CN103898178A - Method for preparing highly chirally pure (S)-3-pipradrol and derivatives of highly chirally pure (S)-3-pipradrol by use of enzymic method - Google Patents
Method for preparing highly chirally pure (S)-3-pipradrol and derivatives of highly chirally pure (S)-3-pipradrol by use of enzymic method Download PDFInfo
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- CN103898178A CN103898178A CN201410016372.3A CN201410016372A CN103898178A CN 103898178 A CN103898178 A CN 103898178A CN 201410016372 A CN201410016372 A CN 201410016372A CN 103898178 A CN103898178 A CN 103898178A
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- 239000012450 pharmaceutical intermediate Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- XUWHAWMETYGRKB-UHFFFAOYSA-N piperidin-2-one Chemical compound O=C1CCCCN1 XUWHAWMETYGRKB-UHFFFAOYSA-N 0.000 description 1
- 150000003053 piperidines Chemical class 0.000 description 1
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Substances [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 description 1
- 235000015320 potassium carbonate Nutrition 0.000 description 1
- 235000011118 potassium hydroxide Nutrition 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 235000017550 sodium carbonate Nutrition 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- LWRYDHOHXNQTSK-UHFFFAOYSA-N thiophene oxide Chemical compound O=S1C=CC=C1 LWRYDHOHXNQTSK-UHFFFAOYSA-N 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 229960004418 trolamine Drugs 0.000 description 1
- 238000003828 vacuum filtration Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Landscapes
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a method for preparing a highly chirally pure (S)-3-pipradrol and derivatives of highly chirally pure (S)-3-pipradrol by use of an enzymic method. The method is characterized in that the reducing (S)-3-pipradrol and the derivatives of reducing (S)-3-pipradrol are prepared at a high yield and high purity under the reaction conditions that pH value ranges from 6.0 to 7.5, and a recombinant carbonylacyl reductase and a recombinant glucose dehydrogenase which are co-expressed efficiently in escherichia coli, and coenzyme are taken as catalysts; the method is short in reaction time and low in preparation cost.
Description
Technical field
The present invention relates to a kind of enzyme process and prepare the method for high chiral purity (S)-3-piperidine alcohols and derivative thereof, belong to the synthesis technical field of medicine intermediate, also belong to Green Chemistry and genetically engineered field.
Background technology
Chirality 3-piperidine alcohols is piperidine derivatives, is the key intermediate of multiple medicine, agricultural chemicals, is one of the focus in the synthetic field of medicine always, is also a kind of very important medicine molecule of the skeleton.Chirality 3-piperidine alcohols derivative has been found to have antidepressant, anti-arrhythmia, antithrombotic and has formed, separated the effects such as spasm, calmness and reduction blood cholesterol activity, also common this class formation in this external antitumour activity medicine.For example, in antitumor field, CDK(mono-class relies on the protein serine/threonine of Cylin) in the regulation and control of cell cycle, play an important role, the functional disorder of CDK and tumour exist close relationship between occurring.Flavopiridol (Flavopiridol) is first CDK inhibitor that enters clinical trial, and chemical structure is flavonoid, derives from first a kind of Indian plant Rohitukin, at present can synthetic.In Flavopiridol structure, contain crucial (S)-3-piperidine alcohols structure.In addition, also have other multi-medicaments to 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 the N-protected such as Boc-, Cbz-form.The synthesis mode of known (S) or (R)-3-piperidine alcohols and derivative thereof has multiplely at present, is roughly divided into following approach:
(1) set out with natural chiral acid such as L MALIC ACID, L-Glu or L-Asp, carry out through condensation, reduction etc., often step complexity, all needs polystep reaction, and the chiral raw material relating to and to go back original reagent expensive, is difficult to industry and amplifies.
(2) split as (+)-dibenzoyl tartaric acid through chiral acid with the 3-piperidine alcohols of racemization, and obtain chiral alcohol through recrystallization repeatedly.This method is easy, but its chiral acid relating to is expensive, and yield is low, is only 24%, and chirality is difficult to be guaranteed relatively.
