Enzymatic synthesis of 12beta-hydroxycinobufagin

Abstract

The application relates to an enzyme synthesis method of 12beta-hydroxycinobufagin and belongs to the technical field of chemical synthesis. In view of the technical problem that the separation yield of the natural product is extremely low in the prior art, and androstenone is used as a raw material to obtain a C17 position Suzuki coupling precursor through three-step chemical conversion; then a specific enzyme is used for an oxidation reaction to stereospecifically introduce C12beta and C15alpha hydroxyl groups; and then the compound is first fully synthesized through steps such as Suzuki coupling, elimination, Diels Alder reaction, rearrangement, reduction, acetylation and deprotection. The application has the advantages of easy-to-obtain raw materials, short synthesis route, high yield, good stereoselectivity and the like, and provides a new way for efficient preparation of active natural products of bufadienolides.

Description

Technical Field

[0001] This invention belongs to the field of chemical synthesis technology and relates to an enzyme synthesis method for 12β-hydroxycinobufagin. Background Technology

[0002] The traditional Chinese medicine "Toad Venom" is made from the skin secretions of the Chinese giant toad (Bufo gargarizans Cantor) or the black-spotted toad (Bufo melanostrictus Schneider). The main bioactive components of Toad Venom are bufo lactones, which have steroidal A / B cis and C / D cis ring structures, containing a 17β-2-pyran ring, and possess activities such as cardiotonic, blood pressure-raising, blood pressure-lowering, anesthetic, analgesic, and antitumor effects. However, due to the complex preparation process of Toad Venom, it is difficult to guarantee reliable quality during production, and the increasingly scarce wild animal resources also significantly restrict the pharmacological research and clinical application of Toad Venom. The composition of Toad Venom is complex, with some active components antagonistic to each other, or possessing good physiological activity but high toxicity. Therefore, the artificial synthesis of certain physiologically active components in Toad Venom is particularly important. Currently, there are two main approaches to obtaining these compounds: one is direct extraction from toad skin secretions; the other is to start from simple steroidal compounds and obtain bufo lactones through multiple chemical transformations.

[0003] 12β-hydroxycinobufagin, isolated from toad venom in 1978, exhibits cytotoxic activity against human liver cancer cells (HepG2, SMMC-7221, and BEL-7402) and human leukemia cells (K562, HL60, and HEL) (steroids 71 ​​(2006) 392–402). However, the isolation yield was only 0.001% (J. Nat. Prod. 2001, 64, 1148-1152), lacking practicality and economic viability. In 2004, Dean Guo et al. (APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2004, p. 3521–3527) obtained this compound using microbial transformation, but the substrate cinobufagin used was also a natural product. To date, no chemical synthesis method for this natural product has been reported. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide an enzyme synthesis method for 12β-hydroxycinobufagin.

[0005] To achieve the above objectives, the present invention provides the following technical solution: The enzyme used in this invention is derived from a published patent (application publication number: CN118360264A).

[0006] This invention provides an enzyme synthesis method for 12β-hydroxycinobufagin, the steps of which are as follows: S1: Compound 4 was dissolved in DMF solvent and added to PBS to re-rotate bacterial cells. The reaction was carried out at 30°C and 250 rpm for 4-5 days. The bacterial cells were extracted with ethyl acetate, and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography to obtain compound 5. The chemical reaction formula is as follows: ; S2: Under nitrogen protection, compound 5, DMF, 2-pyrrole-5-boronate, K3PO4, and dppfCl2Pd were added sequentially to a round-bottom flask. The mixture was stirred at 80°C for 12 hours, and the system was allowed to cool naturally to room temperature. The reaction mixture was filtered, and the resulting mixture was extracted with ethyl acetate. The combined organic layers were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The crude product was subjected to silica gel column chromatography to obtain compound 6, the chemical reaction of which is as follows: ; S3: Compound 6 was dissolved in methanol, p-toluenesulfonic acid was added, the mixture was heated to 50°C and stirred for 2 hours. After the reaction was completed, saturated sodium bicarbonate solution was added to quench the reaction. The mixture was extracted with ethyl acetate and washed with saturated sodium chloride solution. The organic layer was dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain compound 7, whose chemical reaction formula is as follows: ; S4: Compound 7, TBSCl, DMF, and imidazole were added sequentially to a round-bottom flask. The reaction system was stirred at 40°C for 24 hours. After the reactants had completely reacted, the reaction was quenched with saturated NaHCO3 solution. The aqueous phase was extracted with dichloromethane, and the resulting organic phase was washed with saturated sodium chloride, dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the organic solvent. The crude product was purified by silica gel column chromatography to obtain compound 8, whose chemical reaction formula is as follows: ; S5: Add sodium bicarbonate and methylene blue to a round-bottom flask, seal the flask with a rubber stopper, and place the reaction system under a nitrogen atmosphere. Then add compound 8 dissolved in anhydrous dichloromethane. Cool the reaction mixture to -78°C and replace it with oxygen. Irradiate the reaction system with a 60W LED lamp at -78°C until the reactants are completely consumed. After turning off the light source, maintain the reaction system at -78°C and replace it with a nitrogen atmosphere. Dissolve tetraphenylcobalt porphyrin in anhydrous dichloromethane and add it dropwise to the reaction system. Stir at -78°C for 15 minutes, then heat the reaction mixture to room temperature and concentrate it under reduced pressure. The crude product is purified by silica gel column chromatography to obtain compound 9, whose chemical reaction formula is as follows: ; S6: Under nitrogen protection, compound 9, anhydrous dichloromethane, and scandium trifluoromethanesulfonate were added to a round-bottom flask. The reaction mixture was stirred at room temperature for 10 minutes, then filtered through diatomaceous earth, washed with dichloromethane, and concentrated under reduced pressure to obtain the crude product, compound 10. Compound 10 was dissolved in anhydrous tetrahydrofuran and methanol, then sodium borohydride was added. After the reaction was complete, the reaction was quenched with saturated ammonium chloride aqueous solution and extracted with ethyl acetate. The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain compound 11, whose chemical reaction formula is as follows: ; S7: Compound 11 was dissolved in anhydrous pyridine, followed by the addition of anhydrous acetic anhydride. The reaction mixture was reacted at room temperature for 6 hours until the starting material was completely consumed. A saturated aqueous sodium bicarbonate solution was then added, followed by extraction with ethyl acetate. The organic phase was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain compound 12, whose chemical reaction formula is as follows: ; S8: Compound 12 was dissolved in anhydrous pyridine, then transferred to a plastic bottle and cooled to 0°C. A 50% aqueous hydrofluoric acid solution was then added dropwise. The mixture was heated to room temperature for 48 hours, after which the reaction was quenched with a saturated sodium bicarbonate solution. The mixture was extracted with ethyl acetate, the organic phase was washed with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain the natural product 13, 12β-hydroxycinobufagin, with the following chemical reaction formula: .

