CN101574646A - Lipase bonded stationary phase and preparation method and use thereof - Google Patents
Lipase bonded stationary phase and preparation method and use thereof Download PDFInfo
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- CN101574646A CN101574646A CNA2009100868390A CN200910086839A CN101574646A CN 101574646 A CN101574646 A CN 101574646A CN A2009100868390 A CNA2009100868390 A CN A2009100868390A CN 200910086839 A CN200910086839 A CN 200910086839A CN 101574646 A CN101574646 A CN 101574646A
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Abstract
The invention discloses a lipase bonded stationary phase and a preparation method and use thereof in the field of chromatographic stationary phase preparation. The stationary phase is obtained by directly bonding a spherical silica gel filler with an aldehyde group on the surface and the amino group of the lipase by an imine bond, and then reducing the imine into the amino group. The stationary phase has very high chiral recognition capability, not only can be used as the filler of a chiral chromatographic column, but also can be used as the filler of an online liquid-phase chromatographic reactor, and has good chemical stability, therefore, quite high activity can be kept within three months; furthermore, the preparation cost is relatively low, and the application prospect is good no matter in filling analytical columns or preparative columns.
Description
Technical field
The present invention relates to a kind of lipase bonded fixedly phase and its production and use, belong to chromatographic stationary phase preparation field.
Background technology
Split enantiomer mutually bibliographical information is early arranged as liquid chromatogram is fixing with immobilised enzymes.1988, people such as Wainer proposed to come the resolving chiral material with the immobilization chymotrypsin mutually as fixing of liquid chromatogram.A lot of amino acid, amino acid whose derivative and dipeptides have been split.1989, they used the same method and have synthesized the DL-Amino Acid that immobilizing trypsinase has split a large amount of O-derivatizations.1989, people's immobilizations such as Kalbe chymotrypsin and trypsase, split a large amount of aromatic series and aliphatic amino-acid ester.1992, people such as Marl synthesized 4 kinds of immobilization chymotrypsins, had split a large amount of enantiomers.People's immobilizations such as calendar year 2001 G.Fe ' lix penicillase, they are at a large amount of chiral carboxylic acids that fixedly has been separated that makes, and should fixing also have the asymmetric hydrolysis effect as online reactor to some ester.Up to the present, also the someone reports immobilized lipase is used to split enantiomer mutually as liquid chromatogram is fixing.
Summary of the invention
The purpose of this invention is to provide a kind of lipase bonded fixedly phase and its production and use,, keep the activity of enzyme, improve post and imitate to improve the bonded amount of enzyme at carrier surface.
For achieving the above object, technical scheme of the present invention is:
A kind of lipase bonded fixedly phase is characterized in that, this fixing amino that has the spherical silica gel filler of aldehyde radical and lipase by the surface then is reduced to amino to imines and makes with the imine linkage Direct Bonding.
Described fixedly phase preparation method's concrete steps are:
1) preparation of epoxy radicals silica gel: the hydrochloric acid solution that in the 100mL there-necked flask, adds 3.0g spherical silica gel and 50mL 10%, after adding hot reflux 12～14h, stop heating, suction filtration, be washed with distilled water to neutrality, 60 ℃ of vacuum drying 12h, the silica gel that drying is good places the 100mL there-necked flask, adds 5～8mL 3-glycidol propoxyl group trimethoxy silane and 50mL dry toluene then, under the nitrogen protection in 110 ℃ of backflow stirring reaction 12～14h, stop heating then, suction filtration is used toluene, methyl alcohol successively, acetone and n-hexane are cleaned, 60 ℃ of vacuum drying 12h;
2) the epoxy radicals silica gel of preparation places the 500mL there-necked flask the preparation of glycol-based silica gel: with above-mentioned steps 1), the sulfuric acid solution backflow stirring reaction 1.5h that adds 300mL, pH=3～4 then stops heating, suction filtration then, be washed with distilled water to neutrality, vacuum drying 12h under the room temperature;
3) the glycol-based silica gel of preparation places the 100mL there-necked flask the preparation of aldehyde radical silica gel: with above-mentioned steps 2), adds aqueous acetic acid and the 2～3g sodium metaperiodate stirring reaction 2h of 60mL 90% then, and suction filtration is cleaned with distilled water;
4) preparation of lipase bonded fixedly phase: take by weighing candida antarctica lipase B 150～600mg, place the 250mL there-necked flask, the 0.1mol/L acetate buffer that adds 100mL, pH=5～6, stirring and dissolving, add above-mentioned steps 3) middle aldehyde radical silica gel 3g and the 75mg sodium cyanoborohydride for preparing, in 0～4 ℃ of stirring reaction 3～5 days, suction filtration, clean with the 0.1mol/L phosphate buffer of pH=8; Gained is fixing in the 0.1mol/L of 100mL pH=8 phosphate buffer, in 2～3h, divide and add sodium borohydride 75mg altogether 3 times, each 25mg, with the remaining aldehyde radical in reduction silica gel surface, suction filtration, 0.1mol/L phosphate buffer with pH=8 is cleaned, and obtains desired fats enzyme bonded stationary phase.
