CN101805756B - Biological catalysis method for preparing statin medicinal intermediate - Google Patents
Biological catalysis method for preparing statin medicinal intermediate Download PDFInfo
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Abstract
The invention provides a method for preparing and synthesizing a statin medicinal intermediate, namely, an (R)-3-hydroxy-glutaric acid ethyl ester or a hydroxy substituted derivative of the (R)-3-hydroxy-glutaric acid ethyl ester with high optical activity by performing chiral separation on a racemic 4-cyano-group-3-hydroxy butyric acid ethyl ester or a hydroxy substituted derivative of the 4-cyano-group-3-hydroxy butyric acid ethyl ester and a strain. The method comprises the following steps: performing a conversion reaction in a reaction system in which a compound shown in a formula (11) is used as a substrate and an enzyme obtained by fermenting and culturing rhodococcus erythropolis CCTCC No:M 209244 or CCTCC No:M 209194 is used as a catalyst at the temperature of between 10 and 50 DEG C; and after the reaction, separating and purifying reaction liquid to obtain the compound shown in formula (I). The method realizes obtaining the (R)-3-hydroxy-glutaric acid ethyl ester or the hydroxy substituted derivative of the (R)-3-hydroxy-glutaric acid ethyl ester with high optical activity by catalyzing and hydrolyzing the racemic 4-cyano-group-3-hydroxy butyric acid ethyl ester or the hydroxy substituted derivative of the 4-cyano-group-3-hydroxy butyric acid ethyl ester in one step under the condition of not hydrolyzing an ester group in the substrate or producing any byproduct, and overcomes the defects of more byproducts, low target product yield, low optical purity and the like brought by a conventional synthetic method in a hydrolyzing process, such as a chemical method.
Description
(1) technical field
The present invention relates to the method for a kind of biological catalysis method for preparing statin medicinal intermediate (R)-3-hydroxyl pentanedioic acid diethyl ester or its hydroxyl substitutive derivative, and bacterial strain used in production process.
(2) background technology
Statins is to treat at present the most effective medicines of cardiovascular disorder such as hypertension, hyperlipidemia.Rosuvastatin claims again superstatin, and the statins as a kind of novel better efficacy receives people's concern day by day.But due to the synthesis technique of its chiral side chain imperfection also, make its preparation cost compare other statinses high, so selling price is always high, has restricted developing and the development in its market, has also hindered effective control and the treatment of cardiovascular disorder simultaneously.Because in the synthetic and preparation technology of statins, the synthetic of the chiral side chain of high-optical-purity (〉=99%) is committed step, is also difficult point simultaneously, its parent is synthetic relatively simple.(R)-3-hydroxyl pentanedioic acid diethyl ester or its hydroxyl substitutive derivative are as the key intermediate of synthesizing rosuvastatin spit of fland side chain, and the yield that it is synthetic and the height of optical purity have become the bottleneck of whole Rosuvastatin synthesis technique.If synthesize (R)-3-hydroxyl pentanedioic acid diethyl ester or its hydroxyl substitutive derivative of high-optical-purity with less step and lower cost, the synthetic of Rosuvastatin will be more perfect, and cost can reduce greatly.But so far, the synthetic of (R)-3-hydroxyl pentanedioic acid diethyl ester of chiral purity or its hydroxyl substitutive derivative also do not realized.
Utilize the method for traditional chemosynthesis, because the carbon chain lengths of (R)-3-hydroxyl pentanedioic acid diethyl ester or its hydroxyl substitutive derivative is shorter, and contain the reactive groups such as hydroxyl, carboxyl and ester group, and the condition of chemical reaction is harsher, often need High Temperature High Pressure, be easy to destroy the product molecular structures, chemically be not suitable for the synthetic of this product, also have no relevant report.In the Biological preparation of having reported, be mainly to utilize commercial enzyme to carry out catalyzed conversion.For example, Jacobsen etc. utilize commercial lipase, esterase and α-catalytic hydrolysis 3-hydroxyl ethyl glutarates such as rotten galactase to prepare (R)-3-hydroxyl pentanedioic acid diethyl ester (J Mol Catal B-Enzym, 21:55-58; Tetrahedron lett.25:5235-5238), but the ee value (91%) of productive rate (80%) and product is not very high, the acquisition cost of what is more important commercial enzyme is very high, therefore also is not suitable for being applied to the industrialization production of (R)-3-hydroxyl pentanedioic acid diethyl ester.
