Method for synthesizing steroid he3286 by using enzymatic method and chemical method

Abstract

The present invention relates to a method for synthesizing a steroid HE3286 by using an enzymatic method and a chemical method. The method comprises: screening for a cytochrome P450 mutant LG-23 capable of catalyzing 7β-hydroxylation of dehydroepiandrosterone, using a P450BM3 mutant enzyme to catalyze dehydroepiandrosterone to generate 7β-hydroxydehydroepiandrosterone, and then using a chemical method for an alkynylation reaction at the 17-position carbonyl to generate a steroid HE3286. The method for synthesizing HE3286 of the present invention has simple synthesis steps, high catalytic selectivity, reduced by-products, increased yield, mild reaction conditions, low costs, green and environmentally friendly characteristics, and high efficiency, and has important application value for promoting the development process of steroid drugs in China.

Description

Method for synthesizing steroid HE3286 by combining enzymatic method and chemical method TECHNICAL FIELD

[0001] The present application belongs to the technical field of chemical enzymatic synthesis, and particularly relates to a method for synthesizing steroid HE3286 by combining enzymatic method and chemical method. BACKGROUND

[0002] At present, more than 400 kinds of steroid drugs are produced globally, which is the second largest drug family next to antibiotics. Steroid drugs are widely used for the treatment of various clinical diseases, including autoimmune, inflammation, cancer, coronavirus infection, osteoporosis and the like. It is crucial to introduce different functional groups such as hydroxyl into the steroid skeleton in a regionally and stereoselective manner for the physiological and pharmacological activity of steroid drugs.

[0003] HE3286 (CAS: 1001100-69-1), chemically named as 17α-ethynyl androsta-5-ene-3β,7β,17β-triol, can be used for preventing or treating metabolic diseases such as type 2 diabetes, hyperglycemia and autoimmune diseases such as rheumatoid arthritis.

[0004] Patent WO2009149392 reports three synthesis routes of HE3286. In the first synthesis route, DHEA is used as a starting material, 3-hydroxyl TMSCl is protected, 17-carbonyl is acetylated, and then 3-acetyl is protected, and a 7-keto compound is obtained by oxidation. The 7-keto compound intermediate is reduced to obtain a 7β-hydroxyl compound, and finally the 3-hydroxyl is hydrolyzed to obtain the target product HE3286. Through 6-step reactions, the total yield is 15%, and the target product HE3286 is obtained.

[0005] In the second synthesis route, dehydroepiandrosterone acetate is used as a starting material, 17-vicinal acetal protection is first performed, and then 7-oxidation and reduction are sequentially performed to obtain a 7β-hydroxyl intermediate, which is sequentially subjected to 17-vicinal acetal deprotection, 3-hydrolysis, 3,7-hydroxyl TMS protection, 17-alkynylization and finally TMS deprotection to obtain the target product HE3286. Through 8-step reactions, the total yield is 6%, and the target product HE3286 is obtained.

[0006] In the third synthesis route, dehydroepiandrosterone acetate is still used as a starting material, and sequentially subjected to 7-oxidation, 17-hydroxylamination, 7-reduction and 17-hydrolysis to obtain 7β-hydroxyl dehydroepiandrosterone acetate. The intermediate is further subjected to 3-hydrolysis, 3,7-hydroxyl TMS protection, 17-alkynylization and finally TMS deprotection as described in route two to obtain the target product HE3286. Through 8-step reactions, the total yield is 30%, and the target product HE3286 is obtained.

[0007] In patent CN114478672A, trihydroxy (CAS: 2963-69-1; 3β, 7α, 15α-trihydroxyandrostan-5-ene-17-one) is used as a starting material to obtain the target product HE3286 through transposition, double esterification, elimination, hydrogenation, acetylene and hydrolysis. Through 6-step reaction, the target product HE3286 is obtained with a total yield of 80%.

