CN106086148A - A kind of chemical-enzymatic prepares the method for dehydroepiandros-sterone - Google Patents
A kind of chemical-enzymatic prepares the method for dehydroepiandros-sterone Download PDFInfo
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- CN106086148A CN106086148A CN201610439697.1A CN201610439697A CN106086148A CN 106086148 A CN106086148 A CN 106086148A CN 201610439697 A CN201610439697 A CN 201610439697A CN 106086148 A CN106086148 A CN 106086148A
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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- C07J1/0011—Androstane derivatives substituted in position 17 by a keto group
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Abstract
The invention discloses a kind of method that chemical-enzymatic prepares dehydroepiandros-sterone, with 4 androstenedione as initial substrate, carry out chemical method successively and bioanalysis two step prepares dehydroepiandros-sterone, in preparation process, two-step reaction process is optimized, during chemical method prepares 5 androstenedione, reactant liquor is joined and has been mixed with in sodium ascorbate and vinegar aqueous acid, make reaction condition the gentleest, simultaneously during second step bioanalysis prepares dehydroepiandros-sterone, have employed ketoreductase as catalyst, make to prepare the yield of the product of gained and purity is higher, in whole course of reaction, the consumption of the raw material such as coenzyme and potassium tert-butoxide is lower simultaneously, there is higher practical value.
Description
Technical field
The present invention relates to pharmaceutical field, be specifically related to a kind of method that chemo-enzymatic process prepares dehydroepiandros-sterone.
Background technology
Dehydroepiandros-sterone (Dehydroepiandrosterone, DHEA) structure as shown in Equation 1, is human acth cortex
The steroidal compounds of secretion, has slow down aging, resists the effects such as disease, is also the precursor synthesizing other steroid drugss.
At present the most disclosed in prepare in the method for dehydroepiandros-sterone, more for fermentation method and chemical method.Add precursor
The report such as fermentation method such as patent CN 201210316197 grade of bioconversion is carried out to microbial fermentation solution, the longest, product
Content is less than 1g/L, there is the problems such as separation and Extraction.With other steroidal compounds for initial chemical method such as patent CN
102212099, the report such as 102603841,102603839, utilizes protection more, hydrolyzes, hydrogenates and the step such as oximate, yield low and
Environmental problem is serious.
With common 4-AD (4-AD) as initial substrate, the double bond migration reaction sequentially passing through chemical method generates
The carbonyl reduction reaction of 5-androstenedione (5-AD) and biological method can prepare dehydroepiandros-sterone, be the step of at present report
Few, the method that yield is higher.Document Tetrahedron Letters, 1962,15:669-672, wait to report utilizing the tert-butyl alcohol
Potassium realizes the double bond migration reaction of 4-AD;Patent WO 2014188353 reports chemical method double bond migration and follow-up enzyme process also
Former reaction.
In report 4-AD generates 5-AD reaction, the purity of 5-AD be under the influence of the subject matter of single step reaction because its
Impurity is difficult to remove, and it is higher generally to there is potassium tert-butoxide consumption, and the problem that product 5-AD yield is low, such as patent WO
Potassium tert-butoxide consumption is substrate 5 times of equivalents in 2014188353, product yield only has 83%, it is contemplated that product goes hydrogen meter male
In the cost of ketone, substrate accounts for 80%, therefore there is bigger application difficult.It is otherwise noted that the second step of this patent
Biosynthesis is simultaneously used NAD and NADP as coenzyme, and consumption of organic solvent is high, and yield is 70%, equally exists practicality
Problem.
Summary of the invention
It is an object of the invention to provide and overcome the deficiencies in the prior art, it is provided that a kind of chemo-enzymatic process prepares dehydroepiandros-sterone
Method.
