CN101104861B - Method for preparing S-ibuprofen and S-ibuprofen ester by biological catalysis - Google Patents

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

The method that living things catalysis prepares S- brufens and S- ibuprofen esters

Technical field

The invention belongs to biological technical field, it is related to the method for preparing S- brufens and S- ibuprofen esters, more specifically, relates to the use of lipase-catalyzed resolution of racemic brufen, the method that the S- brufens and S- ibuprofen esters of high-optical-purity is made.

Background technology

NSAIDs (non~steroidal anti~inflammatory drugs, NSAIDs it is) that a class has antipyretic, analgesia and most also anti-inflammatory, the medicines of anti rheumatism action, clinically it is widely used in treatment rheumatoid arthritis, osteoarthritis and other rheumatic diseases, is that hundreds of millions of rheumatisants release pain.This kind of medicine mainly includes racemic ibuprofen (racemic -2- (4- isobutyl phenenyls) propionic acid), Flurbiprofen (racemic -2- ((the fluoro- 4- phenyl of 3-) phenyl) propionic acid, fenoprofen (racemic -2- (3- Phenoxyphenyls) propionic acid), suprofen (racemic -2- (4 (2- thienyls oxo) phenyl) propionic acid), Carprofen (racemic -2- (the chloro- 9H- carbazyls of 6-) propionic acid), naproxen (racemic -2- (6- methoxyl group -2- naphthyls) propionic acid), Ketoprofen (racemic-a- methyl -3- benzoyls-phenylacetic acid) etc..The pharmacological activity of these medicines is that they can be by preventing Cycloxygenase from arachidonic C-13 abstract hydrogen atoms, and then the peroxidating of arachidonic C-11 and C-15 positions is prevented, it is finally hindered to the prostaglandin of inflammation and thromboxane A2 bioconversion.

Racemic ibuprofen is the minimum one kind of 11 kinds of Nonsteroidal anti-inflammatory medicine toxic side effects such as naproxen, Ketoprofen, and most western developed countries are all as non-prescribed medicine.Racemic ibuprofen why have higher activity be due to it connect Cycloxygenase affinity it is similar with natural substrate arachidonic acid.Research shows, the two kinds of isomers S- brufens and R~brufen of racemic ibuprofen are different in terms of pharmacological kinetics and biotransformation, S- brufens have significantly higher clinical effectiveness (Adams et al., J.Pharm.Pharakol, 28,257 and Jamali et al., Pharmac.Res.1988,5,44).With racemic compound phase ratio, S- isomers can quickly in blood Reach treatment concentration.Racemic ibuprofen is received by many patients as anti-inflammation analgesia medicine, but racemic ibuprofen is used as the active drug of the treatment chronic disease such as rheumatism and rheumatoid arthritis, long term frequent heavy dose medication can increase the side reaction of intestines and stomach, even cause gastrointestinal bleeding, there is certain infringement to kidney, be greatly limited its clinical practice.To solve the problems, such as dosage, toxicity and the pharmacokinetics of the outer brufen administration presence that disappears, prepare single chiral S- brufens and seem increasingly important.

Current S- brufens preparation method can be divided into stereoselective chemical synthesis and the major class of racemate resolution two, racemate resolution is main to split two kinds using chemical resolution agent fractionation and biotechnology, wherein stereoselective chemical synthesis is related to the chiral auxiliary or catalyst of costliness, or it is related to the preparation technology and various chemicals of complexity, chemical resolution agent is also costly, therefore cost is high in industrial production.Biotechnology, which is split, mainly uses biology enzyme to be split, and biotechnology is split because its technique is simple, production cost is low and is favored, with good prospects for commercial application.There is the lipase from Rhizomucor miehei, Candida rugosa, Candida antarctica (Novozyme 435), Aspergillus niger AC-54 and Thermomyces lanuginosa etc. currently used for the enzyme of racemic ibuprofen Chiral Separation, but the catalytic conversion of these enzymes and stereoselectivity are relatively low, unsatisfactory (referring to：Fabiano Jares Contesini and Patri′ciade Oliveira Carvalho^*Esterification of(RS)-Ibuprofen by native andcommercial lipases in a two-phase system containing ionic liquidsTetrahedron：Asymmetry 17(2006)2069-2073).

The content of the invention

The present invention provides a kind of new method for being split by enzymatic and preparing S- ibuprofen esters on the one hand.In particular, the invention provides a kind of method for preparing a variety of S- ibuprofen esters using the extracellular lipase-catalyzed resolution of racemic brufen from sub- sieve solution fat yeast (Yarrowialipolytica), using the esterification of extracellular lipase-catalyzed promotion racemic ibuprofen and alcohol from sub- sieve solution fat yeast, then separation obtains S- ibuprofen esters from reactant mixture.

Invention further provides the method for preparing S- brufens, the step of being hydrolyzed including the S- ibuprofen esters to being prepared according to the present invention.Ester hydrolysis reacts technology known to those skilled in the art into acid.

