CN113493814B

CN113493814B - Dehydroepiandrosterone biosynthesis method

Info

Publication number: CN113493814B
Application number: CN202010251276.2A
Authority: CN
Inventors: 谢新开; 黄晓飞; 梁晓亮
Original assignee: Hunan Yinhang Biological Technology Co ltd
Current assignee: Hunan Yinhang Biological Technology Co ltd
Priority date: 2020-04-01
Filing date: 2020-04-01
Publication date: 2023-03-31
Anticipated expiration: 2040-04-01
Also published as: CN113493814A

Abstract

The invention discloses a biological preparation method of dehydroepiandrosterone, which starts from a cheap and easily-obtained steroid androstenedione ester and finally obtains a target product with a yield as high as 94 percent through one-step biological enzyme catalytic reaction. The process has no accumulation of the intermediate A, so oxidative impurities generated due to instability of the intermediate A can not be generated, the reaction yield is high, and the product quality is good.

Description

Dehydroepiandrosterone biosynthesis method

Technical Field

The invention belongs to the technical field of biological pharmacy and biochemical engineering, and particularly relates to a biological preparation method of dehydroepiandrosterone.

Background

Dehydroepiandrosterone (DHEA) is a substance with important biological activity and multiple physiological functions. It plays an important role in maintaining normal physiological functions, prolonging life span and the like. The secretion of DHEA in humans peaks at 25-30 years of age and subsequently declines year by year. A large number of clinical experiments prove that the high DHEA level of a human body is beneficial to the health of the human body. The compound has wide application, and has anti-aging and protein assimilation effects as a sex hormone precursor. The main functions are as follows: 1. improving sexual function and increasing libido; 2. regulating immune system function, and improving immunity; 3. enhancing physical ability, improving emotion and sleep, and improving memory; 4. delaying aging and keeping youth activity; 5, the traditional Chinese medicine composition can be used for adjuvant treatment of diseases, and is currently applied to treatment of systemic lupus erythematosus, anti-allergy/asthma and the like.

The prior method for producing dehydroepiandrosterone mainly comprises a chemical method: however, the method generally has the defects of long steps, harsh reaction conditions, environmental friendliness and the like. Such as:

the reported routes of Chinese patent application No. 201110085711.X, publication No. 1022120099A are as follows:

the method uses dehydroepiandrosterone as a target product, which is obtained by taking dehydroepiandrosterone as a raw material and carrying out three chemical reactions such as oximation reaction, beckmann rearrangement reaction, hydrolysis reaction and the like to obtain the target product with a total yield of about 70%. The process route needs to use a large amount of easy-to-poison reagents, namely acetic anhydride and acetone, generates a large amount of waste water and is not environment-friendly. In addition, the overall yield of this route is only 70%, and the atom economy is poor.

The reported routes of the Chinese patent application No. 201210038094.2 and the publication No. 102603841A are as follows:

the method uses steroid androstenedione as a raw material to carry out four-step chemical reactions of esterification reaction, ketal reaction (protecting carbonyl), reduction reaction, hydrolysis reaction (deprotection) and the like, and finally obtains the target product dehydroepiandrosterone with a total yield of about 80%. The method needs two steps of protection and deprotection of a substrate, and is very uneconomical.

Some attempts have been made by biological workers to prepare this compound, and in chinese patent CN105695551B, steroid androstenedione is used as the starting material, and the intermediate a is obtained by double bond displacement under the action of potassium tert-butoxide. Then the ketoreductase is used for carrying out selective asymmetric reduction on the 3-position ketocarbonyl of the intermediate A, and the reaction formula is as follows:

this method, while a great improvement over conventional chemical methods, has several drawbacks. Such as: two equivalents of potassium tert-butoxide, an easily explosive chemical, are required; the medium form A is poorly stable, readily converts to androstenedione and produces a variety of oxidative impurities (Development of a chemoenzymic Process for Dehydroepiandrosterone Synthesis, org. Process Res. Dev.2016,20, 1520-1528) resulting in lower yields and poor product quality.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a simple and feasible biological method for synthesizing dehydroepiandrosterone. The method is simple to operate, mild in condition and environment-friendly, greatly reduces the production cost, and is suitable for large-scale industrial production.

