CN1948499A - Method of preparing (R) 4,4,4-trifluoro 3-hydroxy ethyl butyrate by biocatalytic reaction - Google Patents
Method of preparing (R) 4,4,4-trifluoro 3-hydroxy ethyl butyrate by biocatalytic reaction Download PDFInfo
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Abstract
This invention discloses a preparation of producing (R)-4,4,4-tri-fluorine -3-hydroxyl ethyl butyrate by biocatalysts, belonging to technological domain of chirality's halogen hydroxyl butylate by biological dissymmetry reduction preparation. The invention uses production germ saccharomyces uvarum screening and conserving hypsi-antipode selectivity carbonyl reducase, fermenting production enzyme on optimizing condition and hygro-thalline in single aqueous phase system, utilizing 4,4,4-tri-fluorine ethyl acetoacetate as substrate, and adding glucose and macroporous resin to prepare (R)-4,4,4-tri-fluorine -3-hydroxyl ethyl butyrate. When concentration of substrate 4,4,4-tri-fluorine ethyl acetoacetate is 30g/L, transforming by six hours to obtain the product (R)-4,4,4-tri-fluorine -3-hydroxyl ethyl butyrate whose concentration is 26.2g/L, optical purity 88.9%e.e, molar transformation efficiency 87.3%,needing no additional coenzyme in the course of whole reaction.
Description
Technical field
Biocatalysis preparation (R)-4,4, the method for 4-trifluoro 3-hydroxy ethyl butyrate belongs to biological asymmetric reduction and prepares chirality halogenated hydroxyl butyric ester technical field.
Background technology
(R)-4,4,4-trifluoro 3-hydroxy ethyl butyrate (R)-Ethyl 4,4,4-trifluoro-3-hydroxybutanoate) be a kind of important organic synthesis intermediate, be the chirality stripping and slicing of synthetic antidepressive monoamine oxidase-A inhibitor befloxatone (Befloxatone), still produce the precursor of L-trifluoro Threonine and D-trifluoro Threonine.Because 4,4, the 4-trifluoroacetic ethyl acetoacetate is easy to synthetic and cheap, is that substrate carries out asymmetric reduction reaction and obtains (R)-4 with it, 4, the 4-trifluoro 3-hydroxy ethyl butyrate is very cost-effective preparation approach, and reaction product (R)-4,4, the 4-trifluoro 3-hydroxy ethyl butyrate is difficult for being the microbial metabolism utilization, with micro-organisms living cell catalysis process preparation (R)-4,4, the 4-trifluoro 3-hydroxy ethyl butyrate is highly beneficial.
Known today from 4,4, the method predominating path that the 4-trifluoroacetic ethyl acetoacetate is reduced to chiral alcohol is the microorganism catalysis method of asymmetrically reducing, promptly the stereoselectivity biocatalysis by complete microorganism cells (as bread yeast) realizes, but it is relatively more difficult to screen the good microorganism strains that obtains highly-solid selectively; Simultaneously also need to add coenzyme, energy matter is given in not cut-off, also has substrate in addition to the toxicity of thalline and the instability problem in the aqueous solution.
Summary of the invention
The purpose of this invention is to provide the microorganism that produces the highly-solid selectively carbonyl reductase, a kind of method of effectively catalytic asymmetric reduction prepared in reaction chirality halogenated hydroxyl butyric ester newly is provided, and utilize such microorganism strains catalysis 4,4,4-trifluoroacetic ethyl acetoacetate asymmetric reduction, to obtain (R)-4,4 of high-optical-purity, high reaction yield, high production concentration, 4-trifluoro 3-hydroxy ethyl butyrate.
