CN104388488A - Preparation of (3R,5R)-6-cyan-3,5-dihydroxy ter-butyl caproate employing immobilized whole-cell catalysis - Google Patents
Preparation of (3R,5R)-6-cyan-3,5-dihydroxy ter-butyl caproate employing immobilized whole-cell catalysis Download PDFInfo
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- CN104388488A CN104388488A CN201410587382.2A CN201410587382A CN104388488A CN 104388488 A CN104388488 A CN 104388488A CN 201410587382 A CN201410587382 A CN 201410587382A CN 104388488 A CN104388488 A CN 104388488A
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- 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
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
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Abstract
The invention discloses a method for preparing (3R,5R)-6-cyan-3,5-dihydroxy ter-butyl caproate employing immobilized whole-cell catalysis. The method comprises the following steps: mixing immobilized coexpression whole cells, (R)-6-cyan-5-hydroxy-3-oxo-tert-butyl ester, cofactors, glucose and a buffer liquid according to a certain ratio; reacting at 20-45 DEG C under the condition that pH is 5-8; and obtaining the product by post-treatment. Compared with the prior art, the adopted method has the advantages that a biocatalyst is easy to separate from a reaction liquid, the operation is convenient, the catalyst can be recycled, the product is high in yield and optical purity, and the method has good application value.
Description
Technical field
The invention belongs to pharmaceutical industry biological technical field, be specifically related to the method for a kind of immobilized whole-cell catalysis preparation (3R, 5R)-6-cyano group-3,5-dihydroxyl hecanoic acid t-butyl ester.
Background technology
(3R, 5R)-6-cyano group-3,5-dihydroxyl hecanoic acid t-butyl ester is the crucial chiral intermediate of decreasing cholesterol statins atorvastatin (Atorvastatin), 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 boranes derivative to be obtained by (R)-6-cyano group-5-hydroxyl-3-oxo tert-butyl ester asymmetric reduction at-70 DEG C that chemical process produces (3R, 5R)-6-cyano group-3,5-dihydroxyl hecanoic acid t-butyl ester.This technique needs the reaction conditions of extreme low temperature and anhydrous and oxygen-free, and reductive agent borine used is inflammable, and energy consumption is serious and the stereoisomeric purity of product is only 98% (US2009/0216029A1), is unfavorable for suitability for industrialized production.
CN103911403 discloses biological process synthesis (3R, 5R)-6-cyano group-3,5-dihydroxyl hecanoic acid t-butyl ester, the method with (R)-6-cyano group-5-hydroxyl-3-oxo tert-butyl ester for substrate, Recombinant E. coli Fermentation Broth is catalyzer, concentration of substrate is the highest only has 55g/L, and need add solubility promoter methyl-sulphoxide.The impurity contained in fermented liquid when the method adopts fermented liquid directly to make catalyzer can bring disadvantageous effect to reaction, and solubility promoter methyl-sulphoxide environmental pollution is comparatively large, and concentration of substrate is too low, and these factors are all unfavorable for suitability for industrialized production.
Application number is report a kind of enzyme process preparation (3R in the patent application of 201410462724.8,5R)-6-cyano group-3, the method of 5-dihydroxyl hecanoic acid t-butyl ester, the method biological catalyst used can not recovery, during aftertreatment, the separation of biological catalyst is removed more difficult, virtually adds production time and cost.
In addition, we are disclose one (3R in the application documents of 201410546518.5 at application number, 5R)-6-cyano group-3, the biological preparation method of 5-dihydroxyl hecanoic acid t-butyl ester, the method utilizes immobilized cell composition to catalyze and synthesize target compound, but need use two kinds of cells, operating process shows slightly loaded down with trivial details.
Summary of the invention
In order to overcome the above-mentioned defect that prior art exists, the invention discloses the method for a kind of immobilized whole-cell catalysis preparation (3R, 5R)-6-cyano group-3,5-dihydroxyl hecanoic acid t-butyl ester.
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 (the R)-6-cyano group-5-hydroxyl-3-oxo tert-butyl ester to (3R, 5R)-6-cyano group-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 coexpression full cell of ketoreductase (KRED) with Hexose phosphate dehydrogenase (GDH) by fermentation; Complete for above-mentioned coexpression cell is mixed with fixing agent by a certain percentage, the full cell of being fixed of drying and moulding coexpression.
2), by complete for immobilization coexpression cell, (R)-6-cyano group-5-hydroxyl-3-oxo tert-butyl ester, cofactor, glucose and damping fluid mix by a certain percentage, in pH=5 ~ 8, temperature reacts 1-48 hour at being 20 ~ 45 DEG C; In reaction mixture, the full wet cell weight concentration of immobilization coexpression is 100 ~ 400g/L, is preferably 60 ~ 150g/L, (R) the concentration of-6-cyano group-5-hydroxyl-3-oxo tert-butyl ester is 100 ~ 400g/L, is preferably 200 ~ 250g/L, the concentration of cofactor is 0.001 ~ 1.0g/L, the concentration of glucose is 100 ~ 400g/L, is preferably 200 ~ 250g/L, and the concentration of damping fluid is 10 ~ 100mM/L, is preferably 80mM/L; Reaction terminates the full cell of rear filtered and recycled immobilization coexpression; Filtrate through organic solvent extraction, concentrated obtain the target product that yield is greater than 95%, optical purity is greater than 99.9%, the preparation of downstream intermediate or product can be directly used in.