[0007] Preferably, the method for synthesizing compound 4 is as follows: Compound 3 was dissolved in ethanol, and 85% hydrazine hydrate and triethylamine were added sequentially. The reaction was carried out at 70°C for 12 hours. After the reaction was completed, the solvent was removed by vacuum distillation to obtain a white solid. The white solid was dissolved in anhydrous tetrahydrofuran, and triethylamine was added at 0°C. Then, iodine granules were added to the reaction system one by one until the solution turned reddish-brown and did not fade. The reaction was continued for 30 minutes, and the reaction was quenched by adding saturated sodium thiosulfate solution. The mixture was extracted with ethyl acetate, and the organic phase was dried with anhydrous sodium sulfate. After filtration, the organic solvent was removed by vacuum distillation. The crude product was purified by silica gel column chromatography to obtain compound 4, whose chemical reaction formula is as follows: .

[0008] Preferably, the method for synthesizing compound 3 is as follows: Compound 2 was dissolved in anhydrous tetrahydrofuran. The reaction system was cooled to -78 °C, and 1 mL of Selectride THF solution was added dropwise. The reaction was carried out at this temperature for 3 hours. The reaction was quenched by adding saturated ammonium chloride solution. The reaction solution was extracted with ethyl acetate, and the organic phase was washed with saturated sodium chloride solution. After drying with anhydrous sodium sulfate and filtration, the organic solvent was removed by concentration under reduced pressure. The crude product was purified by silica gel column chromatography to obtain compound 3, whose chemical reaction formula is as follows: .

[0009] Preferably, the method for synthesizing compound 2 is as follows: Compound 1 androstenedione and 5% Pd / C were added to a round-bottom flask. Anhydrous pyridine was added under nitrogen protection, and then the nitrogen was replaced with hydrogen. The reaction was carried out at room temperature for 24 hours. The solution was filtered through a funnel lined with diatomaceous earth and washed with ethyl acetate. The organic phases were combined and concentrated under vacuum. The crude product was purified by silica gel column chromatography to give compound 2, whose chemical reaction formula is as follows: .

[0010] Preferred: In step S1, the ratio of compound 4 to DMF solvent is 150 mg: 1 mL; In step S2, the ratio of compound 5: 2-pyrrole-5-boronate: K3PO4: dppfCl2Pd is 1 mmol: 1.5 mmol: 0.1 mmol; In step S3, compound 6: p-toluenesulfonic acid is 1 mmol: 5 mmol; In step S4, compound 7: TBSCl: imidazole is 1 mmol: 5 mmol: 8 mmol; In step S5, compound 8: methylene blue: sodium bicarbonate: tetraphenylcobalt porphyrin is present in a ratio of 1 mmol: 0.1 mmol: 2 mmol: 0.1 mmol. In step S6, the ratio of compound 9: anhydrous dichloromethane: scandium trifluoromethanesulfonate is 150 mg: 6 mL: 11.5 mg; In step S7, compound 11: anhydrous pyridine: anhydrous acetic anhydride was prepared in a ratio of 1 mmol: 35.7 mL: 5 mmol. In step S8, the ratio of compound 12: anhydrous pyridine: hydrofluoric acid is 60 mg: 6 mL: 1 mL.

[0011] Preferred, In step S1, the purification mobile phase is dichloromethane and methanol in a volume ratio of 30:1. In step S2, the purification mobile phase is dichloromethane and methanol in a volume ratio of 20:1; In step S3, the mobile phase for purification is petroleum ether and ethyl acetate in a volume ratio of 1:1. In step S4, the mobile phase for purification is petroleum ether and ethyl acetate in a volume ratio of 10:1. In step S5, the mobile phase for purification is petroleum ether and ethyl acetate in a volume ratio of 2:1. In step S6, the purification mobile phase is petroleum ether and ethyl acetate in a volume ratio of 2:1; In step S7, the mobile phase for purification is petroleum ether and ethyl acetate in a volume ratio of 4:1. In step S8, the purification mobile phase is dichloromethane and methanol in a volume ratio of 20:1.

[0012] Preferably, the ratio of compound 3, hydrazine hydrate and triethylamine is 1 mmol:10 mmol:10 mmol, the ratio of anhydrous tetrahydrofuran, triethylamine and iodine particles is 100 mL:14.37 mL:13.12 g, and the purification mobile phase is petroleum ether and ethyl acetate in a volume ratio of 8:1.

[0013] Preferably, the ratio of compound 2, anhydrous tetrahydrofuran, and THF solution is 9.87 g: 41.09 mL, and the purification mobile phase is petroleum ether and ethyl acetate in a volume ratio of 2:1.

[0014] Preferably, the ratio of compound 1 to 5% Pd / C is 10 g: 2 g, and the purification mobile phase is petroleum ether and ethyl acetate in a volume ratio of 4:1.

[0015] The beneficial effects of this invention are as follows: The purpose of this invention is to provide a chemical-enzymatic synthesis method for the natural product 12β-hydroxycinobufagin. This invention has advantages such as readily available raw materials, high yield, simple post-processing, and a concise synthetic route. Raw materials are inexpensive and readily available: This invention uses the inexpensive and readily available steroidal compound androstenedione as a raw material. It is inexpensive and widely available, which greatly reduces costs and resource dependence compared to direct extraction from toad secretions or the use of the natural product cinobufagin as a substrate.

[0016] The synthetic route is concise and efficient: This invention obtains a precursor for the Suzuki coupling reaction at C-17 through three simple chemical transformations; then, a stereospecific oxidation reaction with an enzyme yields a dihydroxylated product with β-hydroxylation at C-12 and α-hydroxylation at C-15; a carbon-carbon bond is constructed at C-17 via the Suzuki coupling reaction; after elimination of the hydroxyl group at C-15, a diepoxide is obtained via the Diels-Alder reaction; and subsequent rearrangement, reduction, acetylation, and deprotection achieve the total chemical-enzymatic synthesis of the natural product 12β-hydroxycinobufagin.