Technical scheme of the present invention: producing bacterium with the selectivity carbonyl reductase is starting strain, in single aqueous phase system, with 4,4, the 4-trifluoroacetic ethyl acetoacetate is a substrate, in the enzyme reaction process, replenish and add substrate, and add glucose and macroporous resin, prepare (R)-4,4, the 4-trifluoro 3-hydroxy ethyl butyrate, step is as follows:
(1) bacterial strain: saccharomyces uvarum (Saccharomyces uvarum) ATCC26602, CICC 1023, CICC1328, CICC 1494, CICC 1445, CICC 1625, yeast saccharomyces cerevisiae (Saccharomycescerevisiae) AS2.1429, AS2.23, AS2.146, AS2.240, unusual Hansenula anomala (Saccharomyces Hansenula anomala) AS2300;
(2) cultivation of wet thallus: the substratum composition is counted with g/L: sodium-acetate 10～100, and corn steep liquor 10～100, potassium primary phosphate 1～10, sal epsom 0.1～1, pH are 4～9, and 20～40 ℃ of temperature were cultivated 1～5 day, and filtering fermentation liquor obtains wet thallus;
(3) preparation reaction system: with 4,4, the 4-trifluoroacetic ethyl acetoacetate is a substrate, uses 0.1M, and the citrate buffer solution of pH 5.0 is mixed with reaction system, and concentration of substrate counts 10～500 with g/L;
(4) enzymatic conversion reaction: add wet thallus in reaction system, the humidification biomass is 1～20g/g substrate, and enzyme reaction temperature is 20～50 ℃, and the reaction times is 1～20 hour; Replenish in reaction process and add substrate, and add glucose and macroporous resin, the conversion fluid of gained carries out chirality gas chromatographic analysis (GC) and measures.
(5) conversion fluid aftertreatment: conversion fluid filters or the centrifugation thalline, and clear liquid is with ethyl acetate extraction, polymeric adsorbent eluent ethyl acetate, combining extraction liquid and elutriant, dehydration again, decolouring, solvent is reclaimed in evaporation, obtain colourless oil liquid (R)-4,4, the 4-trifluoro 3-hydroxy ethyl butyrate.
Product (R)-4,4, the concrete operations that the 4-trifluoro 3-hydroxy ethyl butyrate extracts are: after conversion reaction finishes, with reaction solution centrifugal (8,000g * 20min, 4 ℃), supernatant liquor extracts three times with isopyknic ethyl acetate, the polymeric adsorbent eluent ethyl acetate, combined ethyl acetate extraction liquid and elutriant, to the anhydrous magnesium sulfate that wherein adds 1～5% (w/v), stir evenly the back standing over night again, remove remaining moisture content.With the organic phase filter paper filtering, collect organic phase.70 ℃ of water-bath rotary evaporations reclaim solvent, obtain colourless oil liquid (R)-4,4, the 4-trifluoro 3-hydroxy ethyl butyrate.Get (R)-4,4,4-trifluoro 3-hydroxy ethyl butyrate 0.05g is dissolved in the ethyl acetate, is settled to 5mL, carries out qualitative analysis with GC-MASS, and the contrast of sample and standard substance (Sigma Co.) mass spectrum is defined as same substance.
Bacterial strain adopts saccharomyces uvarum (Saccharomyces uvarum) ATCC26602, i.e. SW-58, and its changing effect is more excellent.
The preparation reaction system, or adopt in wet thallus cultivation and fermentation liquid, directly add with g/L count 10～500 4,4, the 4-trifluoroacetic ethyl acetoacetate is a substrate.
Enzymatic conversion reaction, the substrate initial concentration is 10g/L, replenishes in reaction process and adds substrate to 500g/L.
Add glucose in reaction process, its concentration is 10-70g/L.
Add macroporous resin in reaction process, adopt adding resin is to be used for adsorbing substrate, product, to suppress substrate, the product toxicity to thalline.The mass ratio of resin and substrate is 0.6-6: 1, macroporous resin is DA201, Hz816, Hz803, AB-8, D4006, HPD300, HPD100A, NAK-II, X-5 macroporous adsorbent resin, D201GF, D392, D315, D290, D280, D293 large pore anion resin, HD-2, D61, D001, HD-1, D001-CC, D113 macropore cation resin, the aperture of described macroporous resin are 100～180 .Above-mentioned resin is the city beast, as products such as Shanghai East China University of Science China shake scientific and technological development company, Chemical Plant of Nankai Univ..
