CN104328148A - Method for preparing tert-butyl (3R, 5S)-6-chloro-3,5-dihydroxy hexanoate by using enzymatic method - Google Patents
Method for preparing tert-butyl (3R, 5S)-6-chloro-3,5-dihydroxy hexanoate by using enzymatic method Download PDFInfo
- Publication number
- CN104328148A CN104328148A CN201410613335.0A CN201410613335A CN104328148A CN 104328148 A CN104328148 A CN 104328148A CN 201410613335 A CN201410613335 A CN 201410613335A CN 104328148 A CN104328148 A CN 104328148A
- Authority
- CN
- China
- Prior art keywords
- chloro
- coexpression
- concentration
- butyl
- cofactor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/62—Carboxylic acid esters
Landscapes
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for preparing tert-butyl (3R, 5S)-6-chloro-3,5-dihydroxy hexanoate by using an enzymatic method. The method comprises the following steps: mixing coexpression whole-cells, tert-butyl (S)-6-chloro-5-hydroxy-3-oxo oxohexanoate, a cofactor, glucose and a buffer liquid in a certain ratio, reacting under the conditions that the pH is 5-8 and the temperature is 20-45 DEG C, and performing aftertreatment, thereby obtaining a product. Compared with the prior art, the method disclosed by the invention is simple in operation process, high in production efficiency, friendly to the environment, relatively low in production cost and high in reaction conversion rate and product optical purity, and has good industrial application values.
Description
Technical field
The invention belongs to pharmaceutical industry biological technical field, be specifically related to the method for chloro-3, the 5-dihydroxyl hecanoic acid t-butyl esters of a kind of enzyme process preparation (3R, 5S)-6-.
Background technology
Chloro-3, the 5-dihydroxyl hecanoic acid t-butyl esters of (3R, 5S)-6-are crucial chiral intermediates of decreasing cholesterol statins superstatin (Rosuvastatin), and the synthetic technology of this intermediate is subject to studying widely and paying close attention to all the time.The preparation method of current bibliographical information mainly contains chemical method and biological process.
It is utilize inflammable boranes derivative to be obtained by the chloro-5-hydroxyl of (S)-6--3-oxo hecanoic acid t-butyl ester asymmetric reduction at-70 DEG C that chemical method produces chloro-3, the 5-dihydroxyl hecanoic acid t-butyl esters of (3R, 5S)-6-.This technique needs the reaction conditions of extreme low temperature and anhydrous and oxygen-free, and energy consumption is serious, is difficult to practical (Angew.Chem.Int.Ed., 2000,39,4306-4308).
Biocatalysis technology, to have that Substratspezifitaet is strong, catalytic efficiency is high, reaction conditions is gentle, reactions steps is easy and the feature such as environmental pollution is little, is widely used in preparing Statins chiral side chain precursor.
Preparation (the 3R reported, 5S)-6-chloro-3, in the biological process of 5-dihydroxyl hecanoic acid t-butyl ester, major part all needs to use ketoreductase and Hexose phosphate dehydrogenase simultaneously, and be all separately express, separately reinforced, this just makes, and fermentation times increases, cost improves, and also affects use and the efficiency (US7879585B) of production simultaneously.
Summary of the invention
In order to overcome the above-mentioned defect that prior art exists, the invention discloses the method for chloro-3, the 5-dihydroxyl hecanoic acid t-butyl esters of a kind of enzyme process preparation (3R, 5S)-6-.
Concrete technology route is as follows:
For achieving the above object, the technical solution used in the present invention is as follows:
1), can express and be used for reduction (S)-6-chloro-5-hydroxyl-3-oxo hecanoic acid t-butyl ester to (3R, 5S)-6-chloro-3, the ketoreductase gene of 5-dihydroxyl hecanoic acid t-butyl ester and the gene expressing Hexose phosphate dehydrogenase to be recombinated to same plasmid be loaded in genetic engineering bacterium by biotechnology, and gained genetic engineering bacterium obtains a large amount of containing the full cell of ketoreductase (KRED) with Hexose phosphate dehydrogenase (GDH) by fermentation;
2), by complete for above-mentioned coexpression cell, the chloro-5-hydroxyl of (S)-6--3-oxo hecanoic acid t-butyl ester, cofactor, glucose and damping fluid hybrid reaction by a certain percentage, in pH=5 ~ 8, temperature reacts 1-48 hour at being 20 ~ 45 DEG C; Reaction terminates rear centrifugal segregation cell, gained clear liquid organic solvent extraction, namely obtains chloro-3, the 5-dihydroxyl hecanoic acid t-butyl esters of (3R, 5S)-6-, can be directly used in the preparation of downstream intermediate after extraction liquid is concentrated.