Nitrile compound exists in a large amount of form with inorganic nitrile and organic nitrile of occurring in nature.Simultaneously, it also can obtain by the method production of chemosynthesis as a kind of staple commodities.And can prepare high added value, have bioactive organic acid substance from these cheap nitrile compounds.For example the chemical method preparation technology of the 4-cyano-3-hydroxy ethyl butyrate of racemization or its hydroxyl substitutive derivative is quite ripe, and with low cost.And by the cyano group in its molecular structure of hydrolysis, can prepare (R)-3-hydroxyl pentanedioic acid diethyl ester or its hydroxyl substitutive derivative by 4-cyano-3-hydroxy ethyl butyrate or its hydroxyl substitutive derivative.But contain active hydroxyl, carbonyl and carboxyl in this same material molecule, utilize chemical hydrolysis method to be easy to produce the by product of different structure, cause the difficulty of separation.And can prepare (R)-3-hydroxyl pentanedioic acid diethyl ester of high-optical-purity or its hydroxyl substitutive derivative (reaction formula see under) step by microorganism nitrilase cell selective catalytic hydrolysis 4-cyano-3-hydroxy ethyl butyrate or its hydroxyl substitutive derivative, this hydrolysis reaction system is simple, mild condition, and the stereoselectivity of nitrilase is also very high.Yet, because substrate 4-cyano-3-hydroxy ethyl butyrate or its hydroxyl substitutive derivative all contain an ester group, all can contain and generally contain esterase or the lipase that is hydrolyzed ester group in the lytic enzyme microorganism cells, so the screening of this biological catalyst is also a great problem, up to now, also have no the relevant report of this nitrilase bacterial strain.These factors make synthetic being difficult to of optically pure (R)-3-hydroxyl pentanedioic acid diethyl ester or its hydroxyl substitutive derivative realize.
R
1：-H，-C(O)CH
3，-C(O)CH
2OCH
3，-CH
2OCH
3(CH
3)
2，TMDS，TBDMS
(3) summary of the invention
The purpose of this invention is to provide and a kind ofly have (R)-3-hydroxyl pentanedioic acid diethyl ester of high optical activity or the method for its hydroxyl substitutive derivative by the preparation of chiral separation racemic 4-cyano-3-hydroxy ethyl butyrate or its hydroxyl substitutive derivative is synthetic, and provide a strain from the Nature soil and sewage screening obtain containing the new bacterial strain of rhodococcus erythropolis that stereoselectivity nitrilase enzyme is lived and do not contained the esterase of ester group in hydrolysis 4-cyano-3-hydroxy ethyl butyrate or its hydroxyl substitutive derivative.
The technical solution used in the present invention is:
the method of a kind of biological catalysis production (R)-3-hydroxyl pentanedioic acid diethyl ester or its hydroxyl substitutive derivative, described method comprises: the method for a kind of biological catalysis production (R)-3-hydroxyl pentanedioic acid diethyl ester or its hydroxyl substitutive derivative, described (R)-3-hydroxyl pentanedioic acid diethyl ester or its hydroxyl substitutive derivative structure are suc as formula shown in (I), described method comprises: take compound shown in formula (II) as substrate, the enzyme that obtains take rhodococcus erythropolis CCTCC No:M 209244 or CCTCC No:M 209194 fermentation culture is in the reaction system of catalyzer, carry out conversion reaction under 10～50 ℃, reaction finishes afterreaction liquid and obtains (R)-3-hydroxyl pentanedioic acid diethyl ester shown in corresponding formula (I) or its hydroxyl substitutive derivative through separation and purification,
In formula (I), (II): in formula (I), (II): R
1For-H ,-C (O) CH
3,-C (O) CH
2OCH
3,-CH
2OCH
3(CH
3)
2(methyl isopropyl ether), TMDS (tetramethyl-two silicon ethers) or TBDMS (tertiary butyl dimethyl-silicon ether).
Described enzyme contains the enzyme somatic cells from rhodococcus erythropolis CCTCC No:M 209244 or CCTCC No:M209194 through what fermentation culture obtained.Described enzyme can be directly participates in reaction with the fermented liquid form, and the enzyme somatic cells that contains that also filtering fermentation liquor can be obtained adds in reaction system and reacts, and described somatic cells also can be used for reaction after immobilization.
Usually, described conversion reaction is carried out in the damping fluid of pH7.0～8.0.
Preferably, in described reaction system, initial substrate concentration is 1～20mmol/L, and containing enzyme somatic cells starting point concentration is 1～10g DCW/L (dry mycelium concentration).
The described enzyme somatic cells that contains prepares by the following method: rhodococcus erythropolis CCTCC No:M 209244 or CCTCC No:M 209194 are seeded to fermention medium, and 30 ℃ of lower shaking culture 1～3 day obtain to contain the fermented liquid of enzyme somatic cells; Described fermention medium final concentration consists of: glucose 0～20g, peptone 0～10g, hexanolactam 0～5g, K
2HPO
40～2.0g, MgSO
47H
2O 0～0.5g, NaCl 0～2g, FeSO
47H
2O 0～0.2g, solvent are water, pH3～8.
Described separation purification method is as follows: reaction solution is centrifugal, getting supernatant liquor, to transfer pH with the HCl solution of 1～10mol/L be 2～4, again with ethyl acetate or n-hexane extraction 1～5 time, combined ethyl acetate or normal hexane organic phase, underpressure distillation boils off ethyl acetate or normal hexane, namely obtains described (R)-3-hydroxyl pentanedioic acid diethyl ester or its hydroxyl substitutive derivative.
