CN101748087A - Tsukamurella-tyrosinosolvens and application thereof in catalysis preparation of (S) -alpha - ethyl -2-oxo-1-pyrrolidine acetic acid prepared by catalysis - Google Patents

Tsukamurella-tyrosinosolvens and application thereof in catalysis preparation of (S) -alpha - ethyl -2-oxo-1-pyrrolidine acetic acid prepared by catalysis Download PDF

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CN101748087A
CN101748087A CN200910155776A CN200910155776A CN101748087A CN 101748087 A CN101748087 A CN 101748087A CN 200910155776 A CN200910155776 A CN 200910155776A CN 200910155776 A CN200910155776 A CN 200910155776A CN 101748087 A CN101748087 A CN 101748087A
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王普
何军邀
袁帅
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Zhejiang University of Technology ZJUT
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Abstract

The present invention provides a novel strain, tsukamurella-tyrosinosolvens E105, and an application thereof in the chiral biocatalysis preparation of (S)-alpha-ethyl-2-oxo-1-pyrrolidine acetic acid. The tsukamurella-tyrosinosolvens E105 is stored in China Center for Type Culture Collection (CCTCC) at Wuhan University of Science and Technology (postal code: 430072) on Dec.16th, 2009. The preservation series number is CCTCC NO:M209306. The present invention adopts a microorganism preparation method of (S)-alpha-ethyl-2-oxo-1-pyrrolidine acetic acid for the first time and provides a novel strain of high stereoselectivity and capability of preparing (S)-alpha-ethyl-2-oxo-1-pyrrolidine acetic acid of high optical purity. The present invention can make the optical purity of the target product (S)-alpha-ethyl-2-oxo-1-pyrrolidine acetic acid prepared by the chiral biocatalysis of tsukamurella-tyrosinosolvens E105 cells reach 99.4%, and make productivity reach 48.1%. The novel strain obtained by screening provides helpful references in the aspects of research in the microorganism preparation of the key chiral intermediate of levetiracetam and the technique optimization of bioconversion.

Description

Anti-tyrosine tomb village Salmonella and catalytic preparation (S)-α-ethyl thereof-2-oxygen-1-pyrrolidine acetic acid
(1) technical field
The invention belongs to the biocatalysis technology field, relate to the new bacterial classification of a strain---anti-tyrosine tomb village Salmonella (Tsukamurella tyrosinosolvens) E105, and the application in chirality biocatalysis preparation (S)-α-ethyl-2-oxygen-1-pyrrolidine acetic acid.
(2) background technology
(S)-chemical structural formula of α-ethyl-2-oxygen-1-pyrrolidine acetic acid is:
Figure G200910155776XD00011
(S)-α-ethyl-2-oxygen-1-pyrrolidine acetic acid is synthetic Levetiracetam (levetiracetam, LEV, trade(brand)name: Keppra ) important chiral intermediate.The chemistry of Levetiracetam is by name: (S)-α-ethyl-2-oxygen-1-pyrrolidine acetamide, be the nootropics with pyrrolidone structure of Belgian Unichem (UCB) research and development.It is high to the activity of the memory defects that Scopolamine causes, has anticonvulsion and antiepileptic action simultaneously, and its S-isomer (Levetiracetam) has biological activity, and the R-isomer does not have activity.This medicine is stereoselectivity, saturated, the reversible combination with the brain cell membrane of central nervous system.Compare with similar medicine, its therapeutic index is big, and significant quantity and toxic dose differ far, and long-term prescription has no drug resistance or the drug withdrawal syndromes occurs.U.S. FDA is used for the interpolation treatment of adult's partial seizures more than 16 years old in official approval in December, 1999.Discover, LEV is as the child patient assisting therapy of partial seizures safety and can effectively reduce the epileptic seizures frequency not only, confirmed that not only LEV can effectively control children's partial seizures as assisting therapy, and side effect is mutually slight, better tolerance has bigger reference value for the intractable epilepsy of clinical treatment children.Again indication is expanded to children more than 4 years old in June, 2005.Because the clinical effectiveness of its unique anti-epileptic mechanism, comparatively ideal pharmacokinetics, highly effective and safe, it is unique antiepileptic drug of report at present with special performance of prevention epilepsy generation, and can be used for independent treatment, do not interact with other antiepileptic drugs, become one of the most promising novel antiepileptic drug, be extensive use of the America and Europe.Got permission listing in 2006, be applied to clinical in China.China is one of Levetiracetam bulk drug leading exporter, and market outlook are very wide.
The preparation method of the Levetiracetam of bibliographical information mainly contains chemical resolution method and utilizes the synthetic method of chiral amino acid as starting raw material at present.Traditional chemical method for splitting is with racemic Etiracetam intermediate (±)-alpha-ethyl-2-oxo-1-pyrrolidyl acetate and optically pure resolution reagent (R)-(+)-α-phenyl-ethyl amine or chirality phosphine generation salt, crystallisation by cooling is isolated it corresponding 2 enantiomorphs again.Liu Yuejin etc. (synthesizing of Levetiracetam and derivative thereof. Chinese Journal of New Drugs, 2007,16 (11): 860-864) utilize (R)-(+)-α-phenyl-ethyl amine as (2-Oxo-1-pyrroliding derivatives such as chiral selectors and Surtees J, process for preparing them andtheir uses.WO:0164637A1 2001-9-7) utilizes the chirality phosphine to split as chiral reagent.The yield of traditional enantiomorph split process is lower.
