WO2024078646A1 - Method for preparing (r)-tebuconazole by means of enzyme chemical process - Google Patents

Method for preparing (r)-tebuconazole by means of enzyme chemical process Download PDF

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WO2024078646A1
WO2024078646A1 PCT/CN2024/071076 CN2024071076W WO2024078646A1 WO 2024078646 A1 WO2024078646 A1 WO 2024078646A1 CN 2024071076 W CN2024071076 W CN 2024071076W WO 2024078646 A1 WO2024078646 A1 WO 2024078646A1
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tebuconazole
reaction
preparing
rpeh
epoxy intermediate
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胡蝶
何玉财
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常州大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
    • C07D249/02—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms not condensed with other rings
    • C07D249/08—1,2,4-Triazoles; Hydrogenated 1,2,4-triazoles
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the invention relates to a method for preparing (R)-tebuconazole by enzyme chemistry, belonging to the technical field of biochemical engineering.
  • Triazole fungicides are one of the most widely used fungicides in the world. 84% of triazole fungicides are chiral pesticides. Except for diniconazole and diniconazole, most triazole chiral fungicides exist in the form of racemates. Studies have found that single chiral pesticides have high efficacy, low dosage, less waste, safer for crops and environmental ecology, and relatively lower cost. Therefore, developing more high-efficiency and low-toxic single enantiomer pesticide products is of great significance for reducing pesticide residues, protecting the environment, and maintaining human health.
  • Tebuconazole is a typical chiral triazole fungicide that effectively controls a variety of fungal diseases on crops by inhibiting the synthesis of fungal ergosterol. It has the advantages of a broad fungicidal spectrum, high efficiency, low toxicity and long-lasting effect. It is widely used for seed pretreatment or foliar spraying of important crops such as grasses, fruits and vegetables. A large number of studies have shown that there are obvious stereoselective differences in the target biological activity, ecotoxicity and environmental enrichment of tebuconazole enantiomers.
  • the activity of (R)-tebuconazole is more than 100 times that of (S)-tebuconazole, but (S)-tebuconazole has a significant role in regulating plant growth (Stehmann C. et al.
  • the preparation of single enantiomers of tebuconazole is mainly based on the high performance liquid chromatography asymmetric resolution method, but this method has the disadvantages of high cost and low yield, making it difficult to achieve industrial production and large-scale application.
  • Chiral epoxy intermediate-mediated asymmetric catalysis is a promising approach to obtain optically active monomers of some triazole fungicides.
  • chiral epoxy intermediates with large steric hindrance groups make it difficult to achieve high stereoselective synthesis by chemical and biological methods. So far, there has been no report on the chemical or biological asymmetric synthesis of chiral tebuconazole epoxy intermediates with large steric hindrance groups.
  • Chinese patent document (application number 201811621721.9), which discloses an epoxide hydrolase derived from marine red yeast, and the epoxide hydrolase is used for biocatalytic preparation of (R)-phenylethylene glycol from epoxybenzene.
  • the present invention uses it to prepare (R)-tebuconazole, thereby providing a new method for preparing (R)-tebuconazole by enzymatic chemistry, which is green and efficient.
  • the present invention utilizes the recombinant bacteria E. coli/Rpeh whole cells derived from Rhodotorula parudigensis epoxide hydrolase (which is the marine red yeast-derived epoxide hydrolase RpEH1 in the Chinese patent document with application number 201811621721.9) as a biocatalyst to asymmetrically resolve racemic tebuconazole to prepare a highly optically pure (R)-tebuconazole epoxide intermediate, and then uses solid base to catalyze the ring-opening reaction of the (R)-tebuconazole epoxide intermediate with 1,2,4-triazole to prepare (R)-tebuconazole.
  • Rhodotorula parudigensis epoxide hydrolase which is the marine red yeast-derived epoxide hydrolase RpEH1 in the Chinese patent document with application number 201811621721.9
  • the concentration of the recombinant genetically engineered bacteria E. coli/Rpeh in the reaction system is 5 to 200 mg/mL.
  • step 1 of the present invention the usage ratio of the racemic tebuconazole epoxy intermediate substrate and the recombinant genetically engineered bacteria E. coli/Rpeh is 2:1 to 10:1 (mM:mg/mL).
  • the pH of the reaction system is 5.5 to 9.0, preferably 6.5 to 8.5, and more preferably 7.0 to 8.0.
  • the reaction temperature is 20 to 50°C, preferably 20 to 40°C, and more preferably 30 to 35°C.
  • the concentration of racemic tebuconazole epoxy intermediate in the reaction system is 20 to 200 mM.
  • the organic solvent extractant is preferably any one of ethyl acetate, methyl acetate, and dichloromethane, or a mixture of several of them.
  • the organic solvent is preferably any one or more of n-butanol, toluene and dichloromethane.
  • the volume of the organic solvent is such that the molar concentration of the (R)-tebuconazole epoxy intermediate is 100-200 mM.
  • the solid base is preferably any one or more of potassium hydroxide, sodium hydroxide, potassium carbonate or sodium tert-butoxide.
  • step 2 of the present invention the molar ratio of 1,2,4-triazole:solid base:(R)-tebuconazole epoxy intermediate is 4 to 1:1:1.
  • step 2 of the present invention the reaction temperature is 110-140°C.
  • step 2 of the present invention the reaction time is 4 to 24 hours.
  • the present invention also claims protection for: application of the method in the fields of chiral pesticides, chiral drugs, etc.
  • the present invention uses the recombinant bacteria E. coli/Rpeh whole cells derived from the Rhodotorula parudygensis epoxide hydrolase as a catalyst to asymmetric split the racemic tebuconazole epoxide intermediate, successfully obtains the (R)-tebuconazole epoxide intermediate, and then uses solid base to catalyze the ring-opening reaction of the (R)-tebuconazole epoxide intermediate with 1,2,4-triazole to prepare a high-purity (R)-tebuconazole enantiomer.
  • the method disclosed in the present invention provides a new method for preparing (R)-tebuconazole by a green and effective enzymatic chemistry method. Compared with the expensive chiral chromatography separation method, it has the advantages of simpler preparation process, more environmentally friendly, lower production cost, etc., and has broad application prospects.
  • Fig. 1 Effect of pH on the activity and stability of the recombinant E. coli/Rpeh enzyme
  • Figure 5 The effect of different substrate concentrations on the resolution of racemic tebuconazole epoxide intermediates by recombinant bacteria E. coli/Rpeh;
  • FIG6 1 H NMR spectrum of (R)-tebuconazole.
  • Chiral gas chromatography conditions 7820B Agilent gas chromatograph, flame ionization detector, injection port The temperature of the detection port was 250°C, and the temperature was programmed to rise from 100°C to 220°C at 5°C/min, and the temperature was kept isothermal for 2min; the chiral gas chromatography column was CYCLOSIL-B (30m ⁇ 0.25mm ⁇ 0.25 ⁇ m). The retention times of (S)- and (R)-tebuconazole epoxy intermediates were 20.835 and 20.920min, respectively.
  • E.coli/Rpeh add 50 ⁇ L 50mg/mL E.coli/Rpeh bacterial suspension (to a final concentration of 5mg/mL wet bacteria) and 400 ⁇ L potassium phosphate buffer (100mM, pH 7.0) to a 1.5mL EP tube, and preheat at 25°C for 5min; add 50 ⁇ L 200mM racemic tebuconazole epoxy intermediate (to a final concentration of 20mM), react for 10min, take 100 ⁇ L and add it to 900 ⁇ L methanol, mix well, pass through a 0.22 ⁇ m organic membrane, and perform reversed-phase HPLC chromatography analysis.