The structure of dehydroepiandrosterone of the invention is as follows:

the invention is characterized in that: the target product can be obtained with a yield up to 94% by one-step biological enzyme catalytic reaction starting from cheap and easily-obtained steroid androstenedione ester. The process has no accumulation of the intermediate A, so oxidative impurities generated by instability of the intermediate A can not be generated, the reaction yield is high, and the product quality is good.

The reaction formula of the invention is as follows:

the reaction process is as follows:

the ester group of the raw material steroid androstenedione ester is hydrolyzed under the action of hydrolase to form an enol structure, the enol structure and the intermediate A have reaction balance, but because selective ketoreductase aiming at the intermediate A exists in a reaction system at the same time, the intermediate A is quickly reduced into DHEA, so the reaction balance of enol and the intermediate A is broken, the intermediate A is continuously and quickly converted, the amount of the intermediate A in the system is very small, and the accumulation of the intermediate A is reduced and avoided to the maximum extent.

The invention provides a method for synthesizing dehydroepiandrosterone, which is characterized in that steroid androstenedione ester, hydrolase and ketoreductase are dissolved in a solvent together to prepare dehydroepiandrosterone;

further, nicotinamide adenine dinucleotide is added as a coenzyme;

further, adding glucose and glucose dehydrogenase or isopropanol and alcohol dehydrogenase or formate and formate dehydrogenase to realize the regeneration of coenzyme;

further, the solvent is tert-butyl alcohol and phosphate buffer;

further, the pH value of the method is 6-8, and the pH value is kept stable by adding alkali in the reaction process;

further, the steroid androstenedione ester is steroid androstenedione acetate;

it is to be noted that the ketoreductase is a selective ketoreductase which can selectively and asymmetrically reduce the ketocarbonyl group at the 3-position of the substrate, and the ketoreductase is known, and the selective asymmetric reduction can be achieved by the ketoreductase described in CN105695551B, or by the ketoreductase described below; as the glucose dehydrogenase for coenzyme regeneration, the enzyme described in CN105695551B may be used, or a glucose dehydrogenase enzyme described below may be used, and the coenzyme regeneration effect can be achieved. Systems for coenzyme regeneration, not limited to glucose-glucose dehydrogenase systems, isopropanol-isopropanol dehydrogenase and formate-formate dehydrogenase systems are suitable (Biocatalysis and Biotransformation,2004,22 (2), 89-96 adv Biochem Eng Biotechnol, 2010,120, 195-242). The choice of hydrolytic enzymes is likewise known and freely available on the market, and is not restricted here, provided that the esters of steroidal androstenedione can be hydrolyzed efficiently.

Description of the drawings:

in order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the embodiments are briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts

FIG. 1 is a high performance liquid chromatogram of a steroidal androstenedione acetate;

FIG. 2 is a high performance liquid chromatogram of double bond shifted intermediate A;

FIG. 3 is a high performance liquid chromatogram of Dehydroepiandrosterone (DHEA);

FIG. 4 is a high performance liquid chromatogram of a mixture during a reaction.

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.

Example 1:

high performance liquid chromatography detection of androstenedione acetate

Androstenedione acetate source: preparation of androstenedione as raw material

Reference is made to the following documents: journal of Chinese medicine and chemistry, no. 5, vol.23, 368-371

The high performance liquid chromatography analysis method comprises the following steps:

the obtained high performance liquid chromatogram is shown in FIG. 1.

Example 2:

high performance liquid chromatography detection of double bond shift intermediate A

Source of double bond displaced intermediate a: preparation method of androstenedione by using androstenedione as raw material

Reference is made to the following documents: chinese patent CN105695551B

the obtained high performance liquid chromatogram is shown in FIG. 2.

Example 3:

high performance liquid chromatography detection of Dehydroepiandrosterone (DHEA)

Sources of Dehydroepiandrosterone (DHEA): purchased from Shanghai Aladdin Biotechnology Ltd

the obtained high performance liquid chromatogram is shown in FIG. 3.

