A chemical-biological fusion method for preparing cholesterol

Abstract

This invention discloses a chemical-biological fusion method for cholesterol synthesis. Using plant-derived 21-hydroxy-20-methylpregn-4-en-3-one, also known as bis(2-hydroxy-2-yl)-3-one (BA), as a raw material, cholesterol is synthesized through a four-step reaction involving sulfonation, acetylation, enzymatic hydrolysis and reduction, and Grignard coupling. Addressing the drawbacks of traditional animal-derived cholesterol, which carries unpredictable risks of pathogenic bacteria and viruses, this invention uses plant-derived steroidal raw material BA to synthesize cholesterol. This method not only offers high safety, avoiding the risk of pathogenic bacteria and viral infections, but also utilizes the tandem catalysis of esterase and carbonyl reductase during synthesis, along with coenzyme regeneration, to obtain key intermediates with high stereoselectivity and in a safe and clean manner. This significantly shortens the cholesterol synthesis route, increases efficiency, simplifies operation, minimizes solvent recycling losses, reduces waste, and allows for mild reaction conditions. This method is safe, economical, and suitable for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of organic compound preparation technology, and relates to a method for preparing cholesterol through a chemical-biological fusion process. Background Technology

[0002] Cholesterol (Cholest-5-en-3β-ol), also known as cholesterol, is a derivative of cyclopentanoperhydrophenanthrene. As a lipid molecule, cholesterol is not only an important component of cell membranes but can also be transformed into various physiologically active substances, such as corticosteroids, vitamin D3, and bile acids, through side-chain oxidation, reduction, or degradation. Traditionally, cholesterol is mainly obtained through extraction from animal organs. However, due to the emergence of diseases such as mad cow disease in livestock in recent years, the safety of animal-derived cholesterol has been increasingly questioned. Therefore, synthetic methods using plant-derived extracts as starting materials have become a focus of research.

[0003] Traditional cholesterol synthesis methods use diosgenin as a raw material and synthesize cholesterol through a six-step reaction (patent CN1772760A).

[0004]

[0005] In recent years, with the reduction in the production cost of plant-derived steroid raw materials, more and more raw materials are available, resulting in the reporting of many new routes. However, the methods for constructing cholesterol side chains are still mostly limited to the Wittig reaction and organometallic reagent coupling.

[0006] The Wittig reaction is widely used. For example, patent documents CN105218609A and CN104961788A (omitting the step of protecting the 3-hydroxyl group) report synthetic routes using pregnenolone as a starting material. However, in order to ensure the stereoselectivity of the 20-methyl group in the catalytic hydrogenation step, relatively expensive hydrogenation catalysts and ligands are required.

[0007]

[0008] Phytosterols are natural organic compounds that can be produced in large quantities from byproducts of the vegetable oil industry. Among them, stigmasterol and its degradation products have wide applications in steroid synthesis. CN106632565A and CN105237603A report synthetic routes using stigmasterol as a starting material. By using different strategies to protect the 3-position hydroxyl group of the starting material, the original side chain was cleaved using an ozonolysis reaction, and then the cholesterol side chain was constructed through a Wittig reaction. However, the ozonolysis reaction poses a significant risk to the scale-up production of this type of route.

[0009]

[0010] Similarly, patent document CN105218610A reports a route using 3-carbonylpregn-4-ene-22-aldehyde, a degradation product of phytosterols, as a raw material.

[0011]

[0012] With the development of biodegradation technology, the cost of plant-derived steroid raw material 21-hydroxy-20-methylpregn-4-en-3-one (BA or bis-decanoyl alcohol) has decreased significantly in recent years, gradually becoming an economical and practical synthetic intermediate. CN113248557A reports a new synthetic route based on the Wittig reaction using BA as a raw material.

[0013]

[0014] On the other hand, methods involving metal reagent coupling have also been reported. Patent document CN112608361A uses BA as a raw material and employs a copper-lithium reagent method to construct the side chains.

