CN105506022A - Method for preparing ezetimibe chiral intermediate by utilizing whole-cell catalytic synthesis on escherichia coli - Google Patents
Method for preparing ezetimibe chiral intermediate by utilizing whole-cell catalytic synthesis on escherichia coli Download PDFInfo
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- CN105506022A CN105506022A CN201610067795.7A CN201610067795A CN105506022A CN 105506022 A CN105506022 A CN 105506022A CN 201610067795 A CN201610067795 A CN 201610067795A CN 105506022 A CN105506022 A CN 105506022A
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Abstract
The invention discloses a method for preparing ezetimibe chiral intermediate by utilizing whole-cell catalytic synthesis on escherichia coli, and belongs to the field of biocatalysis. Whole-cell catalytic precursor ketone of escherichia coli is synthesized into (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxy valeryl]-4-phenyl-1, 3-oxdiazole cyclopentane-2-ketone, with extremely high stereoselectivity, wherein the value e.e of the (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxy valeryl]-4-phenyl-1, 3-oxdiazole cyclopentane-2-ketone is 99.99 percent, and the conversion rate is about 70 percent.
Description
Technical field
The present invention is specifically related to a kind of method taking intestinal bacteria as biological catalyst and synthesize Ezetimibe intermediate, belongs to biocatalysis field.
Background technology
Ezetimibe is a kind of cholesterol absorption inhibitor, is first ACAT (acetyl-CoA-cholesterol acyltransferase) inhibitor found so far.(4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl base]-4-phenyl-1; 3-oxazolidine-2-ketone is as the crucial chiral intermediate of Ezetimibe, and it can split its precursor ketone by chemical/biological method of asymmetrically reducing or Dynamics of Enzyme Catalysis and prepare.Wherein, the target product optical purity that chemical method of asymmetrically reducing obtains is not high enough, and chemical method cost is higher simultaneously, and chemical catalyst easily causes serious pollution to environment.Compared with chemical method, biological process shows that obvious stereospecificity is strong, catalytic efficiency is high and the advantage such as pollution-free.
The two strain bacterial strains that only have of current report have reducing activity to this precursor ketone, wherein, M.J.Homannhas utilizes microorganism SchizosaccharomycesoctosporusATCC2479 catalytic reduction precursor ketone production Ezetimibe intermediate, and transformation efficiency is about 33% (USPatent5618707).UttamC.Banerjee etc. utilize the full cell of Burkholderiacenocepacia to carry out catalytic reduction, and productive rate is about 54% (JIndMicrobiolBiotechnol (2009) 36:1369 – 1374).In addition; Codexis a kind of R type desaturase from Lactobacillus kefir carries out orthogenesis; obtain the S type desaturase (USPatent8415126B2) that can catalyze and synthesize (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl base]-4-phenyl-1,3-oxazolidine-2-ketone.
The present invention using intestinal bacteria as biological catalyst; oscillatory reaction after mixing with substrate solution; obtain three-dimensional pure (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl base]-4-phenyl-1,3-oxazolidine-2-ketone.
Summary of the invention
Main research of the present invention: the present invention utilizes E. coli whole cell as biological catalyst; Ezetimibe intermediate (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl base]-4-phenyl-1,3-oxazolidine-2-ketone is obtained after asymmetric reduction.
E. coli whole cell is utilized to catalyze and synthesize a method for Ezetimibe chiral intermediate, it is characterized in that comprising the following steps:
(1) bacterial strain is selected: select intestinal bacteria as bacterial classification;
(2) fermented liquid preparation: be seeded to by strain Escherichia coli in Luria-Bertani liquid nutrient medium, shaking culture, obtains bacterial strain fermentation liquor; Centrifugal for bacterial strain fermentation liquor rear removing supernatant liquor is obtained wet thallus, obtains wet thallus suspension with the resuspended wet thallus of phosphate buffered saline buffer that pH value is 7.0;
(3) asymmetric reduction reaction: Ezetimibe intermediate precursor ketone is dissolved in dimethyl sulfoxide (DMSO), is made into substrate solution; Wet thallus suspension step (2) obtained mixes obtained thalline mixed solution with substrate solution, shaking culture, obtains reaction solution.
Further, Luria-Bertani liquid culture based formulas is: in 950ml deionized water, add peptone 10g, yeast extract 5g and sodium-chlor 10g, ultrasonic until solute dissolves. adjust pH to 7.0 with 5mol/LNaOH, be settled to 1L with deionized water, at 121 DEG C, sterilizing 20min.
Further, the strain Escherichia coli described in step (2) is inoculated in Luria-Bertani liquid nutrient medium with the volume ratio of 0.1% ~ 0.5%, 37 DEG C, shaking culture 12h.
Further, the temperature of the shaking culture described in step (3) is 37 DEG C, and the time is 3 ~ 24h.
Further, described pH value is that the phosphate buffered saline buffer of 7.0 is by 0.05MNa
2hPO
4: 0.05MNaH
2pO
4volume ratio 7:3 mixes.