  • enzyme activity unit Under this assay condition, the amount of wet bacteria required to consume 1 ⁇ mol of tebuconazole epoxy intermediate per minute is defined as 1 epoxide hydrolase activity unit (U).
  • U epoxide hydrolase activity unit
  • the gene sequence of the recombinant bacteria E. coli/Rpeh in the following examples is the genetically engineered bacteria shown in SEQ ID NO.2 in the Chinese patent document with application number 201811621721.9.
  • Example 1 Optimal pH and pH stability of racemic tebuconazole epoxide intermediate catalyzed by recombinant E. coli/Rpeh
  • Optimum pH determination Take 50 ⁇ L of 200 mM substrate tebuconazole epoxy intermediate (to a final concentration of 5 mg/mL wet bacteria) were added with 450 ⁇ L potassium phosphate buffer of different pH values (100 mM, pH 5.5-9.0), and preheated at 25°C for 5 min; 50 ⁇ L of E. coli/Rpeh bacterial suspension incubated with different pH values was added, and after reacting for 10 min, 100 ⁇ L was added to 900 ⁇ L methanol and mixed, and the mixture was passed through a 0.22 ⁇ m organic membrane and subjected to reverse phase HPLC chromatography to determine the specific activity of the recombinant bacteria E. coli/Rpeh.
  • pH stability determination take 100 ⁇ L 50 mg/mL E. coli/Rpeh bacterial suspension and incubate it in potassium phosphate buffer with different pH values (20 mM, pH 5.5-9.0) for 1 hour; immediately aspirate 50 ⁇ L of E.
  • coli/Rpeh bacterial suspension incubated with different pH values and add it to 450 ⁇ L potassium phosphate buffer (100 mM, pH 7.0), preheat at 25 °C for 5 minutes; add 50 ⁇ L 200 mM substrate tebuconazole epoxide intermediate (to a final concentration of 5 mg/mL wet bacteria), react for 10 minutes, take 100 ⁇ L and add it to 900 ⁇ L methanol, mix well, pass through a 0.22 ⁇ m organic membrane, and perform reverse phase HPLC chromatography analysis to determine the residual specific activity of the recombinant bacteria E. coli/Rpeh, and define the specific activity of the non-recombinant bacteria E. coli/Rpeh as 100% to calculate the relative enzyme activity.
  • Optimal reaction stability determination add 50 ⁇ L of 200 mM racemic tebuconazole epoxy intermediate (to a final concentration of 20 mM) and 450 ⁇ L of potassium phosphate buffer (100 mM, pH 7.5) to a 1.5 mL EP tube, preheat at 20-50 ° C for 5 min; add 50 ⁇ L of E. coli / Rpeh bacterial suspension (to a final concentration of 5 mg / mL wet bacteria), react at different temperatures of 20-50 ° C for 10 min, take 100 ⁇ L and add 900 ⁇ L of methanol to mix, pass through a 0.22 ⁇ m organic membrane, and perform reversed-phase HPLC chromatography to determine the initial stability of the recombinant bacteria E. coli / Rpeh reaction speed.
  • Temperature stability determination take 100 ⁇ L 50 mg/mL E. coli/Rpeh bacterial suspension and incubate it in a water bath at 20-50°C for 1 hour, then cool it in an ice bath; add 50 ⁇ L of E. coli/Rpeh bacterial suspension incubated at different temperatures (to a final concentration of 5 mg/mL wet bacteria) and 450 ⁇ L potassium phosphate buffer (100 mM, pH 7.5) into a 1.5 mL EP tube, and preheat at 25°C for 5 minutes; add 50 ⁇ L of 200 mM racemic tebuconazole epoxide intermediate (to a final concentration of 20 mM), react for 10 minutes, take 100 ⁇ L and add it to 900 ⁇ L methanol, mix well, pass through a 0.22 ⁇ m organic membrane, and perform reverse-phase HPLC chromatography analysis to determine the residual specific activity of the recombinant bacteria E. coli/Rpeh after incubation, and define the specific activity
  • the optimal reaction temperature of the recombinant bacteria E.coli/Rpeh was 30°C, and it had high catalytic activity in the range of 25-35°C, with a relative enzyme activity of more than 80%.
  • the optimal temperature of 30°C and the optimal pH of 7.5 the specific activity of the E.coli/Rpeh bacterial suspension was 84% higher than that of the initial wet bacteria.
  • the recombinant bacteria E.coli/Rpeh had high thermal stability below 30°C, with a residual relative enzyme activity of >98%. Therefore, 30°C was selected as the optimal reaction temperature in the subsequent catalytic reaction (Figure 2).
  • Example 4 Effect of different concentrations of recombinant bacteria E. coli/Rpeh on the resolution of racemic tebuconazole epoxy intermediate
  • Example 5 Effect of different substrate concentrations on the resolution of racemic tebuconazole epoxy intermediate by recombinant E. coli/Rpeh
  • the concentration of E. coli/Rpeh recombinant bacteria was 200 mg/mL, and the concentrations of racemic tebuconazole epoxide intermediates were 100, 200, 300, 400 and 500 mM, respectively.
  • the reaction was oscillated at 30 °C for 12 h. An appropriate amount of the reaction solution was extracted and quenched in 1 mL of ethyl acetate. The solution was dried over a 0.22 ⁇ m organic film and analyzed by chiral gas chromatography.
  • the recombinant bacteria E. coli/Rpeh catalyzed the conversion of tebuconazole epoxide intermediates with different substrate concentrations (100, 200, 300, 400 and 500 mM, respectively) at 30°C for 12 h, and the enantiomeric excess ee values of the retained (R)-tebuconazole epoxide intermediates were 90.0%, 88.9%, 79.6%, 74.5% and 61.6%, respectively. Therefore, the recombinant bacteria E.
  • 1,2,4-triazole 37.7 mg, 0.55 mmol
  • KOH 10 mg, 0.18 mmol
  • the reaction mixture was heated to 110 ° C for 2 h, and then the (R)-tebuconazole epoxy intermediate (50 mg, 0.18 mmol) prepared above was added, the reaction mixture was heated to 133 ° C, stirred for 4 h, and then cooled to room temperature.
  • the organic layer was washed with water (10 mL ⁇ 3) until the solution was neutral, dried with anhydrous MgSO 4 , filtered, and concentrated in vacuo.

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Abstract

A method for preparing (R)-tebuconazole by means of an enzyme chemical process, comprising: firstly, by taking the whole cell of recombinant bacterium E. coli/Rpeh expressing an epoxide hydrolase as a catalyst, resolving a racemic tebuconazole epoxy intermediate in a mild reaction system to prepare the (R)-tebuconazole epoxy intermediate, wherein the ee value is 92.7% and the yield is 44.0%; and performing a 1,2,4-triazole mediated ring-opening reaction on the (R)-tebuconazole epoxy intermediate under the catalysis of a solid base to prepare (R)-tebuconazole with a high enantiomer purity (＞99% ee).

Description

一种酶化学法制备(R)-戊唑醇的方法A method for preparing (R)-tebuconazole by enzyme chemistry 技术领域Technical Field
本发明涉及一种酶化学法制备(R)-戊唑醇的方法，属于生物化工技术领域。The invention relates to a method for preparing (R)-tebuconazole by enzyme chemistry, belonging to the technical field of biochemical engineering.
