CN101104861A - Method for preparing S-ibuprofen and S-ibuprofen ester by biological catalysis - Google Patents
Method for preparing S-ibuprofen and S-ibuprofen ester by biological catalysis Download PDFInfo
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Abstract
The invention provides a novel catalytic synthesis method to produce s-ibuprofen and various s-ibuprofen esters. S-ibuprofen ester is obtained by esterifying racemic ibuprofen and alcohol with the extracellular lipase deriving from yarrowia lipolytica. The invention further relates to the method of hydrolyzing the synthesized s-ibuprofen ester to prepare s-ibuprofen.
Description
Technical field
The invention belongs to biological technical field, relate to the method for preparing S-Ibuprofen BP/EP and S-ibuprofen ester, more specifically, relate to and utilize lipase-catalyzed resolution of racemic Ibuprofen BP/EP, make the method for the S-Ibuprofen BP/EP and the S-ibuprofen ester of high-optical-purity.
Background technology
NSAID (non-steroidal anti-inflammatory drug) (non～steroidal anti～inflammatory drugs, NSAIDs) to be that a class has analgesic, analgesia and most medicine that also has anti-inflammatory, anti rheumatism action, be widely used in treating rheumatoid arthritis, osteoarthritis and other rheumatisms clinically, for hundreds of millions of rheumatisants removes misery.This class drug main will comprise racemize Ibuprofen BP/EP (racemize-2-(4-isobutyl phenenyl) propionic acid); flurbiprofen (racemize-2-((3-fluoro-4-phenyl) phenyl) propionic acid; fenoprofen (racemize-2-(3-Phenoxyphenyl) propionic acid); sutoprofen (racemize-2-(4 (2-thienyl oxo) phenyl) propionic acid); carprofen (racemize-2-(6-chloro-9H-carbazyl) propionic acid); Naproxen Base (racemize-2-(6-methoxyl group-2-naphthyl) propionic acid); Ketoprofen (racemize-a-methyl-3-benzoyl-toluylic acid) etc.The pharmacologically active of these medicines is that they can capture hydrogen atom from arachidonic C-13 by stoping cyclooxygenase, and then stoped arachidonic C-11 and the peroxidation of C-15 position, finally hindered its bio-transformation to the prostaglandin(PG) and the thromboxane A2 of inflammation.
The racemize Ibuprofen BP/EP is a kind of of 11 kinds of non-steroid antiinflammatory drug thing toxic side effect minimums such as Naproxen Base, Ketoprofen, most western developed countries all with it as nonprescription drugs.Why the racemize Ibuprofen BP/EP has higher activity is because oxidasic avidity of its shack and natural substrate arachidonic acid are similar.Studies show that, two kinds of isomer S-Ibuprofen BP/EPs of racemize Ibuprofen BP/EP and R～Ibuprofen BP/EP are different aspect pharmacological kinetics and biotransformation, the S-Ibuprofen BP/EP has obviously higher clinical effectiveness (Adams et al., J.Pharm.Pharakol, 28,257 and Jamali et al., Pharmac.Res.1988,5,44).Compare with racemic compound, the S-isomer can reach treatment concentration fast in blood.The racemize Ibuprofen BP/EP is accepted by extensive patients as anti-inflammation analgesia medicine, but the racemize Ibuprofen BP/EP is as the active drug of chronic diseases such as treatment rheumatism and rheumatoid arthritis, long-term frequent heavy dose of medication meeting increases GI side reaction, even cause gastrointestinal hemorrhage, kidney there is certain infringement, its clinical application is greatly limited.For solving dosage, toxicity and the pharmacokinetics problem that the outer Ibuprofen BP/EP administration that disappears exists, preparation single chiral S-Ibuprofen BP/EP seems especially important.
S-Ibuprofen BP/EP preparation method can be divided into stereoselectivity chemosynthesis and racemic modification fractionation two big classes at present, racemic modification splits and mainly to comprise and utilize chemical resolving agent to split and two kinds of biotechnology fractionations, wherein the stereoselectivity chemosynthesis relates to expensive chiral auxiliary(reagent) or catalyzer, perhaps relate to complicated preparation technology and various chemical, the chemistry resolving agent is also comparatively expensive, therefore cost height on industrial production.It mainly is to utilize biological enzyme to split that biotechnology splits, and the biotechnology fractionation is simple owing to its technology, production cost is low is favored, and has favorable industrial application prospect.Be used for enzyme that racemize Ibuprofen BP/EP enantiomorph splits at present lipase from Rhizomucor miehei, Candida rugosa, Candida antarctica (Novozyme 435), Aspergillus niger AC-54 and Thermomyces lanuginosa etc. is arranged, but the catalytic conversion of these enzymes and stereoselectivity are all lower, unsatisfactory (referring to: Fabiano Jares Contesini and Patrl ' ciade Oliveira Carvalho
*Esterification of (RS)-Ibuprofen by native andcommercial lipases in a two-phase system containing ionic liquidsTetrahedron:Asymmetry 17 (2006) 2069-2073).
