CN103898177B - Prepare the method for high chiral purity (R)-3-piperidine alcohols and derivative thereof - Google Patents
Prepare the method for high chiral purity (R)-3-piperidine alcohols and derivative thereof Download PDFInfo
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- CN103898177B CN103898177B CN201410016356.4A CN201410016356A CN103898177B CN 103898177 B CN103898177 B CN 103898177B CN 201410016356 A CN201410016356 A CN 201410016356A CN 103898177 B CN103898177 B CN 103898177B
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Abstract
The invention provides a kind of enzyme process and prepare high chiral purity<i>(R)</i>Described in the method for-3-piperidine alcohols and derivative thereof, reaction condition is pH6.0-7.5, taking efficient coexpression restructuring alcohol dehydrogenase and recombinant glucose dehydrogenase and coenzyme in Escherichia coli as catalyst, and high yield, the preparation reduction of high-purity ground<i>(R)</i>-3-piperidine alcohols and derivative thereof, its reaction time is short, and preparation cost is low.
Description
Technical field
The present invention relates to one and prepare the method for high chiral purity (R)-3-piperidine alcohols and derivative thereof,Belong to the synthesis technical field of medicine intermediate, also belong to Green Chemistry and genetic engineering field.
Background technology
Chirality 3-piperidine alcohols is piperidine derivatives, is the key intermediate of multiple medicine, agricultural chemicals,Be one of the focus in the synthetic field of medicine, be also a kind of very important medicine molecule of the skeleton always.Chirality 3-piperidine alcohols derivative be found to have antidepression, anti-arrhythmia, antithrombotic form,Separate the effect such as spasm, calmness and reduction cholesterolemia activity, in this external active anticancer medicine alsoCommon this class formation. For example, in antitumor field, the silk of CDK(mono-class dependence Cylin/Serineprotein kinase) in the regulation and control of cell cycle, play an important role, the function of CDK is lostBetween occurring, the tumour that is in harmonious proportion exists close relationship. Flavopiridol (Flavopiridol) isOne enters the CDK inhibitor of clinical testing, and chemical constitution is flavonoids, derives from firstA kind of Indian plant Rohitukin, can manually synthesize at present. In Flavopiridol structure, contain relevant(S)-3-piperidine alcohols structure of key. In addition, also have other multi-medicaments contain (S)-3-piperidine alcohols or(R)-3-piperidine alcohols structure.
Conventionally in the time using chirality 3-piperidine alcohols, can adopt the form of N-protected, as Boc-,The N-protected forms such as Cbz-. Closing of known (S) or (R)-3-piperidine alcohols and derivative thereof at presentOne-tenth mode has multiple, is roughly divided into following approach:
(1) set out with natural chiral acid such as L MALIC ACID, L-Glu or L-Asp, through condensation,Reduction waits to be carried out, and often step complexity, all needs multistep reaction, and the chiral raw material relating to and alsoOriginal reagent is expensive, is difficult to industry and amplifies.
(2) split as (+)-dibenzoyl tartaric acid through chiral acid with the 3-piperidine alcohols of racemization, andThrough the acquisition chiral alcohol that is repeatedly recrystallized. The method is easy, but its chiral acid relating to is expensive,Yield is low, is only 24%, and chirality is difficult to be guaranteed relatively.
(3) selectively split with lipase, (S)-all can obtain with (R)-type product. ButLipase splits the highest yield of theory only 50% that obtains (S) and (R)-3-piperidine alcohols, product pointFrom and purification difficult, often need silica gel column chromatography etc., be difficult to industry amplify. It is another that enzyme splitsOne large defect is to be difficult to racemization initiation material to obtain higher yield, raw material racemization at presentStill there is no feasible short-cut method, thereby the fractionation cost of lipase is high.
(4) with microorganism or enzymatic hydroxylation mode, directly selective hydroxylating piperidones andThe derivative of its N-protected is corresponding (S)-or (R)-3-piperidine alcohols. This mode can be maximumLimit ground improves substrate utilization degree, and can obtain the chiral alcohol that is close to 100%. But at present,This approach only stays in theoretical research and laboratory stage, and concentration of substrate is less than 5g/L, does not haveActual production meaning.
