CN101805756A - 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 used bacterial strain in the production process.
(2) background technology
Statins is to treat the most effective medicines of cardiovascular disorder such as hypertension, hyperlipidemia at present.Rosuvastatin claims super his spit of fland again, and the statins as a kind of novel better efficacy is subjected to people's attention day by day.But because the synthesis technique of its chiral side chain imperfection also, it is high to make its preparation cost compare other statinses, so selling price is high always, has restricted the developing and the development in its market, has also hindered the 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, also is difficult point simultaneously, its parent is synthetic then relatively simple.(R)-and 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 height of its synthetic yield and optical purity has become the bottleneck of whole Rosuvastatin synthesis technique.If can 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 (R)-carbon chain lengths of 3-hydroxyl pentanedioic acid diethyl ester or its hydroxyl substitutive derivative is shorter, and contain reactive groups such as hydroxyl, carboxyl and ester group, and the condition of chemical reaction is relatively harsher, often need High Temperature High Pressure, be easy to destroy the structure of product molecule, chemically be not suitable for the synthetic of this product, do not see relevant report yet.In the biological process preparation of having reported, mainly be 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 the organic acid substance of high added value, biologically active 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, 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 by the cyano group in its molecular structure of hydrolysis.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 isolating difficulty.And can prepare (R)-3-hydroxyl pentanedioic acid diethyl ester or its hydroxyl substitutive derivative (reaction formula as follows) of high-optical-purity a 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, and generally contain the esterase or the lipase that all can contain the hydrolysis ester group in the lytic enzyme microorganism cells, so the screening of this biological catalyst also is an a great problem, up to now, do not see the relevant report of this nitrilase bacterial strain yet.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 (the 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: be substrate with compound shown in the formula (II), the enzyme that obtains with 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 the corresponding formula (I) or its hydroxyl substitutive derivative through separation and purification;
Among formula (I), (II): among 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 directly participate in reaction with the fermented liquid form, and containing in the enzyme somatic cells adding reaction system of also filtering fermentation liquor can being obtained reacted, 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, the substrate starting point concentration is 1～20mmol/L in the described reaction system, and containing enzyme somatic cells starting point concentration is 1～10g DCW/L (dry mycelium concentration).
The described enzyme somatic cells that contains is prepared by following method: rhodococcus erythropolis CCTCC No:M 209244 or CCTCC No:M 209194 are seeded to fermention medium, and 30 ℃ of following 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, promptly 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 following 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, under 30 ℃, carry out conversion reaction, 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, promptly 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 culture collection center, address: Chinese Wuhan 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 culture collection center; address: Chinese Wuhan Wuhan University; 430072; deposit number CCTCC No:M 209194; preservation date: on September 4th, 2009, in first to file CN200910154891.5, protected as new bacterial strain.
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 that 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 4-cyano-3-hydroxy ethyl butyrate to 0.05～2% of degerming after filtration before the inoculation.Enrichment culture is after a couple of days, and the nutrient solution of getting the generation muddiness circulates successively by last method and cultivates 0～5 time, is applied to the solid plate substratum.The solid culture based component is except that 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 final sample concentration is 1～10g DCW/L (dry mycelium concentration) in 6～9 the Tris-HCl damping fluid; Add substrate 4-cyano-3-hydroxy ethyl butyrate and begin reaction, making its final concentration is 20～150mmol/L.This reaction system behind reaction 30～240min, adds isopyknic Co that contains in 20～50 ℃ of reactions down