Summary of the invention
The present invention provides a kind of new enzyme catalysis of passing through to split the method for preparing the S-ibuprofen ester on the one hand.In particular, the invention provides a kind of method that is used to prepare multiple S-ibuprofen ester from the lipase-catalyzed resolution of racemic Ibuprofen BP/EP in extracellular that inferior sieve is separated fat yeast (Yarrowialipolytica), comprise the esterification that is used to separate fat zymic extracellular lipase-catalyzed promotion racemize Ibuprofen BP/EP and alcohol, from reaction mixture, separate obtaining the S-ibuprofen ester then from inferior sieve.
The present invention further provides the method for preparing the S-Ibuprofen BP/EP, comprising the step that the S-ibuprofen ester according to the present invention's preparation is hydrolyzed.The reaction that ester is hydrolyzed into acid is a technology known in those skilled in the art.
Description of drawings
Fig. 1 has shown under the differential responses temperature that the transformation efficiency c of racemize Ibuprofen BP/EP and Ya Luo separate the change curve of the stereoselectivity E of fat yeast fat enzyme.
Fig. 2 has shown the acid (being the racemize Ibuprofen BP/EP) at various molar ratios: alcohol or alcohol: under the acid, the transformation efficiency c of racemize Ibuprofen BP/EP and Ya Luo separate the change curve of the stereoselectivity E of fat yeast fat enzyme.
Embodiment
In the present invention, can adopt any racemize Ibuprofen BP/EP to prepare S-ibuprofen ester or S-Ibuprofen BP/EP as splitting raw material.The racemize Ibuprofen BP/EP was promptly prepared successfully by Britain Boots company as far back as the sixties in 20th century.Racemize Ibuprofen BP/EP as raw material of the present invention can prepare by technology as known in the art, or can buy on market.
In the present invention, can adopt and separate the external racemic ibuprofen of fat zymic extracellular lipase from inferior sieve and split, thus preparation S-ibuprofen ester or S-Ibuprofen BP/EP.Described enzyme can be buied by market, but and also laboratory self-control.A kind of such preparation method is included in the suitable medium and cultivates inferior sieve separate fat yeast reasonable time under suitable temperature, then the described lipase of separation and purification from substratum.The fermentation of lipase can be with reference to Tan TW, Zhang M, Wang BW, Ying CH, Deng L.Screening of high lipase producing Candida sp.and production of lipase byfermentation.Process Biochem 2003; 39 (4): disclosed method among the 459-65, its extraction and further purifying can be with reference to Mingrui Yu, Shaowei Qin, Tianwei Tan, Purification andcharacterization of the extracellular lipase.Lip2 from Yarrowia lipolytica.Process Biochemistry 42 (2007) 384-391.
Inferior sieve is separated the fat yeast and is belonged to unconventional yeast, and the FDA authentication is security level yeast (GRAS), and this bacterial strain is used to produce citric acid, single cell protein on a large scale, has accumulated a large amount of experiences in the industrial production.In the present invention available lipase can from or separate the fat yeast derived from any inferior sieve, for example comprise that disclosed inferior sieve is separated the fat yeast among the CN200510112638.5, it is deposited in (address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City Institute of Microorganism, Academia Sinica, postcode: 100080), China Committee for Culture Collection of Microorganisms common micro-organisms center with deposit number CGMCCNo.1470.A kind of separate the method for fat yeast scale operation lipase from inferior sieve can be referring to people such as Destain (1997).Can be used for inferior sieve of the present invention separates fat yeast extracellular lipase and can also prepare by gene engineering method.Such method all has instruction in people such as Fignede (2000) and CN200510112638.5.Those of ordinary skill is according to instruction of the prior art, also know the method that inferior sieve of genetically engineered production is separated fat yeast fat enzyme fully, the nucleotide sequence that comprises the described lipase of for example will encoding is inserted in the suitable expression vector, and express in the introducing appropriate host cell, separate this purpose lipase then from substratum or in the cell.Perhaps, being used for lipase of the present invention also can make by the chemosynthesis approach.Such method has sufficient instruction in the prior art.
In the present invention, can the free thick enzyme form of described lipase is used, and promptly this enzyme can or be untreated and uses through simple process, for example thick enzyme powder, even can be directly with fermented liquid (because the enzyme of the present invention's use is an extracellular enzyme).
Enzyme of the present invention can also immobilized enzyme and the form of modifying enzyme etc. use, comprise the enzyme that makes with different fixing means of carrier of the same race and the identical or different fixing means of different carriers.It is to know in the prior art that enzyme is carried out the fixed method.In one embodiment, described immobilized lipase comprises above-mentioned lipase, carrier and common fixing agent.Fixation support activates with the common fixing agent of definite composition and proportioning earlier.Fixation support and common fixing agent are pressed 1: 1～1: 3 (W: V) mixed, drying.The lipase deionized water dissolving mixes with fixation support after the activation in the ratio of 1000～30000 units/gram carriers, dries stand-by.
In lipase immobilization of the present invention, described carrier can be a solid particulate, as silica gel, diatomite.Perhaps, described carrier can be membranaceous textiles, for example natural fabric such as cotton or man made fiber fabric such as terylene, and especially membranaceous textiles, it has, and surface-area is big, adsorptivity strong, low price, good stability and the characteristics that can reuse.
