Use of a cytochrome p450 bm3 mutant in the synthesis of allylprogesterone

Abstract

The application discloses application of a cytochrome P450BM3 mutant in synthesis of allylprogesterone, the cytochrome P450BM3 mutant can catalyze 4, 9 to generate 11 alpha-hydroxy-4, 9, and further synthesis of allylprogesterone is combined with chemical catalysis. That is, the application provides a method for synthesizing allylprogesterone by combining biological enzyme catalysis and chemical catalysis, which not only simplifies a drug synthesis step, improves catalytic selectivity, reduces by-products, improves yield, and 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.

Description

Technical Field

[0001] The invention belongs to the technical field of biocatalytic enzymes, and particularly relates to application of a cytochrome P450 BM3 mutant in the synthesis of allylgesterone. Background Art

[0002] Steroid hormones are the second largest class of drugs in the world, second only to antibiotics. They are widely used to treat inflammation, cardiovascular disease, tumors, skin diseases, endocrine disorders, geriatric diseases, and other ailments. my country has abundant natural steroid resources, and its production scale and product quality are approaching world-leading levels. However, there is still a gap in the technological level of steroid drug research and development. This is primarily due to the cumbersome synthesis steps, complex reactions, significant long-range effects of the groups, low yields, and, in particular, the difficulties of separation and purification.

[0003] Steroid hormones can be divided into three main categories based on their physiological activity: anabolic androgenic hormones, adrenocortical hormones, and sex hormones. Alprogesterone, a natural progesterone analog, can reduce the concentrations of follicular estrogen and luteinizing hormone in animal serum, subsequently causing follicular degeneration and inhibiting normal estrus and ovulation. Upon discontinuation of therapy, luteinizing hormone concentrations in the animal's plasma gradually recover, allowing follicle maturation and ovulation. Alprogesterone is widely used to synchronize estrus and subsequent insemination in animals, facilitating reproductive management in livestock farming, such as sow breeding.

[0004] All compounds of allylprogesterone contain a 4,9,11-conjugated triene structure. Its synthesis process uses estra-4,9-diene-3,17-dione (estra-4,9-diene-3,17-dione, tetra-nine compound) as the raw material. It is necessary to first perform an acetal protection operation on the 3-position carbonyl group to convert the 4,9-conjugated diene structure of 19-norandrostenedione into a 5(10),9(11)-conjugated diene structure. Then, on this basis, DDQ (dichlorodicyanoquinone) chemical dehydrogenation is used to introduce a double bond at the 9,11 position of the steroid nucleus. In the DDQ chemical dehydrogenation process, in order to remove the residual DDQ in the product after the reaction is completed, a large amount of water is required to wash (first wash the product with alkaline water to remove DDQ, and then wash the product with clean water until it is neutral), resulting in a large amount of wastewater and complex preparation of high-quality products. In addition, the entire reaction process involves protection and deprotection steps, which reduces the total yield of the product.

[0005] The combination of enzymatic hydroxylation and chemical dehydration introduces a double bond at the 9,11 position, avoiding both the protection and deprotection steps required for DDQ dehydrogenation and the generation of large amounts of wastewater during the DDQ removal process, thus improving product quality. However, no P450 enzymatic hydroxylation of steroidal compounds containing 4,9-conjugated dienes (such as methyl dienolone and tetranona) has been reported. Summary of the Invention

[0006] The present invention addresses the deficiencies of the prior art and provides a P450 BM3 mutant enzyme that has an 11α-hydroxylation effect on estradiol-4,9-diene-3,17-dione (4,9-compound). Starting from the inexpensive and readily available 4,9-compound, the P450 BM3 mutant enzyme is used to catalyze the 4,9-compound to produce the 11α-hydroxy 4,9-compound, and GDH enzyme is used to regenerate and recycle the cofactor NADPH. This is further combined with a chemical catalytic reaction to produce allergy-progesterone. Enzymatic catalysis has high selectivity, few by-products, mild reaction conditions, low cost, and is environmentally friendly and efficient. By combining enzymatic catalysis with chemical catalysis, allergy-progesterone can be synthesized more efficiently and quickly.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] One of the objects of the present invention is to provide a cytochrome P450 BM3 mutant, or its vector, or the use of a cell containing the vector in the synthesis of allergy-resistant progesterone, wherein the cytochrome P450 BM3 mutant includes: mutant LG-23, and / or mutants LG-23 / T438S, LG-23 / T438A, and LG-23 / T438G obtained by mutating the threonine at position 438 to serine, alanine, or glycine based on the mutant LG-23,

[0009] Furthermore, the amino acid sequence of the mutant LG-23 is shown in SEQ ID NO: 1, the amino acid sequence of the mutant LG-23 / T438S is shown in SEQ ID NO: 2, the amino acid sequence of the mutant LG-23 / T438A is shown in SEQ ID NO: 3, and the amino acid sequence of the mutant LG-23 / T438G is shown in SEQ ID NO: 4.

