CN102676590A - Chiral synthesis of Lipitor intermediate ATS-4 by using bio-enzyme - Google Patents
Chiral synthesis of Lipitor intermediate ATS-4 by using bio-enzyme Download PDFInfo
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- CN102676590A CN102676590A CN2011100624285A CN201110062428A CN102676590A CN 102676590 A CN102676590 A CN 102676590A CN 2011100624285 A CN2011100624285 A CN 2011100624285A CN 201110062428 A CN201110062428 A CN 201110062428A CN 102676590 A CN102676590 A CN 102676590A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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Abstract
The invention discloses an enzymic method for preparing ethyl (S)-4-chloro-3-hydroxybutyrate (ATS-4, Lipitor intermediate) and derivatives thereof. The preparation process is shown as a reaction formula in the specifications, wherein in the formula, R refers to methyl, ethyl (ATS-4), propyl, butyl or benzyl, and other C1-C8 substituted alkyl at any position. The main principle is that in a buffer solution, carbonyl in a compound I is reduced by using gene recombined oxidation-reduction enzyme and related coenzymes to obtain a chiral compound II (ATS-4, R refers to ethyl).
Description
Technical field
The present invention relates to the enzyme process preparation of chirality (S)-4-chloro-3-hydroxyl-ethyl n-butyrate (ATS-4, Lipitor midbody) and verivate thereof, belong to the Application Areas of enzyme in chirality is synthetic, present method also belongs to Green Chemistry and genetically engineered field.
Background technology
(S)-and 4-chloro-3-hydroxyl-ethyl n-butyrate (ATS-4) is one of crucial chiral intermediate of producing Lipitor, China is producing with chemical method mostly.Cost is high, and purity is low, pollute especially serious, very big to the influence of environment.Produce these midbodys with bio-transformation, remarkable advantages is arranged.At present, Ciba, Diverva, DSM, companies such as Codixis adopt enzyme method technique in ATS-4 is synthetic.On the basis of existing technology and knowledge; We obtain key enzyme at screening; Comprise ketoreductase, Hexose phosphate dehydrogenase increases enzymic activity through half with methods such as several saturation mutations and orthomutations on the basis of known protein structure; Chirality Reaction selectivity (rising of optical purity e.e value), thermostability and to the organic solvent tolerance.Therefore, biology catalytic activity is greatly improved, and the chiral selectivity of reaction has also obtained reinforcement, and this just makes the production cost of ATS-4 reduce greatly.
Summary of the invention
Technical problem to be solved by this invention is: the preparation method of will provide that a kind of reactions step is short, cost is lower, optical purity of products is high (S)-4-chloro-3-hydroxyl-ethyl n-butyrate (ATS-4) and verivate thereof.
For solving the problems of the technologies described above, the technical scheme that the present invention adopts is: the preparation method of described chirality (S)-4-chloro-3-hydroxyl-ethyl n-butyrate (ATS-4) and verivate thereof, and its preparation process is represented with following reaction formula:
In the following formula, R is a methyl, ethyl (ATS-4), propyl group, butyl, or benzyl, and any substituted alkyl of other C1-C8.Its cardinal principle is in buffered soln, utilizes the oxydo-reductase of recombination and the carbonyl among the associated coenzymes reducing compound I, obtains chipal compounds II (ATS-4, R are ethyl).
Said preparation method comprises the steps: one mole compound I is dissolved in 500 milliliters to 2000 milliliters the buffered soln and organic solvent, in above-mentioned organic solution, adds weight and be 0.1~20% recombination oxydo-reductase, P-FAD, coenzyme of compound I, and the maintenance system is between 15 to 45 degrees centigrade; Preferentially, stirred stopped reaction 48-120 hour at 25~45 degrees centigrade; Regulate pH; With about 1000 milliliters organic solvent extractions 3 times, merge organic phase, siccative is dry; Organic solvent is removed in underpressure distillation, obtains target compound II.
Oxydo-reductase recited above is the escherichia coli high-level expression ketoreductase.
Oxydo-reductase recited above is used for the direct catalysis of colibacillus engineering of its expression.