Example 4:

synthesis of DHEA of the invention

1500 g of 100mM phosphate buffer (pH = 6.5) was added to a 3-liter reaction flask, and 300 g of glucose and 300 ml of t-butanol were added under stirring and stirred uniformly. 400 g of androstenedione esterified substance as a substrate, 400 mg of nicotinamide adenine dinucleotide, 4 g of ketoreductase enzyme powder (purchased from Suzhou piloting biotechnology limited, product number YH 2046) and 4 g of glucose dehydrogenase enzyme powder (purchased from Suzhou piloting biotechnology limited, product number YH 1901) were added in sequence under stirring, and the reaction temperature was controlled to 30-35 ℃ by 4 g of hydrolase enzyme powder (purchased from Suzhou piloting biotechnology limited, product number YH 1203), and the reaction was started with stirring, and the pH of the system was adjusted to 6.2-6.9 with 6M NaOH during the reaction. After 20 hours of reaction, sampling and HPLC detection are carried out, and the conversion rate is over 99 percent. And (3) completely reacting, distilling under reduced pressure to remove tert-butyl alcohol, filtering, and washing a filter cake with water to obtain a crude DHEA. The crude DHEA was crystallized from a mixed solvent of methanol and water to yield 330 g of pure DHEA in 94% yield. The purity of the product reaches 99.7 percent, and the content of the product reaches 99.6 percent.

Example 5

The method detects the mixture by high performance liquid chromatography in the reaction process.

The reaction time was adjusted to 7 hours according to the method of example 4, after which a sample was taken for HPLC analysis by the following method:

the obtained high performance liquid chromatogram is shown in FIG. 4.

From the analysis of fig. 4, the reaction process has no accumulation of intermediate a, and thus no oxidative impurities due to instability of intermediate a are generated.

Example 6: synthesis of DHEA of the invention

1500 g of 100mM phosphate buffer (pH = 6.5) was added to a 3-liter reaction flask, and 300 g of glucose and 300 ml of t-butanol were added thereto with stirring and stirred uniformly. 400 g of androstenedione esterified substance as a substrate, 400 mg of nicotinamide adenine dinucleotide, 4 g of ketoreductase enzyme powder (purchased from Suzhou piloting Biotechnology Co., ltd., product number YH 2046) and 4 g of glucose dehydrogenase enzyme powder (purchased from Suzhou piloting Biotechnology Co., ltd., product number YH 1901) were added in this order under stirring, 10 g of hydrolase enzyme powder (purchased from Shandong Long Dabiol Ltd., product name: neutral lipase) were added to control the reaction temperature at 30-35 ℃ to start the stirring reaction, and the pH of the system was adjusted to 6.2-6.9 with 6M NaOH during the reaction. After 20 hours, a sample was taken and tested by HPLC, and the conversion rate was 97%. And (3) completely reacting, distilling under reduced pressure to remove tert-butyl alcohol, filtering, and washing a filter cake with water to obtain a crude DHEA. The crude DHEA is crystallized by a mixed solvent of methanol and water to obtain a pure DHEA product with the yield of 88 percent. The purity of the product reaches 99.6 percent, and the content of the product reaches 99.6 percent.

Example 7: synthesis of DHEA

1500 g of 100mM phosphate buffer (pH = 6.5) was added to a 3-liter reaction flask, and 300 g of glucose and 300 ml of t-butanol were added under stirring and stirred uniformly. 400 g of androstenedione esterified substance as a substrate, 400 mg of nicotinamide adenine dinucleotide, 4 g of ketoreductase enzyme powder (purchased from Suzhou piloting biotechnology limited, product number is YH 2046) and 4 g of glucose dehydrogenase enzyme powder (purchased from Suzhou piloting biotechnology limited, product number is YH 1901) were added in sequence under stirring, 8 g of hydrolase enzyme powder (purchased from Suzhou encyclopedia group, product name: lipase) were added to control the reaction temperature to 30-35 ℃ to start the stirring reaction, and the pH of the system was adjusted to 6.2-6.9 with 6M NaOH during the reaction. After 20 hours, sampling and HPLC detection are carried out, and the conversion rate is more than 99%. And (3) completely reacting, distilling under reduced pressure to remove tert-butyl alcohol, filtering, and washing a filter cake with water to obtain a crude DHEA. The crude DHEA is crystallized by a mixed solvent of methanol and water to obtain pure DHEA with the yield of 90 percent. The purity of the product reaches 99.5 percent, and the content of the product reaches 99.3 percent.