[0010] Furthermore, estra-4,9-diene-3,17-dione is treated with the cytochrome P450 BM3 mutant to generate 11α-hydroxymethylestra-4,9-diene-3,17-dione.

[0011] Furthermore, the vector contains a gene encoding a cytochrome P450BM3 mutant as shown in any one of SEQ ID NOs. 5-8; wherein the nucleotide sequence encoding the mutant LG-23 gene is shown in SEQ ID NO: 5, the nucleotide sequence encoding the mutant LG-23 / T438S gene is shown in SEQ ID NO: 6, the nucleotide sequence encoding the mutant LG-23 / T438A gene is shown in SEQ ID NO: 7, and the nucleotide sequence encoding the mutant LG-23 / T438G gene is shown in SEQ ID NO: 8.

[0012] The second object of the present invention is to provide a method for synthesizing alprogesterone, which comprises the steps of combining bioenzyme catalysis with chemical catalysis to produce alprogesterone.

[0013] Furthermore, the bio-enzyme catalysis is performed by the above-mentioned cytochrome P450 BM3 mutant or its carrier, or a cell containing the carrier.

[0014] Furthermore, the bioenzyme catalysis includes: generating 11α-hydroxymethylestradiol-4,9-diene-3,17-dione from estradiol-4,9-diene-3,17-dione under the action of the cytochrome P450 BM3 mutant or its carrier, or cells containing the carrier.

[0015] Furthermore, the bio-enzyme catalysis process is specifically as follows: the genetically engineered bacteria expressing the cytochrome P450 BM3 mutant are dissolved in a buffer solution, glucose dehydrogenase, substrate estradiol-4,9-diene-3,17-dione, and cofactor NADP are added. + , glucose, and react completely at 20-30°C. Ethyl acetate is added to extract the reaction solution to obtain ethyl acetate extract, which is dehydrated with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain crude 11α-hydroxyestra-4,9-diene-3,17-dione.

[0016] Furthermore, during the bio-enzyme catalysis process, the coding gene of the cytochrome P450 BM3 mutant is connected to an expression regulatory sequence to obtain an expression vector.

[0017] Furthermore, the above expression vector is transformed into Escherichia coli to obtain an expression genetically engineered bacterium.

[0018] Furthermore, the OD of the co-expression genetically engineered bacteria 600 is 20-30, the molar concentration of estradiol-4,9-diene-3,17-dione is 1mM, and NADP + The molar concentration is 0.5 mM, the final glucose dehydrogenase activity concentration is 1 U / mL, and the glucose content is 5% (m / v).

[0019] Furthermore, the glucose dehydrogenase encoding gene is connected to the expression regulatory sequence alone to obtain an expression vector of the glucose dehydrogenase gene.

[0020] Furthermore, the chemical catalysis includes chemical dehydration, ketal protection, Grignard reaction, and finally hydrolysis to generate allylgestalone.

[0021] Furthermore, the chemical dehydration comprises: reacting 11α-hydroxyestra-4,9-diene-3,17-dione with p-toluenesulfonic acid monohydrate, and extracting to obtain estra-4,9,11-triene-3,17-dione.

[0022] Furthermore, the chemical dehydration step is specifically as follows: dissolving the crude 11α-hydroxymethyl dienol ketone in chloroform, adding monohydrated p-toluenesulfonic acid, stirring at room temperature until the reaction is complete, adding saturated sodium carbonate to terminate the reaction, and extracting the reaction solution with chloroform to obtain a chloroform extract, dehydrating with anhydrous sodium sulfate, filtering, concentrating under reduced pressure, separating and purifying by column chromatography, and concentrating and drying to obtain estra-4,9,11-triene-3,17-dione.

[0023] Furthermore, in the chemical dehydration step, the mass ratio of 11α-hydroxymethyl dienolone: ​​chloroform: p-toluenesulfonic acid monohydrate is 1:25-35:0.6-0.8.

[0024] Furthermore, the ketal protection comprises: reacting estra-4,9,11-triene-3,17-dione with ethylene glycol, triethyl orthoformate and boron trifluoride etherate to obtain 3-position ketal-protected estra-4,9,11-triene-3,17-dione-3-vinyl ketal.