Xu Baocai etc. (the synthesis technique progress of (S)-alpha-ethyl-2-oxo-1-pyrrolidineacetamide. meticulous and specialty chemicals .2004,12 (24): 15-17) and Zhou Xianbo etc. (Synthesis of Anticonvulsant Drug Levetiracetam. fine-chemical intermediate .2005,35 (2): 27-28) respectively by (S)-(-)-α-[2-(first sulfydryl) ethyl]-2-OXo-1-pyrrolidine ethanamide or contain the amino acid of first sulfydryl, also original reagent of first sulfydryl is removed in employing, as NaBH 4/ NiCl 26H 2O or Raney's nickel (Raney nickel) are sloughed the first sulfydryl, obtain single levo form.But in reduction reaction, sulfocompound makes poisoning of catalyst easily, and therefore the consumption of active Raney's nickel is very big, is generally 7～10 times that are reduced compound weight.Because it is inflammable that active Raney's nickel is exposed in the air in drying regime, therefore, there is certain potential safety hazard in this technology when industrial applications.This technology starting raw material is a kind of sulfur-bearing chiral amino acid, and raw materials cost is higher, and is easy to generate heavy metal contamination.(Oxopyrrolidinecompounds such as Ates C, preparation of said compounds and their use in the manufacturingof levetircetam and analogues[P] .WO:03041080A2,2003-2-20) utilizing (S)-2-aminobutyric acid is raw material, obtain amino ester by esterification, intermolecular nucleophilic substitution takes place with 4-bromo-butyric acid ethyl ester molecule, closed loop is synthetic in the presence of 2 hydroxy pyrimidine, this method exists raw material reagent to cost an arm and a leg equally, loss is big, the problem that productive rate is low.
(3) summary of the invention
The object of the invention provides a strain and can be used for the novel bacterial that biocatalysis asymmetric hydrolysis racemic modification α-ethyl-2-oxygen-1-tetramethyleneimine ethyl ester prepares optically pure (S)-α-ethyl-2-oxygen-1-pyrrolidine acetic acid---anti-tyrosine tomb village Salmonella (Tsukamurella tyrosinosolvens) E105, and the application of this bacterial classification in microbial method preparation (S)-α-ethyl-2-oxygen-1-pyrrolidine acetic acid.
The technical solution used in the present invention is:
Anti-tyrosine tomb village Salmonella (Tsukamurella tyrosinosolvens) E105 is preserved in Chinese typical culture collection center (CCTCC), the address: Chinese Wuhan Wuhan University, 430072, preservation date: on December 16th, 2009, deposit number: CCTCC NO:M 209306.
The bacterial classification source: separation screening obtains near the soil sample of anti-tyrosine tomb village Salmonella (Tsukamurella tyrosinosolvens) E105 bacterial strain system picking up from auspicious symbol town, Hangzhou, Zhejiang province.Concrete screening method is as follows: will gather soil sample and be added in the stroke-physiological saline solution and shake up, absorption 0.5mL soil supension is seeded to the 250mL that the 50mL enrichment medium is housed and shakes in the bottle, 30 ℃, 200rpm, cultivate 4～5d, treat that nutrient solution becomes muddiness after, get the 1mL nutrient solution and be forwarded in the fresh enrichment medium, continue to cultivate 4～5d, so repeat enrichment culture 3～4 times.Be sole carbon source with racemic modification α-ethyl-2-oxygen-1-tetramethyleneimine ethyl ester in the enrichment medium.Concrete prescription is as follows: racemic modification α-ethyl-2-oxygen-1-tetramethyleneimine ethyl ester 10mmol/L, (NH 4) 2SO 42g/L, K 2HPO 42g/L, KH 2PO 41g/L, NaCl 0.5g/L, MgSO 47H 2O 0.5g/L, pH 7.0.
Enrichment culture liquid is applied on the separating plate through gradient dilution subsequently, obtains single bacterium colony after separating for several times.The plate culture medium prescription is the agar that adds 15g/L in the enrichment medium.With single colony inoculation seed culture medium of picking, cultivated 24 hours, be forwarded to again to produce in the enzyme substratum and cultivated 48 hours.Adopt the corresponding body excessive value (e.e. value) of chiral gas chromatography testing goal product (S)-α-ethyl-2-oxygen-1-pyrrolidine acetic acid, screening obtains described microorganism strains---anti-tyrosine tomb village Salmonella (Tsukamurella tyrosinosolvens) E105.
The feature of this novel bacterial is as follows:
Colonial morphology: cultivate 36h for 30 ℃, it is irregular that bacterium colony is, and the edge is the decomposite leaf shape, and is flat, white, slightly jaundice, drying, tarnish.Thalline is thin rod shape.
Cellular form: cell is straight or crooked bacillus [(0.5～0.8) μ m * (1.0～5) μ m], can arrange or be gathered into clump single, in pairs.
Physiological and biochemical property: the new bacterial strain that from soil, screens, Gram-positive has weak extremely strong resistance to acid, and obligate is aerobic.Can utilize beta-cyclodextrin, dextrin, glycogen, inulin, the apricot glycosides, the D-arabitol, the D-cellobiose, the L-trehalose, the D-semi-lactosi, D-glucose, the m-Inositol nf12 99, maltose, the D-glycosides reveals sugar, the D-melizitose, the D-melibiose, Alpha-Methyl-D-galactoside, 3-methyl D-glucose, Alpha-Methyl-D-glucoside, Beta-methyl-D-glucoside, Alpha-Methyl-D-mannoside, the D-psicose, D-ribose, saligenin, stachyose, turanose, the D-wood sugar, acetic acid, beta-hydroxy-butanoic acid, α-Tong Wuersuan, α-ketogulonic acid, lactic amide, the D-oxysuccinic acid, propionic acid, pyruvic acid, succinamic acid, succsinic acid, the succsinic acid methyl ester, the L-Beta Alanine, the L-Serine, uridine-5 '-phosphoric acid salt, the D-fructose-6-phosphate.Can not utilize alpha-cylodextrin, Tween-40, tween-80, L-arabinose, D-fructose, the D-galactosonic acid, gentiobiose, α-D-lactose, lactulose, trisaccharide maltose, D-N.F,USP MANNITOL, Beta-methyl-D-galactoside, the L-rhamnosyl, sedoheptulosan, sucrose, tagatose, the D-Xylitol, the D-wood sugar, gamma-hydroxybutyric acid, D-lactic acid methyl ester, L-lactic acid, the P-HPAA, the D-Beta Alanine, the L-alanyl-glycine, L-L-glutamic acid, glycylalanine, putrescine, 2, the 3-butyleneglycol, the glycyl proline(Pro), 2 '-desoxy sugar, Trophicardyl, uridine, thymidine-5 '-phosphoric acid salt, alpha-D-glucose-1-phosphoric acid salt, D-G-6-P salt.
