Preparation method of estetrol
Through the microbial fermentation conversion method, estritol was prepared from the compounds of formula I, which solved the problems of low yield and difficulty in separation in the prior art, and achieved efficient, safe and environmentally friendly preparation of estritol.
Patent Information
- Application Number
- CN202510357278.2
- 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 preparation methods for estritol have problems with low yields and difficulty in separation, especially because the formation of C15-β and C16-β dihydroxy isomers affects the quality and safety of estritol.
By adopting the microbial fermentation conversion method, estritol is prepared from the compounds of formula I, and C15α hydroxylation and C16α hydroxylation are achieved through the fermentation steps of Saccharomyces cerevisiae, ochrassia and Penicillium Ress, thereby avoiding the formation of isomers.
It improves the yield and purity of estritol, simplifies the separation process, reduces production costs, and meets the requirements of green chemical industry.
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Figure CN120118969A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of compound synthesis, and particularly relates to a method for preparing estetrol. Background Art
[0002] As an important natural estrogen in clinical research, estetrol is produced in the liver of the fetus during pregnancy 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 the impurities of this isomer have a great impact on the quality and safety of estetrol. Therefore, multiple steps of purification are required to remove impurities, which is not only difficult to separate, but also seriously affects the yield, increasing the production cost and generating a large amount of waste and wastewater due to the large consumption of reagent raw materials.
[0003] J.Org.Chem.(1968,33,3133 - 3135) reported a method for synthesizing estetrol from estrone derivative Ⅲ, as shown below:
[0004]
[0005] The total yield of this method is only 7%, and dangerous reagent LiA l H 4 is required. The reaction conditions are harsh, and highly toxic reagent osmium tetroxide is used in the oxidation step, which is very unsuitable for industrial production.
[0006] Steroids.(1976,27,111 - 121) reported another method for synthesizing estetrol starting from estrone, as shown below:
[0007]
[0008] The total yield of this method is only 8%, and dangerous reagent LiA l H 4 and highly toxic reagent osmium tetroxide are also required, which is not suitable for industrial production.
[0009] Therefore, there is an urgent need to provide a green and safe method for preparing estetrol to solve the problems of low yield and difficult separation of estetrol at present. Summary of the Invention
[0010] In view of this, the object of the present invention is to provide a method for preparing estetrol, which is prepared by microbial fermentation transformation method. The transformation site is specific, and the problem of generating C15-β, C16-β dihydroxy isomers is solved.
[0011] In order to achieve the above object of the invention, the present invention provides the following technical solutions:
[0012] A method for preparing estetrol, using the compound shown in formula I as the starting material, and the synthesis route is as follows:
[0013]
[0014] Among them, R 1 is a hydroxyl group or a ketone group, and R 2 is hydrogen or an α-hydroxy group;
[0015] The microorganisms include Saccharomyces cerevisiae, Aspergillus ochraceus and Penicillium raistrickii.
[0016] Preferably, when the R 1 is a ketone group, Saccharomyces cerevisiae is used to ferment and reduce the ketone group to a hydroxyl group; when the R 2 is hydrogen, Aspergillus ochraceus is used to ferment to hydroxylate C16α; Penicillium raistrickii is used to ferment to hydroxylate C15α.
[0017] Preferably, the conditions for fermenting with the Saccharomyces cerevisiae include: the temperature is 30-32 °C, and the pH is 6.2-6.4.
[0018] Preferably, the fermentation medium for using the Saccharomyces cerevisiae is: glucose 20-25 g / L, plant peptone 20-25 g / L, yeast powder 10-15 g / L, antifoam 0.2-0.3 mL / L.
[0019] Preferably, the conditions for fermenting with the Aspergillus ochraceus include: the temperature is 26-28 °C, and the pH is 6.8-7.0.
[0020] Preferably, the fermentation medium for using the Aspergillus ochraceus is: glucose 30-35 g / L, corn steep liquor 20-25 g / L, antifoam 0.3-0.5 mL / L.
[0021] Preferably, the conditions for fermenting with the Penicillium raistrickii include: the temperature is 28-30 °C, and the pH is 7.0-7.5.
[0022] Preferably, the fermentation medium for using the Penicillium raistrickii is: glucose 20-25 g / L, corn steep liquor 20-25 g / L, yeast powder 10-15 g / L, antifoam 0.3-0.5 mL / L.
[0023] Preferably, the Saccharomyces cerevisiae, Aspergillus ochraceus, and Penicillium resedanum can be fermented in any order.
