CN102643879B - Method for preparing duloxetine chiral intermediate through microbial conversion - Google Patents
Method for preparing duloxetine chiral intermediate through microbial conversion Download PDFInfo
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Abstract
The invention provides a method for preparing duloxetine chiral intermediate (S)-3-hydroxyl-3-(2-thineyl) propanenitrile through microbial conversion. The method comprises the following step of: converting 3-hydroxyl-3-(2-thineyl) propanenitrile into the (S)-3-hydroxyl-3-(2-thineyl) propanenitrile under the catalysis of enzyme-containing thallus cells obtained by fermentation of saccharomyces cerevisiae CGMCC No. 2266. The method has the advantages of mild reaction conditions, easiness and convenience for operation, low cost, high product yield, high purity and suitability for large-scale industrial production.
Description
Technical field
The invention belongs to microbial transformation field, be specifically related to one and utilize microbial transformation to prepare the method for duloxetine chiral intermediate (S)-3-hydroxyl-3-(2-thienyl) propionitrile.
Background technology
(S)-3-hydroxyl-3-(2-thienyl) propionitrile ((S)-3-hydroxy-3-(2-thienyl) propanenitrile), CAS accession number: 591727-36-5, molecular formula C
7h
7nOS, molecular weight 153.(S)-3-hydroxyl-3-(2-thienyl) propionitrile is the important intermediate of of synthesis antidepressant drug duloxetine.Duloxetine is a kind of novel antidepressant, is the double inhibitor of serotonin and norepinephrine reuptake.Current discovery not only can Cure of depression, also can be used for treatment stress incontinence and diabetic peripheral neuropathy pain, also has certain curative effect for the dysthymia disorders chronic pain that occurs together.
The people such as Ahmed Kamal, G. B. disclose at Tetrahedron Letters 44 (2003) 4783 – 4,787 one is prepared duloxetine route by (S)-3-hydroxyl-3-(2-thienyl) propionitrile:
。
About the preparation of (S)-3-hydroxyl-3-(2-thienyl) propionitrile, in current bibliographical information, obtain mainly through lipase resolution of racemates.In the US Patent No. 7045341 of authorizing for 2006, the people such as Kamal adopts lipase-catalyzed transesterification fractionations 3-hydroxyl-3-(2-thienyl) propionitrile to synthesize (S)-3-hydroxyl-3-(2-thienyl) propionitrile, immobilization pseudomonas cepacia (
pseudomonas cepacia) lipase 500 mg splits 5 mmoL racemize 3-hydroxyl-3-(2-thienyl) propionitrile in solvent isopropyl ether 100 mL; acry radical donor vinyl-acetic ester 25mmoL; temperature 25 DEG C; time 14 h; product (S)-3-hydroxyl-3-(2-thienyl) propionitrile productive rate is 42%, and optical purity is greater than 99%ee.The fractionation of the substrate of this process is most effective can only reach 50%, and lipase is expensive, preparation process very complicated, and this process has by product shown in following formula (R)-2-cyano group-1-(2-thienyl) ethyl acetate to generate,
。
Therefore, lipase resolution of racemic body method is not the desirable route of preparation of industrialization (S)-3-hydroxyl-3-(2-thienyl) propionitrile.
Summary of the invention
It is gentle that the object of the invention is to provide a kind of reaction conditions, and environmental friendliness, catalytic efficiency is high, with low cost, is easy to the preparation method of (S)-3-hydroxyl-3-(2-thienyl) propionitrile realizing suitability for industrialized production.
The technical solution used in the present invention is:
A kind of method of microbial transformation preparation (S)-3-hydroxyl-3-(2-thienyl) propionitrile, the method with 3-carbonyl-3-(2-thienyl) propionitrile for substrate, with yeast saccharomyces cerevisiae (
saccharomyces cerevisiae) CGMCC No. 2266 ferments, and what obtain is biological catalyst containing enzyme somatic cells, carries out obtained described (S)-3-hydroxyl-3-(2-thienyl) propionitrile of conversion reaction.
Described microbial bacteria Accharomyces cerevisiae CGMCC No.2266; be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms preservation center; be positioned at Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica; preserving number CGMCC No.2266; preservation date on November 26th, 2007; previous granted patent CN200810059686.6(applicant: Zhejiang Polytechnical University, applying date: on February 4th, 2008) in protected as new strains.