Furtherly, the full cell of immobilization coexpression be by the gene of the gene and Hexose phosphate dehydrogenase (GDH) of expressing ketoreductase (KRED) by biotechnology recombinate same plasmid and to be loaded in genetic engineering bacterium through fermenting, immobilization process obtains.
Furtherly, genetic engineering bacterium is intestinal bacteria or yeast, is wherein preferably intestinal bacteria.
Furtherly, utilize the full cell direct catalytic reaction of immobilization coexpression, 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, pH=5 ~ 8 of reaction, 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, fixing agent is a kind of of polyoxyethylene glycol, polyacrylamide, polyvinyl alcohol or alginate calcium or their combination.
Furtherly, the ratio of the full cell of coexpression and fixing agent is: 1:1 ~ 1:2.
Furtherly, the organic solvent extracted is methylene dichloride or ethyl acetate, is wherein preferably ethyl acetate.
Furtherly, the full cell of immobilization coexpression after filtering can recovery, and applying mechanically number of times is 1 ~ 3 time.
Compared with prior art, the present invention utilizes immobilization coexpression whole-cell catalytic to synthesize (3R, 5R)-6-cyano group-3,5-dihydroxyl hecanoic acid t-butyl ester, have that the full cell of immobilization coexpression is easily separated with reaction solution, easy to operate, catalyzer recoverable, product yield and optical purity advantages of higher, there is good using 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.
Embodiment 1
One, the preparation of the full cell of immobilization coexpression
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.
Coexpression cell can cultivate acquisition by fermentation, and fermention medium uses 2*YT substratum, containing 16g/L peptone, and 10g/L yeast powder, 5g/L NaCl, 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, obtain the full cell of coexpression finally by high speed centrifugation.
Under room temperature, 450g polyvinyl alcohol is added in the water of 3.5L, be warming up to 90 ~ 95 DEG C, after polyvinyl alcohol dissolves completely, cool the temperature to 25 ~ 30 DEG C; In above-mentioned solution, add the full cell of 300g coexpression, after mixing, stir 1h; Draw mixed solution with peristaltic pump and the plastics volley of rifle fire, and note forms disc-shaped in the plane; Move into air dry oven, 30 DEG C of dry 1.0-1.5h; Move into 0.1M Na
2sO
4solution-stabilized 2h, is filtered dry and washes twice with clear water, obtains the full cell of 1.3kg immobilization coexpression.
Two, the synthesis of (3R, 5R)-6-cyano group-3,5-dihydroxyl hecanoic acid t-butyl ester
450g (the R)-6-cyano group-5-hydroxyl-3-oxo tert-butyl ester and 450g glucose are added in reactor, add 600ml80mM/L phosphate buffered saline buffer, pH=7.0 is adjusted with saturated aqueous sodium carbonate, add the full cell of 300g immobilization coexpression and 0.3g NADP, continuing to add phosphate buffered saline buffer to reaction system is 3L, stirring at room temperature 24h; Filter, filtrate is extracted with ethyl acetate, and concentrating under reduced pressure ethyl acetate obtains 449g weak yellow liquid, purity ﹥ 95%, optical purity ﹥ 99.9%.
Embodiment 2
One, the preparation of immobilized whole-cell composition
The preparation of the full cell of coexpression is with embodiment one.
Under room temperature, 300g polyoxyethylene glycol is dissolved in 3.5L water, in solution, adds 300g polyvinyl alcohol, be warming up to 90 ~ 95 DEG C, after polyvinyl alcohol dissolves completely, cool the temperature to 25 ~ 30 DEG C; In above-mentioned solution, add the full cell of 300g coexpression, after mixing, stir 1h; Draw mixed solution with peristaltic pump and the plastics volley of rifle fire, and note forms disc-shaped in the plane; Move into air dry oven, 30 DEG C of dry 1.0-1.5h; Move into 0.1M Na
2sO
4solution-stabilized 2h, is filtered dry and washes twice with clear water, obtains the full cell of 1.6kg immobilization coexpression.
Two, the synthesis of (3R, 5R)-6-cyano group-3,5-dihydroxyl hecanoic acid t-butyl ester
500g (the R)-6-cyano group-5-hydroxyl-3-oxo tert-butyl ester and 500g glucose are added in reactor, add 400ml80mM/L Triethanolamine buffer, pH=7.0 is adjusted with saturated aqueous sodium carbonate, add the full cell of 360g immobilization coexpression and 0.4g NADP, continuing to add Triethanolamine buffer to reaction system is 2L, stirring at room temperature 24h; Filter, filtrate is extracted with ethyl acetate, and concentrating under reduced pressure ethyl acetate obtains 494g weak yellow liquid, purity ﹥ 95%, optical purity ﹥ 99.9%.