(3) carry out selectivity fractionation with lipase, (S)-all can obtain with (R)-type product.But lipase splits the highest yield of theory only 50% that obtains (S) and (R)-3-piperidine alcohols, and the separation of product and purification difficult, often need silica gel column chromatography etc., is difficult to industry and amplifies.Another large defect that enzyme splits is to be difficult to racemization starting raw material to obtain higher yield, and raw material racemization at present does not still have feasible short-cut method, thereby the fractionation cost of lipase is high.
(4), with microorganism or enzymatic hydroxylation mode, directly the derivative of selectivity hydroxylation piperidone and N-protected thereof be (S)-or (R)-3-piperidine alcohols accordingly.This mode can improve substrate utilization degree to greatest extent, and can obtain the chiral alcohol that is close to 100%.But at present, this approach only stays in theoretical investigation and laboratory stage, and concentration of substrate is less than 5g/L, there is no actual production meaning.
Carbonyl acyl reductase (Canbonyl reductase; EC1.1.1.184) with alcoholdehydrogenase (Alcohol dehydrogenase; EC1.1.1.1), the alcoholic extract hydroxyl group that ketone carbonyl that all can asymmetric reduction prochiral ketones molecule is chirality, is 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), and reduced coenzyme Ⅰ, II exist as hydrogen donor in reduction reaction, become oxidized coenzyme I (NAD after hydrogen supply
+) or codehydrogenase Ⅱ (NADP
+), oxidized coenzyme can be obtained hydrogen by other oxydase or desaturase effect again, again becomes reduced coenzyme, completes coenzyme circulation.
Carbonyl acyl reductase and alcoholdehydrogenase are mainly derived from the microorganism such as yeast, bacterium, and existing multiple this fermentoid gene is in the news, as Candida magnoliae(Genbank Acc.No.JC7338; And Candida parapsilosis(Genbank Acc.No.BAA24528.1 GI:11360538); GI:2815409) etc.
At present existing multiple important chirality pharmaceutical intermediate compound can be synthetic with carbonyl acyl reductase and alcoholdehydrogenase, comprises multiple synthesis mode such as pure enzyme and microbe whole-cell or immobilized enzyme/cell etc.Conventionally in synthetic system, also need to add regenerating coenzyme enzyme, as Hexose phosphate dehydrogenase (Glucose dehydrogenase, GDH) and hydrogenlyase (Formate dehydrogenase, FDH) etc.For example, the asymmetric reduction of the chloro-methyl aceto acetate 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 one of key intermediate of statins; The asymmetric reduction (as: U.S.Pat.No.6,800,477) of methyl phenyl ketone and derivative thereof; The asymmetric reduction (WO 2005/054491) of thienone.At medicine 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 a kind of enzyme process to prepare the method for high chiral purity (S)-3-piperidine alcohols and derivative thereof.
Object of the present invention is achieved through the following technical solutions:
Enzyme process is prepared the method for high chiral purity (S)-3-piperidine alcohols and derivative thereof, and described reaction process is as follows:
P is hydrogen, tertbutyloxycarbonyl (Boc), carbobenzoxy-(Cbz) (Cbz) or fluorenylmethyloxycarbonyl (Fmoc); described reaction conditions is pH6.0-7.5; recombinate carbonyl acyl reductase and recombinant glucose dehydrogenase and coenzyme as catalyzer take coexpression; described restructuring carbonyl acyl reductase and recombinant glucose dehydrogenase catalyzer are liquor, lyophilized powder, immobilized enzyme or immobilized cell; the aminoacid sequence of described restructuring carbonyl acyl reductase is as shown in sequence table SEQ .ID NO:1, and described recombinant glucose dehydrogenase aminoacid sequence is as shown in sequence table SEQ .ID NO:2.
Preferably, described reaction conditions is pH6.4-6.6, and described restructuring carbonyl acyl reductase and recombinant glucose dehydrogenase be efficient coexpression in genetic engineering bacterium.
Preferably, described genetic engineering bacterium is the intestinal bacteria with recombinant vectors pETDuet-1.