背景技术Background technique
三唑类杀菌剂是目前世界上应用最广泛的杀菌剂之一，84％的三唑类杀菌剂属于手性农药，除烯唑醇和烯效唑外，多数三唑类手性杀菌剂均以外消旋体的形式存在，研究发现，单一手性农药的药效高、用药量省、三废少，对农作物和环境生态更安全，相对成本更低。因此，开发出更多的高效低毒的单一对映体的农药产品，对于降低农药残留，保护环境，维护人类身体健康具有重要意义。Triazole fungicides are one of the most widely used fungicides in the world. 84% of triazole fungicides are chiral pesticides. Except for diniconazole and diniconazole, most triazole chiral fungicides exist in the form of racemates. Studies have found that single chiral pesticides have high efficacy, low dosage, less waste, safer for crops and environmental ecology, and relatively lower cost. Therefore, developing more high-efficiency and low-toxic single enantiomer pesticide products is of great significance for reducing pesticide residues, protecting the environment, and maintaining human health.
戊唑醇为典型手性三唑类杀菌剂，通过抑制真菌麦角甾醇合成有效控制作物上的多种真菌病害，具有杀菌谱广、高效低毒和持效期久等优点，被广泛用于禾本科、果类和蔬菜等重要农作物的种子预处理或叶面喷洒。大量研究表明，戊唑醇对映异构体的靶标生物活性，生态毒性和环境富集等方面存在着明显的立体选择性差异。例如，(R)-戊唑醇的活性是(S)-戊唑醇活性的100多倍，但是(S)-戊唑醇拥有显著的调节植物生长的作用(Stehmann C.et al.Pestic Sci,1995,44:183–195)；(R)-戊唑醇在生菜中的降解快于其对映体(赵刘清,2021,硕士论文，中国农业科学院)；(R)-戊唑醇在兔子脑和心脏部位降解更快(Zhu et al.,2007)，拟南芥中(R)-戊唑醇优先降解，而(S)-戊唑醇在拟南芥中被富集。目前，戊唑醇单一对映体的制备主要为高效液相色谱不对称拆分法，但该方法存在成本高昂和产量低等缺陷，难以实现产业化生产和大规模应用。Tebuconazole is a typical chiral triazole fungicide that effectively controls a variety of fungal diseases on crops by inhibiting the synthesis of fungal ergosterol. It has the advantages of a broad fungicidal spectrum, high efficiency, low toxicity and long-lasting effect. It is widely used for seed pretreatment or foliar spraying of important crops such as grasses, fruits and vegetables. A large number of studies have shown that there are obvious stereoselective differences in the target biological activity, ecotoxicity and environmental enrichment of tebuconazole enantiomers. For example, the activity of (R)-tebuconazole is more than 100 times that of (S)-tebuconazole, but (S)-tebuconazole has a significant role in regulating plant growth (Stehmann C. et al. Pestic Sci, 1995, 44: 183–195); (R)-tebuconazole is degraded faster in lettuce than its enantiomer (Zhao Liuqing, 2021, Master's thesis, Chinese Academy of Agricultural Sciences); (R)-tebuconazole is degraded faster in rabbit brain and heart (Zhu et al., 2007), (R)-tebuconazole is preferentially degraded in Arabidopsis, while (S)-tebuconazole is enriched in Arabidopsis. At present, the preparation of single enantiomers of tebuconazole is mainly based on the high performance liquid chromatography asymmetric resolution method, but this method has the disadvantages of high cost and low yield, making it difficult to achieve industrial production and large-scale application.
手性环氧中间体介导的不对称催化法是获取部分三唑类杀菌剂光学活性单 体的最有效途径之一，但具有大位阻基团的手性环氧中间体(例如戊唑醇环氧中间体)导致化学和生物法均难以实现其高立体选择性合成，迄今为止尚未见具有大位阻基团的手性戊唑醇环氧中间体的化学或生物法不对称合成的报道。中国专利文献(申请号为201811621721.9)，其中公开了一种海洋红酵母来源的环氧化物水解酶，并将该环氧水解酶用于生物催化环氧苯乙烷制备(R)-苯乙二醇。为了拓展这种环氧化物水解酶的应用，本发明将其用于制备(R)-戊唑醇，进而提供一种绿色、高效的酶化学法制备(R)-戊唑醇的新方法。Chiral epoxy intermediate-mediated asymmetric catalysis is a promising approach to obtain optically active monomers of some triazole fungicides. However, chiral epoxy intermediates with large steric hindrance groups (such as tebuconazole epoxy intermediates) make it difficult to achieve high stereoselective synthesis by chemical and biological methods. So far, there has been no report on the chemical or biological asymmetric synthesis of chiral tebuconazole epoxy intermediates with large steric hindrance groups. Chinese patent document (application number 201811621721.9), which discloses an epoxide hydrolase derived from marine red yeast, and the epoxide hydrolase is used for biocatalytic preparation of (R)-phenylethylene glycol from epoxybenzene. In order to expand the application of this epoxide hydrolase, the present invention uses it to prepare (R)-tebuconazole, thereby providing a new method for preparing (R)-tebuconazole by enzymatic chemistry, which is green and efficient.
发明内容Summary of the invention
本发明利用来源于帕鲁迪根红酵母环氧水解酶的重组菌E.coli/Rpeh全细胞(为申请号为201811621721.9的中国专利文献中的海洋红酵母来源的环氧化物水解酶RpEH1)作为生物催化剂，不对称拆分外消旋戊唑醇制备高光学纯的(R)-戊唑醇环氧中间体，再经固体碱催化1,2,4-三氮唑对(R)-戊唑醇环氧中间体开环反应制备(R)-戊唑醇，相较于昂贵的手性色谱分离法，其制备工艺更简单、环境更友好、生产成本更低等优势，具有广阔的应用前景。The present invention utilizes the recombinant bacteria E. coli/Rpeh whole cells derived from Rhodotorula parudigensis epoxide hydrolase (which is the marine red yeast-derived epoxide hydrolase RpEH1 in the Chinese patent document with application number 201811621721.9) as a biocatalyst to asymmetrically resolve racemic tebuconazole to prepare a highly optically pure (R)-tebuconazole epoxide intermediate, and then uses solid base to catalyze the ring-opening reaction of the (R)-tebuconazole epoxide intermediate with 1,2,4-triazole to prepare (R)-tebuconazole. Compared with the expensive chiral chromatography separation method, the preparation process is simpler, more environmentally friendly, and has lower production costs, and has broad application prospects.
为了实现本发明目的采用的技术方案为：利用酶化学法制备(R)-戊唑醇的方法，包括如下步骤：In order to achieve the purpose of the present invention, the technical solution adopted is: a method for preparing (R)-tebuconazole by enzymatic chemistry, comprising the following steps:
(1)酶法制备(R)-戊唑醇环氧中间体：利用含有表达重组帕鲁迪根红酵母环氧水解酶的重组菌基因工程菌E.coli/Rpeh，加入外消旋戊唑醇环氧中间体，在反应温度下，恒温震荡反应，反应结束后加入有机溶剂萃取剂后，充分震荡混匀，收集上清液，旋蒸后获得酶法制备的(R)-戊唑醇环氧中间体和相应(S)-邻二醇的混合物；(1) Enzymatic preparation of (R)-tebuconazole epoxy intermediate: using a recombinant genetically engineered bacterium E. coli/Rpeh containing recombinant Rhodotorula parudygensis epoxide hydrolase, adding racemic tebuconazole epoxy intermediate, and subjecting the mixture to constant temperature shaking reaction at the reaction temperature. After the reaction is completed, an organic solvent extractant is added, and the mixture is thoroughly shaken and mixed. The supernatant is collected, and rotary evaporation is performed to obtain a mixture of the enzymatically prepared (R)-tebuconazole epoxy intermediate and the corresponding (S)-vicinal diol;
(2)化学法催化(R)-戊唑醇环氧中间体开环制备(R)-戊唑醇：在有机溶剂中加入1,2,4-三氮唑和固体碱，加入酶法制备的(R)-戊唑醇环氧中间体和相应(S)-邻二 醇的混合物，加热反应，反应结束后将反应液冷却至室温，有机层用水洗涤至溶液呈中性，有机相经除水、过滤、旋蒸，所得粗制残留物通过硅胶层析柱纯化，获得白色固体状高光学纯度的(R)-戊唑醇。(2) Chemical catalytic ring-opening of (R)-tebuconazole epoxy intermediate to prepare (R)-tebuconazole: 1,2,4-triazole and solid base are added to an organic solvent, and the (R)-tebuconazole epoxy intermediate prepared by the enzymatic method and the corresponding (S)-dihydrotriazole are added to the organic solvent. The mixture of alcohols was heated for reaction. After the reaction was completed, the reaction solution was cooled to room temperature, and the organic layer was washed with water until the solution was neutral. The organic phase was dehydrated, filtered, and rotary evaporated. The obtained crude residue was purified by silica gel chromatography to obtain (R)-tebuconazole with high optical purity as a white solid.