Brief description of the drawings

Fig. 1 shows at a temperature of differential responses that the conversion ratio c and Ya Luo of racemic ibuprofen solve the stereoselectivity E of fat Yeast-lipase change curve.

Fig. 2 shows the acid (i.e. racemic ibuprofen) in various molar ratios：Alcohol or alcohol：Under acid, the stereoselectivity E of conversion ratio c and Ya Luo the solution fat Yeast-lipase of racemic ibuprofen change curve.

Embodiment

In the present invention, S- ibuprofen esters or S- brufens as raw material is split can be prepared using any racemic ibuprofen.Racemic ibuprofen is to be successfully prepared by Boots companies of Britain early in 1960s.It can be prepared, or can be commercially bought by technology as known in the art as the racemic ibuprofen of raw material of the present invention.

In the present invention, racemic ibuprofen can be split using the extracellular lipase from sub- sieve solution fat yeast, so as to prepare S- ibuprofen esters or S- brufens.The enzyme can be commercially available, and also can laboratory self-control.A kind of such preparation method is included in suitable culture medium cultivates sub- sieve solution fat yeast reasonable time at a proper temperature, and the lipase is then isolated and purified from culture medium.The fermentation of lipase refers 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)：Method disclosed in 459-65, it is extracted and further purifying refers to Mingrui Yu, Shaowei Qin, (2007) 384-391 of Tianwei Tan, Purification andcharacterization of the extracellular lipase.Lip2 from Yarrowia lipolytica.Process Biochemistry 42.

Sub- sieve solution fat yeast belongs to unconventional yeast, and FDA certifications are safe level yeast (GRAS), and the bacterial strain is used to produce to have accumulated substantial amounts of experience in citric acid, single cell protein, industrial production by extensive.Available lipase can come from or derived from any sub- sieve solution fat yeast in the present invention, including sub- sieve solution fat yeast for example disclosed in CN200510112638.5, it is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (address with deposit number CGMCCNo.1470：North One No. 13 Institute of Microorganism, Academia Sinica in Jing Shi Haidian District Zhong Guan-cun north, postcode：100080).A kind of method for mass producing lipase from sub- sieve solution fat yeast can be found in Destain et al. (1997).Sub- sieve solution fat yeast extracellular lipase available for the present invention can also be prepared by gene engineering method.Such method has teaching in Fignede et al. (2000) and CN200510112638.5.Those of ordinary skill is according to teaching of the prior art, also the method for the sub- sieve solution fat Yeast-lipase of genetically engineered production is known completely, including for example the nucleotide sequence for encoding the lipase is inserted into suitable expression vector, and be introduced into appropriate host cell and expressed, then from culture medium or intracellular separation purpose lipase.Or, the lipase for the present invention can be also made by chemical synthesis approach.Such method has sufficient teaching in the prior art.

In the present invention, the thick enzyme form that the lipase can dissociate is used, i.e., the enzyme can be through simple process or untreated and use, such as thick enzyme powder, it might even be possible to directly with zymotic fluid (because the enzyme that the present invention is used is ectoenzyme).

The enzyme of the present invention can be used in the form of immobilised enzymes and modification enzyme etc., including with enzyme made from the different fixing means of carrier of the same race and the identical or different fixing means of different carriers.It is well known in the prior art to the method that enzyme is fixed.In one embodiment, described immobilized lipase includes above-mentioned lipase, carrier and common fixative.Fixation support is first activated with definite composition and the common fixative of proportioning.Fixation support and common fixative are mixed in 1: 1~1: 3 (W: V) ratios, dried.Lipase deionized water dissolving, is mixed with the fixation support after activation in the ratio of 1000~30000 units/gram carriers, dried stand-by.

In fatty enzyme immobilizatio of the invention, the carrier can be solid particle, such as silica gel, diatomite.Or, the carrier can be membranaceous textile, for example natural fabric such as cotton or science fabric such as terylene, especially membranaceous textile, and it has the characteristics of surface area is big, adsorptivity is strong, cheap, stability is good and can reuse.

In another preferred embodiment, the fixative altogether be selected from (including) high-molecular compound, surfactant, protein, at least one of inorganic salts.

In a further preferred embodiment, the fixative altogether is selected from least one of PEG6000, coconut oil, Tween 80, gelatin, lecithin and magnesium sulfate, in preferably described fixative altogether The mass ratio of each component is gelatin: lecithin: PEG6000: Tween 80: magnesium sulfate: coconut oil=5: 1: 1: 2: 1: 1.

