A method for the bioproduction of a key chiral intermediate of troxistat ethyl ester

Abstract

This invention discloses a bio-preparation method for a key chiral intermediate of erlotinib ethyl ester. The method utilizes resting cells of *Saccharomyces cerevisiae* ZJPH1807 as a catalyst to prepare (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol via biocatalysis of 1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol. The product obtained using this strain exhibits high optical purity (ee value > 99.9%) and a reaction yield of 86.4%. The process is simple, environmentally friendly, and uses microbial cells as the biocatalyst, resulting in low cost.

Description

(I) Technical Field

[0001] This invention relates to a method for the bio-preparation of (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol, a key chiral intermediate of erlotinib ethyl ester. (II) Background Technology

[0002] The chemical structural formula of (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol is:

[0003]

[0004] (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol is a key chiral intermediate in the synthesis of the drug erlotinib ethyl ester, and its molecular formula is: C 12 H 10 ClF3N2O. Tarrositol ethyl ester is the first and only oral medication developed by Lexicon Pharmaceuticals for the treatment of carcinoid syndrome diarrhea (CSD). It is a tryptophan hydroxylase (TPH) inhibitor that targets and inhibits the excessive production of serotonin by neuroendocrine cells. Its molecular formula is: C 27 H 26ClF3N6O3. CSD (Chronic Serotonin Depression) is caused by the release of large amounts of serotonin (5-hydroxytryptamine) during liver metastasis of neuroendocrine tumors, leading to its accumulation due to the liver's inability to effectively clear it. Conventional treatment for CSD involves injecting somatostatin analogs (SSA) to inhibit the secretion of some growth hormones and alleviate symptoms and signs associated with functional gastrointestinal pancreatic enzyme endocrine tumors. However, this treatment is not specific and only provides temporary relief for CSD, and it can easily cause hormonal imbalances in the patient's body. Tarcesta ethyl ester, on the other hand, can reduce peripheral serotonin levels without affecting serotonin levels in the brain. It specifically reduces serotonin secretion, improves CSD symptoms, has a high response rate, and has fewer adverse reactions. Currently, the methods used for the asymmetric reduction of 1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol to prepare optically pure (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol mainly include chemical and biological methods. The chemical reduction method requires platinum group metal (e.g., iridium, ruthenium, rhodium) catalysts and Noyori-type chiral ligands. For example, (1R,2R)-(-)-N-(4-toluenesulfonyl)-1,2-diphenylethylenediamine is used to reduce 1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol to (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol, with an enantiomeric excess (ee) of 99.9%. This chemical preparation method requires expensive metal catalysts and strict control of reaction conditions, limiting its industrial application. In addition, there are reports of using enzymatic catalysis to prepare (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol. Li Hengyu et al. used recombinant Lactobacillus fermentum short-chain dehydrogenase / reductase 1 (LfSDR1) as a biocatalyst to catalyze the reduction of 60 g / L of 1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol using its mutant. This route has high product selectivity, but the enzyme separation and purification process is complex, and expensive coenzymes need to be added during the catalytic process. (III) Summary of the Invention

[0005] This invention provides a biochemical preparation method for (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol, a key chiral intermediate of erlotinib ethyl ester. Specifically, it utilizes whole-cell biocatalysis of *Cyberlindnera saturnus* ZJPH1807 to prepare (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol. This method, employing whole-cell catalysis by microorganisms, not only provides mild and environmentally friendly reaction conditions but also simplifies operation and reduces catalyst preparation costs. Furthermore, during the bioreduction process, no additional coenzyme is required; in-situ regeneration of the coenzyme can be achieved through coupling with an inexpensive auxiliary substrate.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] This invention provides a bio-preparation method for a chiral intermediate of erlotinib ethyl ester. The method comprises: using wet cells of *Cyberlindnera saturnus* ZJPH1807 obtained through fermentation as a catalyst; using 1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethyl ketone as a substrate; adding an auxiliary substrate; and using a phosphate buffer solution with a pH of 6.0-8.0 as the reaction medium to construct a transformation system. The transformation reaction is carried out at 25-50℃ and 150-250 rpm for 4-48 h (preferably 30-50℃ and 200 rpm for 28 h). After the reaction, an equal volume of n-hexylene is added to the transformation solution. The reaction is terminated with alkyl and extracted. After centrifugation, the supernatant is collected to obtain a hexane extract containing the chiral intermediate of erlotinib ethyl ester. The hexane extract is then separated and purified to obtain the chiral intermediate of erlotinib ethyl ester, namely (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol. The auxiliary substrate is one of the following: glucose, maltose, sucrose, ethanol, isopropanol, glycerol, L-cysteine, L-alanine, L-glutamic acid, or L-lysine, preferably glucose or maltose, and most preferably glucose.

[0008] The amount of wet bacterial cells used is 50–500 g / L (preferably 80 g / L) based on the volume of the phosphate buffer, and the amount of substrate used is 0.5–6 g / L (preferably 1.5 g / L) based on the volume of the phosphate buffer. The amount of auxiliary substrate added is 4–200 g / L, preferably 20–100 g / L, and most preferably 60 g / L, based on the volume of the phosphate buffer. The ee value of the bioreduction product is greater than 99.9%.

[0009] The technical approach of this invention is as follows:

[0010]

[0011] The *Cyberlindnera saturnus* strain ZJPH1807 described in this invention is deposited at the China Center for Type Culture Collection (CCTCC) on March 29, 2019, with accession number CCTCC NO: M 2019215, located at Wuhan University, Wuhan, China, 430072, China. This strain has been disclosed in the applicant's previous patent application (application number CN110283733A, publication date September 27, 2019).

[0012] Further, the n-hexane extract is separated and purified as follows: the n-hexane extract is concentrated by rotary evaporation to remove the solvent n-hexane, thus obtaining the crude product extract; a silica gel chromatography column is prepared by packing hexane-soaked silica gel (preferably 300-400 mesh) into a chromatography column (preferably 40 cm high, 2.6 cm inner diameter, and 28 cm packing height); the silica gel column is equilibrated with a hexane:ethyl acetate ratio of 1:1 to 10:1 (v / v) (preferably 5:1 (v / v)); the crude product extract is loaded onto the silica gel chromatography column; a layer of silica gel is then placed on top of the column; elution is performed with a hexane:ethyl acetate ratio of 5:1 (v / v); the eluent is monitored by TLC thin-layer chromatography; and the eluent is collected and combined with a hexane:ethyl acetate ratio of 5:1 (v / v). f The eluent containing the target product at a concentration of 0.3 g / mL was concentrated to dryness by rotary evaporation to obtain a white powder, which is the product (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol.

[0013] Furthermore, the concentration of the phosphate buffer is 0.01M to 0.2M, preferably 0.1M, pH 7.5.

[0014] Furthermore, to promote mass transfer and coenzyme regeneration during the bioreduction reaction and improve the reaction yield, a surfactant is added to the reaction system. The surfactant is Tween 20, Tween 60, Tween 80, Span 20, Span 60, Span 80, Triton X-100, or Triton X-114, preferably Tween 60. The amount of surfactant added is 2 to 20 g / L based on the volume of the buffer solution, preferably 5 g / L.

[0015] Furthermore, an organic solvent is added to the reaction system. The organic solvent is dimethyl sulfoxide, N,N-dimethylformamide, methanol, ethanol, isopropanol, toluene, or cyclohexane, preferably methanol. The volume of the organic solvent is 1-7% of the buffer volume, preferably 1%.