[0024] Compared with the prior art, the present invention has the following beneficial effects:
[0025] The present invention provides a method for preparing estetrol, and for the first time, estetrol is prepared from Formula I through a microbial fermentation conversion method. By using site-specific C15α-hydroxylation bacteria and C16α-hydroxylation bacteria, the present invention effectively solves the problem of the generation of C15-β, C16-β dihydroxy isomers, thus providing a solution to the problems of difficult separation and purification and low yield caused by the generation of isomers. The reaction process of the present invention is entirely a microbial fermentation process, without involving dangerous reaction steps such as high temperature, high pressure, and explosion, and has low pollution, which better meets the requirements of green chemical industry. Description of the Drawings
[0026] Figure 1 1H NMR spectrum of estetrol prepared in Example 1;
[0027] Figure 2 13C NMR spectrum of estetrol prepared in Example 1;
[0028] Figure 3 Chromatogram of estetrol prepared in Example 2;
[0029] Figure 4 Chromatogram of estetrol prepared in Example 3;
[0030] Figure 5 Chromatogram of estetrol prepared in Example 4. Detailed Embodiments
[0031] The present invention provides a method for preparing estetrol, using the compound shown in Formula I as the starting material, and the synthesis route is as follows:
[0032]
[0033] Among them, R 1 is a hydroxyl group or a ketone group, and R 2 is hydrogen or an α-hydroxy group; the microorganisms include Saccharomyces cerevisiae, Aspergillus ochraceus, and Penicillium resedanum. The present invention discloses for the first time the preparation of estetrol from Formula I through a microbial fermentation conversion method, without involving dangerous reaction steps such as high temperature, high pressure, and explosion, and without the need to add other reagents for the reaction, which is safe and pollution-free.
[0034] In the present invention, when the R 1 is a ketone group, Saccharomyces cerevisiae is used for fermentation to reduce the ketone group to a hydroxyl group; when the R 2When it is hydrogen, Aspergillus ochraceus is used for C16α hydroxylation by fermentation; Penicillium restrictum is used for C15α hydroxylation by fermentation. The present invention studies and finds that Saccharomyces cerevisiae can reduce the ketone group at the C17 position, Aspergillus ochraceus has a specific transformation site and can achieve C16α hydroxylation, and Penicillium restrictum has a specific transformation site and can achieve C15α hydroxylation.
[0035] In the present invention, before using Saccharomyces cerevisiae for fermentation, Saccharomyces cerevisiae is first activated. The activation temperature is 30 - 32°C, and the activation time is 96 - 120 h, preferably 100 - 110 h. After the Saccharomyces cerevisiae is activated, the activated Saccharomyces cerevisiae is eluted with sterile water, and the eluate is inoculated into a liquid medium. The inoculation amount of the eluate is 8% - 10% of the volume of the liquid medium. After inoculation, it is cultured for 12 - 14 h, and Compound I of Formula I is added. The addition amount of Compound I of Formula I is 1% - 3% of the mass of the liquid medium, and the culture is continued until the conversion rate of Compound I of Formula I ≥ 98%. The culture conditions of Saccharomyces cerevisiae in the present invention are: the temperature is 30 - 32°C, and the pH is 6.2 - 6.4; the culture temperature is preferably 31°C, and the pH value is preferably 6.3. As an implementable way, the shaking speed is controlled at 130 - 180 rpm during the culture of Saccharomyces cerevisiae, preferably 150 rpm.
[0036] In the present invention, before using Aspergillus ochraceus for fermentation, Aspergillus ochraceus is first activated. The activation temperature is 26 - 28°C, and the activation time is 96 - 120 h, preferably 100 - 110 h. After the Aspergillus ochraceus is activated, the activated Aspergillus ochraceus is eluted with sterile water, and the eluate is inoculated into a liquid medium. The inoculation amount of the eluate is 8% - 10% of the volume of the liquid medium. After inoculation, it is cultured for 20 - 24 h, and Compound I of Formula I is added. The addition amount of Compound I of Formula I is 1% - 3% of the mass of the liquid medium, and the culture is continued until the conversion rate ≥ 98%. The culture conditions of Aspergillus ochraceus in the present invention are: the temperature is 26 - 28°C, and the pH is 6.8 - 7.0; the culture temperature is preferably 27°C, and the pH value is preferably 6.9. As an implementable way, the shaking speed is controlled at 180 - 220 rpm during the culture of Aspergillus ochraceus, preferably 200 rpm.
