CN106047950A - Biological preparation method of (S)-1-(2,6-dichloro-3-fluorophenyl)ethanol - Google Patents
Biological preparation method of (S)-1-(2,6-dichloro-3-fluorophenyl)ethanol Download PDFInfo
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Abstract
The invention discloses a biological preparation method of (S)-1-(2,6-dichloro-3-fluorophenyl)ethanol. The method comprises the following steps: proportionally mixing co-expressed whole cells, 2,6-dichloro-3-fluoroacetophenone, glucose and a buffer solution, reacting at 30-40 DEG C under the pH value of 5-8, and carrying out after-treatment to obtain the target product, wherein the co-expressed whole cells are gene engineering bacteria containing carbonyl reductase and glucose dehydrogenase. Compared with the prior art, the method disclosed by the invention does not need any exogenous coenzyme, and the catalyst can be easily separated from the reaction solution, so that the technique is greatly simplified and the production cost is lowered. The product has higher yield and ee value, and thus, has favorable industrial application value.
Description
Technical field
The invention belongs to field of biological pharmacy, be specifically related to the life of one (S)-1-(2,6-bis-chloro-3-fluorophenyl) ethanol
Thing preparation method.
Background technology
Gram azoles is that the ATP of the suppression ALK/MET/ROS developed by Pfizer is emulative many for Buddhist nun (Crizotinib)
Target point protein inhibitors of kinases, this medicine is all proved in the tumor patient of ALK, ROS and MET abnormal kinase to be had human body
Notable clinical efficacy.2011 years grams of azoles are listed by FDA (Food and Drug Adminstration) (FDA) approval for Buddhist nun, and in January, 2013 is in China
Import lists, trade name " Sai Kerui ", is currently used primarily in the positive Locally Advanced for the treatment of anaplastic lymphoma kinase ALK Alk receptor tyrosine kinase and turns
The nonsmall-cell lung cancer moved.Its structural formula is as follows:
(S)-1-(2,6-bis-chloro-3-fluorophenyl) ethanol is the synthesis gram azoles key intermediate for Buddhist nun, makes it both at home and abroad
Preparation Method carries out widely studied, and current preparation method includes chemical synthesis and biotransformation method.Chemical synthesis mainly uses
The chloro-3-fluoro acetophenone of chiral catalyst asymmetric reduction 2,6-bis-obtains (S)-1-(2,6-bis-chloro-3-fluorophenyl) ethanol
(CN103319311A, CN201510820824 etc.), there is severe reaction conditions, chiral catalyst costliness, product hands in the method
The problem such as property purity is low and treatment cost of waste liquor is high.
Compared with chemical synthesis, biological synthesis process has that reaction condition is gentle, conversion ratio high and stereo selectivity is strong etc.
Advantage.Patent WO2006021881, WO2007066187, WO2009036404 disclose gram azoles for Buddhist nun and key intermediate (S)-
The preparation method of 1-(2,6-bis-chloro-3-fluorophenyl) ethanol, is mainly synthesized in the middle of crucial by the method for esterase catalyzed hydrolysis
Body, but the method operating process is complicated, and the response time is long, and yield is less than 50%, it is difficult to realize industrialized production.
Patent CN101851237 reports employing Hepar Sus domestica Lipase catalyzed hydrolysis 1-(2,6-bis-chloro-3-fluorophenyl) ethyl
Acetate carries out selectivity fractionation, and in the method, ethyl acetate need to be through multistep reaction for raw material 1-(2,6-bis-chloro-3-fluorophenyl)
Preparing, catalytic process step is more, and final yield is only 49%, and rear extraction process is complicated, is unfavorable for realizing industry metaplasia
Produce.
Patent WO2006021885 reports different biocatalyzer reductase 12, and 6-bis-chloro-3-fluoro acetophenone directly obtains
(S)-1-(2,6-bis-chloro-3-fluorophenyl) ethanol, but the conversion ratio of reaction is relatively low, when with HLADH and coenzyme NAD H,
When NADPH is catalyst, conversion ratio is 55%；When being catalyst with the bacterial strain Y2-UC2387 deriving from Rhodutorula sp.
Time conversion ratio be 57%, the simultaneous reactions time is longer up to 7 days, and the method is difficult to industrialized production at present.