Hexose phosphate dehydrogenase recited above utilizes a kind of glucose dehydrogenase modification of biting hot bacterium efficiently to reduce the NADP coenzyme.Variant has been compared three amino acid difference with wild-type.
Hexose phosphate dehydrogenase recited above, this Hexose phosphate dehydrogenase of escherichia coli high-level expression capable of using.
Oxydo-reductase recited above and Hexose phosphate dehydrogenase utilize intestinal bacteria coexpression S1 ketoreductase and Hexose phosphate dehydrogenase.
Organic solvent recited above is selected from methyl alcohol, ethanol, propyl alcohol, butanols, the trimethyl carbinol, own propyl alcohol, THF, methyl tert-butyl ether.
Buffered soln recited above is inorganic phosphate and inorganic phosphate.
Used mineral alkali recited above is selected from sodium hydroxide, Pottasium Hydroxide, yellow soda ash, salt of wormwood, sodium hydrogencarbonate, saleratus.
Advantage of the present invention is: productive rate is high, and optical purity of products is high, and the reaction times is short, and oxydo-reductase is the recombination product, and preparation cost is lower.
Embodiment
Through embodiment the present invention is done further description below, but do not limit the present invention in any way.
Embodiment 1:
Get 50 milliliters, 0.1M, the Na of pH=7.0
2H
2PO4.Na
2HPO
4Buffered soln adds in the triangular flask, adds compound
1(10 gram) and oxydo-reductase (0.1 gram), Hexose phosphate dehydrogenase (0.5 gram), NADPH coenzyme (0.05 gram) stirred 72 hours down in 40 degrees centigrade, and control pH value is between 7.5~8.5, and performance liquid chromatography monitoring reaction finishes.Ethyl acetate extraction twice, organic phase are revolved the dried 9.2 gram ATS-4 (optical purity>99%, productive rate>90%) that obtain.
Claims (10)
1. the chirality enzyme process synthesizes the preparation method of (S)-4-chloro-3-hydroxyl-ethyl n-butyrate (ATS-4, Lipitor midbody) and verivate thereof, and its preparation process is represented with following reaction formula:
In the following formula, R is a methyl, ethyl (ATS-4), propyl group, butyl, or benzyl, and any substituted alkyl of other C1-C8.Its cardinal principle is in buffered soln, utilizes the oxydo-reductase of recombination and the carbonyl among the associated coenzymes reducing compound I, regulates the pH value through inorganic acid alkali, utilizes organic solvent extraction, obtains chipal compounds II (ATS-4, R are ethyl).
2.Candida the variant gene of magnoliae ketoreductase and protein sequence.The sequence of variant and wild-type sequence have 1-10 amino acid change.The more efficient catalytic reduction of these variants.
3. according to synthetic (the S)-4-chloro-3-hydroxyl of the chirality enzyme process shown in the claim 1-butyric ester verivate, it is characterized in that: utilize the escherichia coli high-level expression ketoreductase.
4. according to synthetic (the S)-4-chloro-3-hydroxyl of the chirality enzyme process shown in the claim 1-butyric ester verivate, it is characterized in that: utilize the direct catalysis of this colibacillus engineering.
5. according to synthetic (the S)-4-chloro-3-hydroxyl of the chirality enzyme process shown in the claim 1-butyric ester verivate, it is characterized in that: utilize a kind of glucose dehydrogenase modification of biting hot bacterium efficiently to reduce the NADP coenzyme.Variant has been compared three amino acid difference with wild-type.
6. according to synthetic (the S)-4-chloro-3-hydroxyl of the chirality enzyme process shown in the claim 1-butyric ester verivate, it is characterized in that: utilize this Hexose phosphate dehydrogenase of escherichia coli high-level expression.
7. according to synthetic (the S)-4-chloro-3-hydroxyl of the chirality enzyme process shown in the claim 1-butyric ester verivate, it is characterized in that: utilize intestinal bacteria coexpression S1 ketoreductase and Hexose phosphate dehydrogenase.
8. according to synthetic (the S)-4-chloro-3-hydroxyl of the chirality enzyme process shown in the claim 1-butyric ester verivate, it is characterized in that: utilize the direct catalysis of this colibacillus engineering.