16S rDNA Sequence Identification: total DNA is a template with the cell that extracts, and utilizes the 16S rDNA gene of universal primer P1 and P2 amplification bacterial strain, the PCR product is carried out 1% agarose gel electrophoresis again.The 16S rDNA gene order of confirming described anti-tyrosine tomb village Salmonella E105 through order-checking is as follows:
ACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGGTAAGGCCCTTCGGGGTACACGAGTGGCGAACGGGTGAGTAACACGTGGGTGACCTGCCCTGTACTTCGGGATAAGCCTGGGAAACTGGGTCTAATACCGGATATGACCTTCCTCTGCATGGGGGTTGGTGGAAAGCTTTTGCGGTACAGGATGGGCCCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGGCCTGAGAGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCTACGACGAAGCGCAAGTGACGGTAGTAGCAGAAGAAGCACCGGCCAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTGTCCGGATTTACTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCACGTCGTCTGTGAAAACCCGAGGCTTAACCTCGGGCCTGCAGGCGATACGGGCAGACTTGAGTACTGTAGGGGAGACTGGAATTCCTGGTGTAGCGGTGGAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGCAGTAACTGACGCTGAGGAGCGAAAGCGTGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGTACTAGGTGTGGGTTTCCTTCCACGGGATCCGTGCCGTAGCTAACGCATTAAGTACCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGTTTGACATATAGAGGATCGCCGGAGAGATTCGGTTTGCCTTGTGCCTTCTATACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCTCATGTTGCCAGCACGTTATGGTGGGGACTCGTGAGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGCGCGTACAGAGGGCTGCGATACCGTGAGGTGGAGCGAATCCCTTAAAGCGCGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAACACCCGAAGCCGGTGGCCTAACCCCTTGTGGGAGGGAGCTGTCGAAGG。
This sequence has been submitted GenBank (the GenBank accession number is No.GU318217) to, carries out similarity analysis with related data among the GenBank, and the result shows: the part bacterial strain sequence homology of E105 bacterial strain and tomb village Bordetella (Tsukamurella sp.) is higher.The Phylogenetic Relationships of E105 bacterial strain and Tsukamurella tyrosinosolvens bacterial strain IFM10913 (the GenBank accession number is No.AB478957, and sequence homology 100%/1458bp is based on 16S rRNA) is nearest.
According to physio-biochemical characteristics binding molecule biological assay, this bacterial strain is accredited as anti-tyrosine tomb village Salmonella (Tsukamurella tyrosinosolvens).
Investigated of the influence of each component of substratum by Plackett-Burman to anti-tyrosine tomb village Salmonella (Tsukamurella tyrosinosolvens) E105 strain enzyme-producing; Adopt rotation center combination experiment design method that yeast powder concentration and these two significant factors of influence of pH are optimized simultaneously, the optimization substratum that obtains anti-tyrosine tomb village Salmonella (Tsukamurella tyrosinosolvens) E105 bacterial strain consists of: glucose 10～15g/L, yeast powder 10～15g/L, NH 4Cl 7.6～9.5g/L, KH 2PO 41.3～2.0g/L, K 2HPO 40.7～1.0g/L, MgSO 40.4～0.6g/L, NaCl 0.4～0.6g/L, pH 6.0～7.0.
The culture condition of anti-tyrosine tomb village Salmonella (Tsukamurella tyrosinosolvens) E105 is: initial pH 7.0, shake bottled liquid measure 60～110mL/250mL Erlenmeyer flask, 30 ℃ of culture temperature, shaking speed 200rpm, inoculum size 2～7%, incubation time 36h.
Microorganism involved in the present invention is to obtain by following program screening:
1) enrichment culture: the soil sample of gathering is inserted in the enrichment medium, put 30 ℃, the shaking table of 200rpm is cultivated 4～5d, after treating that nutrient solution becomes muddiness, get the 1mL nutrient solution and be forwarded in the fresh enrichment medium, continue to cultivate 4～5d, so repeat enrichment culture 3～4 times.The enrichment culture based formulas is as follows: racemic modification α-ethyl-2-oxygen-1-tetramethyleneimine ethyl ester 10mmol/L, (NH 4) 2SO 42g/L, K 2HPO 42g/L, KH 2PO 41g/L, NaCl 0.5g/L, MgSO 47H 2O 0.5g/L, pH 7.0.
2) the dull and stereotyped cultivation: enrichment culture liquid is suitably diluted back spread plate substratum, and plate culture medium consists of the agar that adds 15g/L in the enrichment medium.
3) slant culture: the slant culture based formulas is as follows: glucose 10g/L, peptone 5g/L, yeast powder 2g/L, (NH 4) 2SO 42g/L, K 2HPO 42g/L, KH 2PO 41g/L, NaCl 0.5g/L, MgSO 47H 2O 0.5g/L, agar 15g/L, pH 7.0.Cultivate 2～3d for 30 ℃, 4 ℃ of refrigerators are preserved.
4) seed culture: choose a ring thalline and insert the 250mL that the 75mL seed culture medium is housed and shake the bottle from cultivating sophisticated inclined-plane, 30 ℃, 200r/min cultivated 24 hours.The seed culture based formulas is as follows: glucose 10g/L, peptone 5g/L, yeast powder 2g/L, (NH 4) 2SO 42g/L, K 2HPO 42g/L, KH 2PO 41g/L, NaCl 0.5g/L, MgSO 47H 2O 0.5g/L, pH 7.0.
5) fermentation culture: the inoculum size with 4% is transferred to the 250mL that the 70mL fermention medium is housed with seed liquor and shakes in the bottle, and 30 ℃, 200r/min cultivated 48 hours.Fermentative medium formula is with the seed substratum.