Carbonyl acyl reductase (Canbonylreductase, EC1.1.1.184) and alcohol dehydrogenase(Alcoholdehydrogenase, EC1.1.1.1), all can asymmetric reduction prochiral ketones moleculeThe ketone carbonyl alcoholic extract hydroxyl group that is chirality, be a kind of important way of introducing chirality in drug molecule.This enzymatic asymmetric reduction often needs reduced coenzyme Ⅰ (NADH) or codehydrogenase Ⅱ(NADPH), reduced coenzyme Ⅰ, II exist as hydrogen donor in reduction reaction, hydrogen supplyAfter become oxidized coenzyme I (NAD+) or codehydrogenase Ⅱ (NADP+), oxidized coenzyme again canObtained hydrogen by other oxidizing ferment or dehydrogenase effect, again become reduced coenzyme, complete coenzyme and followRing.
Carbonyl acyl reductase and alcohol dehydrogenase are mainly derived from the microorganism such as yeast, bacterium, existing manyPlant this fermentoid gene and be in the news, as Candidamagnoliae(GenbankAcc.No.JC7338; And Candidaparapsilosis(GenbankAcc.No. GI:11360538)BAA24528.1; GI:2815409) etc.
At present existing multiple important chirality pharmaceutical intermediate compound can be used carbonyl acyl reductase and alcohol dehydrogenaseEnzymatic synthesis, comprises multiple synthesis mode such as pure enzyme and microbe whole-cell or immobilised enzymes/cell etc.Conventionally in synthetic system, also need to add regenerating coenzyme enzyme, as GDH (GlucoseDehydrogenase, GDH) and hydrogenlyase (Formatedehydrogenase, FDH)Deng. For example, the asymmetric reduction of the chloro-ethyl acetoacetate of 4-(as: Zhou, J.Am.Chem.Soc.1983105:5925-5926；Santaniello,J.Chem.Res.(S)1982:132-133；U.S.Pat.No.5,891,685 etc.), its reduzate (S)-4-chloro-3-hydroxyl ethyl butyrate is for heOne of key intermediate of spit of fland class medicine; The asymmetric reduction of acetophenone and derivative thereof (as:U.S.Pat.No.6,800,477); The asymmetric reduction (WO2005/054491) of thienone.At medical industry circle, still have multiple important prochiral ketones compound need to develop enzyme reducing process.
Summary of the invention
The object of the invention is to solve above-mentioned technical problem, provide one to prepare high chiral purity(R) method of-3-piperidine alcohols and derivative thereof.
Object of the present invention is achieved through the following technical solutions:
Prepare the method for high chiral purity (R)-3-piperidine alcohols and derivative thereof, described course of reaction asShown in lower:
P is hydrogen, tertbutyloxycarbonyl (Boc), benzyloxycarbonyl group (Cbz) or fluorenylmethyloxycarbonyl (Fmoc),Described reaction condition is pH6.0-7.5, de-with coexpression restructuring alcohol dehydrogenase and restructuring glucoseHydrogen enzyme and coenzyme are catalyst, described restructuring alcohol dehydrogenase and recombinant glucose dehydrogenase catalystFor liquid solution, freeze-dried powder, immobilised enzymes or immobilized cell, described restructuring alcohol dehydrogenaseAmino acid sequence as shown in sequence table SEQ .IDNO:1, described recombinant glucose dehydrogenase ammoniaBase acid sequence is as shown in sequence table SEQ .IDNO:2.
Preferably, described reaction condition is pH6.4-6.6, described restructuring alcohol dehydrogenase and restructuringGDH is efficient coexpression in genetic engineering bacterium.
Preferably, described genetic engineering bacterium is the Escherichia coli with recombinant vector pETDuet-1.