进一步的，在本发明的步骤1中，反应体系中重组菌基因工程菌E.coli/Rpeh浓度为5～200mg/mL。Furthermore, in step 1 of the present invention, the concentration of the recombinant genetically engineered bacteria E. coli/Rpeh in the reaction system is 5 to 200 mg/mL.
进一步的，在本发明的步骤1中，外消旋戊唑醇环氧中间体底物与重组菌基因工程菌E.coli/Rpeh的用量比例为2:1～10:1(mM:mg/mL)。Furthermore, in step 1 of the present invention, the usage ratio of the racemic tebuconazole epoxy intermediate substrate and the recombinant genetically engineered bacteria E. coli/Rpeh is 2:1 to 10:1 (mM:mg/mL).
进一步的，在本发明的步骤1中，反应体系的pH为5.5～9.0，优选6.5～8.5，更优选7.0～8.0。Furthermore, in step 1 of the present invention, the pH of the reaction system is 5.5 to 9.0, preferably 6.5 to 8.5, and more preferably 7.0 to 8.0.
进一步的，在本发明的步骤1中，反应温度为20～50℃，优选20～40℃，更优选30～35℃。Furthermore, in step 1 of the present invention, the reaction temperature is 20 to 50°C, preferably 20 to 40°C, and more preferably 30 to 35°C.
进一步的，在本发明的步骤1中，反应体系中外消旋戊唑醇环氧中间体浓度为20～200mM。Furthermore, in step 1 of the present invention, the concentration of racemic tebuconazole epoxy intermediate in the reaction system is 20 to 200 mM.
进一步的，在本发明的步骤1中，有机溶剂萃取剂优选为乙酸乙酯、乙酸甲酯、二氯甲烷中的任意一种或几种的混合液。Furthermore, in step 1 of the present invention, the organic solvent extractant is preferably any one of ethyl acetate, methyl acetate, and dichloromethane, or a mixture of several of them.
进一步的，在本发明的步骤2中，有机溶剂优选为正丁醇、甲苯、二氯甲烷中的任意一种或几种。Furthermore, in step 2 of the present invention, the organic solvent is preferably any one or more of n-butanol, toluene and dichloromethane.
进一步的，在本发明的步骤2中，有机溶剂体积量是使(R)-戊唑醇环氧中间体的摩尔浓度为100～200mM。Furthermore, in step 2 of the present invention, the volume of the organic solvent is such that the molar concentration of the (R)-tebuconazole epoxy intermediate is 100-200 mM.
进一步的，在本发明的步骤2中，固体碱优选为氢氧化钾、氢氧化钠、碳酸钾或叔丁醇钠中的任意一种或几种。Furthermore, in step 2 of the present invention, the solid base is preferably any one or more of potassium hydroxide, sodium hydroxide, potassium carbonate or sodium tert-butoxide.
进一步的，在本发明的步骤2中，1,2,4-三氮唑:固体碱:(R)-戊唑醇环氧中间体的摩尔比为4～1:1:1。 Furthermore, in step 2 of the present invention, the molar ratio of 1,2,4-triazole:solid base:(R)-tebuconazole epoxy intermediate is 4 to 1:1:1.
进一步的，在本发明的步骤2中，反应温度为110～140℃。Furthermore, in step 2 of the present invention, the reaction temperature is 110-140°C.
进一步的，在本发明的步骤2中，反应时间为4～24h。Furthermore, in step 2 of the present invention, the reaction time is 4 to 24 hours.
本发明还要求保护：所述方法在手性农药、手性药物等领域的应用。The present invention also claims protection for: application of the method in the fields of chiral pesticides, chiral drugs, etc.
本发明的有益效果：本发明利用来源于帕鲁迪根红酵母环氧水解酶的重组菌E.coli/Rpeh全细胞作为催化剂不对称拆分外消旋戊唑醇环氧中间体，成功获得(R)-戊唑醇环氧中间体，在经固体碱催化1,2,4-三氮唑对(R)-戊唑醇环氧中间体开环反应，制备高纯度(R)-戊唑醇对映体。本发明公开的方法提供一种绿色、有效的酶化学法制备(R)-戊唑醇的新方法，相较于昂贵的手性色谱分离法，其制备工艺更简单、环境更友好、生产成本更低等优势，具有广阔的应用前景。Beneficial effects of the present invention: The present invention uses the recombinant bacteria E. coli/Rpeh whole cells derived from the Rhodotorula parudygensis epoxide hydrolase as a catalyst to asymmetric split the racemic tebuconazole epoxide intermediate, successfully obtains the (R)-tebuconazole epoxide intermediate, and then uses solid base to catalyze the ring-opening reaction of the (R)-tebuconazole epoxide intermediate with 1,2,4-triazole to prepare a high-purity (R)-tebuconazole enantiomer. The method disclosed in the present invention provides a new method for preparing (R)-tebuconazole by a green and effective enzymatic chemistry method. Compared with the expensive chiral chromatography separation method, it has the advantages of simpler preparation process, more environmentally friendly, lower production cost, etc., and has broad application prospects.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1pH对重组菌E.coli/Rpeh酶活力和稳定性的影响；Fig. 1 Effect of pH on the activity and stability of the recombinant E. coli/Rpeh enzyme;
图2温度对重组菌E.coli/Rpeh酶活力和稳定性的影响；Figure 2 Effect of temperature on enzyme activity and stability of recombinant E. coli/Rpeh;
图3重组菌E.coli/Rpeh拆分外消旋戊唑醇环氧中间体的反应进程；Figure 3 Reaction process of recombinant bacteria E. coli/Rpeh in resolving racemic tebuconazole epoxide intermediate;
图4不同浓度重组菌E.coli/Rpeh对外消旋戊唑醇环氧中间体拆分的影响；Figure 4 Effects of different concentrations of recombinant bacteria E. coli/Rpeh on the resolution of racemic tebuconazole epoxy intermediates;
图5重组菌E.coli/Rpeh拆分外消旋戊唑醇环氧中间体不同底物浓度的影响；Figure 5 The effect of different substrate concentrations on the resolution of racemic tebuconazole epoxide intermediates by recombinant bacteria E. coli/Rpeh;
图6(R)-戊唑醇的1H NMR核磁图。FIG6 1 H NMR spectrum of (R)-tebuconazole.
