Composite biological enzyme preparation, application thereof and estetrol preparation method
By using the catalytic synthesis method of compound biological enzyme preparation, the problems of low yield, difficulty in separation and high waste in the preparation process of estritol in the prior art were solved, and efficient and environmentally friendly preparation of estritol was achieved, improving product quality and safety.
Patent Information
- Application Number
- CN202510357008.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-25
- Publication Date
- 2025-06-10
AI Technical Summary
The existing methods for preparing estritol have problems such as low yields, difficulty in separation and the generation of large amounts of waste and wastewater, especially isomer impurities have a great impact on product quality and safety.
Complex biological enzyme preparations, including ketone reductase, P450C15α enzyme and P450C16α enzyme, were prepared by catalytic synthesis method to achieve a controlled directed enzyme catalyzed hydroxylation reaction of C15α and C16α sites to reduce isomer generation.
It improves the conversion efficiency and yield of estritol, simplifies the production process, reduces production costs, reduces the generation of waste and wastewater, and significantly improves product quality and safety.
Smart Images
Figure CN120118869A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of compound synthesis, and particularly relates to a composite bioenzyme preparation, its application, and a preparation method of estetrol. Background Art
[0002] As an important natural estrogen in clinical research, estetrol is produced in the liver of the fetus during pregnancy in pregnant women and can be used as an indicator to monitor the health of the fetus. A series of clinical studies have shown that estetrol can not only be used in hormone replacement therapy and as an oral contraceptive for women, but also has important applications in the treatment of autoimmune diseases, the prevention and treatment of breast tumors and colon tumors, enhancing sexual desire, skin care, and wound healing. Since the discovery of estetrol, how to efficiently prepare estetrol has become a research hotspot. The existing routes for preparing estetrol mainly rely on chemical synthesis, which have problems such as low yield and difficult separation. For example, in the process of synthesizing estetrol from estrone, a key step is the asymmetric dihydroxylation reaction, in which isomers (C15-β, C16-β-dihydroxy) are generated, and these isomer impurities have a great impact on the quality and safety of estetrol. Therefore, multi-step purification to remove impurities is required, which is not only difficult to separate but also seriously affects the yield, increasing both production costs and generating a large amount of waste and wastewater due to the large consumption of reagent raw materials.
[0003] Therefore, providing a method for preparing estetrol with specific transformation sites and high transformation efficiency has good market prospects. Summary of the Invention
[0004] In view of this, the purpose of the present invention is to provide a composite bioenzyme preparation. By using the composite bioenzyme preparation provided by the present invention to prepare estetrol by catalytic synthesis, the transformation efficiency is high.
[0005] To achieve the above invention purpose, the present invention provides the following technical solutions:
[0006] A composite bioenzyme preparation, the composite bioenzyme preparation includes ketoreductase, P450C15α enzyme, and P450C16α enzyme; the amino acid sequence of the ketoreductase is as shown in SEQ ID NO.1, the amino acid sequence of the P450C15α enzyme is as shown in SEQ ID NO.2, and the amino acid sequence of the P450C16α enzyme is as shown in SEQ ID NO.3.
[0007] Another purpose of the present invention is to provide the application of the composite bioenzyme preparation in the preparation of estetrol.
[0008] Another purpose of the present invention is to provide a preparation method of estetrol, using the compound shown in formula I as the starting material, and the synthesis route is as follows:
[0009]
[0010] Among them, R 1 is a hydroxyl group or a ketone group, and R 2 is hydrogen or α-hydroxy; the catalytic synthesis includes biocatalytic enzyme catalysis, and the biocatalytic enzyme used is the above-mentioned composite biocatalytic enzyme preparation.
[0011] Preferably, when the R 1 is a ketone group, a ketoreductase is used to reduce the ketone group to a hydroxyl group; when the R 2 is hydrogen, P450C16α enzyme is used for C16α hydroxylation; P450C15α enzyme is used for C15α hydroxylation.