[0037] In the present invention, before using Penicillium restrictum for fermentation, Penicillium restrictum is first activated. The activation temperature is 28 - 30°C, and the activation time is 96 - 120 h, preferably 100 - 110 h. After the activation of Penicillium restrictum, the activated Penicillium restrictum is eluted with sterile water, and the eluate is inoculated into a liquid medium. The inoculation amount of the eluate is 8% - 10% of the mass of the liquid medium. After inoculation, it is cultured for 20 - 24 h, and a compound of formula I is added. The addition amount of the compound of formula I is 1% - 3% of the mass of the liquid medium, and the culture is continued until the conversion rate ≥ 98%. The culture conditions of Penicillium restrictum in the present invention are: the temperature is 28 - 30°C, and the pH is 7.0 - 7.5; the culture temperature is preferably 29°C, and the pH value is preferably 7.2 - 7.3 h. As an implementable method, when culturing Aspergillus ochraceus, the shaking speed of the shaker is controlled at 180 - 220 rpm, preferably 200 rpm.
[0038] In the specific embodiments of the present invention, the conversion rate is detected by HPLC.
[0039] In the present invention, the activation medium for Saccharomyces cerevisiae is: 2 g of glucose, 2 g of soy peptone, 1 g of yeast powder, 1.5 - 2.0 g of agar, 100 mL of water, and the pH value before sterilization is 6.0 - 6.2. The liquid medium for Saccharomyces cerevisiae is: 2.0 - 2.5 g of glucose, 2.0 - 2.5 g of soy peptone, 1.0 - 1.5 g of yeast powder, 0.02 - 0.03 mL of antifoam agent, 100 mL of water, and the pH value before sterilization is 5.6 - 5.8.
[0040] In the present invention, the activation medium for Aspergillus ochraceus is: 3 g of glucose, 2 g of corn steep liquor, 2.5 - 3.0 g of agar, 100 mL of water, and the pH value before sterilization is 6.8 - 7.0. The liquid medium for Aspergillus ochraceus is: 3.0 - 3.5 g of glucose, 2.0 - 2.5 g of corn steep liquor, 0.03 - 0.05 mL of antifoam agent, 100 mL of water, and the pH value before sterilization is 6.2 - 6.4.
[0041] In the present invention, the activation medium for Penicillium restrictum is: 2 g of glucose, 2 g of corn steep liquor, 1 g of yeast powder, 2.5 - 3.0 g of agar, 100 mL of water, and the pH value before sterilization is 7.0 - 7.2. The liquid medium for Penicillium restrictum is: 2.0 - 2.5 g of glucose, 2.0 - 2.5 g of corn steep liquor, 1.0 - 1.5 g of yeast powder, 0.03 - 0.05 mL of antifoam agent, 100 mL of water, and the pH value before sterilization is 6.6 - 6.8.
[0042] The sterilization method for the medium used in the present invention is: high-temperature moist heat sterilization at 121°C for 20 min.
[0043] In the present invention, the Saccharomyces cerevisiae, Aspergillus ochraceus, and Penicillium raistrickii can be fermented in any order. The microbial transformation sites of the present invention are specific, and fermentation can be carried out in any order to achieve C15α hydroxylation, C16α hydroxylation, and C17 keto reduction.
[0044] In the present invention, Saccharomyces cerevisiae, Aspergillus ochraceus, and Penicillium raistrickii can all be obtained through ordinary commercial channels.
[0045] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.
[0046] Example 1
[0047] A method for preparing estetrol, the synthesis route is as follows:
[0048]
[0049] In this example, the activation medium for Saccharomyces cerevisiae is: 2 g of glucose, 2 g of plant peptone, 1 g of yeast powder, 1.8 g of agar, 100 mL of water, and the pH value before sterilization is 6.2. The liquid medium for Saccharomyces cerevisiae is: 2 g of glucose, 2 g of plant peptone, 1 g of yeast powder, 0.02 mL of antifoam agent, 100 mL of water, and the pH value before sterilization is 5.8. Sterilize at 121 °C by high-temperature moist heat for 20 min.
[0050] The activation medium for Aspergillus ochraceus is: 3 g of glucose, 2 g of corn steep liquor, 3.0 g of agar, 100 mL of water, and the pH value before sterilization is 7.0. The liquid medium for Aspergillus ochraceus is: 3 g of glucose, 2 g of corn steep liquor, 0.03 mL of antifoam agent, 100 mL of water, and the pH value before sterilization is 6.4. Sterilize at 121 °C by high-temperature moist heat for 20 min.