[0017] The post-processing is simple and suitable for scale-up. Most steps use conventional column chromatography for purification, and enzyme conversion uses resting Saccharomyces cerevisiae cells. The operating conditions are mild, and the post-processing extraction, filtration and other operations are simple, making it a potential industrial application.

[0018] In summary, this invention provides an economical, efficient, stereoselective, and scalable 12β... The artificial synthesis method of hydroxycinobufagin solves the problems of scarce natural product sources and extremely low extraction yield, and is of great significance for the pharmacological research and clinical application of toad lactone active compounds.

[0019] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0020] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein: Figure 1 The 1H NMR spectrum (400MHz, CDCl3) of 12β-hydroxycinobufagin of this invention is shown. Detailed Implementation

[0021] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of the present invention. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0022] The accompanying drawings are for illustrative purposes only and are schematic diagrams, not actual pictures. They should not be construed as limiting the invention. To better illustrate the embodiments of the invention, some parts in the drawings may be omitted, enlarged, or reduced, and do not represent the actual product dimensions. It is understandable to those skilled in the art that some well-known structures and their descriptions may be omitted in the drawings.

[0023] In the accompanying drawings of the embodiments of the present invention, the same or similar reference numerals correspond to the same or similar components. In the description of the present invention, it should be understood that if terms such as "upper," "lower," "left," "right," "front," and "rear" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, they are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, the terms used to describe positional relationships in the drawings are only for illustrative purposes and should not be construed as limiting the present invention. For those skilled in the art, the specific meaning of the above terms can be understood according to the specific circumstances.

[0024] Example 1

[0025] Anhydrous pyridine (500 mL) was added to a 1 L round-bottom flask containing compound 1 androstenedione 1 (10.0 g, 34.94 mmol) and 5% Pd / C (2.0 g) under nitrogen protection. The nitrogen atmosphere was then replaced with a hydrogen balloon. The reaction was carried out at room temperature for 24 hours. After the reaction was complete, the solution was filtered through a funnel lined with diatomaceous earth and washed with ethyl acetate (300 mL). The organic phases were combined and concentrated under vacuum. The crude product was subjected to silica gel column chromatography (mobile phase: petroleum ether and ethyl acetate, v / v) to give compound 2 (9.87 g, 98%) as a white solid. 1 H NMR (400 MHz, Chloroform- d )δ 2.73 – 2.61 (m, 1H), 2.46 (dd, J=19.2, 8.2 Hz, 1H), 2.31 (td, J = 14.7, 5.4 Hz, 1H), 2.22 – 2.14 (m, 1H), 2.09 – 1.90 (m, 4H), 1.89 – 1.82 (m, 2H), 1.73 – 1.58 (m, 4H), 1.58 – 1.48 (m, 2H), 1.46 – 1.28 (m, 5H), 1.18 (td, J = 13.3, 3.0 Hz, 1H), 1.05 (s, 3H), 0.89 (s, 3H).

[0026] Compound 2 (9.87 g, 34.24 mmol) was dissolved in 200 mL of anhydrous tetrahydrofuran. The reaction mixture was cooled to -78 °C, and 1 mL of Selectride THF solution (41.09 mL, 41.09 mmol) was added dropwise. The reaction was allowed to proceed at this temperature for 3 hours. Upon completion of the reaction, 50 mL of saturated ammonium chloride solution was added to quench the reaction. The reaction solution was extracted with 300 mL of ethyl acetate, and the organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the organic solvent. The crude product was subjected to silica gel column chromatography (mobile phase: petroleum ether and ethyl acetate, v / v) to give compound 3 (7.25 g, 73%) as a white solid.

[0027] 1 H NMR (400 MHz, Chloroform- d )δ 4.12 (p, J = 2.9 Hz, 1H), 2.44 (ddd, J = 19.2, 8.8, 1.1 Hz, 1H), 2.06 (dt, J = 19.2, 9.0 Hz, 1H), 2.00 – 1.88 (m,3H), 1.83 – 1.74 (m, 2H), 1.62 – 1.44 (m, 9H), 1.34 (tdd, J = 11.6, 5.1, 2.8Hz, 3H), 1.26 – 1.20 (m, 2H), 1.19 – 1.07 (m, 1H), 0.99 (s, 3H), 0.86 (s, 3H).

[0028] Compound 3 (6.0 g, 20.67 mmol) was dissolved in 100 mL of ethanol. Then, 11.86 mL of 85% hydrazine hydrate and 28.74 mL of triethylamine were added sequentially. The reaction was carried out at 70 °C for 12 hours. After the reaction was completed, the solvent was removed by vacuum distillation to obtain a white solid. The white solid was dissolved in 100 mL of anhydrous tetrahydrofuran. 14.37 mL of triethylamine was added at 0 °C, followed by the addition of 13.12 g of iodine granules one by one until the solution turned reddish-brown and did not fade. The reaction was continued for another 30 minutes. The reaction was quenched with saturated sodium thiosulfate solution, extracted with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate. After filtration, the organic solvent was removed under reduced pressure. The crude product was subjected to silica gel column chromatography (mobile phase: petroleum ether and ethyl acetate, volume ratio 8:1) to obtain a white solid compound 4 (6.86 g, 83%).

[0029] 1 H NMR (400 MHz, Chloroform- d )δ 6.11 (dd, J = 3.3, 1.7 Hz, 1H), 4.11(t, J = 2.9 Hz, 1H), 2.12 (ddd, J = 14.9, 6.3, 3.2 Hz, 1H), 2.03 – 1.88 (m,3H), 1.75 (dt, J = 11.7, 3.2 Hz, 1H), 1.61 (dd, J = 11.5, 5.2 Hz, 2H), 1.51(dt, J = 9.1, 4.5 Hz, 5H), 1.45 – 1.31 (m, 5H), 1.24 – 1.09 (m, 3H), 0.99 (s, 3H), 0.71 (s, 3H).