A method for the above-described genetic engineering bacterium of fermented and cultured, comprises structure genetic engineeringThe further fermentation of bacterium and genetic engineering bacterium, the structure of described genetic engineering bacterium comprises the following steps:
To the synthetic restructuring alcohol dehydrogenase encoding gene of full gene and GDH coding baseBecause of respectively through double digestion; Again it is cloned into respectively to many grams of the differences of expression vector pETDuet-1Grand anti-site, recombinant plasmid order-checking is converted into respectively expression coli strain, structure after confirmingBuild corresponding recombinant bacterial strain;
The further fermentation of described genetic engineering bacterium comprises the steps:
Above-described coli strain is seeded to the LB culture medium that contains ampicillinIn, be cultured to OD600=0.8 fresh medium, adds the ampicillin of filtration sterilizationSolution to final concentration is 0.1mg/mL, and 37 DEG C of 800rpm cultivate; Feed supplement after cultivation 2hr,Regulate pH7.0 ± 0.1 by concentrated ammonia liquor/hydrochloric acid, as the OD of nutrient solution600Reach at 25 o'clock, willTank temperature drop to 25 DEG C, adding final concentration is 1mmol/LIPTG, continues to control each condition of cultureInduction 14hr, last centrifugal results thalline.
Beneficial effect of the present invention is mainly reflected in: adopt restructuring alcohol dehydrogenase, glucose dehydrogenationEnzyme is applied to background and reduces, and concentration of substrate is up to 150g/L, and productive rate is high, product opticsPurity is high, and the reaction time is short, and preparation cost is low.
Detailed description of the invention
The present invention has disclosed the method for the high chiral purity of preparation (R)-3-piperidine alcohols and derivative thereof, instituteState course of reaction as follows:
P is hydrogen, tertbutyloxycarbonyl (Boc), benzyloxycarbonyl group (Cbz) or fluorenylmethyloxycarbonyl（-Fmoc），
Described preparation method is as follows: the Compound I of a mole is dissolved in to the slow of 500～2000mlIn dissolved liquid and organic solvent, in above-mentioned solution, adding weight is 0.1～20% of Compound IGenetic recombination alcohol dehydrogenase, GDH and coenzyme, holder tie up to 15～45 DEG C itBetween, preferentially at 25～40 DEG C. Regulate pH 6.0～7.5 with acid/alkali lye, preferably 6.4～6.6.Stir 16-72h, stop reaction, with the organic solvent extraction of 1000 milliliters of left and right 3 times, closeAnd organic phase, desiccant dryness, organic solvent is removed in decompression distillation, obtains homochiral targetCompound III. Conventionally compound III chiral purity is greater than 98%, can be used for the preparation of medicine.
Described organic solvent be selected from methyl alcohol, ethanol, propyl alcohol, butanols, the tert-butyl alcohol, isopropyl alcohol,One in oxolane, methyl tertiary butyl ether, ethyl acetate, butyl acetate and toluene.
Described cushioning liquid is inorganic sulfuric acid, inorganic phosphate or triethanolamine hydrochloric acid buffer saltIn one.
Described inorganic base is selected from NaOH, potassium hydroxide, ammoniacal liquor, sodium carbonate, potashIn one.
P in described Compound I is preferably tertbutyloxycarbonyl.
Restructuring alcohol dehydrogenase and recombinant glucose dehydrogenase are at the efficient coexpression of Escherichia coli, and it canTo be liquid solution, freeze-dried powder, can be also immobilized enzyme or cell.
Described alcohol dehydrogenase is external evolution, utilizes the enzyme of purifying or the large intestine for its expressionThe direct catalysis of bacillus engineering bacteria. It utilizes a kind of hot water high temperature coccus (ThermococcusThe variant of alcohol dehydrogenase hydrothermalis), variant has been compared 1 amino acid with wild typeDifference.