The full cell concn of coexpression that the inventive method is used for catalyzed reaction is 10 ~ 300g/L, is wherein preferably 60 ~ 90g/L; (S) the chloro-5-hydroxyl of-6--3-oxo hecanoic acid t-butyl ester concentration is 10-300g/L, is preferably 200 ~ 250g/L; Cofactor concentration is 0.01 ~ 1.0g/L; Glucose concn is 10 ~ 300g/L, is preferably 200 ~ 250g/L; Buffer concentration is 10 ~ 100mM/L, is preferably 80mM/L.Reaction terminates rear reaction solution organic solvent extraction, concentrated after obtain the target product that transformation efficiency is greater than 95%, optical purity is greater than 99.9%.
Furtherly, the full cell of coexpression is obtained by fermentation culture by the intestinal bacteria or yeast that can express ketoreductase and Hexose phosphate dehydrogenase recombination simultaneously, is wherein preferably intestinal bacteria.
Furtherly, the full cell of coexpression obtained that ferments is utilized directly to carry out catalyzed reaction, without the need to broken and separation and purification.
Furtherly, recombination forms by expressing ketoreductase gene (the ketoreductase gene order used in the present invention derives from Saccharomyces Cerevisiae in S accharomyces cerevisiae) and expressing glucose dehydrogenase gene (the glucose dehydrogenase gene sequence used in the present invention comes from subtilis Bacillus subtilis) restructuring.
Furtherly, reaction pH=5 ~ 8, are preferably pH=7; Temperature of reaction is 20 ~ 45 DEG C, is preferably 30 DEG C.
Furtherly, cofactor is the combination of any one or they of NAD, NADH, NADP and NADPH, is wherein preferably NADP.
Furtherly, cofactor is NADP, can be lyophilized powder or the aqueous solution, is wherein preferably the aqueous solution.
Furtherly, damping fluid is selected from phosphate buffered saline buffer or Triethanolamine buffer, is wherein preferably Triethanolamine buffer.
Furtherly, organic solvent is methylene dichloride or ethyl acetate, is wherein preferably ethyl acetate.
Compared with prior art, operating process of the present invention is simple, production efficiency is high, environmental friendliness, production cost are lower and reaction conversion ratio and product optical purity high, there is good industrial application value.
Embodiment
Below in conjunction with specific embodiment, technology contents of the present invention is further elaborated, its objective is content for a better understanding of the present invention, but protection scope of the present invention is not limited thereto.
The preparation of embodiment 1 coexpression recombination bacillus coli and full cell
Complete synthesis KRED gene is carried out pcr amplification as template and introduces restriction enzyme site (forward primer introducing NcoI restriction enzyme site at two ends, reverse primer introduces HindIII restriction enzyme site), use NcoI and HindIII to cut its enzyme, and recovery obtain KRED gene fragment.Meanwhile, extract the plasmid of pRSF-Duet carrier, also NcoI and HindIII enzyme carried out to it and cut, and reclaim enzyme cut after carrier segments.Use T4 ligase enzyme to be connected KRED gene fragment and carrier segments, and be transformed in E. coli competent BL21 (DE3), coating Kan resistant panel, cultivates in 37 DEG C of incubators.After son to be transformed grows, the some mono-clonals of picking carry out bacterium colony PCR checking, and choosing the result is that positive mono-clonal carries out subsequent experimental, by this bacterial classification called after KRED-1 bacterium.Then extract KRED bacteria plasmid, re-use NdeI and XhoI and enzyme is carried out to it cut, reclaim this carrier segments; On the other hand, pcr amplification is carried out to GDH gene, with complete synthesis gene for template, and at two ends primer restriction enzyme site, (forward primer adds NdeI restriction enzyme site, reverse primer adds XhoI restriction enzyme site) reclaim after, also use NdeI and XhoI to carry out enzyme to it and cut, and be transformed in sky BL21 (DE3) after GDH gene fragment is connected with KRED-1 bacteria plasmid fragment, coating Kan resistant panel, cultivates in 37 DEG C of incubators.After bacterium colony grows, picking mono-clonal carries out bacterium colony PCR checking, chooses positive monoclonal, namely obtains coexpression bacterial classification.