The preparation of immobilised enzymes of the present invention can be found in the co-pending Chinese invention patent application No.200510112638.5 of the applicant.In addition, also teach that the preparation of the zymotic fluid of thick enzyme, immobilised enzymes, modification enzyme and enzyme used in the present invention etc. in the following documents：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, (2007) 384-391 of Tianwei Tan, Purification and characterizationof the extracellular lipase Lip2 from Yarrowia lipolytica, ProcessBiochemistry 42；Kaili Nie, Feng Xie, Fang Wang, TianweiTan, Lipase catalyzed methanolysis to produce biodiesel：Optimization ofthe biodiesel production.Journal of Molecular Catalysis B：Enzymatic 43(2006)142-147；And Chinese invention patent application number 02117614.0, its Publication No. CN1456674；Mingrui Yu, Stefan Lange, Sven Richter, Tianwei Tan, Rolf D.Schmid High~level expression of extracellular lipase Lip2from Yarrowialipolytica in Pichia pastoris and its purification and characterization.Protein Expression and Purification 53 (2007) 255~263.The content of above-mentioned file is incorporated herein by reference of text.

Further, it will be understood by those skilled in the art that the fermentation of enzyme, transformation and immobilization are not limited to the method that above-mentioned document is provided, general knowledge of the those of ordinary skill in this area can extremely be readily determined suitable enzyme preparation method.

In one embodiment, the enzyme for the present invention is the extracellular lipase from Asia sieve solution fat yeast (Yarrowialipolytica) CGMCC No.1470, and its primary amino acid sequences is as follows：

VYTSTETSHIDQESYNFFEKYARLANIGYCVGPGTKIFKPFNCGLQCA

HFPNVELIEEFHDPRLIFDVSGYLAVDHASKQIYLVIRGTHSLEDVITD

IRIMQAPLTNFDLAANISSTATCDDCLVHNGFIQSYNNTYNQIGPKLDS

VIEQYPDYQIAVTGHSLGGAAALLFGINLKVNGHDPLVVTLGQPIVG

NAGFANWVDKLFFGQENPDVSKVSKDRKLYRITHRGDIVPQVPFWD

GYQHCSGEVFIDWPLIHPPLSNVVMCQGQSNKQCSAGNTLLQQVNV

IGNHLQYFVTEGVCGI(SEQ ID NO；1)

Sub- sieve solution fat yeast is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (address on 30th in September in 2005：No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City Institute of Microorganism, Academia Sinica, postcode：100080), deposit number is CGMCC No.1470.

In the present invention, in addition to above-mentioned lipase, their amino acid sequence variation, analog, derivative etc. can also be used.Those of ordinary skill has full knowledge that, after modifying the amino acid sequence in protein, and the protein obtained is likely to still remain original biological activity, it is thus possible among the present invention.The preparation of this protein and activity determination method are known in the prior art.

In the present invention, the extracellular lipase from sub- sieve solution fat yeast plays stereoselectivity catalytic action to racemic mixture, and those skilled in the art can be readily determined its effective dose in the methods of the invention according to general knowledge of the prior art.In general, with the total volume meter of reaction system, thick enzyme powder consumption is 0.001mg/ml~100mg/ml, more preferably preferably 0.01mg/ml~50mg/ml, 0.05mg/ml~10mg/ml, most preferably particularly preferred 0.1~5mg/ml, 0.2~1mg/ml；Its consumption of immobilised enzymes is 5mg/ml~500mg/ml, more preferably 10~50mg/ml, particularly preferred 20~30mg/ml.

According to the present invention, alcohol for preparing ibuprofen ester can be the alcohol of C1~20, such as unitary primary alconol, the alcohol can be saturation, such as methanol, ethanol, normal propyl alcohol, n-butanol, n-amyl alcohol, n-hexyl alcohol, n-heptanol, n-octyl alcohol, n-nonyl alcohol, Decanol, lauryl alcohol, octadecyl alcolol, it can also be undersaturated, such as 2~propenyl, or its any mixture.Preferably, the alcohol is methanol, ethanol, normal propyl alcohol or its any mixture.It is highly preferred that the alcohol is ethanol.

In the method for splitting of the present invention, the ratio between racemic ibuprofen and consumption of alcohol used can be determined arbitrarily.In general, the consumption mol ratio of racemic ibuprofen and alcohol is between 1: 5~5: 1, preferably 1: 3~3: 1, more preferably 1: 2~2: 1, most preferably 1: 2.Certainly, those of ordinary skill is fully able to understand, it would however also be possible to employ the amount ratio outside this scope, simply should suitably adjust the other parameters in esterification, including such as reaction temperature, time and solvent.

According to the present invention, the esterification of the racemic ibuprofen and alcohol is carried out in organic solution.In one embodiment, the organic solvent can be selected from solvents of the Log p not less than 3 such as alkane, halogenated alkane, the example has petroleum ether, pentane, hexamethylene, n-hexane, normal heptane, normal octane, isooctane, n -nonane, n-decane, carbon tetrachloride etc., but is not limited thereto.Wherein p is the hydrophobic constant of organic solvent.It is of course also possible to 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 determine such temperature range according to teaching of the prior art.Usual esterification is carried out at 15~50 DEG C, is preferably carried out at a temperature of 20~45 DEG C, particularly preferred 25~40 DEG C, most preferably from about 40 DEG C of temperature.