A method for the above-described genetic engineering bacterium of fermentation culture, comprises the further fermentation that builds genetic engineering bacterium and genetic engineering bacterium, and the structure of described genetic engineering bacterium comprises the following steps:
To the synthetic restructuring carbonyl acyl reductase encoding gene of full gene and Hexose phosphate dehydrogenase encoding gene respectively through double digestion; It is cloned into respectively to the different Anti-TNF-αs site of expression vector pETDuet-1, recombinant plasmid order-checking is converted into respectively expression coli strain after confirming, builds corresponding recombinant bacterial strain again;
The further fermentation of described genetic engineering bacterium comprises the steps:
Above-described coli strain is seeded in the LB substratum that contains penbritin, is cultured to OD
600=0.8 fresh medium, adding penbritin solution to the final concentration of filtration sterilization is 0.1mg/mL, 37 ℃ of 800rpm cultivate; Feed supplement after cultivation 2hr, regulates pH7.0 ± 0.1 by strong aqua/hydrochloric acid, as the OD of nutrient solution
600reach at 25 o'clock, by tank temperature drop to 25 ℃, adding final concentration is 1mmol/L IPTG, continues to control each culture condition induction 14hr, last centrifugal results thalline.
Beneficial effect of the present invention is mainly reflected in: adopt restructuring carbonyl acyl reductase, Hexose phosphate dehydrogenase to be applied to background and to reduce, concentration of substrate is up to 150g/L, and productive rate is high, and optical purity of products is high, and the reaction times is short, and preparation cost is low.
Embodiment
The present invention has disclosed a kind of method that enzyme process is prepared high chiral purity (S)-3-piperidine alcohols and derivative thereof, and described reaction process is as follows:
P is hydrogen, tertbutyloxycarbonyl (Boc), carbobenzoxy-(Cbz) (Cbz) or fluorenylmethyloxycarbonyl (Fmoc),
Described preparation method is as follows: the Compound I of a mole is dissolved in the buffered soln and organic solvent of 500～2000 milliliters; in above-mentioned solution, adding weight is 0.1～20% gene recombination carbonyl acyl reductase, Hexose phosphate dehydrogenase and coenzyme of Compound I; holder ties up between 15～45 ℃, preferentially at 25～40 ℃.Regulate pH 6.0～7.5 with acid/alkali lye, preferably 6.4～6.6.Stir 16-72h, stopped reaction, with the organic solvent extraction of 1000 milliliters of left and right 3 times, merging organic phase, desiccant dryness, organic solvent is removed in underpressure distillation, obtains homochiral target compound II.Conventionally Compound I I chiral purity is greater than 98%, can be used for the preparation of medicine.
Described organic solvent is selected from the one in methyl alcohol, ethanol, propyl alcohol, butanols, the trimethyl carbinol, Virahol, tetrahydrofuran (THF), methyl tert-butyl ether, ethyl acetate, butylacetate and toluene.
Described buffered soln is the one in inorganic sulfuric acid, inorganic phosphate or trolamine hydrochloric acid buffering salt.
Described mineral alkali is selected from the one in sodium hydroxide, potassium hydroxide, ammoniacal liquor, sodium carbonate, salt of wormwood.
P in described Compound I is preferably tertbutyloxycarbonyl.
Restructuring carbonyl acyl reductase and recombinant glucose dehydrogenase are at the efficient coexpression of intestinal bacteria, and it can be liquor, lyophilized powder, can be also immobilized enzyme or cell.
Described carbonyl acyl reductase is external evolution, utilizes the enzyme of purifying or the direct catalysis of colibacillus engineering for its expression.It utilizes the variant of the carbonyl acyl reductase of a kind of reddish brown shadow yeast (Sporobolomyces salmonicolor), has compared 11 amino acid difference with wild-type.
The sequence optimisation of carbonyl acyl reductase is carried out round increasing activity, thermostability and organic solvent stability on wild-type basis.Main employing take structure evolved and high flux screening as basic semi-directional, and the sequence of final gained has been compared 11 amino acid whose differences with wild-type.Great majority sudden change concentrates on enzyme surface and contacts site with subunit.Gene order is revised according to the codon of intestinal bacteria preference, and eliminates the secondary structure that may affect expression.Carbonyl acyl reductase after optimization high efficient expression in E.coli, enzymic activity is the more than 50 times of wild-type, and stability also significantly increases.Carbonyl acyl reductase high reactivity variant after optimization and Hexose phosphate dehydrogenase, after E.coli coexpression, through thick purifying, can efficient catalytic 3-piperidone be (S)-3-piperidine alcohols.