[0012] Preferably, when using the ketoreductase to reduce the ketone group to a hydroxyl group, the mass of the ketoreductase is 0.1-0.2 times the mass of the compound of formula I.
[0013] Preferably, when using P450C16α enzyme for C16α hydroxylation, the mass of P450C16α enzyme is 0.4-0.5 times the mass of the compound of formula I.
[0014] Preferably, when using P450C15α enzyme for C15α hydroxylation, the mass of P450C15α enzyme is 0.4-0.5 times the mass of the compound of formula I.
[0015] Preferably, the biocatalytic enzyme catalysis is a one-step reaction or a multi-step reaction.
[0016] Preferably, the one-step reaction is to simultaneously add ketoreductase, P450C15α enzyme and P450C16α enzyme for catalytic reaction, and the multi-step reaction is to add any one or two of ketoreductase, P450C15α enzyme and P450C16α enzyme for stepwise catalytic reaction.
[0017] Preferably, when carrying out the biocatalytic enzyme catalysis, glucose, glucose dehydrogenase and nicotinamide adenine dinucleotide are also added for assistance.
[0018] Preferably, the biocatalytic enzyme catalysis conditions are: the reaction temperature is 25-30 °C, and the pH value is 6.5-7.0.
[0019] Preferably, the catalytic synthesis also includes chemical synthesis. When R 1 is a ketone group, the compound of formula I is added to an organic solvent, and under the catalytic condition of pyridine or cerium chloride heptahydrate, potassium borohydride or sodium borohydride is used to reduce the ketone group to a hydroxyl group.
[0020] Compared with the prior art, the present invention has the following beneficial effects:
[0021] The present invention provides a composite bioenzyme preparation. Using the composite bioenzyme preparation provided by the present invention, estetrol is prepared by a catalytic synthesis method, and the conversion efficiency is high. The present invention provides a method for preparing estetrol. For the first time, the synthetic biology method is used to prepare estetrol, creating a brand-new process method for the preparation of estetrol. The present invention uses bioenzymes with specific conversion sites, mild conversion conditions, and high conversion efficiency to achieve a controllable directional enzymatic hydroxylation reaction of C15α and C16α, solving the problem of the formation of C15-β and C16-β dihydroxy isomers. At the same time, the conversion cycle is shortened, the substrate conversion concentration is increased, thereby effectively solving the impurity problem, and improving the conversion efficiency, yield, and product quality. For the first time, the present invention realizes the hydroxylation modification of the steroid parent ring C15α and C16α by an enzymatic method to prepare estetrol, and for the first time realizes the one-step preparation of estetrol by a multi-enzyme system. The reaction process of the present invention is all ordinary reactions, without involving dangerous reaction steps such as high temperature, high pressure, and explosion. The requirements for the reaction device are low, the operating cost is low, the operation is simple, it is suitable for industrial production, and has a good market prospect. Description of the Drawings
[0022] Figure 1 Chromatogram of estetrol prepared in Example 1;
[0023] Figure 2 Chromatogram of estetrol prepared in Example 2;
[0024] Figure 3 Chromatogram of estetrol prepared in Example 3;
[0025] Figure 4 Chromatogram of estetrol prepared in Example 4;
[0026] Figure 5 Chromatogram of estetrol prepared in Example 5;
[0027] Figure 6 Chromatogram of estetrol prepared in Example 6;
[0028] Figure 7 Chromatogram of estetrol prepared in Example 7;
[0029] Figure 8 Chromatogram of estetrol prepared in Example 8. Detailed Description of the Invention
[0030] The present invention provides a composite bioenzyme preparation. The composite bioenzyme preparation includes a ketoreductase, a P450C15α enzyme, and a P450C16α enzyme; the amino acid sequence of the ketoreductase is as shown in SEQ ID NO.1, the amino acid sequence of the P450C15α enzyme is as shown in SEQ ID NO.2, and the amino acid sequence of the P450C16α enzyme is as shown in SEQ ID NO.3.
[0031] The present invention also provides the application of the composite bio-enzyme preparation in the preparation of estetrol.