In another preferred embodiment, described altogether fixing agent is selected from least a in (comprising) macromolecular compound, tensio-active agent, protein, the inorganic salt.
In a further preferred embodiment, described altogether fixing agent is selected from least a in PEG6000, Oleum Cocois, tween 80, gelatin, Yelkin TTS and the sal epsom, and the mass ratio of each component is a gelatin in the preferred described fixing agent altogether: Yelkin TTS: PEG6000: tween 80: sal epsom: Oleum Cocois=5: 1: 1: 2: 1: 1.
The preparation of immobilized enzyme of the present invention can be referring to the applicant's common pending trial Chinese invention patent application No.200510112638.5.In addition, in following document, also instructed the preparation of the fermented liquid etc. of the thick enzyme, immobilized enzyme, modifying enzyme and the enzyme that use in the present invention: Tan TW, Zhang M, Wang BW, Ying CH, Deng L.Screening of high lipase producing Candida sp.andproduction of lipase by fermentation.Process Biochem 2003,39 (4): 459-65; Mingrui Yu, Shaowei Qin, Tianwei Tan, Purification and characterizationof the extracellular lipase Lip2 from Yarrowia lipolytica, ProcessBiochemistry 42 (2007) 384-391; Kaili Nie, Feng Xie, Fang Wang, TianweiTan, Lipase catalyzed methanolysis to produce biodiesel:Optimization ofthe biodiesel production.Journal of Molecular C atalysis B:Enzymatic 43 (2006) 142-147; And Chinese invention patent application number 02117614.0, its publication number is CN1456674; Mingrui Yu, Stefan Lange, Sven Richter, Tianwei Tan, Rolf D.Schmid High～level expression of extracellular lipase Lip2 from Yarrowialipolytica in Pichia pastoris and its purification and characterization.Protein Expression and Purification 53 (2007) 255～263.The content of above-mentioned file is all incorporated this paper by reference in full into.
In addition, it will be understood by those skilled in the art that fermentation, transformation and the immobilization of enzyme is not limited to the method that above-mentioned document provides, those of ordinary skill can very easily be determined suitable enzyme preparation method according to the general knowledge in this area.
In one embodiment, being used for enzyme of the present invention is the extracellular lipase of separating fat yeast (Yarrowialipolytica) CGMCC No.1470 from inferior sieve, and one grade amino acid sequence is as follows:
VYTSTETSHIDQESYNFFEKYARLANIGYCVGPGTKIFKPFNCGLQCA
HFPNVELIEEFHDPRLIFDVSGYLAVDHASKQIYLVIRGTHSLEDVITD
IRIMQAPLTNFDLAANISSTATCDDCLVHNGFIQSYNNTYNQIGPKLDS
VIEQYPDYQIAVTGHSLGGAAALLFGINLKVNGHDPLVVTLGQPIVG
NAGFANWVDKLFFGQENPDVSKVSKDRKLYRITHRGDIVPQVPFWD
GYQHCSGEVFIDWPLIHPPLSNVVMCQGQSNKQCSAGNTLLQQVNV
IGNHLQYFVTEGVCGI(SEQ ID NO；1)
Described inferior sieve is separated the fat yeast and has been deposited in (address: No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City Institute of Microorganism, Academia Sinica, China Committee for Culture Collection of Microorganisms common micro-organisms center on September 30th, 2005, postcode: 100080), deposit number is CGMCC No.1470.
In the present invention, except above-mentioned lipase, can also use their aminoacid sequence variant, analogue, derivative etc.Those of ordinary skill is known fully, and after the aminoacid sequence in the protein was modified, the protein that is obtained had still kept original biologic activity probably, thereby can be used among the present invention.This proteinic preparation and activity determination method are known in the prior art.
In the present invention, separate fat zymic extracellular lipase from inferior sieve racemic mixture is played the stereoselectivity katalysis, those skilled in the art can easily determine its effective level in the methods of the invention according to general knowledge of the prior art.Generally speaking, in the cumulative volume of reaction system, thick enzyme powder consumption is 0.001mg/ml～100mg/ml, preferred 0.01mg/ml～50mg/ml, more preferably 0.05mg/ml～10mg/ml, especially preferred 0.1～5mg/ml, most preferably 0.2～1mg/ml; Its consumption of immobilized enzyme is 5mg/ml～500mg/ml, more preferably 10～50mg/ml, especially preferred 20～30mg/ml.
According to the present invention, the alcohol that is used to prepare ibuprofen ester can be C1～20 alcohol, monobasic primary alconol for example, described alcohol can be saturated, as methyl alcohol, ethanol, n-propyl alcohol, propyl carbinol, Pentyl alcohol, n-hexyl alcohol, n-Heptyl alcohol, n-Octanol, nonanol-, nonylcarbinol, lauryl alcohol, stearyl alcohol etc., also can be undersaturated, as 2～vinylcarbinol, perhaps its any mixture.Preferably, described alcohol is methyl alcohol, ethanol, n-propyl alcohol or its any mixture.More preferably, described alcohol is ethanol.
