A chemical-enzymatic method for preparing dehydroepiandrosterone

A technology for preparing dehydroepiandrosterone and enzymatic method, which is applied in the field of chemical-enzymatic preparation of dehydroepiandrosterone, can solve the problems of high organic solvent consumption, difficult to remove impurities, high consumption of potassium tert-butoxide, etc., and achieve product yield The effect of high efficiency, high practical value and low dosage

Active Publication Date: 2020-10-02
ENZYMEWORKS
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In the reported 4-AD to 5-AD reaction, the purity of 5-AD is the main problem affecting the next step of the reaction, because its impurities are difficult to remove, and there is generally a high amount of potassium tert-butoxide, and the product 5-AD has a high yield. The problem of low yield, such as the amount of potassium tert-butoxide in the patent WO2014188353 is 5 times the equivalent of the substrate, and the product yield is only 83%. Considering that the substrate accounts for about 80% of the cost of the product dehydroepiandrosterone, there is greater difficulty in application
It is also worth noting that the second-step biosynthesis of this patent uses NAD and NADP as coenzymes at the same time, the amount of organic solvent is high, and the yield is 70%, which also has practical problems

Method used

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  • A chemical-enzymatic method for preparing dehydroepiandrosterone
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  • A chemical-enzymatic method for preparing dehydroepiandrosterone

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Experimental program
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Effect test

Embodiment 1

[0023] Embodiment 1 prepares 5-AD

[0024] At 25°C, nitrogen gas was blown into tert-butanol (80 mL) for 15 minutes, and potassium tert-butoxide (6.0 g, 3.0 eq) was added under aeration conditions, and the reactor was sealed, replaced with nitrogen and stirred for half an hour. Substrate (5.0 g, 1.0 eq) was added under nitrogen protection, and reacted at 35° C. for 5 hours. Take 3.5g of sodium ascorbate, 5.7g of acetic acid, and 200mL of water, stir for 15 minutes, pour the above reaction solution into it, stir for half an hour under nitrogen protection, and the conversion rate is 96.5% as detected by HPLC. The mixture was filtered to obtain a white solid, which was washed with water and spin-dried to obtain 4.8 g of a white solid with a purity of 95.5% and a yield of 91.7% by HPLC.

Embodiment 2

[0025] Embodiment 2 prepares dehydroepiandrosterone

[0026] Dissolve the above 4.8g product in 25mL 2-methyltetrahydrofuran, add 25mL 100mM pH6.5 phosphate buffered saline solution and start stirring, add 5g glucose, 80mg magnesium chloride hexahydrate, 60mg ketoreductase (by sequence 26 in Escherichia coli obtained by expression in ), 30mg of glucose dehydrogenase (from Suzhou Hanzyme Biotechnology Co., Ltd., number EW002), 20mg of NAD, started the reaction at 25°C, and controlled the pH to 6.5 with 40% sodium hydroxide. After 6 hours, the sample was taken for detection. The conversion rate is 99%, adjust the pH99%, content>99% , after the mother liquor was evaporated to dryness, 1.4g of solid was obtained, which was recovered and applied mechanically, and the yield after multiple batches of post-treatment was 85%.

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Abstract

The invention discloses a method for preparing dehydroepiandrosterone through a chemical-enzyme method. The method comprises that dehydroepiandrosterone is prepared from 4-androstenedione as an initial substrate orderly through a chemical method and a biological method. In preparation, the two reaction processes are optimized. In the chemical method-based 5-androstenedione preparation process, a reaction solution is added into an aqueous solution with sodium ascorbate and acetic acid so that reaction conditions are mild. In the second biological method-based dehydroepiandrosterone preparation process, a ketoreductase is used as a catalyst so that the product yield and purity are improved. In the whole reaction processes, use amounts of a coenzyme and potassium tert-butoxide are low and a high practical value is obtained.

Description

technical field [0001] The invention relates to the field of pharmacy, in particular to a method for preparing dehydroepiandrosterone by a chemical-enzymatic method. Background technique [0002] The structure of dehydroepiandrosterone (DHEA) is shown in Formula 1. It is a steroidal compound secreted by the human adrenal cortex. It has the functions of delaying aging and resisting diseases, and is also a precursor for the synthesis of other steroidal drugs. [0003] [0004] Among the currently disclosed methods for preparing DHEA, most of them are fermentation methods and chemical methods. The fermentation method of adding precursors to microbial fermentation broth for biotransformation, as reported in patent CN 201210316197, takes a long time, the product content is less than 1g / L, and there are problems such as separation and extraction. The chemical methods starting with other steroidal compounds, such as those reported in patents CN102212099, 102603841, and 10260383...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P33/06C07J1/00
CPCC07J1/0011C12P33/06
Inventor 张跃
Owner ENZYMEWORKS
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