[0025] Furthermore, the ketal protection step is specifically as follows: adding estra-4,9,11-triene-3,17-dione, ethylene glycol, and triethyl orthoformate to a reaction flask, stirring and dissolving, cooling to 0°C, then adding boron trifluoride etherate, maintaining the temperature at 0°C and stirring to react, after the TLC reaction is complete, slowly pouring the reaction solution into the alkaline solution prepared in advance to quench, precipitating with water and stirring, filtering, washing the filter cake with water once, and drying the solid at 40°C with air blast to obtain estra-4,9,11-triene-3,17-dione-3-vinyl ketal.

[0026] Furthermore, in the ketal protection step, the ratio of estra-4,9,11-triene-3,17-dione, ethylene glycol, and triethyl orthoformate is 30 (m):240 (v):83 (m).

[0027] Furthermore, in the ketal protection step, the mass ratio of estra-4,9,11-triene-3,17-dione to boron trifluoride etherate is 187.5:1.

[0028] Furthermore, in the ketal protection step, the stirring reaction time after adding boron trifluoride etherate is 5 hours.

[0029] Furthermore, the alkali solution in the ketal protection step is a 2% NaOH solution.

[0030] Furthermore, the Grignard reaction comprises: reacting estra-4,9,11-triene-3,17-dione-3-vinyl ketal with allyl magnesium chloride to obtain 17α-allyl-17β-hydroxy-3-vinyl ketal-estra-4,9,11-triene.

[0031] Furthermore, the Grignard reaction step is specifically as follows: adding estra-4,9,11-triene-3,17-dione-3-vinyl ketal and tetrahydrofuran to a reaction flask, stirring to dissolve, adding a nitrogen balloon, pulling vacuum for replacement three times, stirring and cooling to 0°C, and then starting to dropwise add allylmagnesium chloride, and after the dropwise addition is completed, stirring and reacting at 0-5°C, heating to 5-10°C and stirring and reacting, after the TLC reaction is complete, slowly pouring the reaction solution into the ammonium chloride aqueous solution prepared in advance to quench, stirring, standing and separating, the aqueous layer is extracted once with tetrahydrofuran, the organic phases are combined, washed once with sodium chloride aqueous solution, separated, and the organic phase is concentrated under reduced pressure to dryness to obtain an oily substance 17α-allyl-17β-hydroxy-3-vinyl ketal-estra-4,9,11-triene.

[0032] Furthermore, in the Grignard reaction step, the mass-to-volume ratio of estrast-4,9,11-triene-3,17-dione-3-vinyl ketal to tetrahydrofuran is 1:10.

[0033] Furthermore, in the Grignard reaction step, the mass volume ratio of estra-4,9,11-triene-3,17-dione-3-vinyl ketal to allyl magnesium chloride is 1:4.01.

[0034] Furthermore, the hydrolysis comprises: hydrolyzing 17α-allyl-17β-hydroxy-3-vinyl ketal-estra-4,9,11-triene in acetone and sulfuric acid aqueous solution to obtain allylgestal.

[0035] Furthermore, the hydrolysis step is specifically as follows: the 17α-allyl-17β-hydroxy-3-vinyl ketal-estrast-4,9,11-triene obtained by the concentration in the previous step is dissolved in acetone and transferred to a reaction bottle, stirred and cooled to 0-5°C, and then a sulfuric acid aqueous solution is added dropwise. After the addition is completed, the mixture is kept at 0-5°C and stirred for reaction. After the TLC reaction is complete, the reaction solution is slowly poured into the quenching solution prepared in advance for quenching, stirred, and allowed to stand for separation. The aqueous layer is back-extracted twice with dichloromethane, the organic phases are combined, washed twice with water, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. Ethyl acetate is entrained twice, and after concentration to dryness, 1.0V of ethyl acetate is added and stirred to dissolve, stirred, cooled to -5°C, and stirred for crystallization. Filter by suction, rinse the filter cake with ice ethyl acetate, and dry the solid at 40°C with air to obtain allylgestal.

[0036] Furthermore, in the hydrolysis step, the mass volume ratio of 17α-allyl-17β-hydroxy-3-vinyl ketal-estrast-4,9,11-triene to acetone is 1:10.

[0037] Furthermore, the aqueous sulfuric acid solution used in the hydrolysis step was prepared by slowly adding 7.2 g of sulfuric acid to 51 g of water.

[0038] Furthermore, in the hydrolysis step, the stirring time after adding the sulfuric acid aqueous solution is 0.5 h.

[0039] Furthermore, the quenching solution used in the hydrolysis step is 7.2 g of sodium carbonate, 300 mL of water is added, stirred to dissolve, and then 300 ml of dichloromethane is added, stirred, and cooled to 5-10°C.

[0040] Furthermore, in the hydrolysis step, the stirring time after adding the quenching solution is 10 minutes.