[0008] It can be seen that the synthesis of HE3286 in patent WO2009149392 not only has a long route but also has a low total yield. Although the total yield of HE3286 in patent CN114478672A is significantly improved, there are still problems such as long reaction path, complicated operation and high synthesis cost, which are not suitable for large-scale industrial production.

[0009] SUMMARY

[0010] To solve the above problems in the prior art for synthesizing HE3286, the present application designs a chemical enzyme method combined synthesis route, that is, first, the C7β hydroxylation of dehydroepiandrosterone is carried out by enzyme method to obtain the hydroxylated steroid 7β-hydroxy dehydroepiandrosterone, and then the steroid HE3286 is obtained by acetylene reaction through chemical method.

[0011] The present application provides a method for synthesizing steroid HE3286 by combining enzyme method and chemical method, which comprises the following steps:

[0012] S1, dehydroepiandrosterone is generated 7β-hydroxy dehydroepiandrosterone under the action of 7β hydroxylase;

[0013] S2, acetylene reaction of C17 carbonyl of 7β-hydroxy dehydroepiandrosterone to generate HE3286.

[0014] Further, the 7β hydroxylase is cytochrome P450 enzyme, preferably P450 BM3 mutant, more preferably P450 BM3 mutant LG-23, wherein the amino acid sequence of the P450 BM3 mutant LG-23 is shown in SEQ ID NO: 1, and the nucleotide sequence is shown in SEQ ID NO: 2; the acetylene reaction is specifically the reaction of 7β-hydroxy dehydroepiandrosterone with ethynyl magnesium bromide or acetylene gas or other ethynyl format reagent.

[0015] Further, the step S1 is specifically: the P450 BM3 mutant LG-23 is completely reacted with isopropanol dehydrogenase or glucose dehydrogenase, dehydroepiandrosterone, coenzyme NADP+, isopropanol or glucose, ethyl acetate is added to extract the reaction liquid to obtain ethyl acetate extract, which is dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure to obtain 7β-hydroxy dehydroepiandrosterone crude product, and then 7β-hydroxy dehydroepiandrosterone pure product is obtained by recrystallization.

[0016] Further, the step S2 is specifically:

[0017] Take 0.5-2g 7β-hydroxy dehydroepiandrosterone, add 3.2-12.8mL THF, ice bath, nitrogen protection, drop 70-100mL 0.3-0.5M acetylene magnesium bromide / tetrahydrofuran solution (18eq), 0-40℃ reaction, TLC (dichloromethane:methanol=15:1) monitoring reaction progress, until the substrate is completely converted. After the reaction is completed, quench the reaction solution with saturated ammonium chloride solution, add equal volume of ethyl acetate, extract three times, combine the organic phase, add anhydrous Na2SO4 drying, filtration, rotary evaporation to remove the organic phase, followed by 2.5mL isopropyl ether slurry, cooling crystallization, suction filtration, oven drying, finally get HE3286 solid product.

[0018] Further, the step S2 is:

[0019] S21, 7β-hydroxy dehydroepiandrosterone is dissolved in an organic solvent, and then an activating agent and tert-butyl dimethylsilyl chloride (TBDMSCl) are added, and the compound is reacted at 85℃ to reflux to obtain a 3,7-hydroxyl protected compound;

[0020] S22, the 3,7-hydroxyl protected compound C17 carbonyl group undergoes acetylene reaction and then deprotection to generate HE3286.

[0021] Further, the step S22 is specifically: adding a cosolvent to the 3,7-hydroxyl protected compound and reacting with acetylene magnesium bromide or acetylene gas or other acetylene group Grignard reagent, then adding p-toluenesulfonic acid and reducing pressure to obtain HE3286.

[0022] Further, the organic solvent in the step S21 is selected from tetrahydrofuran (THF), acetonitrile (MeCN), dichloromethane (DCM) or N,N-dimethylformamide (DMF), preferably THF; the activating agent is selected from imidazole, pyridine, 4-dimethylaminopyridine (DMAP), 2,6-lutidine, triethylamine, diisopropylethylamine (DIPEA) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), preferably imidazole, the molar ratio of TBDMSCl to 7β-hydroxy dehydroepiandrosterone is 3-5:1, the molar ratio of imidazole to TBDMSCl is 1.2-1.5:1, and the reaction temperature is not higher than 50℃.

