An enzyme-catalyzed synthesis system of levonorgestrel intermediate and its application
By using a specific defoaming agent in the enzymatic synthesis of a levonorgestrel intermediate, the problem of low solubility of ethyl hydroxylate was solved, the conversion rate and yield of the product were improved, and high-purity industrial production was achieved.
Patent Information
- Application Number
- CN202210591486.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-05-27
- Publication Date
- 2025-09-26
- Estimated Expiration
- 2042-05-27
AI Technical Summary
In the existing enzymatic synthesis method of levonorgestrel intermediates, the solubility of ethyl hydroxylate in water is low, resulting in insufficient contact between the substrate and the enzyme solution, making it difficult to separate the product from the surfactant, reducing the yield and increasing production costs.
By using specific defoaming agent adjuvants such as polysiloxane defoamers, polyoxypropylene glycerol ether defoamers or composite high-efficiency defoamers, the product is peeled off from the emulsified state into an insoluble powdery solid through emulsification, while keeping the substrate unaffected. Combined with the catalytic reaction of ketoreductase, glucose dehydrogenase and NADP sodium salt, the conversion rate and yield are improved.
The high purity and high yield of the product are achieved, which is suitable for industrial production. The yield is increased to 90-95%, which is significantly better than the system without defoaming agent additives.
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Abstract
Description
Technical Field
[0001] The present invention relates to the pharmaceutical field, and in particular to an enzyme-catalyzed synthesis system of a levonorgestrel intermediate and its application. Background Art
[0002] Levonorgestrel, whose structure is shown in Formula 1, is a synthetic steroid compound primarily used as a contraceptive for visiting relatives and as an emergency contraceptive. It can also be used to treat irregular menstruation, dysfunctional uterine bleeding, and endometriosis.
[0003]
[0004] Formula 1
[0005] Ethyl hydroxylate is a key intermediate of levonorgestrel. Currently, there are two methods for its synthesis: cell-catalyzed and enzyme-catalyzed, as shown in Formula 2. Cell-catalyzed production utilizes brewer's yeast, but this method is not suitable for industrial production due to its poor selectivity, demanding culture conditions, long reaction times, and low yields. Enzyme-catalyzed production utilizes ketoreductase, which offers high selectivity, short reaction times, mild conditions, and high yields.
[0006]
[0007] Formula 2
[0008] However, the solubility of ethyl hydroxylate in water is low, and the enzyme solution mainly exists in the aqueous phase, resulting in insufficient contact between the substrate ethyl condensate and the enzyme. To address this, surfactants are generally added to promote complete substrate conversion, but this increases the solubility of the product in the aqueous phase, making it difficult to separate the product and surfactant, reducing the final yield and increasing production costs. Summary of the Invention
[0009] The purpose of the present invention is to provide an enzyme-catalyzed synthesis system of a levonorgestrel intermediate and a method for catalytically synthesizing the levonorgestrel intermediate ethyl hydroxylate.
[0010] To achieve the above object, the technical solution adopted by the present invention is:
[0011] An enzymatic synthesis system for a levonorgestrel intermediate ethyl hydroxylate, wherein the concentrations of the components are as follows:
[0012]
[0013] According to the above scheme, the defoaming agent auxiliary agent in the above enzyme-catalyzed synthesis system is selected from polyether defoamers, polysiloxane defoamers, esterified defoamers, polyoxypropylene glycerol ether defoamers, and composite high-efficiency defoamers. Preferably, the defoamer is polysiloxane defoamer, polyoxypropylene glycerol ether defoamer, or composite high-efficiency defoamer, and more preferably, the composite high-efficiency defoamer.
[0014] The composite high-efficiency defoamer is a composite defoamer obtained by mixing one or more of polysiloxane defoamers and polyoxypropylene glycerol ether defoamers with one or more of polysiloxane defoamers and esterified defoamers.
[0015] The application of the above enzyme-catalyzed synthesis system in the enzymatic synthesis of levonorgestrel intermediate ethyl hydroxylate is as follows: adding a substrate ethyl condensate to the enzyme-catalyzed synthesis system, adding an enzyme solution for enzymatically synthesizing the levonorgestrel intermediate ethyl hydroxylate, and catalytically synthesizing the levonorgestrel intermediate ethyl hydroxylate.