6) bioconversion reaction: the centrifugal wet thallus that obtains is suspended in the phosphate buffered saline buffer, adds a certain amount of racemic modification α-ethyl-2-oxygen-1-tetramethyleneimine ethyl ester, put in 30 ℃ of shaking tables and react certain hour.Reaction obtains supernatant liquor after finishing behind the centrifugation thalline, adopt the HPLC method to measure the productive rate of purpose product.
7) derivatization method: be about to transform products therefrom alpha-ethyl-2-oxo-1-pyrrolidine acetic acid esterification and become alpha-ethyl-2-oxo-1-tetramethyleneimine methyl esters.Will be after the above-mentioned gained supernatant liquor drying add the dissolving of 1.8mL anhydrous methanol, stir shake up bottle A.In the 0.4mL anhydrous methanol in the SOCl that drips 0.104mL below 0 ℃ gradually 2, strict controlled temperature gets bottle B.Bottle B is splashed into a bottle A gradually, drip off in 18～24s.Control reaction temperature is reacted 3h at 15 ℃.Pour reactant into a bottle C after reaction finishes, drip the ethanolic soln (the hydramine volume ratio is 2: 1) of triethylamine, in and pH to 8, temperature is unsuitable too high when noticing that control drips, and puts into refrigerator after neutralization finishes and spends the night.Take out to filter next day, use methanol wash filtrate again, in 40 ℃ of following vacuum-dryings to constant weight.Behind acetic acid ethyl dissolution, adopt the gas chromatography determination optical purity.
6) analyzing and testing:
HPLC chromatographic condition: adopt the Agilent1200 high performance liquid chromatograph; Chromatographic column: Agilent-C 18Post (5 μ m, 4.6 * 250nm); Detect wavelength: 210nm; Moving phase: methyl alcohol/ammonium acetate buffer (20mmol/L, pH 5.5)=20: 80; Flow velocity: 0.7mL/min; Column temperature: room temperature; Sample size: 10 μ L.The calculation of yield formula is:
Y (productive rate)=N Product/ N Substrate* 100%
In the formula, N ProductFor transforming the volumetric molar concentration of products therefrom alpha-ethyl-2-oxo-1-pyrrolidine acetic acid, N SubstrateThe volumetric molar concentration of substrate alpha-ethyl-2-oxo-1-tetramethyleneimine ethyl ester when reacting initial.
GC conditions: adopt Tianjin, island GC-2014 gas chromatograph to detect chiral chromatographic column HPChiral 10%Cyclodextrin (30m * 0.25mm * 0.25 μ m).Carrier gas is a nitrogen; 250 ℃ of sampler temperature; 120～180 ℃ of chromatogram column temperatures; 250 ℃ of detector temperatures; Heat-up rate: 2.5 ℃/min; Sample size: 0.6 μ L; Splitting ratio 15: 1; The hydrogen ion flame detector.Gas chromatogram is seen Fig. 1～3.The optical purity of product is characterized by enantiomeric excess value (e.e.), and calculating formula is:
e.e.＝(C S-C R)/(C S+C R)×100％
In the formula, C SBe the concentration of (S)-alpha-ethyl-2-oxo-1-tetramethyleneimine methyl esters, C RConcentration for (R)-alpha-ethyl-2-oxo-1-tetramethyleneimine methyl esters.
The invention still further relates to the application of described anti-tyrosine tomb village Salmonella E105 in chirality biocatalysis preparation (S)-α-ethyl-2-oxygen-1-pyrrolidine acetic acid.
Concrete, described being applied as: with racemic modification α-ethyl-2-oxygen-1-tetramethyleneimine ethyl ester is substrate, add anti-tyrosine tomb village Salmonella (Tsukamurella tyrosinosolvens) E105 wet thallus cell, under 25 ℃～35 ℃, carried out conversion reaction 12～60 hours in pH is 6.0～8.0 damping fluid, reaction finishes afterreaction liquid and obtains (S)-α-ethyl-2-oxygen-1-pyrrolidine acetic acid through separation and purification.
Substrate racemic modification α-ethyl in the described damping fluid-2-oxygen-1-tetramethyleneimine ethyl ester starting point concentration is 5.2～31.2g/L, and the starting point concentration of anti-tyrosine tomb village Salmonella E105 wet thallus cell is 10～35g/L with dry weight.
Preferably, described conversion reaction is carried out in pH is 7.0 phosphate buffered saline buffer.
Described anti-tyrosine tomb village Salmonella E105 wet thallus cell is prepared by following method: with anti-tyrosine tomb village Salmonella E105 bacterial classification inoculation to fermention medium, 30 ℃, 200rpm shaking culture 36 hours, centrifugal, the washing precipitation of fermented liquid is collected and is obtained the wet thallus cell; Described fermention medium final concentration is composed as follows: glucose 10～35g/L, yeast powder 5～30g/L, NH 4Cl 1.9～11.4g/L, K 2HPO 40.7～3.3g/L, KH 2PO 40.3～1.7g/L, MgSO 47H 2O 0.2～1.0g/L, NaCl 0.2～1g/L, pH 6.3～7.5.
Concrete, described application method is as follows:
(1) with anti-tyrosine tomb village Salmonella E105 bacterial classification inoculation to fermention medium, 30 ℃, 200rpm shaking culture 36 hours, centrifugal, the washing precipitation of fermented liquid is collected and is obtained the wet thallus cell; Described fermention medium final concentration is composed as follows: glucose 15g/L, yeast powder 12.1g/L, NH 4Cl 9.5g/L, KH 2PO 41g/L, K 2HPO 42g/L, MgSO 40.6g/L, NaCl 0.6g/L, pH 7.0;
(2) getting 100mM pH is 7.0 phosphate buffered saline buffer, add step (1) gained wet thallus cell, described wet thallus cell concn is counted 30g/L with dry weight, and α-ethyl-2-oxygen-1-tetramethyleneimine ethyl ester to concentration of substrate is 15.2g/L to add the substrate racemic modification, 30 ℃ of shaking table oscillatory reactions 36 hours, reaction is centrifugal with reaction solution after finishing, and gets supernatant liquor, add equal volume of ethyl acetate twice, obtain (S)-α-ethyl-2-oxygen-1-pyrrolidine acetic acid.
