CN100549018C - Contain the compound of phosphorylcholine and comprise the surface-modifying agent of this compound - Google Patents
Contain the compound of phosphorylcholine and comprise the surface-modifying agent of this compound Download PDFInfo
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- CN100549018C CN100549018C CNB2004800351159A CN200480035115A CN100549018C CN 100549018 C CN100549018 C CN 100549018C CN B2004800351159 A CNB2004800351159 A CN B2004800351159A CN 200480035115 A CN200480035115 A CN 200480035115A CN 100549018 C CN100549018 C CN 100549018C
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Abstract
The compound that contains phosphorylcholine of following formula (1) expression.In the formula (1), m is 2～6, and n is 1～4.X
1, X
2, X
3Be methoxyl group, oxyethyl group or halogen independently of one another.But, X
1, X
2, X
3In at the most 2 can be in methyl, ethyl, propyl group, sec.-propyl, butyl, the isobutyl-any one.R is any one (in following formula (2)～(4) structure, formula (1) compound is represented with A-R-B) in following formula (2)～(4) structure.In formula (2)～(4), L represents 1～6, and P represents 1～3.In addition, comprise the above-mentioned surface-modifying agent that contains the compound of phosphorylcholine, the modified powder of handling with this surface-modifying agent, the chromatographic grade weighting agent that comprises the modified support of handling with this surface-modifying agent, with the strainer of this surface-modifying agent processing, with the glass wares of this surface-modifying agent processing.
Description
Technical field
The present invention relates to contain the novel cpd of phosphorylcholine, and comprise the surface-modifying agent of this compound, with this surface-modifying agent modification modified powder, utilize this modified powder as the chromatographic grade weighting agent of carrier, with this surface-modifying agent modification strainer and glass experiment utensil.
The surface-modifying agent that comprises The compounds of this invention is given object biocompatibility, moisture retention and multiple other useful function.
Background technology
The polymkeric substance that has carried out containing phosphorylcholine is as the high molecular research of biocompatibility, and developed this polymkeric substance is overlayed on biocompatible material on the various matrix.
For example, in the patent documentation 1, the powder that will be covered with the homopolymer and the multipolymer of 2-methacryloxyethyl phosphorylcholine has improved moisture retention or skin adherence as the used for cosmetic powder makeup are disclosed.
In addition, in patent documentation 2 and the patent documentation 3, medical material or separating agent with the polymkeric substance lining that contains phosphorylcholine are disclosed.
Above-mentioned materials mainly is propylene class monomer and the 2-chloro-1 that has hydroxyl by making, 3,2-two oxa-s phosphorane-2-oxide compound reaction, further form quaternary ammonium by Trimethylamine, synthetic thus monomer with phosphorylcholine structure, this monomer polymerization is obtained polymkeric substance, with the surface (about the preparation method of polymkeric substance, with reference to patent documentation 4 and 5) of this polymkeric substance coating material.
In the patent documentation 4, prepare the multipolymer of 2-methacryloxyethyl phosphorylcholine and methacrylic ester, prepared the homopolymer of 2-methacryloxyethyl phosphorylcholine in the patent documentation 5.
On the other hand, by size exclusion protein or the biological samples such as polypeptide littler than protein molecular weight are carried out isolating GFC and a lot of commercially available products are arranged with weighting agent., exist with the crosslinking hydrophilic polymer with in the weighting agent at this GFC as the weighting agent of carrier with the weighting agent of silica gel as carrier.
The pH scope of the moving phase that can be suitable for as the weighting agent of carrier with the crosslinking hydrophilic polymer is wide, the versatility height.Yet, with the weighting agent of polymer as carrier, than with silica gel as the weighting agent (1) of carrier because the control difficulty of fine pore, and be difficult to obtain high theoretical plate number.In addition, when used in high performance liquid chromatography (HPLC) (2), because the insufficient strength of antagonism condition of high voltage, and particle was because mobile phase solvent and swelling, and the situation of chromatographic column that therefore can not obtain favorable reproducibility is also a lot.
With the weighting agent of silica gel, exist protein or polypeptide to be adsorbed on the problem on silica-gel carrier surface as carrier.Therefore, for the protein in the inhibition analysis sample or polypeptide are adsorbed onto on the silica gel, sell on the market and use the weighting agent of having modified surperficial silica gel with non-dissociating property hydrophilic group.