More specifically:
Above-mentioned intestinal bacteria (Escherichiacoli), for routine, can buy.
Substratum is conventional Luria-Bertani liquid nutrient medium, and filling a prescription is: in 950ml deionized water, add peptone 10g, yeast extract 5g and sodium-chlor 10g, ultrasonic until solute dissolves. adjust pH to 7.0 with 5mol/LNaOH, be settled to 1L with deionized water.At 121 DEG C, sterilizing 20min.Agents useful for same is that routine can be bought, peptone (OXOIDTRYPTONELP0042), yeast powder extract (OXOIDYEASTEXTRACTLP0021), sodium-chlor (analytical pure Chemical Reagent Co., Ltd., Sinopharm Group)
Centrifugal for fermented liquid rear removing supernatant is obtained wet thallus (wet thallus is the thalline that phalangeal cell includes moisture), obtain wet thallus suspension with the resuspended wet thallus of phosphate buffered saline buffer that pH value is 7.0.
Asymmetric reduction reaction described in step (3), is dissolved in dimethyl sulfoxide (DMSO) by Ezetimibe intermediate precursor ketone, is made into substrate solution.Substrate solution is joined in wet thallus suspension, wherein comprise 100mg wet thallus in every 1mL reaction system.Shaking culture after mixing.
Shaking culture terminates, and makes to be extracted with ethyl acetate, and product is dissolved in organic layer.
High-efficient liquid phase chromatogram condition:
Chemical pure analysis condition
(a) chromatographic column: Shiseido C18
(b) column temperature: 25 DEG C
(c) moving phase: A phase: 50mM ammonium acetate solution (second acid for adjusting pH extremely), B phase: methyl alcohol, A phase is 1:2 with B phase velocity ratio
(d) detector: UV-detector 215nm
Optical purity analysis condition
(a) chromatographic column: OJ-H
(b) column temperature: 25 DEG C
C () moving phase: A phase: Virahol, B phase: ethanol, A phase is 7:3 with B phase velocity ratio
(d) detector: UV-detector 215nm
Feature of the present invention:
The present invention adopts intestinal bacteria catalyged precursor ketone to obtain Ezetimibe chiral intermediate first, provides a kind of New biocatalyst.Compared with chemical method, the present invention has that stereospecificity is strong, environmental pollution is little and the advantage such as mild condition.
Accompanying drawing explanation
Fig. 1 is the HPLC collection of illustrative plates of Ezetimibe intermediate precursor ketone
Fig. 2 is the chirality HPLC collection of illustrative plates of standard substance raceme (4S)-3-[5-(4-fluorophenyl)-5-hydroxypentanoyl base]-4-phenyl-1,3-oxazolidine-2-ketone.
Fig. 3 is the HPLC collection of illustrative plates in embodiment 1 after intestinal bacteria catalytic reduction precursor ketone.
Embodiment
Below in conjunction with embodiment, this aspect is described further, but the present invention is not limited to following examples
Embodiment 1
The present embodiment is that a kind of E. coli whole cell catalyzes and synthesizes (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl base]-4-phenyl-1,3-oxazolidine-2-ketone.
The method comprises the steps:
(1) bacterial strain select: select intestinal bacteria (EscherichiacoliBL21) as bacterial classification, this culture presevation with-80 DEG C;
(2) fermented liquid preparation: by described strain Escherichia coli with 0.1% volume ratio be seeded in Luria-Bertani liquid nutrient medium, 37 DEG C, 220rpm shaking culture 24h;
Luria-Bertani liquid culture based formulas is: in 950ml deionized water, add tryptone 10g, yeast extract 5g and sodium-chlor 10g, ultrasonic until solute dissolves. adjust pH to 7.0 with 5mol/LNaOH, be settled to 1L with deionized water.At 121 DEG C, sterilizing 20min.
(3) microorganism collection: get fermented liquid centrifugal 10min under 8000rpm rotating speed, removing supernatant obtains wet thallus, collects wet thallus and uses as biological catalyst;
(4) asymmetric reduction reaction: obtain wet thallus suspension with the resuspended wet thallus of phosphate buffered saline buffer that pH value is 7.0.Described pH value is that the phosphate buffered saline buffer of 7.0 is by 0.05MNa
2hPO
4: 0.05MNaH
2pO
4volume ratio 7:3 mixes.Ezetimibe intermediate precursor ketone is dissolved in dimethyl sulfoxide (DMSO), is made into substrate solution.
Mixed with Ezetimibe intermediate precursor ketone by wet thallus suspension, obtained thalline mixed solution, shaking culture, obtains reaction solution; 20ul substrate solution (in reaction system, Final substrate concentrations is 2mM) and 100mg wet thallus is comprised in its 1ml reaction system.Described shaking culture temperature is 37 DEG C, and rotating speed is 220rpm, time 24h.