Bacterium source: described microbial bacteria Accharomyces cerevisiae CGMCC No.2266 obtains by screening from the soil near Hangzhou West Lake brew-house.
The described enzyme somatic cells that contains is prepared in accordance with the following methods: be seeded in fermention medium by yeast saccharomyces cerevisiae CGMCC No. 2266, shaking speed is 150 ~ 200 r/min, cultivates 18 ~ 30 h for 26 ~ 35 DEG C, and fermented liquid is centrifugal, obtained containing enzyme somatic cells.
The described enzyme somatic cells consumption that contains counts 1 ~ 50 g/1g 3-carbonyl-3-(2-thienyl) propionic acid substrate with dry cell weight.
Further, the described enzyme somatic cells dry weight that contains refers to fermented liquid centrifugation, and abandoning supernatant is little after constant weight 120 DEG C of oven dry 48 by gained wet cell, the weight of gained stem cell.
Further, the described enzyme somatic cells fermented liquid method for determination of amount that contains needed for quantify cellular dry weight is: get centrifugation in Partial fermentation liquid, abandoning supernatant, wet cell is little of constant weight 120 DEG C of oven dry 48, measure gained dry cell weight, calculate unit containing stem cell ratio in enzyme somatic cells fermented liquid, then calculate needed for quantify cellular dry weight containing containing enzyme somatic cells fermented liquid consumption with this ratio.
Further, the method of described microbial transformation preparation (S)-3-hydroxyl-3-(2-thienyl) propionitrile comprises: in the phosphate buffered saline buffer of pH 5.0 ~ 8.0, with 3-carbonyl-3-(2-thienyl) propionitrile for substrate, what obtain with yeast saccharomyces cerevisiae CGMCC No. 2266 fermentation contains enzyme somatic cells for biological catalyst, conversion reaction 8 ~ 40 hours at 25 ~ 45 DEG C, after reaction terminates, conversion fluid obtains described (S)-3-hydroxyl-3-(2-thienyl) propionitrile through separation and purification.
The starting point concentration of described 3-carbonyl-3-(2-thienyl) propionitrile in phosphate buffered saline buffer is 0.1 ~ 1 mol/L.
Can add in described phosphate buffered saline buffer final concentration be the glucose of 10 ~ 100 g/L as cosubstrate, be conducive to improving the molar yield of substrate.
Described (S)-3-hydroxyl-3-(2-thienyl) propionitrile separation purification method is as follows: after reaction terminates, by centrifugal for conversion fluid 4000 r/min 20 minutes, discard bacterial sediment, by supernatant liquor equal-volume ethyl acetate continuous extraction 3 times, combined ethyl acetate extraction liquid, anhydrous sodium sulphate removing moisture is added in acetic acid ethyl acetate extract, suction filtration, filtrate decompression distillation removing ethyl acetate, obtains described (S)-3-hydroxyl-3-(2-thienyl) propionitrile.