For example, as silica type GFC post, Showa Denko K. K has Shodex PROTEINKW-803 (ProductName) commercially available.This silica type post, explanation on the products catalogue be applicable to molecular weight thousands of～post of the silica type GFC pattern of about 1,000,000 protein analysis.
In addition, the ワ of Co., Ltd. イ エ system シ イ has YMC-Pack Diol (ProductName) commercially available.This also is the GFC post of silica type, and it is illustrated as owing to the functional group's chemistry with diol structure is attached on the silica-gel carrier, applicable to molecular weight 10,000～hundreds thousand of proteinic separation.
In the non-patent literature 1, put down in writing the phosphorylcholine by chemical graft on carrier, absorption of proteins reduces.
In addition, in the patent documentation 6 and 7, disclose and had the known organosilicon alkanes surface-modifying agent (silane coupling agent) that shows good hydrophilic betaine structure.In the patent documentation 6, think by make the dimethylamino alkyl silane in organic solvent with 1, the 3-N-morpholinopropanesulfonic acid lactone reaction can obtain having the silane coupling agent of the sultaine that the negative charge by the positive charge of quaternary ammonium and sulfonic acid forms.In the patent documentation 7, put down in writing the preparation method of silane coupling agent with the carboxybetaine that forms by quaternary ammonium and carboxyl.These silane coupling agents can be coated on glass etc., come the modified material surface by making its drying.Yet,,, can not form electric neutrality because positive charge and negative charge intensity in the trimethyl-glycine have deviation although can give the material surface good hydrophilicity with the trimethyl-glycine of these structures.For example, in the sultaine, because the strongly-acid of sulfonic acid and electronegative, in carboxybetaine, show the positive electricity character due to the quaternary ammonium.In such betaine structure, and produce strong ion-exchange interaction between the protein, caused proteinic non-reversibility absorption.
On the other hand, be adsorbed as purpose with biocompatibility or arrestin matter and these silane coupling agents be used for the example of chromatographic grade weighting agent or strainer class and experiment utensil class also do not have.
Patent documentation 1: the spy opens flat 7-118123 communique
Patent documentation 2: the spy opens the 2000-279512 communique
Patent documentation 3: the spy opens the 2002-98676 communique
Patent documentation 4: the spy opens flat 9-3132 communique
Patent documentation 5: the spy opens flat 10-298240 communique
Patent documentation 6: the spy opens flat 5-222064 communique
Patent documentation 7: the spy opens clear 63-295593 communique
Non-patent literature 1:Jian R.Lu etc., Langmuir 2001,17,3382-3389
Summary of the invention
Yet,, be difficult on all surfaces, be covered effectively carrying out in the method for modification with containing the polymkeric substance serving surface of phosphorylcholine.In addition, owing to the polymkeric substance of lining is peeled off from object, so the situation that has the weather resistance aspect to have problems.And, because body surface is aggregated thing lining, also exists and depart from the purpose of giving objective functions such as biocompatibility by phosphorylcholine, and make the situation of the such essential property forfeiture of microtexture such as the desired pore of object self.
In addition, when importing in object as low molecule the phosphorylcholine radical derivative, on object first importing can with the functional group of phosphorylcholine derivatives reaction, make then in the method for phosphorylcholine derivatives reaction, because residual on the body surface have unreacted functional group on body surface, so biocompatibility is also lower.
For example, at first importing on the body surface aminoly, under the situation that the aldehyde derivatives that makes phosphorylcholine then and the amino of body surface react, having a lot of unreacted amino residual.Although these residual amino can be difficult to keep the wetting ability of body surface, and can not block fully by combining and be blocked to a certain extent with other low molecular compound.Under the situation of residual amino on the body surface, because amino has strong basicity, mainly be that acidic protein shows obviously very strong ion-exchange interaction, it nearly all produces absorption.As under the situation of chromatographic grade weighting agent, become the reason that this recovery of protein rate reduces or the peak significantly trails.And absorption of proteins causes its sex change, when using as biocompatible material, becomes the reason of inflammation etc., thereby bad.
The present invention finds: make the compound that contains phosphorylcholine, with have and the functional group of this compound reaction and have object direct reaction with body surface bonded functional group, easy and have high universalizable, can directly give the phosphorylcholine of the desired any amount of body surface.
Find thus, by surface-modifying agent by this compound formation, can easily prepare modified powder, with this modified powder as the chromatographic grade weighting agent of carrier, through the strainer and the glass experiment utensil of this surface-modifying agent modification, thereby finished the present invention.