[0016] The invention discloses an enzymatic synthesis method for a levonorgestrel intermediate ethyl hydroxylate. The method comprises adding ketoreductase, glucose dehydrogenase and NADP sodium salt to the above-mentioned enzymatic synthesis system for the levonorgestrel intermediate ethyl hydroxylate, carrying out a catalytic reaction, and filtering to obtain a solid ethyl hydroxylate product.
[0017] The enzyme-catalyzed reaction is carried out at 25-35° C. for 24-48 hours.
[0018] The invention discloses an enzyme-catalyzed synthesis reaction solution for a levonorgestrel intermediate ethyl hydroxylate, which comprises the above-mentioned enzyme-catalyzed synthesis system, an enzyme solution for enzymatically synthesizing the levonorgestrel intermediate ethyl hydroxylate, and a substrate ethyl condensate.
[0019] According to the above scheme, the substrate concentration in the system is 50-100 g / L.
[0020] The enzyme solution is an aqueous solution of ketoreductase, glucose dehydrogenase and NADP sodium salt, wherein the ketoreductase in the system is 3-4 g / L, the glucose dehydrogenase is 0.5-1.0 g / L and the NADP sodium salt is 0.15-0.20 g / L.
[0021] The preparation steps of the above-mentioned enzyme-catalyzed synthesis reaction solution include: adding dipotassium hydrogen phosphate, potassium dihydrogen phosphate and glucose in solid form, mixing with a certain amount of water, adding Tween-60, Triton X-100 and a defoaming agent under stirring, and finally adding the substrate and enzyme solution to obtain an enzyme-catalyzed synthesis reaction solution for synthesizing the levonorgestrel intermediate ethyl hydroxylate through an enzyme-catalyzed reaction.
[0022] The enzymatic synthesis system and preparation method of the levonorgestrel intermediate described herein have not been reported to date. By selecting a specific defoaming agent and combining it with other components (dipotassium hydrogen phosphate and potassium dihydrogen phosphate are primarily responsible for maintaining pH stability in the enzyme-catalyzed reaction system and buffering pH fluctuations caused by products during the reaction, glucose provides hydrogen power for the reaction, and Tween 60 and Triton X-100 are primarily responsible for emulsifying the substrate), the enzymatic synthesis system for the levonorgestrel intermediate ethyl hydroxylate provided herein achieves high conversion and yield for the synthesis of the levonorgestrel intermediate ethyl hydroxylate.
[0023] Defoamers are commonly used as foam suppressants in fermentation reactions. Therefore, the enzyme-catalyzed reaction system of the present invention does not require the addition of a defoamer. However, we unexpectedly discovered that the addition of a specific defoamer aid to the system of the present invention disrupts the emulsification of the product. This allows the generated ethyl hydroxylate to be separated from the emulsified state under the action of the defoamer aid, forming an insoluble powdery solid product. Simultaneously, the substrate ethyl condensate is unaffected by the defoamer and does not produce other negative effects. This achieves the goal of promoting product migration in the positive direction while simultaneously improving the reaction conversion rate and yield.
[0024] The present invention uses ethyl condensate as a substrate and emulsifies it into emulsion droplets through the emulsification effect of surfactants Tween 60 and Triton X-100, thereby increasing the contact area with the aqueous phase. The catalyst ketoreductase catalyzes the removal of hydrogen ions from NADPH to produce the product ethyl hydroxylate. NADP, which has lost its hydrogen ions, is catalytically oxidized to NADPH under the action of glucose dehydrogenase, completing the cycle of the enzyme-catalyzed reaction. During this process, the generated ethyl hydroxylate is stripped from the emulsified state under the action of a defoamer to form an insoluble powdery solid product. At the same time, the substrate ethyl condensate is not affected by the defoamer, not only not affecting the reaction, but also promoting the reaction. The reaction mechanism is shown in FIG. Figure 1 shown.
[0025] The advantages of the present invention are:
[0026] The enzyme-catalyzed reaction system of the present invention produces a product with high purity and yield, and can be directly used in industrial production. Compared with a system without a defoaming agent (yield of 81.8%), the yield is significantly increased to approximately 90-95%. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 It is a mechanism diagram of the enzyme-catalyzed reaction system.