The particle diameter of described spherical silica gel is 5～30 microns, and the aperture is 200～4000 dusts.
Described spherical silica gel is buied by Japanese fuji company; Described candida antarctica lipase B is buied by Dutch Chiralvision company.
Described lipase bonded fixing mutually as chiral chromatographic column and online liquid chromatogram reactor filler.
Described lipase bonded fixing mutually as the chiral chromatogram column packing, separating chiral compound.
Described lipase bonded fixing mutually as the chiral chromatogram column packing, separate 1-naphthyl ethyl alcohol, 2-naphthyl ethyl alcohol, 1-naphthalene propyl alcohol, 2-naphthalene propyl alcohol or alkene azoles alcohol.
Described lipase bonded fixing mutually as online liquid chromatogram reactor filler, the asymmetric hydrolysis of catalysis chiral, secondary alcohols ester and 3-chloro-1-(2-thiophene) propyl alcohol butyrate.
Advantage of the present invention:
1) this lipase bonded fixing has very high chiral Recognition ability mutually, is filled with the chromatographic column of this fixedly phase, both can be used as chiral chromatographic column, can be used as online liquid chromatogram reactor again;
2) the chemical property quite stable of this lipase bonded fixedly phase can keep quite high activity within three months;
3) preparation cost of this lipase bonded fixedly phase is relatively low, no matter be filling analytical column or preparative column all has good application prospects.
Description of drawings
Fig. 1 is the fractionation spectrogram of 1-naphthyl ethyl alcohol;
Fig. 2 is the fractionation spectrogram of 2-naphthyl ethyl alcohol;
Fig. 3 is the fractionation spectrogram of 1-naphthalene propyl alcohol;
Fig. 4 is the fractionation spectrogram of 2-naphthalene propyl alcohol;
Fig. 5 is the fractionation spectrogram of alkene azoles alcohol;
Fig. 6 is the asymmetric hydrolysis spectrogram of 2-naphthyl ethyl alcohol ethyl ester;
Fig. 7 is the asymmetric hydrolysis spectrogram of 2-naphthyl ethyl alcohol propyl ester;
Fig. 8 is the asymmetric hydrolysis spectrogram of 4-methoxybenzene ethanol propyl ester;
Fig. 9 is the asymmetric hydrolysis spectrogram of 4-methylbenzene ethanol propyl ester;
Figure 10 is the asymmetric hydrolysis spectrogram of 3-chloro-1-(2-thiophene) propyl alcohol butyl ester.
The specific embodiment
Following examples are used to explain the present invention, but the scope that these embodiment do not limit the present invention in any way.
