CN1205228C - Process for synthesizing chiral fixed phase of liquid-phase chromatographic protein - Google Patents
Process for synthesizing chiral fixed phase of liquid-phase chromatographic protein Download PDFInfo
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- CN1205228C CN1205228C CN 99113091 CN99113091A CN1205228C CN 1205228 C CN1205228 C CN 1205228C CN 99113091 CN99113091 CN 99113091 CN 99113091 A CN99113091 A CN 99113091A CN 1205228 C CN1205228 C CN 1205228C
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Abstract
The present invention relates to a method for synthesizing the chiral fixed phase of liquid phase chromatographic protein. The present invention is characterized in that base material with amino or a sulfhydryl group on the surface is used, the base material reacts with cyanuric chloride to be activated. Then, the activated base material is bound with protein, and small molecules with amino are used for sealing a tail. The method is used for synthesizing the fixed phase of the protein within 12 hours, and the protein has the advantages of great bonding amount, high activity maintenance and high successful rate. The protein chiral fixed phase synthesized by the method can be bound and can be then arranged on columns and can also be directly bound on the columns. Consequently, the method can be used for high performance liquid chromatography and is also particularly suitable for electro chromatography.
Description
The present invention is a kind of method of synthetic protein chiral stationary phase, synthetic chiral chromatography medium is because it has the characteristics of bonding on the post in this way, so not only can be applicable to high performance liquid chromatography, also be specially adapted to fields such as electrochromatography, the invention provides a kind of convenience, reliable immobilized method of protein in addition.
Along with the progress of life science and the development of pharmaceutical industry, human more and more deep to optically active enantiomorph role understanding in life entity.For example: the medicine of enantiomorph is different often in the intravital pharmacology of people each other, that have even opposite, in processing bioactive material is arranged, as: VITAMIN, pheromone, coenzyme, medicament production reach during naturally occurring chipal compounds, Separation of Enantiomers and analysis all are all in all problems, therefore the chiral separation of enantiomorph is subject to people's attention day by day, and classical enantiomorph method for splitting comprises: mechanical process, chemical method, biological process, chromatography etc.Chromatography with its have efficiently, advantage easily, have overwhelming superiority in the chiral separation field always, difference according to the mechanism of action can be divided into indirect method: (promptly separating diastereomer with the achirality post) and direct method: (promptly splitting enantiomorph) with chiral column, and the latter's advantage is obvious.Compare liquid chromatography with gas-chromatography because applicable object is wide, variable factor is many, the characteristics that not only can analyze but also can prepare, thereby more wide prospect arranged, the liquid chromatography Chiral Media that has occurred at present is a lot, as: Okamoto propose with Mierocrystalline cellulose and derivative thereof as chiral stationary phase, the cyclodextrins chiral medium of Armstrong development, and the polymkeric substance Chiral Media of the Pirkle type of Pirkle group development and the preparation of employing molecular imprinting technique etc., because these chiral stationary phases are simple in structure, and the reactive force difference between the enantiomorph is little, thereby each chiral column only is applicable to the fractionation of minority enantiomorph.Stewart in 1972 etc. are bonded to bovine serum albumin (BSA) on the agarose first, and finding has the fractionation effect to the DL-tryptophane, can be used for chiral separation.Because protein structure complexity, different with the different substances point of application, and its conformation is with the variation of many factors such as the pH value of solution, ionic strength and difference, thereby has a wider suitability, on other chiral column, be difficult to split or split unfavorable enantiomorph, on the albumen post, often can be separated preferably.In addition owing to there is numerous albumen available, this just might be to different enantiomorphs, select the albumen chiral column an of the best, the phase because albumen fixes, the albumen post also can be used for studying medicine and proteic interaction, albumen and proteic interaction, and the screening of activeconstituents in the medicine.Initial albumen is bonded on the agarose, this is because the advantage that agarose has the feature of surface hydrophilicity and is easy to chemically modified, but agarose is a kind of gel-like substance, very easily produce non-reversible deformation, even also limited by the pressure that crosslinked increase intensity can bear, so can only be used for early stage low-pressure chromatography, silica gel has then overcome above defective and the surface is easy to modify, so always as the stratographic main stuffing, at present nearly all albumen post is all as matrix.Existing several at present about proteic bonding method, as epoxy method, glutaraldehyde method, carbonylic imidazole method, but epoxy method must not be suitable for alkali-sensitive albumen at bonding under the alkaline condition, though can increase ionic strength to produce salting-out effect, bonded amount is less relatively.Glutaraldehyde method bonding step is various, and has used reductive agent in synthetic, and proteic activity is had certain influence, and it is often lower that synthetic albumen post post is imitated.The carbonylic imidazole method at first must be reacted under anhydrous condition in synthetic, the easy hydrolysis of the activated media of generation, and success ratio is not high, so seek a kind of mild condition, efficient, general synthetic method is an important topic always.