Further, the method for described microbial transformation preparation (S)-3-hydroxyl-3-(2-thienyl) propionitrile, carry out according to following steps:
(1) slant culture: yeast saccharomyces cerevisiae CGMCC No. 2266 is inoculated into slant medium, cultivates 4 ~ 6 days to obtain thalline inclined-plane for 26 ~ 35 DEG C; Described slant medium is by forming preparation as follows: wort 5 ~ 15 g/L, yeast powder 2 ~ 4 g/L, peptone 4 ~ 6 g/L, glucose 7 ~ 12 g/L, agar 15 ~ 25 g/L, natural ph, and solvent is water; 121 DEG C of sterilizing 20 min, cool bevel after sterilizing;
(2) seed culture: get a transfering loop thalline from thalline inclined-plane and be transferred to seed culture medium, 26 ~ 35 DEG C, shaking speed is 150 ~ 200 r/min, cultivates 18 ~ 26 h and obtains seed liquor; Described seed culture medium is by forming preparation as follows: glucose 26 ~ 32 g/L, yeast powder 2 ~ 4 g/L, ammonium sulfate 3 ~ 6 g/L, anhydrous MgSO
40. 2 ~ 0. 4 g/L, K
2hPO
43H
20 0. 5 ~ 1.5 g/L, KH
2pO
40. 6 ~ 1.5 g/L, be 7.0 by the pH value of NaOH or HCl solution rearrange liquids substratum, solvent is water;
(3) fermentation culture: get seed liquor, be inoculated in fermention medium with the inoculum size of volume ratio 10 ~ 20%, culture temperature is 26 ~ 35 DEG C, and shaking speed is 150 ~ 200 r/min, cultivating 18 ~ 30 h obtains containing the fermented liquid containing enzyme somatic cells, and centrifugation obtains described containing enzyme somatic cells; Described fermention medium is by forming preparation as follows: glucose 26 ~ 32 g/L, yeast powder 2 ~ 4 g/L, ammonium sulfate 3 ~ 6 g/L, anhydrous MgSO
40. 2 ~ 0. 4 g/L, K
2hPO
43H
20 0. 5 ~ 1.5 g/L, KH
2pO
40. 6 ~ 1.5 g/L, be 7.0 by the pH value of NaOH or HCl solution rearrange liquids substratum, solvent is water;
(4) bio-transformation: in the phosphate buffered saline buffer of pH 5.0 ~ 8.0, add 3-carbonyl-3-(2-thienyl) propionitrile of 0.1 ~ 1 mol/L, the glucose adding 10 ~ 100 g/L is conducive to improving the molar yield of substrate as cosubstrate, and dry cell weight quality be 3-carbonyl-3-(2-thienyl) propionitrile 1 ~ 50 times containing enzyme somatic cells, at 25 ~ 45 DEG C, carry out conversion reaction 8 ~ 40 hours;
(5) separation and purification: by centrifugal for conversion fluid 4000 r/min 20 minutes after reaction terminates, discard bacterial sediment, by supernatant liquor equal-volume ethyl acetate continuous extraction 3 times, combined ethyl acetate extraction liquid, in acetic acid ethyl acetate extract, add anhydrous sodium sulphate remove a small amount of moisture, after suction filtration, namely the ethyl acetate solution underpressure distillation removing ethyl acetate obtained obtains described (S)-3-hydroxyl-3-(2-thienyl) propionitrile.
The present invention can detect with liquid chromatograph-mass spectrometer purity and the molecular weight of determining product (S)-3-hydroxyl-3-(2-thienyl) propionitrile.
The defining method of the Enantiomer excess value (ee%) of molar yield of the present invention and product (S)-3-hydroxyl-3-(2-thienyl) propionitrile:
Employing liquid-phase chromatographic analysis detects.Chromatographic column is Japanese Daicel chiral column OB-H, and moving phase is normal hexane/Virahol (90/10), and ultraviolet detection wavelength is 254nm.Detect the content of (R)-3-hydroxyl-3-(2-thienyl) propionitrile and (S)-3-hydroxyl-3-(2-thienyl) propionitrile two kinds of enantiomorphs with this chiral column, calculate the molar yield of reaction and the Enantiomer excess value (ee%) of (S)-3-hydroxyl-3-(2-thienyl) propionitrile further.
The present invention adopts microbe transformation method asymmetric reduction 3-carbonyl-3-(2-thienyl) propionitrile to prepare higher (S)-3-hydroxyl-3-(2-thienyl) propionitrile of optical purity.The method has reaction conditions gentleness, environmental friendliness, the features such as catalyzer is cheap and easy to get, low production cost compared with reducing with chemical method.Meanwhile, containing a large amount of abundant enzyme system in microorganism cells, being conducive to the in-situ regeneration realizing reductive agent coenzyme in reduction process, being conducive to by adding cosubstrate to realize increasing substantially the object of production efficiency.Compared with lipase method for splitting, the method is with low cost, and the large scale culturing ratio of microorganism is easier to realize, and does not need the technological process that the separation and Extraction of enzyme and purifying etc. are loaded down with trivial details.
Meanwhile, in the preparation process of (S)-3-hydroxyl-3-(2-thienyl) propionitrile, add final concentration be the glucose of 10 ~ 100 g/L as cosubstrate, effectively improve the molar yield of substrate.The transformation efficiency of 3-hydroxyl-3-(2-thienyl) propionitrile reaches more than 95% in preferred embodiment, and Enantiomer excess value reaches 100%.