The sequence optimisation of alcohol dehydrogenase, active, thermally-stabilised round increasing on wild type basisProperty and organic solvent stability are carried out. Main employing taking structure as basic semi-directional evolved and heightFlux screening. The sequence of final gained has been compared 1 amino acid whose difference with wild type. MostlyNumerical mutation concentrates on enzyme surface and contacts site with subunit. Gene order is according to Escherichia coli preferenceCodon amendment, and eliminate the secondary structure that may affect expression. Alcohol dehydrogenase after optimization existsHigh efficient expression in E.coli, enzymatic activity is the more than 2 times of wild type, and stability is also remarkableIncrease. Alcohol dehydrogenase high activity variant after optimization and GDH are at E.coli coexpressionAfter, through thick purifying, can efficient catalytic 3-piperidones be (S)-3-piperidine alcohols.
Described GDH is external evolution, utilizes a kind of bacterium Burkholderiasp.Glucose dehydrogenase modification efficiently reduces NADP+ coenzyme, has compared 3 amino with wild typeAcid difference. Its gene order is according to the codon amendment of Escherichia coli preference, and elimination may shadowRing the secondary structure of expressing, this sequence is at the high efficient expression of E.coli.
The amino acid sequence of described restructuring alcohol dehydrogenase as shown in sequence table SEQ .IDNO:1,Described recombinant glucose dehydrogenase amino acid sequence is as shown in sequence table SEQ .IDNO:2.
Below describe it in detail greatly with restructuring alcohol dehydrogenase and recombinant glucose dehydrogenase respectivelyExpression in enterobacteria and determination of activity.
Embodiment mono-: expression and the determination of activity of alcohol dehydrogenase in E.coli
The synthetic restructuring alcohol dehydrogenase encoding gene of full gene is through NcoI and HindIII double digestionAfter, be cloned into expression vector pETDuet-1(producer: Novagen, production code member: 71146-3)MCS 1, recombinant plasmid, after order-checking is confirmed, is converted into and expresses bacterial strain E.coliBL21(DE3) in, the recombinant bacterial strain called after pETDuet-ADH-BL of structure21(DE3). ?On ampicillin flat board, select single bacterium colony, access contains in corresponding antibiotic LB culture medium,37 degree are fully cultivated, to OD600=0.6,3% ratio is inoculated into the LB training containing ampicillinSupport base. At bacterial growth to OD600=0.7, cool the temperature to 25 degree, add final concentration to beThe IPTG of 1mmol/L induces spend the night (16h). Centrifugal results thalline ,-20 DEG C frozen.SDS-PAGE detects and shows, the about 44.5KDa of this alcohol dehydrogenase, and target protein expression can be extremely55% of bacterial protein.
By the E.coli bacterium mud of above-mentioned results, with 100mM sodium phosphate buffer (+150mMSodium chloride, pH7.0) resuspended to 10g/L, with the ultrasonic 10min of cell Ultrasonic Cell Disruptor ice-water bath(800W, work 1sec stops 3sec), centrifugal (12,000rpm, 4 DEG C, 10min),Cellular lysate liquid supernatant is crude enzyme liquid. The enzyme activity determination system of thick enzyme is as follows: 100mM phosphorusAcid sodium buffer solution (pH7.0), 5mMN-Boc-3-piperidones, 1mMNADPH(orNADH), measure the decline of light absorption value in 340nm place for 30 DEG C. Enzyme work is defined as oxygen per minuteChanging the needed enzyme amount of 1 micromole NADPH is the alcohol dehydrogenase reductase IU of unit alive.Protein content adopts Bradford method to measure, and result shows that this alcohol dehydrogenase enzyme work is about5.6IU/mg。
Embodiment bis-: expression and the determination of activity of GDH in E.coli
The synthetic GDH encoding gene of full gene after NdeI and XhoI double digestion,Be cloned into the MCS 2 of expression vector pETDuet-1, after recombinant plasmid is confirmed through checking order,Be converted into E.coliBL21(DE3) in, the recombinant bacterial strain called after pETDuet-GDH-of structureBL21 (DE3). On ampicillin flat board, select single bacterium colony, access is containing corresponding antibioticIn LB culture medium, 37 degree are fully cultivated, to OD600=0.6,3% ratio is inoculated into containing ammonia benzylThe LB culture medium of penicillin. At bacterial growth to OD600=0.7, cool the temperature to 25 degree,Adding final concentration is that the IPTG induction of 1mmol/L is spent the night. Centrifugal results thalline ,-20 DEG C freezeDeposit. SDS-PAGE detects and shows, the about 27.8KDa of this GDH, target protein tableThe amount of reaching can be to 50% of bacterial protein.