For reaching above-mentioned purpose, the technical solution used in the present invention is: a kind of chemo-enzymatic process prepares the side of dehydroepiandros-sterone
Method, comprises the steps:
1) with 4-AD as substrate, temperature be 20~40 DEG C, under nitrogen protection, with the tert-butyl alcohol as solvent, uncle
React 1~12h under the effect of butanol potassium, then this reactant liquor joined and be mixed with in sodium ascorbate, vinegar aqueous acid,
HPLC detects reaction process, and when conversion ratio reaches 90~99%, reaction terminates, and obtains 5-androstenedione, wherein, potassium tert-butoxide with
The mass ratio that feeds intake of 4-AD is 2~0.5:1;
2) by step 1) the 5-androstenedione that obtains constitutes reaction system with solvent, aqueous phase buffer solution, and add biology and urge
Agent, magnesium chloride hexahydrate, coenzyme and regenerating coenzyme system, react under the temperature conditions of 25~40 DEG C, control whole instead
Answering the pH of process 6.0~7.5, HPLC detects reaction process, and when conversion ratio reaches 90-99%, reaction terminates, and obtains product and goes
Hydrogen meter androsterone, wherein, described solvent is 2-methyltetrahydrofuran, and described biocatalyzer is ketoreductase, and described is auxiliary
Enzyme regenerative system is glucose and glucose dehydrogenase, described glucose: biocatalyzer: glucose dehydrogenase: 5-androstene
The mass ratio that feeds intake of diketone is: 0.8~1.2:0.008~0.016:0.004~0.008:1.
Preferably, step 2) described in aqueous phase buffer solution be pH be the phosphate buffered solution of 6.5.
Preferably, described coenzyme is NAD, and it with the mass ratio that feeds intake of 5-androstenedione is: 0.003~0.005:1.
Preferably, described glucose dehydrogenase is that the trade mark produced purchased from Suzhou Chinese biotechnology of enzymes company limited is
The glucose dehydrogenase of EW002.
Preferably, the gene order of described ketoreductase has at least 90% with any sequence in sequence 1~26
Homology.
It is further preferred that described ketoreductase is the expression product in non-pathogenic microorganism.
It is further preferred that described non-pathogenic microorganism is escherichia coli.
Preferably, specific implementation process is as follows:
1) with 4-AD as substrate, temperature be 20~40 DEG C, under nitrogen protection, with the tert-butyl alcohol as solvent, uncle
React 1~12h under the effect of butanol potassium, then this reactant liquor joined and be mixed with in sodium ascorbate, vinegar aqueous acid,
HPLC detects reaction process, and when conversion ratio reaches 90~99%, reaction terminates, and is filtered by mixed liquor, obtains white solid, water
Wash, be spin-dried for i.e. obtaining 5-androstenedione product;
2) by step 1) the 5-androstenedione that obtains is dissolved in 2-methyltetrahydrofuran, and the aqueous phase buffering described in addition is molten
Liquid, adds described glucose, magnesium chloride hexahydrate, ketoreductase, glucose dehydrogenase, coenzyme, at 25~40 DEG C after stirring
Reacting under temperature conditions, the pH of control reaction system is 6.0~7.5, and HPLC detects reaction process, when conversion ratio reaches 90-
When 99%, reaction terminates, the pH < 2 of regulation reaction system, adds ethyl acetate and activated carbon stirring 1~12h, filters, filter cake
Washing three times by ethyl acetate, merging filtrate and cleaning mixture, rotation is evaporated off organic solvent, and aqueous phase adds ethyl acetate extraction, closes
And ethyl acetate, concentrate, be slowly added to normal heptane under room temperature, overnight crystallize, filter, be dried to obtain dehydroepiandros-sterone product, female
Liquid obtains dehydroepiandros-sterone solid product after being evaporated.
Due to the utilization of technique scheme, the present invention compared with prior art have the advantage that the chemistry of the present invention-
Enzyme process prepares the method for dehydroepiandros-sterone, compared with existing route, by changing first step chemical method and second step bioanalysis
Entering, use the reaction condition of milder and more excellent catalyst, the preparation method of the present invention made achieves product yield
The purpose high, purity is high, the consumption of the raw material such as coenzyme and potassium tert-butoxide is lower simultaneously, has higher practical value.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention will be further described in detail, but the present invention is not limited to following enforcement
Example.The implementation condition used in embodiment can do further adjustment, not marked enforcement according to specifically used different requirement
Condition is the condition in normal experiment.