[0030] Compound 4 was subjected to large-scale fermentation using the method described above. The specific procedures are as follows: 100 mL of 40% sterile glucose solution was added to 2 L of PBS-re-vortexed bacterial cells. Compound 4 (600.0 mg, 1.50 mmol) was dissolved in 4 mL of DMF solvent and added to the PBS-re-vortexed bacterial cells. The reaction was carried out at 30 °C and 250 rpm for four to five days. The bacterial cells were extracted with ethyl acetate (3 × 800 mL), and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography (mobile phase: dichloromethane and methanol, v / v) to give a white solid compound 5 (317.6 mg, 49%). 1 H NMR (400 MHz, Methanol- d 4) δ 6.13 (s, 1H), 4.48 (d, J = 8.9 Hz, 1H), 4.05 (d, J = 2.8 Hz, 1H), 3.66 (dd, J = 10.6, 5.3 Hz, 1H), 1.98 (qd, J = 13.9,6.2 Hz, 2H), 1.86 – 1.65 (m, 4H), 1.59 (td, J = 11.6, 10.6, 4.7 Hz, 2H), 1.48(td, J = 11.1, 10.4, 3.1 Hz, 3H), 1.33 (ddd, J = 18.6, 12.3, 9.9 Hz, 4H), 1.25– 1.17 (m, 1H), 1.03 (s, 3H), 0.87 (s, 3H).

[0031] Under nitrogen protection, compound 5 (280 mg, 0.65 mmol), DMF (6 mL), 2-pyrrole-5-boronate (92.6 mg, 0.98 mmol), K3PO4 (482.9 mg, 2.28 mmol), and dppfCl2Pd (47.4 mg, 0.06 mmol) were added sequentially to a 100 mL round-bottom flask. After the addition was complete, the mixture was stirred at 80 °C for 12 hours, then allowed to cool naturally to room temperature. The reaction mixture was filtered, and the resulting mixture was extracted with ethyl acetate. The combined organic layers were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The crude product was subjected to silica gel column chromatography (mobile phase: dichloromethane and methanol, v / v) to give compound 6 (221.1 mg, 85%) as a white solid.

[0032] 1 H NMR (400 MHz, Methanol- d 4)δ 7.91 (d, J = 2.9 Hz, 1H), 7.71 (dd, J =9.7, 2.6 Hz, 1H), 6.29 (d, J = 9.7 Hz, 1H), 5.78 (d, J = 1.6 Hz, 1H), 4.63 –4.60 (m, 1H), 4.07 (d, J = 2.8 Hz, 1H), 3.76 (dd, J = 10.6, 5.1 Hz, 1H), 2.08 –1.94 (m, 2H), 1.81 (td, J = 9.1, 7.5, 5.1 Hz, 3H), 1.71 (dt, J = 12.8, 4.9 Hz,1H), 1.66 – 1.56 (m, 2H), 1.50 (ddd, J = 11.1, 7.8, 3.1 Hz, 3H), 1.39 (ddd, J =15.5, 12.0, 5.7 Hz, 4H), 1.29 – 1.22 (m, 1H), 1.05 (s, 3H), 1.03 (s, 3H).

[0033] Compound 6 (205.0 mg, 0.51 mmol) was dissolved in methanol (10 mL), and p-toluenesulfonic acid (352.8 mg, 2.05 mmol) was added. The resulting mixture was heated to 50°C and stirred for 2 hours. After the reaction was complete, the reaction was quenched by adding saturated sodium bicarbonate solution (20 mL). The mixture was extracted with ethyl acetate (30 mL) and washed with saturated sodium chloride solution. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was subjected to silica gel column chromatography (mobile phase: petroleum ether and ethyl acetate, v / v) to give compound 7 (179.3 mg, 92%) as a white solid.

[0034] 1 H NMR (400 MHz, Methanol- d 4)δ 8.34 (d, J= 3.1 Hz, 1H), 7.97 (dd, J =9.8, 2.7 Hz, 1H), 6.71 (d, J = 2.6 Hz, 1H), 6.33 (d, J = 9.9 Hz, 1H), 6.03 (d, J = 2.2 Hz, 1H), 4.02 (d, J = 3.1 Hz, 1H), 3.03 (dd, J = 11.2, 4.3 Hz, 1H), 2.32(tt, J = 9.5, 3.4 Hz, 1H), 2.07 (tt, J = 13.2, 5.0 Hz, 1H), 1.97 – 1.87 (m,1H), 1.87 – 1.81 (m, 1H), 1.69 (dd, J = 10.5, 6.2 Hz, 3H), 1.58 – 1.53 (m,1H), 1.46 (s, 3H), 1.36 (d, J = 8.3 Hz, 2H), 1.33 – 1.27 (m, 2H), 1.09 (s, 3H), 1.08 (s, 3H).

[0035] At room temperature, compound 7 (160.0 mg, 0.42 mmol), TBSCl (315.0 mg, 2.09 mmol), DMF (8 mL), and imidazole (228.0 mg, 3.35 mmol) were added sequentially to a 100 mL round-bottom flask. The reaction system was stirred at 40 °C for 24 hours. After the starting materials had completely reacted, the reaction was quenched with saturated NaHCO3 solution, and the aqueous phase was extracted with dichloromethane (45 mL). The resulting organic phase was washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the organic solvent. The crude product was subjected to silica gel column chromatography (mobile phase: petroleum ether and ethyl acetate, v / v) to give compound 8 (202.5 mg, 79%) as a white solid.

[0036] 1 H NMR (400 MHz, Benzene- d 6)δ 7.90 (d, J = 3.2 Hz, 1H), 7.03 (dd, J=9.9, 2.7 Hz, 1H), 6.20 (d, J = 2.3 Hz, 1H), 6.07 (dd, J = 9.9, 1.2 Hz, 1H), 5.91 (t, J = 2.1 Hz, 1H), 3.92 (d, J = 3.1 Hz, 1H), 3.06 (dd, J = 11.1, 4.1 Hz, 1H), 2.13 (dq, J = 11.9, 6.6, 4.3 Hz, 1H), 1.96 (d, J = 16.7 Hz, 1H), 1.88 (dt, J = 13.4, 4.4 Hz, 1H), 1.74 – 1.59 (m, 4H), 1.53 – 1.47 (m, 1H), 1.42 – 1.36(m, 3H), 1.28 – 1.20 (m, 3H), 1.13 (d, J = 2.8 Hz, 1H), 1.06 (s, 3H), 1.01 (s,9H), 0.92 (s, 3H), 0.90 (s, 9H), 0.08 (d, J = 4.4 Hz, 6H), -0.06 (s, 3H), -0.12 (s, 3H).