Patent WO2009036404 reports ketoreductase catalysis 2,6-bis-chloro-3-fluoro acetophenone and obtains (S)-1-(2,6-
Two chloro-3-fluorophenyls) method of ethanol, conversion ratio is 94%.The method employs two kinds of oxidoreductase enzyme powders and outer
Source property coenzyme NAD P, reaction system is complicated, and production cost is high.Additionally, it is tight using enzyme powder as last handling process emulsifying during catalyst
Weight, has adverse effect extraction efficiency and operating process.
Summary of the invention
In order to overcome the drawbacks described above existing for prior art, the invention discloses one (S)-1-(2,6-bis-chloro-3-fluorine
Phenyl) biological preparation method of ethanol, improves the production efficiency of this product with this.
Concrete technology route is as follows:
For achieving the above object, the technical solution used in the present invention is as follows:
1) can reductase 12, the ketone of 6-bis-chloro-3-fluoro acetophenone to (S)-1-(2,6-bis-chloro-3-fluorophenyl) ethanol is also
Protoenzyme encoding gene is connected with the encoding gene of glucose dehydrogenase in the same expression vector of insertion, builds coexpression recombinant bacterium,
The coenzyme carried by coexpression and the cell of two kinds of enzymes realizes substrate reduction and the catalyst system and catalyzing of coenzyme circulation, it is not necessary to outside interpolation
Source property coenzyme；
2) by above-mentioned coexpression free cell or immobilized whole-cell, 2,6-bis-chloro-3-fluoro acetophenone, glucose, buffering
Liquid mixes by a certain percentage, is to react 1～48h at 30～40 DEG C in pH=5～8, temperature.In reaction mixture, coexpression dissociates
Cell concentration is 25～50g/L or immobilized whole-cell concentration is 100～200g/L, 2,6-bis-chloro-3-fluoro acetophenone dense
Degree is 100～200g/L, concentration of glucose is 100～200g/L and the concentration of buffer is 50～100mM.Reaction terminate after from
The heart or cell is recovered by filtration, is extracted with ethyl acetate, concentrates and crystallizes, target product (S)-1-(2,6-bis-chloro-3-fluorophenyl)
Ethanol molar yield more than 90%, ee value be more than 99.9%.
Furtherly, described carbonyl reductase is the Ketoreductase mutant in bacillus caucasicus source, described glucose
Dehydrogenase is the glucose dehydrogenase mutant in bacillus subtilis source.
Furtherly, buffer is phosphate buffer or sodium-acetate buffer, preferably phosphate buffer.
Furtherly, described genetic engineering bacterium is selected from recombination bacillus coli or recombinant yeast pichia pastoris bacterium, the most preferably attaches most importance to
Group e. coli bl21 (DE3).
Furtherly, coexpression cell adds with the form of free cell or immobilized whole-cell, preferably complete with immobilization
Cell adds.
Furtherly, described immobilized whole-cell preparation method is: mixed with fixative by coexpression free cell, be dried
Molding.
Furtherly, one during described fixative is Polyethylene Glycol, polyacrylamide, polyvinyl alcohol or calcium alginate or
Combinations thereof, the most preferably polyvinyl alcohol.
Furtherly, described coexpression free cell is 1:1～1:2 with the mass ratio of described fixative.
Furtherly, described immobilized whole-cell after filtering can be with recovery, and applying mechanically number of times is 1～5 time.
Compared with prior art, it is an advantage of the current invention that: (1) utilizes coexpression cell as catalyst, it is not necessary to add
Exogenous coenzyme, greatly simplifies technique, reduces production cost；(2) utilize immobilized cell to carry out catalytic reaction, cell with
Reactant liquor is easily separated, and operating process is simple, and the repeatable utilization of catalyst improves production efficiency；(3) reaction condition is gentle, produces
Thing yield and ee value are the highest, using glucose as hydrogen donor in course of reaction, cheap, stable in properties, it is simple to industrialization
Produce.
Accompanying drawing explanation
Fig. 1 is the HPLC collection of illustrative plates of the reaction conversion ratio of detection in the embodiment of the present invention 3.
Fig. 2 is the chirality HPLC collection of illustrative plates of the product ee value of detection in the embodiment of the present invention 3.
Detailed description of the invention
Below in conjunction with specific embodiment, the technology contents of the present invention is further elaborated, its purpose is to preferably
Understand present disclosure, but protection scope of the present invention is not limited to this.