[0041] Compared with the prior art, the present invention has the following beneficial effects: the present invention discovers that the cytochrome P450 BM3 mutant LG-23 and mutants LG-23 / T438S, LG-23 / T438A, and LG-23 / T438G all have the function of catalyzing the 11α-hydroxylation of steroid compounds, and can be applied to the synthesis of allergasterone. Starting from the cheap and readily available 4,9-compound (estra-4,9-diene-3,17-dione), the P450 BM3 mutant enzyme is used to catalyze the 4,9-compound to generate the 11α-hydroxy 4,9-compound (11α-hydroxymethylestra-4,9-diene-3,17-dione), and GDH enzyme is used to regenerate and recycle the cofactor NADPH, and further combined with a chemical catalytic reaction to generate allergasterone. That is, the present invention provides a method for synthesizing allergy-resistant progesterone by combining bio-enzyme catalysis and chemical catalysis. The method not only simplifies the synthesis steps of the drug, significantly improves the catalytic selectivity, reduces by-products and increases the yield, but also has mild reaction conditions, low cost, is green, environmentally friendly and efficient, and has important production and application value in promoting the development of steroid drugs in my country. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0043] Figure 1 This is a graph showing the HPLC analysis results of the mutants LG-23 and LG-23 / T438S catalyzing the 11α-hydroxylation of 4,9-compounds or methyldienolone in Example 1 of the present invention;

[0044] Figure 2 This is a reaction flow chart of combining enzyme catalysis and chemical catalysis to produce allylgestalone in Example 3 of the present invention;

[0045] Figure 3 This is the LC / MS analysis spectrum of the allergasteride prepared in Example 3 of the present invention.

[0046] Figure 4 This is the H analysis spectrum of allylgestaltenone prepared in Example 3 of the present invention. DETAILED DESCRIPTION

[0047] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0048] Example 1 Site-directed mutagenesis and functional verification of the P450 BM3 enzyme mutant LG-23

[0049] 1. Site-directed mutagenesis of mutant LG-23

[0050] In this example, based on the P450 BM3 enzyme mutant LG-23 (Li A, Acevedo-Rocha CG, D'Amore L, et al. Regio-and Stereoselective SteroidHydroxylation at the C7-Position by Cytochrome P450 Monooxygenase Mutants [J]. Angewandte Chemie International Edition, 2020) known in the prior art and having steroid 7β-hydroxylation activity, the plasmid vector pRSFDuet-LG-23 expressing the mutant LG-23 was used as a template, and the gene of the mutant LG-23 was subjected to site-directed mutagenesis using primer PCR mutagenesis technology, and the threonine at position 438 was mutated to serine, alanine and glycine, respectively. A pair of primers was designed for the mutation site, wherein the plasmid vector pRSFDuet-LG-23 is already available in this unit.

[0051] This example takes the mutant LG-23 / T438S as an example and provides mutagenesis primers that can be used when obtaining the mutant LG-23 / T438S by site-directed mutagenesis (as shown in SEQ ID NOs: 9-10 in the sequence listing):

[0052] Upstream primer: 5'-TTAAAGAAACTTTATCTTTAAAACCTGAAGGCTTTG-3',

[0053] Downstream primer: 5'-ATTTCCGGCGTGTGAATCAAGCG-3',

[0054] The bold sequences are mutation sites.

[0055] The PCR system (20 μL) was as follows: 0.1-1 ng of template, 1 μL each of a pair of mutation primers (10 μM), 5 μL Prime STARMax DNA polymerase, and sterile distilled water was added to 20 μL.

[0056] The PCR reaction procedure was as follows: (1) pre-denaturation at 98°C for 3 min; (2) denaturation at 98°C for 10 sec, (3) annealing at 56°C for 15 sec, and (4) extension at 72°C for 70 sec. Steps (2) to (4) were repeated 28 times, with a final extension at 72°C for 5 min. The product was stored at 8°C.

[0057] The amplified PCR product was detected by 0.7% agarose gel electrophoresis, and a band of the same length as the plasmid containing the P450 BM3 gene was observed. It was determined that the gene encoding the target mutant had been amplified. Therefore, the restriction endonuclease Dpn I was directly added to the PCR product, digested at 37°C for 3-5 hours, and then transformed into E. coli DH5ɑ competent cells. After adding culture medium for 1 hour, the cells were evenly spread on a solid LB plate containing 50μl / mg kanamycin. After overnight culture at 37°C, a single clone was selected and cultured in 3 mL of LB liquid medium containing 50 μl / mg kanamycin. The mutant was then sent to a sequencing company for sequencing to obtain the correct mutant, which was named LG-23 / T438S. The coding sequence mutation sites included: R47W / S72W / F77Y / V78L / F81I / A82L / F87G / T88S / M177T / M185Q / I209T / A328G / A330W / T438S. The amino acid sequence of the mutant LG-23 / T438S is shown in SEQ ID NO: 2, and the nucleotide sequence is shown in SEQ ID NO: 6.

