CN101892273A - Application of immobilized lipase Lipozyme TLIM as catalyst for unsymmetrical Michael addition reaction - Google Patents
Application of immobilized lipase Lipozyme TLIM as catalyst for unsymmetrical Michael addition reaction Download PDFInfo
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- CN101892273A CN101892273A CN2010102011854A CN201010201185A CN101892273A CN 101892273 A CN101892273 A CN 101892273A CN 2010102011854 A CN2010102011854 A CN 2010102011854A CN 201010201185 A CN201010201185 A CN 201010201185A CN 101892273 A CN101892273 A CN 101892273A
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Abstract
The invention relates to novel application of substance, in particular to application of immobilized lipase Lipozyme TLIM as a catalyst for an unsymmetrical Michael addition reaction. The invention has the advantages of wide substrate applicability, good and catalysis activity and selectivity, easy operation, and can be recycled. The invention further provides a method of using the immobilized lipase Lipozyme TLIM to catalyze the unsymmetrical Michael addition reaction. The method has high yield and favorable selectivity.
Description
Technical field
The present invention relates to the new application of material, particularly immobilized lipase Lipozyme TLIM is as the application of asymmetric Michael addition reaction catalyst.
Background technology
The carbanion that is formed by active methylene compound is to α, and the nucleophilic addition(Adn) of the carbon-carbon double bond of beta-unsaturated carbonyl compound is the alkylating a kind of important method of active methylene compound, and this reaction is called the Michael addition reaction.The Michael addition reaction is normally by base catalysis, or unites the homogeneous catalysis of carrying out by amine and carboxylic acid or Lewis acid, and the use of these catalyzer has caused some environmental problems.Therefore, increasing in recent years diversion is to eco-friendly catalyzer.Biological catalyst just progressively increases in the application of organic synthesis as a kind of green, effective catalyzer.Lytic enzyme (being mainly lipase and proteolytic enzyme) because it all has high stability and high catalytic activity to a lot of substrates, has been used for the Michael addition reaction as a kind of biological catalyst of novelty.But up to now, bibliographical information is not seen in enzymatic asymmetric Michael addition reaction as yet.
Summary of the invention
In view of this, one of purpose of the present invention is to provide the application of immobilized lipase Lipozyme TLIM as asymmetric Michael addition reaction catalyst, have that the substrate suitability is wide, catalytic activity and good, the easy operation of selectivity, can recycling etc. advantage; Two of purpose is to provide the method for utilizing the asymmetric Michael addition reaction of immobilized lipase Lipozyme TLIM catalysis, the product yield height, and selectivity is good.
For achieving the above object, the invention provides following technical scheme:
1, immobilized lipase Lipozyme TLIM is as the application of asymmetric Michael addition reaction catalyst.
2, utilize the method for the asymmetric Michael addition reaction of immobilized lipase Lipozyme TLIM catalysis, be to be catalyzer with immobilized lipase Lipozyme TLIM, in water-containing organic solvent, react to body Michael acceptor and Michael, water is 0～30: 100 with the volume of organic solvent ratio, temperature of reaction is 25 ℃～85 ℃, and it is 1～5: 1～2 that Michael acceptor and Michael give the molar ratio of body.
Further, described organic solvent is tetrahydrofuran (THF), methyl-sulphoxide, N, dinethylformamide or ethanol, and water is 10: 100 or 20: 100 with the volume of organic solvent ratio; Described temperature of reaction is 35 ℃ or 65 ℃; It is 2～5: 1 that described Michael acceptor and Michael give the molar ratio of body;
Further, described organic solvent is a methyl-sulphoxide, and water is 10: 100 with the volume of organic solvent ratio; Described temperature of reaction is 35 ℃; It is 3: 1 that described Michael acceptor and Michael give the molar ratio of body;
Further, described Michael acceptor is the β-nitroolefin of aromatic nucleus or heterocyclic substituted, or α, β-unsaturated cyclic ketones; Described Michael is 1 to body, 3-dicarbonyl compound or cyclic ketones.
Beneficial effect of the present invention is: the invention provides the application of immobilized lipase Lipozyme TLIM as asymmetric Michael addition reaction catalyst, have that the substrate suitability is wide, catalytic activity and good, the easy operation of selectivity, can recycling etc. advantage; The method of utilizing the asymmetric Michael addition reaction of this immobilized lipase Lipozyme TLIM catalysis also is provided, the product yield height, selectivity is good.
Embodiment
In order to make the purpose, technical solutions and advantages of the present invention clearer, below the preferred embodiments of the present invention are described in detail.