The microorganism strains of the effectively catalytic asymmetric reduction prepared in reaction chirality halogenated hydroxyl butyric ester that the present invention's screening obtains is that saccharomyces uvarum (Saccharomyces uvarum) ATCC26602 (is SW-58, the deposit number of Southern Yangtze University biocatalysis research department), CICC 1023, CICC1328, CICC 1494, CICC1445, CICC 1625, yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) AS2.1429, AS2.23, AS2.146, AS2.240, unusual Hansenula anomala (Saccharomyces Hansenula anomala) AS2300 etc., with this bacterial classification culture transferring to containing 1～10 (g/L), 4,4, on the wort agar substratum of 4-trifluoroacetic ethyl acetoacetate, cultivated 3～7 days for 20～40 ℃, detect and containing high density 4,4, the bacterium colony of growing on the 4-trifluoroacetic ethyl acetoacetate substratum.Bacterial strain as catalytic asymmetric reduction prepared in reaction chirality halogenated hydroxyl butyric ester.
Contain 4,4, the wort that the wort agar substratum of 4-trifluoroacetic ethyl acetoacetate is made into 10 ° of Be ' by malt meal adds the agar of 20g/L and makes.Slant culture: preparation contains malt meal and soaks juice 50～300g/L, agar 10～25g/L, the substratum of pH6～9.100～121 ℃ of sterilizations, 20～50 minutes, sterilization postcooling, bevel, inoculation were cultivated 2～7 days for 20～40 ℃.
Substrate 4,4,4-trifluoroacetic ethyl acetoacetate and product (R)-4,4, the gas-chromatography of 4-trifluoro 3-hydroxy ethyl butyrate (GC) is measured: after reaction finishes, with an amount of ethyl acetate extraction, anhydrous MgSO
4Dehydration is filtered, and adds an amount of internal standard substance (n-Hexadecane), analyzes with GC behind the constant volume.Use the VARIAN3900 gas chromatograph, VARIAN CP WAX 52CB polarity chromatographic column (30m * 0.25mm * 0.25 μ m), chromatographic condition is: sample size: 0.5 μ L, injection port: 250 ℃, detector: 250 ℃, flow: 2.0ml/min, adopt temperature programming: 100 ℃ keep 2min, be warming up to 180 ℃ with 5 ℃/min, keep 5min, carrier gas H
2Flow velocity: 30mL/min, tail blows N
2Flow velocity: 25mL/min, burning gas H
2Flow velocity: 30mL/min, air velocity: 300mL/min.
Product (R)-4,4,4-trifluoro 3-hydroxy ethyl butyrate enantiomer excessive value (e.e.) are measured and are adopted chiral gas chromatography.Chiral chromatographic column: CP-Chirasil Dex CB 25m * 0.25mm * 0.25 μ m; Chromatographic condition is: 10 ℃ keep 2min, are warming up to 140 ℃ with 2 ℃/min and keep 2min; Sample size 0.2 μ L; Column flow rate 2.0mL/min; Carrier gas H
2Flow velocity 30mL/min, burning gas H
2Flow velocity 30mL/min, air velocity 300mL/min, tail blows N
2Flow velocity: 25mL/min; Injection port: 250 ℃, detector: 250 ℃, splitting ratio: 50: 1.
Beneficial effect of the present invention: the present invention screens and has obtained optionally saccharomyces uvarum (Saccharomyces uvarum) etc. of high antimer, catalytic asymmetric reduction 4 in single aqueous phase system, 4, (R)-4 that the 4-trifluoroacetic ethyl acetoacetate generates, 4,4-trifluoro 3-hydroxy ethyl butyrate enantiomeric excess value reaches 89%e.e., in reaction system, add resin, the energize material, add high concentration substrate, can obtain high-optical-purity, high reaction yield, high production concentration, main is not need to add coenzyme in entire reaction course.