By the E.coli bacterium mud of above-mentioned results, with 100mM sodium phosphate buffer (+150mMSodium chloride, pH7.0) resuspended to 10g/L, with the ultrasonic 10min of cell Ultrasonic Cell Disruptor ice-water bath(800W, work 1sec stops 3sec), centrifugal (12,000rpm, 4 DEG C, 10min), bacteriumBody lysate supernatant is crude enzyme liquid. The enzyme activity determination system of thick enzyme is as follows: 100mM phosphoric acidSodium buffer solution (pH7.0), 250mM glucose, 1mMNADP+(or NAD+），30℃Measure the increase of light absorption value in 340nm place. Enzyme work is defined as reduction per minute and generates 1 micro-rubbingYour NADPH(or NADH) needed enzyme amount is a GDH Mei Huo unitIU. Protein content adopts Bradford method to measure. Result shows this GDHEnzyme work is about 30IU/mg.
Embodiment tri-: alcohol dehydrogenase and the GDH coexpression in E.coli
GDH encoding gene, after NdeI and XhoI double digestion, is cloned into realityExecute the MCS 2 of the recombinant plasmid pETDuet1-(MCS1) described in example one, gene is through surveyingOrder is converted into E.coliBL after confirming21(DE3) in, the recombinant bacterial strain called after of structurepETDuet-ADH-GDH-BL21(DE3). On ampicillin flat board, select single bacterium colony,Access is containing in corresponding antibiotic LB culture medium, and 37 degree are fully cultivated, to OD600=0.6，3％Ratio is inoculated into the LB culture medium containing ampicillin. At bacterial growth to OD600=0.7，Cool the temperature to 25 degree, add 1mmol/LIPTG induction to spend the night. Centrifugal results thalline,-20 DEG C frozen. SDS-PAGE detects and shows, the expression of alcohol dehydrogenase and GDHQuite, total amount can be to 60% of bacterial protein for amount.
Embodiment tetra-: the fermentation of restructuring alcohol dehydrogenase and crude enzyme liquid preparation
In 100L fermentation tank, add following material: 1Kg peptone, 0.5Kg dusty yeast and0.5Kg sodium chloride, pH nature. 121 DEG C of sterilizing 20min. While being cooled to 37 DEG C, access1L is cultured to OD with LB culture medium (containing ampicillin)600=0.8 freshpETDuet-ADH-BL21(DE3) nutrient solution, add the ampicillin solution of filtration sterilization extremelyFinal concentration is 0.1mg/mL, and 37 DEG C of 800rpm cultivate. Feed supplement after cultivation 2hr, feed supplement trainingFoster base is the solution 15L of 500g/L glycerine, 100g/L peptone and 50g/L dusty yeast,Concentrated ammonia liquor/hydrochloric acid regulates pH7.0 ± 0.1. As the OD of nutrient solution600Reach at 25 o'clock, by tank temperatureBe down to 25 DEG C, adding final concentration is 1mmol/LIPTG, controls each condition of culture induction 14hr.Induction finishes the centrifugal results thalline of rear tube centrifuge maximum (top) speed, weight in wet base 4.21Kg, and 4 DEG C are temporarilyDeposit for subsequent use.