Embodiment 1: the preparation of epoxy radicals silica gel
Add the hydrochloric acid solution of 3.0 gram spherical silica gels (5 microns of particle diameters, aperture 300 dusts, Japanese fuji company) and 50mL 10% in the 100mL there-necked flask, add hot reflux 12h, stop heating then, suction filtration is washed with distilled water to neutrality, 60 ℃ of vacuum drying 12h.The silica gel that drying is good places the 100mL there-necked flask; add 5mL 3-glycidol propoxyl group trimethoxy silane and 50mL dry toluene then; under the nitrogen protection in 110 ℃ of backflow stirring reaction 12h; stop heating then; suction filtration is used toluene, methyl alcohol successively; acetone and n-hexane are cleaned, 60 ℃ of vacuum drying 12h.
Embodiment 2: the preparation of glycol-based silica gel
Above-mentioned epoxy radicals silica gel is placed the 500mL there-necked flask, add the sulfuric acid solution backflow stirring reaction 1.5h of 300mL pH=3 then, stop heating then, suction filtration is washed with distilled water to neutrality, vacuum drying 12h under the room temperature.
Embodiment 3: the preparation of aldehyde radical silica gel
Above-mentioned glycol-based silica gel is placed the 100mL there-necked flask, add aqueous acetic acid and the 3g sodium metaperiodate stirring reaction 2h of 60mL 90% then, suction filtration is cleaned with distilled water.
Embodiment 4: the preparation of lipase bonded fixedly phase
Take by weighing candida antarctica lipase B 300mg (Dutch Chiralvision company), place the 250mL there-necked flask, the 0.1mol/L acetate buffer that adds 100mL pH=6, stirring and dissolving, the aldehyde radical silica gel 3g and the 75mg sodium cyanoborohydride that add preparation in the foregoing description 3, in 2～4 ℃ of stirring reactions 3 days, suction filtration, clean with the 0.1mol/L phosphate buffer of pH=8; Gained is fixing in the 0.1mol/L of 100mL pH=8 phosphate buffer, divides to add sodium borohydride 75mg altogether 3 times each 25mg in 2h, with the remaining aldehyde radical in reduction silica gel surface, suction filtration is cleaned with the 0.1mol/L phosphate buffer of pH=8, obtains required fixedly phase.
Embodiment 5: the chromatographic column filling
Said fixing is a homogenate with the 0.1mol/L phosphate buffer of pH=8, and the stainless steel column of packing under 4000psi pressure is (among the 250mm * 4.6mm).
Embodiment 6: to the separation of chiral, secondary alcohols
The chromatographic column that is filled with the said fixing phase can be used as chiral chromatographic column and uses, and can split multiple chipal compounds.Fig. 1-Fig. 5 is the fractionation chromatogram of chiral, secondary alcohols.The chromatography experiment data are listed in the table 1.
Table 1 splits the chromatography experiment data of part chiral, secondary alcohols
| The sample title | K 1 | α | Phase flows | Flow velocity |
| The 1-naphthyl ethyl alcohol | 0.32 | 1.50 | A | 0.5mL/min |
| The 2-naphthyl ethyl alcohol | 0.37 | 1.21 | A | 0.5mL/min |
| 1-naphthalene propyl alcohol | 1.47 | 1.38 | B | 1mL/min |
| 2-naphthalene propyl alcohol | 1.47 | 1.20 | B | 1mL/min |
| Alkene azoles alcohol | 2.13 | 1.52 | A | 1mL/min |
The 0.1mol/L phosphate buffer of mobile phase A: pH=8: acetonitrile 100/5
The 0.1mol/L phosphate buffer of Mobile phase B: pH=8
Detect wavelength: 230nm
K
1Be retention factors; α is a selectivity factor
Embodiment 7: to the asymmetric hydrolysis of chiral, secondary alcohols ester
The chromatographic column that is filled with the said fixing phase can be used as online liquid chromatogram reactor again and uses, and has possessed the high efficiency of enzymatic highly-solid selectively and chromatographic isolation simultaneously.Therefore, when the substrate sample introduction of racemization in this chromatographic system, in a chromatographic process, just can obtain the catalysate of a certain absolute configuration and the unreacted substrate of another kind of absolute configuration, and product and substrate can separatedly be held also in this process.Fig. 6, Fig. 7, Fig. 8 and Fig. 9 are the asymmetric hydrolysis chromatograms of several chiral, secondary alcohols esters.Wherein, first chromatographic peak of each figure is the peak of hydrolysate-alcohol, and second peak is the peak of unhydrolysed substrate-ester.The chromatography experiment data are listed in the table 2.