Microbe transformation method of the present invention is with respect to traditional chemical asymmetric synthesis method, bread yeast oxide-reduction method or with adding coenzyme NAD H/NAD
+Enzyme catalysis asymmetric reduction method have the following advantages: 1. (R)-4,4 of Sheng Chenging, 4-trifluoro 3-hydroxy ethyl butyrate enantiomeric excess value height reaches 89%e.e.; 2. biological catalyst is a microbial cells, fermentative production voluntarily, and steady quality, with low cost; 3. add resin in reaction system, the energize material adds high concentration substrate, can obtain high-optical-purity, high reaction yield, high production concentration; 4. in entire reaction course, do not need to add coenzyme; 5. reaction conditions gentleness, environmental friendliness.
The biological material specimens explanation
Saccharomyces uvarum (Saccharomyces uvarum) ATCC26602 (being SW-58), CICC 1023, CICC1328, CICC 1494, CICC 1445, CICC 1625 that the present invention is used, yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) AS2.1429, AS2.23, AS2.146, AS2.240; Unusual Hansenula anomala (Saccharomyces Hansenula anomala) AS2300.Wherein ATCC numbering bacterial classification is stored in the US mode bacterial classification and collects the center, and CICC numbering bacterial classification is stored in Chinese industrial microbial strains preservation administrative center, and AS numbering bacterial classification is preserved Chinese microorganism strain preservation administrative center.
Embodiment
The screening that embodiment 1 produces the carbonyl reductase microorganism strains
Slant culture: substratum is 100mL wort (containing malt meal 10g), agar 2g, and 6.0,121 ℃ of sterilizations of pH 20 minutes, the inoculation of sterilization postcooling, bacterial classification is the various microorganism strains shown in the table 1, cultivates 2 days for 28 ℃, as the slant activation seed.
Seed culture and fermentation: sodium-acetate 60g/L, corn steep liquor 30g/L, KH
2PO
46g/L, MgSO
47H
2O1g/L, pH 6.0, and liquid amount is the bottled liquid 50mL of 250mL triangle, sterilized 20 minutes for 120 ℃, sterilization postcooling inoculation inclined-plane seed, the shaking table of 160r/min was cultivated 36 hours for 28 ℃, as seed or fermenting enzyme liquid.
The wet thallus amount is 2.5g/100mL in the fermenting enzyme liquid, centrifugal 10 minutes (8,000 rev/mins) collect thalline, with citrate buffer solution (0.1M, pH 5.0) clean twice, thalline is changed in the identical damping fluid that 25mL contains glucose, and making cell concentration is twice in the fermented liquid, adds 0.25g substrate 4 simultaneously, 4, the 4-trifluoroacetic ethyl acetoacetate, in 30 ℃, 160r/min is reaction down.React end in 6 hours, the centrifugal thalline of removing gets supernatant liquor.The supernatant liquor ethyl acetate extraction, an amount of anhydrous MgSO
4Dried overnight is filtered laggard promoting the circulation of qi analysis of hplc (R)-4,4,4-trifluoro 3-hydroxy ethyl butyrate content and enantiomeric excess value, result such as table 1.