Described Hexose phosphate dehydrogenase is external evolution, utilizes a kind of bacterium Burkholderia sp. glucose dehydrogenase modification efficiently to reduce NADP
+coenzyme, has compared 3 amino acid difference with wild-type.Its gene order is revised according to the codon of intestinal bacteria preference, and eliminates the secondary structure that may affect expression, and this sequence is at the high efficient expression of E.coli.
The aminoacid sequence of described restructuring carbonyl acyl reductase is as shown in sequence table SEQ .ID NO:1, and described recombinant glucose dehydrogenase aminoacid sequence is as shown in sequence table SEQ .ID NO:2.
Below describe its expression and determination of activity in intestinal bacteria in detail with restructuring carbonyl acyl reductase and recombinant glucose dehydrogenase respectively.
Embodiment mono-: expression and the determination of activity of carbonyl acyl reductase in E.coli
The synthetic restructuring carbonyl acyl reductase encoding gene of full gene, after Nco I and Hind III double digestion, is cloned into expression vector pETDuet-1(producer: Novagen
, production code member: 71146-3) multiple clone site 1, recombinant plasmid through order-checking confirm after, be converted in expression strain E.coli BL21 (DE3) the recombinant bacterial strain called after pETDuet-KRED-BL of structure
21(DE
3).On penbritin flat board, select single bacterium colony, access contains in corresponding antibiotic LB substratum, and 37 degree are fully cultivated, to OD
600=0.6,3% ratio is inoculated into the LB substratum containing penbritin.At bacterial growth to OD
600=0.7, cool the temperature to 25 degree, adding final concentration is the IPTG induction of 1mmol/L spend the night (16h).Centrifugal results thalline ,-20 ℃ frozen.SDS-PAGE detects and shows, the about 37.6KDa of this carbonyl acyl reductase, and target protein expression amount can be to 60% of bacterial protein.
By the E.coli bacterium mud of above-mentioned results, with 100mM sodium phosphate buffer (+150mM sodium-chlor, pH7.0) resuspended to 10g/L, with the ultrasonic 10min(800W of cell Ultrasonic Cell Disruptor ice-water bath, work 1sec stops 3sec), centrifugal (12,000rpm, 4 ℃, 10min), cellular lysate liquid supernatant is crude enzyme liquid.The enzyme activity determination system of thick enzyme is as follows: 100mM sodium phosphate buffer (pH7.0), 5mM N-Boc-3-piperidone, 1mM NADPH(or NADH), measure the decline of light absorption value in 340nm place for 30 ℃.Enzyme work is defined as per minute, and to be oxidized the needed enzyme amount of 1 micromole NADPH be a carbonyl acyl reductase IU of Mei Huo unit.Protein content adopts Bradford method to measure, and result shows that this carbonyl acyl reductase enzyme work is about 3.8IU/mg.
Embodiment bis-: expression and the determination of activity of Hexose phosphate dehydrogenase in E.coli
The synthetic Hexose phosphate dehydrogenase encoding gene of full gene, after Nde I and Xho I double digestion, is cloned into multiple clone site 2 recombinant plasmids of expression vector pETDuet-1 after order-checking is confirmed, is converted into E.coli BL
21(DE
3) in, the recombinant bacterial strain called after pETDuet-GDH-BL of structure
21(DE
3).On penbritin flat board, select single bacterium colony, access is containing in corresponding antibiotic LB substratum, and 37 degree are fully cultivated, to OD
600=0.6,3% ratio is inoculated into the LB substratum containing penbritin.At bacterial growth to OD
600=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 ℃ frozen.SDS-PAGE detects and shows, the about 27.8KDa of this Hexose phosphate dehydrogenase, and target protein expression amount can be to 50% of bacterial protein.