In a word, adopt microbe transformation method preparation (S)-3-hydroxyl-3-(2-thienyl) propionitrile compared with chemical synthesis, enzyme catalysis method: (1) produces bacterial strain safety non-toxic, microbial cells is easy to large scale culturing, a large amount of biological catalysts can be obtained, more with low cost than chemical catalyst; (2) easy and simple to handle, do not need in reaction process to add expensive coenzyme, can increase substantially the transformation efficiency of substrate by adding cosubstrate glucose, yield is high; (3) be easy to realize large-scale industrial production; (4) just can realize bioconversion reaction under normal temperature and pressure, reaction conditions is gentle, environmental friendliness.
Embodiment
In order to understand the present invention further, below in conjunction with embodiment, the method that (S)-3-hydroxyl-3-(2-thienyl) propionitrile is prepared in microbial transformation provided by the invention is described in detail.
It is to be appreciated that these embodiments describe just for further describing feature of the present invention, instead of the restriction to the scope of the invention or the claims in the present invention scope.
The preparation of slant medium: wort 10 g/L, yeast powder 3 g/L, peptone 5 g/L, glucose 10 g/L, agar 20 g/L, natural ph, solvent is water; 121 DEG C of sterilizing 20 min, bevel after cooling.
The preparation of seed culture and fermention medium: seed and fermention medium all adopt liquid nutrient medium, by forming preparation as follows: glucose 30 g/L, yeast powder 3 g/L, ammonium sulfate 5 g/L, anhydrous MgSO
40.25 g/L, K
2hPO
43H
2o 1 g/L, KH
2pO
41 g/L, solvent is water, is 7.0,121 DEG C of sterilizing 20 min by the pH value of NaOH or HCl solution rearrange liquids substratum.
Embodiment 1
By CGMCC No. 2266 strain inoculation to slant medium, cultivate 4 ~ 6 days to obtain thalline inclined-plane for 30 DEG C.Get in the 250 mL triangular flasks that a transfering loop thalline is seeded in containing 100 mL liquid nutrient mediums with inoculating needle from thalline inclined-plane, in 30 DEG C, cultivate 24 h under the condition of 180 r/min and obtain seed liquor.10 mL seed liquor (inoculum size is in culture volume mark 10%) are inoculated in the 250 mL triangular flasks containing 100 mL liquid nutrient mediums, in 30 DEG C, cultivate 24 h under the condition of 180 r/min and obtain fermented liquids, fermented liquid is centrifugal, must enzyme somatic cells be contained.
The mensuration of dry cell weight be fermented liquid is centrifugal after from drying 48 little of constant weight at 120 DEG C containing getting little part the wet thallus of enzyme somatic cells, measure the weight of stem cell, calculate unit in fermented liquid and contain stem cell ratio in enzyme somatic cells, then calculate needed for quantify cellular dry weight containing containing enzyme somatic cells fermented liquid consumption with this ratio.In often liter of fermented liquid of the present embodiment, the dry weight contained containing enzyme somatic cells is 50 grams.
The wet cell of the centrifugal rear acquisition of above-mentioned gained 400 milliliters of fermented liquids is added respectively in 10 parts of triangular flasks containing 100 mL pH7.0 phosphate buffered saline buffers, wherein the dry weight contained containing enzyme somatic cells is 20 g, add 3-carbonyl-3-(2-thienyl) propionitrile separately, the final concentration of 3-carbonyl-3-(2-thienyl) propionitrile is made to be respectively 0.1 mol/L, 0.2 mol/L, 0.3 mol/L, 0.4 mol/L, 0.5 mol/L, 0.6 mol/L, 0.7 mol/L, 0.8 mol/L, 0.9 mol/L and 1 mol/L, be placed in 30 DEG C, 24 h are reacted in the shaking table of 180 r/min.After reaction terminates, by conversion fluid in 4000 r/min centrifugal 20 minutes, discard bacterial sediment, by supernatant liquor equal-volume ethyl acetate continuous extraction 3 times, combined ethyl acetate extraction liquid, in acetic acid ethyl acetate extract, add anhydrous sodium sulphate remove a small amount of moisture, suction filtration, filtrate decompression distillation removing ethyl acetate, obtain described (S)-3-hydroxyl-3-(2-thienyl) propionitrile, initial substrate concentration on the molar yield of (S)-3-hydroxyl-3-(2-thienyl) propionitrile and the impact of Enantiomer excess value (ee%) in table 1.