The duration of esterification, those skilled in the art can determine the suitable reaction time by normal experiment depending on many factors, such as by being derived from reaction solution Small Amount sample with being equipped with chiral column liquid chromatographic come monitoring reaction course.The chiral column can be such as CHIRALCELOB~H, 0.46cm

× 150, DAICEL CHEMICAL INDUSTRIES, LID, Jap, the mobile phase used can be such as n-hexanes: isopropanol: trifluoroacetic acid=98: 2: 0.1.Preferably, reaction continues to that the ratio that S- brufens and R~brufen are converted into ester is more than 3: 1, more preferably 4: 1, further preferably 5: 1, particularly preferred 9: 1, most preferably 98: 2.Specifically, the reaction time is usually 12~120 hours, preferably 15~80 hours, more preferably 20~60 hours, most preferably 24~48 hours.Reaction reached after required degree, can terminating reaction in a suitable manner, include the enzymatic activity of for example hot sub- sieve solution fat yeast extracellular lipase of inactivation, or the directly separation of progress ibuprofen ester and reaction substrate.

Appropriate suitable additives, such as such as 18- crown-s 6, crown ether, cyclodextrin, glycerine, the formamide of the crown- 6 of hexichol 18 can be added in the esterification reaction.It is not intended to be limited to theory, it is believed that suitable additives make enzyme be easier to and substrate-function, so as to improve the activity of enzyme, improves the chiral selectivity of enzyme.

After esterification terminates, can by the conventional isolation techniques of this area such as extracting, chromatograph, evaporate separate the ibuprofen ester and unreacted brufen of generation.For example, it is possible to use strong base solution carrys out separating treatment reactant mixture.Preferably, the lower limit of the pH value of the strong base solution is 12,12.5,13,13.5,13.6,13.7,13.8 or 13.9, and the upper limit is the saturated solution of the strong base solution PH value or it is lower by 0.05 than the 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..It is highly preferred that being separated using pH for 13~14 strong base solution, the strong base solution that pH is 14 is most preferably utilized.In one embodiment, the strong base solution can be sodium hydrate aqueous solution or potassium hydroxide aqueous solution, or its mixture.Certainly including others strong base solution is also covered by.After strong base solution processing, to organic phase vacuum distillation under certain vacuum degree, ibuprofen ester is obtained, wherein mainly S- ibuprofen esters, also including a small amount of R- ibuprofen esters.Due to strong selectivity of the sub- sieve solution fat yeast to S- brufens, content of the R- ibuprofen esters in final product is very low.

After S- ibuprofen esters are obtained, ester can be hydrolyzed using conventional method, so that S- brufens are made, reach the purpose of resolution of racemic brufen.How those of ordinary skill is fully aware of selects suitable ester hydrolysis condition.Wherein described hydrolysis can be carried out by conventional chemical method, can also be carried out by enzyme process.It is preferred that enzyme process, wherein utilizing such as lipase or esterase, such as the enzyme derived from sub- sieve solution fat yeast, Candida rugosa or Candida cylindracea etc..They can be prepared by fermentation or gene engineering method, or be bought from the market.In one embodiment, using enzyme process in suitable ph, such as pH=7~10, preferably hydrolyze the ibuprofen ester in pH=8 buffer solution such as phosphate buffer.The concentration of the phosphate buffer can be such as 0.05mol/L~0.5mol/L, more preferably 0.1mol/L~0.2mol/L.

The method catalytic conversion of the present invention is high, stereoselectivity is strong, and obtained S- brufens and S- ibuprofen esters purity is high, and the species of S- ibuprofen esters is more, and important intermediate is provided for medicine；Its preparation process is simple, feasible, and cost is low, easy industrialization.

The present invention will be further illustrated by the detailed description of the specific embodiment of the present invention below, but these embodiments are only illustrative, are not construed as limiting the invention.

Embodiment 1

In 100ml conical flask with stopper, hexane solution 10ml, 0.3mmol of the racemic ibuprofen (being purchased from Sigma companies, hereafter together) that concentration is 0.03mol/L ethanol, 0.2g are sequentially added Immobilised enzymes.Immobilised enzymes is the co-pending Chinese invention patent application No.200510112638.5 and Tan TW with reference to the applicant, 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)：Prepared by the method in 459-65, it is hereinafter identical.The shaking table that reactant mixture is placed at 40 DEG C, vibrates insulation reaction 24 hours under 180 revs/min of rotating speed, obtain containing racemic ibuprofen, S- ibuprofen ethyl esters, R- ibuprofen ethyl esters mixture.20 microlitres of said mixture is taken to be analyzed, analysis method is as follows：