[0051] The activation medium for Penicillium raistrickii is: 2 g of glucose, 2 g of corn steep liquor, 1 g of yeast powder, 2.5 g of agar, 100 mL of water, and the pH value before sterilization is 7.0. The liquid medium for Penicillium raistrickii is: 2 g of glucose, 2 g of corn steep liquor, 1 g of yeast powder, 0.03 mL of antifoam agent, 100 mL of water, and the pH value before sterilization is 6.8. Sterilize at 121 °C by high-temperature moist heat for 20 min.
[0052] Specifically, it includes the following steps:
[0053] (1) C15α-hydroxylation: Penicillium raistrickii was activated in an activation medium at 28 °C for 100 h. The activated Penicillium raistrickii was eluted with sterile water, and the eluate was inoculated into a liquid medium at an inoculation amount of 10% (v / v). After inoculation, it was cultured for 20 h. Estrone was added, and the addition amount of estrone was 3% (w / v). It was continuously cultured until the conversion rate ≥ 98%. The culture conditions were: temperature 29 °C, pH value 7.2, and shaker speed 200 rpm. When the conversion rate of estrone ≥ 98%, the reaction ended. Solid-liquid separation was carried out by suction filtration, and the collected solid was extracted with methanol. The amount of methanol used was 3 times the feeding amount of estrone. Extraction was repeated 3 times, and the 3 extraction solutions were collected. After mixing the extraction solutions, solid-liquid separation was carried out by suction filtration, and the liquid component was collected. The liquid component was concentrated, subjected to water precipitation, and dried to obtain Compound I.
[0054] (2) C17-ketone reduction: Saccharomyces cerevisiae was activated in an activation medium at 32 °C for 100 h. The activated Saccharomyces cerevisiae was eluted with sterile water, and the eluate was inoculated into a liquid medium at an inoculation amount of 10% (v / v). After inoculation, it was cultured for 12 h. Compound I was added, and the addition amount of Compound I was 3% (w / v). It was continuously cultured until the conversion rate ≥ 98%. The culture conditions were: temperature 31 °C, pH value 6.3, and shaker speed 150 rpm. When the conversion rate of Compound I ≥ 98%, the reaction ended. Solid-liquid separation was carried out by suction filtration, and the collected solid was extracted with methanol. The amount of methanol used was 3 times the feeding amount of Compound I. Extraction was repeated 3 times, and the 3 extraction solutions were collected. After mixing the extraction solutions, solid-liquid separation was carried out by suction filtration, and the liquid component was collected. The liquid component was concentrated, subjected to water precipitation, and dried to obtain Compound II.
[0055] (3) C16α-hydroxylation: Aspergillus ochraceus was activated in an activation medium at 27 °C for 100 h. The activated Aspergillus ochraceus was eluted with sterile water, and the eluate was inoculated into a liquid medium at an inoculation amount of 10% (v / v). After inoculation, it was cultured for 20 h. Compound II was added, and the addition amount of Compound II was 3% (w / v). It was continuously cultured until the conversion rate ≥ 98%. The culture conditions were: temperature 27 °C, pH value 6.9, and shaker speed 200 rpm. When the conversion rate of Compound II ≥ 98%, the reaction ended. Solid-liquid separation was carried out by suction filtration, and the collected solid was extracted with methanol. The amount of methanol used was 3 times the feeding amount of Compound II. Extraction was repeated 3 times, and the 3 extraction solutions were collected. After mixing the extraction solutions, solid-liquid separation was carried out by suction filtration, and the liquid component was collected. The liquid component was concentrated, subjected to water precipitation, and dried to obtain the product estetrol.
[0056] The 1H NMR spectrum of the estetrol prepared in this example is as Figure 1 shown, and the 13C NMR spectrum is as Figure 2 shown. HPLC content: 99.81%.
[0057] Example 2
[0058] A preparation method of estetrol, and the synthesis route is as follows:
[0059]
[0060] In this example, the activation medium of Aspergillus ochraceus, the liquid medium of Aspergillus ochraceus, the activation medium of Penicillium raistrickii, and the liquid medium of Penicillium raistrickii are the same as those in Example 1.