By the E.coli bacterium mud of above-mentioned results, with 100mM sodium phosphate buffer (+150mM sodium-chlor, pH7.0) resuspended to 10g/L, with the ultrasonic 10min(800W of cell Ultrasonic Cell Disruptor ice-water bath, work 1sec stops 3sec), centrifugal (12,000rpm, 4 ℃, 10min), cellular lysate liquid supernatant is crude enzyme liquid.The enzyme activity determination system of thick enzyme is as follows: 100mM sodium phosphate buffer (pH7.0), 250mM glucose, 1mM NADP
+(or NAD
+), measure the increase of light absorption value in 340nm place for 30 ℃.Enzyme work is defined as per minute reduction and generates 1 micromole NADPH(or NADH) needed enzyme amount is an IU of Hexose phosphate dehydrogenase Mei Huo unit.Protein content adopts Bradford method to measure.Result shows that this Hexose phosphate dehydrogenase enzyme work is about 30IU/mg.
Embodiment tri-: carbonyl acyl reductase and the Hexose phosphate dehydrogenase coexpression in E.coli
Hexose phosphate dehydrogenase encoding gene, after Nde I and Xho I double digestion, is cloned into the multiple clone site 2 of the recombinant plasmid pETDuet1-(MCS1) described in embodiment mono-, and gene, after order-checking is confirmed, is converted into E.coli BL
21(DE
3) in, the recombinant bacterial strain called after pETDuet-KRED-GDH-BL of structure
21(DE
3).On penbritin flat board, select single bacterium colony, access is containing in corresponding antibiotic LB substratum, and 37 degree are fully cultivated, to OD
600=0.6,3% ratio is inoculated into the LB substratum containing penbritin.At bacterial growth to OD
600=0.7, cool the temperature to 25 degree, add 1mmol/L IPTG induction to spend the night.Centrifugal results thalline ,-20 ℃ frozen.SDS-PAGE detects and shows, the expression amount of carbonyl acyl reductase and Hexose phosphate dehydrogenase is suitable, and total amount can be to 65% of bacterial protein.
Embodiment tetra-: the fermentation of restructuring carbonyl acyl reductase and crude enzyme liquid preparation
In 100L fermentor tank, add following material: 1Kg peptone, 0.5Kg yeast powder and 0.5Kg sodium-chlor, pH nature.121 ℃ of sterilizing 20min.While being cooled to 37 ℃, access 1L is cultured to OD with LB substratum (containing penbritin)
600=0.8 fresh pETDuet-KRED-BL
21(DE
3) nutrient solution, adding penbritin solution to the final concentration of filtration sterilization is 0.1mg/mL, 37 ℃ of 800rpm cultivate.Feed supplement after cultivation 2hr, supplemented medium is the solution 15L of 500g/L glycerine, 100g/L peptone and 50g/L yeast powder, strong aqua/hydrochloric acid regulates pH7.0 ± 0.1.As the OD of nutrient solution
600reach at 25 o'clock, by tank temperature drop to 25 ℃, adding final concentration is 1mmol/L IPTG, controls each culture condition induction 14hr.Induction finishes the centrifugal results thalline of rear tubular-bowl centrifuge maximum speed of revolution, weight in wet base 4.34Kg, and 4 ℃ are temporary for subsequent use.
By above-mentioned 4.34Kg weight in wet base pETDuet-KRED-BL
21(DE
3) by 1:5(v/v) be resuspended in 100mM sodium phosphate (+150mM sodium-chlor, pH7.0) damping fluid under 4 ℃ of low-temperature protections high-pressure homogeneous 2 time: each one time of 800bar+600bar.In above-mentioned cracking, add polymine to final concentration 0.5%(w/v), 4 ℃ are stirred 30min, whizzer 10, the centrifugal 20min of 000rpm, retains supernatant liquor and is restructuring carbonyl acyl reductase crude enzyme liquid, and 4 ℃ keep in Dark Place.The mensuration that carbonyl reductase enzyme is lived press method described in embodiment mono-and is measured, and is 60.5IU/mL, and protein concentration is measured with Bradford method, slightly zymoprotein concentration 24.5mg/mL.
Embodiment five: the fermentation of recombinant glucose dehydrogenase and crude enzyme liquid preparation.