Concrete, described method is as follows:
(1) rhodococcus erythropolis CCTCC No:M 209244 or CCTCC No:M 209194 are seeded to fermention medium, 30 ℃ of lower shaking culture 1～3 day obtain to contain the fermented liquid of enzyme somatic cells; Described fermention medium final concentration consists of: glucose 0～20g, peptone 0～10g, hexanolactam 0～5g, K
2HPO
40～2.0g, MgSO
47H
2O0～0.5g, NaCl 0～2g, FeSO
47H
2O 0～0.2g, solvent are water, and pH 3～8;
(2) step (1) fermented liquid is centrifugal, gets precipitation with physiological saline washing 1～3 time, the bacteria suspension that the somatic cells that obtains is mixed with 1～10g DCW/L with Tris-HCl damping fluid or the phosphate buffered saline buffer of 10～50mmol/L, pH 6～9;
(3) get step (2) bacteria suspension, adding substrate (II) (4-cyano-3-hydroxy ethyl butyrate or derivatives thereof) to concentration is 1～20mmol/L, carry out conversion reaction under 30 ℃, after reaction finishes, reaction solution is centrifugal, getting supernatant liquor, to transfer pH with the HCl solution of 1～10mol/L be 2～4, again with ethyl acetate or n-hexane extraction 1～5 time, combined ethyl acetate or normal hexane organic phase, underpressure distillation boils off ethyl acetate or normal hexane, namely obtains described (R)-3-hydroxyl pentanedioic acid diethyl ester or its hydroxyl substitutive derivative.
The new bacterial strain that the present invention screens acquisition is: rhodococcus erythropolis (Rhodococcus erythropolis) ZJB-0910, be preserved in Chinese Typical Representative culture collection center, address: Wuhan, China Wuhan University, 430072, deposit number CCTCC No:M 209244, preservation date: on October 26th, 2009.
Another strain bacterial strain that the present invention relates to is: rhodococcus erythropolis (Rhodococcus erythropolis) ZJB-09149; be preserved in Chinese Typical Representative culture collection center; address: Wuhan, China Wuhan University; 430072; deposit number CCTCC No:M 209194; preservation date: on September 4th, 2009, protected as new bacterial strain in first to file CN200910154891.5.
The present invention utilizes a kind of rapid screening method of nitrilase, i.e. the two strain target nitrilase bacterial classifications that cobalt ion development process screening obtains, i.e. Rhodococcus erythropolis ZJB-0910 and Rhodococcus erythropolis ZJB-09149.Wherein the ZJB-0910 enzyme is lived higher.Identify by morphology, Physiology and biochemistry and 16S rDNA, red flat (erythropolis) that two bacterial strains all belong to Rhod (Rhodococcus) plants.
Bacterial strain screening method of the present invention is as follows: with aqua sterilisa, sample is made into suspension, gets 0.5～10.0ml suspension in the triangular flask of the enrichment medium that 1～100ml is housed, at 20～40 ℃, cultivate on the shaking table of 100～300rpm.The composition of every liter of enrichment medium is: glucose 0～20g, K
2HPO
40～2.0g, MgSO
47H
2O 0～0.5g, NaCl 0～2g, FeSO
47H
2O 0～0.2g, pH 3～8, add the 4-cyano-3-hydroxy ethyl butyrate to 0.05 of degerming after filtration～2% before inoculation.Enrichment culture is after a couple of days, gets to produce muddy nutrient solution again by upper method Cyclic culture successively 0～5 time, is applied to the solid plate substratum.The solid culture based component is except containing 1～3% agar, and all the other are identical with the enrichment culture based component.Picking list bacterium colony from the flat board is forwarded to 1～100ml fermention medium, at 20～40 ℃, cultivates a couple of days on the shaking table of 100～300rpm.The composition of every liter of fermention medium is: glucose 0～20g, peptone 0～10g, hexanolactam 0～5g, K
2HPO
40～2.0g, MgSO
47H
2O 0～0.5g, NaCl 0～2g, FeSO
47H
2O 0～0.2g, solvent are water, pH3～8.After cultivate finishing, with the centrifugal 0～20min of 0～10000rpm whizzer, the centrifugal sample that obtains obtains sample with 0～0.8% physiological saline rinse 1～3 time.Getting this sample, to be dissolved in pH be that in 6～9 Tris-HCl damping fluid, final sample concentration is 1～10g DCW/L (dry mycelium concentration); Add substrate 4-cyano-3-hydroxy ethyl butyrate and begin reaction, making its final concentration is 20～150mmol/L.This reaction system is reacted under 20～50 ℃, after reaction 30～240min, adds isopyknic Co of containing
2+Ionic concn is that the Tris-HCl buffered soln (pH6～9) of 10～100mmol/L carries out color reaction, and the reaction times is 1～20min.If reaction system becomes yellow by magenta, show that this sample contains the nitrilase of energy catalytic hydrolysis 4-cyano-3-hydroxy ethyl butyrate cyano group; As constant in color, do not contain this nitrilase in sample.Positive strain uses high performance liquid chromatography (HPLC) to carry out multiple sieve again, determines that through multiple sieve this nitrilase whole cell does not have the ester bond in hydrolysate.Concrete operation method and condition are: extract reaction solution 1ml, 10000rpm is centrifugal abandons the somatic cells termination reaction, and obtains supernatant liquor.Get this supernatant liquor sample introduction of 10～20 μ l and do the HPLC analysis.Analysis condition is: moving phase: the triethylamine solution of 0～5% (v/v): acetonitrile (4: 1), and the detection wavelength is 220～275nm, flow velocity is 1～2ml/min.Under this condition, the retention time of product (R)-3-hydroxyl pentanedioic acid diethyl ester is 4.05min (seeing Fig. 1 for details).After multiple sieve and checking by HPLC, determine that above two strains have positive strain Rhodococcuserythropolis ZJB-0910 and the ZJB-09149 of target nitrilase vigor.