[0023] Further, the cosolvent is selected from tetrahydrofuran, diethyl ether, isopropyl ether, methyl tert-butyl ether, ethylene glycol dimethyl ether, 2-methyltetrahydrofuran, 1,4-dioxane, the acetylene group Grignard reagent is acetylene magnesium bromide, and the molar ratio of the acetylene group magnesium bromide to the 3,7-hydroxyl protected compound is 1.05-30:1.

[0024] The present application also provides the use of cytochrome P450 enzyme and / or a carrier expressing the same, and / or a cell containing the same, and / or a composition containing the same, and / or an immobilized enzyme product in the production of a steroid compound, including steroid HE3286.

[0025] Further, the P450 enzyme is a P450 BM3 mutant, preferably P450 BM3 mutant LG-23, the amino acid sequence of which is shown as SEQ ID NO: 1, and the nucleotide sequence of which is shown as SEQ ID NO: 2.

[0026] Compared with the prior art, the present application has the beneficial effect that the P450 BM3 mutant enzyme is found for the first time to have the effect of catalyzing the 7β-hydroxylation of dehydroepiandrosterone, and can be applied to the synthesis of steroid HE3286. That is, the P450 BM3 mutant enzyme is first used to catalyze the generation of 7β-hydroxydehydroepiandrosterone from dehydroepiandrosterone in one step, and isopropyl alcohol dehydrogenase is used for the regeneration and circulation of coenzyme NADPH, and further combined with a chemical method for alkyne reaction to generate steroid HE3286. That is, the present application provides a strategy for the synthesis of HE3286 by combining chemical and enzymatic methods, which not only simplifies the synthesis steps of the drug, significantly improves the catalytic selectivity, reduces the by-products and improves the yield, but also has mild reaction conditions, low cost, green environmental protection and high efficiency, and has important application value for promoting the development process of steroid drugs in China. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be considered as limiting the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0028] Figure 1 shows the route for the synthesis of steroid HE3286 by combining the enzyme method with the chemical method according to the present application.

[0029] Figure 2 shows the C7β-hydroxydehydroepiandrosterone 1 H NMR (CHCl3-d, 100MHz) spectrum.

[0030] Figure 3 shows the C7β-hydroxydehydroepiandrosterone 13 C NMR (CHCl3-d, 400MHz) spectrum. DETAILED DESCRIPTION

[0031] As used in this specification and the claims that follow, the words "comprising", "or" and variations of the words, such as, mean "including but not limited to", "and", etc., and are not intended to exclude, for example, other additives, components, integers or steps. As used in this specification and the claims that follow, the words "etc.", "others" and variations of the words, all mean that not only those described in this patent are included, but also other methods, principles, reagents that can be easily replaced. When an element is described as comprising multiple components, steps or conditions, it should be understood that the element can also be described as comprising any combination of such multiple components, steps or conditions, or "consisting of" or "consisting essentially of" multiple such components, steps or conditions or their combination.

[0032] In order to discover a P450 enzyme with 7β-hydroxylation activity on dehydroepiandrosterone, the inventors screened the existing P450 strains in the laboratory and found that the P450BM3 mutant LG-23 had the best 7β-hydroxylation activity on dehydroepiandrosterone.

[0033] These genes, expression cassettes, plasmids, and transformants can be obtained through genetic engineering construction methods well known to those skilled in the art.

[0034] The transformant host can be any microorganism suitable for expressing cytochrome P450BM3, including bacteria and fungi. Preferably, the microorganism is Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, or Escherichia coli, preferably Escherichia coli, more preferably Escherichia coli BL21 (DE3).