In method for splitting of the present invention, the racemize Ibuprofen BP/EP can at random be determined with the ratio of the consumption of used alcohol.Generally speaking, the consumption mol ratio of racemize Ibuprofen BP/EP and alcohol was preferably 1: 3～3: 1 between 1: 5～5: 1, more preferably 1: 2～2: 1, most preferably be 1: 2.Certainly, those of ordinary skill can be understood fully, also can adopt this extraneous amount ratio, just should suitably adjust other parameter in the esterification, comprises for example temperature of reaction, time and solvent etc.
According to the present invention, the esterification of described racemize Ibuprofen BP/EP and alcohol is carried out in organic solution.In one embodiment, described organic solvent can be selected from Log p and be not less than 3 solvent for example alkane, halogenated alkane etc., the example has sherwood oil, Skellysolve A, hexanaphthene, normal hexane, normal heptane, octane, octane-iso, positive nonane, n-decane, tetracol phenixin etc., but is not limited thereto.Wherein p is the hydrophobic constant of organic solvent.Certainly, also can use the mixture of above-mentioned organic solvent.
In the present invention, esterification can be carried out in any suitable temperature range.Those of ordinary skill can reasonably be determined such temperature range according to instruction of the prior art.Usually esterification is carried out under 15～50 ℃, preferably under 20～45 ℃ temperature, carry out, and especially preferred 25～40 ℃, 40 ℃ temperature most preferably from about.
The time length of esterification decides according to multiple factor, and those skilled in the art can determine the suitable reaction times by normal experiment, for example by a small amount of sample comes monitoring reaction course with being equipped with the chiral column liquid chromatography in the reaction solution to taking from.Described chiral column can be CHIRALCELOB～H for example, 0.46cm * 150, and DAICEL CHEMICAL INDUSTRIES, LID, Jap, the moving phase that is adopted can be normal hexane for example: Virahol: trifluoroacetic acid=98: 2: 0.1.Preferably, reaction lasts till that the ratio that S-Ibuprofen BP/EP and R～Ibuprofen BP/EP are converted into ester is more than 3: 1, more preferably 4: 1, and further preferably 5: 1, especially preferred 9: 1, most preferably 98: 2.Particularly, the reaction times is generally 12～120 hours, and preferred 15～80 hours, more preferably 20～60 hours, most preferably 24～48 hours.After reaction reaches required degree, can the suitable method termination reaction, comprise the enzymic activity that inferior sieve of for example hot deactivation is separated fat yeast extracellular lipase, or directly carry out separating of ibuprofen ester and reaction substrate.
Can in esterification, add an amount of suitable additives, as such as the crown ether of 18-hat-6, hexichol 18 hats-6, cyclodextrin, glycerine, methane amide etc.Do not wish to be subject to theory, we are easier to enzyme and substrate-function at the additive of thinking fit, thereby improve the activity of enzyme, improve the chiral selectivity of enzyme.
After esterification finishes, can wait ibuprofen ester and the unreacted Ibuprofen BP/EP that separates generation by for example extraction of conventional isolation technique, chromatography, the evaporation of this area.For example, can utilize strong base solution to come the separating treatment reaction mixture.Preferably, the following of the pH value of described strong base solution is limited to 12,12.5,13,13.5,13.6,13.7,13.8 or 13.9, on be limited to this strong base solution saturated solution the pH value or than this saturated solution pH value low 0.05,0.1,0.15,0.2,0.25,0.3,0.35,0.4,0.45,0.5,0.55,0.6,0.65,0.7,0.75,0.8,0.85,0.9,0.95,1.0,1.05,1.1,1.15,1.2,1.25 or 1.3 etc.More preferably, utilizing pH is that 13～14 strong base solution separates, and most preferably utilizing pH is 14 strong base solution.In one embodiment, this strong base solution can be aqueous sodium hydroxide solution or potassium hydroxide aqueous solution, perhaps its mixture.Certainly in other strong base solution also is encompassed in.After strong base solution is handled, under the certain vacuum degree,, obtain ibuprofen ester to the organic phase underpressure distillation, wherein mainly be the S-ibuprofen ester, also comprise a spot of R-ibuprofen ester.Because inferior sieve separates the strong selectivity of fat yeast to the S-Ibuprofen BP/EP, the R-ibuprofen ester in the end content in the product is very low.
After obtaining the S-ibuprofen ester, can adopt ordinary method that ester is hydrolyzed, thereby make the S-Ibuprofen BP/EP, reach the purpose of resolution of racemic Ibuprofen BP/EP.How those of ordinary skill fully aware of selects suitable ester hydrolysising condition if being.Wherein said hydrolysis can be undertaken by the chemical method of routine, also can be undertaken by enzyme process.Preferred enzyme process wherein utilizes for example lipase or esterase, such as from or separate the enzyme of fat yeast, Candida rugosa or Candida cylindracea etc. derived from inferior sieve.They can perhaps be buied from the market by fermentation or gene engineering method preparation.In one embodiment, adopt enzyme process at suitable pH value, for example pH=7～10, the preferred damping fluid described ibuprofen ester of hydrolysis in the phosphoric acid buffer for example of pH=8.The concentration of described phosphoric acid buffer can be for example 0.05mol/L～0.5mol/L, more preferably 0.1mol/L～0.2mol/L.