(derive from and dredge the thermophilic hyphomycete of cotton shape (Thermomyces lanuginosus), EC 3.1.1.3 is 0.25U/mg) available from letter (China) Investment Co., Ltd of Novi for the immobilized lipase Lipozyme TLIM that uses in the preferred embodiment.Unless otherwise indicated, all reagent all derive from the commercial channel and without being further purified.
Adopt tlc (GF254 silica-gel plate) monitoring reaction process in the preferred embodiment; Flash column chromatography adopts 100～200 order silica gel pressured columns; Diastereomer ratio (dr) employing proton nmr spectra (
1HNMR) and high performance liquid chromatography chiral stationary phase method measure,
1HNMR adopts Bruker AV-300 type nuclear magnetic resonance analyser (is interior mark with TMS), and chiral stationary phase adopts AD-H, OD-H, AS-H or OJ-H chiral column; The ee value adopts high performance liquid chromatography chiral stationary phase method to measure, and the absolute configuration of product compares definite by the chromatographic peak retention time with bibliographical information.
Embodiment 1～11, different enzymatic Michael addition reaction
In round-bottomed flask, add anti--2,4-two chloro-beta-nitrostyrenes (120mg, 0.55mmol), diethyl malonate (177mg, 1.1mmol), deionized water (1mL), methyl-sulphoxide (DMSO, 5mL) and enzyme (400mg), room temperature (25～30 ℃) stirring reaction down; After reaction finishes, filter, the filter cake washed with dichloromethane, washing lotion and filtrate merge, behind the thin up, use dichloromethane extraction, the combined dichloromethane extraction liquid, behind anhydrous sodium sulfate drying, underpressure distillation remove desolvate crude product, with crude product flash column chromatography purifying, promptly get target product.Reaction conditions and the results are shown in Table 1.
The different enzymatic Michael addition reactions of table 1
As shown in Table 1, anti--2, the Michael addition reaction of 4-two chloro-beta-nitrostyrenes and diethyl malonate does not detect product generation (embodiment 11) under no enzyme situation; In 10 kinds of tested enzymes, the catalytic activity of immobilized lipase Lipozyme TLIM is best, product yield the highest (embodiment 1); Ash wheel silk streptoverticillium produces the catalytic activity of Transglutaminase EC2.3.2.13, papoid, chymopapain and wooden mould cellulase-producing and takes second place product yield medium (embodiment 2～5); The extremely low or catalytically inactive of the catalytic activity of other 5 kinds of enzymes only detects the trace product or does not detect product (embodiment 6～10).
The asymmetric Michael addition reaction of embodiment 12～46, Lipozyme TLIM catalysis 2-cyclonene and methyl ethyl diketone
In round-bottomed flask, add 2-cyclonene, methyl ethyl diketone, deionized water, organic solvent and Lipozyme TLIM, stirring reaction at a certain temperature; After reaction finishes, filter, the filter cake washed with dichloromethane, washing lotion and filtrate merge, behind the thin up, use dichloromethane extraction, the combined dichloromethane extraction liquid, behind anhydrous sodium sulfate drying, underpressure distillation remove desolvate crude product, with crude product flash column chromatography purifying, promptly get target product.Reaction conditions and the results are shown in Table 2.
The asymmetric Michael addition reaction of table 2, Lipozyme TLIM catalysis 2-cyclonene and methyl ethyl diketone
Annotate: embodiment 35～46 does not detect the ee value.
As shown in Table 2, the catalytic asymmetric Michael addition reaction of immobilized lipase Lipozyme TLIM can be carried out under different condition smoothly.Reaction with 2-cyclonene and methyl ethyl diketone is an example, and reaction solvent preferably is made up of organic solvent and water, the preferred tetrahydrofuran (THF) of organic solvent (THF), DMSO, N, dinethylformamide (DMF) or ethanol, more preferably DMSO (embodiment 12～21); Water is preferably 10: 100 or 20: 100 with the volume of organic solvent ratio, more preferably 10: 100 (embodiment 22～28); Preferred 35 ℃ or 65 ℃ of temperature of reaction, more preferably 35 ℃ (embodiment 29～34); The molar ratio of 2-cyclonene and methyl aceto acetate is preferably 2～5: 1, more preferably 3: 1 (embodiment 35～40); In addition, the enzyme amount is also influential to product yield, can be by the regulatory enzyme amount to obtain higher product yield (embodiment 41～46).