[0016] Furthermore, the reaction system also contains cyclodextrin, which includes α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, methyl-β-cyclodextrin, (2-hydroxypropyl)-β-cyclodextrin or (2-hydroxypropyl)-γ-cyclodextrin, preferably β-cyclodextrin; the amount of cyclodextrin added is 10-150 g / L based on the buffer volume, preferably 50 g / L.

[0017] Furthermore, a eutectic solvent is added to the reaction solution system. The eutectic solvent includes choline chloride / alanine, choline chloride / isopropanol, choline chloride / glycine, choline chloride / fructose, choline chloride / trehalose, carnitine / alanine, carnitine / isopropanol, carnitine / glycine, carnitine / fructose, and carnitine / trehalose, preferably carnitine / isopropanol. The amount of the eutectic solvent added is 0.5–2.5 g / L based on the buffer volume, preferably 1.5 g / L. Taking the preparation of choline chloride / alanine as an example, the method for preparing the eutectic solvent of this invention involves adding 0.1 mol of choline chloride and 0.1 mol of alanine to a 500 mL round-bottom flask, adding 200 mL of anhydrous methanol, stirring at 50°C for 24 h, and then vacuum rotary drying at 75°C for 24–48 h to obtain the solid product choline chloride / alanine. The preparation methods for other types of eutectic solvents are similar. The biopreparation method of the chiral intermediate of erlotinib ethyl ester according to the present invention is carried out according to the following steps: using wet cells obtained by fermentation culture of Saturn Rhizopus ZJPH1807 as catalyst, 1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethyl ketone as substrate, and adding auxiliary substrate, surfactant, organic solvent, cyclodextrin and eutectic solvent, and using phosphate buffer with pH 6.0-8.0 as reaction medium to form a conversion system, the conversion reaction is carried out at 25-50℃ and 150-250rpm for 4-48h (preferably 30-50℃ and 200rpm for 28h). After the reaction is completed, an equal volume of n-hexane is added to the conversion solution to terminate the reaction and extract. After centrifugation, the supernatant is taken to obtain the n-hexane extract containing the chiral intermediate of erlotinib ethyl ester. After separation and purification of the n-hexane extract, the chiral intermediate of erlotinib ethyl ester is obtained. The auxiliary substrate is glucose, the surfactant is Tween 60, the organic solvent is methanol, the cyclodextrin is β-cyclodextrin, and the eutectic solvent is carnitine / isopropanol.

[0018] The wet mycelium of this invention is prepared by the following method:

[0019] (1) Plate culture: Single colonies of Cyberlindnera saturnus ZJPH1807 were picked and inoculated into plate culture medium and cultured at 30℃ for 2 days for the first activation. Single colonies were picked from the plate after the first activation and inoculated into plate culture medium again. The plate was activated at 30℃ for 2 days for the second activation (the resulting plates were stored in a refrigerator at 4℃). The final concentration of each component in the plate culture medium was as follows: glucose 15g / L, peptone 20g / L, yeast extract 10g / L, (NH4)2SO4 2g / L, KH2PO4 2g / L, NaCl 1g / L, MgSO4·7H2O 0.5g / L, agar 20g / L, water as solvent, pH 6.5.

[0020] (2) Seed culture: A loopful of bacterial cells was picked from the plate activated for the second time in step (1) and inoculated into the seed culture medium. The culture was carried out at 30°C and 200 rpm for 12 hours to obtain the seed solution. The final concentrations of the components in the seed culture medium were as follows: glucose 15–50 g / L, peptone 5–15 g / L, yeast extract 5–15 g / L, (NH4)2SO4 1–5 g / L, KH2PO4 0.2–1.2 g / L, NaCl 0.2–1.2 g / L, MgSO4·7H2O 0.1–1.0 g / L, with water as the solvent and pH 5–8. Preferably, the final concentrations of the components in the seed culture medium were as follows: glucose 15 g / L, peptone 20 g / L, yeast extract 10 g / L, (NH4)2SO4 2 g / L, KH2PO4 2 g / L, NaCl 1 g / L, MgSO4·7H2O 0.5 g / L, solvent: water, pH 6.5;

[0021] (3) Fermentation culture: Inoculate the seed culture into the fermentation medium at an inoculation amount of 6-12% (preferably 8%), and culture at 30℃ and 200rpm for 10-48h (preferably 28h) to obtain the fermentation broth. Centrifuge the fermentation broth, and wash the resulting precipitate with 0.1M, pH 7.5 K2HPO4-KH2PO4 buffer. After centrifugation, wet cells are obtained, which are the enzyme-containing cells for biotransformation. The final concentration composition of each component in the fermentation medium is as follows: carbon source 15-25g / L, nitrogen source 15-25g / L, KH2PO4 0.2-1.2g / L, MgCl2·6H2O 0.2-1.0g / L, solvent is water, pH 6.0-9.0. The carbon source is one of the following: glucose, maltose, glycerol, lactose, sucrose or dextrin, preferably glucose. The nitrogen source is one of the following: yeast extract, peptone, beef extract, ammonium chloride or ammonium sulfate, preferably ammonium sulfate.

[0022] Furthermore, the final concentrations of each component in the fermentation medium are as follows: glucose 15 g / L, ammonium sulfate 20 g / L, KH2PO4 1.0 g / L, MgCl2·6H2O 0.5 g / L, with water as the solvent and pH 7.5.

[0023] Compared with existing technologies, the beneficial effects of this invention are mainly reflected in the following: This invention utilizes resting cells of *Cyberlindnera saturnus* ZJPH1807 as a catalyst to prepare (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol via biocatalysis of 1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol. The product obtained by this strain has high optical purity, with an ee value >99.9%, and a reaction yield of 86.4%. This invention utilizes a biocatalytic method to prepare (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol. Compared with reported chemical synthesis routes, this process is simple, environmentally friendly, and uses microbial cells as the biocatalyst, resulting in low cost. This provides a useful reference for the preparation of the key chiral intermediate (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol by whole-cell catalysis of microorganisms. (iv) Description of the attached drawings

[0024] Figure 1 The high-performance liquid chromatography chromatogram of the substrate 1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethyl ketone standard.

[0025] Figure 2 The high-performance liquid chromatography (HPLC) chromatograms of the product (S)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol and the standard (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol are shown.

[0026] Figure 3 High-performance liquid chromatography (HPLC) chromatogram of the bioreduction reaction extract of Cyberlindnera saturnus strain ZJPH1807. (V) Detailed Implementation

[0027] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0028] Example 1: Preparation and detection method of (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol

[0029] 1. Preparation method

[0030] (1) Plate culture: Single colonies of Cyberlindnera saturnus ZJPH1807 were picked and inoculated into plate culture medium. The culture was carried out at 30℃ for 2 days for the first activation. Single colonies were picked from the plate after the first activation and inoculated into plate culture medium again. The culture was carried out at 30℃ for 2 days for the second activation (the resulting plates were stored in a refrigerator at 4℃). The final concentration of each component in the plate culture medium was as follows: glucose 15g / L, peptone 20g / L, yeast extract 10g / L, (NH4)2SO4 2g / L, KH2PO4 2g / L, NaCl 1g / L, MgSO4·7H2O 0.5g / L, agar 20g / L, water as solvent, pH 6.5, sterilized at 121℃ for 20min, and after sterilization and cooling, plates were prepared.