In 100L fermentor tank, add following material: 1Kg peptone, 0.5Kg yeast powder and 0.5Kg sodium-chlor, pH nature.121 ℃ of sterilizing 20min.While being cooled to 37 ℃, access 1L is cultured to OD with LB substratum (containing penbritin)
600=0.8 fresh pETDuet-GDH-BL
21(DE
3) nutrient solution, adding penbritin solution to the final concentration of filtration sterilization is 0.1mg/mL, 37 ℃ of 800rpm cultivate.Feed supplement after cultivation 2hr, supplemented medium is the solution 15L of 500g/L glycerine, 100g/L peptone and 50g/L yeast powder, strong aqua/hydrochloric acid regulates pH7.0 ± 0.1.As the OD of nutrient solution
600reach at 25 o'clock, by tank temperature drop to 25 ℃, adding final concentration is 1mmol/L IPTG, controls each culture condition induction 14hr.Induction finishes the centrifugal results thalline of rear tubular-bowl centrifuge maximum speed of revolution, weight in wet base 3.52Kg, and 4 ℃ are temporary for subsequent use.
By above-mentioned 3.52Kg weight in wet base pETDuet-KRED-BL
21(DE
3) by 1:5(v/v) be resuspended in 100mM sodium phosphate (+150mM sodium-chlor, pH7.0) damping fluid under 4 ℃ of low-temperature protections high-pressure homogeneous 2 time: each one time of 800bar+600bar.In above-mentioned cracking, add polymine to final concentration 0.5%(w/v), 4 ℃ are stirred 30min, whizzer 10, the centrifugal 20min of 000rpm, retains supernatant liquor and is recombinant glucose dehydrogenase crude enzyme liquid, and 4 ℃ keep in Dark Place.The mensuration that Hexose phosphate dehydrogenase enzyme is lived press method described in embodiment bis-and is measured, and is 720IU/mL, and protein concentration is measured with Bradford method, slightly zymoprotein concentration 31.2mg/mL.
Embodiment six: restructuring carbonyl acyl reductase is to 3-piperidone and derivative reductive action thereof
Press the enzyme activity determination system of embodiment mono-and embodiment bis-, 3-piperidone and derivative thereof are carried out to the screening of enzyme reduction vigor, result is as follows:
| Ketosubstrate | Restructuring carbonyl acyl reductase |
| N-Boc-3-piperidone | 3.85IU/mg |
| N-3-piperidone | 0.24IU/mg |
| N-Cbz-3-piperidone | 2.71IU/mg |
| N-Fmoc-3-piperidone | 0.86IU/mg |
Embodiment seven: the chiral analysis method of (S)-N-Boc-3-piperidine alcohols
Ee(chirality HPLC): Chiralpak IC150mm × 4.6mm chiral chromatographic column; Moving phase: normal hexane (95%)/IPA(5%); Flow velocity: 0.6mL/min; Wavelength: 210nm; Retention time: (S)-N-Boc-3-piperidine alcohols 30.78min, another enantiomorph (R)-N-Boc-3-piperidine alcohols 33.66min.The reduzate of restructuring carbonyl acyl reductase is (S)-type.
Embodiment eight: the enzymatic conversion method of (S)-N-Boc-3-piperidine alcohols is synthetic
(S) the synthetic reaction formula of pressing of-3-N-Boc-piperidine alcohols carries out:
In a 250mL three-necked bottle, add successively 100mL, 0.2mol/LNaH
2pO
4na
2hPO
4(pH7.0) buffered soln, chemical compounds I (10g), glucose (12g) and 50mL butylacetate, magnetic agitation 10min makes to mix, then adds carbonyl acyl reductase (10mL), Hexose phosphate dehydrogenase (5mL) and coenzyme (NADP
+, 0.01g), at 30 ℃, stir 16 hours, control pH value between 6.5～7.0, high-performance liquid chromatogram determination shows 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 9.4g compound ii (N-Boc-(S)-3-piperidine alcohols), molar yield 93.0%.Optical purity is measured by embodiment seven, product ee value >99.9%.