Embodiment 1 prepares 5-AD
At 25 DEG C, be passed through nitrogen 15min to the tert-butyl alcohol (80mL), under aeration condition add potassium tert-butoxide (6.0g,
3.0eq), seal reactor afterwards, carry out nitrogen displacement and stir half an hour.Add under nitrogen protection substrate (5.0g,
1.0eq), 35 DEG C are reacted 5 hours.Take 3.5g sodium ascorbate, 5.7g acetic acid, 200mL water, stir 15 minutes, by above-mentioned reaction
Liquid is poured into, stirred under nitrogen atmosphere half an hour, and HPLC detects conversion ratio 96.5%.Mixed liquor is filtered, obtains white solid, water
Wash, obtain white solid 4.8g, HPLC after being spin-dried for and detect purity 95.5%, yield 91.7%.
Embodiment 2 prepares dehydroepiandros-sterone
Above-mentioned 4.8g product is dissolved in 25mL 2-methyltetrahydrofuran, adds the phosphoric acid of 25mL 100mM pH6.5
Salt buffer solution starts stirring, is sequentially added into 5g glucose, 80mg magnesium chloride hexahydrate, and 60mg ketoreductase is (by sequence 26 greatly
Enterobacteria is expressed and obtains), 30mg glucose dehydrogenase (from Suzhou Chinese biotechnology of enzymes company limited, numbered EW002),
20mg NAD, starts reaction, and controls pH 6.5 with 40% sodium hydroxide at 25 DEG C.Sampling detection, conversion ratio after 6 hours
99%, with salt acid for adjusting pH, < 2, addition 30mL ethyl acetate and 2g activated carbon stir 1 hour, filter, filter cake 15mL acetic acid second
Ester washs three times, merging filtrate and cleaning mixture, and rotation is evaporated off organic solvent, and aqueous phase adds 40mL ethyl acetate and extracts three times, closes
And ethyl acetate, it is concentrated into 20mL volume, to room temperature, is slowly added to 40mL normal heptane, overnight crystallize, filter, be dried,
3.5g product, purity > 99%, content > 99%, after mother solution is evaporated, obtain 1.4g solid, through recovery again, after many batches
Processing yield is 85%.
Above-described embodiment, only for technology design and the feature of the explanation present invention, its object is to allow person skilled in the art
Scholar will appreciate that present disclosure and is carried out, and can not limit the scope of the invention with this, all according to the present invention
The equivalence that spirit is made changes or modifies, and all should contain within the scope of the present invention.
Claims (8)
1. the method that a chemo-enzymatic process prepares dehydroepiandros-sterone, it is characterised in that comprise the steps:
1) with 4-AD as substrate, temperature be 20 ~ 40 DEG C, under nitrogen protection, with the tert-butyl alcohol as solvent, at potassium tert-butoxide
Effect under reaction 1 ~ 12h, then this reactant liquor is joined and is mixed with in sodium ascorbate, vinegar aqueous acid, HPLC examine
Measured reaction process, when conversion ratio reaches 90 ~ 99%, reaction terminates, and obtains 5-androstenedione, wherein, potassium tert-butoxide and 4-androstene two
The mass ratio that feeds intake of ketone is 2 ~ 0.5:1;
2) 5-androstenedione step 1) obtained and solvent, aqueous phase buffer solution composition reaction system, addition biocatalyzer,
Magnesium chloride hexahydrate, coenzyme and regenerating coenzyme system, react under the temperature conditions of 25 ~ 40 DEG C, controls whole course of reaction
PH 6.0 ~ 7.5, HPLC detects reaction process, and when conversion ratio reaches 90-99%, reaction terminates, and obtains product and goes hydrogen meter male
Ketone, wherein, described solvent is 2-methyltetrahydrofuran, and described biocatalyzer is ketoreductase, described regenerating coenzyme
System is glucose and glucose dehydrogenase, described glucose: biocatalyzer: glucose dehydrogenase: 5-androstenedione
The mass ratio that feeds intake is: 0.8 ~ 1.2:0.008 ~ 0.016:0.004 ~ 0.008:1.
Chemo-enzymatic process the most according to claim 1 prepares the method for dehydroepiandros-sterone, it is characterised in that step 2) in institute
The aqueous phase buffer solution stated be pH be the phosphate buffered solution of 6.5.
Chemo-enzymatic process the most according to claim 1 prepares the method for dehydroepiandros-sterone, it is characterised in that described coenzyme
For NAD, it with the mass ratio that feeds intake of 5-androstenedione is: 0.003 ~ 0.005:1.