Method catalytic conversion height of the present invention, stereoselectivity are strong, S-Ibuprofen BP/EP that makes and S-ibuprofen ester purity height, and the kind of S-ibuprofen ester is many, for medicine provides important intermediate; Its preparation process is simple, feasible, and cost is low, easily industrialization.
To further specify the present invention by the detailed description of specific embodiments of the invention below, but these embodiment carry out exemplary illustration, are not construed as limiting the invention.
Embodiment 1
In the tool plug triangular flask of 100ml, add concentration successively and be 0.03mol/L the racemize Ibuprofen BP/EP (available from Sigma company, the ethanol of Hereinafter the same) hexane solution 10ml, 0.3mmol, 0.2g immobilized enzyme.Immobilized enzyme is REFERENCE TO RELATED people's common pending trial Chinese invention patent application No.200510112638.5 and Tan TW, Zhang M, Wang BW, Ying CH, Deng L.Screening of high lipase producing Candida sp.and production of lipase byfermentation.Process Biochem 2003; 39 (4): the method preparation among the 459-65, hereinafter identical.Reaction mixture is placed shaking table under 40 ℃, and the vibration insulation reaction is 24 hours under 180 rev/mins rotating speed, obtains to contain the mixture of racemize Ibuprofen BP/EP, S-Ibuprofen BP/EP ethyl ester, R-Ibuprofen BP/EP ethyl ester.Get said mixture 20 microlitres and analyze, analytical procedure is as follows:
Detect (chiral column: CHIRALCEL OB～H, 15cm * 4.6mm, DAICEL CHEMICAL INDUSTRIES, LID, Jap by high performance liquid chromatography; Moving phase: normal hexane: Virahol: trifluoroacetic acid=98: 2: 0.1) above-mentioned sample is analyzed, the result is as follows: transformation efficiency c is 0.366, the excessive value ee of substrate enantiomorph
s=0.546, the stereoselectivity E=62.25 of enzyme (optimal selection of enzyme is the S type), E is calculated by following formula:
Wherein, c is a transformation efficiency, and R is the amount of reaction t unreacted R-Ibuprofen BP/EP after the time, and S is the amount of reaction t unreacted S-Ibuprofen BP/EP after the time.
Add 10 milliliters of pH and be 14 aqueous sodium hydroxide solution and handle above-mentioned reaction mixture, by stirring at room, static layering, acquisition contains the solution of organic solvent and S-Ibuprofen BP/EP ethyl ester, utilize conventional rotary evaporation to handle then, obtain S-Ibuprofen BP/EP ethyl ester.
For the further hydrolysis of the S-Ibuprofen BP/EP ethyl ester that obtains is generated the S-Ibuprofen BP/EP, adopt according to the preceding method preparation to be used for esterolytic microbe-derived lipase immobilization form in the hydrolysis of the 0.1mol/L of PH=8 phosphoric acid buffer.
Embodiment 2
In the tool plug triangular flask of 100ml, add concentration successively and be ethanol, the 0.2g immobilized enzyme of hexane solution 10ml, 0.6mmol of the racemize Ibuprofen BP/EP of 0.03mol/L.React, detect, separate and hydrolysis under the condition identical with embodiment 1, the result is as follows: transformation efficiency c is 0.396, the excessive value ees=0.606 of substrate enantiomorph, the stereoselectivity E=47.22 of enzyme (optimal selection of enzyme is the S type).
Embodiment 3
Except that esterification carrying out under 30 ℃ rather than 40 ℃, repeat embodiment 1, the result is as follows: transformation efficiency c is 0.342, the excessive value ees=0.492 of substrate enantiomorph, the stereoselectivity E=59.55 of enzyme (optimal selection of enzyme is the S type).
Embodiment 4
Except that esterification is carried out in carbon tetrachloride solution rather than in hexane solution, repeat embodiment 1, the result is as follows: transformation efficiency c is 0.374, the excessive value ees=0.566 of substrate enantiomorph, the stereoselectivity E=65.55 of enzyme (optimal selection of enzyme is the S type).
Embodiment 5
In the tool plug triangular flask of 100ml, adding concentration successively is racemize Ibuprofen BP/EP hexane solution 10ml, the n-Heptyl alcohol of 0.3mmol, the thick enzyme powder of 50mg of 0.03mol/L.Thick enzyme powder is with reference to Mingrui Yu, Shaowei Qin, Tianwei Tan, Purification and characterizationof the extracellular lipase.Lip2 from Yarrowia lipolytica, the method preparation of putting down in writing among ProcessBiochemistry 42 (2007) 384-391, hereinafter identical.Reaction mixture is placed 40 ℃ of shaking tables, and the vibration insulation reaction is 24 hours under 180 rev/mins rotating speed.Detect under the condition identical with embodiment 1, separate, the result is as follows: transformation efficiency c is 0.325, the excessive value ees=0.460 of substrate enantiomorph, and the stereoselectivity E=69.13 of enzyme (optimal selection of enzyme is the S type) obtains the positive heptyl ester of S-Ibuprofen BP/EP.Adopt the method identical through this ester acquisition of the further hydrolysis of enzyme process S-Ibuprofen BP/EP with embodiment 1.