[0058] Mutation primers were designed using a method similar to the above method, and the threonine at position 438 was mutated to alanine to obtain the mutant LG-23 / T438A, whose amino acid sequence is shown in SEQ ID NO: 3 in the sequence listing, and the nucleotide sequence is shown in SEQ ID NO: 7 in the sequence listing; the threonine at position 438 was mutated to glycine to obtain the mutant LG-23 / T438G, whose amino acid sequence is shown in SEQ ID NO: 4 in the sequence listing, and the nucleotide sequence is shown in SEQ ID NO: 8 in the sequence listing.

[0059] 2. Functional Verification

[0060] The plasmids pRSFDuet-LG-23 / T438S, pRSFDuet-LG-23 / T438A and pRSFDuet-LG-23 / T438G obtained above were transformed into E.coli BL21 competent cells respectively, and after 1 hour of recovery with culture medium, they were evenly spread on a solid LB plate containing 50μl / mg kanamycin. The E.coli BL21 glycerol tube bacteria containing LG-23 stored at -80°C were streaked on a solid LB plate containing 50μl / mg kanamycin and cultured at 37°C overnight. A single clone was selected and transferred to 2mL of liquid LB medium containing 50μl / mg kanamycin, and cultured at 37°C with shaking overnight. 500μL of the bacterial solution was inoculated into a 100mL triangular flask containing 50mL of TB medium, and cultured on a shaker at 37°C and 220rpm. When the absorbance of the culture solution reached OD 600 When the p-value reached 0.8, IPTG was added to a final concentration of 0.2 mM to induce expression at 25°C for 16-20 hours. The culture was centrifuged at 4000 rpm for 10 minutes at 4°C, and the cells were harvested, washed once with 100 mM potassium phosphate buffer (pH 8.0), and stored at -80°C.

[0061] The cells were washed with 10 mL of 100 mM potassium phosphate buffer (pH 8.0, containing 5% glucose, 5% glycerol, 0.5 mM NADP) + , 10U GDH) was resuspended in a 50mL centrifuge tube and immediately quick-frozen in liquid nitrogen. It was then placed in water and thawed at room temperature until thawed. 5mL of the bacterial suspension was taken into a 50mL Erlenmeyer flask, 50uL of the steroid substrate mother solution (prepared to 100mM mother solution in DMF) was added, and the reaction was carried out at 25°C and 220rpm for 5 hours. Samples were taken at intervals, and the reaction solution was extracted with an equal volume of ethyl acetate and centrifuged at high speed for 3min. The upper layer of ethyl acetate was transferred to a clean EP tube. After complete evaporation, it was resuspended with an equal volume of acetonitrile and filtered through a 0.22μm filter membrane into a sample injection bottle. The conversion rate and product distribution of the reaction were detected by HPLC.

[0062] The chromatographic column was a ZORBAX SB C18 (250 × 4.6 mm) column, and the mobile phase consisted of acetonitrile / ultrapure water (10:90) for 2 minutes, 70:20 for 2–15 minutes, and 10:90 for 15–17 minutes. The column temperature was 40°C, the flow rate was 1.5 mL / min, and the injection volume was 10 μL. The UV detection wavelength for estradiol-4,9-diene-3,17-dione (4,9-propionate) and the hydroxylated product was 310 nm.

[0063] The data of catalytic 4,9-substance 11α hydroxylation of 1 mM (0.27 g / L) 4,9-substance by P450 BM3 mutants LG-23, LG-23 / T438S, LG-23 / T438A and LG-23 / T438G are shown in Table 1. The HPLC analysis results are shown in Figure 1 shown.

[0064] Table 1 Cytochrome P450 BM3 mutant catalyzes steroid 11α-hydroxylation

[0065]

[0066] according to Figure 1 The equation for the 4,9-catalysis of P450 BM3 mutants LG-23, LG-23 / T438S, LG-23 / T438S, LG-23 / T438A or LG-23 / T438G is shown in (1).

[0067] (1)

[0068] According to the results in Table 1, the conversion rate and selectivity of mutant LG-23 / T438S for 11α-hydroxylation of 4,9 compounds were improved compared to mutant LG-23. The selectivity and conversion rates of LG-23 / T438A and LG-23 / T438G were comparable to those of LG-23. The selectivity of mutant LG-23 / T438S for 11α-hydroxylation of 4,9 compounds was increased to 94%. These results indicate that mutant LG-23 / T438S is relatively more effective in catalyzing the 11α-hydroxylation of steroids. Subsequently, mutant LG-23 / T438S was used as an example in the synthesis of allylgesterone.