The object of the present invention is to provide a kind of synthetic method of liquid-phase chromatographic protein chiral stationary phase, this method reaction conditions gentleness, the success ratio height, the used time is short and suitability is wide.
The invention provides a kind of synthetic method of liquid-phase chromatographic protein chiral stationary phase, it is characterized in that: adopt the surface to have the matrix of amido or sulfydryl; Matrix and cyanuric chloride reaction activate; Activatory matrix again with the albumen bonding; Carry out sealing with the small molecules that has amido.The albumen of institute's bonding both can be albumin, also can be other albumen (comprising enzyme).
Specifically, react with cyanuric chloride in organic solvent or in the water through the matrix that amido or sulfydryl modification are crossed, the add-on of cyanuric chloride is 1～10 times of amido mole number, reaction can be adopted common inert solvent in organic solvent, as: N, dinethylformamide (DMF), tetrahydrofuran (THF) (THF), dioxane, acetone etc., temperature of reaction can be carried out under room temperature or low temperature; When reacting in water, constantly add mineral alkali, keeping the pH value of solution value in slightly acidic, and temperature is not too high in the reaction process as yellow soda ash, sodium bicarbonate.The medium that is activated then in the aqueous solution with albumen test, the pH value maintains neutrality in the reaction process, reaction can at room temperature be carried out, and also can (20～60 ℃) carry out under heating condition a little.Last bonding has proteic medium to carry out sealing with the small molecules that has amido such as thanomin, glycine ethyl ester reaction in the aqueous solution, and the concentration of sealing agent is 1%, and the pH value maintains neutrality in the reaction process.
Among the present invention when matrix is silica gel, the modification of silica gel can adopt amine propyl silane reagent (for example: the 3-aminopropyl triethoxysilane), epoxypropyl silylating reagent or mercapto propyl silane reagent, reaction conditions can adopt ordinary skill to carry out.Process is as follows:
1. the reaction of silica gel and silylating reagent makes the surface by the silica gel of amido or sulfydryl modification;
2. surperficial silica gel and the cyanuric chloride reaction that has amido or sulfydryl makes activated silica gel;
3. the silica gel that is activated in the aqueous solution with albumen test;
4. carry out sealing with the small molecules that has amido.
Synthetic route can be represented by the formula:
Use this bonding method both can refill post by first bonding albumen, again can be earlier activated media prepacked column bonding albumen on post then, bonding reaction remains under neutrality, the room temperature and carries out, and the general 1～2h that only needs that is swift in response can finish the synthetic of chiral chromatography separating medium within the 12h.
Zhi Bei chiral column success ratio is high as stated above, and protein-active is kept better, and through some months hundreds of time sample introduction, its post is imitated almost and do not changed.Be described further below by preparation and the application of the example of bonding bovine serum albumin on silica gel albumen chiral column of the present invention.
Fig. 1 is the chiral separation spectrogram of DL-tryptophane.Moving phase is: 50mmol/L phosphoric acid salt (pH=7.4)+5% acetonitrile, and flow velocity is 1.0ml/min, and sample size is 1 μ l, and the detection wavelength is 279nm.
Fig. 2 is a DL-dansyl Threonine chiral separation spectrogram.Moving phase is: 50mmol/L phosphoric acid salt (pH=7.4)+10% acetonitrile+1% n-propyl alcohol, and flow velocity is 0.8ml/min, and sample size is 0.5 μ l, and the detection wavelength is 460nm.