Table 1: the screening of producing the carbonyl reductase microorganism strains
| Microorganism strains | Enantiomeric excess value (% e.e.) | Transformation efficiency (%) | The product configuration |
| Yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) AS2.1429 (SW-724) | 34.50 | 22.69 | R |
| Yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) AS2.23 (SW30) | 48.34 | 40.41 | R |
| Yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) AS2.146 (SW24) | 47.56 | 35.37 | R |
| Yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) AS2.240 (SW22) | 47.76 | 34.99 | R |
| Unusual Hansenula anomala (Hansenula anomala) AS2300 (SW-410) | 28.68 | 34.17 | R |
| Saccharomyces uvarum (Saccharomyces uvarum) ATCC26602 (SW-58) | 88.80 | 35.02 | R |
| Saccharomyces uvarum (Saccharomyces uvarum) CICC 1023 | 68.20 | 35.08 | R |
| Saccharomyces uvarum (Saccharomyces uvarum) CICC 1328 | 62.67 | 35.57 | R |
| Saccharomyces uvarum (Saccharomyces uvarum) CICC 1494 | 60.85 | 30.03 | R |
| Saccharomyces uvarum (Saccharomyces uvarum) CICC 1445 | 78.80 | 33.50 | R |
| Saccharomyces uvarum (Saccharomyces uvarum) CICC 1625 | 70.30 | 31.66 | R |
Embodiment 2 adds the influence of different glucose concn to transforming
Saccharomyces uvarum (Saccharomyces uvarum) ATCC 26602 (SW-58), press embodiment 1 method and produce the enzyme cultivation after 36 hours, take by weighing the 2g wet thallus and be added on the bottled 50mL 0.1M of 250mL triangle, in the pH5.0 citrate buffer solution, contain substrate 4,4,4-trifluoroacetic ethyl acetoacetate amount is the 0.5g/ bottle, adds the glucose of different concns respectively, mass concentration is 0g/L, 10g/L, 20g/L, 30g/L, 40g/L, 50g/L, 60g/L, 70g/L, in 30 ℃, carry out conversion reaction under the 160r/min, react after 6 hours and finish, measure its product amount and enantiomeric excess value.The centrifugal thalline of removing of reaction solution gets supernatant liquor.The supernatant liquor ethyl acetate extraction, an amount of anhydrous MgSO
4Drying is filtered laggard promoting the circulation of qi analysis of hplc (R)-4,4,4-trifluoro 3-hydroxy ethyl butyrate content and enantiomeric excess value, result such as table 2.
Table 2: add of the influence of different glucose concn to transforming
| Glucose concn (g/L) | Molar yield (%) | Enantiomeric excess value (%e.e.) |
| 0 | 38.1 | 88.7 |
| 10 | 63.4 | 88.9 |
| 20 | 62.4 | 89.0 |
| 30 | 62.9 | 88.8 |
| 40 | 65.7 | 88.5 |
| 50 | 70 | 88.9 |
| 60 | 70.8 | 87.4 |
| 70 | 71.4 | 88.2 |
As can be seen from Table 2, add the enantiomeric excess value that glucose does not influence product, its productive rate then increases to some extent with the increase of glucose concn.
The influence of biomass that embodiment 3 is different to transforming
Saccharomyces uvarum (Saccharomyces uvarum) SW-58, press embodiment 1 method and produce the enzyme cultivation after 36 hours, the wet thallus amount is 1.5g/100mL in the fermenting enzyme liquid, take by weighing 0.25～2.0g wet thallus and be added on the bottled 0.1M of 250mL triangle, pH 5.0 citrate buffer solution 25mL, initial substrate 4,4,4-trifluoroacetic ethyl acetoacetate amount is the 0.25g/ bottle, in 30 ℃, carries out conversion reaction under the 160r/min.Afterreaction finished in 6 hours, and the centrifugal thalline of removing gets supernatant liquor.The supernatant liquor ethyl acetate extraction, an amount of anhydrous MgSO
4Drying is filtered laggard promoting the circulation of qi analysis of hplc (R)-4,4,4-trifluoro 3-hydroxy ethyl butyrate content and enantiomeric excess value, result such as table 3.
Table 3: the influence of different biomasses to transforming
| Biomass (wet thallus/g) | Molar yield (%) | Enantiomeric excess value (%e.e.) |
| 0.25 | 40.0 | 87.7 |
| 0.5 | 58.2 | 88.0 |
| 1.0 | 68.6 | 88.5 |
| 1.5 | 70.3 | 88.3 |
| 2.0 | 71.6 | 88.9 |
As can be seen from Table 3, along with the increase of biomass, the transformation efficiency of reaction rises gradually, and enantiomeric excess value does not have to change substantially.