[0032] The present invention also provides a method for preparing estetrol, using the compound shown in Formula I as the starting material, and the synthesis route is as follows:
[0033]
[0034] wherein, R 1 is a hydroxyl group or a keto group, and R 2 is hydrogen or α-hydroxy; the catalytic synthesis includes bio-enzyme catalysis, and the bio-enzyme used in the bio-enzyme catalysis is the above-mentioned composite bio-enzyme preparation.
[0035] In the present invention, when the said R 1 is a keto group, the keto group is reduced to a hydroxyl group by using a ketoreductase; when the said R 2 is hydrogen, C16α hydroxylation is carried out by using P450C16α enzyme; C15α hydroxylation is carried out by using P450C15α enzyme.
[0036] In the present invention, the conditions for bio-enzyme catalysis are: the reaction temperature is 25 - 30 °C, and the pH value is 6.5 - 7.0. The reaction temperature is preferably 26 - 28 °C, and the pH value is preferably 6.8.
[0037]
[0038]
[0039]
[0040] The ketoreductase, P450C15α enzyme and P450C16α enzyme are all expression products in non-pathogenic microorganisms, and the non-pathogenic microorganism is Escherichia coli.
[0041] In the present invention, when using the ketoreductase to reduce the keto group to a hydroxyl group, the mass of the ketoreductase is 0.1 - 0.2 times the mass of the compound of formula I.
[0042] In the present invention, when using the P450C16α enzyme for C16α hydroxylation, the mass of the P450C16α enzyme is 0.4 - 0.5 times the mass of the compound of formula I.
[0043] In the present invention, when using the P450C15α enzyme for C15α hydroxylation, the mass of the P450C15α enzyme is 0.4 - 0.5 times the mass of the compound of formula I.
[0044] In the present invention, when performing the biocatalytic reaction, glucose, glucose dehydrogenase (GDH) and nicotinamide adenine dinucleotide (coenzyme) can also be added for auxiliary reaction. Among them, the added mass of glucose is 0.3 - 0.8 times the mass of the compound of formula I, the added mass of GDH is 0.01 - 0.02 times the mass of the compound of formula I, and the added mass of the coenzyme is 0.002 - 0.004 times the mass of the compound of formula I.
[0045] In the present invention, the biocatalytic reaction is a one-step reaction or a multi-step reaction.
[0046] The one-step reaction in the present invention is to simultaneously add the ketoreductase, P450C15α enzyme and P450C16α enzyme for catalytic reaction. The present invention realizes the one-step preparation of estetrol by a multi-enzyme system for the first time, significantly improving the production efficiency of estetrol.
[0047] The multi-step reaction in the present invention is to add any one or two of the ketoreductase, P450C15α enzyme and P450C16α enzyme for step-by-step catalytic reaction.
[0048] In the present invention, when the biocatalytic reaction adopts a multi-step reaction, the method for preparing estetrol by catalytic synthesis in the present invention further includes chemical synthesis: R 1When the group is a keto group, the compound of formula I is added to an organic solvent, and in the presence of pyridine or cerium chloride heptahydrate as a catalyst, the keto group is reduced to a hydroxyl group using potassium borohydride or sodium borohydride. Among them, the organic solvent can be any one of methanol, ethanol, and isopropanol, and the added mass of the organic solvent is 5 - 15 times the weight of the compound of formula I; the added mass of pyridine or cerium chloride heptahydrate is 0.05 - 0.35 times the weight of the compound of formula I; the added mass of potassium borohydride or sodium borohydride is 0.5 - 1.5 times the weight of the compound of formula I. The chemical synthesis described in the present invention is carried out at 0 - 40 °C.
[0049] The biocatalysis and the chemical synthesis described in the present invention can be carried out in any order.
[0050] In the specific examples of the present invention, the conversion rate during the biocatalytic reaction is detected by HPLC.
[0051] The technical solutions provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the protection scope of the present invention.