That is, the invention provides the compound that contains phosphorylcholine of following formula (1) expression.
In the formula, m is 2～6, and n is 1～4.
X
1, X
2, X
3Be methoxyl group, oxyethyl group or halogen independently of one another.But, X
1, X
2, X
3In at the most 2 can be in methyl, ethyl, propyl group, sec.-propyl, butyl, the isobutyl-any one.
R is any one (in following formula (2)～(4) structure, formula (1) compound is represented with A-R-B) in following formula (2)～(4) structure.
A-(CH
2)
L-B
(2)
In formula (2)～(4), L represents 1～6, and P represents 1～3.
In addition, the invention provides the compound that contains phosphorylcholine of following formula (5) or (6) expression.
In the formula, m is 2～6, and n is 1～4.X
1, X
2, X
3Be methoxyl group, oxyethyl group or halogen independently of one another.But, X
1, X
2, X
3In at the most 2 can be in methyl, ethyl, propyl group, sec.-propyl, butyl, the isobutyl-any one.
In addition, the invention provides and comprise the above-mentioned surface-modifying agent that contains the compound of phosphorylcholine.
In addition, the invention provides the preparation method of compound shown in the aforementioned formula (6), it is characterized in that:, synthesize the compound shown in the above-mentioned formula (6) with compound by condensing agent with phosphorylcholine and carboxyl by having amino organic silane compound by with the synthetic compound of the reaction of sodium periodate and ruthenium trichloride oxidation Glycerophosphorylcholine with phosphorylcholine and carboxyl.
In addition, the invention provides the modified powder of handling with above-mentioned surface-modifying agent.
In addition, the invention provides the chromatographic grade weighting agent that comprises the modified support of handling with above-mentioned surface-modifying agent.
Also have, the invention provides the strainer of handling with above-mentioned surface-modifying agent.
In addition, the invention provides with above-mentioned surface-modifying agent and carried out surface-treated glass experiment utensil.
The invention effect
If use compound of the present invention, comprise the surface-modifying agent of this compound,, can give the phosphorylcholine of the required any amount of various body surfaces by extremely easy reaction of a step.Its result, by phosphorylcholine can easily prepare modified powder with required function, with this modified powder as the chromatographic grade weighting agent of carrier, with the strainer and the glass experiment utensil of this surface-modifying agent modification.
In more detail, according to the present invention, phosphorylcholine that can the absorption of protein or polypeptide is few is easy and import and do not damage the microtexture of body surface quantitatively.In addition, owing to do not import phosphorylcholine unreacted functional group in addition, can provide biocompatibility high material.
Be applicable to that the silica gel isochromatic spectrum with under the situation of carrier, is extremely good GFC weighting agent.Feature with surface-modifying agent synthetic chromatographic grade weighting agent of the present invention is: it is self-evident that the molecular weight difference that utilizes GFC pattern (GFC mode) to cause separates, and owing to iso-electric point or the hydrophobicity difference that protein or polypeptide had has good separating power.
Further, because ion exchangeable or hydrophobicity can be regulated according to the salt concn or the pH of moving phase, therefore can carry out unique separation according to protein.And, therefore because do not cause protein irreversibly absorption on powder surface, can not be attended by proteinic sex change, inactivation and separate, divide and get, analyze.
When strainer and glass experiment utensil is handled with surface-modifying agent of the present invention, can obtain few strainer of protein adsorption and glass and test utensil.
Description of drawings
Fig. 1: the structural formula and the 1H-NMR collection of illustrative plates of the compound of preparation in the synthesis example 1.
The 13C-CPMAS collection of illustrative plates of the modified powder of preparation among Fig. 2: the embodiment 2.
The 31P-CPMAS collection of illustrative plates of the modified powder of preparation among Fig. 3: the embodiment 2.
The FT-IR collection of illustrative plates of the modified powder of preparation among Fig. 4: the embodiment 2.
Fig. 5: the calibration curve when the phase chromatography-use weighting agent for preparing among the embodiment 3 is used with the GFC pattern.
Fig. 6: the color atlas when using the phase chromatography-use weighting agent separation of human serum protein for preparing among the embodiment 3.