The influence of embodiment 4 different concentration of substrate to transforming
Saccharomyces uvarum (Saccharomyces uvarum) SW-58, press embodiment 1 method and produce the enzyme cultivation after 36 hours, the wet thallus amount is 1.5g/100mL in the fermenting enzyme liquid, take by weighing the 2.0g wet thallus and be added on the bottled 0.1M of 250mL triangle, pH 5.0 citrate buffer solution 25mL, initial substrate 4,4,4-trifluoroacetic ethyl acetoacetate amount be 0.13～1.13g/ bottle (5～45g/L), in 30 ℃, carry out conversion reaction under the 160r/min.Afterreaction finished in 6 hours, and the centrifugal thalline of removing gets supernatant liquor.The supernatant liquor ethyl acetate extraction, an amount of anhydrous MgSO
4Drying is filtered laggard promoting the circulation of qi analysis of hplc (R)-4,4,4-trifluoro 3-hydroxy ethyl butyrate content and enantiomeric excess value, result such as table 4.
The influence of the different concentration of substrate of table 4 to transforming
| Concentration of substrate (g/L) | Molar yield (%) | Enantiomeric excess value (%e.e.) |
| 5 | 90.4 | 88.1 |
| 10 | 77.0 | 88.5 |
| 15 | 63.2 | 87.7 |
| 20 | 53.0 | 85.8 |
| 25 | 57.4 | 84.6 |
| 30 | 52.4 | 83.2 |
| 35 | 33.4 | 79.8 |
| 40 | 20.6 | 70.4 |
| 45 | 1.2 | 59.0 |
As shown in Table 4, the transformation efficiency of reaction reduces with the increase of concentration of substrate, and concentration of substrate surpasses after the 30g/L, and the transformation efficiency and the enantiomeric excess value of reaction all significantly decrease.
Embodiment 5 transformation time curves
Saccharomyces uvarum (Saccharomyces uvarum) SW-58 presses embodiment 1 method enzymatic production, carries out conversion reaction by embodiment 2 methods, contains substrate 4,4 in the 25mL reaction system, and 4-trifluoroacetic ethyl acetoacetate amount is 0.6g (24g/L).In the sampling of reaction different time, carry out gas chromatographic analysis (R)-4,4,4-trifluoro 3-hydroxy ethyl butyrate content also calculates conversion yield, result such as table 5.
Table 5: transformation time curve
| Time (min) | Production concentration (g/L) | Molar yield (%) | Enantiomeric excess value (%e.e.) |
| 0 | 0 | 0 | 0 |
| 15 | 4.1 | 17.1 | 88.9 |
| 30 | 5.8 | 24.3 | 88.7 |
| 45 | 8.0 | 33.4 | 88.6 |
| 60 | 10.7 | 44.7 | 88.7 |
| 120 | 12.6 | 52.5 | 88.6 |
| 180 | 13.4 | 55.8 | 88.5 |
| 240 | 13.7 | 57.1 | 87.9 |
| 300 | 14.0 | 58.4 | 87.7 |
| 360 | 14.4 | 60.1 | 87.7 |
| 420 | 13.4 | 55.8 | 88.0 |
| 480 | 12.7 | 48.9 | 88.1 |
| 600 | 11.1 | 46.3 | 87.6 |
| 720 | 11.0 | 46.0 | 87.3 |
As shown in Table 5, transformation time is that the 6h production concentration is the highest, and enantiomeric excess value does not change with the increase of transformation time.
Embodiment 6 adds the influence of resin to transforming
Saccharomyces uvarum (Saccharomyces uvarum) SW-58, press embodiment 1 method and produce the enzyme cultivation after 36 hours, the wet thallus amount is 1.5g/100mL in the fermenting enzyme liquid, takes by weighing the 2.0g wet thallus and is added on the bottled 0.1M of 250mL triangle, pH 5.0 citrate buffer solution 25mL, initial substrate 4,4,4-trifluoroacetic ethyl acetoacetate amount are 0.75g/ bottle (30g/L), add resin HPD300 in the reaction system, the described scope of addition by specification in 30 ℃, is carried out conversion reaction under the 160r/min.Afterreaction finished in 6 hours, filter paper filtering, and the centrifugal thalline of removing gets supernatant liquor.The supernatant liquor ethyl acetate extraction, an amount of anhydrous MgSO
4Drying is filtered laggard promoting the circulation of qi analysis of hplc (R)-4,4,4-trifluoro 3-hydroxy ethyl butyrate content and enantiomeric excess value, result such as table 6.