[0052] Example 1
[0053] A method for preparing estetrol, the synthesis route is as follows:
[0054]
[0055] Specifically, it includes the following steps:
[0056] (1) C15α hydroxylation: Using estrone as the reaction substrate, estrone is added to a phosphate buffer system with a pH of 6.5 according to 5% (w / v) of the reaction system, and P450C15α enzyme (SEQ ID NO.2), glucose, GDH, and coenzyme are added. Among them, the mass ratio of the substrate, P450C15α enzyme, glucose, GDH, and coenzyme is 1:0.4:0.3:0.02:0.003. The biocatalytic reaction is carried out at 26 °C. When the conversion rate reaches 99% after 18 h of reaction, the reaction ends. Filtration is carried out for solid-liquid separation, and the solid is collected and extracted with methanol. The amount of methanol used is 5 times the feeding amount of estrone. The extract is collected, and solid-liquid separation is carried out on the extract by filtration. The liquid component is collected, and the liquid component is concentrated, subjected to water precipitation, and dried to obtain compound I.
[0057] (2) Reduction of the C17 ketone group: The compound I obtained in step (1) is added to ethanol, where the weight of ethanol is 10 times that of compound I. Pyridine with a weight 0.2 times that of compound I is added, and then potassium borohydride with a weight 0.8 times that of compound I is added. The reaction is carried out at 25 °C. After the reaction conversion is detected to be complete by HPLC, acetic acid is added to neutralize to pH = 7. Water with a weight 10 times that of compound II is added for water precipitation. Solid-liquid separation is carried out by suction filtration. The collected solid is extracted with methanol, and the amount of methanol used is 5 times the feeding amount of compound I. The extract is collected, and solid-liquid separation is carried out on the extract by suction filtration. The liquid component is collected, and the liquid component is concentrated and subjected to water precipitation. After drying, compound II is obtained.
[0058] (3) C16α hydroxylation: Using the compound II obtained in step (2) as the substrate, the compound II is put into a phosphate buffer salt system with a pH of 6.5 according to the amount of 5% (w / v) of the reaction system. P450C16α enzyme (SEQ ID NO.3), glucose, GDH, and coenzyme are added. Among them, the mass ratio of the substrate, P450C16α enzyme, glucose, GDH, and coenzyme is 1:0.4:0.3:0.02:0.003. The biocatalytic reaction is carried out at 26 °C. When the conversion rate reaches 99% after reacting for 20 h, the reaction ends. Solid-liquid separation is carried out by suction filtration. The collected solid is extracted with methanol, and the amount of methanol used is 5 times the feeding amount of compound II. The extract is collected, and solid-liquid separation is carried out on the extract by suction filtration. The liquid component is collected, and the liquid component is concentrated and subjected to water precipitation. After drying, the product estetrol is obtained.
[0059] The chromatogram of the estetrol prepared in this example is as Figure 1 shown, and the HPLC content is 99.87%.
[0060] Example 2
[0061] A preparation method of estetrol, and the synthetic route is as follows:
[0062]
[0063] Specifically, it includes the following steps:
[0064] (1) C15α-Hydroxylation: Using estradiol as the substrate, add estradiol into the phosphate buffered saline system with a pH of 6.5 according to 5% (w / v) of the reaction system. Then add P450C15α enzyme (SEQ ID NO.2), glucose, GDH, and coenzyme. Among them, the mass ratio of the substrate, P450C15α enzyme, glucose, GDH, and coenzyme is 1:0.5:0.4:0.01:0.002. Conduct the biocatalytic reaction at 28 °C. When the conversion rate reaches 99% after 18 h of reaction, the reaction ends. Perform solid-liquid separation by suction filtration, collect the solid and extract it with methanol. The amount of methanol used is 5 times the feeding amount of estradiol. Collect the extract, perform solid-liquid separation on the extract by suction filtration, collect the liquid component, concentrate and precipitate the liquid component with water, and obtain Compound II after drying.