Beneficial effect of the present invention is mainly reflected in: (S)-α-ethyl-2-oxygen-1-pyrrolidine acetic acid preparation method of bibliographical information mainly contains chemical resolution method and utilizes the chemical synthesis process of chiral amino acid as starting raw material at present.The present invention adopts microbial method preparation (S)-α-ethyl-2-oxygen-1-pyrrolidine acetic acid first, and provides a strain to have highly-solid selectively, can prepare the novel bacterial of (S)-α-ethyl-2-oxygen-1-pyrrolidine acetic acid of high-optical-purity.The present invention can make the optical purity of purpose product (S)-α-ethyl-2-oxygen-1-pyrrolidine acetic acid reach 99.4%e.e by the chirality biocatalysis that adopts anti-tyrosine tomb village Salmonella (Tsukamurella tyrosinosolvens) E105 cell, and productive rate reaches 48.1%.The present invention obtains novel bacterial by screening, thereby prepares the crucial chiral intermediate of Levetiracetam medicine at the research microbial method, and is providing beneficial reference aspect the process optimization of bio-transformation.
(4) description of drawings
Fig. 1 is racemic modification α-ethyl-2-oxygen-1-pyrrolidine acetic acid reference substance derivative color atlas;
Fig. 2 is (S)-α-ethyl-2-oxygen-1-pyrrolidine acetic acid reference substance derivative color atlas;
Fig. 3 is the derivative color atlas of bio-transformation gained high-optical-purity product;
In Fig. 1～3: 1 is (S)-α-ethyl-2-oxygen-1-pyrrolidine acetic acid derivative; 2 is (R)-α-ethyl-2-oxygen-1-pyrrolidine acetic acid derivative.
(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:
Productive rate and optical purity detection method are as follows among the embodiment:
1) the HPLC method is measured productive rate: bio-transformation extracts reaction solution through the centrifugal supernatant liquor that obtains after finishing, and adopts the HPLC method to measure productive rate.
HPLC chromatographic condition: adopt the Agilent1200 high performance liquid chromatograph; Chromatographic column: Agilent-C 18Post (5 μ m, 4.6 * 250nm); Detect wavelength: 210nm; Moving phase: methyl alcohol/ammonium acetate buffer (20mmol/L, pH=5.5)=20: 80; Flow velocity: 0.7ml/min; Column temperature: room temperature; Sample size: 10 μ L.The calculation of yield formula is:
Y (productive rate)=N Product/ N Substrate* 100%
In the formula, N ProductBe the volumetric molar concentration of purpose product alpha-ethyl-2-oxo-1-pyrrolidine acetic acid, N The end ThingThe volumetric molar concentration of substrate alpha-ethyl-2-oxo-1-tetramethyleneimine ethyl ester when reacting initial.
2) the GC method is measured the product optical purity: the bio-transformation products therefrom is earlier through derivatization treatment, and promptly elder generation adopts gas chromatography determination product optical purity after product alpha-ethyl-2-oxo-1-pyrrolidine acetic acid esterification is become alpha-ethyl-2-oxo-1-tetramethyleneimine methyl esters.
Derivatization method: after treating that biological conversion reaction finishes, extract reaction solution, will add the dissolving of 1.8mL anhydrous methanol after the above-mentioned gained supernatant liquor drying through the centrifugal supernatant liquor that obtains, stir shake up bottle A.In the 0.4mL anhydrous methanol in the SOCl that drips 0.104mL below 0 ℃ gradually 2, strict controlled temperature gets bottle B.Bottle B is splashed into a bottle A gradually, drip off in 18～24s.Control reaction temperature is reacted 3h at 15 ℃.Reaction is poured reactant into a bottle C after finishing, and drips the ethanolic soln (the hydramine volume ratio is 2: 1) of triethylamine, and being neutralized to pH is 8, and temperature was unsuitable too high when attention control dripped, and puts into refrigerator after neutralization finishes and spends the night.Take out to filter next day, use methanol wash filtrate again, in 40 ℃ of following vacuum-dryings to constant weight.Behind acetic acid ethyl dissolution, adopt gas chromatography determination product optical purity.
GC chromatographic condition: adopt Tianjin, island GC-2014 gas chromatograph to detect chiral chromatographic column HPChiral 10%Cyclodextrin (30m * 0.25mm * 0.25 μ m).Carrier gas is a nitrogen; 250 ℃ of sampler temperature; 120～180 ℃ of chromatogram column temperatures; 250 ℃ of detector temperatures; Heat-up rate: 2.5 ℃/min; Sample size: 0.6 μ L; Splitting ratio 15: 1; The hydrogen ion flame detector.The optical purity of product is characterized by enantiomeric excess value (e.e.), and calculating formula is:
e.e.＝(C S-C R)/(C S+C R)×100％
In the formula, C SBe the concentration of (S)-alpha-ethyl-2-oxo-1-tetramethyleneimine methyl esters, C RConcentration for (R)-alpha-ethyl-2-oxo-1-tetramethyleneimine methyl esters.
Embodiment 1: the separation of bacterial strain
In 10mL 0.85% physiological saline, add the 1g soil sample, shake up, make into uniform suspension; Draw the 0.5mL soil supension and be inoculated in the 250mL triangular flask that the 50mL enrichment medium is housed, 30 ℃, 200rpm, shake-flask culture 4～5 days, treat that nutrient solution becomes muddiness after, get the 1mL nutrient solution and be forwarded in the fresh enrichment medium, continue to cultivate 4～5 days, so repeat enrichment culture 3～4 times.Pregnant solution is applied on the separating plate through gradient dilution, obtains single bacterium colony after separating for several times.
Enrichment medium is a sole carbon source with racemic modification α-ethyl-2-oxygen-1-tetramethyleneimine ethyl ester, and its prescription is as follows: racemic modification α-ethyl-2-oxygen-1-tetramethyleneimine ethyl ester 10mmol/L, (NH 4) 2SO 42g/L, K 2HPO 42g/L, KH 2PO 41g/L, NaCl 0.5g/L, MgSO 47H 2O 0.5g/L, pH 7.0, prepare with distilled water.