The asymmetric Michael addition reaction of embodiment 47～51,4-chloro-beta-nitrostyrene and methyl aceto acetate
In round-bottomed flask, add 4-chloro-beta-nitrostyrene (3.0mmol), methyl aceto acetate (1.0mmol), deionized water (0.5mL), DMSO (5mL) and catalyzer (200mg), 35 ℃ of stirring reactions; After reaction finishes, filter, the filter cake washed with dichloromethane, washing lotion and filtrate merge, behind the thin up, use dichloromethane extraction, the combined dichloromethane extraction liquid, behind anhydrous sodium sulfate drying, underpressure distillation remove desolvate crude product, with crude product flash column chromatography purifying, promptly get target product.Reaction conditions and the results are shown in Table 3.
The asymmetric Michael addition reaction of table 3,4-chloro-beta-nitrostyrene and methyl aceto acetate
As shown in Table 3, the Michael addition reaction of 4-chloro-beta-nitrostyrene and methyl aceto acetate, product yield under immobilized lipase Lipozyme TLIM catalysis is 90%, product yield under non-zymoprotein such as ovalbumin catalysis only 30%, (embodiment 47 and do not detect the product generation under no enzyme situation, 50～51), illustrate that the right title of Lipozyme TLIM Michael addition reaction has catalytic effect, and this catalytic effect is not to be realized by protein merely, but depends on catalytic site and the three-dimensional structure of Lipozyme TLIM.When using serine protease and thiol protease inhibitor phenylmethylsulfonyl fluoride (PMSF), or when tryptophane and the pretreated Lipozyme TLIM of tyrosine residues modifier N-bromosuccinimide (NBS) catalysis, do not detect product and generate (embodiment 48～49), illustrate that PMSF or NBS processing suppresses the catalytic activity of Lipozyme TLIM fully, promptly Serine and/or sulfydryl and tryptophane and/or tyrosine are the requisite catalytic site of Lipozyme TLIM.
The catalytic asymmetric Michael addition reaction of embodiment 52～66, Lipozyme TLIM
In round-bottomed flask, add Michael acceptor (3.0mmol), Michael to body (1.0mmol), deionized water (0.5mL), DMSO (5mL) and Lipozyme TLIM (200mg), 35 ℃ of stirring reactions; After reaction finishes, filter, the filter cake washed with dichloromethane, washing lotion and filtrate merge, behind the thin up, use dichloromethane extraction, the combined dichloromethane extraction liquid, behind anhydrous sodium sulfate drying, underpressure distillation remove desolvate crude product, with crude product flash column chromatography purifying, promptly get target product.Reaction conditions and the results are shown in Table 4.
The catalytic asymmetric Michael addition reaction of table 4Lipozyme TLIM
Annotate:
*Absolute configuration is not determined.
As shown in Table 4, β-the nitroethylene of Michael acceptor such as aromatic nucleus or heterocyclic substituted, 2-cyclonene and Michael give body such as methyl ethyl diketone, methyl aceto acetate, diethyl malonate, the asymmetric Michael addition reaction of pimelinketone all can be carried out under the catalysis of immobilized lipase Lipozyme TLIM smoothly, obtain medium product to higher yields (30%～90%), and demonstrate selectivity (embodiment 52～66) preferably, illustrate that the catalytic asymmetric Michael addition reaction of Lipozyme TLIM has the substrate general applicability, the Michael acceptor can be the β-nitroolefin of aromatic nucleus or heterocyclic substituted, or α, β-unsaturated cyclic ketones; It can be 1 that Michael gives body, 3-dicarbonyl compound or cyclic ketones etc.
The recycling of embodiment 67, Lipozyme TLIM
In round-bottomed flask, add 4-chloro-beta-nitrostyrene (3.0mmol), methyl aceto acetate (1.0mmol), deionized water (0.5mL), DMSO (5mL) and Lipozyme TLIM (200mg), 35 ℃ of stirring reactions; After reaction finishes, filter, filter cake is the enzyme washed with dichloromethane, and washing lotion and filtrate merge, behind the thin up, use dichloromethane extraction, the combined dichloromethane extraction liquid, behind anhydrous sodium sulfate drying, underpressure distillation remove desolvate crude product, with crude product flash column chromatography purifying, promptly get target product; Enzyme is used methanol wash 2 times again, and washing with acetone 3 times is used for the next round reaction after the drying under the room temperature, and substrate consumption and first round reacting phase are together.Reaction conditions and the results are shown in Table 5.
The recycling of table 5Lipozyme TLIM
As shown in Table 5, immobilized lipase Lipozyme TLIM catalytic activity and cis-selectivity in circulation 3 times reduce gradually, and the 4th circulation productive rate sharply reduces, and illustrate that Lipozyme TLIM can recycling 3 times.