Table 1: initial substrate concentration is on the impact of transformation efficiency and (S)-3-hydroxyl-3-(2-thienyl) propionitrile
| Concentration of substrate (mol/L) | Transformation efficiency (%) | (S) Enantiomer excess value (ee%) of-3-hydroxyl-3-(2-thienyl) propionitrile |
| 0.1 | 95.8 | 100 |
| 0.2 | 87.2 | 100 |
| 0.3 | 72.5 | 100 |
| 0.4 | 61.6 | 100 |
| 0.5 | 55.2 | 100 |
| 0.6 | 42.6 | 100 |
| 0.7 | 32.1 | 100 |
| 0.8 | 23.5 | 100 |
| 0.9 | 10.2 | 100 |
| 1 | 3.7 | 100 |
Embodiment 2:
By CGMCC No. 2266 strain inoculation to slant medium, cultivate 4 ~ 6 days to obtain thalline inclined-plane for 30 DEG C.Get in the 250 mL triangular flasks that a transfering loop thalline is seeded in containing 100 mL liquid nutrient mediums with inoculating needle from thalline inclined-plane, in 30 DEG C, cultivate 24 h under the condition of 180 r/min and obtain seed liquor.10 mL seed liquor (inoculum size is in culture volume consumption 10%) are inoculated in the 250 mL triangular flasks containing 100 mL liquid nutrient mediums, in 30 DEG C, cultivate 24 h under the condition of 180 r/min and obtain fermented liquids, fermented liquid is centrifugal, must contain enzyme somatic cells, in often liter of fermented liquid, the dry weight contained containing enzyme somatic cells is 50 grams.
The wet cell adding the centrifugal rear acquisition of above-mentioned 400 milliliters of fermented liquids in the triangular flask of 100 mL pH7.0 phosphate buffered saline buffers is respectively contained separately at ten parts, wherein the dry weight contained containing enzyme somatic cells is 20 g, add 3-carbonyl-3-(2-thienyl) propionitrile that final concentration is 0.5 mol/L, add cosubstrate glucose, glucose final concentration is made to be respectively 10 g/L, 30 g/L, 50 g/L, 70 g/L, 100 g/L, be placed in 30 DEG C, in the shaking table of 180 r/min, react 24 h.After reaction terminates, by conversion fluid in 4000 r/min centrifugal 20 minutes, discard bacterial sediment, by supernatant liquor equal-volume ethyl acetate continuous extraction 3 times, combined ethyl acetate extraction liquid, in acetic acid ethyl acetate extract, add anhydrous sodium sulphate remove a small amount of moisture, suction filtration, filtrate decompression distillation removing ethyl acetate, obtain described (S)-3-hydroxyl-3-(2-thienyl) propionitrile, substrate glucose concentration on the molar yield of (S)-3-hydroxyl-3-(2-thienyl) propionitrile and the impact of Enantiomer excess value (ee%) in table 2.
Table 2: glucose concn is on the impact of transformation efficiency and (S)-3-hydroxyl-3-(2-thienyl) propionitrile Enantiomer excess value
| Glucose (g/L) | Transformation efficiency (%) | (S) Enantiomer excess value (ee%) of-3-hydroxyl-3-(2-thienyl) propionitrile |
| 10 | 88.7 | 100 |
| 30 | 98.5 | 100 |
| 50 | 80.2 | 100 |
| 70 | 72.7 | 100 |
| 100 | 65.9 | 100 |
| 150 | 60.1 | 100 |
Embodiment 3:
By yeast saccharomyces cerevisiae CGMCC No. 2266 strain inoculation to slant medium, cultivate 4 ~ 6 days to obtain thalline inclined-plane for 30 DEG C.Get in the 250 mL triangular flasks that a transfering loop thalline is seeded in containing 100 mL liquid nutrient mediums with inoculating needle from thalline inclined-plane, in 30 DEG C, cultivate 24 h under the condition of 180 r/min and obtain seed liquor.10 mL seed liquor (inoculum size is in culture volume consumption 10%) are inoculated in the 250 mL triangular flasks containing 100 mL liquid nutrient mediums, in 30 DEG C, cultivate 24 h under the condition of 180 r/min and obtain fermented liquids, fermented liquid is centrifugal, must contain enzyme somatic cells, in often liter of fermented liquid, the dry weight contained containing enzyme somatic cells is 50 grams.