The coexpression cell of gained can cultivate acquisition by fermentation, and fermention medium uses 2*YT substratum, containing 16g/L peptone, and 10g/L yeast powder, 5g/LNaCl, 2g/L glycerine.Concrete steps are as follows: first by this coexpression strain inoculation in the little shaking flask (capacity 250ml) that 50ml substratum is housed, cultivate 12h in 37 DEG C of shaking tables to activate bacterial classification, then the thalline after activation is inoculated large shaking flask (the capacity 1L into being equipped with 400ml substratum, band baffle plate) in, inoculum size 8ml, in 37 DEG C of shaking tables be cultured to OD600nm reach 1.5-2 time, add aseptic IPTG induction, IPTG final concentration is made to reach 0.1mM, and in 25 DEG C of shaking table induction 20h.Coexpression cell is obtained finally by high speed centrifugation.
The preparation of chloro-3, the 5-dihydroxyl hecanoic acid t-butyl esters of embodiment 2 (3R, 5S)-6-
The chloro-5-hydroxyl of (S)-6--3-oxo hecanoic acid t-butyl ester 150g and glucose 150g is added in reactor, add 80mM/L phosphate buffered saline buffer 200ml, pH=7.0 is adjusted with saturated aqueous sodium carbonate, add full cell 60g and NADP0.12g of coexpression, continuing to add phosphate buffered saline buffer to reaction system is 1L, stirring at room temperature 24h; Centrifugal segregation cell, clear liquid, with dichloromethane extraction (500ml*3), merges concentrated organic phase and obtains (3R, 5S)-6-chloro-3,5-dihydroxyl hecanoic acid t-butyl ester, vapor detection transformation efficiency is greater than 95%, optical purity >99.9%.
The preparation of chloro-3, the 5-dihydroxyl hecanoic acid t-butyl esters of embodiment 3 (3R, 5S)-6-
The chloro-5-hydroxyl of (S)-6--3-oxo hecanoic acid t-butyl ester 250g and glucose 250g is added in reactor, add 80mM/L Triethanolamine buffer 200ml, pH=7.0 is adjusted with saturated aqueous sodium carbonate, add full cell 180g and NADP0.20g of coexpression, continuing to add Triethanolamine buffer to reaction system is 1L, stirring at room temperature 24h; Centrifugal segregation cell, clear liquid ethyl acetate extracts (500ml*3), merges concentrated organic phase and obtains (3R, 5S)-6-chloro-3,5-dihydroxyl hecanoic acid t-butyl ester, vapor detection transformation efficiency is greater than 95%, optical purity >99.9%.
Claims (8)
1. an enzyme process preparation (3R, 5S)-6-chloro-3, the method of 5-dihydroxyl hecanoic acid t-butyl ester, is characterized in that: be obtained by reacting product after complete for coexpression cell, the chloro-5-hydroxyl of (S)-6--3-oxo hecanoic acid t-butyl ester, cofactor, glucose and damping fluid being mixed by a certain percentage.
2. the method for claim 1, is characterized in that: the full cell of described coexpression is containing the genetic engineering bacterium of carbonyl reductase (KRED) with Hexose phosphate dehydrogenase (GDH).
3. method as claimed in claim 1 or 2, it is characterized in that: the full cell of described coexpression is obtained by fermentation culture by the intestinal bacteria or yeast that can express carbonyl reductase (KRED) and Hexose phosphate dehydrogenase (GDH) recombination simultaneously, is wherein preferably intestinal bacteria.
4. the method as described in claim 1,2 or 3, is characterized in that: utilize the full cell direct catalytic reaction of described coexpression.