Explanation is at last, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although by invention has been described with reference to the preferred embodiments of the present invention, but those of ordinary skill in the art is to be understood that, can make various changes to it in the form and details, and the spirit and scope of the present invention that do not depart from appended claims and limited.
Claims (5)
1. immobilized lipase Lipozyme TLIM is as the application of asymmetric Michael addition reaction catalyst.
2. utilize the method for the asymmetric Michael addition reaction of the described immobilized lipase Lipozyme of claim 1 TLIM catalysis, it is characterized in that: with immobilized lipase Lipozyme TLIM is catalyzer, in water-containing organic solvent, react to body Michael acceptor and Michael, water is 0～30: 100 with the volume of organic solvent ratio, temperature of reaction is 25 ℃～85 ℃, and it is 1～5: 1～2 that Michael acceptor and Michael give the molar ratio of body.
3. according to the described method of utilizing the asymmetric Michael addition reaction of immobilized lipase Lipozyme TLIM catalysis of claim 2, it is characterized in that: described organic solvent is tetrahydrofuran (THF), methyl-sulphoxide, N, dinethylformamide or ethanol, water is 10: 100 or 20: 100 with the volume of organic solvent ratio; Described temperature of reaction is 35 ℃ or 65 ℃; It is 2～5: 1 that described Michael acceptor and Michael give the molar ratio of body.
4. according to the described method of utilizing the asymmetric Michael addition reaction of immobilized lipase Lipozyme TLIM catalysis of claim 3, it is characterized in that: described organic solvent is a methyl-sulphoxide, and water is 10: 100 with the volume of organic solvent ratio; Described temperature of reaction is 35 ℃; It is 3: 1 that described Michael acceptor and Michael give the molar ratio of body.
5. according to claim 2 or the 3 or 4 described methods of utilizing the asymmetric Michael addition reaction of immobilized lipase Lipozyme TLIM catalysis, it is characterized in that: described Michael acceptor is the β-nitroolefin of aromatic nucleus or heterocyclic substituted, or α, β-unsaturated cyclic ketones; Described Michael is 1 to body, 3-dicarbonyl compound or cyclic ketones.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106811456A (en) * | 2015-11-27 | 2017-06-09 | 南京威安新材料科技有限公司 | A kind of preparation method of production of biodiesel catalyst |
| CN109593804A (en) * | 2018-12-24 | 2019-04-09 | 浙江工业大学 | A kind of method of enzymatic rapid synthesis nitrobenzimidazole analog derivative |
| CN109988794A (en) * | 2018-12-24 | 2019-07-09 | 浙江农林大学 | A method of the Enzyme catalyzed synthesis nitrobenzimidazole analog derivative in continuous flow reactor |
| CN110408660A (en) * | 2019-07-09 | 2019-11-05 | 南京工业大学 | A kind of method of lipase-catalyzed synthesis bis-indole compounds |
Citations (1)
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| CN101642717A (en) * | 2009-08-26 | 2010-02-10 | 西南大学 | Application of L-tryptophane being taken as catalyst of Knoevenagel reaction |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101642717A (en) * | 2009-08-26 | 2010-02-10 | 西南大学 | Application of L-tryptophane being taken as catalyst of Knoevenagel reaction |
Non-Patent Citations (2)
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| 《Journal of Biotechnology》 20061231 Cai et al. Hydrolase-catalyzed Michael addition of imidazoles to acrylic monomers in organic medium. 330-337 1-5 , 2 * |
| 《有机化学》 20031231 段新方 硝基化合物的不对称迈克尔加成反应 776-783 1-5 , 2 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106811456A (en) * | 2015-11-27 | 2017-06-09 | 南京威安新材料科技有限公司 | A kind of preparation method of production of biodiesel catalyst |
| CN109593804A (en) * | 2018-12-24 | 2019-04-09 | 浙江工业大学 | A kind of method of enzymatic rapid synthesis nitrobenzimidazole analog derivative |
| CN109988794A (en) * | 2018-12-24 | 2019-07-09 | 浙江农林大学 | A method of the Enzyme catalyzed synthesis nitrobenzimidazole analog derivative in continuous flow reactor |
| CN109988794B (en) * | 2018-12-24 | 2023-06-13 | 浙江农林大学 | Method for synthesizing nitrobenzimidazole derivatives through enzyme catalysis in continuous flow reactor |
| CN110408660A (en) * | 2019-07-09 | 2019-11-05 | 南京工业大学 | A kind of method of lipase-catalyzed synthesis bis-indole compounds |
| CN110408660B (en) * | 2019-07-09 | 2022-09-16 | 南京工业大学 | Method for synthesizing bisindole compound under catalysis of lipase |
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