[0031] (2) Seed culture: Pick a loopful of bacterial cells from the plate cultured in step (1) and inoculate it into 100 mL of seed culture medium. Culture at 30℃ and 200 rpm for 12 h to obtain seed liquid. The final concentration of each component in the seed culture medium is as follows: glucose 15 g / L, peptone 20 g / L, yeast extract 10 g / L, (NH4)2SO4 2 g / L, KH2PO4 2 g / L, NaCl 1 g / L, MgSO4·7H2O 0.5 g / L, solvent is water, pH 6.5, sterilize at 115℃ for 30 min.

[0032] (3) Fermentation culture: The seed culture was transferred to a 250 mL Erlenmeyer flask containing 100 mL of initial fermentation culture medium at an inoculation volume of 10%. The culture was carried out at 30 °C and 200 rpm for 24 h to obtain the fermentation broth. The fermentation broth was centrifuged, and the resulting precipitate was washed with 0.1 M, pH 7.0 K2HPO4-KH2PO4 buffer. After centrifugation, wet cells were obtained, which are the enzyme source cells. The final concentration composition of each component in the initial fermentation culture medium was: glucose 15 g / L, NH4Cl 15 g / L, KH2PO4 1 g / L, solvent was water, pH 6.5, sterilized at 115 °C for 30 min.

[0033] (4) In 10 mL of 0.1 M, pH 7.0 phosphate buffer, 15 mg of 1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethyl ketone was added as substrate, 1.0 g of glucose as co-substrate, and 1.0 g of the enzyme-source microbial cells obtained in step (3) as catalyst. Biotransformation was performed at 30 °C and 200 rpm for 24 h. After the reaction was completed, 10 mL of n-hexane was added to the transformation solution to terminate the reaction and the solution was extracted for 30 min. After centrifugation, the supernatant was collected, and the yield and ee value of the target product (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol in the transformation solution were determined by chiral high-performance liquid chromatography. The yield was 5.7%, and the ee value was greater than 99.9%.

[0034] 2. Detection Method

[0035] High-performance liquid chromatography (HPLC) detection conditions: Shimadzu SPD-10A HPLC system, Daicel CHIRALPAK (Japan) AD-H normal-phase polysaccharide derivative chiral column (4.6 mm × 250 mm × 5 μm), UV detection wavelength: 254 nm; mobile phase: hexane (V): ethanol (V) = 97:3; flow rate: 1 mL / min; injection volume: 5 μL. Quantification was performed using the external standard method.

[0036] Based on the liquid chromatography chromatogram, the yield and ee value of the product in the conversion solution were calculated.

[0037] The yield calculation method is as follows:

[0038] Standard curve construction: Substrate and product standard solutions with concentrations of 5, 10, 15, 20, and 25 mM were prepared using a mobile phase of hexane (v):ethanol (v) = 97:3. These solutions were then detected by high-performance liquid chromatography (HPLC). The peak areas were obtained by integrating the chromatograms. A plot was constructed with the substrate or product concentration on the x-axis and the peak area as a fraction of 1 / 10,000 on the y-axis. Linear regression was then applied to obtain the standard curve. The equation for the substrate standard curve was: y = 283.81x + 48.839, R0 2 =0.999; R-type product standard curve equation: y = 223.9x - 63.432, R 2 =0.9996.

[0039] The formula for calculating product yield is as follows:

[0040] Yield = C p / C0

[0041] In the formula C pThe concentration of (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol is given, and the initial concentration of 1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethyl ketone is given.

[0042] The optical purity of the product is characterized by the enantiomeric excess (ee). The calculation formula is as follows:

[0043] ee = (C R -C S ) / (Cs+C R )×100%

[0044] In the formula C S and C R The molar concentrations of 1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol are for S-type and R-type, respectively.

[0045] Example 2: Optimization of initial fermentation medium composition and fermentation conditions using single-factor experimental design

[0046] The fermentation enzyme production conditions of Cyberlindnera saturnus strain ZJPH1807 were optimized using a single-factor experimental method, including optimizing the types and amounts of nitrogen source, carbon source and inorganic salt in the initial fermentation medium, the initial pH of the fermentation medium, the fermentation temperature, the volume of liquid in the shake flask, the inoculum size, and the fermentation time.

[0047] 1. Optimization of nitrogen source

[0048] (1) Types of nitrogen sources

[0049] The NH4Cl in the initial fermentation medium of step (3) in Example 1 was replaced with (NH4)2SO4, peptone, yeast extract and beef extract, respectively, and the nitrogen content was controlled at 3.9 g / L. Other operations were the same as in steps (3) and (4) of Example 1. The effects of different types of nitrogen sources on the biotransformation reaction of the obtained cells were investigated. The results are shown in Table 1. (NH4)2SO4 was selected as the best nitrogen source.

[0050] Table 1. Effect of nitrogen source type on bacterial catalytic results

[0051]

[0052] (2) (NH4)2SO4 dosage increase

[0053] After determining (NH4)2SO4 as the optimal nitrogen source, the NH4Cl in the initial fermentation medium of step (3) in Example 1 was replaced with different amounts of (NH4)2SO4. The effects of the obtained cells on the biotransformation reaction under (NH4)2SO4 additions of 10, 15, 20, 25, and 30 g / L were investigated. Other operations were the same as in steps (3) and (4) of Example 1. The results are shown in Table 2. 20 g / L of (NH4)2SO4 was selected as the optimal addition amount.

[0054] Table 2. Effect of (NH4)2SO4 dosage on bacterial catalysis results.

[0055]

[0056] 2. Optimization of carbon sources

[0057] (1) Types of carbon sources

[0058] Based on the determination that the optimal nitrogen source is 20 g / L (NH4)2SO4, the NH4Cl in the initial fermentation medium in step (3) of Example 1 was replaced with 20 g / L (NH4)2SO4. At the same time, glucose was replaced with sucrose, maltose, corn dextrin and glycerol respectively. The effects of different carbon sources such as glucose, sucrose, maltose, corn dextrin and glycerol on the biotransformation reaction of the obtained cells were investigated. Other operations were the same as steps (3) and (4) of Example 1. The results are shown in Table 3. Glucose was selected as the best carbon source.

[0059] Table 3. Effects of carbon source type on cell catalysis results

[0060]

[0061] (2) Increase glucose dosage

[0062] After determining glucose as the optimal carbon source, the effects of the bacterial cells obtained under glucose addition of 10, 15, 20, 25, and 30 g / L on the biotransformation reaction were further investigated. Specifically, the NH4Cl in the initial fermentation medium of step (3) in Example 1 was replaced with 20 g / L (NH4)2SO4, and different amounts of glucose were added. Other operations were the same as steps (3) and (4) in Example 1. The results are shown in Table 4. 15 g / L was selected as the optimal amount of glucose to be added.

[0063] Table 4. Effect of glucose dosage on bacterial catalysis results

[0064]

[0065] 3. Inorganic salts

[0066] (1) Types of inorganic salts

[0067] Based on the determination of the optimal nitrogen and carbon sources, the effects of different types of inorganic salts, such as KH2PO4, CaCl2, NaCl, ZnCl2, MgCl2·6H2O, and FeCl3·6H2O, were investigated. The dosage was 1 g / L, that is, NH4Cl in the initial fermentation medium of step (3) in Example 1 was replaced with 20 g / L (NH4)2SO4, and KH2PO4 was replaced with CaCl2, NaCl, ZnCl2, MgCl2·6H2O, and FeCl3·6H2O. Other operations were the same as in steps (3) and (4) of Example 1. The results are shown in Table 5. The results show that MgCl2·6H2O is the most suitable inorganic salt. Considering that KH2PO4 can not only provide inorganic nutrients for cell growth, but its weak acidity also has a buffering effect on the pH value of the medium, the combination of MgCl2·6H2O and KH2PO4 was selected as the most suitable inorganic salt.