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 then this sample contains the nitrilase of energy catalytic hydrolysis 4-cyano-3-hydroxy ethyl butyrate cyano group; Constant as color, then do not contain this nitrilase in the 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 the hydrolysate.Concrete operation method and condition are: extract reaction solution 1ml, 10000rpm is centrifugal to abandon 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).Behind the multiple sieve and checking by HPLC, determine that above two strains have the 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, under 10～50 ℃, 100～200rpm condition, react 60～300min.After reaction finishes, the centrifugal supernatant liquor that obtains of 10000rpm.HPLC analyzes the transformation efficiency that obtains product.The HCl solution of using 1～10N is then used ethyl acetate extraction 1～5 time again with supernatant liquor furnishing acidity, merges organic phase.Under 0～60 ℃ of condition, boil off ethyl acetate with Rotary Evaporators then and obtain yellow liquid.The specific rotatory power of product ([α]
D 22) be that-1.9 (c 11.5, acetone) and literature value contrast determine that its steric configuration is R type (J Am Chem Soc, 83:4228-4233,1961) after measured.This product molecular structure is by UV spectrum, nucleus magnetic resonance
1H and
13C spectrum, ESI-MS and infrared spectra 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 behind the 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 0.5～2% soil sample suspension and sewage by suspended liquid with sterilized water, inoculate 1～5ml respectively in the 50ml enrichment medium,, cultivate on the shaking table of 150rpm 1～3 day at 30 ℃.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 the packing, the 10～30min that sterilizes under 0.1MPa adds through the 4-of Sterile Filtration cyano-3-hydroxy ethyl butyrate to 0.05～2% (v/v) before the inoculation.After cultivating end, get the nutrient solution that produces muddiness and inoculate 1～5ml more respectively in the 50ml enrichment medium,, cultivated on the shaking table of 150rpm 1～3 day, so circulate 3～5 times at 30 ℃.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 that the agar of adding 1～3%, and all the other are identical with the enrichment culture based component.
(2) picking list bacterium colony, was cultivated on the shaking table of 150rpm 1～3 day at 30 ℃ to the 50ml fermention medium 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 operations step of ion development process is as follows: bacteria suspension 10～100 μ l that get 1～10g DCW/L formulated in the step (2) (dry mycelium concentration) are 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 behind reaction 30～240min, adds isopyknic Co that contains in 20～50 ℃ of reactions down
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,, show that then this sample contains the nitrilase of energy catalytic hydrolysis 4-cyano-3-hydroxy ethyl butyrate cyano group if reaction system becomes yellow by magenta; Constant as color, then do not contain this nitrilase in the 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 (2) is set by step made bacteria suspension.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 got this reaction solution of 1ml centrifugal somatic cells termination reaction of abandoning under 10000rpm, and is obtained supernatant liquor behind reaction 60～240min in 20～50 ℃ of reactions down.Get this supernatant liquor sample introduction of 10～20 μ l and do the HPLC analysis.The model of high performance liquid chromatograph be LC-10 A7 (VP) (Shimadzu, Japan), the chromatographic column model is Hypersil ODS C18 (250mm * 4.6mm, 5 μ m), the UV-detector model be 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.After identify and be Rhodococcus erythropolis.
(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, under 10～50 ℃, 100～200rpm condition, react 60～300min.After reaction finishes, the centrifugal supernatant liquor that obtains of 10000rpm.HPLC analyzes the transformation efficiency that obtains product.The HCl solution of using 1～10N is then used ethyl acetate extraction 1～5 time again with supernatant liquor furnishing acidity, merges organic phase.Under 0～60 ℃ of condition, boil off ethyl acetate with Rotary Evaporators then and obtain yellow liquid.The specific rotatory power of product ([α]
D 22) be that-1.9 (c 11.5, acetone) and literature value contrast determine that it is configured as R type (J Am Chem Soc, 83:4228-4233,1961) after measured.The molecular structure of this product is by UV spectrum, nucleus magnetic resonance
1H and
13C spectrum, ESI-MS and infrared spectra 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), get 0.5ml then 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.Under 10～50 ℃, 100～200rpm condition, react 6～12h.After reaction finishes, the centrifugal supernatant liquor that obtains of 10000rpm.GC analyzes the transformation efficiency that obtains product.
(8) gas-chromatography (GC) analytical procedure of the various products of reaction is as follows in the step (7): 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 ℃, keeps 10min then.Flow rate of carrier gas is 1ml/min.The measuring method of the ee value of product is identical with step (6).