Pass through high performance liquid chromatography detection (chiral column：CHIRALCEL OB~H, 15cm × 4.6mm, DAICEL CHEMICAL INDUSTRIES, LID, Jap；Mobile phase：N-hexane: isopropanol: trifluoroacetic acid=98: 2: 0.1) above-mentioned sample is analyzed, it is as a result as follows：Conversion ratio c is 0.366, the excessive value ee of substrate enantiomer_s=0.546, the stereoselectivity E=62.25 (optimal selectivity towards of enzyme are S types) of enzyme, E is calculated by formula below：

$E=\frac{\ln \left[(1-c)(1-{\mathrm{ee}}_s)\right]}{\ln \left[(1-c)(1+{\mathrm{ee}}_s)\right]}$

${\mathrm{ee}}_s=\frac{R-S}{R+S},(R>S)$

$c=1-\frac{{(R+S)}_t}{{(R+S)}_{t=0}}$

Wherein, c is conversion ratio, and R is the amount of unreacted R- brufens after the reaction t times, and S is the amount of unreacted S- brufens after the reaction t times.

Add 10 milliliters of pH to be 14 sodium hydrate aqueous solution to handle above-mentioned reactant mixture, by being stirred at room temperature, static layering obtains the solution containing organic solvent and S- ibuprofen ethyl esters, is then handled using conventional rotary evaporation, obtains S- ibuprofen ethyl esters.

In order to which the S- ibuprofen ethyl esters of acquisition are further hydrolyzed into generation S- brufens, hydrolyzed using being prepared according to preceding method for the microbe-derived fatty enzyme immobilizatio form of ester hydrolysis in PH=8 0.1mol/L phosphate buffers.

Embodiment 2

In 100ml conical flask with stopper, hexane solution 10ml, 0.6mmol of the racemic ibuprofen that concentration is 0.03mol/L ethanol, 0.2g immobilised enzymes are sequentially added.React, detect, separate and hydrolyze under the same conditions as example 1, it is as a result as follows：Conversion ratio c is 0.396, the excessive value ees=0.606 of substrate enantiomer, the stereoselectivity E=47.22 of enzyme (optimal selectivity towards of enzyme are S types).

Embodiment 3

In addition to esterification is carried out at 30 DEG C rather than 40 DEG C, embodiment 1 is repeated, it is as a result as follows：Conversion ratio c is 0.342, the excessive value ees=0.492 of substrate enantiomer, the stereoselectivity E=59.55 of enzyme (optimal selectivity towards of enzyme are S types).

Embodiment 4

In addition to esterification is carried out in carbon tetrachloride solution rather than in hexane solution, embodiment 1 is repeated, it is as a result as follows：Conversion ratio c is 0.374, the excessive value ees=0.566 of substrate enantiomer, the stereoselectivity E=65.55 of enzyme (optimal selectivity towards of enzyme are S types).

Embodiment 5

In 100ml conical flask with stopper, racemic ibuprofen hexane solution 10ml, 0.3mmol that concentration is the 0.03mol/L thick enzyme powder of n-heptanol, 50mg are sequentially added.Thick enzyme powder is with reference to Mingrui Yu, Shaowei Qin, Tianwei Tan, Purification and characterizationof the extracellular lipase.Lip2from Yarrowia lipolytica, prepared by the method described in ProcessBiochemistry 42 (2007) 384-391, it is hereinafter identical.Reactant mixture is placed in 40 DEG C of shaking tables, insulation reaction is vibrated under 180 revs/min of rotating speed 24 hours.Detect, separate under the same conditions as example 1, it is as a result as follows：Conversion ratio c is 0.325, the excessive value ees=0.460 of substrate enantiomer, the stereoselectivity E=69.13 (optimal selectivity towards of enzyme are S types) of enzyme, obtains the positive heptyl ester of S- brufens.The ester is further hydrolyzed through enzyme process using method same as Example 1 and obtains S- brufens.

Embodiment 6

In 100ml conical flask with stopper, racemic ibuprofen hexane solution 10ml, 0.3mmol that concentration is 0.03mol/L Decanol and the thick enzyme powders of 50mg are sequentially added.40 DEG C of shaking tables are placed in, oscillating reactions is incubated 16 hours under the rotating speed of 180 revs/min of rotating speed.Detect, separate under the same conditions as example 1, it is as a result as follows：Conversion ratio c is 0.401, the excessive value ees=0.629 of substrate enantiomer, the stereoselectivity E=61.31 (optimal selectivity towards of enzyme are S types) of enzyme, obtains the positive last of the ten Heavenly stems ester of S- brufens.This ester is further hydrolyzed using method same as Example 1 and obtains S- brufens.

Embodiment 7

In addition to adding 20mg 18~hat~6 as additive, embodiment 5 is repeated, it is as a result as follows：Conversion ratio c is 0.504, the excessive value ees=0.942 of substrate enantiomer, the stereoselectivity E=94.87 (optimal selectivity towards of enzyme are S types) of enzyme, obtains the positive last of the ten Heavenly stems ester of S- brufens.The ester is further hydrolyzed using method same as Example 1 and obtains S- brufens.