By above-mentioned 4.21Kg weight in wet base pETDuet-ADH-BL21(DE3) press 1:5(v/v) heavyBe suspended from 100mM sodium phosphate (+150mM sodium chloride, pH7.0) buffer solution, 4 DEG C lowLower high-pressure homogeneous 2 times of temperature protection: each one time of 800bar+600bar. In above-mentioned cracking, addEnter polymine to final concentration 0.5%(w/v), 4 DEG C are stirred 30min, centrifuge 10,000The centrifugal 20min of rpm, retains supernatant and is restructuring alcohol dehydrogenase crude enzyme liquid, and 4 DEG C of lucifuges are protectedDeposit. The mensuration that alcohol dehydrogenase enzyme is lived press method mensuration described in embodiment mono-, is 120.5IU/mL,Protein concentration is measured with Bradford method, thick zymoprotein concentration 27.5mg/mL.
Embodiment five: the fermentation of recombinant glucose dehydrogenase and crude enzyme liquid preparation.
In 100L fermentation tank, add following material: 1Kg peptone, 0.5Kg dusty yeast and0.5Kg sodium chloride, pH nature. 121 DEG C of sterilizing 20min. While being cooled to 37 DEG C, access1L is cultured to OD with LB culture medium (containing ampicillin)600=0.8 freshpETDuet-GDH-BL21(DE3) nutrient solution, add the ampicillin solution of filtration sterilization extremelyFinal concentration is 0.1mg/mL, and 37 DEG C of 800rpm cultivate. Feed supplement after cultivation 2hr, feed supplement trainingFoster base is the solution 15L of 500g/L glycerine, 100g/L peptone and 50g/L dusty yeast,Concentrated ammonia liquor/hydrochloric acid regulates pH7.0 ± 0.1. As the OD of nutrient solution600Reach at 25 o'clock, by tank temperatureBe down to 25 DEG C, adding final concentration is 1mmol/LIPTG, controls each condition of culture induction 14hr.Induction finishes the centrifugal results thalline of rear tube centrifuge maximum (top) speed, weight in wet base 3.52Kg, and 4 DEG C are temporarilyDeposit for subsequent use.
By above-mentioned 3.52Kg weight in wet base pETDuet-KRED-BL21(DE3) press 1:5(v/v)Be resuspended in 100mM sodium phosphate (+150mM sodium chloride, pH7.0) buffer solution 4 DEG CUnder low-temperature protection high-pressure homogeneous 2 times: each one time of 800bar+600bar. In above-mentioned crackingAdd polymine to final concentration 0.5%(w/v), 4 DEG C are stirred 30min, centrifuge 10,000The centrifugal 20min of rpm, retains supernatant and is recombinant glucose dehydrogenase crude enzyme liquid, keeps away for 4 DEG CLight is preserved. The mensuration that GDH enzyme is lived press method mensuration described in embodiment bis-, is 720IU/mL, protein concentration is measured with Bradford method, thick zymoprotein concentration 31.2mg/mL.
Embodiment six: restructuring alcohol dehydrogenase is to 3-piperidones and derivative reduction thereof
Press the enzyme activity determination system of embodiment mono-and embodiment bis-, to 3-piperidones and derivative thereofCarry out the screening of enzyme reduction vigor, result is as follows:
| Ketosubstrate | Restructuring alcohol dehydrogenase |
| N-Boc-3-piperidones | 5.63IU/mg |
| N-3-piperidones | 0.45IU/mg |
| N-Cbz-3-piperidones | 3.84IU/mg |
| N-Fmoc-3-piperidones | 0.53IU/mg |
Embodiment seven: the chiral analysis method of (R)-N-Boc-3-piperidine alcohols
Ee(chirality HPLC): ChiralpakIC150mm × 4.6mm chiral chromatographic column; StreamMoving phase: n-hexane (95%)/IPA(5%); Flow velocity: 0.6mL/min; Wavelength: 210nm;Retention time: (R)-N-Boc-3-piperidine alcohols 33.66min, another enantiomer (S)-N-Boc-3-Piperidine alcohols 30.78min. The reduzate of restructuring alcohol dehydrogenase is (R)-type.