Table 6 adds the influence of resin to transforming
| Transformation time (h) | Do not add resin | Add resin | ||||
| Production concentration (g/L) | Molar yield (%) | Enantiomeric excess value (%e.e.) | Production concentration (g/L) | Molar yield (%) | Enantiomeric excess value (%e.e.) | |
| 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 1 | 6.0 | 20.0 | 83.1 | 8.6 | 28.7 | 87.4 |
| 2 | 12.1 | 40.3 | 82.6 | 16.9 | 56.3 | 87.8 |
| 3 | 12.7 | 42.3 | 83.5 | 19.2 | 64.0 | 88.1 |
| 4 | 14.1 | 47.0 | 83.9 | 20.8 | 69.3 | 88.6 |
| 5 | 14.9 | 49.7 | 84.5 | 23.6 | 78.7 | 88.7 |
| 6 | 15.7 | 52.3 | 84.1 | 26.2 | 87.3 | 88.9 |
| 7 | 15.2 | 50.7 | 83.8 | 25.3 | 84.3 | 88.5 |
| 8 | 14.7 | 49.0 | 83.1 | 25.6 | 85.3 | 87.8 |
| 9 | 14.3 | 47.7 | 83.2 | 26.2 | 87.3 | 88.0 |
| 10 | 14.0 | 46.7 | 83.4 | 26.5 | 88.3 | 87.9 |
As shown in Table 6, after in reaction system, adding resin HPD300, transform after product concentration and be significantly improved, and enantiomeric excess value also there is certain rising.
Embodiment 7
With saccharomyces uvarum (Saccharomyces uvarum) SW-58, press embodiment 1 method enzymatic production, transform, initial substrate 4 by embodiment 5 methods, 4,4-trifluoroacetic ethyl acetoacetate concentration 30g/L is after conversion 6h reaction finishes, with reaction solution centrifugal (5,000rpm, 20min, 4 ℃), the faint yellow supernatant 300mL of transparent and homogeneous, measure (R)-4,4, the concentration of 4-trifluoro 3-hydroxy ethyl butyrate is 15.01g/L, and supernatant liquor extracts three times with isopyknic ethyl acetate, the combined ethyl acetate extraction liquid, to the anhydrous magnesium sulfate that wherein adds 1～3% (w/v), stir the back standing over night again, remove remaining moisture content.With gained organic phase filter paper filtering, collect organic phase.80 ℃ of water-bath rotation solvent evaporated get colourless thick shape product crude product 6.31g, and it is 71.55% that GC analyzes its purity.
Thick product 6.31g carries out underpressure distillation with silicone oil bath, under 5～7mmHg condition, collects cut and obtain (R)-4,4 in the time of 80～83 ℃, and 4-trifluoro 3-hydroxy ethyl butyrate product 3.44g analyzes content 92.4%, yield 70.6% with GC.
Get product of distillation 3.44g and dissolve in pentane/ether, in-20 ℃ of crystallizations, recrystallization twice, get refined products 2.82g, with chirality GC stratographic analysis, content 99.2%, enantiomeric excess value reaches 97.3%e.e., and polarimetry is measured [α]
D 24=+12.40 (c=1.0, CHCl
3), yield 88.01%.Carry out qualitative analysis with GC-MS,, determine that sample and standard substance are same substance (collection of illustrative plates slightly) with the contrast of standard specimen mass spectrum.