[0068] Table 5. Effects of Inorganic Salt Species on Cell Catalysis Results

[0069]

[0070] (2) Inorganic salt addition ratio

[0071] After determining MgCl2·6H2O and KH2PO4 as the optimal inorganic salts, the effects of different addition ratios of MgCl2·6H2O and KH2PO4, namely KH2PO4:MgCl2·6H2O at ratios of 1:1, 1:2, 1:3, 2:1, and 3:1, on the biotransformation reaction were further investigated. In the initial fermentation medium of step (3) of Example 1, NH4Cl was replaced with 20 g / L (NH4)2SO4, and KH2PO4 was replaced with 1 g / L KH2PO4 + 1 g / L MnO. The following solutions were used: 1 g / L KH2PO4 + 2 g / L MgCl2·6H2O, 1 g / L KH2PO4 + 3 g / L MgCl2·6H2O, 2 g / L KH2PO4 + 1 g / L MgCl2·6H2O, and 3 g / L KH2PO4 + 1 g / L MgCl2·6H2O. Other operations were the same as steps (3) and (4) in Example 1. The results are shown in Table 6. The optimal inorganic salt addition ratio was selected as KH2PO4:MgCl2·6H2O = 2:1.

[0072] Table 6. Effect of Inorganic Salt Addition Ratio on Cell Catalysis Results

[0073]

[0074] (3) Inorganic salt addition

[0075] After determining that the optimal inorganic salt addition ratio of KH2PO4:MgCl2·6H2O = 2:1 was determined, the experiment of adding MgCl2·6H2O and KH2PO4 was further investigated. That is, when the addition amount of KH2PO4 was 0.5, 1.0, 1.5, 2.0, and 2.5 g / L, the addition amount of MgCl2·6H2O was proportionally converted to 0.25, 0.5, 0.75, 1.0, and 1.25 g / L. The effect of the resulting bacterial cells on the biotransformation reaction was investigated. In the initial fermentation medium of step (3) of Example 1, NH4Cl was replaced with 20 g / L (NH4)2SO4, and KH2PO4 was changed to 0.5-2.5 g / L KH2PO4 + 0.25-1.25 g / L MgCl2·6H2O. Other operations were the same as in steps (3) and (4) of Example 1. The results are shown in Table 7. 1.0 g / L was selected. The optimal amount of inorganic salt to add is KH2PO4 at 0.5 g / L MgCl2·6H2O.

[0076] The fermentation medium consisted of: 15 g / L glucose, 20 g / L (NH4)2SO4, 1 g / L KH2PO4, 0.5 g / L MgCl2·6H2O, with water as the solvent and pH 6.5.

[0077] Table 7. Effect of inorganic salt dosage on bacterial catalysis results.

[0078]

[0079] 4. The effect of initial pH value of fermentation medium

[0080] Based on the determination of the optimal nitrogen source, carbon source and inorganic salt, the effect of the bacterial cells obtained under different initial pH values ​​(5.0, 6.0, 6.5, 7.0, 7.5, 8.0 and 9.0) of the fermentation medium in step 3 (3) on the biotransformation reaction was investigated. Other operations were the same as steps (3) and (4) in Example 1. The results are shown in Table 8. pH 7.5 was selected as the optimal initial pH value of the fermentation medium.

[0081] Table 8. Effects of initial pH values ​​of different fermentation media on cell catalysis results.

[0082]

[0083] 5. The effect of fermentation temperature

[0084] The fermentation medium consisted of: 15 g / L glucose, 20 g / L (NH4)2SO4, 1 g / L KH2PO4, 0.5 g / L MgCl2·6H2O, with water as the solvent and pH 7.5.

[0085] Based on the determination of the optimal fermentation medium composition and initial pH value, the effect of the bacterial cells obtained under the fermentation temperature of 20, 25, 30, 35 and 40℃ in step (3) of Example 1 on the biotransformation reaction was investigated. Other operations were the same as steps (3) and (4) of Example 1. The results are shown in Table 9. 30℃ was selected as the optimal fermentation temperature.

[0086] Table 9. Effects of different fermentation temperatures on cell catalysis results.

[0087]

[0088] 6. Effect of flask volume

[0089] The fermentation medium consisted of: 15 g / L glucose, 20 g / L (NH4)2SO4, 1 g / L KH2PO4, 0.5 g / L MgCl2·6H2O, with water as the solvent and pH 7.5.

[0090] Based on the determination of the optimal fermentation medium composition and initial pH value, the effect of the bacterial cells obtained under the conditions of 50, 70, 90, 100, 110 and 130 mL of shake flask liquid volume in step (3) of Example 1 on the biotransformation reaction was investigated. Other operations were the same as steps (3) and (4) of Example 1. The results are shown in Table 10. 90 mL / 250 mL was selected as the optimal fermentation medium liquid volume.

[0091] Table 10 Effect of different liquid volumes in shake flasks on bacterial catalytic results.

[0092]

[0093]

[0094] 7. Inoculation volume

[0095] The fermentation medium consisted of: 15 g / L glucose, 20 g / L (NH4)2SO4, 1 g / L KH2PO4, 0.5 g / L MgCl2·6H2O, with water as the solvent and pH 7.5.

[0096] Based on the determination of the optimal fermentation medium composition and initial pH value, the effect of different inoculum amounts in step (3) of Example 1 on the biotransformation reaction of the obtained cells was investigated. The volume of the shake flask was 90 mL / 250 mL, and other operations were the same as steps (3) and (4) of Example 1. The results are shown in Table 11. 8% was selected as the optimal inoculum amount.

[0097] Table 11 Effect of different inoculum sizes on bacterial catalytic results

[0098]

[0099] 8. The effect of fermentation time

[0100] The fermentation medium consisted of: 15 g / L glucose, 20 g / L (NH4)2SO4, 1 g / L KH2PO4, 0.5 g / L MgCl2·6H2O, with water as the solvent and pH 7.5.

[0101] Based on the determination of the optimal fermentation medium composition and initial pH value, the effect of the bacterial cells obtained by different fermentation times in step (3) of Example 1 on the biotransformation reaction was investigated. The seed liquid was transferred to a 250 mL Erlenmeyer flask containing 90 mL of fermentation medium at an inoculation amount of 8% by volume. Other operations were the same as steps (3) and (4) of Example 1. The results are shown in Table 12. 28 h was selected as the optimal fermentation time.

[0102] Table 12 Effects of different fermentation times on cell catalysis results

[0103]

[0104] In summary, the final concentrations of the components in the optimal culture medium are: glucose 15 g / L, (NH4)2SO4 20 g / L, KH2PO4 1.0 g / L, MgCl2·6H2O 0.5 g / L, with water as the solvent and a pH of 7.5. The optimal fermentation conditions for enzyme production are: fermentation temperature 30℃, 90 mL / 250 mL liquid volume in shake flasks, 8% inoculum size, and fermentation time 28 h.

[0105] 9. Biotransformation results of cells obtained under optimal fermentation enzyme production conditions

[0106] The seed culture prepared in step (2) of Example 1 was transferred to a 250 mL Erlenmeyer flask containing 90 mL of fermentation medium at an inoculation rate of 8% by volume. The flask was incubated at 30 °C and 200 rpm for 24 h to obtain the fermentation broth. The fermentation broth was centrifuged, and the resulting precipitate was washed with 0.1 M, pH 7.0 K2HPO4-KH2PO4 buffer. After centrifugation, wet cells were obtained.

