / L NADP+, pH 7.5 triethanolamine-hydrochloric acid buffer solution was used to make up to 10 mL, 20°C, 800 rpm reaction for 24 h, the process was intermittently adjusted to pH 7.5, and the reaction was sampled and analyzed at the end.

[0053] Glucose dehydrogenase enzyme solution: raw material (glucose dehydrogenase enzyme solution addition amount) Conversion rate Product ee% value 1: 10 (5 g / L) 45.2% 99.8% 1: 5 (10 g / L) 90.3% 99.5% 1: 2 (25 g / L) 90.7% 99.3% 1: 1 (50 g / L) 91.5% 99.8%

[0054] Example 8 Alcohol dehydrogenase catalyzed synthesis of (9S)-6,7,8,9-tetrahydro-9-hydroxy-5H-cyclohepta[b]pyridin-5-one under different NADP + Amount of alcohol dehydrogenase catalyzed synthesis of (9S)-6,7,8,9-tetrahydro-9-hydroxy-5H-cyclohepta[b]pyridin-5-one

[0055] 10 mL reaction system, 50 g / L raw material, 100 g / L glucose, 10 g / L glucose dehydrogenase enzyme solution, 25 g / L alcohol dehydrogenase enzyme solution, 0.3 g / L NADP+, pH 7.5 triethanolamine-hydrochloric acid buffer solution was used to make up to 10 mL, 20°C, 800 rpm reaction for 24 h, the process was intermittently adjusted to pH 7.5, and the reaction was sampled and analyzed at the end.

[0056] NADP + (g / L)] Conversion rate Product ee% value 0.1 85.3% 99.8% 0.3 90.2% 99.8% 0.5 90.8% 99.1% 1.0 91.6% 99.5%

[0057] Example 9 Alcohol dehydrogenase catalyzed synthesis of (9S)-6,7,8,9-tetrahydro-9-hydroxy-5H-cyclohepta[b]pyridin-5-one under different enzyme substrate ratios

[0058] 10 mL reaction system, 50 g / L raw material, 100 g / L glucose, 10 g / L glucose dehydrogenase enzyme solution, 25 g / L alcohol dehydrogenase enzyme solution, 0.3 g / L NADP+, pH 7.5 triethanolamine-hydrochloric acid buffer solution was used to make up to 10 mL, 20°C, 800 rpm reaction for 24 h, the process was intermittently adjusted to pH 7.5, and the reaction was sampled and analyzed at the end.

[0059] Enzyme substrate ratio Alcohol dehydrogenase enzyme solution (g / L) Conversion rate Product ee% 1:0.5 100 100% 99.8% 1:1 50 99.3% 99.8% 1:2 25 90.5% 99.8% 1:5 10 85.5% 99.5% 1:10 5 80.1% 99.3%

[0060] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced equivalently without departing from the purpose and scope of the technical solutions of the present application, and they should be covered in the scope of the claims of the present application.