Single colony inoculation seed culture medium with picking, cultivated 24 hours, being forwarded to produce cultivates after 48 hours again in the enzyme substratum, detect the enantiomeric excess value (e.e. value) of product (S)-α-ethyl-2-oxygen-1-pyrrolidine acetic acid with chiral gas chromatography, screening obtains described microorganism strains---anti-tyrosine tomb village Salmonella (Tsukamurella tyrosinosolvens) E105.
Embodiment 2: the acquisition of wet thallus cell
Seed culture based formulas: glucose 15g/L, yeast powder 12.1g/L, NH 4Cl 9.5g/L, KH 2PO 41g/L, K 2HPO 42g/L, MgSO 40.6g/L, NaCl 0.6g/L, pH 7.0;
Fermentative medium formula: glucose 15g/L, yeast powder 12.1g/L, NH 4Cl 9.5g/L, KH 2PO 41g/L, K 2HPO 42g/L, MgSO 40.6g/L, NaCl 0.6g/L, pH 7.0;
Choose a ring thalline and insert the 250mL that the 50mL seed culture medium is housed and shake the bottle from cultivating sophisticated inclined-plane, 30 ℃, 200rpm cultivates and got seed liquor in 24 hours, with the inoculum size of volume ratio 4% seed liquor being transferred to the 250mL that the 100mL fermention medium is housed again shakes in the bottle, 30 ℃, 200rpm cultivated 36 hours.It is centrifugal and with the phosphoric acid buffer washing of pH8.0 once cultivate to finish secondary fermentation liquid, and collection wet thallus cell is standby.
Embodiment 3:
The wet thallus of embodiment 2 gained is suspended in the phosphate buffered saline buffer (0.1mol/L, pH 6); Racemic modification α-ethyl-2-oxygen-1-tetramethyleneimine the ethyl ester that adds 10.4g/L is as substrate, and cell concn (with dry weight basis) is 20g/L, places 30 ℃, reacts 24h in the shaking table of 200rpm.After reaction finished, reaction solution was centrifugal, get supernatant liquor, adopted aforementioned detection method to measure the productive rate and the optical purity of product (S)-α-ethyl-2-oxygen-1-pyrrolidine acetic acid, and productive rate is 37.7%, optical purity 64%e.e..
Embodiment 4:
The wet thallus of embodiment 2 gained is suspended in the phosphate buffered saline buffer (0.1mol/L, pH 6.8); Add 10.4g/L racemic modification α-ethyl-2-oxygen-1-tetramethyleneimine ethyl ester is as substrate, cell concn is 20g/L, places 30 ℃, isothermal reaction 24h in the shaking table of 200rpm.After reaction finished, reaction solution was centrifugal, get supernatant liquor, adopted aforementioned detection method to measure the productive rate and the optical purity of product (S)-α-ethyl-2-oxygen-1-pyrrolidine acetic acid, and productive rate is 45.3%, optical purity 98.3%e.e..
Embodiment 5:
The wet thallus of embodiment 2 gained is suspended in the phosphate buffered saline buffer (0.1mol/L, pH 8.0); Add 10.4g/L racemic modification α-ethyl-2-oxygen-1-tetramethyleneimine ethyl ester is as substrate, cell concn is 20g/L, places 30 ℃, isothermal reaction 24h in the shaking table of 200rpm.After reaction finished, reaction solution was centrifugal, get supernatant liquor, adopted aforementioned detection method to measure the productive rate and the optical purity of product (S)-α-ethyl-2-oxygen-1-pyrrolidine acetic acid, and productive rate is 46.2%, optical purity 90.3%e.e..
Embodiment 6:
The wet thallus of embodiment 2 gained is suspended in the phosphate buffered saline buffer (0.1mol/L, pH 7.0); Add 10.4g/L racemic modification α-ethyl-2-oxygen-1-tetramethyleneimine ethyl ester is as substrate, cell concn is 10g/L, places 30 ℃, isothermal reaction 24h in the shaking table of 200rpm.After reaction finished, reaction solution was centrifugal, get supernatant liquor, adopted aforementioned detection method to measure the productive rate and the optical purity of product (S)-α-ethyl-2-oxygen-1-pyrrolidine acetic acid, and productive rate is 43.2%, optical purity 85.5%e.e..
Embodiment 7:
The wet thallus of embodiment 2 gained is suspended in the phosphate buffered saline buffer (0.1mol/L, pH 7.0); Add 10.4g/L racemic modification α-ethyl-2-oxygen-1-tetramethyleneimine ethyl ester is as substrate, cell concn is 35g/L, places 30 ℃, isothermal reaction 24h in the shaking table of 200rpm.After reaction finished, reaction solution was centrifugal, get supernatant liquor, adopted aforementioned detection method to measure the productive rate and the optical purity of product (S)-α-ethyl-2-oxygen-1-pyrrolidine acetic acid, and productive rate is 54.2%, optical purity 87.7%e.e..
Embodiment 8:
The wet thallus of embodiment 2 gained is suspended in the phosphate buffered saline buffer (0.1mol/L, pH 7.0); Add 15.2g/L racemic modification α-ethyl-2-oxygen-1-tetramethyleneimine ethyl ester is as substrate, cell concn is 30g/L, places 30 ℃, isothermal reaction 36h in the shaking table of 200rpm.After reaction finished, reaction solution was centrifugal, get supernatant liquor, adopted aforementioned detection method to measure the productive rate and the optical purity of product (S)-α-ethyl-2-oxygen-1-pyrrolidine acetic acid, and productive rate is 48.1%, optical purity 99.4%e.e..