Embodiment eight: the enzymatic conversion method of (R)-N-Boc-3-piperidine alcohols is synthetic
(R) the synthetic reaction equation of pressing of-3-N-Boc-piperidine alcohols carries out:
In a 250mL three-necked bottle, add successively 100mL, 0.2mol/LNaH2PO4·Na2HPO4(pH7.0) cushioning liquid, chemical compounds I (10g), glucose (12G) with 50mL butyl acetate, magnetic agitation 10min makes to mix, then adds alcohol dehydrogenaseEnzyme (8mL), GDH (5mL) and coenzyme (NADP+, 0.01g), in 30Under degree Celsius, stir 16 hours, control pH value between 6.5～7.0, high performance liquid chromatography is surveyedSurely show that reaction conversion ratio is more than 99.5%. Filtration adds 100mL ethyl acetate after dezymotizing,Re-extract three times, is spin-dried for after organic phase is dry, obtains 9.5 and digests compound III ((R)-N-Boc-3-Piperidine alcohols), molar yield 94.6%. Optical purity is pressed method described in embodiment seven and is measured, and producesThing ee value > 99.4%.
The present invention still has multiple concrete embodiment, and all employings are equal to replaces or equivalent transformationAnd all technical schemes that form, within all dropping on the scope of protection of present invention.
<110>Suzhou Guodie Pharmaceuticals Science & Technology Co., Ltd
<120>prepare the method for high chiral purity (R)-3-piperidine alcohols and derivative thereof
<140>201410016356.4
<160>2
<210>1
<211>406
<212>PRT
<213>thermophilic coccus (Thermococcushydrothermalis)
<220>0
<221>VARIANT
<400>1
MetValTrpGluSerHisValSerIleAsnGlnValPheGluMet
151015
ArgCysLysThrThrAsnTyrPheGlyLeuCysAlaIleHisLys
202530
PheAsnAspIleValArgGluLeuLysGlyLysGlyValAspLys
354045
ValIleLeuValThrGlyThrSerSerTyrLysLysCysGlyGly
505560
AlaTrpValValArgProAlaLeuGluGluAsnGlyValGluLys
657075
ValHisTyrAspLysValGlyAlaAsnProThrValAspMetIle
808590
AspGluAlaAlaGluMetGlyArgGluPheGlyAlaGlnAlaVal
95100105
IleGlyIleGlyGlyGlySerProIleAspSerAlaLysSerVal
110115120
AlaIleLeuLeuGluTyrProAspLysThrAlaArgAspLeuTyr
125130135
GluPheArgPheThrProValLysAlaLysProIleIleAlaIle
140145150
AsnThrThrHisGlyThrGlyThrGluValAspArgPheAlaVal
155160165
AlaSerIleProGluLysGluTyrLysProAlaIleAlaTyrAsp
170175180
CysIleTyrProLeuTyrAlaIleAspAspProAlaLeuThrThr
185190195
LysLeuProProGluGlnThrLeuTyrValThrIleAspAlaLeu
200205210
AsnHisIleThrGluAlaAlaThrThrLysValAlaAsnProTyr
215220225
SerIleLeuLeuAlaLysGluAlaAlaArgLeuIlePheThrTyr
230235240
LeuProGluAlaLeuAsnAsnProAspAsnLeuGlnAlaArgTyr
245250255
AlaLeuLeuTyrAlaSerAlaIleAlaGlyIleSerPheAspAsn
260265270
GlyLeuLeuAsnPheThrHisAlaLeuGluHisProLeuSerAla
275280285
ValLyeProAspLeuProHisGlyLeuGlyLeuAlaMetLeuLeu
290295300
ProAlaValIleArgGlnIleTyrProAlaThrAlaLysIleLeu
305310315
AlaGluValTyrArgProLeuValProGluAlaLysGlyValPro
320325330
GlyGluValGluLeuValAlaArgArgValGluGluTrpLeuPhe
335340345
SerIleGlyIleThrGluLysLeuAlaAspValGlyPheThrGlu
350355360
GlyAspValAspLysLeuThrGlnLeuAlaMetThrThrProSer
365370375
LeuAspLeuLeuLeuSerMetAlaProValGluAlaThrLysGlu
380385390
ArgIleAlaAlaIleTyrArgAspSerLeuTyrProIleGlyArg
395400405
Gly
<210>2
<211>245
<212>PRT
<213>burkholderia (Burkholderiasp.)
