CN1215085A - Enzyme method separation process for racemic ketoprofen - Google Patents
Enzyme method separation process for racemic ketoprofen Download PDFInfo
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- CN1215085A CN1215085A CN98121942A CN98121942A CN1215085A CN 1215085 A CN1215085 A CN 1215085A CN 98121942 A CN98121942 A CN 98121942A CN 98121942 A CN98121942 A CN 98121942A CN 1215085 A CN1215085 A CN 1215085A
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- ketoprofen
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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
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
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Abstract
The present invention discloses an enzymatic resolution process of recemic ketoprofen. Said invention uses the Lipase OF crude enzyme produced by Japan Meito Sangyo Co., Ltd. as enzymatic hydrolysis reaction catalyst to directly make resolution of ketoprofen receme, and implement the enzymatic resolution reaction under the condition of high acidity and in the presence of surfactant, for example Tween-80, so that it not only can obtain the (S)-ketoprofen and (R)-ketoprofen with high optical purity, but also can greatly reduce production cost.
Description
The invention belongs to technical field of biochemical industry, relate to the enzymic resolution craft of left-handed-Ketoprofen BP 93.
The dextrorotation Ketoprofen BP 93, or to be called (S)-Ketoprofen BP 93 be a kind of important 2-aryl propionic non-steroid antiphlogistic, has very to use widely in pharmaceutical industries; And left-handed Ketoprofen BP 93, or be called (R)-Ketoprofen BP 93 and then can make an addition in the toothpaste, be used to prevent and treat osteoporosis.At present, the product that the method by chemosynthesis obtains is racemic modification, need by optical resolution could obtain highly purified (S)-Ketoprofen BP 93 with (R)-Ketoprofen BP 93.Traditional method for splitting has chemical processes such as stereoselectivity crystallization, and utilize microorganism or biocatalysis disassemble technique that enzyme carried out in recent years because it is easy and simple to handle, environmental pollution is little and obtained increasing development and application, becomes the problem that many scientific and technical personnel pay close attention to.Many documents and patent have all related to the technology of this respect, comparatively typically have following several:
(1) document J.Am.Chem.Soc.1990,112:1990-1995 disclose the method for a kind of employing lipase from candida sp (Candida lipase, Sigma L1754, Type VII) catalytic hydrolysis Ketoprofen BP 93 (2-(3-benzoyl benzene)-propionic acid) chloroethene ester.The stereoselectivity of this method is very low, and the optical purity of product one (the S)-Ketoprofen BP 93 of gained only is 52%ee.The document has improved the stereoselectivity of enzymatic resolution reaction by the method that the lipase purifying is also handled with deoxidation cholate and organic solvent, but treating processes is very complicated, the loss of activity of enzyme is very big, and the cost of catalyzer is increased considerably, and has brought certain difficulty to industrial applications.
(2) United States Patent (USP) U.S.Pat.5 108 916, Apr.28, and 1992 have invented a kind of comparatively practicable method for splitting.This method adopts Lipase MY (day real name sugar industry company produces) and Lipase AY (Japanese amano pharmaceutical company produce) the enzyme source as 2-arylpropionic acid and the fractionation of 2-aryloxy propionic acid, from MY or AY crude zyme preparation, separate to obtain two kinds of lipase isozymes (CSC-1 and CSC-2) by methods such as ion exchange chromatography or isoelectrofocusing, be respectively applied for catalysis and split 2-arylpropionic acid and 2-aryloxy propionic acid.This method exists the very complicated defective for the treatment of processes equally;
This patent also adopts the method that adds hard ester acid, oleic acid and various oleic acid esters in system to strengthen the catalytic activity and the stereoselectivity of enzyme, since these hydrophobic fat acids additives be that hydrophobic product 2-arylpropionic acid and 2-aryloxy propionic acid are mixed in together easily equally, give product separation and refiningly brought very big difficulty;
This patent has been investigated oleic acid esters surface active agent such as tween (Tween) series and sapn (Span) series simultaneously to the esterolytic influence of CSC-2 enzyme catalysis Ketoprofen BP 93 monoacylglycerol, but the result shows these tensio-active agents the selectivity of enzyme be there is no enhancement, and is also not remarkable to the active facilitation effect of enzyme.