Embodiment 9:
The wet thallus of embodiment 2 gained is suspended in the phosphate buffered saline buffer (0.1mol/L, pH 8.0); Add 5.2g/L racemic modification α-ethyl-2-oxygen-1-tetramethyleneimine ethyl ester is as substrate, cell concn is 20g/L, places 35 ℃, isothermal reaction 48h in the shaking table of 200rpm.After reaction finished, reaction solution was centrifugal, get supernatant liquor, adopted aforementioned detection method to measure the productive rate and the optical purity of product (S)-α-ethyl-2-oxygen-1-pyrrolidine acetic acid, and productive rate is 24.8%, optical purity 71.9%e.e..
Embodiment 10:
The wet thallus of embodiment 2 gained is suspended in the phosphate buffered saline buffer (0.1mol/L, pH 8.0); Add 31.2g/L racemic modification α-ethyl-2-oxygen-1-tetramethyleneimine ethyl ester is as substrate, cell concn is 20g/L, places 35 ℃, isothermal reaction 48h in the shaking table of 200rpm.After reaction finished, reaction solution was centrifugal, get supernatant liquor, adopted aforementioned detection method to measure the productive rate and the optical purity of product (S)-α-ethyl-2-oxygen-1-pyrrolidine acetic acid, and productive rate is 49.8%, optical purity 24.6%e.e..
SEQUENCELISTING
＜110〉Zhejiang Polytechnical University
＜120〉anti-tyrosine tomb village Salmonella and catalytic preparation (S)-α-ethyl thereof-2-oxygen-1-pyrrolidine acetic acid
<130>
<160>1
<170>PatentIn?version?3.4
<210>1
<211>1416
<212>DNA
<213>Tsukamurella?tyrosinosolvens
<400>1
acgctggcgg?cgtgcttaac?acatgcaagt?cgaacggtaa?ggcccttcgg?ggtacacgag 60
tggcgaacgg?gtgagtaaca?cgtgggtgac?ctgccctgta?cttcgggata?agcctgggaa 120
actgggtcta?ataccggata?tgaccttcct?ctgcatgggg?gttggtggaa?agcttttgcg 180
gtacaggatg?ggcccgcggc?ctatcagctt?gttggtgggg?taatggccta?ccaaggcgac 240
gacgggtagc?cggcctgaga?gggcgaccgg?ccacactggg?actgagacac?ggcccagact 300
cctacgggag?gcagcagtgg?ggaatattgc?acaatgggcg?caagcctgat?gcagcgacgc 360
cgcgtgaggg?atgacggcct?tcgggttgta?aacctctttc?agctacgacg?aagcgcaagt 420
gacggtagta?gcagaagaag?caccggccaa?ctacgtgcca?gcagccgcgg?taatacgtag 480
ggtgcgagcg?ttgtccggat?ttactgggcg?taaagagctc?gtaggcggtt?tgtcacgtcg 540
tctgtgaaaa?cccgaggctt?aacctcgggc?ctgcaggcga?tacgggcaga?cttgagtact 600
gtaggggaga?ctggaattcc?tggtgtagcg?gtggaatgcg?cagatatcag?gaggaacacc 660
ggtggcgaag?gcgggtctct?gggcagtaac?tgacgctgag?gagcgaaagc?gtgggtagcg 720
aacaggatta?gataccctgg?tagtccacgc?cgtaaacggt?gggtactagg?tgtgggtttc 780
cttccacggg?atccgtgccg?tagctaacgc?attaagtacc?ccgcctgggg?agtacggccg 840
caaggctaaa?actcaaagga?attgacgggg?gcccgcacaa?gcggcggagc?atgtggatta 900
attcgatgca?acgcgaagaa?ccttacctgg?gtttgacata?tagaggatcg?ccggagagat 960
tcggtttgcc?ttgtgccttc?tatacaggtg?gtgcatggct?gtcgtcagct?cgtgtcgtga 1020
gatgttgggt?taagtcccgc?aacgagcgca?acccttgtct?catgttgcca?gcacgttatg 1080
gtggggactc?gtgagagact?gccggggtca?actcggagga?aggtggggat?gacgtcaagt 1140
catcatgccc?cttatgtcca?gggcttcaca?catgctacaa?tggcgcgtac?agagggctgc 1200
gataccgtga?ggtggagcga?atcccttaaa?gcgcgtctca?gttcggattg?gggtctgcaa 1260
ctcgacccca?tgaagtcgga?gtcgctagta?atcgcagatc?agcaacgctg?cggtgaatac 1320
gttcccgggc?cttgtacaca?ccgcccgtca?cgtcatgaaa?gtcggtaaca?cccgaagccg 1380
gtggcctaac?cccttgtggg?agggagctgt?cgaagg 1416

Claims (8)

1. anti-tyrosine tomb village Salmonella (Tsukamurella tyrosinosolvens) E105, be preserved in Chinese typical culture collection center (CCTCC), the address: Chinese Wuhan Wuhan University, 430072, preservation date: on December 16th, 2009, deposit number: CCTCC NO:M 209306.
2. anti-tyrosine tomb as claimed in claim 1 village Salmonella E105 is characterized in that the 16S rDNA gene order of described anti-tyrosine tomb village Salmonella E105 is as follows:
ACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGGTAAGGCCCTTCGGGG
TACACGAGTGGCGAACGGGTGAGTAACACGTGGGTGACCTGCCCTGTACTTC
GGGATAAGCCTGGGAAACTGGGTCTAATACCGGATATGACCTTCCTCTGCATG
GGGGTTGGTGGAAAGCTTTTGCGGTACAGGATGGGCCCGCGGCCTATCAGCT
TGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGGCCTGAGA
GGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGG
CAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGC
GTGAGGGATGACGGCCTTCGGGTTGTAAACCTCTTTCAGCTACGACGAAGCG
CAAGTGACGGTAGTAGCAGAAGAAGCACCGGCCAACTACGTGCCAGCAGCC
GCGGTAATACGTAGGGTGCGAGCGTTGTCCGGATTTACTGGGCGTAAAGAGC
TCGTAGGCGGTTTGTCACGTCGTCTGTGAAAACCCGAGGCTTAACCTCGGGC
CTGCAGGCGATACGGGCAGACTTGAGTACTGTAGGGGAGACTGGAATTCCTG
GTGTAGCGGTGGAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCG
GGTCTCTGGGCAGTAACTGACGCTGAGGAGCGAAAGCGTGGGTAGCGAACA
GGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGTACTAGGTGTGGG
TTTCCTTCCACGGGATCCGTGCCGTAGCTAACGCATTAAGTACCCCGCCTGGG
GAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAA
GCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGT
TTGACATATAGAGGATCGCCGGAGAGATTCGGTTTGCCTTGTGCCTTCTATAC
AGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCC
CGCAACGAGCGCAACCCTTGTCTCATGTTGCCAGCACGTTATGGTGGGGACT
CGTGAGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAGT
CATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGCGCGTACAGA
GGGCTGCGATACCGTGAGGTGGAGCGAATCCCTTAAAGCGCGTCTCAGTTCG
GATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAG
ATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTC
ACGTCATGAAAGTCGGTAACACCCGAAGCCGGTGGCCTAACCCCTTGTGGGA
GGGAGCTGTCGAAGG。
3. the application of anti-tyrosine tomb as claimed in claim 1 village Salmonella E105 in chirality biocatalysis preparation (S)-α-ethyl-2-oxygen-1-pyrrolidine acetic acid.