具体实施方式Detailed ways
本发明不局限于下列具体实施方式，本领域一般技术人员根据本发明公开的内容，可以采用其他多种具体实施方式实施本发明的，或者凡是采用本发明的设计结构和思路，做简单变化或更改的，都落入本发明的保护范围。需要说明的是，在不冲突的情况下，本发明中的实施例及实施例中的特征可以相互组合。The present invention is not limited to the following specific embodiments. A person skilled in the art can implement the present invention in various other specific embodiments according to the contents disclosed in the present invention, or any design structure and idea of the present invention with simple changes or modifications shall fall within the protection scope of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention can be combined with each other without conflict.
手性气相色谱条件：7820B安捷伦气相色谱仪，火焰离子化检测器，进样口 和检测口温度为250℃，从100℃以5℃/min程序升温至220℃，等温2min；手性气相色谱柱CYCLOSIL-B(30m×0.25mm×0.25μm)。(S)-和(R)-戊唑醇环氧中间体的保留时间分别为20.835和20.920min。Chiral gas chromatography conditions: 7820B Agilent gas chromatograph, flame ionization detector, injection port The temperature of the detection port was 250℃, and the temperature was programmed to rise from 100℃ to 220℃ at 5℃/min, and the temperature was kept isothermal for 2min; the chiral gas chromatography column was CYCLOSIL-B (30m×0.25mm×0.25μm). The retention times of (S)- and (R)-tebuconazole epoxy intermediates were 20.835 and 20.920min, respectively.
反相HPLC色谱条件：1260Infinity II安捷伦高效液相色谱仪，ProntoSIL C18柱(150×4.6mm)，检测柱温为30℃，流动相流速为0.8mL/min，流动相为甲醇:水＝90:10，紫外检测器在220nm监测，戊唑醇环氧中间体和相应邻二醇的保留时间分别为3.115和5.162min。Reverse phase HPLC chromatographic conditions: 1260 Infinity II Agilent high performance liquid chromatograph, ProntoSIL C18 column (150×4.6mm), detection column temperature of 30°C, mobile phase flow rate of 0.8mL/min, mobile phase of methanol: water = 90:10, UV detector monitoring at 220nm, retention times of tebuconazole epoxy intermediate and corresponding vicinal diol were 3.115 and 5.162min, respectively.
正相HPLC色谱条件：1260Infinity II安捷伦高效液相色谱仪，采用手性Chiralcel OD-H柱(250mm×4.6mm)，检测柱温为30℃，以0.6mL/min的流速，流动相为正己烷/异丙醇(90:10，v/v)，紫外检测器在220nm监测，(S)-和(R)-戊唑醇环氧中间体的保留时间分别为6.9min和7.5min；(S)-邻二醇中间体的保留时间11.4min；(S)-和(R)-戊唑醇的保留时间分别为33.2min和36.9min。Normal phase HPLC chromatographic conditions: Agilent 1260 Infinity II high performance liquid chromatograph, chiral Chiralcel OD-H column (250 mm × 4.6 mm), detection column temperature of 30 ° C, flow rate of 0.6 mL / min, mobile phase of n-hexane / isopropanol (90:10, v / v), UV detector monitoring at 220 nm, (S) - and (R) -tebuconazole epoxy intermediates retention time of 6.9 min and 7.5 min, respectively; (S) -vicinal diol intermediate retention time 11.4 min; (S) - and (R) -tebuconazole retention time 33.2 min and 36.9 min, respectively.
重组菌E.coli/Rpeh比活力测定方法：在1.5mL的EP管中加入50μL 50mg/mL E.coli/Rpeh菌悬液(至终浓度5mg/mL湿菌体)和400μL磷酸钾缓冲液(100mM，pH 7.0)，25℃预热5min；加入50μL的200mM消旋戊唑醇环氧中间体(至终浓度20mM)，反应10min后，取100μL加入900μL甲醇中混匀，过0.22μm的有机膜，进行反相HPLC色谱分析。酶活力单位定义：在此测定条件下，以每分钟消耗1μmol戊唑醇环氧中间体所需的湿菌体的量，定义为1个环氧水解酶活力单位(U)。以下实施例中重组菌E.coli/Rpeh的基因序列为申请号为201811621721.9的中国专利文献中的SEQ ID NO.2所示的基因工程菌。Method for determining the relative activity of recombinant bacteria E.coli/Rpeh: add 50μL 50mg/mL E.coli/Rpeh bacterial suspension (to a final concentration of 5mg/mL wet bacteria) and 400μL potassium phosphate buffer (100mM, pH 7.0) to a 1.5mL EP tube, and preheat at 25℃ for 5min; add 50μL 200mM racemic tebuconazole epoxy intermediate (to a final concentration of 20mM), react for 10min, take 100μL and add it to 900μL methanol, mix well, pass through a 0.22μm organic membrane, and perform reversed-phase HPLC chromatography analysis. Definition of enzyme activity unit: Under this assay condition, the amount of wet bacteria required to consume 1μmol of tebuconazole epoxy intermediate per minute is defined as 1 epoxide hydrolase activity unit (U). The gene sequence of the recombinant bacteria E. coli/Rpeh in the following examples is the genetically engineered bacteria shown in SEQ ID NO.2 in the Chinese patent document with application number 201811621721.9.
实施例1重组菌E.coli/Rpeh催化外消旋戊唑醇环氧中间体的最适pH和pH稳定性Example 1 Optimal pH and pH stability of racemic tebuconazole epoxide intermediate catalyzed by recombinant E. coli/Rpeh
最适pH测定：取50μL 200mM的底物戊唑醇环氧中间体(至终浓度5 mg/mL湿菌体)加入450μL不同pH值(100mM，pH 5.5～9.0)磷酸钾缓冲液，25℃预热5min；加入50μL不同pH值孵育的E.coli/Rpeh菌悬液，反应10min后，取100μL加入900μL甲醇中混匀，过0.22μm的有机膜，进行反相HPLC色谱分析，测定重组菌E.coli/Rpeh的比活力。Optimum pH determination: Take 50 μL of 200 mM substrate tebuconazole epoxy intermediate (to a final concentration of 5 mg/mL wet bacteria) were added with 450 μL potassium phosphate buffer of different pH values (100 mM, pH 5.5-9.0), and preheated at 25°C for 5 min; 50 μL of E. coli/Rpeh bacterial suspension incubated with different pH values was added, and after reacting for 10 min, 100 μL was added to 900 μL methanol and mixed, and the mixture was passed through a 0.22 μm organic membrane and subjected to reverse phase HPLC chromatography to determine the specific activity of the recombinant bacteria E. coli/Rpeh.
pH稳定性测定：取100μL 50mg/mL E.coli/Rpeh菌悬液于不同pH值(20mM，pH 5.5～9.0)的磷酸钾缓冲液中孵育1h；立即吸取50μL不同pH值孵育的E.coli/Rpeh菌悬液加入450μL磷酸钾缓冲液(100mM，pH 7.0)，25℃预热5min；加入50μL 200mM的底物戊唑醇环氧中间体(至终浓度5mg/mL湿菌体)，反应10min后，取100μL加入900μL甲醇中混匀，过0.22μm的有机膜，进行反相HPLC色谱分析，测定重组菌E.coli/Rpeh的残余比活力，将未经重组菌E.coli/Rpeh的比活力定义为100％计算相对酶活力。pH stability determination: take 100 μL 50 mg/mL E. coli/Rpeh bacterial suspension and incubate it in potassium phosphate buffer with different pH values (20 mM, pH 5.5-9.0) for 1 hour; immediately aspirate 50 μL of E. coli/Rpeh bacterial suspension incubated with different pH values and add it to 450 μL potassium phosphate buffer (100 mM, pH 7.0), preheat at 25 ℃ for 5 minutes; add 50 μL 200 mM substrate tebuconazole epoxide intermediate (to a final concentration of 5 mg/mL wet bacteria), react for 10 minutes, take 100 μL and add it to 900 μL methanol, mix well, pass through a 0.22 μm organic membrane, and perform reverse phase HPLC chromatography analysis to determine the residual specific activity of the recombinant bacteria E. coli/Rpeh, and define the specific activity of the non-recombinant bacteria E. coli/Rpeh as 100% to calculate the relative enzyme activity.