Claims (6)
1. biocatalysis prepares (R)-4,4, the method for 4-trifluoro 3-hydroxy ethyl butyrate, it is characterized in that producing bacterium with the selectivity carbonyl reductase is starting strain, in single aqueous phase system, with 4,4, the 4-trifluoroacetic ethyl acetoacetate is a substrate, replenishes in the enzyme reaction process and adds substrate, and add glucose and macroporous resin, preparation (R)-4,4, the 4-trifluoro 3-hydroxy ethyl butyrate, step is as follows:
(1) bacterial strain: saccharomyces uvarum (Saccharomyces uvarum) ATCC26602, CICC 1023, CICC 1328, CICC 1494, CICC 1445, CICC 1625, yeast saccharomyces cerevisiae (Saccharomycescerevisiae) AS2.1429, AS2.23, AS2.146, AS2.240, unusual Hansenula anomala (Saccharomyces Hansenula anomala) AS2300;
(2) cultivation of wet thallus: the substratum composition is counted with g/L: sodium-acetate 10～100, and corn steep liquor 10～100, potassium primary phosphate 1～10, sal epsom 0.1～1, pH are 4～9, and 20～40 ℃ of temperature were cultivated 1～5 day, and filtering fermentation liquor obtains wet thallus;
(3) preparation reaction system: with 4,4, the 4-trifluoroacetic ethyl acetoacetate is a substrate, uses 0.1M, and the citrate buffer solution of pH 5.0 is mixed with reaction system, and concentration of substrate counts 10～500 with g/L;
(4) enzymatic conversion reaction: add wet thallus in reaction system, the humidification biomass is 1～20g/g substrate, and enzyme reaction temperature is 20～50 ℃, and the reaction times is 1～20 hour; In the enzyme reaction process, replenish and add substrate, and add glucose and macroporous resin;
(5) conversion fluid aftertreatment: conversion fluid filters or the centrifugation thalline, and clear liquid is with ethyl acetate extraction, polymeric adsorbent eluent ethyl acetate, combining extraction liquid and elutriant, dehydration again, decolouring, solvent is reclaimed in evaporation, obtain colourless oil liquid (R)-4,4, the 4-trifluoro 3-hydroxy ethyl butyrate.
2. method according to claim 1 is characterized in that described bacterial strain adopts saccharomyces uvarum (Saccharomyces uvarum) ATCC26602, i.e. SW-58.
3. method according to claim 1 is characterized in that described preparation reaction system, or adopt in wet thallus cultivation and fermentation liquid, directly add with g/L count 10～500 4,4, the 4-trifluoroacetic ethyl acetoacetate is a substrate.
4. method according to claim 1 is characterized in that described enzymatic conversion reaction, substrate 4,4, and 4-trifluoroacetic ethyl acetoacetate initial concentration is 10g/L, replenishes in reaction process and adds substrate to 500g/L.
5. method according to claim 1 is characterized in that described enzymatic conversion reaction, adds glucose in reaction process, and its concentration is 10～70g/L.
6. method according to claim 1, it is characterized in that described enzymatic conversion reaction, in reaction process, add macroporous resin, the mass ratio of resin and substrate is 0.6～6: 1, macroporous resin is DA201, Hz816, Hz803, AB-8, D4006, HPD300, HPD100A, NAK-II, X-5 macroporous adsorbent resin, D201GF, D392, D315, D290, D280, D293 large pore anion resin, HD-2, D61, D001, HD-1, D001-CC, D113 macropore cation resin, the aperture of described macroporous resin are 100～180 .
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| JP2566962B2 (en) * | 1987-06-11 | 1996-12-25 | 電気化学工業株式会社 | Method for producing γ-substituted-β-hydroxybutyric acid ester |
| US5413921A (en) * | 1992-05-28 | 1995-05-09 | Ajinomoto Co., Ltd. | Method of the production of (s)-gamma-halogenated-γ-hydroxybutyric acid esters |
| CZ111197A3 (en) * | 1994-10-12 | 1997-07-16 | Du Pont | Process of enzymatic preparation of chiral esters of alpha tertiary carboxylic acids |
| KR100657212B1 (en) * | 2004-04-29 | 2006-12-14 | 엔자이텍 주식회사 | The method of making optically active ester derivatives and their acids from racemic esters |
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