Embodiment 3
With the full cell synthesis of the immobilization coexpression reclaimed (3R, 5R)-6-cyano group-3,5-dihydroxyl hecanoic acid t-butyl ester.
500g (the R)-6-cyano group-5-hydroxyl-3-oxo tert-butyl ester and 500g glucose are added in reactor, add 400ml80mM/L Triethanolamine buffer, pH=7.0 is adjusted with saturated aqueous sodium carbonate, add the full cell of immobilization coexpression and the 0.4g NADP of 360g embodiment 2 recovery, continuing to add Triethanolamine buffer to reaction system is 2L, stirring at room temperature 24h; Filter, filtrate is extracted with ethyl acetate, and concentrating under reduced pressure ethyl acetate obtains 489g weak yellow liquid, purity ﹥ 95%, optical purity ﹥ 99.9%.
Claims (10)
1. an immobilized whole-cell catalysis preparation (3R, 5R)-6-cyano group-3, the method of 5-dihydroxyl hecanoic acid t-butyl ester, is characterized in that: be obtained by reacting product after complete for immobilization coexpression cell, (R)-6-cyano group-5-hydroxyl-3-oxo tert-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 immobilization coexpression be by the gene of the gene and Hexose phosphate dehydrogenase (GDH) of expressing ketoreductase (KRED) by biotechnology recombinate same plasmid and to be loaded in genetic engineering bacterium through fermenting, immobilization process obtains.
3. method as claimed in claim 2, is characterized in that: described genetic engineering bacterium is intestinal bacteria or yeast, is wherein preferably intestinal bacteria.
4. method as claimed in claim 1 or 2, is characterized in that: utilize described immobilization coexpression whole-cell catalytic to react.
5. the method for claim 1, is characterized in that: the concentration that the concentration of the full cell of described immobilization coexpression is 100 ~ 400g/L, the concentration of described (R)-6-cyano group-5-hydroxyl-3-oxo tert-butyl ester is 100 ~ 400g/L, the concentration of described cofactor is 0.001 ~ 1.0g/L, the concentration of described glucose is 100 ~ 400g/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.
9. the method for claim 1, it is characterized in that, the preparation method of the full cell of described immobilization coexpression is: mixed with fixing agent by a certain percentage by complete for coexpression cell, drying and moulding, and described fixing agent is a kind of of polyoxyethylene glycol, polyacrylamide, polyvinyl alcohol or alginate calcium or their combination.
10. method as claimed in claim 9, is characterized in that: the ratio of the full cell of described coexpression and described fixing agent is: 1:1 ~ 1:2.
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| CN201410587382.2A CN104388488A (en) | 2014-10-28 | 2014-10-28 | Preparation of (3R,5R)-6-cyan-3,5-dihydroxy ter-butyl caproate employing immobilized whole-cell catalysis |
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| CN201410587382.2A CN104388488A (en) | 2014-10-28 | 2014-10-28 | Preparation of (3R,5R)-6-cyan-3,5-dihydroxy ter-butyl caproate employing immobilized whole-cell catalysis |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104830921A (en) * | 2015-04-27 | 2015-08-12 | 上海工业生物技术研发中心 | Enzymatic method for preparing statins intermediates |
| CN105713884A (en) * | 2014-12-02 | 2016-06-29 | 安琪酵母股份有限公司 | Biocatalytic hydrogenation composition and method for synthesis of Rosuvastatin chiral intermediate |
| CN106047950A (en) * | 2016-06-30 | 2016-10-26 | 尚科生物医药（上海）有限公司 | Biological preparation method of (S)-1-(2,6-dichloro-3-fluorophenyl)ethanol |
| CN112501223A (en) * | 2020-11-23 | 2021-03-16 | 江苏阿尔法药业有限公司 | Process for the preparation of 2- ((2R, 4R) -4-hydroxy-6-oxotetrahydro-2H-pyran-2-yl) acetonitrile |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105713884A (en) * | 2014-12-02 | 2016-06-29 | 安琪酵母股份有限公司 | Biocatalytic hydrogenation composition and method for synthesis of Rosuvastatin chiral intermediate |
| CN104830921A (en) * | 2015-04-27 | 2015-08-12 | 上海工业生物技术研发中心 | Enzymatic method for preparing statins intermediates |
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| CN106047950A (en) * | 2016-06-30 | 2016-10-26 | 尚科生物医药（上海）有限公司 | Biological preparation method of (S)-1-(2,6-dichloro-3-fluorophenyl)ethanol |
| CN112501223A (en) * | 2020-11-23 | 2021-03-16 | 江苏阿尔法药业有限公司 | Process for the preparation of 2- ((2R, 4R) -4-hydroxy-6-oxotetrahydro-2H-pyran-2-yl) acetonitrile |
| CN112501223B (en) * | 2020-11-23 | 2022-08-12 | 江苏阿尔法药业股份有限公司 | Process for the preparation of 2- ((2R, 4R) -4-hydroxy-6-oxotetrahydro-2H-pyran-2-yl) -acetonitrile |
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