Embodiment 8

In 100ml conical flask with stopper, racemic ibuprofen hexane solution 10ml, 0.3mmol that concentration is 0.03mol/L propenyl, 0.2g immobilised enzymes are sequentially added.40 DEG C of shaking tables are placed in, vibration insulation reaction 24 hours under the rotating speed of 180 revs/min of rotating speed.Detect, separate under the same conditions as example 1, it is as a result as follows：Conversion ratio c is 0.408, the excessive value ees=0.632 of substrate enantiomer, the stereoselectivity E=44.80 (optimal selectivity towards of enzyme are S types) of enzyme, obtains S- brufen propylenes.The ester is further hydrolyzed using method same as Example 1 and obtains S- brufens.

Embodiment 9

In 100ml conical flask with stopper, racemic ibuprofen hexane solution 10ml, 0.6mmol that concentration is 0.03mol/L octadecyl alcolol and 0.2g immobilised enzymes are sequentially added.40 DEG C of shaking tables are placed in, vibration insulation reaction 24 hours under the rotating speed of 180 revs/min of rotating speed.Detect, separate under the same conditions as example 1, it is as a result as follows：Conversion ratio c is 0.375, the excessive value ees of substrate enantiomer =0.594, the stereoselectivity E=364 (optimal selectivity towards of enzyme are S types) of enzyme, obtain S- brufen octadecyl esters.The ester is further hydrolyzed using method same as Example 1 and obtains S- brufens.

Embodiment 10：The influence that organic solvent is esterified to racemic ibuprofen

According to the method for embodiment 1, using 0.3mmol brufens, 0.3mmol isobutanols, the lipase LIP2 of 0.2g immobilizations, and the various solvents of 10ml, in 40 DEG C, the lower reaction of 180rpm stirrings 24 hours.Reaction determines c, ee after terminating_sAnd E.

The influence that the organic solvent of table 1 is esterified to racemic ibuprofen

Organic solvent	Log P	C (%)	ees(%)	E
					Carbon tetrachloride	3.0	40	59	32.4
Pentane	3.0	43	56	11.6
					Petroleum ether	/	49	64	9.7
N-hexane	3.5	48	74	21.2
					Normal heptane	4.0	47	62	10.1
Isooctane	4.5	56	96	46.1

Explanation：/ represent undetermined

It can be seen that above-mentioned organic solvent can be effectively used to the esterification of racemic ibuprofen and alcohol from the result in above-mentioned table 1.

Embodiment 11：The enantio-selectivity that the lipase of various separate sources is esterified to racemic ibuprofen

The different choice that the lipase of separate sources is esterified to racemic ibuprofen is compared in the present embodiment, specific test condition is as described in following table.There it can be seen that the extracellular lipase from sub- sieve solution fat yeast has extraordinary selectivity to S- brufens, hence it is evident that better than the lipase in other sources.

The enantio-selectivity that the lipase in the various different microorganisms of table 2 source is esterified to racemic ibuprofen

^aReaction condition：(RS)-brufen (0.3mmol), 1- propyl alcohol (0.3mmol), lipase (Candidaantarctica, 100mg；Other lipase, 200mg), solvent be n-hexane (10ml), 40 DEG C, 180rpm, 24 hours (/：Undetermined)；

^bReaction condition：(RS)~brufen (4mM), 1- propyl alcohol (12mM), lipase (5.0%w/v), solvent is isooctane (10ml), 35 DEG C, 300rpm, [result comes from Fabiano Jares Contesini and Patri ' ciade Oliveira Carvalho to 48 hours^*Esterification of (RS)~Ibuprofen by native and commerciallipases in a two-phase system containing ionic liquids.Tetrahedron：Asymmetry 17(2006)2069-2073]；

^cReaction condition：Fat-free enzyme, reaction is determined after 108 hours, and other conditions are with more than^aIt is described.

Embodiment 12：Using the suitable reactions time of different substrate alcohol

Determining different alcohol according to the method in embodiment 1 is used to be esterified racemic ibuprofen to split the optimum reacting time of S- brufens.As a result show, using different alcohol, its reaction rate is different, the reaction rate of wherein isobutanol is very fast, and esterification 24 hours is appropriate, and octadecyl alcolol reaction rate is slower, 48 hours appropriate, and with the extension in reaction time, R- types start conversion, by 72 hours, R- types have converted half, by 108 hours, up to poised state, no longer convert, conversion ratio is up to 94%.