[0035] When used as a biocatalyst, the cytochrome P450BM3 of the present invention can be in the form of an enzyme or a bacterial cell. The enzyme forms include free enzymes, immobilized enzymes, including purified enzymes, crude enzymes, fermentation broths, and enzymes immobilized on carriers; the bacterial cell forms include living cells, dead cells, and immobilized cells.

[0036] As another optional embodiment, microbial cells expressing the aforementioned cytochrome P450BM3 can be used as biocatalysts for enzyme-catalyzed reactions. The microorganisms can be in the form of cells or cell fragments thereof, including both living and dead cells. This is because when microorganisms such as Bacillus subtilis, Pichia pastoris, Saccharomyces cerevisiae, or Escherichia coli are no longer fermented and proliferated but are instead used for enzyme-catalyzed reactions, they themselves become natural immobilized enzymes and do not require fragmentation or even extraction and purification processes to be used as enzyme preparations for catalytic reactions. Because both the reaction substrates and reaction products are small molecules that can easily pass through the cell membrane, the biological barrier of the cell, fragmentation of the cell is not necessary, which is economically advantageous.

[0037] More advantageously, many microbial cells contain some coenzymes such as NADP + (Nicotinamide adenine dinucleotide phosphate, coenzyme II) or NAD + (Nicotinamide adenine dinucleotide, coenzyme I) which can effectively promote the redox reaction to proceed without or with reduced need for additional addition of expensive coenzymes in the enzyme catalyzed reaction system.

[0038] In the application of cytochrome P450 BM3 mutants for catalyzing the synthesis of steroid compounds, a co-factor regeneration system can be added to the reaction system. As a preferred embodiment, when cytochrome P450 BM3 and glucose dehydrogenase (GDH) are used for combined catalysis, glucose can be added to the reaction system as the substrate of glucose dehydrogenase. During the reaction, glucose dehydrogenase catalyzes the oxidation of glucose, while (NADP + ) NAD + is reduced to (NADPH) NADH, and cytochrome P450 BM3 and NAD(P)H catalyze the hydroxylation of the substrate. The added amounts of glucose dehydrogenase and glucose can be determined by simple experiments.

[0039] As another alternative embodiment, when cytochrome P450 BM3 mutants and alcohol dehydrogenase are used for combined catalysis, isopropanol can be added to the reaction system as the substrate of alcohol dehydrogenase. During the reaction, alcohol dehydrogenase catalyzes the oxidation of isopropanol, while (NADP + ) NAD + is reduced to (NADPH) NADH, and cytochrome P450 BM3 and NAD(P)H catalyze the hydroxylation of the substrate. The added amounts of alcohol dehydrogenase and isopropanol can be determined by simple experiments.

[0040] It is easy for those skilled in the art to understand that the above-mentioned glucose dehydrogenase and alcohol dehydrogenase can be provided in the form of enzymes or in the form of microbial cells expressing the enzymes.

[0041] In an alternative embodiment, cytochrome P450 BM3 can be co-expressed with glucose dehydrogenase (GDH) or alcohol dehydrogenase (ADH) in the same strain, thereby eliminating the trouble of adding the two enzymes or expressing cells in proportion in the catalytic reaction system.

[0042] In addition to the above mutants, the present patent also provides a new technical solution using a chemical enzyme method strategy to synthesize the steroid HE3286 using dehydroepiandrosterone as a substrate, including the following steps: resuspending the co-expressed or separately expressed P450 enzyme mutant with buffer, adding isopropyl alcohol dehydrogenase or glucose dehydrogenase, substrate dehydroepiandrosterone, co-factor NADP+, isopropyl alcohol or glucose, reacting completely under the condition of 20-30°C, adding ethyl acetate to extract the reaction liquid to obtain ethyl acetate extract, drying with anhydrous sodium sulfate, suction filtration, and concentrating under reduced pressure to obtain 7β-hydroxydehydroepiandrosterone crude product, followed by recrystallization to obtain 7β-hydroxydehydroepiandrosterone pure product. Take 1g of 7β-hydroxydehydroepiandrosterone and add it to a two-necked flask, then add 6.4mL of THF, ice bath, and drop 85mL of 0.33M ethynyl magnesium bromide / tetrahydrofuran solution (18eq) under nitrogen protection, react at 0-40°C, monitor the reaction progress by TLC (dichloromethane:methanol = 15:1), until the substrate is completely converted. After the reaction is completed, quench the reaction liquid with saturated ammonium chloride solution. Add an equal volume of ethyl acetate and extract three times, combine the organic phase, add anhydrous Na2SO4 to dry, filter, remove the organic phase by rotary evaporation, add 2.5mL of isopropyl ether to pulp, cool and crystallize, suction filter, and dry to obtain HE3286 solid product.