[0028] Figure 2 It is the enzyme catalytic system without adding defoamer and with different types of defoamers, and the foaming and sedimentation of the material after the reaction. DETAILED DESCRIPTION
[0029] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content of the present invention record, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the application's appended claims.
[0030] Comparative Example 1
[0031] Weigh 30.8 g of glucose, 2.36 g of dipotassium hydrogen phosphate, and 0.62 g of potassium dihydrogen phosphate into a three-necked flask. Add 400 mL of water and stir for 0.5 h. Add 0.05 g of Tween 60 and 10.8 g of Triton X-100 to the stirring system. After the reaction mixture is thoroughly stirred, add 20 g of ethyl condensate, 1.2 g of ketoreductase, 0.2 g of glucose dehydrogenase, and 0.06 g of NADP sodium salt. React at 30°C for 24 h. Filter the product solid and dry it to obtain 16.36 g of the product with an 81.8% yield and 98.4% purity.
[0032] Example 1
[0033] Weigh 30.8 g of glucose, 2.36 g of dipotassium hydrogen phosphate, and 0.62 g of potassium dihydrogen phosphate into a three-necked flask. Add 400 mL of water and stir for 0.5 h. Add 0.05 g of Tween 60 and 10.8 g of Triton X-100 to the stirring system, followed by 0.04 g of a polyether defoamer. After the reaction mixture is thoroughly stirred, add 20 g of ethyl condensate, 1.2 g of ketoreductase, 0.2 g of glucose dehydrogenase, and 0.06 g of NADP sodium salt. Reaction is carried out at 30°C for 24 h. The product solid is filtered and dried to yield 17.80 g of the product with an 89.0% yield and 98.6% purity.
[0034] Example 2
[0035] Weigh 30.8 g of glucose, 2.36 g of dipotassium hydrogen phosphate, and 0.62 g of potassium dihydrogen phosphate into a three-necked flask. Add 400 mL of water and stir for 0.5 h. Add 0.05 g of Tween 60 and 10.8 g of Triton X-100 to the stirring system, followed by 0.04 g of a polysiloxane defoamer. After the reaction mixture is thoroughly stirred, add 20 g of ethyl condensate, 1.2 g of ketoreductase, 0.2 g of glucose dehydrogenase, and 0.06 g of NADP sodium salt. Reaction is carried out at 30°C for 24 h. The product solid is filtered and dried to yield 18.58 g of the product with a yield of 92.9% and a purity of 98.7%.
[0036] Example 3
[0037] Weigh 30.8 g of glucose, 2.36 g of dipotassium hydrogen phosphate, and 0.62 g of potassium dihydrogen phosphate into a three-necked flask. Add 400 mL of water and stir for 0.5 h. Add 0.05 g of Tween 60 and 10.8 g of Triton X-100 to the stirring system, followed by 0.04 g of an esterified defoamer. After the reaction mixture is thoroughly stirred, add 20 g of ethyl condensate, 1.2 g of ketoreductase, 0.2 g of glucose dehydrogenase, and 0.06 g of NADP sodium salt. React at 30°C for 24 h. Filter the resulting solid and dry it to obtain 18.00 g of the product, with a yield of 90.0% and a purity of 98.9%.
[0038] Example 4
[0039] Weigh 30.8 g of glucose, 2.36 g of dipotassium hydrogen phosphate, and 0.62 g of potassium dihydrogen phosphate into a three-necked flask. Add 400 mL of water and stir for 0.5 h. Add 0.05 g of Tween 60 and 10.8 g of Triton X-100 to the stirring system, followed by 0.04 g of polyoxypropylene glycerol ether defoamer. After the reaction mixture is thoroughly stirred, add 20 g of ethyl condensate, 1.2 g of ketoreductase, 0.2 g of glucose dehydrogenase, and 0.06 g of NADP sodium salt. React at 30°C for 24 h. Filter the product solid and dry it to obtain 19.00 g of the product with a yield of 95.0% and a purity of 98.9%.