[0015]

[0016] In summary, existing methods suffer from drawbacks such as cumbersome operation, expensive catalysts, high safety risks associated with chemical hydrogenation reactions, the generation of a certain proportion of 3α-stereoisomers, reduced yields due to separation and purification, and the production of large amounts of solid waste (such as triphenylphosphine). Therefore, researching new synthetic methods is of great significance for the industrial production of cholesterol. Summary of the Invention

[0017] The technical problem to be solved by the present invention is to provide a chemical-biological fusion method for preparing cholesterol, which uses plant-derived 21-hydroxy-20-methylpregn-4-en-3-one as raw material and synthesizes cholesterol through a four-step reaction of sulfonation, acetylation, enzymatic hydrolysis and reduction, and Grignard coupling.

[0018] The reaction process of the above method of the present invention is shown in reaction formula (I):

[0019]

[0020] In the reaction formula (I), G is a sulfonyl group.

[0021] The specific steps are as follows:

[0022] (a) Sulfonation: BA, represented by compound (1), and sulfonyl chloride undergo sulfonation under alkaline conditions to yield compound (2); the reaction process is shown in reaction (1):

[0023]

[0024] (b) Carbonyl protection reaction: The compound of formula (2) obtained in step (a) is subjected to an acid-catalyzed acetylation reaction with an acid anhydride; the reaction process is shown in reaction formula (2):

[0025]

[0026] (c) Enzymatic hydrolysis and reduction reaction: The compound of formula (3) obtained in step (b) is regenerated by coupling a coenzyme under the tandem catalysis of esterase and carbonyl reductase to obtain compound (4); the reaction process is shown in reaction formula (3):

[0027]

[0028] (d) Grignard coupling reaction: The compound of formula (4) obtained in step (c) is dissolved in an organic solvent, and a Grignard reagent is added dropwise to perform coupling to obtain the target product cholesterol; the reaction process is shown in reaction formula (4):

[0029]

[0030] In step (a) of the present invention, the sulfonation reaction specifically involves the following: the compound of formula (1) reacts with a sulfonyl chloride reagent in an organic solvent under the action of an alkali to obtain the compound of formula (2).

[0031] The sulfonyl chloride reagent is one or more of methanesulfonyl chloride, p-toluenesulfonyl chloride, etc.; preferably, it is methanesulfonyl chloride.

[0032] The organic solvent is selected from one or more of ethyl acetate, dichloromethane, toluene, tetrahydrofuran, 2-methyltetrahydrofuran, etc.; preferably, it is dichloromethane.

[0033] The base is selected from one or more of triethylamine, imidazole, pyridine, etc.; preferably, it is pyridine.

[0034] Wherein, the molar ratio of the compound of formula (1), the sulfonyl chloride reagent, and the base is 1:(1-4):(1-5); preferably, it is 1:1.5:2.

[0035] The sulfonation reaction is carried out at a temperature of 0–50°C; preferably, it is carried out at a temperature of 15–25°C.

[0036] The sulfonation reaction takes 2 to 24 hours; preferably, it takes 2 to 3 hours.

[0037] In step (b) of this invention, the carbonyl protection reaction specifically involves the acetylation reaction of the compound of formula (2), acetic anhydride, and methanesulfonic acid to obtain the compound of formula (3).

[0038] Wherein, the molar ratio of the compound of formula (2), acetic anhydride and methanesulfonic acid is 1:(25-62.5):(0.1-6); preferably, it is 1:25:0.5.

[0039] The temperature of the acetylation reaction is -20 to 110°C; preferably, it is 40°C.

[0040] The acetylation reaction takes 3-24 hours; preferably, it takes 4 hours.

[0041] In step (c) of the present invention, the esterase and carbonyl reductase hydrolyze and reduce the compound of formula (3) to the compound of formula (4) in an organic solvent.

[0042] The organic solvent is one or more selected from methanol, ethanol, propanol, isopropanol, butanol, isobutanol, tert-butanol, tetrahydrofuran, and methyl tert-butyl ether. Preferably, the organic solvent is methanol, isopropanol, or methyl tert-butyl ether.