In sum, when existing commercialization crude zyme preparation splits the Ketoprofen BP 93 racemic modification in catalysis, stereoselectivity is all not ideal enough, and currently used effective ways are the purifying of thick enzyme, though can improve the stereoselectivity of the short fractionation of enzyme, the purge process of enzyme is quite complicated, and the vigor loss of enzyme is also bigger, the cost of catalyzer is increased substantially, limited this method in industrial applying.
The objective of the invention is to overcome the above-mentioned defective of prior art, disclose a kind of directly with the thick enzyme of Lipase OF (day real name sugar company product, derive from Zou's pleat candiyeast Candida rugosa, the olive oil hydrolysis vigor is 360u/mg) as the resolution process of the Ketoprofen BP 93 racemic modification of enzymically hydrolyse catalysts.
Design of the present invention is such:
(1) because the thick enzyme of Lipase OF of day real name sugar company production is the product that activity is the highest in a kind of lipase from candida sp, price is cheaper, therefore, when the thick enzyme of Lipase OF is directly used in fractionation Ketoprofen BP 93 racemic modification as the enzymically hydrolyse catalysts, not only can obtain higher stereoselectivity, and its production cost can reduce significantly;
(2) owing to the reduction along with the pH value of solution value of the steric configuration of the thick enzyme of Lipase OF changes, the reaction of (the S)-substrate that makes the selectivity of enzyme help more, therefore the contriver carries out enzymatic resolution reaction process under the environment of high acidity, then can further improve the optical purity of product (S)-Ketoprofen BP 93;
(3) because the viscosity of enzyme reaction substrate (ester of Ketoprofen BP 93) is higher, low in aqueous phase solubleness, dispersed relatively poor, therefore can in reaction process, add a kind of to enzyme gentleness, no deactivation with to human body nontoxic tensio-active agent such as Tween-80, to improve the dispersion situation of substrate, quicken the speed of response of enzymically hydrolyse, improve the stereoselectivity of enzyme.
According to above-mentioned design, the contriver has proposed realization as described below technical scheme of the present invention:
(1) Zou's pleat lipase from candida sp Lipase OF and the Ketoprofen BP 93 chloroethene ester racemic modification with day real name sugar industry company production places reaction flask, adds buffered soln, insulation reaction, and the processing condition of reaction are such:
Temperature of reaction is 20～50 ℃, and optimum temps is 30～40 ℃; Reaction times is 1～10 day, and the pH of solution is 2～5, and optimal ph is 3～4, and the add-on of enzyme and substrate is:
Lipase: Ketoprofen BP 93 chloroethene ester=1: (1～10) (weight)
In order to quicken the speed of response of enzymically hydrolyse, improve the stereoselectivity of enzyme, can when reaction, add an amount of nonionogenic tenside Tween-80, its add-on is: 1～100 grams per liter reaction solution, optimal dose are 10～50 grams per liter reaction solutions.
(2) adopt conventional separation method as the said product of collection the present invention-(S)-Ketoprofen BP 93 and (R)-Ketoprofen BP 93 chloroethene ester from reaction mixture such as extraction and chromatography, and (R)-Ketoprofen BP 93 chloroethene ester can be converted into (R)-Ketoprofen BP 93 by the method for general chemical hydrolysis.
Adopt the said resolution process of the present invention, not only can obtain highly purified (R)-Ketoprofen BP 93 and (S)-Ketoprofen BP 93, wherein the optical purity of (S)-Ketoprofen BP 93 can reach more than the 95%ee, and production cost is more much lower than prior art, be a kind of production method, can satisfy the needs of the medicine industry of fast development with wide application prospect.Below will be further elaborated technology contents of the present invention by embodiment.