4. application as claimed in claim 3, it is characterized in that described being applied as: with racemic modification α-ethyl-2-oxygen-1-tetramethyleneimine ethyl ester is substrate, add anti-tyrosine tomb village Salmonella (Tsukamurellatyrosinosolvens) E105 wet thallus cell, under 25 ℃～35 ℃, carried out conversion reaction 12～60 hours in pH is 6.0～8.0 damping fluid, reaction finishes afterreaction liquid and obtains (S)-α-ethyl-2-oxygen-1-pyrrolidine acetic acid through separation and purification.
5. application as claimed in claim 4, it is characterized in that substrate racemic modification α-ethyl in the described damping fluid-2-oxygen-1-tetramethyleneimine ethyl ester starting point concentration is 5.2～31.2g/L, the starting point concentration of anti-tyrosine tomb village Salmonella E105 wet thallus cell is 10～35g/L with dry weight.
6. application as claimed in claim 4 is characterized in that described conversion reaction carries out in pH is 7.0 phosphate buffered saline buffer.
7. application as claimed in claim 4, it is characterized in that: described anti-tyrosine tomb village Salmonella E105 wet thallus cell is prepared by following method: with anti-tyrosine tomb village Salmonella E105 bacterial classification inoculation to fermention medium, 30 ℃, 200rpm shaking culture 36 hours, centrifugal, the washing precipitation of fermented liquid is collected and is obtained the wet thallus cell; Described fermention medium final concentration is composed as follows: glucose 10～35g/L, yeast powder 5～30g/L, NH 4Cl1.9～11.4g/L, K 2HPO 40.7～3.3g/L, KH 2PO 40.3～1.7g/L, MgSO 47H 2O 0.2～1.0g/L, NaCl 0.2～1g/L, pH 6.3～7.5.
8. application as claimed in claim 4 is characterized in that described application is as follows:
(1) with anti-tyrosine tomb village Salmonella E105 bacterial classification inoculation to fermention medium, 30 ℃, 200rpm shaking culture 36 hours, centrifugal, the washing precipitation of fermented liquid is collected and is obtained the wet thallus cell; Described fermention medium final concentration is composed as follows: glucose 15g/L, yeast powder 12.1g/L, NH 4Cl 9.5g/L, KH 2PO 41lgL, K 2HPO 42g/L, MgSO 40.6g/L, NaCl 0.6g/L, pH 7.0;
(2) getting 100mM pH is 7.0 phosphate buffered saline buffer, add step (1) gained wet thallus cell, described wet thallus cell concn is counted 30g/L with dry weight, and α-ethyl-2-oxygen-1-tetramethyleneimine ethyl ester to concentration of substrate is 15.2g/L to add the substrate racemic modification, 30 ℃ of shaking table oscillatory reactions 36 hours, reaction is centrifugal with reaction solution after finishing, and gets supernatant liquor, add equal volume of ethyl acetate twice, obtain (S)-α-ethyl-2-oxygen-1-pyrrolidine acetic acid.
CN200910155776XA 2009-12-25 2009-12-25 Tsukamurella-tyrosinosolvens and application thereof in catalysis preparation of (S) -alpha - ethyl -2-oxo-1-pyrrolidine acetic acid prepared by catalysis Expired - Fee Related CN101748087B (en)

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CN103667073A (en) * 2013-09-27 2014-03-26 浙江工业大学 Huperzia serrata endophytic fungus and application in preparation of pyrrole type liver-protecting medicines
CN106591179A (en) * 2016-12-05 2017-04-26 长兴制药股份有限公司 Methylopila sp.cxzy-L013 and application thereof to preparation of (S)-alpha-ethyl-2-oxo-1-pyrrolidine acetate by selective resolution
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CN103667073A (en) * 2013-09-27 2014-03-26 浙江工业大学 Huperzia serrata endophytic fungus and application in preparation of pyrrole type liver-protecting medicines
CN103667073B (en) * 2013-09-27 2016-04-13 浙江工业大学 Huperzia serrata endogenetic epiphyte and the application in preparation pyroles liver-protecting medicine thereof
CN106591179A (en) * 2016-12-05 2017-04-26 长兴制药股份有限公司 Methylopila sp.cxzy-L013 and application thereof to preparation of (S)-alpha-ethyl-2-oxo-1-pyrrolidine acetate by selective resolution
WO2018103409A1 (en) 2016-12-05 2018-06-14 长兴制药股份有限公司 METHYLOCYSTIS AND USE THEREOF IN SELECTIVE RESOLUTION AND PREPARATION OF (S)-α-ETHYL-2-OXO-1-PYRROLIDINE ACETATE
CN106591179B (en) * 2016-12-05 2018-07-03 长兴制药股份有限公司 Methyl packing bacterium and its prepare the application on (S)-α-ethyl -2- oxygen -1- pyrrolidine acetic acid salt in selective fractionation
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