[0107] In 10 mL of 0.1 M, pH 7.0 phosphate buffer, 15 mg of 1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethyl ketone was added as a substrate, 1.0 g of glucose as an auxiliary substrate, and 1.0 g of the wet bacterial cells prepared in the above steps as a catalyst. Biotransformation was carried out at 30 °C and 200 rpm for 28 h. After the reaction was completed, 10 mL of n-hexane was added to the transformation solution to terminate the reaction and extraction was performed for 30 min. After centrifugation, the supernatant was collected, and the yield and ee value of the target product (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol in the transformation solution were determined by chiral high-performance liquid chromatography as described in Example 1. The yield of the biotransformed product was increased to 13.6%, and the ee value was greater than 99.9%.

[0108] Example 3: Preparation of wet bacterial cells

[0109] (1) Plate culture: Single colonies of Cyberlindnera saturnus ZJPH1807 were picked and inoculated into plate culture medium. The culture was carried out at 30℃ for 2 days for the first activation. Single colonies were picked from the plate after the first activation and inoculated into plate culture medium again. The culture was carried out at 30℃ for 2 days for the second activation (the resulting plates were stored in a refrigerator at 4℃). The final concentration of each component in the plate culture medium was as follows: glucose 15g / L, peptone 20g / L, yeast extract 10g / L, (NH4)2SO4 2g / L, KH2PO4 2g / L, NaCl 1g / L, MgSO4·7H2O 0.5g / L, agar 20g / L, water as solvent, pH 6.5, sterilized at 121℃ for 20min, and after cooling, plates were prepared.

[0110] (2) Seed culture: Pick a loopful of bacterial cells from the plate cultured in step (1) and inoculate it into 100 mL of seed culture medium. Culture at 30℃ and 200 rpm for 12 h to obtain seed liquid. The final concentration of each component in the seed culture medium is as follows: glucose 15 g / L, peptone 20 g / L, yeast extract 10 g / L, (NH4)2SO4 2 g / L, KH2PO4 2 g / L, NaCl 1 g / L, MgSO4·7H2O 0.5 g / L, solvent is water, pH 6.5, sterilize at 115℃ for 30 min.

[0111] (3) Fermentation culture: The seed culture was transferred to a 250 mL Erlenmeyer flask containing 90 mL of fermentation medium at an inoculation volume of 8%. The culture was carried out at 30 °C and 200 rpm for 28 h to obtain the fermentation broth. The fermentation broth was centrifuged, and the resulting precipitate was washed with K2HPO4-KH2PO4 buffer at pH 7.0. After centrifugation, wet cells were obtained, which are the enzyme source cells. The final concentration composition of each component in the fermentation medium is as follows: glucose 15 g / L, (NH4)2SO4 20 g / L, KH2PO4 1.0 g / L, MgCl2·6H2O 0.5 g / L, solvent is water, pH 7.5, sterilized at 115 °C for 30 min.

[0112] Example 4: Effect of the type of auxiliary substrate on catalytic results

[0113] 1.0 g (100 g / L) of wet bacterial cells obtained according to the method in Example 3 was washed with distilled water and resuspended in 10 mL of phosphate buffer (0.1 M, pH 7.0). 15 mg of 1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethyl ketone was added as a substrate, along with 1.0 g (100 g / L) of different auxiliary substrates. The mixture was reacted in a shaker at 30 °C and 200 rpm for 24 h. The analysis was performed using the detection method in Example 1, and the yield and ee value of product (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol were calculated. The results are shown in Table 13.

[0114] Table 13 Effects of different types of auxiliary substrates on catalytic results

[0115]

[0116] Glucose was preferred as an auxiliary substrate, and under these conditions, the yield was 12.7% and the ee value was >99.9%.

[0117] Example 5: Effect of glucose dosage on catalytic results

[0118] 1.0 g (100 g / L) of wet bacterial cells obtained according to the method in Example 3 was washed with distilled water and resuspended in 10 mL of phosphate buffer (0.1 M, pH 7.0). 15 mg of 1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethyl ketone was added as a substrate, followed by 0.2–1.0 g (20–100 g / L) of glucose as an auxiliary substrate. The mixture was reacted in a shaker at 30 °C and 200 rpm for 24 h. The analysis was performed using the detection method in Example 1, and the yield and ee value of the product (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol were calculated. The results are shown in Table 14.

[0119] Table 14 Effect of glucose dosage on catalytic results

[0120]

[0121] The preferred glucose dosage is 60 g / L. Under these conditions, the yield is 14.5% and the ee value is >99.9%.

[0122] Example 6: Effect of conversion reaction temperature on catalytic results

[0123] 1.0 g (100 g / L) of wet bacterial cells obtained according to the method in Example 3 was washed with distilled water and resuspended in 10 mL of K2HPO4-KH2PO4 buffer (0.1 M, pH 7.5). 15 mg of 1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol was added as a substrate, and 0.6 g (60 g / L) of glucose was added as an auxiliary substrate. The mixture was reacted for 24 h at different temperatures (20–60 °C) and 200 rpm in a shaker. The analysis was performed using the detection method in Example 1, and the yield and ee value of the product (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol were calculated. The results are shown in Table 15.

[0124] Table 15 Effect of different conversion reaction temperatures on catalytic results

[0125]

[0126] The preferred conversion reaction temperature is 45°C. Under these conditions, the yield is 18.8% and the ee value is >99.9%.

[0127] Example 7: Effects of buffer type and initial pH value on catalytic results

[0128] 1.0 g (100 g / L) of wet bacterial cells obtained according to the method in Example 3 was washed with distilled water and resuspended in 10 mL of phosphate buffer (0.1 M) at pH 6.0-8.0, Tris-HCl buffer (0.1 M) at pH 8.0-9.0, or glycine-NaOH buffer (0.1 M) at pH 9.0-10.5. 15 mg of 1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethyl ketone was added as a substrate, and 0.6 g (60 g / L) of glucose was added as an auxiliary substrate. The mixture was then reacted in a shaker at 45 °C and 200 rpm for 24 h. The detection method of Example 1 was used for analysis and detection, and the yield and ee value of product (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol were calculated. The results are shown in Table 16.

[0129] Table 16. Effects of different buffer types and initial pH values ​​on catalytic results.

[0130]

[0131]

[0132] The preferred buffer is a K2HPO4-KH2PO4 buffer with an initial pH of 7.5. Under these conditions, the yield is 20.7% and the ee value is >99.9%.

[0133] Example 8: Effect of Cell Dosage on Catalytic Results

[0134] The wet bacterial cells obtained according to the method in Example 3 were washed with distilled water and resuspended in 10 mL of K2HPO4-KH2PO4 buffer (0.1 M, pH 7.5) according to different wet cell additions as shown in Table 17. 15 mg of 1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethyl ketone was added as a substrate, and 0.6 g (60 g / L) of glucose was added as an auxiliary substrate. The mixture was reacted in a shaker at 45 °C and 200 rpm for 24 h. The analysis was performed using the detection method in Example 1, and the yield and ee value of the product (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol were calculated. The results are shown in Table 17.

[0135] Table 17 Effect of wet cell dosage on catalytic results

[0136]

[0137] The optimal wet cell concentration is 80 g / L. Under these conditions, the yield is 25.6% and the ee value is >99.9%.