Embodiment nine: utilize the E.coli cell transformation of carbonyl acyl reductase and Hexose phosphate dehydrogenase coexpression to produce (S)-N-Boc-3-piperidine alcohols
In a 250mL three-necked bottle, add successively 100mL, 0.2mol/LNaH
2pO
4na
2hPO
4(pH7.0) buffered soln, chemical compounds I (10g), glucose (15g) and 50mL butylacetate; magnetic agitation 10min makes to mix, then adds 1.0g to utilize to express described in embodiment tri-the carbonyl acyl reductase that obtains and the E.coli cell (pETDuet-KRED-GDH-BL of Hexose phosphate dehydrogenase coexpression
21(DE
3)), and coenzyme (NADP
+, 0.01g), stir 16 hours under 30 degrees Celsius, control pH value between 6.5～7.0, high-performance liquid chromatogram determination shows reaction end.Filtration dezymotize with cell debris after, add 100mL ethyl acetate, re-extract three times, after organic phase is dry, be spin-dried for, obtain 8.3 grams of compound iis ((S)-N-Boc-3-piperidine alcohols), optical purity ee value >99.9%, molar yield 82.2%.
Embodiment ten: the feather weight enzyme process of (S)-N-Boc-3-piperidine alcohols synthesizes and purifying
In a 10L glass reaction still, add successively 2.0L, 0.2mol/LNaH
2pO
4na
2hPO
4(pH6.5) buffered soln, chemical compounds I (1.05Kg), glucose (1.15Kg) and 2.0L deionized water, make to react each mixing of materials with the mechanical stirring stirring 10min of 700 revs/min even, adjust pH to 6.5 ± 0.1 of reaction solution, temperature to 28 ℃ ± 2 ℃.After pH and temperature-stable, the Hexose phosphate dehydrogenase enzyme liquid that adds carbonyl acyl reductase enzyme liquid prepared by above-mentioned 1.0L embodiment tetra-and 0.5L embodiment five to prepare adds the codehydrogenase Ⅱ (NADP of 0.5g simultaneously
+).Reaction is omnidistance controls 28 ℃ ± 2 ℃ of temperature, and with pH controller automatic dripping liquid caustic soda, makes reaction solution pH between 6.3～6.5.After reaction 24hr, high-performance liquid chromatogram determination shows that reaction conversion ratio is more than 99.5%, stopped reaction.
Reaction solution pH is adjusted to 7.0, removes pH probe, add 150g diatomite, stir 10min.Make diatomite filter cake, vacuum filtration is removed the solid such as enzyme and cell debris impurity, and filter residue divides and soaks filter wash 3 times with 5L ethyl acetate.Filtrate, with the extraction of 5L ethyl acetate, in triplicate, merges all organic layers simultaneously, and is spin-dried for after anhydrous sodium sulphate is fully dry, obtains the crude product ((S)-N-Boc-3-piperidine alcohols) of 1.02Kg, and this is pale red brown oil.It is fully drawn to desolventizing with high vacuum oil pump, in n-heptane solution with 60 ℃ of reflux decolours of 0.5% gac, and in normal heptane, carry out crystallization and recrystallization, recrystallisation solvent 10:1, room temperature crystallization after 45 ℃ of thermosols, recrystallization 4 times, finally obtains colourless near-white crystal 854g, HPLC>99.5%, optical purity ee value >99.9%.
The present invention still has multiple concrete embodiment, and all employings are equal to replacement or equivalent transformation and all technical schemes of forming, within all dropping on the scope of protection of present invention.
Claims (4)
1. enzyme process is prepared the method for high chiral purity (S)-3-piperidine alcohols and derivative thereof, and described reaction process is as follows:
It is characterized in that: P is hydrogen, tertbutyloxycarbonyl (Boc), carbobenzoxy-(Cbz) (Cbz) or fluorenylmethyloxycarbonyl (Fmoc), described reaction conditions is pH6.0-7.5, recombinate carbonyl acyl reductase and recombinant glucose dehydrogenase and coenzyme as catalyzer take coexpression, described restructuring carbonyl acyl reductase and recombinant glucose dehydrogenase catalyzer are liquor, lyophilized powder, immobilized enzyme or immobilized cell, the aminoacid sequence of described restructuring carbonyl acyl reductase is as shown in sequence table SEQ .ID NO:1, described recombinant glucose dehydrogenase aminoacid sequence is as shown in sequence table SEQ .ID NO:2.