[0069] Example 2 Preparation of GDH crude enzyme freeze-dried powder

[0070] The mutants LG-23 / T438S, LG-23 / T438A, LG-23 / T438G or LG-23 require the coenzyme NADPH for the 11α-hydroxylation reaction of 4,9 compounds, and use glucose dehydrogenase to regenerate and recycle the cofactor NADPH.

[0071] Glucose dehydrogenase is a glucose dehydrogenase (GDH enzyme) from Bacillus megaterium, and its catalytic equation is shown in (5):

[0072] (5) Glucose + NADP + →Gluconolactone + NADPH

[0073] In order to catalyze the generation of 11α-hydroxy 4,9-compound from 4,9-compound, the GDH enzyme gene can be expressed alone, and the crude enzyme from the supernatant of broken bacteria can be used to catalyze the regeneration and recycling of the cofactor NADPH.

[0074] Preparation of Glucose Dehydrogenase (GDH) Lyophilized Powder

[0075] The GDH gene from Bacillus megaterium was transferred into E. coli BL21 using conventional methods in the art. Expression was induced with IPTG at 25°C for 14-16 hours. The culture was centrifuged at 4000 rpm for 10 minutes at 4°C, and the cells were harvested and washed once with 100 mM potassium phosphate buffer (pH 8.0). The cells were then resuspended in a 50 mL shake tube and ultrasonically disrupted in an ice-water mixture (power 350 W, 2 s on, 4 s off, 15 minutes). Once the solution became clear, it was centrifuged at 9000 rpm at 4°C for 30 minutes to separate the supernatant and cell pellet. Small aliquots were distributed in Petri dishes and lyophilized to a powder in a vacuum freeze dryer. The powder can be stored at -20°C for a long time. If there is no long-term demand for large quantities, the GDH lysis supernatant can be used directly for experiments and stored at -80°C for short-term use.

[0076] The enzyme activity of the obtained GDH crude enzyme freeze-dried powder was determined as follows: 1 mg of GDH crude enzyme freeze-dried powder was dissolved in 940 μL of 100 mM potassium phosphate buffer (pH 8.0) in a cuvette, and 20 μL of 50 mM NADP was added. + A 1-minute time scan at 340 nm was performed using a spectrophotometer as a blank control. Add 20 μL of 50% glucose to the cuvette, mix quickly, and then perform a time scan immediately. Calculate the amount of NADPH generated in 1 minute (μmol) based on the NADPH concentration and the absorbance at 340 nm. Repeat the experiment three times. Calculate the GDH enzyme activity. If the amount of NADPH generated is 1 μmol, the activity of the lyophilized GDH powder is 1 U / mg.

[0077] Example 3 Synthesis of allylgestalone

[0078] This embodiment uses a combination of bioenzyme catalysis and chemical catalysis to generate allylgestalone. First, bioenzyme catalyzes the 4,9 compound to generate the 11α-4,9 compound. Then, chemical dehydration, 3-ketal protection, Grignard reaction, and finally hydrolysis to generate allylgestalone are performed. The reaction flow chart is shown in FIG. Figure 2 shown.

[0079] The specific experimental process includes:

[0080] 1. Hydroxylation reaction

[0081] The whole E. coli cells of BM3-LG-23 / T438S co-expressing P450 obtained by culture in Example 1 were resuspended in 4 L of 100 mM potassium phosphate buffer with a pH of 8.0 (OD 600 20~30), add to 10L reaction tank, 25℃, 400rpm stirring. 1g of 4,9 was dissolved in 40mL DMF and put into the reaction tank, add 0.7g NADP + , 100g glucose, 2000U GDH crude enzyme, after addition, stir and react at 25℃ for 2-3 hours until the raw material reaction is complete according to TLC analysis. Then add 4L ethyl acetate to extract the reaction solution three times, combine all the ethyl acetate extracts, dehydrate with anhydrous sodium sulfate, filter, and concentrate under reduced pressure until there is no distillate to obtain the crude product of 11α-4,9 compound (II). Its LC / MS analysis spectrum is as follows Figure 3 shown.

[0082] 2. Dehydration reaction

[0083] The crude 11α-4,9-component (II) was dissolved in 25 mL of chloroform, and 0.66 g of p-toluenesulfonic acid monohydrate was added. The reaction was stirred at room temperature until complete reaction was confirmed by TLC analysis. The reaction was terminated by the addition of 10 mL of saturated sodium carbonate solution. The aqueous phase was then extracted twice with 10 mL of chloroform. The combined chloroform solutions were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to a small volume. The desired product was then isolated and purified by silica gel column chromatography. The collected chromatographic solution was concentrated under reduced pressure to dryness to obtain 0.729 g of estra-4,9,11-triene-3,17-dione (III), with a mass yield of 72.9% (based on the 4,9-component) and an HPLC assay of 98.3%.