The above two kinds of thalline through the fermention medium cultivation of learning from else's experience are some, use 50mmol L
-1, pH 7.5 phosphate buffered saline buffer be made into the bacteria suspension of 1～10g DCW/L.Compound concentration is 20mmol L
-14-cyano-3-hydroxy ethyl butyrate aqueous solution 100ml, get this substrate solution of 5～20ml and join in 5～20ml thalline solution, react 60～300min under 10～50 ℃, 100～200rpm condition.React complete after, the centrifugal supernatant liquor that obtains of 10000rpm.HPLC analyzes the transformation efficiency that obtains product.Then use the HCl solution of 1～10N that the supernatant liquor furnishing is acid, then use ethyl acetate extraction 1～5 time, merge organic phase.Then boil off ethyl acetate with Rotary Evaporators under 0～60 ℃ of condition and obtain yellow liquid.The specific rotatory power of product ([α]
D 22) be-1.9 (c 11.5, acetone) after measured, and the literature value contrast determines that its steric configuration is R type (J Am Chem Soc, 83:4228-4233,1961).This product molecular structure is by ultraviolet spectra, nuclear magnetic resonance
1H and
13C spectrum, ESI-MS and infrared spectrum are identified.Determine that from above result this product is (R)-3-hydroxyl pentanedioic acid diethyl ester.The ee value (〉=99%) of this product utilizes normal-phase chromatography to measure (Chromatographia, 71:85-89,2010) after separating through the epimer after derivatize.This somatic cells is suitable for the stereo selective hydrolysis of 4-cyano-3-hydroxy ethyl butyrate β position hydroxyl substitutive derivative, the productive rate gc analysis of product equally.
Concrete, screening method is as follows:
(1) will prepare with sterilized water 0.5～2% soil sample suspension and sewage by suspended liquid, inoculate respectively 1～5ml in the 50ml enrichment medium, at 30 ℃, cultivate on the shaking table of 150rpm 1～3 day.The composition of enrichment medium is: glucose 0～20g/L, K
2HPO
40～2.0g/L, MgSO
47H
2O0～0.5g/L, NaCl 0～2g/L, FeSO
47H
2O 0～0.2g/L, pH 3～8.After packing, the 10～30min that sterilizes under 0.1MPa adds before inoculation through the 4-of Sterile Filtration cyano-3-hydroxy ethyl butyrate to 0.05～2% (v/v).After cultivating end, get the nutrient solution that produces muddiness and inoculate respectively again 1～5ml in the 50ml enrichment medium, at 30 ℃, cultivated on the shaking table of 150rpm 1～3 day, so circulate 3～5 times.Get growing way nutrient solution coating separating plate relatively preferably, 30 ℃ of static cultivations of incubator 1～3 day.The plate culture medium composition is except adding 1～3% agar, and all the other are identical with the enrichment culture based component.
(2) picking list bacterium colony to the 50ml fermention medium, at 30 ℃, was cultivated on the shaking table of 150rpm 1～3 day on plate culture medium.The composition of every L fermention medium is: glucose 0～20g, peptone 0～10g, hexanolactam 0～5g, K
2HPO
40～2.0g, MgSO
47H
2O 0～0.5g, NaCl0～2g, FeSO
47H
2O 0～0.2g, solvent are water, and pH 3～8.After cultivating end, with the centrifugal 0～20min of 0～10000rpm whizzer, the centrifugal sample that obtains is with 0～0.8% physiological saline rinse 1～3 time, and the thalline that obtains is mixed with the bacteria suspension of 1～10g DCW/L (dry mycelium concentration) with the Tris-HCl damping fluid (pH 6～9) of 10～50mmol/L.