[0065] (2) C16α-Hydroxylation: Using Compound II obtained in step (1) as the substrate, add Compound II into the phosphate buffered saline system with a pH of 6.5 according to 5% (w / v) of the reaction system. Then add P450C16α enzyme (SEQ ID NO.3), glucose, GDH, and coenzyme. Among them, the mass ratio of the substrate, P450C16α enzyme, glucose, GDH, and coenzyme is 1:0.5:0.4:0.01:0.002. Conduct the biocatalytic reaction at 28 °C. When the conversion rate reaches 99% after 20 h of reaction, the reaction ends. Perform solid-liquid separation by suction filtration, collect the solid and extract it with methanol. The amount of methanol used is 5 times the feeding amount of Compound II. Collect the extract, perform solid-liquid separation on the extract by suction filtration, collect the liquid component, concentrate and precipitate the liquid component with water, and obtain the product estetrol after drying.
[0066] The chromatogram of estetrol prepared in this example is as Figure 2 shown, and the HPLC content is 99.88%.
[0067] Example 3
[0068] A method for preparing estetrol, the synthesis route is as follows:
[0069]
[0070] Specifically, it includes the following steps:
[0071] (1) C15α-hydroxylation: Using estrone as a substrate, add estrone into a phosphate buffer salt system with a pH of 6.5 according to 5% (w / v) of the reaction system. Then add P450C15α enzyme (SEQ ID NO.2), glucose, GDH, and coenzyme. Among them, the mass ratio of the substrate, P450C15α enzyme, glucose, GDH, and coenzyme is 1:0.45:0.5:0.02:0.004. Conduct a biocatalytic reaction at 30°C. When the conversion rate reaches 99% after 19 hours of reaction, the reaction ends. Use suction filtration for solid-liquid separation, collect the solid and extract it with methanol. The amount of methanol used is 5 times the feeding amount of estrone. Collect the extract, use suction filtration for solid-liquid separation again, collect the liquid component, concentrate and water-extract the liquid component, and obtain Compound I after drying.
[0072] (2) C17-keto reduction: Using Compound I obtained in step (1) as a substrate, add Compound I into a phosphate buffer salt system with a pH of 6.5 according to 5% (w / v) of the reaction system. Then add ketoreductase (SEQ ID NO.1), glucose, GDH, and coenzyme. Among them, the mass ratio of ketoreductase, glucose, GDH, and coenzyme is 1:0.15:0.5:0.02:0.004. Conduct a biocatalytic reaction at 30°C. When the conversion rate reaches 99% after 22 hours of reaction, the reaction ends. Use suction filtration for solid-liquid separation, collect the solid and extract it with methanol. The amount of methanol used is 5 times the feeding amount of Compound I. Collect the extract, use suction filtration for solid-liquid separation again, collect the liquid component, concentrate and water-extract the liquid component, and obtain Compound II after drying.
[0073] (3) C16α-hydroxylation: Using Compound II obtained in step (2) as a substrate, add Compound II into a phosphate buffer salt system with a pH of 6.5 according to 5% (w / v) of the reaction system. Then add P450C16α enzyme (SEQ ID NO.3), glucose, GDH, and coenzyme. Among them, the mass ratio of the substrate, P450C16α enzyme, glucose, GDH, and coenzyme is 1:0.45:0.5:0.02:0.004. Conduct a biocatalytic reaction at 28°C. When the conversion rate reaches 99% after 20 hours of reaction, the reaction ends. Use suction filtration for solid-liquid separation, collect the solid and extract it with methanol. The amount of methanol used is 5 times the feeding amount of Compound II. Collect the extract, use suction filtration for solid-liquid separation again, collect the liquid component, concentrate and water-extract the liquid component, and obtain the product estetrol after drying.
[0074] The chromatogram of estetrol prepared in this example is as Figure 3 shown, and the HPLC content is 99.89%.