Embodiment 6
In the tool plug triangular flask of 100ml, adding concentration successively is nonylcarbinol and the thick enzyme powder of 50mg of racemize Ibuprofen BP/EP hexane solution 10ml, the 0.3mmol of 0.03mol/L.Place 40 ℃ of shaking tables, the oscillatory reaction insulation is 16 hours under the rotating speed of 180 rev/mins of rotating speeds.Detect under the condition identical with embodiment 1, separate, the result is as follows: transformation efficiency c is 0.401, the excessive value ees=0.629 of substrate enantiomorph, and the stereoselectivity E=61.31 of enzyme (optimal selection of enzyme is the S type) obtains S-Ibuprofen BP/EP ester in the positive last of the ten Heavenly stems.Adopt method further hydrolysis this ester acquisition S-Ibuprofen BP/EP identical with embodiment 1.
Embodiment 7
Except adding 20mg 18～hat～6 as the additive, repeat embodiment 5, the result is as follows: transformation efficiency c is 0.504, the excessive value ees=0.942 of substrate enantiomorph, the stereoselectivity E=94.87 of enzyme (optimal selection of enzyme is the S type) obtains S-Ibuprofen BP/EP ester in the positive last of the ten Heavenly stems.Adopt method further hydrolysis this ester acquisition S-Ibuprofen BP/EP identical with embodiment 1.
Embodiment 8
In the tool plug triangular flask of 100ml, adding concentration successively is racemize Ibuprofen BP/EP hexane solution 10ml, the vinylcarbinol of 0.3mmol, the 0.2g immobilized enzyme of 0.03mol/L.Place 40 ℃ of shaking tables, the vibration insulation reaction is 24 hours under the rotating speed of 180 rev/mins of rotating speeds.Detect under the condition identical with embodiment 1, separate, the result is as follows: transformation efficiency c is 0.408, the excessive value ees=0.632 of substrate enantiomorph, and the stereoselectivity E=44.80 of enzyme (optimal selection of enzyme is the S type) obtains S-Ibuprofen BP/EP propylene ester.Adopt method further hydrolysis this ester acquisition S-Ibuprofen BP/EP identical with embodiment 1.
Embodiment 9
In the tool plug triangular flask of 100ml, adding concentration successively is the racemize Ibuprofen BP/EP hexane solution 10ml of 0.03mol/L, stearyl alcohol and the 0.2g immobilized enzyme of 0.6mmol.Place 40 ℃ of shaking tables, the vibration insulation reaction is 24 hours under the rotating speed of 180 rev/mins of rotating speeds.Detect under the condition identical with embodiment 1, separate, the result is as follows: transformation efficiency c is 0.375, the excessive value ees=0.594 of substrate enantiomorph, and the stereoselectivity E=364 of enzyme (optimal selection of enzyme is the S type) obtains S-Ibuprofen BP/EP octadecyl ester.Adopt method further hydrolysis this ester acquisition S-Ibuprofen BP/EP identical with embodiment 1.
Embodiment 10: the influence of the external racemic ibuprofen esterification of organic solvent
According to the method for embodiment 1, adopt the 0.3mmol Ibuprofen BP/EP, the 0.3mmol isopropylcarbinol, the immobilized lipase LIP2 of 0.2g, and 10ml all kinds of SOLVENTS, at 40 ℃, 180rpm stirs reaction down 24 hours.Reaction finishes the back and measures c, ee
sAnd E.
The influence of the external racemic ibuprofen esterification of table 1 organic solvent
| Organic solvent | Log P | c(％) | ee s(％) | E |
| Tetracol phenixin | 3.0 | 40 | 59 | 32.4 |
| Skellysolve A | 3.0 | 43 | 56 | 11.6 |
| Sherwood oil | / | 49 | 64 | 9.7 |
| Normal hexane | 3.5 | 48 | 74 | 21.2 |
| Normal heptane | 4.0 | 47 | 62 | 10.1 |
| Octane-iso | 4.5 | 56 | 96 | 46.1 |
Illustrate :/expression undetermined
Result from above-mentioned table 1 can find out that above-mentioned organic solvent all can be effectively used to racemize Ibuprofen BP/EP and pure esterification.
Embodiment 11: the enantio-selectivity of the external racemic ibuprofen esterification of the lipase of various different sourcess
The different choice that has compared the external racemic ibuprofen esterification of lipase of different sources in the present embodiment is described in concrete test condition such as the following table.Therefrom as can be seen, be derived from inferior sieve and separate fat zymic extracellular lipase the S-Ibuprofen BP/EP is had extraordinary selectivity, obviously be better than the lipase in other source.