[0043] The amount of organic solvent used is 3.0-50.0 times (molar ratio) of the compound of formula (3); preferably, it is 20-25 times (molar ratio).

[0044] The esterase mentioned includes all enzymes that can hydrolyze the compound of formula (3). Preferably, it is commercially available esterase 1745 (Huzhou Yihui Biotechnology Co., Ltd.).

[0045] The carbonyl reductase includes all enzymes that can reduce the esterase hydrolysis product of formula (3) to the compound of formula (4); preferably, the carbonyl reductase in this invention is commercially available carbonyl reductase 1603 (Huzhou Yihui Biotechnology Co., Ltd.).

[0046] Furthermore, the carbonyl reductase uses reduced nicotinamide adenine dinucleotide as a coenzyme, and the present invention also provides a method for regenerating the coenzyme.

[0047] The regeneration method for the reduced nicotinamide adenine dinucleotide can utilize any one of the following systems: formate dehydrogenase / formate system, glucose dehydrogenase / glucose system, or alcohol dehydrogenase / isopropanol system. All three methods can reduce the oxidized nicotinamide adenine dinucleotide back to its reduced form.

[0048] Preferably, the glucose dehydrogenase used is glucose dehydrogenase 901 from Huzhou Yihui Biotechnology Co., Ltd., the alcohol dehydrogenase used is alcohol dehydrogenase 1853 from Huzhou Yihui Biotechnology Co., Ltd., and the formate dehydrogenase used is formate dehydrogenase 385 from Huzhou Yihui Biotechnology Co., Ltd.

[0049] Specifically, glucose dehydrogenase 901 reduces oxidized nicotinamide adenine dinucleotide to reduced nicotinamide adenine dinucleotide in the presence of glucose in the system. Alternatively, alcohol dehydrogenase 1853 reduces oxidized nicotinamide adenine dinucleotide to reduced nicotinamide adenine dinucleotide in the presence of isopropanol in the system. Or, formate dehydrogenase 385 reduces oxidized nicotinamide adenine dinucleotide to reduced nicotinamide adenine dinucleotide in the presence of formate in the system.

[0050] By using these two coenzyme regeneration systems in combination with the esterase and carbonyl reductase of the present invention, a high conversion rate of compound (4) can be obtained.

[0051] In this invention, the coenzyme may include oxidized nicotinamide adenine dinucleotide, reduced nicotinamide adenine dinucleotide, and a mixture of their salts.

[0052] In this invention, the weight ratio of the compound of formula (3), the esterase, and the carbonyl reductase is 1:(0.001-0.2):(0.001-0.2); preferably, it is 1:(0.01-0.2):(0.01-0.2).

[0053] In this invention, the reaction temperature is 20-37°C; preferably, it is 25-30°C.

[0054] In this invention, the reaction time is 10-48 hours; preferably, it is 18-30 hours.

[0055] Furthermore, in this invention, during the process of hydrolyzing and reducing the compound of formula (3) to the compound of formula (4) in an organic solvent, the pH of the system is adjusted by an inorganic base.

[0056] The pH value is 6.0-8.0; preferably, it is 7.5±0.5.

[0057] The alkali is one or more of sodium hydroxide, sodium carbonate, potassium carbonate, potassium hydroxide, and ammonia; preferably, it is sodium carbonate.

[0058] In step (d) of this invention, the Grignard coupling reaction specifically involves adding a Grignard reagent to the compound of formula (4) in an organic solvent to obtain cholesterol through a coupling reaction.

[0059] The organic solvent is one or more of diethyl ether, tetrahydrofuran, methyltetrahydrofuran, toluene, etc.; preferably, it is tetrahydrofuran.

[0060] The Grignard reagent is one or more of isopentyl magnesium chloride and isopentyl magnesium bromide; preferably, it is isopentyl magnesium bromide.