[0075] Example 4
[0076] A preparation method of estetrol, and the synthesis route is as follows:
[0077]
[0078] Specifically, it includes the following steps:
[0079] Using estrone as a raw material, add estrone into a phosphate buffer salt system with pH 6.5 according to 5% (w / v) of the reaction system, and add ketoreductase (SEQ ID NO.1), P450C15α enzyme (SEQ ID NO.2), P450C16α enzyme (SEQ ID NO.3), glucose, GDH, and coenzyme. Among them, the mass ratio of the substrate, ketoreductase, P450C15α enzyme, P450C16α enzyme, glucose, GDH, and coenzyme is 1:0.15:0.45:0.45:0.6:0.015:0.003. Conduct a multi-enzyme system biocatalytic reaction at a temperature of 28°C. After reacting for 22 hours, the conversion rate reaches 99%. After the reaction ends, perform solid-liquid separation by suction filtration, collect the solid and extract it with methanol. The amount of methanol used is 5 times the feeding amount of estrone. Collect the extract, perform solid-liquid separation on the extract by suction filtration, collect the liquid component, concentrate and water-extract the liquid component, and obtain the product estetrol by one-step enzyme catalysis after drying.
[0080] The chromatogram of estetrol prepared in this example is as Figure 4 shown, and the HPLC content is 99.89%.
[0081] Example 5
[0082] A preparation method of estetrol, and the synthesis route is as follows:
[0083]
[0084] Specifically, it includes the following steps:
[0085] Using estradiol as a raw material, add estradiol into a phosphate buffer salt system with pH 6.5 according to 5% (w / v) of the reaction system, and add P450C15α enzyme (SEQ ID NO.2), P450C16α enzyme (SEQ ID NO.3), glucose, GDH, and coenzyme. Among them, the mass ratio of the substrate, P450C15α enzyme, P450C16α enzyme, glucose, GDH, and coenzyme is 1:0.45:0.45:0.5:0.015:0.003. Conduct a multi-enzyme system biocatalytic reaction at a temperature of 28°C. After reacting for 20 hours, the conversion rate reaches 99%. After the reaction ends, perform solid-liquid separation by suction filtration, collect the solid and extract it with methanol. The amount of methanol used is 5 times the feeding amount of estradiol. Collect the extract, perform solid-liquid separation on the extract by suction filtration, collect the liquid component, concentrate and water-extract the liquid component, and obtain the product estetrol by one-step enzyme catalysis after drying.
[0086] The chromatogram of estetrol prepared in this example is as Figure 5 shown, and the HPLC content is 99.83%.
[0087] Example 6
[0088] A preparation method of estetrol, the synthetic route is as follows:
[0089]
[0090] Specifically, it includes the following steps:
[0091] Using estriol as the raw material, add estriol into the phosphate buffer system with pH 6.5 according to 5% (w / v) of the reaction system, and add P450C15α enzyme (SEQ ID NO.2), glucose, GDH, and coenzyme. Among them, the mass ratio of the substrate, P450C15α enzyme, glucose, GDH, and coenzyme is 1:0.45:0.5:0.015:0.003. Carry out the multi-enzyme system biocatalytic reaction at a temperature of 28°C. After reacting for 20 h, the conversion rate reaches 99%. After the reaction ends, carry out solid-liquid separation by suction filtration, collect the solid and extract it with methanol. The dosage of methanol is 5 times the feeding amount of estriol. Collect the extract, carry out solid-liquid separation on the extract by suction filtration, collect the liquid component, concentrate and water-precipitate the liquid component, and obtain the product estetrol by one-step enzyme catalysis after drying.
[0092] The chromatogram of estetrol prepared in this example is as Figure 6 shown, and the HPLC content is 99.49%.
[0093] Example 7
[0094] A preparation method of estetrol, the synthetic route is as follows:
[0095]
[0096] Specifically, it includes the following steps:
[0097] (1) C15α-Hydroxylation: Using estrone as the substrate, add estrone into the phosphate buffered saline system with a pH of 6.5 according to 5% (w / v) of the reaction system. Then add P450C15α enzyme (SEQ ID NO.2), glucose, GDH, and coenzyme. Among them, the mass ratio of the substrate, P450C15α enzyme, glucose, GDH, and coenzyme is 1:0.45:0.5:0.015:0.003. Conduct the biocatalytic reaction at 28 °C. When the conversion rate reaches 99% after 23 hours of reaction, the reaction ends. Use suction filtration for solid-liquid separation, collect the solid and extract it with methanol. The amount of methanol used is 5 times the feeding amount of estrone. Collect the extract, use suction filtration for solid-liquid separation again, collect the liquid component, concentrate and precipitate the liquid component with water, and obtain Compound I after drying.