The enantio-selectivity of the external racemic ibuprofen esterification of lipase in the various different microorganisms of table 2 source
| Lipase | C(％) | ee s(％) | E | The enantiomorph preference |
| Inferior sieve is separated the fat yeast a Rhizomucor miehei b Candida antarctica b Candida rugosa b Aspergillus niger b Thermomyces lanuginosa b No enzyme c | 43 20 29 42 8 12 0 | 64 10 18 35 3 3 / | 26.3 2.6 3.1 4.1 2.1 1.7 / | S R R S R R / |
aReaction conditions: (RS)-Ibuprofen BP/EP (0.3mmol), 1-propyl alcohol (0.3mmol), lipase (Candidaantarctica, 100mg; Other lipase, 200mg), solvent is normal hexane (10ml), 40 ℃, 180rpm, 24 hours (/: undetermined);
bReaction conditions: (RS)～Ibuprofen BP/EP (4mM), 1-propyl alcohol (12mM), lipase (5.0%w/v), solvent are octane-iso (10ml), 35 ℃, 300rpm, 48 hours.[result is from Fabiano Jares Contesini and Patrl ' ciade Oliveira Carvalho
*Esterification of (RS)～Ibuprofen by native and commerciallipases in a two-phase system containing ionic liquids.Tetrahedron:Asymmetry 17 (2006) 2069-2073];
cReaction conditions: fat-free enzyme, react after 108 hours and measure, more than other condition is same
aDescribed.
Embodiment 12: the suitable reactions time of adopting different substrate alcohol
Measure different alcohol according to the method among the embodiment 1 and be used for esterification racemize Ibuprofen BP/EP to split the optimum reacting time of S-Ibuprofen BP/EP.The result shows, adopts different alcohol, and its speed of reaction is different, wherein the speed of reaction of isopropylcarbinol is very fast, and esterification 24 hours is more suitable, and the stearyl alcohol speed of reaction is slower, 48 hours more suitable, and along with the prolongation in reaction times, the R-type begins to transform, by 72 hours, the R-type has transformed half, by 108 hours, reaches equilibrium state, no longer transform, transformation efficiency reaches 94%.
Embodiment 13: the transformation efficiency c of racemize Ibuprofen BP/EP and Ya Luo separate the stereoselectivity E of fat yeast fat enzyme and the relation of temperature of reaction
Be the influence that the transformation efficiency c and the Ya Luo of the external racemic ibuprofen of test reaction temperature separates the stereoselectivity E of fat yeast fat enzyme, carried out following serial experiment according to the method for embodiment 1:
Under the differential responses temperature, adopt LIP2 200mg, racemize Ibuprofen BP/EP 0.3mmol, isopropylcarbinol 0.3mmol, and solvent is normal hexane 10ml, reaction is 24 hours under 180rpm.The sampling and testing Ibuprofen BP/EP transforms situation, and calculates the transformation efficiency c of racemize Ibuprofen BP/EP and the stereoselectivity E that Ya Luo separates fat yeast fat enzyme according to the method among the embodiment 1.Calculation result is mapped to temperature of reaction, and the result as shown in Figure 1.
Embodiment 14: the transformation efficiency c of racemize Ibuprofen BP/EP and Ya Luo separate the stereoselectivity E and the reaction raw materials Ibuprofen BP/EP of fat yeast fat enzyme: the relation of pure molar ratio
Be test reaction raw material Ibuprofen BP/EP: the transformation efficiency c of the external racemic ibuprofen of pure mol ratio (i.e. acid/pure mol ratio) and the influence that Ya Luo separates the stereoselectivity E of fat yeast fat enzyme, carried out following serial experiment:
Set up following reaction system: the 0.03mol/L substrate, LIP2 200mg, the 10ml normal hexane reacted 24 hours with 180rpm at 40 ℃.The sampling and testing Ibuprofen BP/EP transforms situation, and calculates the transformation efficiency c of racemize Ibuprofen BP/EP and the stereoselectivity E that Ya Luo separates fat yeast fat enzyme according to the method among the embodiment 1.To acid/pure mol ratio (or alcohol/sour mol ratio) mapping, the result as shown in Figure 2 with calculation result.
Though with several embodiment the present invention is described, can make variations and modifications in the spirit and scope of the invention that limit by claims not departing from, this will be apparent to those skilled in the art.
Claims (12)
1. a method for preparing the S-ibuprofen ester comprises the steps:
(1) be used to from or derived from inferior sieve separate fat yeast (Yarrowia lipolytica) the lipase-catalyzed racemize Ibuprofen BP/EP in extracellular and alcohol esterification and
(2) post reaction mixture is carried out separating treatment, obtain the S-ibuprofen ester.
2. the preparation method of S-ibuprofen ester according to claim 1, it is characterized in that, described esterification is carried out in organic solution, described organic solvent is preferably selected from Log p and is not less than 3 organic solvent for example alkane, halogenated alkane etc., more preferably is selected from sherwood oil, Skellysolve A, hexanaphthene, normal hexane, normal heptane, octane, octane-iso, positive nonane, n-decane, tetracol phenixin or its mixture.
3. the preparation method of S-ibuprofen ester according to claim 1 and 2 is characterized in that, the mol ratio of described racemize Ibuprofen BP/EP and described alcohol is 1: 5～5: 1, is preferably 1: 3～3: 1, more preferably 1: 2～2: 1, most preferably is 1: 2.