Fig. 3 is a warfarin chiral separation spectrogram.Moving phase is: 50mmol/L phosphoric acid salt (pH=7.4)+10% acetonitrile+2% Virahol, and flow velocity is 1.8ml/min, and sample size is 1.5 μ l, and the detection wavelength is 280nm.
Example 1: the modification of silica gel
1) with the reaction of amine propyl silane reagent
Get 5g spherical silica gel (5 μ 300A) and place the 150ml there-necked flask, 20% hydrochloric acid soln that adds the new preparation of 100ml, in 110～120 ℃ of following backflow 4h, filter, be washed till neutrality with deionized water, more than 120 ℃ of following vacuum-drying 12h, get 3g dry silica gel add the 150ml there-necked flask fill the 50ml dry toluene, heating under nitrogen atmosphere, stirring, when temperature is raised to 120 ℃, add amine propyl-triethylsilicane alkane 5ml,, lower the temperature then, filter in 120 ℃ of backflow 24h, silica gel is with toluene, acetone thorough washing, 60 ℃ of vacuum-dryings in the presence of Vanadium Pentoxide in FLAKES.
2) with the reaction of cyclopropyl and mercapto propyl silane reagent
Get example 1) in acidifying silica gel 5g add in the 150ml there-necked flask, add exsiccant toluene 50ml, heating under nitrogen atmosphere, stirring, when temperature is raised to 90 ℃, add epoxypropyl Trimethoxy silane 5ml,, filter at 90～95 ℃ of reaction 10h, silica gel is with toluene, acetone thorough washing, 60 ℃ of vacuum-dryings in the presence of Vanadium Pentoxide in FLAKES, exsiccant silica gel reacts 16h in the ammoniacal liquor damping fluid of pH value=9, promptly obtains the silica gel that end has amido.
The preparation of mercapto propyl group silica gel is the same, just with behind the mercapto propyl silane reagent react can directly use.
Example 2: the preparation of activated silica gel
1) in organic solvent, reacts
Get in the example 1 activated silica gel 2g, add and fill in 50ml DMF, the 2ml pyridine 100ml small beaker, under stirring at room, slowly add cyanuric chloride 0.37g, the 2h after-filtration, silica gel is with the acetone thorough washing.
2) in water, react
Get in the example 1 activated silica gel 2g, add in the 100ml small beaker, measure the 100ml deionized water under agitation ice bath be cooled to 4 ℃, slowly add cyanuric chloride 0.37g, constantly add yellow soda ash therebetween to keep the pH value of solution value about 5, temperature does not surpass 5 ℃ in the reaction process, reacts 2h after cyanuric chloride adds again, filter, with the deionized water thorough washing.
Example 3: silica gel albumen chiral column synthetic
1) usual manner
Getting in the example 2, activatory silica gel 2g adds (50mmol/L in the phosphatic damping fluid of 30ml, pH=7), at room temperature slowly stir, add bovine serum albumin 150mg, in 15～45 ℃ of reactions, in different time, get solution and under 280nm, detect absorption value, approximately 2h post-absorption value no longer changes, stop behind the 4h stirring, filter, silica gel is prepared to react under phosphate buffered saline buffer (pH=7) room temperature that contains 1% glycine ethyl ester and is spent the night with above-mentioned phosphate buffered saline buffer thorough washing, and the silica gel that reaction is good promptly can ordinary skill dress post.
2) deriving mode on the post
Get in the example 2 activatory silica gel 2g dress post, pillar is connected on the high performance liquid chromatograph, dash steady with the pH=7 phosphate buffered saline buffer to the 205nm baseline, getting the 150mg bovine serum albumin is dissolved in the above-mentioned phosphate buffered saline buffer, under 15～45 ℃ in the post cocycle, detect absorption value down in 280nm, approximately 2h post-absorption value no longer changes, dash steady with the pH=7 phosphate buffered saline buffer after the circulation 4h to the 280nm baseline, the phosphate buffered saline buffer (pH=7) that preparation contains 1% glycine ethyl ester carries out sealing, has promptly finished the synthetic of albumen post with the flushing of pH=7 phosphate buffered saline buffer behind the 2h.