9. according to synthetic (the S)-4-chloro-3-hydroxyl of the chirality enzyme process shown in the claim 1-butyric ester verivate, it is characterized in that: described organic solvent is selected from methyl alcohol, ethanol, propyl alcohol, butanols, the trimethyl carbinol, own propyl alcohol, THF, methyl tert-butyl ether.
10. according to synthetic (the S)-4-chloro-3-hydroxyl of the chirality enzyme process shown in the claim 1-butyric ester verivate, it is characterized in that: used buffered soln is inorganic phosphate and inorganic phosphate.
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103388010A (en) * | 2013-08-14 | 2013-11-13 | 苏州卡耐博生物技术有限公司 | Enzymatic method for preparing (S)-3-hydroxy-4-chlorobutyric acid ethyl ester |
CN104342411A (en) * | 2013-07-26 | 2015-02-11 | 南京朗恩生物科技有限公司 | Activity enhanced ketoreductase mutant, coding sequence and preparation method thereof |
CN104372041A (en) * | 2013-08-12 | 2015-02-25 | 南京朗恩生物科技有限公司 | Whole cell catalytic preparation method of (S)-4-chloro-3-hydroxyethyl butyrate |
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CN101613672A (en) * | 2009-08-04 | 2009-12-30 | 南京工业大学 | A kind of recombination bacillus coli and construction process thereof of asymmetric conversion preparation (S)-4-chloro-ethyl 3-hydroxybutanoate |
CN101962661A (en) * | 2010-06-29 | 2011-02-02 | 南京工业大学 | Application of carbonyl acyl reductase in preparing (S)-4-chlorine-3 hydroxyl ethyl butyrate |
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2011
- 2011-03-16 CN CN2011100624285A patent/CN102676590A/en active Pending
Patent Citations (2)
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CN101613672A (en) * | 2009-08-04 | 2009-12-30 | 南京工业大学 | A kind of recombination bacillus coli and construction process thereof of asymmetric conversion preparation (S)-4-chloro-ethyl 3-hydroxybutanoate |
CN101962661A (en) * | 2010-06-29 | 2011-02-02 | 南京工业大学 | Application of carbonyl acyl reductase in preparing (S)-4-chlorine-3 hydroxyl ethyl butyrate |
Non-Patent Citations (2)
Title |
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N. KIZAKI ET AL: "Synthesis of optically pure ethyl (S)-4-chloro-3-hydroxybutanoate by Escherichia coli transformant cells coexpressing the carbonyl reductase and glucose dehydrogenase genes", 《APPL MICROBIOL BIOTECHNOL》, vol. 55, 7 April 2001 (2001-04-07), pages 590 - 595, XP002370726, DOI: doi:10.1007/s002530100599 * |
SOUICHI MORIKAWA ET AL: "Highly Active Mutants of Carbonyl Reductase S1 with Inverted Coenzyme Specificity and Production of Optically Active Alcohols", 《BIOSCI. BIOTECHNOL. BIOCHEM.》, vol. 69, no. 3, 31 December 2005 (2005-12-31), pages 544 - 552 * |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104342411A (en) * | 2013-07-26 | 2015-02-11 | 南京朗恩生物科技有限公司 | Activity enhanced ketoreductase mutant, coding sequence and preparation method thereof |
CN104372041A (en) * | 2013-08-12 | 2015-02-25 | 南京朗恩生物科技有限公司 | Whole cell catalytic preparation method of (S)-4-chloro-3-hydroxyethyl butyrate |
CN104372041B (en) * | 2013-08-12 | 2018-03-16 | 南京朗恩生物科技有限公司 | A kind of method that whole-cell catalytic prepares the 3-hydroxyethyl butyrate of (S) 4 chlorine 3 |
CN103388010A (en) * | 2013-08-14 | 2013-11-13 | 苏州卡耐博生物技术有限公司 | Enzymatic method for preparing (S)-3-hydroxy-4-chlorobutyric acid ethyl ester |
CN103388010B (en) * | 2013-08-14 | 2015-12-02 | 苏州卡耐博生物技术有限公司 | A kind of method of enzymatic preparation (S)-3-hydroxyl-4-neoprene acid ethyl ester |
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