[0040] Example 5
[0041] 30.8 g of glucose, 2.36 g of dipotassium hydrogen phosphate, and 0.62 g of potassium dihydrogen phosphate were weighed and added to a three-necked flask. 400 mL of water was then added and stirred for 0.5 h. 0.05 g of Tween 60 and 10.8 g of Triton X-100 were added to the stirring system, followed by 0.04 g of a composite high-efficiency defoamer (THI X-298, purchased from Yantai Hengxin Chemical Technology Co., Ltd.). After the reaction mixture was thoroughly stirred, 20 g of ethyl condensate, 1.2 g of ketoreductase, 0.2 g of glucose dehydrogenase, and 0.06 g of NADP sodium salt were added. The reaction was carried out at 30°C for 24 h. The product solid was filtered and dried to obtain 19.00 g of the product with a yield of 95.5% and a purity of 99.5%.
[0042] Example 6
[0043] 46.2 g of glucose, 0.72 g of dipotassium hydrogen phosphate, and 0.93 g of potassium dihydrogen phosphate were weighed and added to a three-necked flask. 400 mL of water was then added and stirred for 0.5 h. 0.075 g of Tween 60 and 16.2 g of Triton X-100 were added to the stirring system, followed by 0.06 g of a composite high-efficiency defoamer (THI X-298, purchased from Yantai Hengxin Chemical Technology Co., Ltd.). After the reaction mixture was thoroughly stirred, 40 g of ethyl condensate, 1.2 g of ketoreductase, 0.2 g of glucose dehydrogenase, and 0.06 g of NADP sodium salt were added. The reaction was carried out at 30°C for 24 h. The product solid was filtered and dried to obtain 38.08 g of the product with a yield of 95.2% and a purity of 99.6%.
[0044] Example 7
[0045] 46.2 g of glucose, 0.72 g of dipotassium hydrogen phosphate, and 0.93 g of potassium dihydrogen phosphate were weighed and added to a three-necked flask. 400 mL of water was then added and stirred for 0.5 h. 0.075 g of Tween 60 and 16.2 g of Triton X-100 were added to the stirring system, followed by 0.06 g of a composite high-efficiency defoamer (THI X-298, purchased from Yantai Hengxin Chemical Technology Co., Ltd.). After the reaction mixture was thoroughly stirred, 20 g of ethyl condensate, 1.6 g of ketoreductase, 0.4 g of glucose dehydrogenase, and 0.08 g of NADP sodium salt were added. The reaction was carried out at 30°C for 24 h. The product solid was filtered and dried to obtain 19.08 g of the product with a yield of 95.4% and a purity of 99.5%.
[0046] Comparative Example 2
[0047] Weigh 30.8 g of glucose, 2.36 g of dipotassium hydrogen phosphate, and 0.62 g of potassium dihydrogen phosphate into a three-necked flask. Add 400 mL of water and stir for 0.5 h. Add 0.05 g of Tween 60 and 10.8 g of Triton X-100 to the stirring system, followed by 0.04 g of a natural oil-based defoamer. After the reaction mixture is thoroughly stirred, add 20 g of ethyl condensate, 1.2 g of ketoreductase, 0.2 g of glucose dehydrogenase, and 0.06 g of NADP sodium salt. Reaction is carried out at 30°C for 24 h. The product solid is filtered and dried to yield 16.03 g of the product with an 80.2% yield and 98.4% purity.
[0048] Comparative Example 3
[0049] Weigh 30.8 g of glucose, 2.36 g of dipotassium hydrogen phosphate, and 0.62 g of potassium dihydrogen phosphate into a three-necked flask. Add 400 mL of water and stir for 0.5 h. Add 0.05 g of Tween 60 and 10.8 g of Triton X-100 to the stirring system, followed by 0.04 g of polyaluminum chloride (PAC). After the reaction mixture is thoroughly stirred, add 20 g of ethyl condensate, 1.2 g of ketoreductase, 0.2 g of glucose dehydrogenase, and 0.06 g of NADP sodium salt. Stop stirring and allow the substrate to completely precipitate. React at 30°C for 24 h. Filter the solid product and dry it to yield 19.8 g of the product with a purity of 4%.
[0050] Comparison revealed that different defoamer additives added to the system resulted in different foaming and settling behaviors after the reaction. Foaming clearly detrimentally impacted product yield, while better settling performance indicated improved product separation from the emulsified system. Natural oil-based defoamers exhibited the worst demulsification performance, leaving the product in an emulsified state.