The structure of embodiment 1 coexpression recombinant bacterium
Complete synthesis KRED182 gene is carried out PCR amplification as template and introduces restriction enzyme site (forward primer at two ends
Introducing NcoI restriction enzyme site, reverse primer introduces HindIII restriction enzyme site), NcoI and HindIII is to its enzyme action in use, and returns
Receipts obtain KRED genetic fragment.Meanwhile, extract the plasmid of pRSF-Duet carrier, also it carried out NcoI and HindIII enzyme action,
And reclaim the carrier segments after enzyme action.Use T4 ligase to be attached KRED genetic fragment and carrier segments, and be transformed into
In E.coli BL21 (DE3), it is coated with Kan resistant panel, cultivates in 37 DEG C of incubators.After son to be transformed grows, the some lists of picking
Clone carries out bacterium colony PCR checking, chooses the monoclonal that the result is positive and carries out subsequent experimental, by named for this strain
KRED-1 bacterium.Then extract KRED bacteria plasmid, re-use NdeI and XhoI and it is carried out enzyme action, reclaim this carrier segments；Another
Aspect, carries out PCR amplification to GDH105 gene, and with complete synthesis gene as template, and (forward draws at two ends primer restriction enzyme site
Thing add NdeI restriction enzyme site, reverse primer add XhoI restriction enzyme site) reclaim after, also use NdeI and XhoI it is carried out enzyme
Cut, and by Transformed E .coli BL21 (DE3) after GDH genetic fragment and the connection of KRED-1 bacteria plasmid fragment, be coated with Kan resistance
Flat board, cultivates in 37 DEG C of incubators.After bacterium colony grows, picking monoclonal carries out bacterium colony PCR checking, chooses positive monoclonal, i.e.
Obtain coexpression recombinant bacterium.
Embodiment 2 coexpression recombinant bacterium free cell and the preparation of immobilized whole-cell
Coexpression free cell can cultivate acquisition by fermentation, and fermentation medium uses 2YT culture medium, containing 16g/L
Peptone, 10g/L yeast powder, 5g/L NaCl, 2g/L glycerol.Specifically comprise the following steps that and first this strain is inoculated in equipped with 50mL
In the little shaking flask (capacity 250ml) of culture medium, cultivate 12h in 37 DEG C of shaking tables and strain is activated, then by the bacterium after activation
Body is inoculated into equipped with in the big shaking flask (capacity 1L, band baffle plate) of 400mL culture medium, and inoculum concentration 8ml is cultivated extremely in 37 DEG C of shaking tables
When OD600nm reaches 0.6-1.0, add IPTG to final concentration of 0.1mM, centrifugal collection after 25 DEG C of shaking table abduction delivering 20h
Coexpression cell, is free cell.
Weighing 300g polyvinyl alcohol to add in 3.5L water, be warming up to 90-95 DEG C, stirring is until after polyvinyl alcohol is completely dissolved
It is cooled to 25-30 DEG C.In above-mentioned solution, add 200g coexpression cell, after stirring 1h, draw with peristaltic pump and the plastics volley of rifle fire
Mixed liquor note forms disc-shaped, 30 DEG C of dry 1h of drying baker in the plane, moves into 0.1M Na2SO4It is filtered dry after solution-stabilized 2h, clearly
Water washes twice, and obtains 800g immobilized whole-cell.
(S)-1-(2,6-bis-chloro-3-fluorophenyl) ethanol is prepared in embodiment 3 coexpression free cell conversion
Add in 25mL reaction vessel the phosphate buffer (10mL, pH=6.0) of 100mM, glucose (3.0g) and
Substrate 2,6-bis-chloro-3-fluoro acetophenone (3.0g), it is settled to 15mL, coexpression free cell (750mg) after stirring, 35 DEG C
Lower magnetic agitation is reacted, Na2CO3(20%, w/v) control reaction pH is about 6.0, and TLC detects reaction process.After reaction terminates
Centrifugal segregation cell, equal-volume ethyl acetate extracts three times, merges organic facies, and anhydrous sodium sulfate is dried, and decompression is spin-dried for i.e. obtaining product
Product (S)-1-(2,6-bis-chloro-3-fluorophenyl) ethanol 2.84g, product molar yield is 94%, ee value is more than 99.9%.HPLC examines
Survey conversion ratio and product ee value, conversion ratio > 99%, S type product ee value > 99%, testing result is shown in that Fig. 1 is (when substrate retains respectively
Between be 6.7min, product retention time is 4.0min) and Fig. 2 (S type product retention time be 16.3min, R type product retain time
Between be 17.6min).
