CN106086148B

CN106086148B - Method for preparing dehydroepiandrosterone by chemical-enzymatic method

Info

Publication number: CN106086148B
Application number: CN201610439697.1A
Authority: CN
Inventors: 张跃
Original assignee: ENZYMEWORKS Inc
Current assignee: ENZYMEWORKS Inc
Priority date: 2016-06-20
Filing date: 2016-06-20
Publication date: 2020-10-02
Anticipated expiration: 2036-06-20
Also published as: CN106086148A

Abstract

The invention discloses a method for preparing dehydroepiandrosterone by a chemical-enzymatic method, which takes 4-androstenedione as an initial substrate and sequentially carries out two steps of a chemical method and a biological method to prepare the dehydroepiandrosterone, wherein the two-step reaction process is optimized in the preparation process, in the process of preparing 5-androstenedione by the chemical method, reaction liquid is added into an aqueous solution mixed with sodium ascorbate and acetic acid, so that the reaction condition is milder, and simultaneously, in the process of preparing the dehydroepiandrosterone by the second biological method, ketoreductase is adopted as a catalyst, so that the yield and the purity of the prepared product are higher, and simultaneously, the dosage of coenzyme, potassium tert-butoxide and other raw materials in the whole reaction process is lower, so that the method has higher practical value.

Description

Method for preparing dehydroepiandrosterone by chemical-enzymatic method

Technical Field

The invention relates to the field of pharmacy, in particular to a method for preparing dehydroepiandrosterone by a chemical-enzymatic method.

Background

Dehydroepiandrosterone (DHEA) has a structure shown in formula 1, is a steroid compound secreted by human adrenal cortex, has effects of delaying aging, resisting diseases, etc., and is also a precursor for synthesizing other steroid medicines.

Among the methods for preparing dehydroepiandrosterone that have been disclosed so far, many are fermentation methods and chemical methods. The fermentation method of adding the precursor into the microbial fermentation liquid for biotransformation, as reported in patent CN 201210316197, etc., takes a long time, has a product content less than 1g/L, and has the problems of separation and extraction, etc. Other steroids are used as initial chemical methods, such as patents CN 1022120099, 102603841, 102603839 and the like, and the steps of protection, hydrolysis, hydrogenation, oximation and the like are mostly used, so that the yield is low and the environmental problem is serious.

Common 4-androstenedione (4-AD) is taken as an initial substrate, 5-androstenedione (5-AD) is generated through double bond shift reaction of a chemical method in sequence, and dehydroepiandrosterone can be prepared through carbonyl reduction reaction of a biological method, so that the method is the method which has the fewest steps and high yield at present. Literature Tetrahedron Letters, 1962, 15: 669-672 et al report the use of potassium tert-butoxide for the double bond shift reaction of 4-AD; patent WO2014188353 reports chemical double bond displacement and subsequent enzymatic reduction.

In the reported reaction of generating 5-AD from 4-AD, the purity of 5-AD is a main problem influencing the next reaction, because the impurities are difficult to remove, and the problems of high using amount of potassium tert-butoxide and low yield of the product 5-AD are generally existed, for example, in patent WO2014188353, the using amount of potassium tert-butoxide is 5 times equivalent of the substrate, the product yield is only 83%, and considering that the substrate accounts for about 80% in the cost of the product dehydroepiandrosterone, the application is difficult. It is also noted that the second step of biosynthesis in this patent uses NAD and NADP as coenzymes, and that the amount of organic solvent is high, the yield is 70%, and there is also a problem of practicality.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a method for preparing dehydroepiandrosterone by a chemical-enzymatic method.

In order to achieve the purpose, the invention adopts the technical scheme that: a method for preparing dehydroepiandrosterone by a chemical-enzymatic method comprises the following steps:

1) reacting 4-androstenedione serving as a substrate for 1-12 hours at the temperature of 20-40 ℃ under the protection of nitrogen and with tert-butyl alcohol serving as a solvent under the action of potassium tert-butoxide, then adding the reaction solution into an aqueous solution mixed with sodium ascorbate and acetic acid, detecting the reaction process by HPLC, and finishing the reaction when the conversion rate reaches 90-99% to obtain 5-androstenedione, wherein the feeding mass ratio of the potassium tert-butoxide to the 4-androstenedione is 2-0.5: 1;

2) forming a reaction system by the 5-androstenedione obtained in the step 1), a solvent and a water phase buffer solution, adding a biocatalyst, magnesium chloride hexahydrate, a coenzyme and a coenzyme regeneration system, reacting at the temperature of 25-40 ℃, controlling the pH value of the whole reaction process to be 6.0-7.5, detecting the reaction process by HPLC, and finishing the reaction when the conversion rate reaches 90-99% to obtain a product dehydroepiandrosterone, wherein the solvent is 2-methyltetrahydrofuran, the biocatalyst is ketone reductase, the coenzyme regeneration system is glucose and glucose dehydrogenase, and the glucose: biocatalyst glucose dehydrogenase: the feeding mass ratio of the 5-androstenedione is as follows: 0.8-1.2: 0.008-0.016: 0.004-0.008: 1.