<220>0
<221>VARIANT
<400>2
MetArgAsnSerAlaLeuValIleGlyValGlyAlaGluLeuGly
151015
LeuGlyAlaAlaLeuCysArgLysIleAlaAlaAsnGlyTyrHis
202530
ValTyrValAlaGlyArgThrGlnAlaLysLeuAspIleValThr
354045
AsnGlyIleAlaGlyGlyGlySerAlaGluSerAlaPheAlaMet
505560
AspGlyThrSerGluAlaAspIleMetArgLeuPheAspArgAla
657075
MetSerProProAspLysIleAspValProSerLeuValIleTyr
808590
AsnValGlyAsnAsnArgHisValProPheArgAspLeuThrGlu
95100105
AlaGlnMetGlnAspPheLeuArgSerGlyProValGlyGlyPhe
110115120
LeuValGlyGlyArgAlaAlaArgArgLeuAlaProLeuGlyArg
125130135
GlyThrValIlePheThrGlyAlaTyrAlaSerLeuArgGlyLys
140145150
ProGlyPheAlaHisPheAlaAlaAlaLysAlaGlyLeuArgMet
155160165
ValAlaGlnSerMetAlaArgGluPheGlyProLeuGlyLeuHis
170175180
ValAlaHisValValIleAspGlyGlyIleAspGlyGluArgLeu
185190195
HisValSerArgProGlnAlaAlaAlaGluArgGlyGluAsnGly
200205210
LeuLeuAsnValAspLeuIleAlaGluAlaTyrTrpGlnLeuHis
215220225
LeuGlnHisProSerAlaTrpThrHisGluIleAspLeuArgPro
230235240
PheLysGluProPhe
245
Claims (3)
1. the method for the high chiral purity of preparation (R)-3-piperidine alcohols and derivative thereof, described course of reactionAs follows:
It is characterized in that: P is hydrogen, tertbutyloxycarbonyl, benzyloxycarbonyl group or fluorenylmethyloxycarbonyl, described anti-The condition of answering is pH6.0-7.5, with coexpression restructuring alcohol dehydrogenase and recombinant glucose dehydrogenase andCoenzyme is catalyst, described restructuring alcohol dehydrogenase and recombinant glucose dehydrogenase and coenzyme catalystFor liquid solution, freeze-dried powder, immobilised enzymes or immobilized cell, described restructuring alcohol dehydrogenaseAmino acid sequence as shown in sequence table SEQ .IDNO:1, described recombinant glucose dehydrogenase ammoniaBase acid sequence is as shown in sequence table SEQ .IDNO:2.
2. the high chiral purity of preparation according to claim 1 (R)-3-piperidine alcohols and derivative thereofMethod, it is characterized in that: described reaction condition is PH6.4-6.6 described restructuring alcohol dehydrogenaseEnzyme and recombinant glucose dehydrogenase be efficient coexpression in genetic engineering bacterium.
3. the high chiral purity of preparation according to claim 2 (R)-3-piperidine alcohols and derivative thereofMethod, it is characterized in that: described genetic engineering bacterium is to have recombinant vector pETDuet-1'sEscherichia coli.
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| CN201410016356.4A CN103898177B (en) | 2014-01-14 | 2014-01-14 | Prepare the method for high chiral purity (R)-3-piperidine alcohols and derivative thereof |
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| CN201410016356.4A CN103898177B (en) | 2014-01-14 | 2014-01-14 | Prepare the method for high chiral purity (R)-3-piperidine alcohols and derivative thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103898177A CN103898177A (en) | 2014-07-02 |
| CN103898177B true CN103898177B (en) | 2016-05-11 |
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