(S)-1-(2,6-bis-chloro-3-fluorophenyl) ethanol is prepared in embodiment 4 coexpression free cell conversion
The phosphate buffer (10mL, pH=8.0) of 70mM, glucose (2g) and substrate is added in 25mL reaction vessel
2,6-bis-chloro-3-fluoro acetophenones (2g), are settled to 15mL, coexpression free cell (375mg) after stirring, magnetic force at 35 DEG C
Stirring reaction, Na2CO3(20%, w/v) control reaction pH is about 8.0, and TLC detects reaction process.Reaction is centrifugal after terminating goes
Except cell, equal-volume ethyl acetate extracts three times, merges organic facies, and anhydrous sodium sulfate is dried, decompression be spin-dried for i.e. obtaining product (S)-
1-(2,6-bis-chloro-3-fluorophenyl) ethanol 1.88g, product purity is 97%, and product molar yield is 93%, ee value is more than
99.9%.
(S)-1-(2,6-bis-chloro-3-fluorophenyl) ethanol is prepared in embodiment 5 immobilized whole-cell conversion
Add in 25mL reaction vessel the phosphate buffer (10mL, pH=6.5) of 100mM, glucose (3.0g) and
Substrate 2,6-bis-chloro-3-fluoro acetophenone (3.0g), it is settled to 15mL, is stirring evenly and then adding into immobilized whole-cell (3g), 30 DEG C
Lower magnetic agitation is reacted, Na2CO3(20%, w/v) control reaction pH is about 6.5, and TLC detects reaction process.After reaction terminates
Immobilized whole-cell is recovered by filtration, and filtrate adds equal-volume ethyl acetate and extracts three times, merges organic facies, and anhydrous sodium sulfate is done
Dry, decompression is spin-dried for i.e. obtaining product (S)-1-(2,6-bis-chloro-3-fluorophenyl) ethanol 2.89g, and product purity is 96%, product molar
Yield is 95%, ee value is more than 99.9%.
The preparation of embodiment 6 feather weight (S)-1-(2,6-bis-chloro-3-fluorophenyl) ethanol
The phosphate buffer (10L, pH=6.0) of 50mM, glucose is added in the glass reaction still (25L) of jacketed
(3.0kg) and substrate 2,6-bis-chloro-3-fluoro acetophenone (3.0kg), it is settled to 15L, is stirring evenly and then adding into immobilized whole-cell
(3kg), 35 DEG C of stirring reactions, NaOH (0.5M) control reaction pH is about 6.0, and TLC detection conversion ratio terminates after reaching 99%
Reaction, is recovered by filtration immobilized whole-cell.Filtrate regulation pH to about 2.0,60 DEG C of insulation 1h, add kieselguhr stirring 15min
Rear filtration, filtrate adds equal-volume ethyl acetate and extracts three times, merges organic facies, and anhydrous sodium sulfate is dried, and decompression is spin-dried for, and to obtain final product
Product 2.8kg, molar yield is 92%, and product purity is 97%, product ee value > 99.9%.
The preparation of embodiment 7 feather weight (S)-1-(2,6-bis-chloro-3-fluorophenyl) ethanol
The phosphate buffer (10L, pH=7.0) of 80mM, glucose is added in the glass reaction still (25L) of jacketed
(2kg) and substrate 2,6-bis-chloro-3-fluoro acetophenone (1.5kg), it is settled to 15L, is stirring evenly and then adding into immobilized whole-cell
(1.5kg), 35 DEG C of stirring reactions, NaOH (0.5M) control reaction pH is about 7.0, and TLC detection conversion ratio is tied after reaching 99%
Shu Fanying, is recovered by filtration immobilized whole-cell.Filtrate regulation pH to about 2.0,60 DEG C of insulation 1h, add kieselguhr stirring
Filtering after 15min, filtrate adds equal-volume ethyl acetate and extracts three times, merges organic facies, and anhydrous sodium sulfate is dried, decompression rotation
Dry, obtain product 1.38kg, molar yield is 91%, and product purity is 98%, product ee value > 99.9%.
Embodiment 8 prepares (S)-1-(2,6-bis-chloro-3-fluorophenyl) ethanol with reclaiming immobilized whole-cell conversion
The phosphate buffer (10L, pH=6.0) of 50mM, glucose is added in the glass reaction still (25L) of jacketed
(3.0kg) and substrate 2,6-bis-chloro-3-fluoro acetophenone (3.0kg), it is settled to 15L, is stirring evenly and then adding into embodiment 6 recovery set
Immobilized whole-cell (3kg), 35 DEG C stirring reaction, NaOH (0.5M) control reaction pH about 6.0, TLC detects conversion
Rate terminates reaction after reaching 99%, and immobilized cell is recovered by filtration.Filtrate regulation pH to about 2.0,60 DEG C of insulation 1h, add silicon
Filtering after diatomaceous earth stirring 15min, filtrate equal-volume ethyl acetate extracts three times, merges organic facies, and anhydrous sodium sulfate is dried, decompression
Being spin-dried for, obtain product 2.73kg, molar yield is 90%, product purity 96%, product ee value > 99.9%.