[0061] Amino acid sequence of alcohol dehydrogenase

[0062] SEQ ID NO. 1

[0063] MARVAGKVAIVSGAANGIGKATAQLLAKEGAKVVIGDLKEEDGQKAVAEIKNAGGEATFV

[0064] KLNVTDEAEWKAAIAQTLKLYGRLDIAVNNAGIAYSGSVESTPLEEWRRVQSINLDGVFL

[0065] GTQVAIEAMKKSGGGSIVNLSSIEGLVGDPTLAAYNASKGGVRLFTKSAALHCAKSGYKI

[0066] RVNSVHPGYIWTPMVAGLTKEDAVARQKLVDLHPIGHLGEPNDIAYGILYLASDESKFVT

[0067] GSELVIDGGYTAQ

[0068] Nucleotide sequence of an alcohol dehydrogenase

[0069] SEQ ID NO. 2

[0070] atggcacgtgtagcaggtaaggttgcaattgtttccggcgcagcgaatggaataggtaaa

[0071] gccacagcgcagcttttggccaaggagggtgcaaaagttgttattggtgatttaaaagaa

[0072] gaagatggtcaaaaagctgttgcagaaattaaaaatgcaggtggtgaggccacatttgtt

[0073] aaattaaacgtgacagatgaggcggaatggaaagcagctatcgcgcaaacacttaaattg

[0074] tatgggcggttagatattgcagtaaataatgcaggtattgcgtattctggcagcgtagaa

[0075] agcacgccgctggaagagtggcgccgtgttcagtctatcaatttggatggtgtatttctg

[0076] GGCACGCAGGTGGCTATTGAAGCCATGAAGAAGTCTGGTG GTGGCTCCATTGTCAACCTG

[0077] TCTTCTATTGAGGGGTTGGTAGGAGATCCAACGCTGGCAGCCTATAACGCCAGTAAAGGG

[0078] GGCgtgcggctgtttacaaaatctgcggctctacactgtgcgaaatctggttataaaatt

[0079] Cgggtgaattcagtgcatcccggatatatctggacacctatggttgccggtttaaccaag

[0080] GAAGATGCAGTTGCACGCCAAAAGCTGGTTGATTTGCACCCCATAGGCCATTTGGGTGAA

[0081] CCAAACGATATTGCCTATGgtattttatatcttgcttctgatgaatccaagtttgttacg

[0082] GGAAGTGAACtGgttattgatggtggctacacagcacaataa

Claims

1. A method for the catalytic preparation of (9S)-6,7,8,9-tetrahydro-9-hydroxy- 5H-cyclohepta[b]pyridin-5-one by means of an alcohol dehydrogenase, characterized in that The alcohol dehydrogenase from Acetobacter aceti is used to catalyze the reaction of raw material cycloheptapyridine-5, 9-dione to generate (9S)-6, 7, 8, 9-tetrahydro-9-hydroxy-5H-cycloheptapyridine-5-ketone, and the reaction formula is as follows: 。 2. A method of catalytic preparation of (9S)-6,7,8,9-tetrahydro-9-hydroxy- 5H-cyclohepta[b]pyridin-5-one by alcohol dehydrogenase according to claim 1, characterized in that, The amino acid sequence of the alcohol dehydrogenase is shown in SEQ ID NO.

1.

3. A method of catalytic preparation of (9S)-6,7,8,9-tetrahydro-9-hydroxy-5H- cyclohepta[b]pyridin-5-one by alcohol dehydrogenase according to claims 1 and 2, characterized in that, The alcohol dehydrogenase is prepared by the following method: (1) Synthesizing the nucleotide sequence encoding the alcohol dehydrogenase and constructing an engineering bacterium; (2) Inducing the culture of the engineering bacterium, centrifuging to collect the bacterial body, and then preparing the required enzyme preparation; The nucleotide sequence is shown in SEQ ID NO.

2.

4. A method of catalytic preparation of (9S)-6,7,8,9-tetrahydro-9-hydroxy-5H- cyclohepta[b]pyridin-5-one by alcohol dehydrogenase according to claims 1 and 2, characterized in that, In a reaction medium, a co-substrate, a coenzyme, a coenzyme regenerating enzyme, a substrate cycloheptapyridine-5, 9-dione, and an alcohol dehydrogenase are added to carry out a reaction to obtain (9S)-6, 7, 8, 9-tetrahydro-9-hydroxy-5H-cycloheptapyridine-5-ketone.

5. A method of catalytic preparation of (9S)-6,7,8,9-tetrahydro-9-hydroxy- 5H-cyclohepta[b]pyridin-5-one by alcohol dehydrogenase according to claim 4, characterized in that, The concentration of the substrate is 10-200 g / L.

6. A method of catalytic production of (9S)-6,7,8,9-tetrahydro-9-hydroxy- 5H-cyclohepta[b]pyridin-5-one by an alcohol dehydrogenase according to claim 4, characterized in that, The reaction medium is triethanolamine-hydrochloric acid buffer, Tris-HCl buffer, phosphate buffer, or boric acid-borax buffer.

7. A method of catalytic preparation of (9S)-6,7,8,9-tetrahydro-9-hydroxy- 5H-cyclohepta[b]pyridin-5-one by alcohol dehydrogenase according to claim 4, characterized in that, The reaction temperature is 5-50℃.

8. The method for preparing (9S)-6,7,8,9-tetrahydro-9-hydroxy-5H-cycloheptane[b]pyridin-5-one by alcohol dehydrogenase catalysis according to claim 4, characterized in that, The pH value of the initial reaction is 6-10.

9. The method for preparing (9S)-6,7,8,9-tetrahydro-9-hydroxy-5H-cycloheptane[b]pyridin-5-one by alcohol dehydrogenase catalysis according to claim 4, characterized in that, The co-substrate is glucose.

10. A process according to claims 4 and 9 for (9S)-6,7,8,9-tetrahydro-9-hydroxy- 5H-cyclohepta[b]pyridin-5-one, characterized in that, The added amount of the glucose: substrate is 1: 1-2.5: 1 (W / W).

11. A method of catalytic preparation of (9S)-6,7,8,9-tetrahydro-9-hydroxy- 5H-cyclohepta[b]pyridin-5-one by alcohol dehydrogenase according to claim 4, characterized in that, The coenzyme regenerating enzyme is glucose dehydrogenase.

12. A method of catalytic preparation of (9S)-6,7,8,9-tetrahydro-9-hydroxy- 5H-cyclohepta[b]pyridin-5-one according to claim 4 and 11, characterized in that, The use amount of the coenzyme regenerating enzyme enzyme solution: raw material is 1: 10-1: 1 (W / W).

13. A method of catalytic preparation of (9S)-6,7,8,9-tetrahydro-9-hydroxy- 5H-cyclohepta[b]pyridin-5-one by alcohol dehydrogenase according to claim 4, characterized in that, The coenzyme is NADP + .

14. A method of catalytic preparation of (9S)-6,7,8,9-tetrahydro-9-hydroxy- 5H-cyclohepta[b]pyridin-5-one according to claim 4 and 13, characterized in that, The added amount of the coenzyme is 0.1 g / L-1.0 g / L.

15. The method for preparing (9S)-6,7,8,9-tetrahydro-9-hydroxy-5H-cycloheptane[b]pyridin-5-one by alcohol dehydrogenase catalysis according to claim 4, characterized in that, The mass ratio of the alcohol dehydrogenase enzyme solution to the substrate is 1: 0.5-1: 10.

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