The wet cell adding the centrifugal rear acquisition of above-mentioned 400 milliliters of fermented liquids in the triangular flask of 100 mL pH7.0 phosphate buffered saline buffers is respectively contained separately at five parts, wherein the dry weight contained containing enzyme somatic cells is 20 g, add 3-carbonyl-3-(2-thienyl) propionitrile that final concentration is 0.5 mol/L separately, adding final concentration is 30 g/L cosubstrate glucose, all in the shaking table of 180 r/min, reacts 24 h at 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C.After reaction terminates, by conversion fluid in 4000 r/min centrifugal 20 minutes, discard bacterial sediment, by supernatant liquor equal-volume ethyl acetate continuous extraction 3 times, combined ethyl acetate extraction liquid, in acetic acid ethyl acetate extract, add anhydrous sodium sulphate remove a small amount of moisture, suction filtration, filtrate decompression distillation removing ethyl acetate, obtain described (S)-3-hydroxyl-3-(2-thienyl) propionitrile, temperature of reaction on the molar yield of (S)-3-hydroxyl-3-(2-thienyl) propionitrile and the impact of Enantiomer excess value (ee%) in table 3.
Table 3: temperature of reaction is on the impact of transformation efficiency and (S)-3-hydroxyl-3-(2-thienyl) propionitrile Enantiomer excess value
| Invert point (DEG C) | Transformation efficiency (%) | (S) Enantiomer excess value (ee%) of-3-hydroxyl-3-(2-thienyl) propionitrile |
| 25 | 82.2 | 100 |
| 30 | 98.5 | 100 |
| 35 | 80.6 | 100 |
| 40 | 73.6 | 100 |
| 45 | 62.8 | 100 |
Embodiment 4:
By yeast saccharomyces cerevisiae CGMCC No. 2266 strain inoculation to slant medium, cultivate 4 ~ 6 days to obtain thalline inclined-plane for 30 DEG C.Get the 250 mL triangular flasks that a transfering loop thalline is seeded in containing 100 mL liquid nutrient mediums with inoculating needle from thalline inclined-plane, in 30 DEG C, cultivate 24 h under the condition of 180 r/min and obtain seed liquor.10 mL seed liquor (inoculum size is in culture volume consumption 10%) are inoculated in the 250 mL triangular flasks containing 100 mL liquid nutrient mediums, in 30 DEG C, cultivate 24 h under the condition of 180 r/min and obtain fermented liquids, fermented liquid is centrifugal, must contain enzyme somatic cells, in often liter of fermented liquid, the dry weight contained containing enzyme somatic cells is 50 grams.
In the triangular flask containing 100 mLpH7.0 phosphate buffered saline buffers, the wet cell of the centrifugal rear acquisition of above-mentioned 400 milliliters of fermented liquids is added respectively at each five parts, wherein the dry weight contained containing enzyme somatic cells is 20 g, add 3-carbonyl-3-(2-thienyl) propionitrile that final concentration is 0.5 mol/L separately, adding final concentration is more separately 30 g/L cosubstrate glucose, all be placed in 30 DEG C, in the shaking table of 180 r/min, react 8 h, 16 h, 24 h, 32 h, 40 h respectively.After reaction terminates, by conversion fluid in 4000 r/min centrifugal 20 minutes, discard bacterial sediment, by supernatant liquor equal-volume ethyl acetate continuous extraction 3 times, combined ethyl acetate extraction liquid, in acetic acid ethyl acetate extract, add anhydrous sodium sulphate remove a small amount of moisture, suction filtration, filtrate decompression distillation removing ethyl acetate, obtain described (S)-3-hydroxyl-3-(2-thienyl) propionitrile, the reaction times on the molar yield of (S)-3-hydroxyl-3-(2-thienyl) propionitrile and the impact of Enantiomer excess value (ee%) in table 4.