[0061] The coupling reaction temperature is between 0 and 50°C; preferably, it is between 20 and 25°C.

[0062] The coupling reaction time is 2-12 hours; preferably, it is 5-8 hours.

[0063] Wherein, the molar ratio of the compound of formula (4) and the Grignard reagent is 1:(1-8); preferably, it is 1:(4-5).

[0064] The present invention also proposes cholesterol synthesized by the method described above.

[0065] The beneficial effects of this invention include: the hydrolysis-stereoselective reduction reaction of compound (3) via esterase-reductase tandem catalysis, and the one-pot method of the two-step ester hydrolysis-reduction reaction, which has the advantages of not requiring the separation of intermediates, simple operation, high efficiency, and low waste. Compared with the existing method of preparing compound (4) by reducing with sodium borohydride, the method of this invention has the advantages of high stereoselectivity of carbonyl reductase in reducing the 3-position carbonyl group to 3β-hydroxyl, no 3α-hydroxy isomer, convenient purification, and high yield. To address the drawbacks of traditional animal-derived cholesterol, such as the unpredictable risks of pathogenic bacteria and viruses, this invention synthesizes cholesterol using plant-derived steroidal raw material BA. This method not only offers high safety by avoiding the risk of pathogenic bacteria and viral infections, but also utilizes the tandem catalysis of esterase and carbonyl reductase during synthesis, along with coenzyme regeneration, to prepare key intermediates. This significantly shortens the cholesterol synthesis route. Furthermore, the synthesis method is simple to operate, has a high yield, is environmentally friendly, minimizes solvent recycling losses, generates less waste, operates under mild reaction conditions, is economical, and is suitable for industrial production. Detailed Implementation

[0066] The invention will be further described in detail below with reference to specific embodiments. The processes, conditions, and experimental methods for implementing this invention, except as specifically mentioned below, are all common knowledge and general knowledge in the art, and this invention does not have any particular limitations. The results shown in the embodiments are obtained under optimized experimental conditions and have potential application value.

[0067] Example 1: Sulfonation to prepare compound (2) (methanesulfonyl protected)

[0068]

[0069] Add 60g of solid BA, 57g of pyridine, and 250mL of dichloromethane to a 500mL three-necked flask. After purging with nitrogen, add 41g of methanesulfonyl chloride dropwise at 25℃ over 30 minutes. After the addition is complete, maintain the temperature at 25℃ for 3 hours. After the reaction is complete, concentrate the dichloromethane under reduced pressure. Add the mother liquor to 800mL of ice water, stir for 1 hour, filter, wash with water, and dry the solid in a 60℃ hot air circulating oven for 6 hours to obtain 73.5g of white powder solid, with a molar yield of 99.0%.

[0070] Example 2: Acetylation to prepare compound (3)

[0071]

[0072] 73.5 g of solid compound (2), 503 mL of acetic anhydride, and 8.5 g of methanesulfonic acid were added to a 500 mL three-necked flask. The temperature was controlled at 38–42 °C, and the reaction was maintained at this temperature for 4 h. After the reaction was complete, the reaction solution was added to 630 mL of ice water, stirred for 1 h, filtered, washed with water, and the solid was dried under vacuum and placed in a 60 °C hot air circulating oven for 6 h to obtain 80.0 g of white powder solid, with a molar yield of 98.6%.

[0073] Example 3: Screening of esterases

[0074] Add 15g of compound (3) to a 500mL beaker, add 80mL of tert-butanol, then add 200mL of purified water, stir and heat to 30℃, adjust the pH to 7.0 with 1mol / L sodium hydroxide solution, add 1.5g of various esterases, and stir to react for 30 minutes. Maintain the pH at 7.0 with NaOH solution throughout, and calculate the hydrolytic activity of various esterases on compound (3) based on the volume of sodium hydroxide solution added. The activities of various esterases are shown in Table 1.

[0075] Table 1 Hydrolytic activity of various esterases on compounds of formula (3)

[0076]

[0077] *The activity of 1745 is taken as 100%, and "—" indicates that the activity is less than 5%.