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, do not appear in the newspapers 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 (R)-3-hydroxyl pentanedioic acid diethyl ester or its hydroxyl substitutive derivative that catalytic hydrolysis obtains high-optical-purity from the 4-cyano-3-hydroxy ethyl butyrate of racemization or its hydroxyl substitutive derivative one, the ester group in the hydrolysis substrate not, do not produce any by product, overcome that the by product that traditional synthetic method such as chemical method brought is many, target product yield is low and the not high shortcoming of optical purity in hydrolytic process.Because product (R)-3-hydroxyl pentanedioic acid diethyl ester can't buy 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 the structure evaluation.
(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: utilization Co
2+The screening of ion development process can selectivity catalytic hydrolysis 4-cyano-3-hydroxy ethyl butyrate be the nitrilase bacterial strain of (R)-3-hydroxyl pentanedioic acid diethyl ester
(1) will prepare 1% soil sample suspension and sewage by suspended liquid with sterilized water, inoculate 5ml respectively in the 45ml enrichment medium,, cultivate 2 days on the shaking table of 150rpm at 30 ℃.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 the packing, the 20min that sterilizes under 0.1MPa adds through the 4-of Sterile Filtration cyano-3-hydroxy ethyl butyrate to 1% (v/v) before the inoculation.After cultivating end, get the nutrient solution that produces muddiness and inoculate 1ml more respectively in the 49ml enrichment medium, 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 that the agar of adding 2%, and all the other are identical with the enrichment culture based component.
(2) picking list bacterium colony, was cultivated 2 days on the shaking table of 150rpm at 30 ℃ to the 50ml fermention medium 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 cultivate finishing, 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 operations step of ion development process is as follows: the bacteria suspension 100 μ l that get 10g DCW/L formulated in the step (2) (dry mycelium concentration) add substrate 4-cyano-3-hydroxy ethyl butyrate and begin reaction in 96 orifice plates, and making its final concentration is 40mmol/L.This reaction system behind the reaction 240min, adds isopyknic Co that contains in 35 ℃ of reactions down
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,, show that then this sample contains the nitrilase of energy catalytic hydrolysis 4-cyano-3-hydroxy ethyl butyrate cyano group if reaction system becomes yellow by magenta; Constant as color, then do not contain this nitrilase in the 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 (2) is set by step made bacteria suspension.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 got this reaction solution of 1ml centrifugal somatic cells termination reaction of abandoning under 10000rpm, and is obtained supernatant liquor behind the reaction 240min in 35 ℃ of reactions down.Get this supernatant liquor sample introduction of 10 μ l and do the HPLC analysis.The model of high performance liquid chromatograph be LC-10 A7 (VP) (Shimadzu, Japan), the chromatographic column model is Hypersil ODS C18 (250mm * 4.6mm, 5 μ m), the UV-detector model be 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.After identify and be Rhodococcus erythropolis.
Embodiment 2: biological catalyst R.erythropolis CCTCC NO:M209244 cell preparation
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 the 50ml aseptic seed substratum, at 30 ℃, cultivates 24h on the shaking table of 150rpm, 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 does not have in the 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: this whole cell Preparation of catalysts mode of biological catalyst R.erythropolis CCTCC NO:M 209149 cell preparation is 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 in the thalline solution that 10ml prepares by embodiment 2 methods, at 30 ℃, react 240min under the 180rpm condition.Reaction finishes after testing, and (R)-transformation efficiency of 3-hydroxyl pentanedioic acid diethyl ester (ee 〉=99%) is 46.5%, illustrate that basically (R)-4-cyano-3-hydroxy ethyl butyrate has been converted into product, and what be left is 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 in the thalline solution that 10ml prepares by embodiment 3 methods, at 30 ℃, react 240min under the 180rpm condition.Reaction finishes after testing, (R)-and the transformation efficiency of 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 behind the ethyl acetate extraction, do UV spectrum, nucleus magnetic resonance respectively
1H and
13C spectrum, ESI-MS and infrared spectra 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, does not have 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: utilizing the various hydroxyl substitutive derivatives of M209244 somatic cells stereoselectivity catalytic hydrolysis 4-cyano-3-hydroxy ethyl butyrate is 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), get 0.5ml then 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.Under 30 ℃, 180rpm condition, react 6～12h.After reaction finishes, the centrifugal supernatant liquor that obtains of 10000rpm.GC analyzes the transformation efficiency that obtains product.The result is as shown in the table.