[0037] Sodium bicarbonate (49.5 mg, 0.59 mmol) and methylene blue (9.4 mg, 0.03 mmol) were added to a 100 mL round-bottom flask. The flask was sealed with a rubber stopper, and the reaction mixture was placed under a nitrogen atmosphere. Compound 8 (180.0 mg, 0.29 mmol) dissolved in anhydrous dichloromethane (10 mL) was then added. The reaction mixture was cooled to -78 °C and purged with oxygen. The reaction mixture was irradiated with an LED lamp at -78 °C until the reactants were completely consumed. After turning off the light source, the reaction mixture was maintained at -78 °C under a nitrogen atmosphere. Tetraphenylcobalt porphyrin (20.1 mg, 0.03 mmol) was dissolved in anhydrous dichloromethane (2 mL). It was then added dropwise to the reaction mixture using a syringe. After stirring at -78 °C for 15 minutes, the reaction mixture was heated to room temperature and concentrated under reduced pressure. The crude product was subjected to silica gel column chromatography (mobile phase: petroleum ether and ethyl acetate, volume ratio 2:1) to give compound 9 (163.9 mg, 88%) as a white solid.

[0038] 1 H NMR (400 MHz, Chloroform- d )δ 7.43 (d, J = 2.7 Hz, 1H), 7.36 (dd, J =9.6, 2.6 Hz, 1H), 6.27 (d, J = 9.7 Hz, 1H), 4.04 (t, J = 2.8 Hz, 1H), 3.74 (dd, J = 11.6, 4.0 Hz, 1H), 3.55 (s, 2H), 1.88 – 1.69 (m, 5H), 1.67 – 1.60 (m,1H), 1.56 – 1.48 (m, 1H), 1.45 – 1.33 (m, 4H), 1.29 – 1.23 (m, 1H), 1.24 (s,3H), 1.21 – 1.13 (m, 2H), 0.90 (s, 3H), 0.88 (s, 9H), 0.83 – 0.80 (m,1H),0.78 (s, 9H), 0.02 (d, J = 2.1 Hz, 6H), -0.05 (s, 3H), -0.11 (s, 3H).

[0039] Under nitrogen protection, compound 9 (150.0 mg, 0.23 mmol), anhydrous dichloromethane (6 mL), and scandium trifluoromethanesulfonate (11.5 mg, 0.02 mmol) were added to a 100 mL round-bottom flask. The reaction mixture was stirred at room temperature for 10 minutes, filtered through diatomaceous earth, washed with dichloromethane, and concentrated under reduced pressure to give crude product 10. The crude product was dissolved in anhydrous tetrahydrofuran (8 mL) and methanol (8 mL), and sodium borohydride (13.1 mg, 0.35 mmol) was added. After the reaction was complete, the reaction was quenched with saturated ammonium chloride aqueous solution and extracted with ethyl acetate (3 × 15 mL). The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure. The crude product was subjected to silica gel column chromatography (mobile phase: petroleum ether and ethyl acetate, volume ratio 2:1) to give a white solid compound 11 (137.8 mg, 93%).

[0040] 1 H NMR (400 MHz, Chloroform- d)δ 7.92 (dd, J = 9.7, 2.6 Hz, 1H), 7.22(s, 1H), 6.27 – 6.16 (m, 1H), 4.67 (dd, J = 9.4, 1.3 Hz, 1H), 4.17 – 3.95 (m,1H), 3.60 – 3.42 (m, 1H), 3.42 – 3.26 (m, 1H), 3.05 (d, J = 9.4 Hz, 1H), 1.98(td, J = 11.9, 3.7 Hz, 1H), 1.87 – 1.78 (m, 2H), 1.74 (t, J = 11.9 Hz, 1H),1.67 – 1.58 (m, 2H), 1.55 (dd, J = 11.8, 3.6 Hz, 1H), 1.37 (d, J = 12.2 Hz, 4H), 1.18 (d, J = 15.5 Hz, 3H), 0.98 – 0.92 (m, 1H), 0.95 (s, 3H), 0.89 (d, J =7.5 Hz, 18H), 0.73 (s, 3H), 0.09 (s, 3H), 0.06 (s, 3H), 0.02 (d, J = 1.5 Hz, 6H).

[0041] Compound 11 (90 mg, 0.14 mmol) was dissolved in anhydrous pyridine (5 mL) at room temperature, followed by the addition of anhydrous acetic anhydride (66.0 μL, 0.70 mmol). The reaction mixture was reacted at room temperature for 6 hours until the starting material was completely consumed. A saturated aqueous sodium bicarbonate solution was then added, followed by extraction with ethyl acetate. The organic phase was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was subjected to silica gel column chromatography (mobile phase: petroleum ether and ethyl acetate, v / v) to give compound 12 (92.3 mg, 96%) as a white solid.

[0042] 1 H NMR (400 MHz, Chloroform- d )δ 7.99 (s, 1H), 7.08 (s, 1H), 6.20 (d, J= 9.8 Hz, 1H), 5.39 (d, J = 9.3 Hz, 1H), 4.05 (s, 1H), 3.62 (s, 1H), 3.38 (dd, J = 11.1, 4.3 Hz, 1H), 3.21 (d, J = 9.4 Hz, 1H), 2.00 (td, J = 11.9, 3.7 Hz,1H), 1.90 (s, 3H), 1.84 (d, J = 14.7 Hz, 2H), 1.73 (ddd, J = 14.8, 12.9, 2.4Hz, 1H), 1.68 – 1.57 (m, 3H), 1.53 (dd, J = 12.1, 2.9 Hz, 1H), 1.50 – 1.33 (m,5H), 1.26 – 1.14 (m, 2H), 0.95 (s, 3H), 0.90 (s, 9H), 0.88 (s, 9H), 0.74 (s,3H), 0.13 (s, 3H), 0.07 (s, 3H), 0.02 (d, J = 2.1 Hz, 6H).

[0043] Compound 12 (60.0 mg, 0.09 mmol) was dissolved in 6 mL of anhydrous pyridine, then transferred to a plastic bottle with a screw cap and cooled to 0 °C. 1 mL of hydrofluoric acid (50% in water) was then added dropwise, and the mixture was heated to room temperature after complete addition. After 48 hours, the consumption of the starting material was confirmed by thin-layer chromatography, and the reaction was quenched with 50 mL of saturated sodium bicarbonate solution. The mixture was extracted with ethyl acetate, the organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was subjected to silica gel column chromatography (mobile phase: dichloromethane and methanol, v / v) to give a white solid, natural product 13 (29.7 mg, 72%).