Fig. 7: in the moving phase salt concn is the color atlas during the separation of human serum protein under the condition of 500mM.(a) with surface-modifying agent synthetic weighting agent of the present invention.(b)Shodex PROTEINKW-803。
Fig. 8: in the moving phase salt concn is the color atlas during the separation of human serum protein under the condition of 150mM.(a) with surface-modifying agent synthetic weighting agent of the present invention.(b)Shodex PROTEINKW-803。
Fig. 9: the color atlas when using by 5 kinds of organic acids of surface-modifying agent synthetic weighting agent separation of the present invention.
Figure 10: the compartment at surface-modifying agent of the present invention inserts under the situation of secondary amine the color atlas during the separation of human serum protein.
The FT-IR collection of illustrative plates of synthetic modified powder among Figure 11: the embodiment 9.
Figure 12: the color atlas that uses the phase chromatography-use weighting agent of preparation among the embodiment 10.
Figure 13: the color atlas that uses the phase chromatography-use weighting agent of preparation among the embodiment 3.
Figure 14: use the compound of representing with the formula (14) of preparation in the Comparative Examples 2 to carry out the color atlas of the phase chromatography-use weighting agent of surface modification.
Figure 15: use the compound of representing with the formula (15) of preparation in the Comparative Examples 2 to carry out the color atlas of the phase chromatography-use weighting agent of surface modification.
The 1H-NMR collection of illustrative plates of the compound of preparation among Figure 16: the embodiment 7.
The Mass collection of illustrative plates of the compound of preparation among Figure 17: the embodiment 7.
Figure 18: the Mass collection of illustrative plates of the compound of preparation in the synthesis example 1.
Embodiment
In addition, The compounds of this invention no matter purifying or not purifying can carry out surface modification, thereby can access effects such as protein adsorption inhibition.
The compound that contains phosphorylcholine of following formula (1) or (5) or (6) expression is a novel cpd.
In the formula, m is 2～6, and n is 1～4.
X
1, X
2, X
3Be methoxyl group, oxyethyl group or halogen independently of one another.But, X
1, X
2, X
3In at the most 2 can be in methyl, ethyl, propyl group, sec.-propyl, butyl, the isobutyl-any one.
R is any one (in following formula (2)～(4) structure, formula (1) compound is represented with A-R-B) in following formula (2)～(4) structure.
A-(CH
2)
L-B
(2)
In formula (2)～(4), L represents 1～6, and P represents 1～3.
In the formula, m is 2～6, and n is 1～4.X
1, X
2, X
3Be methoxyl group, oxyethyl group or halogen independently of one another.But, X
1, X
2, X
3In at the most 2 can be in methyl, ethyl, propyl group, sec.-propyl, butyl, the isobutyl-any one.
[preparation method of the compound that contains phosphorylcholine of formula (1) or (5) or (6)]
The phosphoryl choline derivative of following formula (7) expression is dissolved in the distilled water.The phosphoryl choline derivative of following formula (7) is a known compound, can obtain from commercially available product.
The aqueous solution of formula (7) compound is cooled off in ice-water bath, add sodium periodate, stirred 5 hours.With reaction solution concentrating under reduced pressure, drying under reduced pressure, with the phosphoryl choline derivative shown in the methanol extraction following formula (8) with aldehyde radical.Structural formula and NMR collection of illustrative plates as shown in Figure 1, the Mass collection of illustrative plates as shown in figure 18.
Then, in the methanol solution of formula (8), add 0.5 normal 3-TSL 8330.This mixture solution is in stirring at room after the specified time, ice-cold, add an amount of sodium cyanoborohydride, return to room temperature, stirred 16 hours.Also in reaction vessel, continue to feed drying nitrogen during this period.Behind the filtering-depositing, obtain the methanol solution of formula (5) and/or formula (6).
The following describes the purification process of The compounds of this invention.The purification process of The compounds of this invention is not limited to following.
With the methanol solution concentrating under reduced pressure that obtains, and residue is dissolved in the distilled water.With this aqueous solution as sample.(size: 4.6mm i.d. * 250mm) (Shiseido) is connected in the HPLC device will to have performance liquid chromatographic column カ プ セ Le パ Star Network SCX UG 80 S-5 of hydrophobic interaction and cation exchange capacity (CEC), the flow velocity that the phosphoric acid buffer (pH3.5) that makes 0.2mmol/L divides with 1mL/ injects 10 μ L samples after flowing through and carrying out balance.By using the differential refractometer to obtain color atlas, can separate the purpose compound as detector.
Yet, comprise the surface-modifying agent of The compounds of this invention, can directly use with the state in the methanol solution stage before the purifying.