The immobilized cell of embodiment 6 is carried out recovery successively, reaction condition and last handling process with embodiment 6,
Apply mechanically effect as shown in the table:
Claims (9)
1. the biological preparation method of (S)-1-(2,6-bis-chloro-3-fluorophenyl) ethanol, it is characterised in that: coexpression is the thinnest
Born of the same parents, 2,6-bis-chloro-3-fluoro acetophenone, glucose and buffer mix by a certain percentage after reaction obtain product, described coexpression
Full cell is the genetic engineering bacterium containing carbonyl reductase and glucose dehydrogenase.
2. the method for claim 1, it is characterised in that: the full cell of described coexpression is immobilized whole-cell, in reaction
Described immobilized whole-cell concentration is 100～200g/L.
3. the method for claim 1, it is characterised in that: the full cell of described coexpression is free cell, described in reaction
Free cell concentration is 25～50g/L.
4. the method for claim 1, it is characterised in that: described carbonyl reductase be bacillus caucasicus source ketone also
Protoenzyme mutant, described glucose dehydrogenase is the glucose dehydrogenase mutant in bacillus subtilis source.
5. the method for claim 1, it is characterised in that: the concentration of described 2,6-bis-chloro-3-fluoro acetophenone be 100～
200g/L, described concentration of glucose are 100～200g/L, the concentration of described buffer is 50～100mM, react at pH=5～
8, carry out at temperature is 30～40 DEG C.
6. the method for claim 1, it is characterised in that: described buffer is phosphate buffer or sodium acetate buffer
Liquid.
7. method as claimed in claim 3, it is characterised in that: described free cell is obtained by engineering bacteria fermentation, described
Genetic engineering bacterium is selected from escherichia coli or yeast.
8. method as claimed in claim 2, it is characterised in that: the preparation method of described immobilized whole-cell is to be swum by coexpression
Mix with fixative from cell, drying and moulding, the mass ratio of described coexpression free cell and described fixative is 1:1～1:2.
9. method as claimed in claim 8, it is characterised in that: described fixative is Polyethylene Glycol, polyacrylamide, polyethylene
One in alcohol or calcium alginate or combinations thereof.
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| CN107904269A (en) * | 2017-12-29 | 2018-04-13 | 安徽联创生物医药股份有限公司 | A kind of method that engineering strain conversion prepares (S) (+) 3 hydroxyl tetrahydrofuran |
| CN108285908A (en) * | 2017-12-26 | 2018-07-17 | 杭州师范大学 | A kind of method that immobilized bi-enzyme catalyzes and synthesizes (S) -1- (bis- chloro- 3- fluoro-phenyls of 2,6-) ethyl alcohol |
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| CN106636248A (en) * | 2016-12-21 | 2017-05-10 | 浙江海洋大学 | Method for preparing crizotinib intermediate by using carbonyl reductase |
| CN108285908A (en) * | 2017-12-26 | 2018-07-17 | 杭州师范大学 | A kind of method that immobilized bi-enzyme catalyzes and synthesizes (S) -1- (bis- chloro- 3- fluoro-phenyls of 2,6-) ethyl alcohol |
| CN108285908B (en) * | 2017-12-26 | 2021-02-09 | 杭州师范大学 | Method for catalytic synthesis of (S) -1- (2, 6-dichloro-3-fluoro-phenyl) ethanol by using immobilized double enzymes |
| CN107904269A (en) * | 2017-12-29 | 2018-04-13 | 安徽联创生物医药股份有限公司 | A kind of method that engineering strain conversion prepares (S) (+) 3 hydroxyl tetrahydrofuran |
| CN110004162A (en) * | 2019-04-10 | 2019-07-12 | 长兴制药股份有限公司 | A kind of carbonyl reductase, gene and its application on methoxyphenamine hydrochloride key intermediate |
| CN112552144A (en) * | 2020-12-25 | 2021-03-26 | 中山奕安泰医药科技有限公司 | Crizotinib intermediate and refining method thereof |
| CN112552144B (en) * | 2020-12-25 | 2023-02-28 | 中山奕安泰医药科技有限公司 | Crizotinib intermediate and refining method thereof |
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