Preferably, the aqueous phase buffer solution in step 2) is a phosphate buffer solution with a pH of 6.5.

Preferably, the coenzyme is NAD, and the feeding mass ratio of the coenzyme to 5-androstenedione is as follows: 0.003-0.005: 1.

Preferably, the glucose dehydrogenase is a glucose dehydrogenase available from Suzhou Han enzyme Biotechnology Limited under the designation EW 002.

Preferably, the ketoreductase gene sequence has at least 90% homology with any sequence in the sequences 1-26.

Further preferably, the ketoreductase is the expression product in a non-pathogenic microorganism.

Still more preferably, the non-pathogenic microorganism is Escherichia coli.

Preferably, the specific implementation process is as follows:

1) reacting 4-androstenedione serving as a substrate for 1-12 hours at the temperature of 20-40 ℃ under the protection of nitrogen and with tert-butyl alcohol serving as a solvent under the action of potassium tert-butoxide, then adding the reaction solution into an aqueous solution mixed with sodium ascorbate and acetic acid, detecting the reaction process by HPLC (high performance liquid chromatography), when the conversion rate reaches 90-99%, ending the reaction, filtering the mixed solution to obtain a white solid, washing with water, and spin-drying to obtain a 5-androstenedione product;

2) dissolving the 5-androstenedione obtained in the step 1) in 2-methyltetrahydrofuran, adding the aqueous phase buffer solution, stirring, adding the glucose, magnesium chloride hexahydrate, ketoreductase, glucose dehydrogenase and coenzyme, carrying out reaction at the temperature of 25-40 ℃, controlling the pH of a reaction system to be 6.0-7.5, detecting the reaction process by HPLC, when the conversion rate reaches 90-99%, finishing the reaction, adjusting the pH value of a reaction system to be less than 2, adding ethyl acetate and activated carbon, stirring for 1-12 h, filtering, washing a filter cake for three times by using ethyl acetate, combining filtrate and washing liquid, removing an organic solvent by rotary evaporation, adding ethyl acetate into a water phase for extraction, combining ethyl acetate, concentrating, slowly adding n-heptane at room temperature, standing overnight for crystallization, filtering, drying to obtain a dehydroepiandrosterone product, and evaporating a mother liquor to dryness to obtain the dehydroepiandrosterone solid product.

Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages: compared with the existing route, the method for preparing dehydroepiandrosterone by the chemical-enzymatic method realizes the purposes of high product yield and high purity by improving the first-step chemical method and the second-step biological method and adopting milder reaction conditions and better catalysts, and simultaneously has lower consumption of raw materials such as coenzyme, potassium tert-butoxide and the like and higher practical value.

Detailed Description

The present invention will be described in further detail with reference to specific examples, but the present invention is not limited to the following examples. The implementation conditions adopted in the examples can be further adjusted according to different requirements of specific use, and the implementation conditions not indicated are those in routine experiments.

EXAMPLE 1 preparation of 5-AD

Nitrogen was introduced into t-butanol (80mL) at 25 ℃ for 15min, and potassium t-butoxide (6.0g, 3.0eq) was added under aeration conditions, and after that, the reactor was sealed, purged with nitrogen and stirred for half an hour. The substrate (5.0g, 1.0eq) was added under nitrogen and reacted at 35 ℃ for 5 hours. 3.5g of sodium ascorbate, 5.7g of acetic acid and 200mL of water are taken and stirred for 15 minutes, the reaction solution is poured into the mixture and stirred for half an hour under the protection of nitrogen, and the conversion rate is 96.5 percent by HPLC. The mixture was filtered to give a white solid, which was washed with water and dried by spinning to give 4.8g of a white solid with a purity of 95.5% by HPLC and a yield of 91.7%.