Embodiment 1
(1) be that 2.2 10ml buffered soln (0.1mol/L citric acid-0.2mol/L Sodium phosphate dibasic) places reaction flask with 100 milligrams of Ketoprofen BP 93 chloroethene ester racemic modifications, 20 milligrams of Lipase OF crude zyme preparations and pH, on shaking table (40 ℃, 160r/min) insulation reaction is 6 days, can obtain (S)-Ketoprofen BP 93 with (R)-mixture of Ketoprofen BP 93 chloroethene ester;
(2) with the 60ml ethyl acetate divide three times to above-mentioned (S)-Ketoprofen BP 93 and the mixture of (R)-Ketoprofen BP 93 chloroethene ester extract, merge organic phase, sampling 1ml analyzes, analytical procedure is as follows:
With chiral chromatographic column Chiracel OJ (Japanese Daicel company, φ 0.45 * 25cm), with normal hexane-Virahol-acetate (volume ratio is 90: 10: 0.2) is moving phase (1ml/min), in high performance liquid chromatograph (Waters510 type, UV-detector wavelength 254nm) upward above-mentioned sample is analyzed, the result is as follows: the hydrolysis conversion of Ketoprofen BP 93 chloroethene ester is 10.5%, (S)-and the optical purity of Ketoprofen BP 93 is 86%ee (promptly (S)-enantiomorph accounts for 93%).(S)-Ketoprofen BP 93 can adopt following method to separate with (R)-Ketoprofen BP 93:
(1) sodium bicarbonate aqueous solution with 30ml10% carries out back extraction to organic phase, make (S)-Ketoprofen BP 93 be transferred to water, two remove organic phase distillation after being separated and desolvate, and can obtain (R)-Ketoprofen BP 93 chloroethene ester, can obtain (R)-Ketoprofen BP 93 by chemical hydrolysis again;
(2) with ethyl acetate water is extracted, distillation can obtain (S)-Ketoprofen BP 93 after removing and desolvating.
Embodiment 2
Adopt raw material and the analytical procedure identical, when reaction, add 0.8 and restrain Tween-80 with embodiment 1, on shaking table (30 ℃, 160r/min) insulation reaction is 2 days, and the result is as follows:
The hydrolysis conversion of Ketoprofen BP 93 chloroethene ester is 37%, (S)-and the optical purity of Ketoprofen BP 93 is 96%ee (promptly (S)-enantiomorph accounts for 98%).
Embodiment 3
(1) be that 3 10ml buffered soln (0.1mol/L citric acid-0.2mol/L Sodium phosphate dibasic) places reaction flask with 200 milligrams of Ketoprofen BP 93 chloroethene ester racemic modifications, 20 milligrams of Lipase OF crude zyme preparations, 0.5 gram Tween-80 and pH, on shaking table (35 ℃, 160r/min) insulation reaction is 4 days, adopt the analytical procedure identical with embodiment, its result is as follows:
The hydrolysis conversion of Ketoprofen BP 93 chloroethene ester is 43%, (S)-and the optical purity of Ketoprofen BP 93 is 90%ee (promptly (S)-enantiomorph accounts for 95%).
Technical scheme disclosed according to the present invention and embodiment, relevant engineering technical personnel can be easily lift one with the present invention's said Lipase OF crude zyme preparation and reaction process and return three ground and be used for the 2-arylpropionic acid of other type such as the fractionation of Naproxen Base, Ibuprofen BP/EP etc.
Claims (5)
1. an enzymatic resolution process of recemic ketoprofen mainly comprises the enzymically hydrolyse reaction of racemize Ketoprofen BP 93 and separating of reaction mixture, it is characterized in that:
Zou's pleat candiyeast (Candida rugosa) lipase Lipase OF of real name sugar industry (Meito Sangyo) company production makes catalyzer with day, be directly used in the enzymically hydrolyse reaction of Ketoprofen BP 93 racemic modification, adopt then conventional separation method from reaction-ure mixture, collects this (S)-Ketoprofen BP 93 with (R)-Ketoprofen BP 93.