[0043] As another alternative embodiment, the P450 includes but is not limited to P450BM3 mutants, and also includes other P450s capable of hydroxylating dehydroepiandrosterone at C7β, among which the P450BM3 mutants reported in the present patent are optimal. The reagent selected in the alkyne reaction includes but is not limited to ethynyl magnesium bromide reagent, and also includes other reagents capable of adding alkyne to C17 of dehydroepiandrosterone, such as ethynyl magnesium chloride, acetylene, trimethyl ethynyl silicon, calcium carbide, etc., among which ethynyl magnesium bromide is optimal.

[0044] The substrate involved in the hydroxylation reaction described herein not only includes this specific compound dehydroepiandrosterone, but also includes its compound precursors or key intermediates or similar compounds, such as androstenedione (CAS:63-05-8), dehydroepiandrosterone acetate (CAS:1239-31-2), androstenediol (CAS:521-17-5), ethynyl androstenediol, epiandrosterone analog substrates, etc.

[0045] The "P450 7β-hydroxylase system" of the present application refers to a cytochrome P450 enzyme system capable of hydroxylating the 7β position of dehydroepiandrosterone. The hydroxylase catalyzed reactions described herein are typically carried out in a solvent. While water is most preferred, in some cases, organic solvents such as ethyl acetate, butyl acetate, 1-octanol, heptane, octane, methyl tert-butyl ether (MTBE), toluene, and the like, as well as ionic liquids such as 1-ethyl 4-methylimidazole tetrafluoroborate, 1-butyl-3-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium hexafluorophosphate, and the like, can be used, alone or in combination with water. In preferred embodiments, aqueous solvents are used, including water and aqueous co-solvent systems. The solvent system is preferably greater than 50%, 75%, 90%, 95%, or 98% water, and in one embodiment is 100% water.

[0046] The reagent selected for the hydroxyl protection reaction in the present application includes, but is not limited to, TBDMSCl, and also includes other silane reagents capable of protecting hydroxyl groups, such as TBDMSOTf, TMSCl, TESCl, TBDPSCl, and TIPSCl, and the like, of which TBDMSCl is the most preferred.

[0047] The amounts, contents, and concentrations of various substances referred to herein are all by weight unless otherwise specified.

[0048] Materials and Methods

[0049] The total gene synthesis, primer synthesis, and sequencing in the examples were completed by Shengong Bioengineering (Shanghai) Co., Ltd.

[0050] The molecular biology experiments in the examples, including plasmid construction, enzyme digestion, ligation, preparation of competent cells, transformation, medium preparation, and the like, were mainly performed according to the Molecular Cloning Laboratory Manual, Third Edition (J. Sambrook, D. W. Russell (USA) edited, Huang Peitang et al. translated, Science Press, Beijing, 2002). The specific experimental conditions can be determined by simple tests if necessary.

[0051] The PCR amplification experiments were performed according to the reaction conditions or kit instructions provided by the plasmid or DNA template supplier. The conditions can be adjusted by simple tests if necessary.

[0052] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.2. (LB solid medium additionally contains 20 g / L agar powder.)