Embodiment 13：The stereoselectivity E and the relation of reaction temperature of conversion ratio c and Ya Luo the solution fat Yeast-lipase of racemic ibuprofen

The vertical of fat Yeast-lipase is solved to the conversion ratio c and Ya Luo of racemic ibuprofen for test reaction temperature Body selectivity E influence, following serial experiment has been carried out according to the method for embodiment 1：

At a temperature of differential responses, using LIP2200mg, racemic ibuprofen 0.3mmol, isobutanol 0.3mmol, and solvent is n-hexane 10ml, is reacted 24 hours under 180rpm.Sampling and testing brufen converts situation, and the conversion ratio c and Ya Luo for calculating racemic ibuprofen according to the method in embodiment 1 solve the stereoselectivity E of fat Yeast-lipase.Result of calculation is mapped to reaction temperature, as a result as shown in Figure 1.

Embodiment 14：Stereoselectivity E and reaction raw materials brufen: the relation of alcohol molar ratio of conversion ratio c and Ya Luo the solution fat Yeast-lipase of racemic ibuprofen

For test reaction raw material brufen: alcohol mol ratio (i.e. acid/alcohol mol ratio) solves the stereoselectivity E of fat Yeast-lipase influence to the conversion ratio c and Ya Luo of racemic ibuprofen, has carried out following serial experiment：

Set up following reaction system：0.03mol/L substrates, LIP2200mg, 10ml n-hexanes are reacted 24 hours at 40 DEG C with 180rpm.Sampling and testing brufen converts situation, and the conversion ratio c and Ya Luo for calculating racemic ibuprofen according to the method in embodiment 1 solve the stereoselectivity E of fat Yeast-lipase.Result of calculation is mapped to acid/alcohol mol ratio (or alcohol/acid mol ratio), as a result as shown in Figure 2.

Although illustrating the present invention with several embodiments, can various changes and modifications may be made in without departing from the spirit and scope of the invention being defined by the appended claims, this will be apparent to those skilled in the art.

Claims (51)

1. a kind of method for preparing S- ibuprofen esters, comprises the following steps：

(1) esterification from the extracellular lipase-catalyzed racemic ibuprofen and alcohol for being deposited in sub- sieve solution fat yeast that Chinese microorganism strain preservation administrative center, deposit number are CGMCC No.1470 is utilized, the primary amino acid sequences of the extracellular lipase as shown in SEQ ID No.1, and

(2) separating treatment is carried out to reacted mixture, obtains S- ibuprofen esters.

2. the preparation method of S- ibuprofen esters according to claim 1, it is characterised in that the esterification is carried out in organic solution.

3. the preparation method of S- ibuprofen esters according to claim 2, it is characterised in that the organic solvent is selected from the organic solvent that Log p are not less than 3.

4. the preparation method of S- ibuprofen esters according to claim 2, it is characterised in that the organic solvent is selected from alkane or halogenated alkane.

5. the preparation method of S- ibuprofen esters according to claim 2, it is characterised in that the organic solvent is selected from petroleum ether, pentane, hexamethylene, n-hexane, normal heptane, normal octane, isooctane, n -nonane, n-decane, carbon tetrachloride or its mixture.

6. the preparation method of the S- ibuprofen esters according to claim any one of 1-5, it is characterised in that the mol ratio of the racemic ibuprofen and the alcohol is 1: 5~5: 1.

7. the preparation method of S- ibuprofen esters according to claim 6, it is characterised in that the mol ratio of the racemic ibuprofen and the alcohol is 1: 3~3: 1.

8. the preparation method of S- ibuprofen esters according to claim 6, it is characterised in that the mol ratio of the racemic ibuprofen and the alcohol is 1: 2~2: 1.

9. the preparation method of S- ibuprofen esters according to claim 1, it is characterised in that the esterification reaction temperature is 15~50 DEG C.

10. the preparation method of S- ibuprofen esters according to claim 9, it is characterised in that the esterification reaction temperature is 20~45 DEG C.

11. the preparation method of S- ibuprofen esters according to claim 9, it is characterised in that the esterification reaction temperature is 25~40 DEG C.

12. the preparation method of S- ibuprofen esters according to claim 9, it is characterised in that the esterification reaction temperature is 40 DEG C.

13. the preparation method of S- ibuprofen esters according to claim 1, it is characterised in that the alcohol is C1~C20 alcohol.

14. the preparation method of S- ibuprofen esters according to claim 13, it is characterised in that the alcohol is unitary primary alconol.

15. the preparation method of S- ibuprofen esters according to claim 13, it is characterised in that the alcohol is saturation.

16. the preparation method of S- ibuprofen esters according to claim 14, characterized in that, the alcohol is methanol, ethanol, normal propyl alcohol, n-butanol, n-amyl alcohol, n-hexyl alcohol, n-heptanol, n-octyl alcohol, n-nonyl alcohol, Decanol, lauryl alcohol, octadecyl alcolol or its any mixture.

17. the preparation method of S- ibuprofen esters according to claim 13, it is characterised in that the alcohol is undersaturated.

18. the preparation method of S- ibuprofen esters according to claim 17, it is characterised in that the alcohol is 2~propenyl.

19. the preparation method of S- ibuprofen esters according to claim 16, it is characterised in that the alcohol is methanol, ethanol, normal propyl alcohol or its any mixture.