[0138] Example 9: Effect of conversion time on catalytic results

[0139] 0.8 g (80 g / L) of wet bacterial cells obtained according to the method in Example 3 was washed with distilled water and resuspended in 10 mL of K2HPO4-KH2PO4 buffer (0.1 M, pH 7.5). 15 mg of 1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethyl ketone was added as a substrate, and 0.6 g (60 g / L) of glucose was added as an auxiliary substrate. The mixture was reacted in a shaker at 45 °C and 200 rpm for 16-40 h. The analysis was performed using the detection method in Example 1, and the yield and ee value of product (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol were calculated. The results are shown in Table 18.

[0140] Table 18 Effect of conversion time on catalytic results

[0141]

[0142]

[0143] The optimal conversion time is 28 hours. Under these conditions, the yield is 28.3% and the ee value is >99.9%.

[0144] Example 10: The effect of different types of surfactants on catalytic results

[0145] 0.8 g (80 g / L) of wet bacterial cells obtained according to the method in Example 3 was washed with distilled water and resuspended in 10 mL of K2HPO4-KH2PO4 buffer (0.1 M, pH 7.5). 15 mg of 1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethyl ketone was added as a substrate, 0.6 g (60 g / L) of glucose was added as an auxiliary substrate, and 0.05 g (5 g / L) of different types of surfactants were added. The mixture was then reacted in a shaker at 45 °C and 200 rpm for 28 h. The analysis was performed using the detection method in Example 1, and the yield and ee value of product (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol were calculated. The results are shown in Table 19.

[0146] Table 19 Effect of different surfactant types on catalytic results

[0147]

[0148] The preferred surfactants are Tween 20, Tween 60 and Tween 80, with Tween 60 being the most preferred.

[0149] Example 11: Effect of surfactant dosage on catalytic results

[0150] 0.8 g (80 g / L) of wet bacterial cells obtained according to the method in Example 3 was washed with distilled water and resuspended in 10 mL of K2HPO4-KH2PO4 buffer (0.1 M, pH 7.5). 15 mg of 1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethyl ketone was added as a substrate, 0.6 g (60 g / L) of glucose was added as an auxiliary substrate, and 0.02 g–0.20 g (2–20 g / L) of Tween 60 surfactant was added. The mixture was reacted in a shaker at 45 °C and 200 rpm for 28 h. The analysis was performed using the detection method in Example 1, and the yield and ee value of product (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol were calculated. The results are shown in Table 20.

[0151] Table 20 Effect of Tween 60 dosage on catalytic results

[0152]

[0153] The preferred dosage is 0.05 g (5 g / L) of surfactant Tween 60. Under these conditions, the yield is 51.2% and the ee value is >99.9%.

[0154] Example 12: Effect of different types of organic solvents on catalytic results

[0155] 0.8 g (80 g / L) of wet bacterial cells obtained according to the method in Example 3 was washed with distilled water and resuspended in 10 mL of K2HPO4-KH2PO4 buffer (0.1 M, pH 7.5). 15 mg of substrate 1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethyl ketone was dissolved in 100 μL (1% v / v) of different organic solvents and added to the reaction system. At the same time, 0.6 g (60 g / L) of glucose was added as an auxiliary substrate and 0.05 g (5 g / L) of surfactant Tween 60 was added. The reaction was carried out in a shaker at 45 °C and 200 rpm for 28 h. The detection method of Example 1 was used for analysis and detection, and the yield and ee value of product (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol were calculated. The results are shown in Table 21.

[0156] Table 21 Effect of Organic Solvent Type on Catalytic Results

[0157]

[0158] Methanol is the preferred organic solvent. Under these conditions, the yield is 61.0% and the ee value is >99.9%.

[0159] Example 13: Effect of different methanol dosages on catalytic results

[0160] 0.8 g (80 g / L) of wet bacterial cells obtained according to the method in Example 3 was washed with distilled water and resuspended in 10 mL of K2HPO4-KH2PO4 buffer (0.1 M, pH 7.5). 15 mg of substrate 1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroacetone was dissolved in 100 μL-700 μL (1%-7%) methanol and added to the reaction system. At the same time, 0.6 g (60 g / L) of glucose was added as an auxiliary substrate and 0.05 g (5 g / L) of surfactant Tween 60 was added. The reaction was carried out in a shaker at 45 °C and 200 rpm for 28 h. The analysis and detection were performed using the detection method of Example 1, and the yield and ee value of product (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol were calculated. The results are shown in Table 22.

[0161] Table 22 Effect of different methanol dosages on catalytic results

[0162]

[0163] The preferred methanol dosage is 100 μL (1%). Under these conditions, the yield is 61.6% and the ee value is >99.9%.

[0164] Example 14: Effect of different types of cyclodextrins on catalytic results

[0165] 0.8 g (80 g / L) of wet bacterial cells obtained according to the method in Example 3 was washed with distilled water and resuspended in 10 mL of K2HPO4-KH2PO4 buffer (0.1 M, pH 7.5). 15 mg of substrate 1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroacetone was dissolved in 100 μL (1% v / v) methanol and added to the reaction system. At the same time, 0.6 g (60 g / L) of glucose was added as an auxiliary substrate, 0.05 g (5 g / L) of surfactant Tween 60 was added, and 0.1 g (10 g / L) of different types of cyclodextrin were added. The mixture was placed in a shaker at 45 °C and 200 rpm for 28 h. The detection method of Example 1 was used for analysis and detection, and the yield and ee value of product (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol were calculated. The results are shown in Table 23.

[0166] Table 23 Effect of Cyclodextrin Type on Catalytic Results

[0167]

[0168] β-cyclodextrin is preferred; under these conditions, the yield is 71.8% and the ee value is >99.9%.

[0169] Example 15: Effect of β-cyclodextrin dosage on catalytic results

[0170] 0.8 g (80 g / L) of wet bacterial cells obtained according to the method in Example 3 was washed with distilled water and resuspended in 10 mL of K2HPO4-KH2PO4 buffer (0.1 M, pH 7.5). 15 mg of substrate 1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroacetone was dissolved in 100 μL (1%) methanol and added to the reaction system. At the same time, 0.6 g (60 g / L) of glucose was added as an auxiliary substrate, 0.05 g (5 g / L) of surfactant Tween 60 was added, and 0.1 g-1.5 g (10-150 g / L) of β-cyclodextrin was added. The mixture was placed in a shaker at 45 °C and 200 rpm for 28 h. The detection method of Example 1 was used for analysis and detection, and the yield and ee value of product (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol were calculated. The results are shown in Table 24.

[0171] Table 24 Effect of β-cyclodextrin dosage on catalytic results

[0172]

[0173] The preferred dosage of β-cyclodextrin is 0.5 g (50 g / L). Under these conditions, the yield is 80.6% and the ee value is >99.9%.

[0174] Example 16: Effect of different types of eutectic solvents on catalytic results

[0175] 0.8 g (80 g / L) of wet bacterial cells obtained according to the method in Example 3 was washed with distilled water and resuspended in 10 mL of K2HPO4-KH2PO4 buffer (0.1 M, pH 7.5). 15 mg of substrate 1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroacetone was dissolved in 100 μL (1% v / v) methanol and added to the reaction system. At the same time, 0.6 g (60 g / L) of glucose was added as an auxiliary substrate, 0.05 g (5 g / L) of surfactant Tween 60 was added, 0.5 g (50 g / L) of β-cyclodextrin was added, and 0.01 g (1 g / L) of different kinds of eutectic solvents were added. The mixture was placed in a shaker at 45 °C and 200 rpm for 28 h. The detection method of Example 1 was used for analysis and detection, and the yield and ee value of product (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol were calculated. The results are shown in Table 25.