结果表明，重组菌E.coli/Rpeh的最适反应pH为7.5，在中性pH环境下(pH6.5～8.5)保持较高催化活性，相对酶活力大于80％，而当pH<6.0酶活力快速下降。重组菌E.coli/Rpeh在pH 7.0～8.0下具有高pH稳定性，残余相对酶活力可以大于95％。以上结果表明：RpEH在中性pH范围内高的催化活力和稳定性(图1)。The results showed that the optimal reaction pH of the recombinant bacteria E. coli / Rpeh was 7.5. It maintained a high catalytic activity in a neutral pH environment (pH6.5-8.5), with a relative enzyme activity greater than 80%, and the enzyme activity dropped rapidly when pH <6.0. The recombinant bacteria E. coli / Rpeh had high pH stability at pH 7.0-8.0, and the residual relative enzyme activity could be greater than 95%. The above results show that RpEH has high catalytic activity and stability in the neutral pH range (Figure 1).
实施例2重组菌E.coli/Rpeh催化外消旋戊唑醇环氧中间体的最适温度和温度稳定性Example 2 Optimal Temperature and Temperature Stability of Racemic Tebuconazole Epoxide Intermediate Catalyzed by Recombinant E. coli/Rpeh
最适反应稳定性测定：在1.5mL的EP管中加入50μL的200mM消旋戊唑醇环氧中间体(至终浓度20mM)和450μL磷酸钾缓冲液(100mM，pH 7.5)，在20～50℃预热5min；加入50μL E.coli/Rpeh菌悬液(至终浓度5mg/mL湿菌体)，在20～50℃不同温度下反应10min后，取100μL加入900μL甲醇中混匀，过0.22μm的有机膜，进行反相HPLC色谱分析，测定重组菌E.coli/Rpeh初始 反应速度。Optimal reaction stability determination: add 50 μL of 200 mM racemic tebuconazole epoxy intermediate (to a final concentration of 20 mM) and 450 μL of potassium phosphate buffer (100 mM, pH 7.5) to a 1.5 mL EP tube, preheat at 20-50 ° C for 5 min; add 50 μL of E. coli / Rpeh bacterial suspension (to a final concentration of 5 mg / mL wet bacteria), react at different temperatures of 20-50 ° C for 10 min, take 100 μL and add 900 μL of methanol to mix, pass through a 0.22 μm organic membrane, and perform reversed-phase HPLC chromatography to determine the initial stability of the recombinant bacteria E. coli / Rpeh reaction speed.
温度稳定性测定：取100μL 50mg/mL E.coli/Rpeh菌悬液于20～50℃温度水浴下孵育1h后，至于冰浴中冷却；在1.5mL的EP管中加入50μL不同温度下孵育的E.coli/Rpeh菌悬液(至终浓度5mg/mL湿菌体)和450μL磷酸钾缓冲液(100mM，pH 7.5)，25℃预热5min；加入50μL的200mM消旋戊唑醇环氧中间体(至终浓度20mM)，反应10min后，取100μL加入900μL甲醇中混匀，过0.22μm的有机膜，进行反相HPLC色谱分析，测定经孵育处理后的重组菌E.coli/Rpeh残余比活力，将未经过处理的E.coli/Rpeh菌悬液的比活力定义为100％计算相对酶活力。Temperature stability determination: take 100 μL 50 mg/mL E. coli/Rpeh bacterial suspension and incubate it in a water bath at 20-50°C for 1 hour, then cool it in an ice bath; add 50 μL of E. coli/Rpeh bacterial suspension incubated at different temperatures (to a final concentration of 5 mg/mL wet bacteria) and 450 μL potassium phosphate buffer (100 mM, pH 7.5) into a 1.5 mL EP tube, and preheat at 25°C for 5 minutes; add 50 μL of 200 mM racemic tebuconazole epoxide intermediate (to a final concentration of 20 mM), react for 10 minutes, take 100 μL and add it to 900 μL methanol, mix well, pass through a 0.22 μm organic membrane, and perform reverse-phase HPLC chromatography analysis to determine the residual specific activity of the recombinant bacteria E. coli/Rpeh after incubation, and define the specific activity of the untreated E. coli/Rpeh bacterial suspension as 100% to calculate the relative enzyme activity.
研究表明：重组菌E.coli/Rpeh最适反应温度为30℃，在25～35℃范围内具有较高的催化活性，其相对酶活力为80％以上，在最适温度30℃和最适pH 7.5条件下，E.coli/Rpeh菌悬液的比活力较初始湿菌体提高了84％。重组菌E.coli/Rpeh在低于30℃具有较高热稳定性，残余相对酶活>98％。因此，在后续的催化反应中选择30℃为最佳反应温度(图2)。The study showed that the optimal reaction temperature of the recombinant bacteria E.coli/Rpeh was 30°C, and it had high catalytic activity in the range of 25-35°C, with a relative enzyme activity of more than 80%. At the optimal temperature of 30°C and the optimal pH of 7.5, the specific activity of the E.coli/Rpeh bacterial suspension was 84% higher than that of the initial wet bacteria. The recombinant bacteria E.coli/Rpeh had high thermal stability below 30°C, with a residual relative enzyme activity of >98%. Therefore, 30°C was selected as the optimal reaction temperature in the subsequent catalytic reaction (Figure 2).
实施例3重组菌E.coli/Rpeh不同反应温度拆分20mM外消旋戊唑醇环氧中间体Example 3 Resolving 20 mM racemic tebuconazole epoxy intermediates by recombinant E. coli/Rpeh at different reaction temperatures
在2mL反应体系中，包含20mM外消旋戊唑醇环氧中间体和200μL适当100mg/mL E.coli/Rpeh重组菌(至终浓度10mg/mL)和1.6mL的磷酸钾缓冲液(100mM，pH 7.5)，并分别在10℃、20℃、25℃和30℃条件下，振荡反应3～6h，定时吸取100μL加入800μL乙酸乙酯中萃灭，干燥过0.22μm的有机膜，进行手性气相色谱分析。In a 2mL reaction system, 20mM racemic tebuconazole epoxide intermediate and 200μL of appropriate 100mg/mL E.coli/Rpeh recombinant bacteria (to a final concentration of 10mg/mL) and 1.6mL of potassium phosphate buffer (100mM, pH 7.5) were included. The reaction was oscillated at 10℃, 20℃, 25℃ and 30℃ for 3-6h. 100μL was periodically drawn out and added to 800μL of ethyl acetate for extraction. The mixture was dried over a 0.22μm organic membrane and analyzed by chiral gas chromatography.