[0051] Further comparison shows that the defoamer auxiliary agent of the present invention is preferably polysiloxane defoamer, polyoxypropylene glycerol ether defoamer, or composite high-efficiency defoamer, and more preferably composite high-efficiency defoamer. The composite high-efficiency defoamer has the best product sedimentation effect and the highest yield.
[0052] The ketoreductase, glucose dehydrogenase and NADP sodium salt used in the present invention were purchased from Suzhou Hanzyme Biotechnology Co., Ltd.
Claims
1. An enzyme-catalyzed synthesis system for levonorgestrel intermediate ethyl hydroxylate, characterized in that: The concentrations of the components are as follows: Among them: dipotassium hydrogen phosphate 1.800-5.900 g / L; Potassium dihydrogen phosphate 0.775-2.325 g / L; Glucose 38.500-115.500 g / L; Tween 600.100-0.188 g / L; Triton X-100 13.500-40.500 g / L; Defoamer additive 0.050-0.150 g / L; The enzymatic synthesis of the intermediate ethyl hydroxylate of levonorgestrel uses the ethyl condensate as a substrate; the enzymes for the enzymatic synthesis of the intermediate ethyl hydroxylate of levonorgestrel are ketoreductase, glucose dehydrogenase and NADP sodium salt; The defoamer auxiliary agent is selected from polyether defoamers, polysiloxane defoamers, esterified defoamers, polyoxypropylene glycerol ether defoamers or composite high-efficiency defoamers. The composite high-efficiency defoamer is a composite defoamer obtained by mixing one or more of polysiloxane defoamers and polyoxypropylene glycerol ether defoamers with one or more of polysiloxane defoamers and esterified defoamers.
2. The enzyme-catalyzed synthesis system according to claim 1, characterized in that: The defoamer auxiliary agent is a polysiloxane defoamer, a polyoxypropylene glycerol ether defoamer or a composite high-efficiency defoamer.
3. Use of the enzymatic synthesis system of claim 1 in the enzymatic synthesis of levonorgestrel intermediate ethyl hydroxylate, the application method comprising: adding a substrate ethyl condensate to the enzymatic synthesis system of claim 1, and adding an enzyme solution for enzymatically synthesizing the levonorgestrel intermediate ethyl hydroxylate, to catalyze the synthesis of the levonorgestrel intermediate ethyl hydroxylate.
4. An enzymatic synthesis method of levonorgestrel intermediate ethyl hydroxylate, characterized in that: The substrate ethyl condensate, ketoreductase, glucose dehydrogenase and NADP sodium salt are added to the enzyme catalytic synthesis system according to claim 1, catalyzed reaction is carried out, and the product ethyl hydroxylate solid is obtained by filtration.
5. The enzymatic synthesis method according to claim 4, wherein: The enzyme-catalyzed reaction is carried out at 25-35° C. for 24-48 hours.
6. A reaction solution for the enzymatic synthesis of levonorgestrel intermediate ethyl hydroxylate, characterized in that: The invention comprises the enzyme catalytic synthesis system according to claim 1, an enzyme solution for enzymatically synthesizing the levonorgestrel intermediate ethyl hydroxylate, and a substrate ethyl condensate.
7. The enzyme-catalyzed synthesis reaction solution according to claim 6, characterized in that The enzyme solution for the enzyme-catalyzed synthesis of the levonorgestrel intermediate ethyl hydroxylate is an aqueous solution of ketone reductase, glucose dehydrogenase and NADP sodium salt, wherein the ketone reductase in the system is 3-4 g / L, the glucose dehydrogenase is 0.5-1.0 g / L, and the NADP sodium salt is 0.15-0.20 g / L; and the substrate concentration in the system is 50-100 g / L.
8. The enzyme-catalyzed synthesis reaction solution according to claim 6, characterized in that: Dipotassium hydrogen phosphate, potassium dihydrogen phosphate and glucose are added in solid form, mixed with a certain amount of water, and then Tween-60, Triton X-100 and a defoaming agent are added under stirring. Finally, the substrate ethyl condensate and enzyme solution are added to obtain an enzyme-catalyzed synthesis reaction solution for synthesizing the levonorgestrel intermediate ethyl hydroxylate through an enzyme-catalyzed reaction.
Citation Information
Patent Citations
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