20. the preparation method of S- ibuprofen esters according to claim 13, it is characterised in that the alcohol is ethanol.

21. the preparation method of S- ibuprofen esters according to claim 1, wherein being additionally added additive in the esterification.

22. the preparation method of S- ibuprofen esters according to claim 21, it is characterised in that the additive is selected from 18- crown-s 6, the crown- 6 of hexichol 18, cyclodextrin, glycerine, formamide or its any mixture.

23. the preparation method of S- ibuprofen esters according to claim 1, it is characterised in that the separating treatment is carried out under highly basic reaction condition.

24. the preparation method of S- ibuprofen esters according to claim 23, it is characterised in that utilize strong base solution reaction mixture.

25. the preparation method of S- ibuprofen esters according to claim 24, it is characterised in that the lower limit of the pH value of the strong base solution is 12,12.5,13,13.5,13.6,13.7,13.8 or 13.9.

26. the preparation method of S- ibuprofen esters according to claim 24, characterized in that, the upper limit of the pH value of the strong base solution for the strong base solution saturated solution pH value or it is lower by 0.05 than the 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.

27. the preparation method of S- ibuprofen esters according to claim 24, it is characterised in that the pH value of the strong base solution is 13~14.

28. the preparation method of S- ibuprofen esters according to claim 24, it is characterised in that the pH value of the strong base solution is 14.

29. the preparation method of S- ibuprofen esters according to claim 24, it is characterised in that the strong base solution is selected from sodium hydroxide, the solution of potassium hydroxide or its mixture.

30. the preparation method of S- ibuprofen esters according to claim 1, it is characterised in that sub- sieve solution fat yeast cells external fat enzyme is immobilised enzymes.

31. the preparation method of S- ibuprofen esters according to claim 30, it is characterised in that the immobilised enzymes consumption is 5mg/ml~500mg/ml.

32. the preparation method of S- ibuprofen esters according to claim 30, it is characterised in that the immobilised enzymes consumption is 10~50mg/ml.

33. the preparation method of S- ibuprofen esters according to claim 30, it is characterised in that the immobilised enzymes consumption is 20~30mg/ml.

34. the preparation method of S- ibuprofen esters according to claim 1, it is characterised in that sub- sieve solution fat yeast cells external fat enzyme is thick enzyme powder.

35. the preparation method of S- ibuprofen esters according to claim 34, it is characterised in that the thick enzyme powder consumption is 0.001mg/ml~100mg/ml.

36. the preparation method of S- ibuprofen esters according to claim 34, it is characterised in that the thick enzyme powder consumption is 0.01mg/ml~50mg/ml.

37. the preparation method of S- ibuprofen esters according to claim 34, it is characterised in that the thick enzyme powder Consumption is 0.05mg/ml~10mg/ml.

38. the preparation method of S- ibuprofen esters according to claim 34, it is characterised in that the thick enzyme powder consumption is 0.1~5mg/ml.

39. the preparation method of S- ibuprofen esters according to claim 34, it is characterised in that the thick enzyme powder consumption is 0.2~1mg/ml.

40. the preparation method of the S- ibuprofen esters according to claim any one of 30-39, it is characterised in that sub- sieve solution fat yeast cells external fat enzyme is fixed on silica gel, diatomite or membranaceous textile.

41. the preparation method of S- ibuprofen esters according to claim 40, it is characterised in that sub- sieve solution fat yeast cells external fat enzyme is fixed on natural fabric.

42. the preparation method of S- ibuprofen esters according to claim 41, it is characterised in that the natural fabric is cotton and science fabric.

43. the preparation method of S- ibuprofen esters according to claim 1, wherein the esterification continues to that the ratio that S- brufens and R~brufen are converted into ester is more than 3: 1.

44. the preparation method of S- ibuprofen esters according to claim 1, wherein the esterification continues to that the ratio that S- brufens and R~brufen are converted into ester is 4: 1.

45. the preparation method of S- ibuprofen esters according to claim 1, wherein the esterification continues to that the ratio that S- brufens and R~brufen are converted into ester is 5: 1.

46. the preparation method of S- ibuprofen esters according to claim 1, wherein the esterification continues to that the ratio that S- brufens and R~brufen are converted into ester is 9: 1.

47. the preparation method of S- ibuprofen esters according to claim 1, wherein the esterification continues to that the ratio that S- brufens and R~brufen are converted into ester is 98: 2.

48. the preparation method of S- ibuprofen esters according to claim 1, wherein the reaction time of the esterification is 12~120 hours.

49. the preparation method of S- ibuprofen esters according to claim 1, wherein the reaction time of the esterification is 15~80 hours.

50. the preparation method of S- ibuprofen esters according to claim 1, wherein the reaction time of the esterification is 20~60 hours.

51. the preparation method of S- ibuprofen esters according to claim 1, wherein the reaction time of the esterification is 24~48 hours.

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