[0053] TB medium: 24 g / L yeast extract, 12 g / L tryptone, 16.43 g / L K2HPO4·3H2O, 2.31 g / L KH2PO4, 5 g / L glycerol, pH 7.0-7.5. (TB solid medium plus 20 g / L agar powder.) Dehydroepiandrosterone was purchased from Sigma-Aldrich.

[0054] All samples were analyzed by Shimadzu high performance liquid chromatography (LC-2030 or LC-2030C). Agilgnt, ZORBAX SB C18 (250 x 4.6 mm) chromatographic column was used, the detection wavelength was 210 nm, and acetonitrile and ultrapure water were used as mobile phases to analyze dehydroepiandrosterone.

[0055] It should be noted that, for the sake of convenience of description, in the examples, the strain number, plasmid number, enzyme number, and enzyme coding gene number can share a number, which is easily understood by those skilled in the art, i.e., the same number can refer to different biological forms in different environments.

[0056] The plasmids expressing cytochrome P450BM3, such as pRSFDuet-LG-23, and the plasmids used for gene editing operations in the examples were constructed and preserved by the Li Aitao research group of the School of Life Sciences, Hubei University, and any unit and individual can obtain the plasmids for verifying the present application, but without the permission of Hubei University, they cannot be used for other purposes, including development and utilization, scientific research, and teaching.

[0057] The present application will be further described in detail below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present application and not used to limit the scope of the present application.

[0058] Example 1 Screening of dehydroepiandrosterone 7β-hydroxylase

[0059] In order to obtain an enzyme capable of 7β-hydroxylating dehydroepiandrosterone, we screened the P450 enzyme library in the laboratory. First, the E. coli of all P450 mutants preserved in the laboratory was streaked on a solid LB plate containing 50 μg / mL kanamycin and incubated at 37°C overnight. Single colonies were selected and inoculated into 2 mL of liquid LB medium containing 50 μg / mL kanamycin and incubated at 37°C with shaking overnight. 500 μL of bacterial solution was inoculated into a 100 mL triangular flask containing 50 mL of TB medium and incubated at 37°C with shaking at 220 rpm. When the absorbance OD600 of the culture reached 0.8, 0.2 mM IPTG was added to induce expression, and the induction temperature was 25°C, and the induction time was 16-20 hours. The culture was centrifuged at 4000 rpm and 4°C for 10 min, the cells were collected and washed once with 100 mM potassium phosphate buffer (pH 8.0), and the cells were stored at -80°C. 600 The culture was centrifuged at 4000 rpm and 4°C for 10 min, the cells were collected and washed once with 100 mM potassium phosphate buffer (pH 8.0), and the cells were stored at -80°C.

[0060] The cells were resuspended in 50 mL centrifuge tubes with 10 mL of 100 mM potassium phosphate buffer, pH 8.0 (containing 5% w / v glucose, 5% v / v glycerol, 0.2 mM NADP + The cells were resuspended in 50 mL centrifuge tubes with 10 mL of 100 mM potassium phosphate buffer, pH 8.0 (containing 5% w / v glucose, 5% v / v glycerol, 0.2 mM NADP

[0061] (1)

[0062] Table 1: Catalytic activity of P450BM3 mutants on dehydroepiandrosterone

[0063] The P450BM3 mutant LG-23 with steroid C7β-hydroxylation activity in this example was constructed by our research group and a related patent has been applied. The amino acid sequence is shown in SEQ ID NO: 1 and the nucleotide sequence is shown in SEQ ID NO: 2.

[0064] Example 2 Two-step synthesis of HE3286

[0065] In this example, a strategy combining biocatalysis and chemical synthesis was used to generate HE3286. First, the substrate enzyme catalyzed dehydroepiandrosterone to generate 7β-hydroxydehydroepiandrosterone in one step, and then chemical acetylene reaction was used to generate HE3286. The reaction flow is shown in (2).