Claims (8)
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 (R)-3-hydroxyl pentanedioic acid diethyl ester or its hydroxyl substitutive derivative structure are suc as formula shown in (I), described method comprises: be substrate with compound shown in the formula (II), the enzyme that obtains with 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 the corresponding formula (I) or its hydroxyl substitutive derivative through separation and purification;
Among formula (I), (II): R
1For-H ,-C (O) CH
3,-C (O) CH
2OCH
3,-CH
2OCH
3(CH
3)
2, TMDS or TBDMS.
2. the method for claim 1 is characterized in that: described enzyme contains the enzyme somatic cells from rhodococcus erythropolis CCTCCNo:M 209244 or CCTCC No:M 209194 through what fermentation culture obtained.
3. the method for claim 1 is characterized in that described conversion reaction carries out in the damping fluid of pH7.0～8.0.
4. method as claimed in claim 2 is characterized in that the substrate starting point concentration is 1～20mmol/L in the described reaction system, and containing enzyme somatic cells starting point concentration is 1～10g DCW/L.
5. method as claimed in claim 2, it is characterized in that the described enzyme somatic cells that contains is prepared by following method: rhodococcus erythropolis CCTCC No:M 209244 or CCTCC No:M209194 are seeded to fermention medium, 30 ℃ of following 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, and pH 3～8.
6. the method for claim 1, it is characterized in that 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, merge organic phase, underpressure distillation boils off ethyl acetate or normal hexane obtains described (R)-3-hydroxyl pentanedioic acid diethyl ester or its hydroxyl substitutive derivative.
7. the method for claim 1 is characterized in that described method is as follows:
(1) rhodococcus erythropolis CCTCC No:M 209244 or CCTCC No:M 209194 are seeded to fermention medium, 30 ℃ of following 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 compounds (II) to concentration is 1～20mmol/L, under 30 ℃, carry out conversion reaction, 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
Described (R)-3-hydroxyl pentanedioic acid diethyl ester or its hydroxyl substitutive derivative.
8. rhodococcus erythropolis (Rhodococcus erythropolis) ZJB-0910 is preserved in Chinese typical culture collection center, the address: Chinese Wuhan Wuhan University, 430072, deposit number CCTCCNo:M 209244, preservation date: on October 26th, 2009.
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| WO2014205917A1 (en) * | 2013-06-28 | 2014-12-31 | 苏州汉酶生物技术有限公司 | Method for preparing rosuvastatin intermediate |
| CN103361386B (en) * | 2013-06-28 | 2015-04-15 | 苏州汉酶生物技术有限公司 | Method for preparing rosuvastatin intermediate |
| CN104372040A (en) * | 2013-08-12 | 2015-02-25 | 南京朗恩生物科技有限公司 | Continuous preparation method of statin pharmaceutical intermediate namely (R)-3-hydroxyethyl glutarate |
| CN104372040B (en) * | 2013-08-12 | 2018-07-03 | 南京朗恩生物科技有限公司 | A kind of method for continuously preparing statins intermediate (R) -3- hydroxyl pentanedioic acid diethyl esters |
| CN104152500A (en) * | 2014-08-27 | 2014-11-19 | 中国科学院天津工业生物技术研究所 | New method of biologically synthesizing (R)-3-hydroxylglutarate monoester |
| CN106676141A (en) * | 2015-11-06 | 2017-05-17 | 浙江京新药业股份有限公司 | Enzymatic preparation method of chiral intermediate (S)-3-hydroxyglutaric acid monoester |
| CN111100856A (en) * | 2020-01-13 | 2020-05-05 | 浙江工业大学 | Nitrilase mutant and application thereof in synthesis of pregabalin chiral intermediate |
| CN111100856B (en) * | 2020-01-13 | 2021-12-07 | 浙江工业大学 | Nitrilase mutant and application thereof in synthesis of pregabalin chiral intermediate |
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