Claims (7)
1. the synthetic method of a liquid-phase chromatographic protein chiral stationary phase is characterized in that: adopt the surface to have the silica matrix of amido or sulfydryl; Silica matrix and cyanuric chloride reaction activate; The activatory silica matrix again with the bovine serum albumin bonding; Carry out sealing with thanomin or the glycine ethyl ester that has amido.
2. according to the synthetic method of the described liquid-phase chromatographic protein chiral stationary phase of claim 1, it is characterized in that: the mass ratio of cyanuric chloride add-on and silica matrix is 0.37: 2 in the priming reaction.
3. according to the synthetic method of the described liquid-phase chromatographic protein chiral stationary phase of claim 2, it is characterized in that: priming reaction carries out in organic solvent, organic solvent is selected from following any one inert solvent: N, dinethylformamide, tetrahydrofuran (THF), dioxane, acetone.
4. according to the synthetic method of the described liquid-phase chromatographic protein chiral stationary phase of claim 2, it is characterized in that: priming reaction carries out in water, constantly add mineral alkali to keep the pH value in slightly acidic.
5. according to the synthetic method of the described liquid-phase chromatographic protein chiral stationary phase of claim 1, it is characterized in that: bonding reaction carries out under ℃ condition of room temperature～60, and the pH value is kept neutrality in the reaction process.
6. according to the synthetic method of the described liquid-phase chromatographic protein chiral stationary phase of claim 1, it is characterized in that: the sealing agent is selected from amine acetate or glycine ethyl ester, and the sealing agent concentration is 1%, and the pH value is kept neutrality in the reaction process.
7. according to the synthetic method of the arbitrary described liquid-phase chromatographic protein chiral stationary phase of claim 1-6, it is characterized in that: when matrix is silica gel, adopt 3-aminopropyl triethoxysilane, epoxypropyl silylating reagent or mercapto propyl silane reagent to modify.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100506919C (en) * | 2006-02-23 | 2009-07-01 | 中国科学院化学研究所 | Method for conducting chiral induction to non-chiral cyanine dye aggregate using human serum protein |
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| CN100386342C (en) * | 2005-11-24 | 2008-05-07 | 复旦大学 | Method for preparing carbon nanotube-protein stationary phase |
| CN100471556C (en) * | 2005-11-25 | 2009-03-25 | 中国科学院生态环境研究中心 | Nano metal chiral identification solid phase, chiral separation chromatograph and its preparation method |
| CN104813165B (en) * | 2012-12-06 | 2019-10-08 | 株式会社大赛璐 | Release agent |
| CN103512854B (en) * | 2013-09-23 | 2015-06-17 | 桂林理工大学 | Flame atomic absorption spectrometry method for separating, enriching and detecting trace plumbum in water sample by utilizing thiobarbituric-acid-modified silica gel |
| CN104353442A (en) * | 2014-11-04 | 2015-02-18 | 华文蔚 | Preparation method of nanosilicone-gold chiral separation stationary phase |
| CN105618013B (en) * | 2014-11-24 | 2017-12-15 | 中国科学院大连化学物理研究所 | A kind of preparation method using silica gel as the agglutinin high-effective affinity chromatography material of matrix |
| CN108114706A (en) * | 2016-11-29 | 2018-06-05 | 中国科学院大连化学物理研究所 | A kind of silica matrix taurine bonded stationary phase and its preparation and application |
| CN112387262B (en) * | 2020-11-10 | 2021-12-07 | 泰州学院 | Preparation method of chiral stationary phase based on photocatalytic cross-linked protein, chiral stationary phase and application |
| CN115463647A (en) * | 2021-06-11 | 2022-12-13 | 苏州瑞恒嘉航医疗科技有限公司 | Special stationary phase for protein separation and preparation and application thereof |
| CN114100584A (en) * | 2021-11-23 | 2022-03-01 | 赣江中药创新中心 | Triazine-based bonded chromatographic stationary phase and preparation method thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100506919C (en) * | 2006-02-23 | 2009-07-01 | 中国科学院化学研究所 | Method for conducting chiral induction to non-chiral cyanine dye aggregate using human serum protein |
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