Table 4: the reaction times is on the impact of transformation efficiency and (S)-3-hydroxyl-3-(2-thienyl) propionitrile Enantiomer excess value
| Transformation time (h) | Transformation efficiency (%) | (S) Enantiomer excess value (ee%) of-3-hydroxyl-3-(2-thienyl) propionitrile |
| 8 | 76.2 | 100 |
| 16 | 82.9 | 100 |
| 24 | 98.5 | 100 |
| 32 | 100 | 100 |
| 40 | 100 | 100 |
Embodiment 5:
By yeast saccharomyces cerevisiae CGMCC No. 2266 strain inoculation to slant medium, cultivate 4 ~ 6 days obtained thalline inclined-planes for 30 DEG C.Get the 250 mL triangular flasks that a ring thalline is seeded in containing 100 mL liquid nutrient mediums with inoculating needle from thalline inclined-plane, in 30 DEG C, cultivate 24 h under the condition of 180 r/min and obtain seed liquor.10 mL seed liquor (inoculum size is in culture volume consumption 10%) are inoculated in the 250 mL triangular flasks containing 100 mL liquid nutrient mediums, in 30 DEG C, cultivate 24 h under the condition of 180 r/min and obtain fermented liquids, fermented liquid is centrifugal, must contain enzyme somatic cells, in often liter of fermented liquid, the dry weight contained containing enzyme somatic cells is 50 grams.
Five parts separately containing the triangular flask of 100 mL pH7.0 phosphate buffered saline buffers in add the wet cell of the centrifugal rear acquisition of fermented liquid of above-mentioned gained 40 milliliters, 100 milliliters, 200 milliliters, 300 milliliters and 400 milliliters respectively, be wherein respectively 2 g, 5 g, 10 g, 15 g and 20 g containing the dry weight containing enzyme somatic cells.In above-mentioned five triangular flasks, add final concentration is respectively 0.5 mol/L 3-carbonyl-3-(2-thienyl) propionitrile, and final concentration is 30 g/L cosubstrate glucose, is placed in 30 DEG C, reacts 24 h in the shaking table of 180 r/min.By conversion fluid in 4000 r/min centrifugal 20 minutes after reaction terminates, discard bacterial sediment, by supernatant liquor equal-volume ethyl acetate continuous extraction 3 times, combined ethyl acetate extraction liquid, in acetic acid ethyl acetate extract, add anhydrous sodium sulphate remove a small amount of moisture, suction filtration, filtrate decompression distillation removing ethyl acetate, obtain described (S)-3-hydroxyl-3-(2-thienyl) propionitrile, biomass on the molar yield of (S)-3-hydroxyl-3-(2-thienyl) propionitrile and the impact of Enantiomer excess value (ee%) in table 5.
Table 5: biomass is on the impact of transformation efficiency and (S)-3-hydroxyl-3-(2-thienyl) propionitrile Enantiomer excess value
| Biomass (dry weight) (g) | Transformation efficiency (%) | (S) Enantiomer excess value (ee%) of-3-hydroxyl-3-(2-thienyl) propionitrile |
| 2 | 22.0 | 100 |
| 5 | 45.2 | 100 |
| 10 | 67.5 | 100 |
| 15 | 82.5 | 100 |
| 20 | 98.5 | 100 |
Claims (6)
1. the method for duloxetine chiral intermediate (S)-3-hydroxyl-3-(2-thienyl) propionitrile is prepared in a microbial transformation, it is characterized in that, described method with 3-carbonyl-3-(2-thienyl) propionitrile for substrate, what obtain with yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC No.2266 fermentation contains enzyme somatic cells for biological catalyst, carry out obtained described (S)-3-hydroxyl-3-(2-thienyl) propionitrile of conversion reaction, the starting point concentration of wherein said 3-carbonyl-3-(2-thienyl) propionitrile is 0.1-0.6mol/L, the described enzyme somatic cells consumption that contains counts 5-20g/g substrate with dry cell weight.