[0098] (2) C16α-Hydroxylation: Using Compound I obtained in step (1) as the substrate, add Compound I into the phosphate buffered saline system with a pH of 6.5 according to 5% (w / v) of the reaction system. Then add P450C16α enzyme (SEQ ID NO.3), glucose, GDH, and coenzyme. Among them, the mass ratio of the substrate, P450C16α enzyme, glucose, GDH, and coenzyme is 1:0.45:0.5:0.015:0.003. Conduct the biocatalytic reaction at 28 °C. When the conversion rate reaches 99% after 20 hours of reaction, the reaction ends. Use suction filtration for solid-liquid separation, collect the solid and extract it with methanol. The amount of methanol used is 5 times the feeding amount of Compound I. Collect the extract, use suction filtration for solid-liquid separation again, collect the liquid component, concentrate and precipitate the liquid component with water, and obtain Compound III after drying.
[0099] (3) C17-Keto Reduction: Conduct chemical reduction using Compound III obtained in step (2) as the raw material. Add Compound III into isopropanol, where the weight of isopropanol is 10 times the weight of Compound III. Then add cerium chloride heptahydrate, and the weight of cerium chloride heptahydrate is 0.25 times the weight of Compound III. Then add sodium borohydride for reduction, and the weight of sodium borohydride is 0.75 times the weight of Compound III. React at 25 °C. After detecting that the reaction is completely converted by HPLC, add acetic acid to neutralize to pH = 7, add water 10 times the weight of Compound II for precipitation, use suction filtration for solid-liquid separation, collect the solid and extract it with methanol. The amount of methanol used is 5 times the feeding amount of Compound III. Collect the extract, use suction filtration for solid-liquid separation again, collect the liquid component, concentrate and precipitate the liquid component with water, and obtain the product estetrol after drying.
[0100] The chromatogram of estetrol prepared in this example is as Figure 7 shown, and the HPLC content is 99.40%.
[0101] Example 8
[0102] A method for preparing estetrol, and the synthesis route is as follows:
[0103]
[0104] Specifically, it includes the following steps:
[0105] (1) C15α hydroxylation: Using estrone as the substrate, add estrone into the phosphate buffer salt system with a pH of 6.5 according to the amount of 5% (w / v) of the reaction system, and add P450C15α enzyme (SEQ ID NO.2), glucose, GDH, and coenzyme. Among them, the mass ratio of the substrate, P450C15α enzyme, glucose, GDH, and coenzyme is 1:0.5:0.4:0.015:0.003. Carry out the biocatalytic reaction at 25°C. When the conversion rate reaches 99% after 18 hours of reaction, the reaction ends. Use suction filtration for solid-liquid separation, collect the solid and extract it with methanol. The amount of methanol used is 5 times the feeding amount of estrone. Collect the extract, use suction filtration for solid-liquid separation, collect the liquid component, concentrate and water-separate the liquid component, and obtain Compound I after drying.
[0106] (2) C16α hydroxylation: Using Compound I obtained in step (2) as the substrate, add Compound II into the phosphate buffer salt system with a pH of 6.5 according to the amount of 5% (w / v) of the reaction system, and add P450C16α enzyme (SEQ ID NO.3), glucose, GDH, and coenzyme. Among them, the mass ratio of the substrate, P450C16α enzyme, glucose, GDH, and coenzyme is 1:0.5:0.4:0.015:0.003. Carry out the biocatalytic reaction at 25°C. When the conversion rate reaches 99% after 20 hours of reaction, the reaction ends. Use suction filtration for solid-liquid separation, collect the solid and extract it with methanol. The amount of methanol used is 5 times the feeding amount of Compound I. Collect the extract, use suction filtration for solid-liquid separation, collect the liquid component, concentrate and water-separate the liquid component, and obtain Compound III after drying.