[0044] 1 H NMR (400 MHz, Methanol- d 4)δ 7.99 (s, 1H), 7.37 (s, 1H), 6.24 (d, J =9.8 Hz, 1H), 5.46 (d, J= 9.3 Hz, 1H), 4.05 (s, 1H), 3.73 (s, 1H), 3.48 (dd, J = 11.4, 4.1 Hz, 1H), 3.43 (d, J = 9.4 Hz, 1H), 2.07 – 1.89 (m, 3H), 1.86 (s, 3H), 1.83 – 1.71 (m, 3H), 1.67 – 1.58 (m, 1H), 1.57 – 1.43 (m, 4H), 1.42 –1.29 (m, 2H), 1.25 – 1.17 (m, 1H), 1.11 – 1.02 (m, 1H), 1.00 (s, 3H), 0.74(s, 3H). Example 2 Anhydrous pyridine (500 mL) was added to a 1 L round-bottom flask containing compound 1 androstenedione 1 (10.0 g, 34.92 mmol) and 5% Pd / C (2.0 g) under nitrogen protection. The nitrogen atmosphere was then replaced with a hydrogen balloon. The reaction was carried out at room temperature for 24 hours. After the reaction was complete, the solution was filtered through a funnel lined with diatomaceous earth and washed with ethyl acetate (300 mL). The organic phases were combined and concentrated under vacuum. The crude product was subjected to silica gel column chromatography (mobile phase: petroleum ether and ethyl acetate, v / v) to give compound 2 (9.86 g, 98%) as a white solid. Compound 2 (9.87 g, 34.24 mmol) was dissolved in 200 mL of anhydrous tetrahydrofuran. The reaction mixture was cooled to -78 °C, and 1 mL of Selectride THF solution (41.09 mL, 41.09 mmol) was added dropwise. The reaction was allowed to proceed at this temperature for 3 hours. Upon completion of the reaction, 50 mL of saturated ammonium chloride solution was added to quench the reaction. The reaction solution was extracted with 300 mL of ethyl acetate, and the organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the organic solvent. The crude product was subjected to silica gel column chromatography (mobile phase: petroleum ether and ethyl acetate, v / v) to give compound 3 (7.25 g, 73%) as a white solid.

[0045] Compound 3 (6.0 g, 20.67 mmol) was dissolved in 100 mL of ethanol. Then, 11.86 mL of 85% hydrazine hydrate and 28.74 mL of triethylamine were added sequentially. The reaction was carried out at 70 °C for 12 hours. After the reaction was completed, the solvent was removed by vacuum distillation to obtain a white solid. The white solid was dissolved in 100 mL of anhydrous tetrahydrofuran. 14.37 mL of triethylamine was added at 0 °C, followed by the addition of 13.12 g of iodine granules one by one until the solution turned reddish-brown and did not fade. The reaction was continued for another 30 minutes. The reaction was quenched with saturated sodium thiosulfate solution, extracted with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate. After filtration, the organic solvent was removed under reduced pressure. The crude product was subjected to silica gel column chromatography (mobile phase: petroleum ether and ethyl acetate, volume ratio 8:1) to obtain a white solid compound 4 (6.86 g, 83%).

[0046] Compound 4 was subjected to large-scale fermentation using the method described above. The specific procedures are as follows: 150 mL of 40% sterile glucose solution was added to 3 L of PBS-re-vortexed bacterial cells. Compound 4 (900.0 mg, 2.25 mmol) was dissolved in 6 mL of DMF solvent and added to the PBS-re-vortexed bacterial cells. The reaction was carried out at 30 °C and 250 rpm for four to five days. The bacterial cells were extracted with ethyl acetate (3 × 1000 mL), and the solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography (mobile phase: dichloromethane and methanol, v / v ratio 30:1) to give a white solid compound 5 (476.4 mg, 49%). Under nitrogen protection, compound 5 (420 mg, 0.98 mmol), DMF (9 mL), 2-pyrrole-5-boronate (138.9 mg, 1.47 mmol), K3PO4 (725.4 mg, 3.42 mmol), and dppfCl2Pd (71.1 mg, 0.09 mmol) were added sequentially to a 100 mL round-bottom flask. After the addition was complete, the mixture was stirred at 80 °C for 12 hours, then allowed to cool naturally to room temperature. The reaction mixture was filtered, and the resulting mixture was extracted with ethyl acetate. The combined organic layers were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The crude product was subjected to silica gel column chromatography (mobile phase: dichloromethane and methanol, v / v) to give compound 6 (331.7 mg, 85%) as a white solid.

[0047] Compound 6 (307.5 mg, 0.77 mmol) was dissolved in methanol (15 mL), and p-toluenesulfonic acid (529.2 mg, 3.08 mmol) was added. The resulting mixture was heated to 50°C and stirred for 2 hours. After the reaction was complete, the reaction was quenched with saturated sodium bicarbonate solution (30 mL). The mixture was extracted with ethyl acetate (45 mL) and washed with saturated sodium chloride solution. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was subjected to silica gel column chromatography (mobile phase: petroleum ether and ethyl acetate, v / v) to give compound 7 (269 mg, 92%) as a white solid.

[0048] At room temperature, compound 7 (240.0 mg, 0.63 mmol), TBSCl (472.5.0 mg, 3.14 mmol), DMF (12 mL), and imidazole (342.0 mg, 5.03 mmol) were added sequentially to a 100 mL round-bottom flask. The reaction system was stirred at 40 °C for 24 hours. After the starting materials had completely reacted, the reaction was quenched with saturated NaHCO3 solution, and the aqueous phase was extracted with dichloromethane (68 mL). The resulting organic phase was washed with saturated sodium chloride, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the organic solvent. The crude product was subjected to silica gel column chromatography (mobile phase: petroleum ether and ethyl acetate, v / v) to give compound 8 (303.8 mg, 79%) as a white solid.