[0078] Example 4: Preparation of compound (4) by enzymatic hydrolysis and reduction (glucose dehydrogenase / glucose system)

[0079]

[0080] Add 600 mL of water, 1 g of potassium dihydrogen phosphate, and 50 g of glucose monohydrate to a 2000 mL three-necked flask. After stirring until completely dissolved, add 300 g of tert-butanol and adjust the pH to 7.0. Then add 100.0 g of the compound from step (3), 100 mg of nicotinamide adenine dinucleotide, 160 g of carbonyl reductase, 174 g of esterase, and 90 g of glucose dehydrogenase. Maintain the internal temperature at 30 ± 2 °C and control the pH at 7.0 with 1 mol / L sodium carbonate solution. Keep the reaction at this temperature for 24 h. After the reaction is complete, cool to 15 °C and let stand for 1 h. Filter and wash with water. Add 300 g of ethanol to the solid and reflux to dissolve and filter. Cool the filtrate to 5 °C, filter and wash. Place the solid in a 60 °C hot air circulating oven and dry for 6 h to obtain 82 g of white powder solid with a molar yield of 90.0%.

[0081] Example 5: Preparation of compound (4) by enzymatic hydrolysis and reduction (alcohol dehydrogenase / isopropanol system)

[0082] Add 850 mL of water and 2.5 g of potassium dihydrogen phosphate to a 2000 mL three-necked flask, add 100 g of isopropanol, adjust the pH to 7.0, then add 150 g of compound (3), 40 mg of nicotinamide adenine dinucleotide, 160-350 g of carbonyl reductase, and 174-550 g of esterase. Maintain the internal temperature at 30 ± 2 °C, and use 1 mol / L sodium carbonate solution to control the pH at 7.0 and keep the reaction at this temperature for 24 h. After the reaction is complete, cool to 15 °C and let stand for 1 h. Filter, wash with water, add 300 g of ethanol to dissolve the solid under reflux and filter, cool the filtrate to 5 °C, filter, rinse, and place the solid in a 60 °C hot air circulating oven for 6 h to obtain 126.7 g of white powder solid, with a molar yield of 92.7%.

[0083] Example 6: Preparation of compound (4) by hydrolysis and reduction (formate dehydrogenase / formate system)

[0084] Add 800 mL of water, 200 g of tert-butanol, and 35 g of ammonium formate to a 2000 mL three-necked flask to adjust the pH to 7.0. Then add 200 g of compound (3), 150 mg of nicotinamide adenine dinucleotide, 160-370 g of carbonyl reductase, 174-560 g of esterase, and 38-550 g of formate dehydrogenase. Maintain the internal temperature at 30 ± 2 °C and control the pH at 7.0 with formic acid solution. Keep the reaction at this temperature for 24 h. After the reaction is complete, cool to 15 °C and let stand for 1 h. Filter and wash with water. Dissolve the solid in 300 g of ethanol under reflux and filter. Cool the filtrate to 5 °C, filter and wash. Place the solid in a 60 °C hot air circulating oven and dry for 6 h to obtain 173.1 g of white powder solid, with a molar yield of 95.0%.

[0085] Example 7: Cholesterol Synthesis

[0086]

[0087] In a three-necked flask, 20 g of compound (4) and 75 mL of tetrahydrofuran were added, purged with nitrogen, and isopentyl magnesium bromide Grignard reagent was added dropwise under ice bath conditions. The reaction was maintained at 20 °C for 8 hours. TLC analysis showed that the starting material reacted completely. The reaction solution was poured into 100 mL of ammonium chloride solution, the organic phase was separated, and the aqueous phase was extracted with toluene (50 mL × 2). The organic phases were combined, washed once with water, filtered, and evaporated to dryness to obtain a crude product. 100 mL of ethanol was added to the crude product for purification to obtain 15.5 g of a white solid, with a molar yield of 82.0%.