[0061] Specifically, it includes the following steps:
[0062] (1) C15α hydroxylation: Activate Penicillium raistrickii at 30 °C in the activation medium for 105 h. Wash the activated Penicillium raistrickii with sterile water, and inoculate the eluate into the liquid medium with an inoculation amount of 10% (v / v). After inoculation, incubate for 20 h, then add estradiol with an input amount of 3% (w / v), and continue to incubate until the conversion rate ≥ 98%. The incubation conditions are: temperature 28 °C, pH value 7.0, and shaker speed 200 rpm. When the conversion rate ≥ 98%, the reaction ends. Perform solid-liquid separation by suction filtration through a 0.22 μm filter membrane, collect the solid, extract it with methanol, and the amount of methanol used is 3 times the input amount of estradiol. Repeat the extraction 3 times, collect the 3 extraction solutions, mix the extraction solutions, perform solid-liquid separation by suction filtration through a 0.22 μm filter membrane, collect the liquid component, concentrate the liquid component by water precipitation, and obtain Compound II after drying.
[0063] (2) C16α hydroxylation: Activate Aspergillus ochraceus at 26 °C in the activation medium for 110 h. Wash the activated Aspergillus ochraceus with sterile water, and inoculate the eluate into the liquid medium with an inoculation amount of 10% (v / v). After inoculation, incubate for 20 h, then add Compound II with an input amount of 3% (w / v), and continue to incubate until the conversion rate ≥ 98%. The incubation conditions are: temperature 26 °C, pH value 6.8, and shaker speed 200 rpm. When the conversion rate ≥ 98%, the reaction ends. Perform solid-liquid separation by suction filtration, collect the solid, extract it with methanol, and the amount of methanol used is 3 times the input amount of Compound II. Repeat the extraction 3 times, collect the 3 extraction solutions, mix the extraction solutions, perform solid-liquid separation by suction filtration, collect the liquid component, concentrate the liquid component by water precipitation, and obtain the product estetrol after drying.
[0064] The chromatogram of estetrol prepared in this example is as Figure 3 shown, HPLC content: 99.88%.
[0065] Example 3
[0066] A preparation method of estetrol, and the synthesis route is as follows:
[0067]
[0068] In this example, the activation medium and liquid medium of Penicillium raistrickii are the same as those in Example 1.
[0069] Specifically, it includes the following steps:
[0070] C15α hydroxylation: Activate Penicillium raistrickii at 29 °C in the activation medium for 96 h. Wash the activated Penicillium raistrickii with sterile water, and inoculate the eluate into the liquid medium with an inoculation amount of 10% (v / v). After inoculation, incubate for 24 h, then add estriol with an input amount of 3% (w / v), and continue to incubate until the conversion rate ≥ 98%. The incubation conditions are: temperature 30 °C, pH value 7.4, and shaker speed 200 rpm. When the conversion rate ≥ 98%, the reaction ends. Perform solid-liquid separation by suction filtration, collect the solid and extract it with methanol. The amount of methanol used is 3 times the input amount of estriol. Repeat the extraction 3 times, collect the 3 extraction solutions, mix the extraction solutions and then perform solid-liquid separation by suction filtration to collect the liquid component. Concentrate and precipitate the liquid component with water, and dry it to obtain the product estetrol.
[0071] The chromatogram of estetrol prepared in this example is as Figure 4 shown, HPLC content: 99.82%.
[0072] Example 4
[0073] A method for preparing estetrol, the synthesis route is as follows:
[0074]
[0075] In this example, the activation medium of Saccharomyces cerevisiae, the liquid medium of Saccharomyces cerevisiae, the activation medium of Aspergillus ochraceus, the liquid medium of Aspergillus ochraceus, the activation medium of Penicillium raistrickii, and the liquid medium of Penicillium raistrickii are the same as those in Example 1.
[0076] Specifically, it includes the following steps:
[0077] (1) C15α hydroxylation: Activate Penicillium raistrickii at 28 °C in the activation medium for 100 h. Wash the activated Penicillium raistrickii with sterile water, and inoculate the eluate into the liquid medium with an inoculation amount of 10% (v / v). After inoculation, incubate for 20 h, then add estrone with an input amount of 3% (w / v), and continue to incubate until the conversion rate ≥ 98%. The incubation conditions are: temperature 29 °C, pH value 7.2, and shaker speed 200 rpm. When the conversion rate ≥ 98%, the reaction ends. Perform solid-liquid separation by suction filtration, collect the solid and extract it with methanol. The amount of methanol used is 3 times the input amount of estrone. Repeat the extraction 3 times, collect the 3 extraction solutions, mix the extraction solutions and then perform solid-liquid separation by suction filtration to collect the liquid component. Concentrate and precipitate the liquid component with water, and dry it to obtain Compound I.