EXAMPLE 2 preparation of dehydroepiandrosterone

4.8g of the above product was dissolved in 25mL of 2-methyltetrahydrofuran, 25mL of 100mM phosphate buffer solution pH6.5 was added to start stirring, and 5g of glucose, 80mg of magnesium chloride hexahydrate, 60mg of ketoreductase (obtained by expression of SEQ ID NO: 26 in E.coli), 30mg of glucose dehydrogenase (from Han enzyme Biotechnology Co., Ltd., Suzhou, No. EW002), 20mg of NAD were added in this order to start the reaction at 25 ℃ and the pH was controlled to 6.5 with 40% sodium hydroxide. Sampling and detecting after 6 hours, wherein the conversion rate is 99%, adjusting the pH value to be less than 2 by using hydrochloric acid, adding 30mL of ethyl acetate and 2g of activated carbon, stirring for 1 hour, filtering, washing a filter cake for three times by using 15mL of ethyl acetate, combining filtrate and washing liquid, removing an organic solvent by rotary evaporation, adding 40mL of ethyl acetate into a water phase, extracting for three times, combining ethyl acetate, concentrating to 20mL of volume, slowly adding 40mL of n-heptane at room temperature, crystallizing overnight, filtering, drying to obtain 3.5g of a product with the purity of more than 99% and the content of more than 99%, evaporating a mother solution to dryness to obtain 1.4g of a solid, and recycling and applying the solid again to obtain 85% of a multi-batch post-.

The above-mentioned embodiments are merely illustrative of the technical idea and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the scope of the present invention, and all equivalent changes or modifications made according to the spirit of the present invention should be covered in the scope of the present invention.

Claims

1. A method for preparing dehydroepiandrosterone by a chemical-enzymatic method is characterized by comprising the following steps:

2) forming a reaction system by the 5-androstenedione obtained in the step 1), a solvent and a water phase buffer solution, adding a biocatalyst, magnesium chloride hexahydrate, a coenzyme and a coenzyme regeneration system, reacting at the temperature of 25-40 ℃, controlling the pH value of the whole reaction process to be 6.0-7.5, detecting the reaction process by HPLC, and finishing the reaction when the conversion rate reaches 90-99% to obtain a product dehydroepiandrosterone, wherein the solvent is 2-methyltetrahydrofuran, the biocatalyst is ketone reductase, the coenzyme regeneration system is glucose and glucose dehydrogenase, and the glucose: biocatalyst glucose dehydrogenase: the feeding mass ratio of the 5-androstenedione is as follows: 0.8-1.2: 0.008-0.016: 0.004-0.008: 1; the ketoreductase gene sequence has at least 90% of homology with any sequence in the sequences 2-26.

2. The chemo-enzymatic method for preparing dehydroepiandrosterone according to claim 1, wherein the aqueous buffer solution in step 2) is a phosphate buffer solution with pH 6.5.

3. The chemo-enzymatic method for preparing dehydroepiandrosterone according to claim 1, wherein the coenzyme is NAD and the mass ratio of the coenzyme to 5-androstenedione is: 0.003-0.005: 1.

4. The chemo-enzymatic method of preparing dehydroepiandrosterone according to claim 1 wherein the glucose dehydrogenase is a glucose dehydrogenase having the designation EW002, available from sehan enzyme biotechnology limited, su.

5. The chemo-enzymatic process for preparing dehydroepiandrosterone according to claim 1 wherein the ketoreductase is the product of expression in a non-pathogenic microorganism.

6. The chemo-enzymatic method for preparing dehydroepiandrosterone according to claim 5, wherein the non-pathogenic microorganism is Escherichia coli.

7. The method for preparing dehydroepiandrosterone by the chemo-enzymatic method according to claim 1, comprising the following steps:

2) dissolving the 5-androstenedione obtained in the step 1) in 2-methyltetrahydrofuran, adding the aqueous phase buffer solution, stirring, adding the glucose, magnesium chloride hexahydrate, ketoreductase, glucose dehydrogenase and coenzyme, carrying out reaction at the temperature of 25-40 ℃, controlling the pH of a reaction system to be 6.0-7.5, detecting the reaction process by HPLC, when the conversion rate reaches 90-99%, finishing the reaction, adjusting the pH value of a reaction system to be less than 2, adding ethyl acetate and activated carbon, stirring for 1-12 h, filtering, washing a filter cake with ethyl acetate for three times, combining filtrate and washing liquid, removing an organic solvent by rotary evaporation, adding ethyl acetate into a water phase for extraction, combining ethyl acetate, concentrating, slowly adding n-heptane at room temperature, standing overnight for crystallization, filtering, drying to obtain a dehydroepiandrosterone product, and evaporating a mother liquor to dryness to obtain the dehydroepiandrosterone solid product.