5. the method for claim 1, is characterized in that: the concentration that the concentration of the full cell of described coexpression is 10 ~ 300g/L, the concentration of described (S)-6-chloro-5-hydroxyl-3-oxo hecanoic acid t-butyl ester is 10 ~ 300g/L, the concentration of described cofactor is 0.01 ~ 1.0g/L, the concentration of described glucose is 10 ~ 300g/L and described damping fluid is 10 ~ 100mM/L.
6. the method for claim 1, is characterized in that: described reaction in pH=5 ~ 8, temperature carries out at being 20 ~ 45 DEG C.
7. the method for claim 1, is characterized in that: described cofactor is the combination of any one or they of NAD, NADH, NADP and NADPH, is wherein preferably NADP.
8. the method for claim 1, is characterized in that: described damping fluid is selected from phosphate buffered saline buffer or Triethanolamine buffer, is wherein preferably Triethanolamine buffer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410613335.0A CN104328148A (en) | 2014-11-04 | 2014-11-04 | Method for preparing tert-butyl (3R, 5S)-6-chloro-3,5-dihydroxy hexanoate by using enzymatic method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410613335.0A CN104328148A (en) | 2014-11-04 | 2014-11-04 | Method for preparing tert-butyl (3R, 5S)-6-chloro-3,5-dihydroxy hexanoate by using enzymatic method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104328148A true CN104328148A (en) | 2015-02-04 |
Family
ID=52402952
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410613335.0A Pending CN104328148A (en) | 2014-11-04 | 2014-11-04 | Method for preparing tert-butyl (3R, 5S)-6-chloro-3,5-dihydroxy hexanoate by using enzymatic method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104328148A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104726506A (en) * | 2015-03-17 | 2015-06-24 | 苏州汉酶生物技术有限公司 | Preparation method of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate |
| CN104789505A (en) * | 2015-04-23 | 2015-07-22 | 苏州东和盛昌生物科技有限公司 | Method and strain for preparing cis-3,5-dihydroxyhexanoate compound by reduction and strain |
| CN105087684A (en) * | 2015-09-16 | 2015-11-25 | 连云港宏业化工有限公司 | Preparation method of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate |
| CN105087685A (en) * | 2015-09-16 | 2015-11-25 | 连云港宏业化工有限公司 | Method for synthesizing (3R,5S)-6-chloro-3,5-dihydroxyhexanoate |
| CN114875081A (en) * | 2022-06-07 | 2022-08-09 | 湖北迅达药业股份有限公司 | Green industrial production method of rosuvastatin key intermediate |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6645746B1 (en) * | 1999-12-03 | 2003-11-11 | Kaneka Corporation | Carbonyl reductase, gene thereof and method of using the same |
| CN103695486A (en) * | 2014-01-03 | 2014-04-02 | 黄冈华阳药业有限公司 | Biological preparation method of (3R, 5R)-6-cyano-3,5-dyhydroxytert-butylhexanoate |
| CN104087546A (en) * | 2014-07-03 | 2014-10-08 | 浙江大学 | Engineering bacteria and method for preparing tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate |
-
2014
- 2014-11-04 CN CN201410613335.0A patent/CN104328148A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6645746B1 (en) * | 1999-12-03 | 2003-11-11 | Kaneka Corporation | Carbonyl reductase, gene thereof and method of using the same |
| CN103695486A (en) * | 2014-01-03 | 2014-04-02 | 黄冈华阳药业有限公司 | Biological preparation method of (3R, 5R)-6-cyano-3,5-dyhydroxytert-butylhexanoate |
| CN104087546A (en) * | 2014-07-03 | 2014-10-08 | 浙江大学 | Engineering bacteria and method for preparing tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate |
Non-Patent Citations (2)
| Title |
|---|
| 宿宇宁: "不对称还原制备光学纯(R)-2-羟基-4-苯基丁酸乙酯的双酶共表达重组菌的构建", 《催化学报》 * |
| 曹政: "羰基还原酶不对称还原(R)-6-氰基-5-羟基-3-羰基己酸叔丁酯", 《生物加工过程》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104726506A (en) * | 2015-03-17 | 2015-06-24 | 苏州汉酶生物技术有限公司 | Preparation method of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate |
| CN104789505A (en) * | 2015-04-23 | 2015-07-22 | 苏州东和盛昌生物科技有限公司 | Method and strain for preparing cis-3,5-dihydroxyhexanoate compound by reduction and strain |
| CN104789505B (en) * | 2015-04-23 | 2018-03-06 | 苏州东和盛昌生物科技有限公司 | Reduction prepares the method and strain of cis 3,5 dihydroxyhexanoate compound |
| CN105087684A (en) * | 2015-09-16 | 2015-11-25 | 连云港宏业化工有限公司 | Preparation method of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate |
| CN105087685A (en) * | 2015-09-16 | 2015-11-25 | 连云港宏业化工有限公司 | Method for synthesizing (3R,5S)-6-chloro-3,5-dihydroxyhexanoate |
| CN114875081A (en) * | 2022-06-07 | 2022-08-09 | 湖北迅达药业股份有限公司 | Green industrial production method of rosuvastatin key intermediate |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ravella et al. | Extracellular polysaccharide (EPS) production by a novel strain of yeast-like fungus Aureobasidium pullulans | |
| CN104328148A (en) | Method for preparing tert-butyl (3R, 5S)-6-chloro-3,5-dihydroxy hexanoate by using enzymatic method | |
| CN103497911B (en) | Application of Chryseobacterium sp. and carbonyl reductase thereof in production of aprepitant chiral intermediate | |
| Xie et al. | The kinetic characterization of photofermentative bacterium Rhodopseudomonas faecalis RLD-53 and its application for enhancing continuous hydrogen production | |
| CN104372017A (en) | Method for enhancing yields of gene engineering bacterium isoprene and derivatives thereof and application thereof | |
| CN102965403A (en) | Biological preparation method of tert-butyl(3R,5S)-6-chloro-3,5-dihydroxyhexanoate | |
| CN102925501A (en) | Biological preparation method of (S)-4-chloro-3-hydroxybutyrate ethyl | |
| CN106544284A (en) | A kind of restructuring Yarrowia lipolytica engineered strain and its construction method and application | |
| WO2022188403A1 (en) | Strain of enterobacter chengduensis for producing nicotinamide mononucleotide and use thereof | |
| CN115820527B (en) | Recombinant halomonas for producing mevalonate and construction method and application thereof | |
| CN104371966A (en) | Gene engineering strain capable of synthesizing phloroglucinol from acetic acid and construction method and application thereof | |
| CN115975832B (en) | Use of formate dehydrogenase in increasing resistance of microbial fermentation strains to formic acid and acetic acid in cellulose hydrolysates | |
| CN105177078A (en) | Preparation method of hydroxyectoine | |
| CN104388488A (en) | Preparation of (3R,5R)-6-cyan-3,5-dihydroxy ter-butyl caproate employing immobilized whole-cell catalysis | |
| CN107400652A (en) | A kind of engineering bacteria construction method of the hydracrylic acid of dynamic regulation 3 synthesis | |
| CN102978249A (en) | Biological preparation method of 6-cyan-(3R, 5R)- dihydroxy-hexanoic acid tert-butyl ester | |
| CN112063532B (en) | Geotrichum linum and application thereof in preparation of (S) -1- (2-trifluoromethylphenyl) ethanol | |
| CN103695486A (en) | Biological preparation method of (3R, 5R)-6-cyano-3,5-dyhydroxytert-butylhexanoate | |
| CN104726355A (en) | Method for converting and preparing (S)-phenylglycol asymmetrically through (S)-carbonyl reductase II expressed by Saccharomyces cerevisiae | |
| CN104195191B (en) | A kind of method that enzyme process prepares (3R, 5R)-6-cyano group-3,5-dihydroxy hecanoic acid t-butyl ester | |
| CN109694892B (en) | Method and kit for preparing salidroside | |
| CN101469318B (en) | Synthesis of (R)-styrene glycol by coupling acceleration of (R)-carbonyl reduction enzyme and formic dehydrogenase | |
| CN105274041B (en) | One plant of recombination bacillus coli and its application in production 2- butanol | |
| CN104357526B (en) | The method for preparing cholestenone using conversion of resting cells using eutectic as chaotropic agent | |
| CN108004275B (en) | Escherichia coli recombinant bacterium for producing adipic acid and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150204 |