[0049] Sodium bicarbonate (1.37 mg, 0.89 mmol) and methylene blue (14.1 mg, 0.045 mmol) were added to a 100 mL round-bottom flask. The flask was sealed with a rubber stopper, and the reaction mixture was placed under a nitrogen atmosphere. Compound 8 (270.0 mg, 0.44 mmol) dissolved in anhydrous dichloromethane (15 mL) was then added. The reaction mixture was cooled to -78 °C and purged with oxygen. The reaction mixture was irradiated with a 60W LED lamp at -78 °C until the reactants were completely consumed. After turning off the light source, the reaction mixture was maintained at -78 °C under a nitrogen atmosphere. Tetraphenylcobalt porphyrin (30.2 mg, 0.045 mmol) was dissolved in anhydrous dichloromethane (3 mL). It was then added dropwise to the reaction mixture using a syringe. After stirring at -78 °C for 15 minutes, the reaction mixture was heated to room temperature and concentrated under reduced pressure. The crude product was subjected to silica gel column chromatography (mobile phase: petroleum ether and ethyl acetate, volume ratio 2:1) to give compound 9 (245.9 mg, 88%) as a white solid.

[0050] Under nitrogen protection, compound 9 (225 mg, 0.35 mmol), anhydrous dichloromethane (9 mL), and scandium trifluoromethanesulfonate (17.3 mg, 0.03 mmol) were added to a 100 mL round-bottom flask. The reaction mixture was stirred at room temperature for 10 minutes, filtered through diatomaceous earth, washed with dichloromethane, and concentrated under reduced pressure to give crude product 10. The crude product was dissolved in anhydrous tetrahydrofuran (12 mL) and methanol (12 mL), and sodium borohydride (19.7 mg, 0.53 mmol) was added. After the reaction was complete, the reaction was quenched with saturated ammonium chloride aqueous solution and extracted with ethyl acetate (3 × 25 mL). The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure. The crude product was subjected to silica gel column chromatography (mobile phase: petroleum ether and ethyl acetate, volume ratio 2:1) to give a white solid compound 11 (206.7 mg, 93%).

[0051] Compound 11 (135 mg, 0.21 mmol) was dissolved in anhydrous pyridine (7.5 mL) at room temperature, followed by the addition of anhydrous acetic anhydride (99.0 μL, 1.05 mmol). The reaction mixture was reacted at room temperature for 6 hours until the starting material was completely consumed. A saturated aqueous sodium bicarbonate solution was then added, followed by extraction with ethyl acetate. The organic phase was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was subjected to silica gel column chromatography (mobile phase: petroleum ether and ethyl acetate, v / v) to give compound 12 (138.5 mg, 96%) as a white solid.

[0052] Compound 12 (90.0 mg, 0.135 mmol) was dissolved in 9 mL of anhydrous pyridine, then transferred to a plastic bottle with a screw cap and cooled to 0 °C. 1.5 mL of hydrofluoric acid (50% in water) was then added dropwise, and the mixture was heated to room temperature after complete addition. After 48 hours, the consumption of the starting material was confirmed by thin-layer chromatography, and the reaction was quenched with 80 mL of saturated sodium bicarbonate solution. The mixture was extracted with ethyl acetate, the organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was subjected to silica gel column chromatography (mobile phase: dichloromethane and methanol, v / v) to give a white solid, natural product 13 (44.6 mg, 72%).