(3) Co
2+The concrete operation step of ion development process is as follows: get bacteria suspension 10～100 μ l of 1～10g DCW/L formulated in step (2) (dry mycelium concentration) in 96 orifice plates, add substrate 4-cyano-3-hydroxy ethyl butyrate and begin reaction, making its final concentration is 20～150mmol/L.This reaction system is reacted under 20～50 ℃, after reaction 30～240min, adds isopyknic Co of containing
2+Ionic concn is that the Tris-HCl buffered soln (pH 6～9) of 10～100mmol/L carries out color reaction, and the reaction times is 1～20min.In reaction process, if reaction system becomes yellow by magenta, show that this sample contains the nitrilase of energy catalytic hydrolysis 4-cyano-3-hydroxy ethyl butyrate cyano group; As constant in color, do not contain this nitrilase in sample.The reaction solution that contains positive bacteria can be used ultraviolet spectrophotometer its absorbancy of continuously measured under 370nm, and deducts the blank value, and the absorbance of its positive bacteria is between 0.2～0.8, with not adding the reaction solution of thalline as blank.
(4) HPLC sieves again and verifies: through Co
2+The bacterial strain that the screening of ion development process obtains is made bacteria suspension by step (2).Add this bacteria suspension 5～20ml in the triangular flask of 50ml, add substrate 4-cyano-3-hydroxy ethyl butyrate and begin reaction, making its final concentration is 20～150mmol/L.This reaction system is reacted under 20～50 ℃, after reaction 60～240min, gets that this reaction solution of 1ml is centrifugal under 10000rpm abandons the somatic cells termination reaction, and obtains supernatant liquor.Get this supernatant liquor sample introduction of 10～20 μ l and do the HPLC analysis.The model of high performance liquid chromatograph is LC-10 A7 (VP) (Shimadzu, Japan), the chromatographic column model is Hypersil ODS C18 (250mm * 4.6mm, 5 μ m), the UV-detector model is Spd-10 A Vp plus (Shimadzu, Japan).Analysis condition is: moving phase: the triethylamine solution of 0～5% (v/v): acetonitrile (4: 1), and the detection wavelength is 220～275nm, flow velocity is 1～2ml/min.Under this condition, the retention time of product (R)-3-hydroxyl pentanedioic acid diethyl ester is 4.05min (seeing Fig. 1 for details).If have the product absorption peak to occur, the certain positive bacterium of this bacterial strain so just be described.
(5) get the two strain bacterial strains that obtain of screening and do Physiology and biochemistry and Molecular Identification.Be Rhodococcus erythropolis finally by evaluation.
(6) the above two kinds of thalline cultivated through fermention medium of learning from else's experience are some, use 50mmol L
-1, pH 7.5 phosphate buffered saline buffer be made into the bacteria suspension of 1～10g DCW/L.Compound concentration is 20mmol L
-14-cyano-3-hydroxy ethyl butyrate aqueous solution 100ml, get this substrate solution of 5～20ml and join in 5～20ml thalline solution, react 60～300min under 10～50 ℃, 100～200rpm condition.React complete after, the centrifugal supernatant liquor that obtains of 10000rpm.HPLC analyzes the transformation efficiency that obtains product.Then use the HCl solution of 1～10N that the supernatant liquor furnishing is acid, then use ethyl acetate extraction 1～5 time, merge organic phase.Then boil off ethyl acetate with Rotary Evaporators under 0～60 ℃ of condition and obtain yellow liquid.The specific rotatory power of product ([α]
D 22) be-1.9 (c 11.5, acetone) after measured, and the literature value contrast determines that it is configured as R type (J Am Chem Soc, 83:4228-4233,1961).The molecular structure of this product is by ultraviolet spectra, nuclear magnetic resonance
1H and
13C spectrum, ESI-MS and infrared spectrum are identified.Determine that from above result this product is (R)-3-hydroxyl pentanedioic acid diethyl ester.The ee value of product is (Chromatographia, 71:85-89,2010) more than 99% after measured.
(7) the hydroxyl substitutive derivative of the various 4-cyano-3-hydroxy ethyl butyrate of preparation is dissolved in dehydrated alcohol (concentration is 0～100mmolL
-1), then get 0.5ml and join the phosphate buffered saline buffer (pH 7.5, and cell concentration is 1～10g DCW/L) that 9.5ml contains thalline and begin reaction.React 6～12h under 10～50 ℃, 100～200rpm condition.After reaction finishes, the centrifugal supernatant liquor that obtains of 10000rpm.GC analyzes the transformation efficiency that obtains product.
(8) in step (7), gas-chromatography (GC) analytical procedure of the various products of reaction is as follows: the model of gas chromatograph is HP 6890N, the chromatographic column model is the AT.FFAP capillary column, injector temperature is 320 ℃, detector temperature is 320 ℃, the column temperature heating schedule is 80 ℃ of beginnings, speed with 10 ℃/min rises to 220 ℃, then keeps 10min.Flow rate of carrier gas is 1ml/min.The measuring method of the ee value of product is identical with step (6).