[0088] Example 8: Sulfonation preparation of compound (2) (p-Toluenesulfonyl group protected)

[0089]

[0090] Add 60g of solid BA, 57g of pyridine, and 250mL of dichloromethane to a 500mL three-necked flask. After purging with nitrogen, add 41g of methanesulfonyl chloride dropwise at 25℃ over 30 minutes. After the addition is complete, maintain the temperature at 25℃ for 3 hours. Once the reaction is complete, concentrate the dichloromethane under reduced pressure. Add the mother liquor to 800mL of ice water, stir for 1 hour, filter, and wash with water. Place the solid in a 60℃ hot air circulating oven for 6 hours to obtain 85.4g of white powder solid, with a molar yield of 97.0%.

[0091] Example 9: Acetylation to prepare compound (3)

[0092]

[0093] 85.4 g of solid compound (2), 503 mL of acetic anhydride, and 8.5 g of methanesulfonic acid were added to a 500 mL three-necked flask. The temperature was controlled at 38–42 °C, and the reaction was maintained at this temperature for 4 h. After the reaction was complete, the reaction solution was added to 630 mL of ice water, stirred for 1 h, filtered, washed with water, and the solid was dried under vacuum. The solid was then placed in a 60 °C hot air circulating oven and dried for 6 h to obtain 91.0 g of white powder solid, with a molar yield of 96.0%.

[0094] Example 10: Preparation of compound (4) by enzymatic hydrolysis and reduction

[0095] Add 800 mL of water, 200 g of tert-butanol, and 35 g of ammonium formate to a 2000 mL three-necked flask to adjust the pH to 7.0. Then add 234 g of compound (3), 150 mg of nicotinamide adenine dinucleotide, 160-370 g of carbonyl reductase, 174-560 g of esterase, and 38-550 g of formate dehydrogenase. Maintain the internal temperature at 30 ± 2 °C and control the pH at 7.0 with formic acid solution. Keep the reaction at this temperature for 24 h. After the reaction is complete, cool to 15 °C and let stand for 1 h. Filter and wash with water. Dissolve the solid in 300 g of ethanol under reflux and filter. Cool the filtrate to 5 °C, filter and wash. Place the solid in a 60 °C hot air circulating oven and dry for 6 h to obtain 201.0 g of white powder solid, with a molar yield of 93.0%.

[0096] Example 11: Cholesterol Synthesis

[0097]

[0098] In a three-necked flask, 23.7 g of compound (4) and 75 mL of tetrahydrofuran were added, purged with nitrogen, and isopentylmagnesium bromide Grignard reagent was added dropwise under ice bath conditions. The reaction was maintained at 20 °C for 8 hours. TLC analysis showed that the starting material reacted completely. The reaction solution was poured into 100 mL of ammonium chloride solution, the organic phase was separated, and the aqueous phase was extracted with toluene (50 mL × 2). The organic phases were combined, washed once with water, filtered, and evaporated to dryness to obtain the crude product. 100 mL of ethanol was added to the crude product for purification to obtain 15.1 g of white solid, with a molar yield of 80.0%.

[0099] The above description is merely a specific embodiment of the present invention and is not intended to limit the present invention. Any variations and advantages that can be conceived by those skilled in the art without departing from the spirit and principles of the present invention are included in the present invention and are protected by the appended claims.