[0084] The product was structurally characterized using a Bruker AVANCE AV400 nuclear magnetic resonance spectrometer to collect data. The 1H spectrum data are as follows:

[0085] 1 H NMR(400MHz,Chloroform-d)δ6.54–6.42(m,2H),5.80(s,1H),2.81(dddd,J=17.2,15.2,8.1,4.1Hz,2H),2.70–2.39(m,6 H),2.26–2.12(m,1H),2.03(ddt,J=16.2,12.0,4.5Hz,2H),1.85–1.60(m,2H),1.40(qd,J=12.5,4.8Hz,1H),1.01(s,3H).

[0086] 3. Ketal protection

[0087] Prepare lye: Add 20g of sodium hydroxide and 1000mL of water to a beaker, stir and dissolve until clear;

[0088] Take a 500mL reaction bottle, add 30g of compound III, 240mL of ethylene glycol, 83g of triethyl orthoformate, stir and dissolve, cool to 0°C, then add 0.16g of boron trifluoride etherate, keep stirring at 0°C and react for 5h. After the TLC reaction is complete, the reaction solution is slowly poured into the alkaline solution prepared in advance to quench, precipitate and stir for 30min, filter, wash the filter cake with water once, and dry the solid at 40°C with air to obtain 31.5g of compound IV with a mass yield of 105%.

[0089] The product was characterized by using a Bruker AVANCE AV400 NMR spectrometer to collect data. 1 The H spectrum data are as follows:

[0090] 1 H NMR (400MHz, DMSO-d6) δ6.45(d,J=9.8Hz,1H),6.15(d,J=9.8Hz,1H),5.41(s,1H),3.94–3.81(m,4H),2.49(d,J=19.9Hz,7H),2.46–2.28 (m,3H),2.20–2.06(m,1H),1.91(dq,J=11.9,5.0,3.6Hz,2H),1.71(t,J=6.5Hz,2H),1.68–1.53(m,2H),1.26–1.11(m,1H),0.91(s,3H).

[0091] 4. Grignard reaction

[0092] Take a 500mL reaction bottle, add 30 g of compound IV and 300mL of tetrahydrofuran, stir to dissolve, add a nitrogen balloon, pull vacuum and replace three times, stir and cool to 0°C, then start to add 120.2mL of allylmagnesium chloride dropwise, keep warm at 0-5°C and stir to react for 1h, raise the temperature to 5-10°C and stir to react for 2.0h. After the TLC reaction is complete, slowly pour the reaction solution into the previously prepared aqueous ammonium chloride solution to quench, stir for 10min, let stand and separate, extract the aqueous layer once with tetrahydrofuran, combine the organic phases, wash once with sodium chloride aqueous solution, separate the layers, and concentrate the organic phase under reduced pressure to dryness to obtain compound V as an oil.

[0093] 5. Hydrolysis reaction

[0094] Prepare sulfuric acid aqueous solution: slowly add 7.2g sulfuric acid dropwise to 51g water, stir and cool to room temperature for later use;

[0095] Prepare quenching solution: add 7.2g sodium carbonate to the reaction flask, add 300mL water, stir to dissolve, then add 300ml dichloromethane, stir and cool to 5-10℃ for later use;

[0096] The compound V oil obtained by the previous step of concentration was dissolved in 300 mL of acetone and transferred to a 500 mL reaction bottle, stirred and cooled to 0-5 ° C, and then sulfuric acid aqueous solution was added dropwise. After the addition was completed, the temperature was kept at 0-5 ° C and stirred for 0.5 h. After the TLC reaction was complete, the reaction solution was slowly poured into the quenching solution prepared in advance to quench, stirred for 10 min, and allowed to stand for stratification. The aqueous layer was back-extracted twice with dichloromethane, the organic phases were combined, washed twice with water, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. Ethyl acetate was entrained twice, and after concentration to dryness, 1.0 V of ethyl acetate was added and stirred to dissolve, stirred and cooled to -5 ° C and stirred for crystallization. Filtered with suction, the filter cake was rinsed with ice ethyl acetate, and the solid was dried with air at 40 ° C to obtain 25.5 g of allylgestaltenes with a mass yield of 85%.