Top step even m, n in the compound shown in formula (5) or (6) change, also can similarly be carried out.The step of Xian Shiing is the situation of m=3, n=2 herein.In addition, can have amino silane compound, between silane position and phosphorylcholine, insert secondary amine, also can carry out with step same as described above about this point by using conducts such as 3-(2-aminoethylamino propyl group) Trimethoxy silane.Reaction solvent is not particularly limited, and except above-mentioned methyl alcohol, can also make alcohol such as water or ethanol, propyl alcohol, butanols, N, non-protonic solvents such as dinethylformamide or methyl-sulphoxide.But, for the polymerization of organic silane compound in preventing to react, preferred dehydrated solvent.
In addition, the methoxyl group (OCH in formula (5) or (6)
3) be oxyethyl group (OC
2H
5) situation under, methyl alcohol is replaced by ethanol reacts, under the situation for Cl, be replaced by dimethyl formamide or methyl-sulphoxide.
In addition, among Si bonded methoxy or ethoxy or Cl, 2 or 1 are arranged, also can similarly be prepared with aforesaid method by under any one displaced situation of institute in methyl, ethyl, propyl group, sec.-propyl, butyl, the isobutyl-.
Surface-modifying agent
The compound of above-mentioned formula (5) and (6) can be used as the surface-modifying agent of material.That is, import the phosphorylcholine of aequum and carry out modification at material surface easily.Particularly, have on the surface under the situation of material of hydroxyl, by the hydroxyl of this material surface and the Si-OCH of formula (5) and (6) compound
3Carry out dehydration reaction and form chemical bond.This chemical reaction quantitatively carries out in 10 ℃～250 ℃ temperature range in nearly all organic solvent easy as can.By this dehydration reaction, can carry out chemistry, the surface modification of passing through phosphorylcholine of stabilizer pole physically.
In addition, do not exist on the material surface under the situation of hydroxyl, following method is effective: the compound dissolution of formula (5) and (6) in volatile solvent, to material surface, and is made solvent seasoning with this solution coat.As concrete example, formula (5) and (6) compound dissolution in methyl alcohol, and are applied to it on material surface.In 10 ℃～250 ℃ temperature range, make methanol gasifying then, further carry out heat treated as required.At this moment, the Si-OCH in formula (5) and (6) compound
3Between dehydration reaction takes place, generate the Si-O-Si key, thereby can be covered material surface.Si-OCH
3Between the reaction that dehydration reaction generates the Si-O-Si key takes place is known.The film that when methyl alcohol volatilizees, forms like this and since with nearly all material surface on the hydroxyl that exists of trace produce throughout and combine, therefore become the surface-modification method that has good stability.This law to there not being the material of hydroxyl, also is the surface-modification method of very imitating to the material that hydroxyl is arranged not only.
With surface-modifying agent modification of the present invention material (or material), become biocompatibility and wetting ability excellent material and molding.As on material surface, directly having the material that biocompatible phosphorylcholine is arranged, can use in the purposes widely at makeup, medical material (artificial organ, operational tool etc.), chromatographic grade weighting agent, coating etc.
In addition, surface-modifying agent of the present invention, method of modifying as the pin of the pipe arrangement that separates or analytical equipment is used, tubing connection portion part, sampling, sampling jug, detection cell etc. and the contacted parts of experimental liquid is useful, especially is preferred for the modification of the materials such as capillary pipe arrangement of the connecting pipings of HPLC, MS, NMR or electrophoresis apparatus.Materials such as teflon (registered trademark) pipe, テ Off ゼ Le pipe, ピ one Network pitch tube, fused quartz tube.
[use has the method for compound shown in the phosphoryl choline derivative preparation formula (6) of carboxyl]
The phosphoryl choline derivative wetting ability is high, and the solubleness in organic solvent is extremely low.Phosphoryl choline derivative synthetic roughly is divided into two oxa-phosphorane classes as the complete synthesizing process of initial substance with use by being included in the phosphatide in the soybean etc.---and Glycerophosphorylcholine that the phosphatidylcholine hydrolysis obtains is as the synthesis method of initial substance.Because the organic solvent of dissolving phosphoric acid choline derivative is limited, causes complex synthetic route, preparation cost is high and hinder practical application.This synthetic complicacy and cost problem are had outstanding performance in complete synthesizing process.But preparation in accordance with the present invention, can in the good solvent of phosphoryl choline derivative, extremely simply and with high yield prepare and have the phosphoryl choline derivative of carboxyl, and can be from by being prepared by the cheap a large amount of phosphatidylcholine deutero-Glycerophosphorylcholines that obtain, also very favourable aspect cost.Finally, the compound shown in the formula (6) also can obtain easy and with high yield.