The rhodococcus erythropolis Rhodococcus erythropolis ZJB-0910 that the present invention's screening obtains and ZJB-09149 are the nitrilase bacterial strain that the 4-cyano-3-hydroxy ethyl butyrate of newfound energy chirality resolution of racemic prepares (R)-3-hydroxyl pentanedioic acid diethyl ester, have no report both at home and abroad.Simultaneously, also reported first utilize the fats beta-hydroxy nitrile compound or derivatives thereof of the nitrilase cell stereoselectivity catalytic hydrolysis racemization of rhodococcus erythropolis to be chiral organic acid class material.The discovery of this nitrilase has realized going on foot from the 4-cyano-3-hydroxy ethyl butyrate of racemization or its hydroxyl substitutive derivative one (R)-3-hydroxyl pentanedioic acid diethyl ester or its hydroxyl substitutive derivative that catalytic hydrolysis obtains high-optical-purity, the ester group in hydrolysis substrate not, do not produce any by product, overcome that the by product that traditional synthetic method such as chemical method bring is many, target product yield is low and the shortcoming such as optical purity is not high in hydrolytic process.Because product (R)-3-hydroxyl pentanedioic acid diethyl ester can't be bought and obtain, so this patent prepared (R)-3-hydroxyl pentanedioic acid diethyl ester (ee 〉=99%) of high-optical-purity first, and this product has been carried out Structural Identification.
(4) description of drawings
Fig. 1 is the high-efficient liquid phase chromatogram of (R)-3-hydroxyl pentanedioic acid diethyl ester.
(5) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1: use Co
2+The screening of ion development process can selective catalysis hydrolysis 4-cyano-3-hydroxy ethyl butyrate be the nitrilase bacterial strain of (R)-3-hydroxyl pentanedioic acid diethyl ester
(1) will prepare with sterilized water 1% soil sample suspension and sewage by suspended liquid, inoculate respectively 5ml in the 45ml enrichment medium, at 30 ℃, cultivate 2 days on the shaking table of 150rpm.The composition of every liter of enrichment medium is: glucose 5g, K
2HPO
40.5g, MgSO
47H
2O 0.5g, NaCl 0.1g, FeSO
47H
2O 0.02g, solvent are water, and pH 6.5.After packing, the 20min that sterilizes under 0.1MPa adds before inoculation through the 4-of Sterile Filtration cyano-3-hydroxy ethyl butyrate to 1% (v/v).After cultivating end, get the nutrient solution that produces muddiness and inoculate respectively 1ml in the 49ml enrichment medium again, at 30 ℃, cultivated 2 days on the shaking table of 150rpm, enrichment 4 times so circulates.Get growing way nutrient solution coating separating plate relatively preferably, 30 ℃ of static cultivations of incubator 2 days.The plate culture medium composition is except adding 2% agar, and all the other are identical with the enrichment culture based component.
(2) picking list bacterium colony to the 50ml fermention medium, at 30 ℃, was cultivated 2 days on the shaking table of 150rpm on plate culture medium.The composition of every liter of fermention medium is: glucose 10g, peptone 5g, hexanolactam 2g, K
2HPO
40.5g, MgSO
47H
2O 0.2g, NaCl 1g, FeSO
47H
2O0.02g, solvent are water, and pH 6.8.20min sterilizes under 0.1MPa.After cultivating end, with the centrifugal 10min of 10000rpm whizzer, the centrifugal sample that obtains is with 0.8% physiological saline rinse 2 times, and the thalline that obtains is mixed with the bacteria suspension of 10gDCW/L (dry mycelium concentration) with the Tris-HCl damping fluid (pH 7.0) of 40mmol/L.
(3) Co
2+The concrete operation step of ion development process is as follows: get the bacteria suspension 100 μ l of 10g DCW/L formulated in step (2) (dry mycelium concentration) in 96 orifice plates, add substrate 4-cyano-3-hydroxy ethyl butyrate and begin reaction, making its final concentration is 40mmol/L.This reaction system is reacted under 35 ℃, after reaction 240min, adds isopyknic Co of containing
2+Ionic concn is that the Tris-HCl buffered soln (pH 7.0) of 10mmol/L carries out color reaction, and the reaction times is 5min.In reaction process, if reaction system becomes yellow by magenta, show that this sample contains the nitrilase of energy catalytic hydrolysis 4-cyano-3-hydroxy ethyl butyrate cyano group; As constant in color, do not contain this nitrilase in sample.The reaction solution that contains positive bacteria can be used ultraviolet spectrophotometer its absorbancy of continuously measured under 370nm, and deducts the blank value, gets the absorbancy maximum sample and makes HPLC and sieve again and verify, with not adding the reaction solution of thalline as blank.The model of ultraviolet spectrophotometer is Beckman DU 800.