The chromatography experiment data of table 2 asymmetric hydrolysis part chiral, secondary alcohols ester
| The sample title | The product absolute configuration | The ee value |
| 2-naphthyl ethyl alcohol ethyl ester | R | ＞99％ |
| 2-naphthyl ethyl alcohol propyl ester | R | ＞99％ |
| 2-naphthyl ethyl alcohol ethyl ester | R | ＞99％ |
| 2-naphthyl ethyl alcohol propyl ester | R | ＞99％ |
The 0.1mol/L phosphate buffer of mobile phase: pH=8: acetonitrile 100/5
Flow velocity: 1mL/min
Detect wavelength: 230nm
The asymmetric hydrolysis of embodiment 8:3-chloro-1-(2-thiophene) propyl alcohol butyrate
The intermediate of antidepressant drug Duloxetine, the butyrate of 3-chloro-1-(2-thiophene) propyl alcohol also can be this fixing going up by asymmetric hydrolysis mutually, and conversion ratio and optical purity are all very high.Figure 10 is the asymmetric hydrolysis chromatogram of the butyrate of 3-chloro-1-(2-thiophene) propyl alcohol.Wherein, first chromatographic peak is the peak of hydrolysate-alcohol, and second peak is the peak of unhydrolysed substrate-ester, and the absolute configuration of alcohol is R, ee value＞99%.
Claims (8)
1. a lipase bonded fixedly phase is characterized in that, this fixing amino that has the spherical silica gel filler of aldehyde radical and lipase by the surface then is reduced to amino to imines and makes with the imine linkage Direct Bonding.
2. the preparation method of the described lipase bonded fixedly phase of claim 1 is characterized in that, described method concrete steps are:
1) preparation of epoxy radicals silica gel: the hydrochloric acid solution that in the 100mL there-necked flask, adds 3.0g spherical silica gel and 50mL 10%, after adding hot reflux 12～14h, stop heating, suction filtration, be washed with distilled water to neutrality, 60 ℃ of vacuum drying 12h, the silica gel that drying is good places the 100mL there-necked flask, adds 5～8mL 3-glycidol propoxyl group trimethoxy silane and 50mL dry toluene then, under the nitrogen protection in 110 ℃ of backflow stirring reaction 12～14h, stop heating then, suction filtration is used toluene, methyl alcohol successively, acetone and n-hexane are cleaned, 60 ℃ of vacuum drying 12h;
2) the epoxy radicals silica gel of preparation places the 500mL there-necked flask the preparation of glycol-based silica gel: with above-mentioned steps 1), the sulfuric acid solution backflow stirring reaction 1.5h that adds 300mL, pH=3～4 then stops heating, suction filtration then, be washed with distilled water to neutrality, vacuum drying 12h under the room temperature;
3) the glycol-based silica gel of preparation places the 100mL there-necked flask the preparation of aldehyde radical silica gel: with above-mentioned steps 2), adds aqueous acetic acid and the 2～3g sodium metaperiodate stirring reaction 2h of 60mL 90% then, and suction filtration is cleaned with distilled water;
4) preparation of lipase bonded fixedly phase: take by weighing candida antarctica lipase B 150～600mg, place the 250mL there-necked flask, the 0.1mol/L acetate buffer that adds 100mL, pH=5～6, stirring and dissolving, add above-mentioned steps 3) middle aldehyde radical silica gel 3g and the 75mg sodium cyanoborohydride for preparing, in 0～4 ℃ of stirring reaction 3～5 days, suction filtration, clean with the 0.1mol/L phosphate buffer of pH=8; Gained is fixing in the 0.1mol/L of 100mL pH=8 phosphate buffer, in 2～3h, divide and add sodium borohydride 75mg altogether 3 times, each 25mg, with the remaining aldehyde radical in reduction silica gel surface, suction filtration, 0.1mol/L phosphate buffer with pH=8 is cleaned, and obtains desired fats enzyme bonded stationary phase.