[0066] (2)

[0067] 1. Hydroxylation reaction

[0068] The whole cells of E. coli co-expressing LG-23 mutant and isopropanol dehydrogenase were resuspended with 2 L of 100 mM potassium phosphate buffer, pH 8.0 (containing 5% w / v glucose, 5% v / v glycerol, 0.2 mM NADP 600To a 5L reactor, 2g of dehydroepiandrosterone was added, and 10 mL of isopropanol was added. The reaction was stirred at 25°C for 6-8h until TLC analysis showed that the starting material was completely converted to 7β-hydroxydehydroepiandrosterone. Then, 2L of ethyl acetate was added to extract the reaction solution four times, and the combined ethyl acetate extract was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrated product was recrystallized to obtain 1.9g of 7β-hydroxydehydroepiandrosterone with a molar yield of 90%.

[0069] 2. Alkyne reaction

[0070] To a 5L reactor, 2g of dehydroepiandrosterone was added, and 10 mL of isopropanol was added. The reaction was stirred at 25°C for 6-8h until TLC analysis showed that the starting material was completely converted to 7β-hydroxydehydroepiandrosterone. Then, 2L of ethyl acetate was added to extract the reaction solution four times, and the combined ethyl acetate extract was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrated product was recrystallized to obtain 1.9g of 7β-hydroxydehydroepiandrosterone with a molar yield of 90%.

[0071] Example 3. Three-step synthesis of HE3286

[0072] This example uses a combination of biocatalysis and chemical synthesis to produce HE3286. First, the starting material dehydroepiandrosterone is biocatalytically converted to 7β-hydroxydehydroepiandrosterone. Then, the 3,7-hydroxyl groups are protected, and a chemical alkyne reaction and deprotection are used to produce HE3286. The reaction scheme is shown in (3).

[0073] (3)

[0074] 1. Hydroxylation reaction

[0075] To a 5L reactor, 2g of dehydroepiandrosterone was added, and 10 mL of isopropanol was added. The reaction was stirred at 25°C for 6-8h until TLC analysis showed that the starting material was completely converted to 7β-hydroxydehydroepiandrosterone. Then, 2L of ethyl acetate was added to extract the reaction solution four times, and the combined ethyl acetate extract was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The concentrated product was recrystallized to obtain 1.9g of 7β-hydroxydehydroepiandrosterone with a molar yield of 90%.

[0076] 2. Hydroxyl protection reaction

[0077] Dissolve 7β-hydroxy dehydroepiandrosterone (Compound II) (1.80 g) in THF (18 mL), add imidazole (1.94 g) and TBDMSCl (3.56 g) sequentially, stir at room temperature, dilute with water (18 mL) after reaction is complete, extract with ethyl acetate (3 x 20 mL), dry over anhydrous sodium sulfate, filter, concentrate, add n-hexane to slurry, filter and dry to obtain 3.60 g of 3,7-hydroxyl protected Compound III with a molar yield of 99%.

[0078] 3. Acetylene reaction

[0079] Dissolve 3.6 g of 3,7-hydroxyl protected Compound III in tetrahydrofuran (THF), add ethynyl magnesium bromide (18 eq, 0.33 M in THF) dropwise under nitrogen protection and ice bath conditions, then warm to 35°C and react for 5 h. After TLC detection shows that the reaction is complete, add p-TsOH (2.47 g), stir the reaction solution under reflux, neutralize with saturated NaHCO3 solution (20 mL) after reaction is complete, extract with DCM (3 x 20 mL), dry over anhydrous sodium sulfate, filter, concentrate, recrystallize in acetonitrile (28 mL), filter and dry to obtain 2.2 g of HE3286 with a molar yield of 95%. Dehydroepiandrosterone is converted to HE3286 through three steps with a total yield of 85%.

[0080] The above detailed description of the embodiments of the present application is not intended to limit the present application to the specific details described above. Various modifications and changes can be made to the technical solution of the present application within the scope of the claims and technical concept of the present application, and these simple modifications all belong to the protection scope of the present application.