(4) HPLC sieves again and verifies: through Co
2+The bacterial strain that the screening of ion development process obtains is made bacteria suspension by step (2).Add this bacteria suspension 9ml in the triangular flask of 50ml, add substrate 4-cyano-3-hydroxy ethyl butyrate and begin reaction, making its final concentration is 100mmol/L.This reaction system is reacted under 35 ℃, after reaction 240min, gets that this reaction solution of 1ml is centrifugal under 10000rpm abandons the somatic cells termination reaction, and obtains supernatant liquor.Get this supernatant liquor sample introduction of 10 μ l and do the HPLC analysis.The model of high performance liquid chromatograph is LC-10 A7 (VP) (Shimadzu, Japan), the chromatographic column model is Hypersil ODS C18 (250mm * 4.6mm, 5 μ m), the UV-detector model is Spd-10 A Vp plus (Shimadzu, Japan).Analysis condition is: moving phase: the triethylamine solution of 0～5% (v/v): acetonitrile (4: 1), and the detection wavelength is 260nm, flow velocity is 1ml/min.Under this condition, the retention time of product (R)-3-hydroxyl pentanedioic acid diethyl ester is 4.05 (seeing Fig. 1 for details).If have the product absorption peak to occur, the certain positive bacterium of this bacterial strain so just be described.
(5) get the two strain bacterial strains that obtain of screening and do Physiology and biochemistry and Molecular Identification.Be Rhodococcus erythropolis finally by evaluation.
Embodiment 2: the preparation of biological catalyst R.erythropolis CCTCC NO:M209244 cell
The new nitrilase that obtains from seed selection of the present invention produces picking one ring thalline on the test tube slant of bacterial classification R.erythropolis CCTCCNO:M 209244, is seeded in 50ml aseptic seed substratum, at 30 ℃, cultivates 24h on the shaking table of 150rpm, gets seed liquor.The composition of every liter of seed culture medium is: glucose 8g, peptone 5g, K
2HPO
40.5g, MgSO
47H
2O 0.2g, solvent are water, the pH nature, and 20min sterilizes under 0.1MPa.
Seed liquor is forwarded to 3% (v/v) inoculum size and 100.0ml is housed without in bacteria fermentation culture medium, at 30 ℃, cultivates 48h on the shaking table of 150rpm.The composition of every liter of fermention medium is: glucose 20g, peptone 10g, hexanolactam 1g, K
2HPO
40.5g, MgSO
47H
2O 0.2g, solvent are water, and pH 7.0.20min sterilizes under 0.1MPa.After cultivating end, make bacteria suspension by embodiment 1, stem cell concentration is (3.0g/L), and wherein buffered soln is changed into the phosphate buffered saline buffer (50mmol/L) of pH 7.5 by the Tris-HCl of pH 7.0.
Embodiment 3: biological catalyst R.erythropolis CCTCC NO:M 209149 cells prepare the preparation method of this whole cell catalyzer with embodiment 2.
Embodiment 4: utilizing CCTCC NO:M209244 cell selective catalytic hydrolysis racemize 4-cyano-3-hydroxy ethyl butyrate is (R)-3-hydroxyl pentanedioic acid diethyl ester
Compound concentration is 20mmol L
-14-cyano-3-hydroxy ethyl butyrate aqueous solution 100ml, get this substrate solution of 10ml and join 10ml by in the standby thalline solution that obtains of embodiment 2 legal systems, at 30 ℃, react 240min under the 180rpm condition.Reaction finishes after testing, the transformation efficiency of (R)-3-hydroxyl pentanedioic acid diethyl ester (ee 〉=99%) is 46.5%, illustrate that (R)-4-cyano-3-hydroxy ethyl butyrate has been converted into product basically, and remaining be exactly (S)-4-cyano-3-hydroxy ethyl butyrate.
Embodiment 5: utilizing CCTCC NO:M209149 cell selective catalytic hydrolysis racemize 4-cyano-3-hydroxy ethyl butyrate is (R)-3-hydroxyl pentanedioic acid diethyl ester
Compound concentration is 20mmol L
-14-cyano-3-hydroxy ethyl butyrate aqueous solution 100ml, get this substrate solution of 10ml and join 10ml by in the standby thalline solution that obtains of embodiment 3 legal systems, at 30 ℃, react 240min under the 180rpm condition.Reaction finishes after testing, and the transformation efficiency of (R)-3-hydroxyl pentanedioic acid diethyl ester (ee 〉=99%) is 35.6%.The fractionation that utilizes this somatic cells also can realize the 4-cyano-3-hydroxy ethyl butyrate of racemization is described, but transformation efficiency is not as CCTCC No:M209244.