3. the preparation method of lipase bonded fixedly phase according to claim 2 is characterized in that, the particle diameter of described spherical silica gel is 5～30 microns, and the aperture is 200～4000 dusts.
4. the preparation method of lipase bonded fixedly phase according to claim 2 is characterized in that, described spherical silica gel is buied by Japanese fuji company; Described candida antarctica lipase B is buied by Dutch Chiralvision company.
5. the purposes of the described lipase bonded fixedly phase of claim 1 is used as chiral chromatographic column and online liquid chromatogram reactor filler.
6. the purposes of lipase bonded fixedly phase according to claim 5 is characterized in that, and is described lipase bonded fixing mutually as the chiral chromatogram column packing, separating chiral compound.
7. the purposes of lipase bonded fixedly phase according to claim 5 is characterized in that, and is described lipase bonded fixing mutually as the chiral chromatogram column packing, separates 1-naphthyl ethyl alcohol, 2-naphthyl ethyl alcohol, 1-naphthalene propyl alcohol, 2-naphthalene propyl alcohol or alkene azoles alcohol.
8. the purposes of lipase bonded fixedly phase according to claim 5 is characterized in that, and is described lipase bonded fixing mutually as online liquid chromatogram reactor filler, the asymmetric hydrolysis of catalysis chiral, secondary alcohols ester and 3-chloro-1-(2-thiophene) propyl alcohol butyrate.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101766993B (en) * | 2009-12-31 | 2012-04-18 | 广西师范大学 | Overall chiral stationary phase of silica gel capillary and preparation method thereof |
| CN103055832A (en) * | 2012-12-31 | 2013-04-24 | 浙江月旭材料科技有限公司 | Chromatographic packing for separating water soluble polymer and protein and preparation method of same |
| CN104745646A (en) * | 2013-12-31 | 2015-07-01 | 丰益(上海)生物技术研发中心有限公司 | Apparatus for esterifying oils and fats by use of enzymic method |
| CN105385675A (en) * | 2015-12-31 | 2016-03-09 | 厦门大学 | Immobilization method of candida antarctica lipase B |
| CN115672295A (en) * | 2022-11-05 | 2023-02-03 | 中国科学院兰州化学物理研究所 | Preparation and application of imine column [5] arene modified silica gel chromatographic packing |
-
2009
- 2009-06-08 CN CN2009100868390A patent/CN101574646B/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101766993B (en) * | 2009-12-31 | 2012-04-18 | 广西师范大学 | Overall chiral stationary phase of silica gel capillary and preparation method thereof |
| CN103055832A (en) * | 2012-12-31 | 2013-04-24 | 浙江月旭材料科技有限公司 | Chromatographic packing for separating water soluble polymer and protein and preparation method of same |
| CN103055832B (en) * | 2012-12-31 | 2015-04-01 | 浙江月旭材料科技有限公司 | Chromatographic packing for separating water soluble polymer and protein and preparation method of same |
| CN104745646A (en) * | 2013-12-31 | 2015-07-01 | 丰益(上海)生物技术研发中心有限公司 | Apparatus for esterifying oils and fats by use of enzymic method |
| CN104745646B (en) * | 2013-12-31 | 2019-12-10 | 丰益(上海)生物技术研发中心有限公司 | Equipment for treating grease by enzymatic esterification |
| CN105385675A (en) * | 2015-12-31 | 2016-03-09 | 厦门大学 | Immobilization method of candida antarctica lipase B |
| CN115672295A (en) * | 2022-11-05 | 2023-02-03 | 中国科学院兰州化学物理研究所 | Preparation and application of imine column [5] arene modified silica gel chromatographic packing |
| CN115672295B (en) * | 2022-11-05 | 2023-12-15 | 中国科学院兰州化学物理研究所 | Preparation and application of imine type column [5] arene modified silica gel chromatographic packing |
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