2. enzyme process according to claim 1 is prepared the method for high chiral purity (S)-3-piperidine alcohols and derivative thereof, it is characterized in that: described restructuring carbonyl acyl reductase and recombinant glucose dehydrogenase be efficient coexpression in genetic engineering bacterium.
3. enzyme process according to claim 1 is prepared the method for high chiral purity (S)-3-piperidine alcohols and derivative thereof, it is characterized in that: described genetic engineering bacterium is the intestinal bacteria with recombinant vectors pETDuet-1.
4. a method for the genetic engineering bacterium of fermentation culture as shown in claim 3, is characterized in that: comprise the further fermentation that builds genetic engineering bacterium and genetic engineering bacterium, the structure of described genetic engineering bacterium comprises the following steps:
To the synthetic restructuring carbonyl acyl reductase encoding gene of full gene and Hexose phosphate dehydrogenase encoding gene respectively through double digestion; It is cloned into respectively to the different multiple clone site of expression vector pETDuet-1, recombinant plasmid order-checking is converted into respectively coli strain after confirming, builds corresponding recombinant bacterial strain again;
The further fermentation of described genetic engineering bacterium comprises the steps:
Above-described coli strain is seeded in the LB substratum that contains penbritin, is cultured to OD
600=0.8 fresh medium, adding penbritin solution to the final concentration of filtration sterilization is 0.1mg/mL, 37 ℃ of 800rpm cultivate; Feed supplement after cultivation 2hr, regulates pH7.0 ± 0.1 by strong aqua/hydrochloric acid, as the OD of nutrient solution
600reach at 25 o'clock, by tank temperature drop to 25 ℃, adding final concentration is 1mmol/L IPTG, continues to control each culture condition induction 14hr, last centrifugal results thalline.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105420307A (en) * | 2015-12-02 | 2016-03-23 | 中国科学院成都生物研究所 | Method for preparing (S)-N-t-butyloxycarboryl-3-hydroxypiperidine |
| CN105671014A (en) * | 2016-03-09 | 2016-06-15 | 浙江工业大学 | Recombinant carbonyl reductase ReCR, encoding gene, vector, engineering bacterium and application thereof |
| CN106520855A (en) * | 2016-11-10 | 2017-03-22 | 中国科学院成都生物研究所 | Method for preparing stereoscopic complementary N-heterocycle alcohol compounds by conducting biological catalysis through carbonyl reductase |
| CN107586798A (en) * | 2017-09-27 | 2018-01-16 | 上海合全药物研发有限公司 | The method that living things catalysis prepares the hydroxy piperidine of (S) 1 N benzene methoxycarbonyl group 3 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104603278A (en) * | 2012-06-18 | 2015-05-06 | 化学实验室国际股份公司 | Process for producing chiral 1 - substituted 2 - piperidinols employing oxidoreductases |
| CN103276027A (en) * | 2013-05-10 | 2013-09-04 | 苏州汉酶生物技术有限公司 | Method for biologically preparing chiral N-protective pipradrol |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105420307A (en) * | 2015-12-02 | 2016-03-23 | 中国科学院成都生物研究所 | Method for preparing (S)-N-t-butyloxycarboryl-3-hydroxypiperidine |
| CN105671014A (en) * | 2016-03-09 | 2016-06-15 | 浙江工业大学 | Recombinant carbonyl reductase ReCR, encoding gene, vector, engineering bacterium and application thereof |
| CN106520855A (en) * | 2016-11-10 | 2017-03-22 | 中国科学院成都生物研究所 | Method for preparing stereoscopic complementary N-heterocycle alcohol compounds by conducting biological catalysis through carbonyl reductase |
| CN107586798A (en) * | 2017-09-27 | 2018-01-16 | 上海合全药物研发有限公司 | The method that living things catalysis prepares the hydroxy piperidine of (S) 1 N benzene methoxycarbonyl group 3 |
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