Chemo-enzymatic process the most according to claim 1 prepares the method for dehydroepiandros-sterone, it is characterised in that described Fructus Vitis viniferae
Glucocorticoid dehydrogenase is the glucose dehydrogenase that the trade mark is EW002 produced purchased from Suzhou Chinese biotechnology of enzymes company limited.
Chemo-enzymatic process the most according to claim 1 prepares the method for dehydroepiandros-sterone, it is characterised in that described ketone is also
The gene order of protoenzyme has the homology of at least 90% with any sequence in sequence 1 ~ 26.
Chemo-enzymatic process the most according to claim 5 prepares the method for dehydroepiandros-sterone, it is characterised in that described ketone is also
Protoenzyme is the expression product in non-pathogenic microorganism.
Chemo-enzymatic process the most according to claim 6 prepares the method for dehydroepiandros-sterone, it is characterised in that described non-cause
Sick microorganism is escherichia coli.
Chemo-enzymatic process the most according to claim 1 prepares the method for dehydroepiandros-sterone, it is characterised in that be embodied as
Journey is as follows:
1) with 4-AD as substrate, temperature be 20 ~ 40 DEG C, under nitrogen protection, with the tert-butyl alcohol as solvent, at potassium tert-butoxide
Effect under reaction 1 ~ 12h, then this reactant liquor is joined and is mixed with in sodium ascorbate, vinegar aqueous acid, HPLC examine
Measured reaction process, when conversion ratio reaches 90-99%, reaction terminates, and is filtered by mixed liquor, obtains white solid, wash, be spin-dried for i.e.
Obtain 5-androstenedione product;
2) 5-androstenedione step 1) obtained is dissolved in 2-methyltetrahydrofuran, the aqueous phase buffer solution described in addition,
Described glucose, magnesium chloride hexahydrate, ketoreductase, glucose dehydrogenase, coenzyme is added, the temperature of 25 ~ 40 DEG C after stirring
Under the conditions of react, control reaction system pH 6.0 ~ 7.5, HPLC detects reaction process, when conversion ratio reaches 90 ~ 99%,
Reaction terminates, the pH < 2 of regulation reaction system, adds ethyl acetate and activated carbon stirring 1 ~ 12h, filters, filter cake acetic acid second
Ester washs three times, merging filtrate and cleaning mixture, and rotation is evaporated off organic solvent, and aqueous phase adds ethyl acetate extraction, merges acetic acid second
Ester, concentrates, is slowly added to normal heptane, overnight crystallize, filters, be dried to obtain dehydroepiandros-sterone product, after mother solution is evaporated under room temperature
Obtain dehydroepiandros-sterone solid product.
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CN109750051A (en) * | 2017-11-01 | 2019-05-14 | 中国科学院天津工业生物技术研究所 | 3 beta-hydroxy steroid dehydrogenase types prepare dehydrobenzene (DHEA) |
CN110590889A (en) * | 2019-10-05 | 2019-12-20 | 湖北竹溪人福药业有限责任公司 | Method for recycling dehydroepiandrosterone intermediate mother liquor |
CN112795457A (en) * | 2021-01-07 | 2021-05-14 | 陈广会 | Organic anti-aging beverage and preparation method thereof |
CN113621672A (en) * | 2021-07-30 | 2021-11-09 | 浙江神洲药业有限公司 | Novel method for preparing dehydroepiandrosterone |
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Cited By (8)
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CN109750051A (en) * | 2017-11-01 | 2019-05-14 | 中国科学院天津工业生物技术研究所 | 3 beta-hydroxy steroid dehydrogenase types prepare dehydrobenzene (DHEA) |
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CN110590889A (en) * | 2019-10-05 | 2019-12-20 | 湖北竹溪人福药业有限责任公司 | Method for recycling dehydroepiandrosterone intermediate mother liquor |
CN110590889B (en) * | 2019-10-05 | 2022-02-22 | 湖北竹溪人福药业有限责任公司 | Method for recycling dehydroepiandrosterone intermediate mother liquor |
CN112795457A (en) * | 2021-01-07 | 2021-05-14 | 陈广会 | Organic anti-aging beverage and preparation method thereof |
CN113621672A (en) * | 2021-07-30 | 2021-11-09 | 浙江神洲药业有限公司 | Novel method for preparing dehydroepiandrosterone |
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