Example 8: synthesis of DHEA

1500 g of 100mM phosphate buffer (pH = 6.5) was added to a 3-liter reaction flask, and 300 g of glucose and 300 ml of t-butanol were added thereto with stirring and stirred uniformly. 400 g of substrate androstenedione esterified ester, 400 mg of nicotinamide adenine dinucleotide, 4 g of ketoreductase enzyme powder (purchased from Suzhou piloting Biotechnology Co., ltd., product number YH 2046) and 4 g of glucose dehydrogenase enzyme powder (purchased from Suzhou piloting Biotechnology Co., ltd., product number YH 1901) were added in sequence under stirring, 5 g of hydrolase enzyme powder (purchased from Novoxil, product number: 06-3140) were reacted at 30-35 ℃ with stirring, and the pH of the system was adjusted to 6.2-6.9 with 6M NaOH during the reaction. After 20 hours, sampling and HPLC detection are carried out, and the conversion rate is more than 99%. And (3) completely reacting, distilling under reduced pressure to remove tert-butyl alcohol, filtering, and washing a filter cake with water to obtain a crude DHEA product. The crude DHEA is crystallized by a mixed solvent of methanol and water to obtain a pure product with the yield of 96 percent. The purity of the product reaches 99.8 percent, and the content reaches 99.5 percent.

Example 9: synthesis of DHEA

1500 g of 100mM phosphate buffer (pH = 6.5) was added to a 3-liter reaction flask, and 300 g of glucose and 300 ml of t-butanol were added under stirring and stirred uniformly. 400 g of androstenedione esterified substance as a substrate, 400 mg of nicotinamide adenine dinucleotide, 4 g of ketoreductase enzyme powder (purchased from Suzhou piloting biotechnology limited, and the product number is YH 2046) and 4 g of glucose dehydrogenase enzyme powder (purchased from Suzhou piloting biotechnology limited, and the product number is YH 1901) are added in turn under stirring, 4 g of hydrolase enzyme powder (purchased from Tianye enzyme preparation (Jiangsu) limited, and the product name is Lipase PS "Amano" IM), the reaction temperature is controlled at 30-35 ℃, the stirring reaction is started, and the pH of a 6M NaOH regulation system is kept at 6.2-6.9 in the reaction process. After 20 hours, sampling and HPLC detection are carried out, and the conversion rate is more than 99%. And (3) completely reacting, distilling under reduced pressure to remove tert-butyl alcohol, filtering, and washing a filter cake with water to obtain a crude DHEA. The crude DHEA is crystallized by a mixed solvent of methanol and water to obtain the pure DHEA with the yield of 92 percent. The purity of the product reaches 99.6 percent, and the content of the product reaches 99.4 percent.

Claims

1. Dehydrogenation meterThe synthesis method of androsterone is characterized in that steroid androstenedione ester, hydrolase and ketoreductase are dissolved in solvent together to prepare dehydroepiandrosterone, wherein the ketoreductase is selective ketoreductase which can selectively and asymmetrically reduce 3-position ketocarbonyl of intermediate A, and the structure of the intermediate A is

2. The method of synthesis according to claim 1, wherein nicotinamide adenine dinucleotide is added as a coenzyme.

3. The method of claim 2, wherein the regeneration of the coenzyme is effected by the addition of glucose and glucose dehydrogenase or isopropanol and alcohol dehydrogenase or formate and formate dehydrogenase.

4. The synthetic method of any one of claims 1-3 wherein the solvent is t-butanol and phosphate buffer.

5. The method of claim 4, wherein the pH of the method is from 6 to 8 and the pH is maintained constant during the reaction by addition of a base.

6. A method according to any one of claims 1 to 3, wherein the steroid androstenedione ester is a steroid androstenedione acetate.