结果表明：重组菌E.coli/Rpeh催化(S)-戊唑醇环氧中间体优先水解为(S)-邻二醇，保留(R)-戊唑醇环氧中间体。在10℃、20℃、25℃和30℃的转化时间分 别为6h、5h、3h和3h；保留(R)-戊唑醇环氧中间体的对映体过量ee值分别为49.5％、74.4％、86.6％和92.7％；(R)-戊唑醇环氧中间体得率分别为30.3％、20.0％、41.5％和44.0％。因此，重组菌E.coli/Rpeh在30℃条件下拆分20mM外消旋戊唑醇环氧中间体，制备(R)-戊唑醇环氧中间体的ee值和得率最高为92.7％和44.0％(图3)。The results showed that the recombinant E. coli/Rpeh catalyzed the preferential hydrolysis of (S)-tebuconazole epoxy intermediate to (S)-vicinal diol, while retaining (R)-tebuconazole epoxy intermediate. The conversion time at 10℃, 20℃, 25℃ and 30℃ was significantly higher than that at 10℃, 20℃, 25℃ and 30℃. The enantiomeric excess ee values of the retained (R)-tebuconazole epoxy intermediate were 49.5%, 74.4%, 86.6% and 92.7%, respectively; the yields of (R)-tebuconazole epoxy intermediate were 30.3%, 20.0%, 41.5% and 44.0%, respectively. Therefore, the recombinant bacteria E. coli/Rpeh split 20mM racemic tebuconazole epoxy intermediate at 30°C, and the ee value and yield of the (R)-tebuconazole epoxy intermediate were as high as 92.7% and 44.0% (Figure 3).
实施例4不同浓度重组菌E.coli/Rpeh对外消旋戊唑醇环氧中间体拆分的影响Example 4 Effect of different concentrations of recombinant bacteria E. coli/Rpeh on the resolution of racemic tebuconazole epoxy intermediate
在2mL反应体系中，包含200mM外消旋戊唑醇环氧中间体和适量的磷酸钾缓冲液(100mM，pH 7.5)，在酶浓度分别为50、100、150和200mg/mL重组菌E.coli/Rpeh在30℃反应条件下，振荡反应6h，吸取20μL加入800μL乙酸乙酯中萃灭，同时取10μL加入800μL甲醇中萃灭，过0.22μm的有机膜，分别进行手性气相色谱分析和HPLC分析。In a 2 mL reaction system, containing 200 mM racemic tebuconazole epoxide intermediate and an appropriate amount of potassium phosphate buffer (100 mM, pH 7.5), the recombinant bacteria E. coli/Rpeh was oscillated at 30 °C for 6 h at enzyme concentrations of 50, 100, 150 and 200 mg/mL. 20 μL was extracted with 800 μL of ethyl acetate, and 10 μL was extracted with 800 μL of methanol. The samples were passed through a 0.22 μm organic membrane and subjected to chiral gas chromatography and HPLC analysis, respectively.
结果表明：在50(4:1)、100(2:1)、150(4:3)和200(1:1)mg/mL重组菌E.coli/Rpeh催化外消旋戊唑醇环氧中间体，保留(R)-戊唑醇环氧中间体的对映体过量ee值分别为86％、89％、84％和82％；(R)-戊唑醇环氧中间体得率分别为43.8％、46.5％、46.0％和46.3％。因此，重组菌E.coli/Rpeh在不同工程菌浓度条件下拆分200mM外消旋戊唑醇环氧中间体，制备(R)-戊唑醇环氧中间体的最优的外消旋戊唑醇环氧中间体底物与重组菌基因工程菌E.coli/Rpeh湿菌体的用量比例为2:1(mM:mg/mL)(图4)。The results showed that the enantiomeric excess ee values of (R)-tebuconazole epoxide intermediates were 86%, 89%, 84% and 82% respectively when the recombinant bacteria E. coli/Rpeh catalyzed the racemic tebuconazole epoxide intermediate at 50 (4:1), 100 (2:1), 150 (4:3) and 200 (1:1) mg/mL; the yields of (R)-tebuconazole epoxide intermediates were 43.8%, 46.5%, 46.0% and 46.3% respectively. Therefore, the optimal ratio of racemic tebuconazole epoxide intermediate substrate to recombinant bacteria genetically engineered bacteria E. coli/Rpeh wet cells for the reconstitution of 200mM racemic tebuconazole epoxide intermediates under different engineered bacteria concentrations to prepare (R)-tebuconazole epoxide intermediates was 2:1 (mM:mg/mL) (Figure 4).
实施例5重组菌E.coli/Rpeh拆分外消旋戊唑醇环氧中间体不同底物浓度的影响Example 5 Effect of different substrate concentrations on the resolution of racemic tebuconazole epoxy intermediate by recombinant E. coli/Rpeh
在2mL反应体系中，E.coli/Rpeh重组菌浓度为200mg/mL，外消旋戊唑醇环氧中间体的浓度分别为100、200、300、400和500mM，在30℃反应条件下，振荡反应12h，吸取适量反应液加入1mL乙酸乙酯中萃灭，干燥过0.22μm的有机膜，进行手性气相色谱分析。 In a 2 mL reaction system, the concentration of E. coli/Rpeh recombinant bacteria was 200 mg/mL, and the concentrations of racemic tebuconazole epoxide intermediates were 100, 200, 300, 400 and 500 mM, respectively. The reaction was oscillated at 30 °C for 12 h. An appropriate amount of the reaction solution was extracted and quenched in 1 mL of ethyl acetate. The solution was dried over a 0.22 μm organic film and analyzed by chiral gas chromatography.
结果表明：重组菌E.coli/Rpeh催化不同底物浓度戊唑醇环氧中间体(分别为100、200、300、400和500mM)在30℃条件下转化12h，保留(R)-戊唑醇环氧中间体的对映体过量ee值分别为90.0％、88.9％、79.6％、74.5％和61.6％。因此，重组菌E.coli/Rpeh在30℃条件下拆分高浓度外消旋戊唑醇环氧中间体，底物浓度在100mM可以得到对映体过量ee值最优的(R)-戊唑醇环氧中间体(图5)。The results showed that the recombinant bacteria E. coli/Rpeh catalyzed the conversion of tebuconazole epoxide intermediates with different substrate concentrations (100, 200, 300, 400 and 500 mM, respectively) at 30°C for 12 h, and the enantiomeric excess ee values of the retained (R)-tebuconazole epoxide intermediates were 90.0%, 88.9%, 79.6%, 74.5% and 61.6%, respectively. Therefore, the recombinant bacteria E. coli/Rpeh could resolve high-concentration racemic tebuconazole epoxide intermediates at 30°C, and the substrate concentration was 100 mM to obtain the (R)-tebuconazole epoxide intermediate with the best enantiomeric excess ee value (Figure 5).
实施例6化学法转化(R)-戊唑醇环氧中间体制备(R)-戊唑醇Example 6 Chemical Conversion of (R)-Tebuconazole Epoxy Intermediate to Prepare (R)-Tebuconazole
在正丁醇(10mL)的溶液中加入1,2,4-三氮唑(37.7mg，0.55mmol)和KOH(10mg，0.18mmol)，将反应混合物加热至110℃反应2h，然后加入上述制备得到的(R)-戊唑醇环氧中间体(50mg，0.18mmol)，将反应混合物加热至133℃，搅拌4h后，将反应混合物冷却至室温，有机层用水(10mL×3)洗涤至溶液呈中性，用无水MgSO4干燥，过滤，真空浓缩。所得粗制残留物通过硅胶层析柱纯化，得到白色固体状(R)-戊唑醇，产品1H NMR核磁图谱分析(图6)。(R)-戊唑醇:>99％ee；1H NMR(400MHz,CDCl3,ppm)δ8.14(s,1H),7.88(s,1H),7.09(d,J＝8.0Hz,2H),6.86(d,J＝8.0Hz,2H),4.26(s,2H),3.36(s,1H),2.40–2.33(m,1H),1.73–1.59(m,3H),0.94(s,9H)。1,2,4-triazole (37.7 mg, 0.55 mmol) and KOH (10 mg, 0.18 mmol) were added to a solution of n-butanol (10 mL), the reaction mixture was heated to 110 ° C for 2 h, and then the (R)-tebuconazole epoxy intermediate (50 mg, 0.18 mmol) prepared above was added, the reaction mixture was heated to 133 ° C, stirred for 4 h, and then cooled to room temperature. The organic layer was washed with water (10 mL × 3) until the solution was neutral, dried with anhydrous MgSO 4 , filtered, and concentrated in vacuo. The obtained crude residue was purified by silica gel chromatography to obtain (R)-tebuconazole as a white solid, and the product 1 H NMR nuclear magnetic spectrum analysis (Figure 6). (R)-Tebuconazole:>99%ee; 1 H NMR (400 MHz, CDCl 3 , ppm) δ8.14 (s, 1H), 7.88 (s, 1H), 7.09 (d, J＝8.0 Hz, 2H), 6.86 (d, J＝8.0 Hz, 2H), 4.26 (s, 2H), 3.36 (s, 1H), 2.40–2.33 (m, 1H), 1.73–1.59 (m, 3H), 0.94 (s, 9H).