Claims

1. A chemo-bio-fusion method for cholesterol synthesis, characterized by, Cholesterol was synthesized from plant-derived 21-hydroxy-20-methylpregn-4-en-3-one via a four-step reaction involving sulfonation, acetylation, enzymatic hydrolysis and reduction, and Grignard coupling. The reaction process of the method is shown in reaction formula (I). Reaction formula (I) In the reaction formula (I), G is a sulfonyl group; The specific steps of the method are as follows: Step (a) Sulfonation: BA, represented by compound (1), and sulfonyl chloride reagent undergo sulfonation reaction under alkaline conditions to obtain compound (2); the reaction process is shown in reaction formula (1): Reaction formula (1); Step (b) Carbonyl protection reaction: The compound of formula (2) obtained in step (a) is acetylated with an acid anhydride under acid catalysis; the reaction process is shown in reaction formula (2): Reaction formula (2); Step (c) Enzymatic hydrolysis and reduction reaction: The compound of formula (3) obtained in step (b) is regenerated by simultaneously coupling the coenzymes glucose dehydrogenase / formate dehydrogenase under the tandem catalysis of esterase 1745 and carbonyl reductase, to prepare compound (4); the reaction process is shown in reaction formula (3): Reaction formula (3); Step (d) Grignard coupling reaction: The compound of formula (4) obtained in step (c) is dissolved in an organic solvent, and a Grignard reagent is added dropwise to perform coupling to obtain the target product cholesterol; the reaction process is shown in reaction formula (4): Reaction formula (4).

2. The method as described in claim 1, characterized in that, In step (a), the sulfonation reaction specifically involves reacting the compound of formula (1) with a sulfonyl chloride reagent in an organic solvent under the action of a base to obtain the compound of formula (2).

3. The method as described in claim 2, characterized in that, The sulfonyl chloride reagent is selected from one or two of methanesulfonyl chloride and p-toluenesulfonyl chloride; and / or, The organic solvent is selected from one or more of ethyl acetate, dichloromethane, toluene, tetrahydrofuran, and 2-methyltetrahydrofuran; and / or the base is selected from one or more of triethylamine, imidazole, and pyridine; and / or the molar ratio of the compound of formula (1), sulfonyl chloride reagent, and base is 1:(1-4):(1-5); and / or the temperature of the sulfonation reaction is 0-50°C; and / or the time of the sulfonation reaction is 2-24 h.

4. The method as described in claim 1, characterized in that, In step (b), the carbonyl protection reaction is as follows: the compound of formula (2), acetic anhydride and methanesulfonic acid undergo an acetylation reaction to obtain the compound of formula (3).

5. The method as described in claim 4, characterized in that, The molar ratio of the compound of formula (2), acetic anhydride, and methanesulfonic acid is 1:(25-62.5):(0.1-6); and / or, the acetylation reaction temperature is -20~110℃; and / or, the acetylation reaction time is 3-24h.

6. The method as described in claim 1, characterized in that, In step (c), the esterase and carbonyl reductase hydrolyze and reduce the compound of formula (3) to the compound of formula (4) in an organic solvent.

7. The method as described in claim 6, characterized in that, The organic solvent is methanol, ethanol, propanol, isopropanol, butanol, isobutanol, tert-butanol, tetrahydrofuran, methyl tert-butyl ether; and / or, The amount of organic solvent used is 3.0-50.0 times the molar ratio of the amount of compound in formula (3); and / or, The temperature for the enzymatic hydrolysis-reduction reaction is 20-37℃; and / or, The enzymatic hydrolysis-reduction reaction takes 10-48 hours; and / or, The weight ratio of the compound of formula (3), esterase, and carbonyl reductase is 1:(0.001-0.2):(0.001-0.2); and / or, Step (c) further includes adjusting the pH with an alkali, wherein the pH is 6.0-8.0; the alkali is one or more of sodium hydroxide, sodium carbonate, potassium carbonate, potassium hydroxide, and ammonia.

8. The method as described in claim 1, characterized in that, In step (d), the Grignard coupling reaction is as follows: the compound of formula (4) is added dropwise to an organic solvent with a Grignard reagent to undergo a coupling reaction and obtain cholesterol.

9. The method as described in claim 8, characterized in that, The organic solvent is one or more selected from diethyl ether, tetrahydrofuran, methyltetrahydrofuran, and toluene; and / or, The Grignard reagent is one or more of isopentyl magnesium chloride and isopentyl magnesium bromide; and / or The molar ratio of the compound of formula (4) to the Grignard reagent is 1:(1-8); and / or, The Grignard coupling reaction temperature is between 0-50°C; and / or, The Grignard coupling reaction time is 2-12 hours.

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