[0097] The product was characterized by using a Bruker AVANCE AV400 NMR spectrometer to collect data. 1 The H spectrum data are as follows:

[0098] 1 H NMR (400MHz, Chloroform-d) δ6.46 (d, J = 10.0 Hz, 1H), 6.33 (d, J = 10.0 Hz, 1H), 5.95 (ddt, J = 17. 3,10.2,7.2Hz,1H),5.76(s,1H),5.25–5.10(m,2H),2.80(tdd,J=7.0,4.9,2.0Hz,2H),2.65–2. 51(m,2H),2.45(t,J=7.3Hz,3H),2.35–2.25(m,1H),2.17(dd,J=13.8,7.1Hz,1H),2.10–1.97(m ,1H),1.95–1.86(m,2H),1.82–1.59(m,3H),1.57–1.41(m,1H),1.38–1.20(m,1H),1.02(s,3H).

[0099] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. Use of a cytochrome P450 BM3 mutant, or a vector thereof, or a cell comprising the vector in the synthesis of naprogesterone, characterized in that: The cytochrome P450 BM3 mutants include: mutant LG-23, and / or mutants LG-23 / T438S, LG-23 / T438A, and LG-23 / T438G obtained by mutating the threonine at position 438 to serine, alanine, or glycine on the basis of mutant LG-23, The amino acid sequence of the mutant LG-23 is shown in SEQ ID NO: 1, the amino acid sequence of the mutant LG-23 / T438S is shown in SEQ ID NO: 2, the amino acid sequence of the mutant LG-23 / T438A is shown in SEQ ID NO: 3, and the amino acid sequence of the mutant LG-23 / T438G is shown in SEQ ID NO: 4; The synthesis of allylgestalone adopts a combination of biological enzyme catalysis and chemical catalysis, wherein the biological enzyme catalysis includes: generating 11α-hydroxymethyl-estra-4,9-diene-3,17-dione by reacting estra-4,9-diene-3,17-dione with the cytochrome P450 BM3 mutant or its carrier, or cells containing the carrier; The chemical catalysis steps include: chemical dehydration, ketal protection, Grignard reaction, and finally hydrolysis to generate allylgestalone; The chemical dehydration comprises: reacting 11α-hydroxy estradiol-4,9-diene-3,17-dione with p-toluenesulfonic acid monohydrate, and extracting to obtain estradiol-4,9,11-triene-3,17-dione; the ketal protection comprises: reacting estradiol-4,9,11-triene-3,17-dione with ethylene glycol, triethyl orthoformate and boron trifluoride etherate to obtain 3-position ketal-protected estradiol-4,9,11-triene-3,17-dione. Ketone-3-vinyl ketal; the Grignard reaction includes: reacting estra-4,9,11-triene-3,17-dione-3-vinyl ketal with allyl magnesium chloride to obtain 17α-allyl-17β-hydroxy-3-vinyl ketal-estra-4,9,11-triene; the hydrolysis includes: hydrolyzing 17α-allyl-17β-hydroxy-3-vinyl ketal-estra-4,9,11-triene in acetone and sulfuric acid aqueous solution to obtain allylgestal.

2. The use according to claim 1, characterized in that The vector contains a gene encoding a cytochrome P450 BM3 mutant as shown in any one of SEQ ID NOs. 5-8.

3. A method for synthesizing alprogesterone, characterized in that: Allylgestalone is produced by combining biological enzyme catalysis and chemical catalysis, wherein the biological enzyme catalysis is carried out by the cytochrome P450 BM3 mutant or its carrier, or a cell containing the carrier, according to claim 1, comprising: generating 11α-hydroxymethylestradiol-4,9-diene-3,17-dione by the action of the cytochrome P450 BM3 mutant or its carrier, or a cell containing the carrier; The chemical catalysis steps include: chemical dehydration, ketal protection, Grignard reaction, and finally hydrolysis to generate allylgestalone; The chemical dehydration comprises: reacting 11α-hydroxy estradiol-4,9-diene-3,17-dione with p-toluenesulfonic acid monohydrate, and extracting to obtain estradiol-4,9,11-triene-3,17-dione; the ketal protection comprises: reacting estradiol-4,9,11-triene-3,17-dione with ethylene glycol, triethyl orthoformate and boron trifluoride etherate to obtain 3-position ketal-protected estradiol-4,9,11-triene-3,17-dione. Ketone-3-vinyl ketal; the Grignard reaction includes: reacting estra-4,9,11-triene-3,17-dione-3-vinyl ketal with allyl magnesium chloride to obtain 17α-allyl-17β-hydroxy-3-vinyl ketal-estra-4,9,11-triene; the hydrolysis includes: hydrolyzing 17α-allyl-17β-hydroxy-3-vinyl ketal-estra-4,9,11-triene in acetone and sulfuric acid aqueous solution to obtain allylgestal.

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