[0176] Table 25 Effects of different types of eutectic solvents on catalytic results

[0177]

[0178]

[0179] The preferred eutectic solvent is carnitine / isopropanol, under which the yield is 86.2% and the ee value is >99.0%.

[0180] Example 17: Effect of different dosages of carnitine / isopropanol on catalytic results

[0181] 0.8 g (80 g / L) of wet bacterial cells obtained according to the method in Example 3 was washed with distilled water and resuspended in 10 mL of K2HPO4-KH2PO4 buffer (0.1 M, pH 7.5). 15 mg of substrate 1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroacetone was dissolved in 100 μL (1%) methanol and added to the reaction system. At the same time, 0.6 g (60 g / L) of glucose was added as an auxiliary substrate, 0.05 g (5 g / L) of surfactant Tween 60 was added, 0.5 g (50 g / L) of β-cyclodextrin was added, and 0.005 g-0.025 g (0.5-2.5 g / L) of carnitine / isopropanol was added. The mixture was placed in a shaker at 45 °C and 200 rpm for 28 h. The detection method of Example 1 was used for analysis and detection, and the yield and ee value of product (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol were calculated. The results are shown in Table 26.

[0182] Table 26 Effect of different carnitine / isopropanol dosages on catalytic results

[0183]

[0184] The preferred dosage is 0.015 g (1.5 g / L) of carnitine / isopropanol. Under these conditions, the yield is 86.4% and the ee value is >99.0%.

[0185] Example 18: Preparation of product (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol under optimal conditions

[0186] 8.0 g (80 g / L) of wet bacterial cells obtained according to the method in Example 3 was washed with distilled water and resuspended in 100 mL of K2HPO4-KH2PO4 buffer (0.1 M, pH 7.5). 150 mg of substrate 1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroacetone was dissolved in 1 mL (1%) of methanol and added to the reaction system. At the same time, 6.0 g (60 g / L) of glucose was added as an auxiliary substrate, 0.5 g (5 g / L) of surfactant Tween 60 was added, 5.0 g (50 g / L) of β-cyclodextrin was added, and 0.15 g (1.5 g / L) of carnitine / isopropanol was added. The mixture was placed in a shaker at 45 °C and 200 rpm for 28 h. After the reaction was completed, 100 mL of n-hexane was added to the conversion solution to terminate the reaction and the mixture was extracted for 30 min. After centrifugation, the supernatant was collected and concentrated using a rotary evaporator to remove the solvent n-hexane, yielding the crude product extract. A silica gel chromatography column was prepared by packing hexane-soaked silica gel (300-400 mesh) into a chromatography column (40 cm high, 2.6 cm inner diameter, 28 cm packing height). The silica gel column was then equilibrated with a hexane:ethyl acetate eluent at a volume ratio of 5:1. The crude product extract was loaded onto the silica gel column, and a layer of silica gel was placed on top of the column. Elution was then performed with a hexane:ethyl acetate ratio of 5:1 (v / v). The composition of the eluent was monitored by TLC thin-layer chromatography with a hexane:ethyl acetate ratio of 5:1 (v / v) as the developing solvent. The eluent was collected and combined. f The eluent containing the target product at a concentration of 0.3 was concentrated to dryness by rotary evaporation to obtain a white powder, yielding 75.6 mg of (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol product with an ee value >99.9%.

[0187] Example 19: Conversion capacity study of (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol as a key chiral intermediate in the biocatalytic preparation of teroseltate ethyl ester by *Geotrichum candidum* ZJPH1907.

[0188] (1) *Geotrichum candidum* ZJPH1907, deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on July 6, 2020, accession number: CCTCCNO:M 2020282. This strain has been disclosed in a previous patent application (Publication No.: CN112063531A, Publication Date: December 11, 2020). The culture method of the strain and the preparation process of the enzyme source cells are based on the previous patent application (Publication No.: CN112063531A, Publication Date: December 11, 2020).

[0189] (2) Biocatalytic preparation of key chiral intermediate (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol for erlotinib ethyl ester

[0190] The wet cell mass of *Geotrichum candidum* ZJPH1907, prepared by fermentation, was suspended in 10 mL of potassium phosphate buffer (pH 7.0, 0.1 M). 1.0 g of glucose was used as an auxiliary substrate, and 15 mg of 1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethyl ketone was added to form the transformation system. The wet cell mass was 1.0 g by wet weight. The system was incubated at 30°C and 200 rpm for 24 h in a shaker. The product was detected using the high-performance liquid chromatography method described in Example 1. The product had an (S)-configuration, an ee value of 97.7%, and a yield of 12.3%.

[0191] The chemical structural formula of (S)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol is:

[0192]

[0193] Conclusion: The transformation of *Geotrichum candidum* strain ZJPH1907 with 1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol yielded (S)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol, with an ee value of 97.7% and a yield of 12.3%.

[0194] Example 20: Conversion capacity study of (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol, a key chiral intermediate for the biocatalytic preparation of teroseltate from *Candida palrapsilosis* ZJPH1305.

[0195] (1) *Candida palrapsilosis* ZJPH1305, deposited at the China Center for Type Culture Collection (CCTCC) on November 8, 2013, at Wuhan University, Wuhan, China, accession number CCTCC NO: M2013559. This strain has been disclosed in a previous patent application (Publication No.: CN103849574A, Publication Date: June 11, 2014). The culture method of the strain and the preparation process of the enzyme source cells are based on the previous patent application (Publication No.: CN103849574A, Publication Date: June 11, 2014).

[0196] (2) Biocatalytic preparation of key chiral intermediate (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol for erlotinib ethyl ester

[0197] (3) The wet cell mass of *Candida palrapsilosis* ZJPH1305, prepared by fermentation, was suspended in 10 mL of potassium phosphate buffer (pH 7.0, 0.1 M). 1.0 g of glucose was used as an auxiliary substrate, and 15 mg of substrate 1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol was added to form the transformation system. The wet cell mass was 1.0 g by wet weight. The system was incubated at 30 °C and 200 rpm for 24 h. The product (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol was detected using the high-performance liquid chromatography method described in Example 1, and the yield and ee value of the product were calculated.

[0198] Conclusion: Candida palrapsilosis ZJPH1305 cannot catalyze the yield of (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol from 1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol.

[0199] Example 21: Conversion capacity study of (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol as a key chiral intermediate for the biocatalytic preparation of teroseltate from Candida tropicalis 104.

[0200] (1) *Candida tropicalis* 104, deposited at the China Center for Type Culture Collection (CCTCC) on November 8, 2013, Wuhan University, Wuhan, China, accession number: CCTCC No: M 209034, deposited on February 27, 2009. This strain was previously disclosed in a patent application (publication number: CN 101519674A, publication date: September 2, 2009). The culture method and enzyme source cell preparation process of the strain are based on the previous patent application (publication number: CN 101519674A, publication date: September 2, 2009).

[0201] (2) Biocatalytic preparation of key chiral intermediate (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol for erlotinib ethyl ester

[0202] The wet cell mass of *Candida tropicalis* 104, prepared by fermentation, was suspended in 10 mL of potassium phosphate buffer (pH 7.0, 0.1 M). 1.0 g of glucose was used as an auxiliary substrate, and 15 mg of 1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol was added to form the transformation system. The wet cell mass was 1.0 g by wet weight. The system was incubated at 30 °C and 200 rpm for 24 h in a shaker. The product (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol was detected using the high-performance liquid chromatography method described in Example 1, and the yield and ee value of the product (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol were calculated.