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. 12 β-hydroxycinobufagin enzyme synthesis method, characterized by: The steps are as follows: S1: Compound 4 was dissolved in DMF solvent and added to PBS to re-vortex bacterial cells. The reaction was carried out at 30°C and 250 rpm for 4-5 days. After centrifugation of the fermentation broth, the bacterial cells were extracted with ethyl acetate, and the organic solvent was removed by rotary evaporation. The crude product was purified by silica gel column chromatography to obtain compound 5. The chemical reaction formula is as follows: ； S2: Under nitrogen protection, compound 5, DMF, 2-pyrrole-5-boronate, K3PO4, and dppfCl2Pd were added sequentially to a round-bottom flask. The mixture was stirred at 80°C for 12 hours, and the system was allowed to cool naturally to room temperature. The reaction mixture was filtered, and the resulting mixture was extracted with ethyl acetate. The combined organic layers were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the solvent was removed under reduced pressure. The crude product was subjected to silica gel column chromatography to obtain compound 6, the chemical reaction of which is as follows: ； S3: Compound 6 was dissolved in methanol, p-toluenesulfonic acid was added, the mixture was heated to 50°C and stirred for 2 hours. After the reaction was completed, saturated sodium bicarbonate solution was added to quench the reaction. The mixture was extracted with ethyl acetate and washed with saturated sodium chloride solution. The organic layer was dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain compound 7, whose chemical reaction formula is as follows: ； S4: Compound 7, TBSCl, DMF, and imidazole were added sequentially to a round-bottom flask. The reaction system was stirred at 40°C for 24 hours. After the reactants had completely reacted, the reaction was quenched with saturated NaHCO3 solution. The aqueous phase was extracted with dichloromethane, and the resulting organic phase was washed with saturated sodium chloride, dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the organic solvent. The crude product was purified by silica gel column chromatography to obtain compound 8, whose chemical reaction formula is as follows: ； S5: Add sodium bicarbonate and methylene blue to a round-bottom flask, seal the flask with a rubber stopper, and place the reaction system under a nitrogen atmosphere. Then add compound 8 dissolved in anhydrous dichloromethane. Cool the reaction mixture to -78°C and replace it with oxygen. Irradiate the reaction system with a 60W LED lamp at -78°C until the reactants are completely consumed. After turning off the light source, maintain the reaction system at -78°C and replace it with a nitrogen atmosphere. Dissolve tetraphenylcobalt porphyrin in anhydrous dichloromethane and add it dropwise to the reaction system. Stir at -78°C for 15 minutes, then heat the reaction mixture to room temperature and concentrate it under reduced pressure. The crude product is purified by silica gel column chromatography to obtain compound 9, whose chemical reaction formula is as follows: ； S6: Under nitrogen protection, compound 9, anhydrous dichloromethane, and scandium trifluoromethanesulfonate were added to a round-bottom flask. The reaction mixture was stirred at room temperature for 10 minutes, then filtered through diatomaceous earth, washed with dichloromethane, and concentrated under reduced pressure to obtain the crude product, compound 10. Compound 10 was dissolved in anhydrous tetrahydrofuran and methanol, then sodium borohydride was added. After the reaction was complete, the reaction was quenched with saturated ammonium chloride aqueous solution and extracted with ethyl acetate. The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and then concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain compound 11, whose chemical reaction formula is as follows: ； S7: Compound 11 was dissolved in anhydrous pyridine, followed by the addition of anhydrous acetic anhydride. The reaction mixture was reacted at room temperature for 6 hours until the starting material was completely consumed. A saturated aqueous sodium bicarbonate solution was then added, followed by extraction with ethyl acetate. The organic phase was washed with a saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain compound 12, whose chemical reaction formula is as follows: ； S8: Compound 12 was dissolved in anhydrous pyridine, then transferred to a plastic bottle and cooled to 0°C. A 50% aqueous hydrofluoric acid solution was then added dropwise. The mixture was heated to room temperature for 48 hours, after which the reaction was quenched with a saturated sodium bicarbonate solution. The mixture was extracted with ethyl acetate, the organic phase was washed with a saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography to obtain the natural product 13, 12β-hydroxycinobufagin, with the following chemical reaction formula: 。 2. The enzymatic synthesis method for 12β-hydroxycinobufagin according to claim 1, characterized in that, The method for synthesizing compound 4 is as follows: Compound 3 was dissolved in ethanol, and 85% hydrazine hydrate and triethylamine were added sequentially. The reaction was carried out at 70°C for 12 hours. After the reaction was completed, the solvent was removed by vacuum distillation to obtain a white solid. The white solid was dissolved in anhydrous tetrahydrofuran, and triethylamine was added at 0°C. Then, iodine granules were added to the reaction system one by one until the solution turned reddish-brown and did not fade. The reaction was continued for 30 minutes, and the reaction was quenched by adding saturated sodium thiosulfate solution. The mixture was extracted with ethyl acetate, and the organic phase was dried with anhydrous sodium sulfate. After filtration, the organic solvent was removed by vacuum distillation. The crude product was purified by silica gel column chromatography to obtain compound 4, whose chemical reaction formula is as follows: 。 3. The enzymatic synthesis method for 12β-hydroxycinobufagin according to claim 2, characterized in that, The method for synthesizing compound 3 is as follows: Compound 2 was dissolved in anhydrous tetrahydrofuran. The reaction system was cooled to -78ºC, and 1 mL of Selectride THF solution was added dropwise. The reaction was carried out at this temperature for 3 hours. The reaction was quenched by adding saturated ammonium chloride solution. The reaction solution was extracted with ethyl acetate, and the organic phase was washed with saturated sodium chloride solution. After drying with anhydrous sodium sulfate and filtration, the organic solvent was removed by concentration under reduced pressure. The crude product was purified by silica gel column chromatography to obtain compound 3, whose chemical reaction formula is as follows: 。 4. The enzymatic synthesis method for 12β-hydroxycinobufagin according to claim 3, characterized in that, The method for synthesizing compound 2 is as follows: Compound 1 androstenedione and 5% Pd / C were added to a round-bottom flask. Anhydrous pyridine was added under nitrogen protection, and then the nitrogen was replaced with hydrogen. The reaction was carried out at room temperature for 24 hours. The solution was filtered through a funnel lined with diatomaceous earth and washed with ethyl acetate. The organic phases were combined and concentrated under vacuum. The crude product was purified by silica gel column chromatography to give compound 2, whose chemical reaction formula is as follows: 。 5. The enzymatic synthesis method for 12β-hydroxycinobufagin according to claim 1, characterized in that, In step S1, the ratio of compound 4 to DMF solvent is 150 mg: 1 mL; In step S2, the ratio of compound 5: 2-pyrrole-5-boronate: K3PO4: dppfCl2Pd is 1 mmol: 1.5 mmol: 0.1 mmol; In step S3, compound 6: p-toluenesulfonic acid is 1 mmol: 5 mmol; In step S4, compound 7: TBSCl: imidazole is 1 mmol: 5 mmol: 8 mmol; In step S5, compound 8: methylene blue: sodium bicarbonate: tetraphenylcobalt porphyrin is in the ratio of 1 mmol: 0.1 mmol: 2 mmol: 0.1 mmol; In step S6, the ratio of compound 9: anhydrous dichloromethane: scandium trifluoromethanesulfonate is 150 mg: 6 mL: 11.5 mg; In step S7, compound 11: anhydrous pyridine: anhydrous acetic anhydride was prepared at a ratio of 1 mmol: 35.7 mL: 5 mmol. In step S8, the ratio of compound 12: anhydrous pyridine: hydrofluoric acid is 60 mg: 6 mL: 1 mL.

6. The enzymatic synthesis method for 12β-hydroxycinobufagin according to claim 1, characterized in that, In step S1, the purification mobile phase is dichloromethane and methanol in a volume ratio of 30:

1. In step S2, the purification mobile phase is dichloromethane and methanol in a volume ratio of 20:1; In step S3, the mobile phase for purification is petroleum ether and ethyl acetate in a volume ratio of 1:

1. In step S4, the mobile phase for purification is petroleum ether and ethyl acetate in a volume ratio of 10:

1. In step S5, the mobile phase for purification is petroleum ether and ethyl acetate in a volume ratio of 2:

1. In step S6, the purification mobile phase is petroleum ether and ethyl acetate in a volume ratio of 2:1; In step S7, the mobile phase for purification is petroleum ether and ethyl acetate in a volume ratio of 4:

1. In step S8, the purification mobile phase is dichloromethane and methanol in a volume ratio of 20:

1.

7. The enzymatic synthesis method for 12β-hydroxycinobufagin according to claim 2, characterized in that, The ratio of compound 3, hydrazine hydrate, and triethylamine is 1 mmol:10 mmol:10 mmol, and the ratio of anhydrous tetrahydrofuran, triethylamine, and iodine particles is 100 mL:14.37 mL:13.12 g. The purification mobile phase is petroleum ether and ethyl acetate in a volume ratio of 8:

1.

8. The enzymatic synthesis method for 12β-hydroxycinobufagin according to claim 3, characterized in that, The ratio of compound 2 to anhydrous tetrahydrofuran was 9.87 g: 41.09 mL, and the purification mobile phase was petroleum ether and ethyl acetate in a volume ratio of 2:

1.

9. The enzymatic synthesis method for 12β-hydroxycinobufagin according to claim 4, characterized in that, The ratio of compound 1 to 5% Pd / C is 10 g: 2 g, and the purification mobile phase is petroleum ether and ethyl acetate in a volume ratio of 4:1.

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