(5) HPLC measures: get the above-mentioned reaction of 1ml and the abundant oscillation extraction of 1ml ethyl acetate, make target product be dissolved in ethyl acetate, then at the centrifugal 1min of 12000rpm, get organic layer 10ul and carry out Liquid Detection.Liquid phase result shows, product (4S)-3-[(5S)-5-(4-fluorophenyl)-5-hydroxypentanoyl base]-4-phenyl-1,3-oxazolidine-2-ketone e.e value is 99.99%, and substrate conversion efficiency is about 70%
The reagent adopted in embodiment 1 and instrument such as non-specified otherwise are market conventional products.
Obviously, the above embodiment of the present invention, just in order to the citing that the present invention does clearly is described, is not the restriction to embodiment of the present invention.For those of ordinary skill in the field, other multi-form change or variations can also be made on the basis of the above description, such as, the above-mentioned substrate of catalytic reduction after clonal expression be carried out to enzyme corresponding in intestinal bacteria.Here cannot give exhaustive to all embodiments.Every belong to technical scheme of the present invention the apparent change of amplifying out or variation be still in the row of protection scope of the present invention.
Claims (5)
1. utilize E. coli whole cell to catalyze and synthesize a method for Ezetimibe chiral intermediate, it is characterized in that comprising the following steps:
(1) bacterial strain is selected: select intestinal bacteria as bacterial classification;
(2) fermented liquid preparation: be seeded to by strain Escherichia coli in Luria-Bertani liquid nutrient medium, shaking culture, obtains bacterial strain fermentation liquor; Centrifugal for bacterial strain fermentation liquor rear removing supernatant liquor is obtained wet thallus, obtains wet thallus suspension with the resuspended wet thallus of phosphate buffered saline buffer that pH value is 7.0;
(3) asymmetric reduction reaction: Ezetimibe intermediate precursor ketone is dissolved in dimethyl sulfoxide (DMSO), is made into substrate solution; Wet thallus suspension step (2) obtained mixes obtained thalline mixed solution with substrate solution, shaking culture, obtains reaction solution.
2. according to the method for claim 1, it is characterized in that, Luria-Bertani liquid culture based formulas is: in 950ml deionized water, add peptone 10g, yeast extract 5g and sodium-chlor 10g, ultrasonic until solute dissolves. adjust pH to 7.0 with 5mol/LNaOH, 1L is settled to deionized water, at 121 DEG C, sterilizing 20min.
3. according to the method for claim 1, it is characterized in that, the strain Escherichia coli described in step (2) is inoculated in Luria-Bertani liquid nutrient medium with the volume ratio of 0.1% ~ 0.5%, 37 DEG C, shaking culture 12h.
4. according to the method for claim 1, it is characterized in that, the temperature of the shaking culture described in step (3) is 37 DEG C, and the time is 3 ~ 24h.
5. according to the method for claim 1, it is characterized in that, described pH value is that the phosphate buffered saline buffer of 7.0 is by 0.05MNa
2hPO
4: 0.05MNaH
2pO
4volume ratio 7:3 mixes.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109097412A (en) * | 2018-07-24 | 2018-12-28 | 江苏理工学院 | A kind of method of bioanalysis synthesis Ezetimibe intermediate |
| CN112458143A (en) * | 2020-12-15 | 2021-03-09 | 江苏阿尔法药业有限公司 | Method for synthesizing ezetimibe chiral intermediate through whole-cell catalysis |
Citations (3)
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| CN103864708A (en) * | 2012-12-12 | 2014-06-18 | 天津市医药集团技术发展有限公司 | Preparation method of ezetimibe intermediate |
| CN103911403A (en) * | 2014-04-28 | 2014-07-09 | 中国药科大学 | Method for preparing chiral intermediate of atorvastatin |
| CN104342460A (en) * | 2013-08-09 | 2015-02-11 | 南京朗恩生物科技有限公司 | Method for preparing statin side chain intermediate by means of whole-cell catalysis |
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- 2016-01-30 CN CN201610067795.7A patent/CN105506022A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103864708A (en) * | 2012-12-12 | 2014-06-18 | 天津市医药集团技术发展有限公司 | Preparation method of ezetimibe intermediate |
| CN104342460A (en) * | 2013-08-09 | 2015-02-11 | 南京朗恩生物科技有限公司 | Method for preparing statin side chain intermediate by means of whole-cell catalysis |
| CN103911403A (en) * | 2014-04-28 | 2014-07-09 | 中国药科大学 | Method for preparing chiral intermediate of atorvastatin |
Non-Patent Citations (1)
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109097412A (en) * | 2018-07-24 | 2018-12-28 | 江苏理工学院 | A kind of method of bioanalysis synthesis Ezetimibe intermediate |
| CN112458143A (en) * | 2020-12-15 | 2021-03-09 | 江苏阿尔法药业有限公司 | Method for synthesizing ezetimibe chiral intermediate through whole-cell catalysis |
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Application publication date: 20160420 |