4. the preparation method of S-ibuprofen ester according to claim 1 is characterized in that, described esterification reaction temperature is 15～50 ℃, preferred 20～45 ℃, and more preferably 25～40 ℃, most preferably from about 40 ℃.
5. the preparation method of S-ibuprofen ester according to claim 1, it is characterized in that, described alcohol is C1～C20 alcohol, monobasic primary alconol for example, described alcohol is saturated, as methyl alcohol, ethanol, n-propyl alcohol, propyl carbinol, Pentyl alcohol, n-hexyl alcohol, n-Heptyl alcohol, n-Octanol, nonanol-, nonylcarbinol, lauryl alcohol, stearyl alcohol etc., or it is undersaturated, as 2～vinylcarbinol, perhaps its any mixture, preferred described alcohol is methyl alcohol, ethanol, n-propyl alcohol or its any mixture, and more preferably described alcohol is ethanol.
6. the preparation method of S-ibuprofen ester according to claim 1 wherein also adds additive in described esterification, preferred described additive is selected from 18-hat-6, hexichol 18 hats-6, cyclodextrin, glycerine, methane amide or its any mixture.
7. the preparation method of S-ibuprofen ester according to claim 1, it is characterized in that, described separating treatment is carried out under the highly basic reaction conditions, preferably include and utilize the strong base solution reaction mixture, the following of the pH value of described strong base solution is limited to 12,12.5,13,13.5,13.6,13.7,13.8 or 13.9, on be limited to the pH value of saturated solution of this strong base solution or lower by 0.05 than this saturated solution pH value, 0.1,0.15,0.2,0.25,0.3,0.35,0.4,0.45,0.5,0.55,0.6,0.65,0.7,0.75,0.8,0.85,0.9,0.95,1.0,1.05,1.1,1.15,1.2,1.25 or 1.3 etc., more preferably its pH value is 13～14, especially 14, described strong base solution can be selected from sodium hydroxide, the solution of potassium hydroxide etc. or its any mixture.
8. the preparation method of S-ibuprofen ester according to claim 1, it is characterized in that it is immobilized enzyme that described inferior sieve is separated fat yeast cell external fat enzyme, preferably its consumption is 5mg/ml～500mg/ml, more preferably 10～50mg/ml, especially preferred 20～30mg/ml, perhaps it is thick enzyme powder, consumption is 0.001mg/ml～100mg/ml, preferred 0.01mg/ml～50mg/ml, more preferably 0.05mg/ml～10mg/ml, especially preferred 0.1～5mg/ml, most preferably 0.2～1mg/ml.
9. the preparation method of S-ibuprofen ester according to claim 8, it is characterized in that, described inferior sieve is separated fat yeast cell external fat enzyme and is fixed on silica gel, diatomite or membranaceous textiles, comprises natural fabric such as cotton and man made fiber fabric such as terylene, on the especially membranaceous textiles.
10. the preparation method of S-ibuprofen ester according to claim 1, wherein said esterification lasts till that the ratio that S-Ibuprofen BP/EP and R～Ibuprofen BP/EP are converted into ester is more than 3: 1, more preferably 4: 1, further preferably 5: 1, especially preferred 9: 1, most preferably 98: 2, more specifically, reaction time of esterification is generally 12～120 hours, preferred 15～80 hours, more preferably 20～60 hours, most preferably 24～48 hours.
11. a method for preparing the S-Ibuprofen BP/EP comprises the step according to the further hydrolysis acquisition of the S-ibuprofen ester that each obtained in aforementioned claim S-Ibuprofen BP/EP.
12. method according to claim 11, wherein said hydrolysis adopts conventional chemical method or enzyme process to carry out, preferred lipase or the esterase of adopting carries out, more preferably described lipase from or separate fat yeast, Candida rugosa and Candida cylindracea etc. derived from inferior sieve.
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| CN102174633A (en) * | 2011-01-20 | 2011-09-07 | 蚌埠丰原医药科技发展有限公司 | Method for splitting ibuprofen |
| CN103194511A (en) * | 2013-03-06 | 2013-07-10 | 清华大学 | Method of lipase-catalyzed synthesis of fatty acid ester of clindamycin |
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| CN100424170C (en) * | 2005-10-11 | 2008-10-08 | 北京化工大学 | Lipase, its gene, yalulipolytic geast for producing said enzyme and its application |
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| CN102174633A (en) * | 2011-01-20 | 2011-09-07 | 蚌埠丰原医药科技发展有限公司 | Method for splitting ibuprofen |
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| CN109295153A (en) * | 2018-09-14 | 2019-02-01 | 湖南理工学院 | A method of loxoprofen enantiomer is split using biological enzyme stereoselectivity |
| CN109295153B (en) * | 2018-09-14 | 2021-11-05 | 湖南理工学院 | Method for stereoselectively splitting loxoprofen enantiomer by adopting bio-enzyme catalysis |
| CN109295154A (en) * | 2018-09-21 | 2019-02-01 | 湖南理工学院 | A kind of method that stereoselectivity lipase-catalyzed esterification splits brufen enantiomer |
| PL441467A1 (en) * | 2022-06-13 | 2023-12-18 | Politechnika Śląska | Method of obtaining (S)-(+)-ibuprofen |
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