[0078] (2) C16α-Hydroxylation: Aspergillus ochraceus was activated in the activation medium at 28 °C for 115 h. The activated Aspergillus ochraceus was eluted with sterile water, and the eluate was inoculated into the liquid medium at an inoculation amount of 10% (v / v). After inoculation, after culturing for 22 h, Compound I was added, and the addition amount of Compound I was 3% (w / v). Then, the culture was continued until the conversion rate ≥ 98%. The culture conditions were: temperature 28 °C, pH value 7.0, and shaker speed 200 rpm. When the conversion rate ≥ 98%, the reaction ended. Solid-liquid separation was carried out by suction filtration, and the collected solid was extracted with methanol. The amount of methanol used was 3 times the feeding amount of Compound I. The extraction was repeated 3 times, and the 3 extraction solutions were collected. After mixing the extraction solutions, solid-liquid separation was carried out by suction filtration, and the liquid component was collected. The liquid component was concentrated, subjected to water precipitation, and dried to obtain Compound III.
[0079] (3) C17-Ketone Reduction: Saccharomyces cerevisiae was activated in the activation medium at 30 °C for 96 h. The activated Saccharomyces cerevisiae was eluted with sterile water, and the eluate was inoculated into the liquid medium at an inoculation amount of 10% (v / v). After inoculation, after culturing for 14 h, Compound III was added, and the addition amount of Compound III was 3% (w / v). Then, the culture was continued until the conversion rate ≥ 98%. The culture conditions were: temperature 30 °C, pH value 6.2, and shaker speed 150 rpm. When the conversion rate ≥ 98%, the reaction ended. Solid-liquid separation was carried out by suction filtration, and the collected solid was extracted with methanol. The amount of methanol used was 3 times the feeding amount of Compound III. The extraction was repeated 3 times, and the 3 extraction solutions were collected. After mixing the extraction solutions, solid-liquid separation was carried out by suction filtration, and the liquid component was collected. The liquid component was concentrated, subjected to water precipitation, and dried to obtain the product estetrol.
[0080] The chromatogram of estetrol prepared in this example is as Figure 5 shown, HPLC content: 99.45%.
[0081] The above is only the preferred embodiment of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.
Claims
1. 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 microorganisms include Saccharomyces cerevisiae, Aspergillus ochraceus and Penicillium reis.
2. The method for preparing estetrol according to claim 1, characterized in that: When the R1 is a keto group, the keto group is reduced to a hydroxyl group by fermentation with Saccharomyces cerevisiae; when the R2 is hydrogen, Aspergillus ochraceus is used for fermentation to hydroxylate C16α; and Penicillium reis is used for fermentation to hydroxylate C15α.
3. The method for preparing estetrol according to claim 1, characterized in that: The fermentation conditions using the brewer's yeast include: a temperature of 30-32° C. and a pH of 6.2-6.
4.
4. The method for preparing estetrol according to claim 3, characterized in that: The brewer's yeast fermentation medium includes: 20-25 g / L glucose, 20-25 g / L soy peptone, 10-15 g / L yeast powder, and 0.2-0.3 mL / L buprenorphine.
5. The method for preparing estetrol according to claim 1, characterized in that: The conditions for fermenting with the Aspergillus ochraceus include: a temperature of 26-28° C. and a pH of 6.8-7.
0.
6. The method for preparing estetrol according to claim 5, characterized in that: The Aspergillus ochraceus fermentation medium used is: 30-35 g / L of glucose, 20-25 g / L of corn steep liquor, and 0.3-0.5 mL / L of buprofezin.
7. The method for preparing estetrol according to claim 1, characterized in that: The conditions for fermentation using the Penicillium rapae include: a temperature of 28-30° C. and a pH of 7.0-7.
5.
8. The method for preparing estetrol according to claim 7, characterized in that: The fermentation medium of Penicillium reishi is as follows: 20-25 g / L glucose, 20-25 g / L corn steep liquor, 10-15 g / L yeast powder and 0.3-0.5 mL / L foetida.
9. The method for preparing estetrol according to claim 1, characterized in that: The Saccharomyces cerevisiae, Aspergillus ochraceus and Aspergillus ochraceus may be fermented in any order.