Embodiment 6: the structural characterization of product (R)-3-hydroxyl pentanedioic acid diethyl ester
Get the product after ethyl acetate extracts, do respectively ultraviolet spectra, nuclear magnetic resonance
1H and
13C spectrum, ESI-MS and infrared spectrum are identified.The steric configuration of product adopts the method for measuring specific rotatory power to determine.Ultraviolet maximum absorption wavelength is 220nm (in the aqueous solution), and the peak shape rule is smooth, there is no other assorted peaks.
1H?NMR：1.30(3H，t)，2.58(2H，d)，2.62(2H，d)，4.20(2H，q)，4.50(1H，m)，
13C?NMR：13.8，40.3，40.5，60.8，64.5，171.8，175.7。ESI-MS (positive ion mode): [M+H]
+, 177.5; [M+Na]
+, 199.1; [M+K]
+, 214.0, the actual molecular weight of product is 176.19.The result of infrared spectra (IR) is: 3443.6 (σ
OH), 2985,2938 (σ
C-H), 1726 (σ
C=O), 1412 (δ
OH), 1213,1085 (σ
C-O).Specific rotatory power [[α] from the product that records
D 22-1.9 (c 11.5, acetone)] can determine that this product is configured as R type (J Am Chem Soc, 83:4228-4233,1961).From above qualification result, the structure of this product is identical with (R)-3-hydroxyl pentanedioic acid diethyl ester, proves to be (R)-3-hydroxyl pentanedioic acid diethyl ester really.
Embodiment 7～11: the various hydroxyl substitutive derivatives that utilize M209244 somatic cells stereoselectivity catalytic hydrolysis 4-cyano-3-hydroxy ethyl butyrate are corresponding product acid
The hydroxyl substitutive derivative of preparing various 4-cyano-3-hydroxy ethyl butyrate is dissolved in dehydrated alcohol, and (concentration is 100mmol L
-1), then get 0.5ml and join the phosphate buffered saline buffer (pH 7.5, and cell concentration is 5g DCW/L) that 9.5ml contains thalline and begin reaction.React 6～12h under 30 ℃, 180rpm condition.After reaction finishes, the centrifugal supernatant liquor that obtains of 10000rpm.GC analyzes the transformation efficiency that obtains product.Result is as shown in the table.
Claims (2)
1. the method for a biological catalysis method for preparing statin medicinal intermediate (R)-3-hydroxyl pentanedioic acid diethyl ester or its hydroxyl substitutive derivative, described method is as follows:
(1) rhodococcus erythropolis CCTCC No:M 209244 or CCTCC No:M 209194 are seeded to fermention medium, 30 ℃ of lower shaking culture 1 ~ 3 day obtain to contain the fermented liquid of enzyme somatic cells; Every liter of fermention medium consists of: glucose 20g, peptone 10 g, hexanolactam 1 g, K
2HPO
40.5 g, MgSO
47H
2O 0.2g, solvent are water, and pH 7.0;
(2) step (1) fermented liquid is centrifugal, gets precipitation with physiological saline washing 1 ~ 3 time, the bacteria suspension that the somatic cells that obtains is mixed with 1 ~ 10 g DCW/L with Tris-HCl damping fluid or the phosphate buffered saline buffer of 10 ~ 50 mmol/L, pH 6 ~ 9;
(3) get step (2) bacteria suspension, adding substrate compounds (II) to concentration is 1 ~ 20mmol/L, carry out conversion reaction under 30 ℃, after reaction finished, reaction solution was centrifugal, and getting supernatant liquor is 2 ~ 4 with the HCl solution accent pH of 1 ~ 10mol/L, again with ethyl acetate or n-hexane extraction 1 ~ 5 time, merge organic phase, underpressure distillation boils off ethyl acetate or normal hexane, obtains the hydroxyl pentanedioic acid diethyl ester of (R)-3-shown in corresponding formula I or its hydroxyl substitutive derivative;
Product (I) structure of each group reaction substrate (II) and correspondence is as shown in the table:
。
2. rhodococcus erythropolis (Rhodococcus erythropolis) ZJB-0910, be preserved in Chinese Typical Representative culture collection center, address: Wuhan, China Wuhan University, 430072, deposit number CCTCC No:M 209244, preservation date: on October 26th, 2009.
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Non-Patent Citations (4)
| Title |
|---|
| Li-Qun Jin et al..Identification and characterization of Serratia marcescens ZJB-09104, a nitrile-converting bacterium..《World J Microbiol Biotechnol》.2009,817–823. |
| Li-Qun Jin et al..Identification and characterization of Serratia marcescens ZJB-09104, a nitrile-converting bacterium..《World J Microbiol Biotechnol》.2009,817–823. * |
| Yu-Guo Zheng et al..Isolation, identification and characterization of Bacillus subtilis ZJB-063, a versatile nitrile-converting bacterium..《Appl Microbiol Biotechnol》.2007,985–993. * |
| 郑裕国 等.腈转化酶在精细化学品生产中的应用.《生物工程学报》.2009,1795-1807. * |
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