2. the method for claim 1, the pH that it is characterized in that solution when enzymically hydrolyse is reflected at is 2～5.
3. as the described method of claim 1-2, it is characterized in that: add tween-80 during reaction, its add-on is 1～100 grams per liter reaction solution.
4. as the described method of claim 1～2, it is characterized in that: the enzymically hydrolyse temperature of reaction is 20～50 ℃, reaction times is 1-10 days, and the add-on of lipase Lipase OF and Ketoprofen BP 93 racemic modification is: lipase: Ketoprofen BP 93 racemic modification=1: (1～10) (weight).
5. as the described method of claim 1～2, it is characterized in that: the optimal ph of reaction is 3～4, and the optimum temps of reaction is 30～40 ℃, and the tween-80 optimal addn is 10～50 grams per liter reaction solutions.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN98121942A CN1065917C (en) | 1998-10-12 | 1998-10-12 | Enzyme method separation process for racemic ketoprofen |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN98121942A CN1065917C (en) | 1998-10-12 | 1998-10-12 | Enzyme method separation process for racemic ketoprofen |
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| CN1215085A true CN1215085A (en) | 1999-04-28 |
| CN1065917C CN1065917C (en) | 2001-05-16 |
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| CN98121942A Expired - Fee Related CN1065917C (en) | 1998-10-12 | 1998-10-12 | Enzyme method separation process for racemic ketoprofen |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101225427B (en) * | 2007-12-14 | 2011-07-20 | 华南理工大学 | Method for improving reaction rate of enzymatic ibuprofen chiral separation |
| CN108251493A (en) * | 2017-12-23 | 2018-07-06 | 湖南理工学院 | A kind of method of stereoselectivity enzymatic hydrolysis fractionation -2- (3- chlorphenyls) propionic acid enantiomer |
| CN110818550A (en) * | 2019-11-25 | 2020-02-21 | 东华理工大学 | Method for preparing dextro-ibuprofen through selective enzymatic hydrolysis reaction |
| CN113088553A (en) * | 2021-03-30 | 2021-07-09 | 东华理工大学 | Method for preparing S-ibuprofen by enzymatic resolution-chemical racemization tandem |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5322791A (en) * | 1985-12-20 | 1994-06-21 | Wisconsin Alumni Research Foundation | Process for preparing (S)-α-methylarylacetic acids |
| US5108916A (en) * | 1989-06-05 | 1992-04-28 | Rhone-Poulenc Rorer, S.A. | Process for stereoselectively hydrolyzing, transesterifying or esterifying with immobilized isozyme of lipase from candida rugosa |
-
1998
- 1998-10-12 CN CN98121942A patent/CN1065917C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101225427B (en) * | 2007-12-14 | 2011-07-20 | 华南理工大学 | Method for improving reaction rate of enzymatic ibuprofen chiral separation |
| CN108251493A (en) * | 2017-12-23 | 2018-07-06 | 湖南理工学院 | A kind of method of stereoselectivity enzymatic hydrolysis fractionation -2- (3- chlorphenyls) propionic acid enantiomer |
| CN108251493B (en) * | 2017-12-23 | 2021-10-15 | 湖南理工学院 | Method for splitting-2- (3-chlorphenyl) propionic acid enantiomer through stereoselective enzymatic hydrolysis |
| CN110818550A (en) * | 2019-11-25 | 2020-02-21 | 东华理工大学 | Method for preparing dextro-ibuprofen through selective enzymatic hydrolysis reaction |
| CN113088553A (en) * | 2021-03-30 | 2021-07-09 | 东华理工大学 | Method for preparing S-ibuprofen by enzymatic resolution-chemical racemization tandem |
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| CN1065917C (en) | 2001-05-16 |
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