[0107] (3) C17 keto group reduction: Using Compound III obtained in step (2) as the substrate, add Compound III into the phosphate buffer salt system with a pH of 6.5 according to the amount of 3% (w / v) of the reaction system, and add ketoreductase (SEQ ID NO.1), glucose, GDH, and coenzyme. Among them, the mass ratio of ketoreductase, glucose, GDH, and coenzyme is 1:0.15:0.3:0.01:0.002. Carry out the biocatalytic reaction at 25°C. When the conversion rate reaches 99% after 21 hours of reaction, the reaction ends. Use suction filtration for solid-liquid separation, collect the solid and extract it with methanol. The amount of methanol used is 5 times the feeding amount of Compound III. Collect the extract, use suction filtration for solid-liquid separation, collect the liquid component, concentrate and water-separate the liquid component, and obtain the product estetrol after drying.
[0108] The chromatogram of estetrol prepared in this example is as Figure 8 shown, and the HPLC content is 99.74%.
[0109] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.
Claims
1. A composite bio-enzyme preparation, characterized in that: The composite bio-enzyme preparation comprises ketoreductase, P450C15α enzyme and P450C16α enzyme; the amino acid sequence of the ketoreductase is shown in SEQ ID NO.1, the amino acid sequence of the P450C15α enzyme is shown in SEQ ID NO.2, and the amino acid sequence of the P450C16α enzyme is shown in SEQ ID NO.
3.
2. Use of the composite bio-enzyme preparation according to claim 1 in the preparation of estetrol.
3. A method for preparing estetrol, characterized in that: Using the compound shown in formula I as the starting material, the synthesis route is as follows: Wherein, R1 is hydroxyl or keto, and R2 is hydrogen or α-hydroxyl; The catalytic synthesis includes bio-enzyme catalysis, and the bio-enzyme used in the bio-enzyme catalysis is the composite bio-enzyme preparation according to claim 1.
4. The method for preparing estetrol according to claim 3, characterized in that: When the R1 is a keto group, the keto group is reduced to a hydroxyl group using ketoreductase; when the R2 is hydrogen, C16α hydroxylation is performed using P450C16α enzyme; and C15α hydroxylation is performed using P450C15α enzyme.
5. The method for preparing estetrol according to claim 4, characterized in that: When the ketoreductase is used to reduce the keto group to the hydroxyl group, the mass of the ketoreductase is 0.1-0.2 times the mass of the compound of formula I.
6. The method for preparing estetrol according to claim 4, characterized in that: When the P450C16α enzyme is used for C16α hydroxylation, the mass of the P450C16α enzyme is 0.4-0.5 times the mass of the compound of formula I.
7. The method for preparing estetrol according to claim 4, characterized in that: When the P450C15α enzyme is used for C15α hydroxylation, the mass of the P450C15α enzyme is 0.4-0.5 times the mass of the compound of formula I.
8. The method for preparing estetrol according to claim 3, characterized in that: The bio-enzyme catalysis is a one-step reaction or a multi-step reaction.
9. The method for preparing estetrol according to claim 8, characterized in that: The one-step reaction is to add ketoreductase, P450C15α enzyme and P450C16α enzyme at the same time for catalytic reaction, and the multi-step reaction is to add any one or two enzymes of ketoreductase, P450C15α enzyme and P450C16α enzyme for stepwise catalytic reaction.
10. The method for preparing estetrol according to claim 3, characterized in that: The catalytic synthesis also includes chemical synthesis. When R1 is a keto group, the compound of formula I is added to an organic solvent, and the keto group is reduced to a hydroxyl group using potassium borohydride or sodium borohydride under the catalytic conditions of pyridine or cerium chloride heptahydrate.