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，均属于本发明的保护范围应该以权利要求书所界定的为准。 Although the present invention has been disclosed as above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology may make various changes and modifications without departing from the spirit and scope of the present invention. The scope of protection of the present invention should be based on the definition of the claims.

Claims (8)

  1. 一种酶化学法制备(R)-戊唑醇的方法，其特征在于，包括如下步骤：A method for preparing (R)-tebuconazole by enzymatic chemistry, characterized in that it comprises the following steps:
    (1)酶法制备(R)-戊唑醇环氧中间体：利用含有表达重组帕鲁迪根红酵母环氧水解酶的重组菌E.coli/Rpeh，加入外消旋戊唑醇环氧中间体，在反应温度下，恒温震荡反应，反应结束后加入有机溶剂萃取剂后，充分震荡混匀，收集上清液，旋蒸后获得酶法制备的(R)-戊唑醇环氧中间体和相应(S)-邻二醇的混合物；(1) Enzymatic preparation of (R)-tebuconazole epoxy intermediate: using recombinant bacteria E. coli/Rpeh expressing recombinant Rhodotorula parudygensis epoxide hydrolase, adding racemic tebuconazole epoxy intermediate, and subjecting to constant temperature shaking reaction at reaction temperature. After the reaction is completed, adding an organic solvent extractant, shaking and mixing thoroughly, collecting the supernatant, and rotary evaporating to obtain a mixture of (R)-tebuconazole epoxy intermediate and corresponding (S)-vicinal diol prepared by the enzymatic method;
    (2)化学法催化(R)-戊唑醇环氧中间体开环制备(R)-戊唑醇：在有机溶剂中加入1,2,4-三氮唑和固体碱，加入酶法制备的(R)-戊唑醇环氧中间体和相应(S)-邻二醇的混合物，加热反应，反应结束后将反应液冷却至室温，有机层用水洗涤至溶液呈中性，有机相经除水、过滤、旋蒸，所得粗制残留物通过硅胶层析柱纯化，获得白色固体状高光学纯度的(R)-戊唑醇。(2) Chemical catalysis of the ring-opening of the (R)-tebuconazole epoxy intermediate to prepare (R)-tebuconazole: 1,2,4-triazole and a solid base are added to an organic solvent, and a mixture of the (R)-tebuconazole epoxy intermediate prepared by an enzymatic method and the corresponding (S)-vicinal diol is added, and the reaction is heated. After the reaction is completed, the reaction solution is cooled to room temperature, and the organic layer is washed with water until the solution is neutral. The organic phase is dehydrated, filtered, and rotary evaporated. The resulting crude residue is purified by silica gel chromatography to obtain white solid (R)-tebuconazole with high optical purity.
  2. 根据权利要求1所述的酶化学法制备(R)-戊唑醇的方法，其特征在于，步骤(1)反应体系中外消旋戊唑醇环氧中间体底物与重组菌E.coli/Rpeh湿菌体的用量比例为2:1～10:1(mM:mg/mL)The method for preparing (R)-tebuconazole by enzymatic chemistry according to claim 1, characterized in that the ratio of the racemic tebuconazole epoxy intermediate substrate to the recombinant bacteria E. coli/Rpeh wet bacteria in the reaction system of step (1) is 2:1 to 10:1 (mM:mg/mL).
  3. 根据权利要求1所述的酶化学法制备(R)-戊唑醇的方法，其特征在于，步骤(1)反应体系中重组菌E.coli/Rpeh浓度为5～200mg/mL，外消旋戊唑醇环氧中间体底物浓度为20～200mM。The method for preparing (R)-tebuconazole by enzymatic chemistry according to claim 1, characterized in that the concentration of the recombinant bacteria E. coli/Rpeh in the reaction system of step (1) is 5 to 200 mg/mL, and the concentration of the racemic tebuconazole epoxy intermediate substrate is 20 to 200 mM.
  4. 根据权利要求1所述的酶化学法制备(R)-戊唑醇的方法，其特征在于，步骤(1)反应体系pH为6.5～9.0，反应温度为20～50℃。The method for preparing (R)-tebuconazole by enzymatic chemistry according to claim 1, characterized in that the pH of the reaction system in step (1) is 6.5 to 9.0 and the reaction temperature is 20 to 50°C.
  5. 根据权利要求1所述的酶化学法制备(R)-戊唑醇的方法，其特征在于，步骤(1)反应体系pH为7.0～8.0，反应温度为30～35℃。The method for preparing (R)-tebuconazole by enzymatic chemistry according to claim 1, characterized in that the pH of the reaction system in step (1) is 7.0-8.0 and the reaction temperature is 30-35°C.
  6. 根据权利要求1所述的酶化学法制备(R)-戊唑醇的方法，其特征在于，步骤(1)有机溶剂萃取剂为乙酸乙酯、乙酸甲酯、二氯甲烷中的任意一种或几种的混合液。 The method for preparing (R)-tebuconazole by enzymatic chemistry according to claim 1, characterized in that the organic solvent extractant in step (1) is any one of ethyl acetate, methyl acetate, and dichloromethane, or a mixture of several thereof.
  7. 根据权利要求1所述的酶化学法制备(R)-戊唑醇的方法，其特征在于，步骤(2)有机溶剂为正丁醇、甲苯、二氯甲烷中的任意一种或几种。The method for preparing (R)-tebuconazole by enzymatic chemistry according to claim 1, characterized in that the organic solvent in step (2) is any one or more of n-butanol, toluene, and dichloromethane.
  8. 根据权利要求1所述的酶化学法制备(R)-戊唑醇的方法，其特征在于，步骤(2)有机溶剂体积量是使(R)-戊唑醇环氧中间体的摩尔浓度为100～200mM，固体碱为氢氧化钾、氢氧化钠、碳酸钾或叔丁醇钠，1,2,4-三氮唑:固体碱:(R)-戊唑醇环氧中间体的摩尔比为4～1:1:1，反应温度为110～140℃，反应时间为4～24h。 The method for preparing (R)-tebuconazole by enzymatic chemistry according to claim 1, characterized in that the volume of the organic solvent in step (2) is such that the molar concentration of the (R)-tebuconazole epoxy intermediate is 100 to 200 mM, the solid base is potassium hydroxide, sodium hydroxide, potassium carbonate or sodium tert-butoxide, the molar ratio of 1,2,4-triazole: solid base: (R)-tebuconazole epoxy intermediate is 4 to 1:1:1, the reaction temperature is 110 to 140° C., and the reaction time is 4 to 24 h.
PCT/CN2024/071076 2023-07-18 2024-01-08 Method for preparing (r)-tebuconazole by means of enzyme chemical process WO2024078646A1 (en)

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