2. the method for claim 1, it is characterized in that, described method is: in the phosphate buffered saline buffer of pH 5.0-8.0, with 3-carbonyl-3-(2-thienyl) propionitrile for substrate, what obtain with yeast saccharomyces cerevisiae CGMCC No.2266 fermentation contains enzyme somatic cells for biological catalyst, conversion reaction 8-40 hour at 25-45 DEG C, after reaction terminates, conversion fluid obtains described (S)-3-hydroxyl-3-(2-thienyl) propionitrile through separation and purification.
3. method as claimed in claim 2, is characterized in that, be also added with the glucose that final concentration is 10-100g/L in described phosphate buffered saline buffer.
4. the method for claim 1, it is characterized in that, the described enzyme somatic cells that contains is prepared in accordance with the following methods: be seeded in fermention medium by yeast saccharomyces cerevisiae CGMCC No.2266, shaking speed is 150-200r/min, cultivate 18-30h for 26-35 DEG C, fermented liquid is centrifugal, obtained containing enzyme somatic cells.
5. method as claimed in claim 2, it is characterized in that, the method of described separation and purification is as follows: after reaction terminates, by centrifugal for conversion fluid 4000r/min 20 minutes, discard bacterial sediment, by supernatant liquor equal-volume ethyl acetate continuous extraction 3 times, combined ethyl acetate extraction liquid, adds anhydrous sodium sulphate removing moisture, suction filtration in acetic acid ethyl acetate extract, filtrate decompression distillation removing ethyl acetate, obtains described (S)-3-hydroxyl-3-(2-thienyl) propionitrile.
6. the method for claim 1, is characterized in that, described method is carried out according to following steps:
(1) slant culture: yeast saccharomyces cerevisiae CGMCC No.2266 is inoculated into slant medium, 26-35 DEG C of cultivation obtains thalline inclined-plane in 4-6 days; Described slant medium final concentration consists of: wort 5-15g/L, yeast powder 2-4g/L, peptone 4-6g/L, glucose 7-12g/L, agar 15-25g/L, natural ph, and solvent is water;
(2) seed culture: get a transfering loop thalline from thalline inclined-plane and be transferred to seed culture medium, 26-35 DEG C, shaking speed is 150-200r/min, cultivates 18-26h and obtains seed liquor; Described seed culture medium final concentration consists of: glucose 26-32g/L, yeast powder 2-4g/L, ammonium sulfate 3-6g/L, anhydrous MgSO
40.2-0.4g/L, K
2hPO
43H
2o 0.5-1.5g/L, KH
2pO
40.6-1.5g/L, be 7.0 by the pH value of NaOH or HCl solution rearrange liquids substratum, solvent is water;
(3) fermentation culture: get seed liquor, be inoculated in fermention medium with the inoculum size of volume ratio 10-20%, culture temperature is 26-35 DEG C, shaking speed is 150-200r/min, cultivating 18-30h obtains containing the fermented liquid containing enzyme somatic cells, and centrifugation obtains described containing enzyme somatic cells; Described fermention medium final concentration consists of: glucose 26-32g/L, yeast powder 2-4g/L, ammonium sulfate 3-6g/L, anhydrous MgSO
40.2-0.4g/L, K
2hPO
43H
2o 0.5-1.5g/L, KH
2pO
40.6-1.5g/L, be 7.0 by the pH value of NaOH or HCl solution rearrange liquids substratum, solvent is water;
(4) bio-transformation: in the phosphate buffered saline buffer of pH 5.0-8.0, add 3-carbonyl-3-(2-thienyl) propionitrile of 0.1-0.6mol/L, add the glucose of final concentration 10-100g/L again as cosubstrate, and dry cell weight be 3-carbonyl-3-(2-thienyl) propionitrile 1-20 doubly containing enzyme somatic cells, conversion reaction 8-40 hour at 25-45 DEG C, reaction terminates obtained conversion fluid;
(5) separation and purification: by centrifugal for conversion fluid 4000r/min 20 minutes after reaction terminates, discard bacterial sediment, by supernatant liquor equal-volume ethyl acetate continuous extraction 3 times, combined ethyl acetate extraction liquid, anhydrous sodium sulphate removing moisture is added in acetic acid ethyl acetate extract, suction filtration, filtrate decompression distillation removing ethyl acetate, obtains described (S)-3-hydroxyl-3-(2-thienyl) propionitrile.
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