Claims

1. A method for the synthesis of the steroid HE3286 by a combination of enzymatic and chemical methods, characterized in that, Comprising the following steps: S1, dehydroepiandrosterone is generated 7β-hydroxydehydroepiandrosterone under the action of 7β-hydroxylase; S2, the carbonyl group at C17 of 7β-hydroxydehydroepiandrosterone is subjected to acetylene reaction to generate steroid HE3286.

2. The method of claim 1, wherein, The 7β-hydroxylase is cytochrome P450 enzyme, preferably P450 BM3 mutant, more preferably P450 BM3 mutant LG-23, wherein the amino acid sequence of the P450 BM3 mutant LG-23 is shown as SEQ ID NO: 1; the acetylene reaction is specifically a reagent reaction of 7β-hydroxydehydroepiandrosterone with ethynyl magnesium bromide or acetylene gas or other ethynyl Grignard reagent.

3. The method of claim 2, wherein, The step S1 is: reacting P450 BM3 mutant LG-23 with isopropanol dehydrogenase or glucose dehydrogenase, dehydroepiandrosterone, co-factor NADP + , isopropanol or glucose completely, and then extracting with ethyl acetate to obtain 7β-hydroxydehydroepiandrosterone crude product, followed by recrystallization to obtain 7β-hydroxydehydroepiandrosterone pure product.

4. The method according to any one of claims 1 to 3, characterized in that, The step S2 is specifically: 7β-hydroxydehydroepiandrosterone is taken, tetrahydrofuran is added, ice bath, ethynyl magnesium bromide / tetrahydrofuran solution is added dropwise under nitrogen protection, reaction is carried out at 0-40℃, after completion, saturated ammonium chloride solution is added to quench the reaction solution, ethyl acetate is added for extraction to obtain HE3286 product.

5. The method of any one of claims 1-3, wherein, The step S2 is: S21, 7β-hydroxydehydroepiandrosterone is dissolved in an organic solvent, an activating agent and TBDMSCl are added in sequence to obtain a 3,7-position hydroxyl-protected compound; S22, the carbonyl group at C17 of the 3,7-position hydroxyl-protected compound is subjected to acetylene reaction followed by deprotection to generate HE3286.

6. The method of claim 5, wherein, The step S22 is specifically: the 3,7-position hydroxyl-protected compound is added with a cosolvent and reacted with ethynyl magnesium bromide or acetylene gas or other ethynyl Grignard reagent, after the reaction is completed, p-toluenesulfonic acid is added, and HE3286 is obtained by vacuum concentration.

7. The method of claim 5, wherein, The organic solvent in the step S21 is selected from tetrahydrofuran, acetonitrile, dichloromethane or N,N-dimethylformamide, preferably tetrahydrofuran; the activating agent is selected from imidazole, pyridine, 4-dimethylaminopyridine, 2,6-dimethylpyridine, triethylamine, diisopropylethylamine or 1,8-diazobicyclo[5.4.0]undec-7-ene, preferably imidazole; the molar ratio of TBDMSCl to 7β-hydroxydehydroepiandrosterone is 3-5:1, the molar ratio of imidazole to TBDMSCl is 1.2-1.5:1, and the reaction temperature is not higher than 50℃.

8. The method of claim 6, wherein, The cosolvent is selected from tetrahydrofuran, diethyl ether, isopropyl ether, methyl tert-butyl ether, ethylene glycol dimethyl ether, 2-methyltetrahydrofuran, 1,4-dioxane, the ethynyl Grignard reagent is ethynyl magnesium bromide, and the molar ratio of the ethynyl magnesium bromide to the 3,7-position hydroxyl-protected compound is 1.05-30:

1.

9. Application of cytochrome P450 enzyme and / or carrier expressing the same, and / or cell containing the same, and / or composition containing the same, and / or immobilized enzyme product thereof in production of steroid compound, the steroid compound including steroid HE3286.

10. Use according to claim 9, characterized in that, The P450 enzyme is P450 BM3 mutant, preferably P450 BM3 mutant LG-23, and the amino acid sequence of the P450 BM3 mutant LG-23 is shown as SEQ ID NO: 1.

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