[0203] Conclusion: Candida tropicalis 104 cannot catalyze the yield of (R)-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol from 1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol.

Claims

1. A method for the biopreparation of a chiral intermediate of erlotinib ethyl ester, characterized in that, The method is as follows: using Saturn Rhizome Yeast ( Cyberlindnera saturnus Using wet cells obtained from fermentation culture of ZJPH1807 as a catalyst, and 1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethyl ketone as a substrate, with the addition of an auxiliary substrate, and phosphate buffer solution with a pH of 6.0-8.0 as the reaction medium, the transformation system was constructed. The transformation reaction was carried out at 25-50℃ and 150-250 rpm for 4-48 h. After the reaction was completed, an equal volume of n-hexane was added to the transformation solution to terminate the reaction and extract. After centrifugation, the supernatant was collected to obtain the n-hexane extract containing the chiral intermediate of erlotinib ethyl ester. The n-hexane extract was separated and purified to obtain the chiral intermediate of erlotinib ethyl ester, namely ( R )-1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethanol; the auxiliary substrate is one of the following: glucose, maltose, sucrose, ethanol, isopropanol, glycerol, L-cysteine, L-alanine, L-glutamic acid or L-lysine; The amount of wet bacterial cells used is 50–500 g / L based on the volume of the phosphate buffer, the amount of substrate used is 0.5–6 g / L based on the volume of the phosphate buffer, and the amount of auxiliary substrate added is 4–200 g / L based on the volume of the phosphate buffer.

2. The method for biopreparing the chiral intermediate of erlotinib ethyl ester as described in claim 1, characterized in that, The phosphate buffer solution is a 0.1 M, pH 7.5 phosphate buffer solution.

3. The method for biopreparing the chiral intermediate of erlotinib ethyl ester as described in claim 1, characterized in that, The reaction system also contains a surfactant, which is Tween 20, Tween 60, Tween 80, Span 20, Span 60, Span 80, Triton X-100, or Triton X-114; the amount of surfactant added is 2-20 g / L based on the volume of the buffer solution.

4. The method for biopreparing the chiral intermediate of erlotinib ethyl ester as described in claim 1, characterized in that, An organic solvent is also added to the reaction system. The organic solvent is dimethyl sulfoxide, N,N-dimethylformamide, methanol, ethanol, isopropanol, toluene, or cyclohexane. The volume of the organic solvent is 1-7% of the volume of the buffer solution.

5. The method for biopreparing the chiral intermediate of erlotinib ethyl ester as described in claim 1, characterized in that, The reaction system also contains cyclodextrin, including α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, methyl-β-cyclodextrin, (2-hydroxypropyl)-β-cyclodextrin, or (2-hydroxypropyl)-γ-cyclodextrin; the amount of cyclodextrin added is 10-150 g / L based on the volume of the buffer solution.

6. The method for biopreparing the chiral intermediate of erlotinib ethyl ester as described in claim 1, characterized in that, The reaction system also contains a eutectic solvent, which includes choline chloride / alanine, choline chloride / isopropanol, choline chloride / glycine, choline chloride / fructose, choline chloride / trehalose, carnitine / alanine, carnitine / isopropanol, carnitine / glycine, carnitine / fructose, and carnitine / trehalose; the amount of the eutectic solvent added is 0.5-2.5 g / L based on the volume of the buffer solution.

7. The method for biopreparing the chiral intermediate of erlotinib ethyl ester as described in any one of claims 1-6, characterized in that, The biopreparation method of the chiral intermediate of teroseltartine ethyl ester is carried out according to the following steps: Wet cells obtained by fermentation culture of *Saccharomyces cerevisiae* ZJPH1807 are used as catalysts; 1-[4-chloro-2-(3-methyl-1H-pyrazol-1-yl)phenyl]-2,2,2-trifluoroethyl ketone is used as a substrate; auxiliary substrates, surfactants, organic solvents, cyclodextrin, and eutectic solvents are added; and a phosphate buffer solution with a pH of 6.0-8.0 is used as the reaction medium to form the transformation system. The transformation reaction is carried out at 25-50℃ and 150-250 rpm for 4-48 minutes. h. After the reaction is complete, an equal volume of n-hexane is added to the conversion solution to terminate the reaction and extract. After centrifugation, the supernatant is collected to obtain the n-hexane extract containing the chiral intermediate of erlotinib ethyl ester. The n-hexane extract is then separated and purified to obtain the chiral intermediate of erlotinib ethyl ester. The auxiliary substrate is glucose, the surfactant is Tween 60, the organic solvent is methanol, the cyclodextrin is β-cyclodextrin, and the eutectic solvent is carnitine / isopropanol.

8. The method for biopreparing the chiral intermediate of erlotinib ethyl ester as described in claim 1, characterized in that, The wet bacterial cells were prepared according to the following method: (1) Plate culture: Select Saturn Rhizoma Saccharomyces cerevisiae ( Cyberlindnera saturnus ZJPH1807 single colonies were inoculated into agar plates and incubated at 30 °C for 2 days for the first activation. Single colonies were then picked from the first activated plates and inoculated into agar plates again for a second activation at 30 °C for 2 days. The final concentrations of the components in the agar plates were as follows: glucose 15 g / L, peptone 20 g / L, yeast extract 10 g / L, (NH4)2SO4 2 g / L, KH2PO4 2 g / L, NaCl 1 g / L, MgSO4·7H2O 0.5 g / L, agar 20 g / L, water as solvent, pH 6.

5. (2) Seed culture: Pick a loopful of bacterial cells from the plate activated for the second time in step (1) and inoculate it into the seed culture medium. Culture at 30℃ and 200 rpm for 12 h to obtain seed liquid. The final concentration of each component in the seed culture medium is as follows: glucose 15-50 g / L, peptone 5-15 g / L, yeast extract 5-15 g / L, (NH4)2SO4 1-5 g / L, KH2PO4 0.2-1.2 g / L, NaCl 0.2-1.2 g / L, MgSO4·7H2O 0.1-1.0 g / L, solvent is water, pH 5-8. (3) Fermentation culture: Inoculate the seed culture into the fermentation medium at an inoculum concentration of 6-12% (v / v), and culture at 30 ℃ and 200 rpm for 10-48 h to obtain the fermentation broth. Centrifuge the fermentation broth, and wash the resulting precipitate with 0.1 M K2HPO4-KH2PO4 buffer solution at pH 7.

5. After centrifugation, obtain wet cells. The final concentration composition of each component in the fermentation medium is as follows: carbon source 15-25 g / L, nitrogen source 15-25 g / L, KH2PO4 0.2-1.2 g / L, MgCl2·6H2O 0.2-1.0 g / L, solvent is water, pH 6.0-9.

0. The carbon source is one of the following: glucose, maltose, glycerol, lactose, sucrose or dextrin. The nitrogen source is one of the following: yeast extract, peptone, beef extract, ammonium chloride or ammonium sulfate.

9. The method for biopreparing the chiral intermediate of erlotinib ethyl ester as described in claim 8, characterized in that, The final concentrations of the components in the fermentation medium are as follows: glucose 15 g / L, ammonium sulfate 20 g / L, KH2PO4 1.0 g / L, MgCl2·6H2O 0.5 g / L, with water as the solvent and pH 7.5.

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