Glycerophosphorylcholine, sodium periodate, ruthenium trichloride (hydrate) are joined in the acetonitrile solution.After stirring at room, filter, from filtrate, remove and desolvate.From the solids that obtains, use the methanol extraction object, by distillation for removing methanol, obtain the phosphoryl choline derivative shown in the following formula (9) then with carboxyl.Structural formula and NMR collection of illustrative plates as shown in figure 16, the Mass collection of illustrative plates as shown in figure 17.
In addition, reaction solvent also can be a water, and can use other periodates beyond the Periodic acid or Periodic acid etc., also can use other divalence beyond the ruthenium trichloride and/or the hydrate of trivalent ruthenium compound or these materials etc.
Then, in the methanol solution of the compound shown in the formula (9), add each 1 equivalent of 3-TSL 8330 0.5 equivalent and N-hydroxy-succinamide (NHS) and N-ethyl-N '-3-diamino propyl carbodiimide diimine (EDC).By with this mixing solutions stirring at room 3 hours, obtain compound shown in the formula (6).
In addition, reaction solvent also can be the N beyond the methyl alcohol, and dinethylformamide, methyl-sulphoxide, chloroform etc. can use two cyclohexyl carbodiimide diimines (DCC) beyond NHS, the EDC, carboxyl diimidazole (CDI) etc. in addition.
Top step even m, n in the compound shown in the formula (6) change, also can fully similarly be carried out.The step of Xian Shiing is the situation of m=3, n=2 herein.But, for the polymerization of organic silane compound in preventing to react, preferred dehydrated solvent.
In addition, the methoxyl group (OCH in formula (6)
3) become oxyethyl group (OC
2H
5) situation under, methyl alcohol is replaced by ethanol reacts, become under the situation of Cl, be replaced by dimethyl formamide or methyl-sulphoxide.
In addition, in Si bonded methoxy or ethoxy or Cl, 2 or 1 are arranged, also can fully similarly be prepared with aforesaid method by under any one the displaced situation of institute in methyl, ethyl, propyl group, sec.-propyl, butyl, the isobutyl-.
[modified powder]
The powder that surface-modifying agent of the present invention can be preferred for having hydroxyl carries out modification.
Modified powder of the present invention prepares in order to the below method.By this method, can easily prepare the modified powder that directly has phosphorylcholine at powder surface, that is, import the modified powder of phosphorylcholine by Chemical bond at powder surface.
This modified powder, the powder that imports phosphorylcholine with the polymkeric substance lining that has phosphorylcholine by usefulness is compared, and has the advantage that can not lose phosphorylcholine because of peeling off of polymkeric substance.In addition, owing to be not aggregated the thing lining, thereby have the advantage of not damaging powder self microtexture.Particularly, the surface-modifying agent of the application of the invention can not bury number nm other microtextures of level (minute aperture etc.) on the solid that powder surface has and with phosphorylcholine lining surface.
In addition, the phosphorylcholine radical derivative is being imported under the situation of object as low molecule, at first import on this object and can make then with the functional group of phosphorylcholine derivatives reaction in the method for phosphorylcholine derivatives reaction, because unreacted functional group remains on the body surface on the body surface, so biocompatibility is low.For example, at first importing aminoly on body surface, under the situation that the aldehyde derivatives that makes phosphorylcholine then and the amino of body surface react, a lot of unreacted amino are residual.Although these residual amino can block to a certain extent by combining with other low molecular compound, be difficult to keep the wetting ability of body surface, and can not block fully.Under the situation of residual a lot of amino on the body surface, because amino has strong basicity, mainly be that acidic protein shows obviously very strong electricity interaction, its nearly all generation absorption.As under the situation of chromatographic grade weighting agent, become the reason that this recovery of protein rate reduces or the peak significantly trails.And absorption of proteins causes its sex change, as under the situation of biocompatible material, becomes the reason of inflammation etc., thereby bad.
When synthesizing surface-modifying agent of the present invention, with respect to having amino organic silane compound, add the aldehyde derivatives of excessive phosphorylcholine, the two is reacted in liquid phase.The reactivity of known amino and aldehyde radical is very high, is adding under the situation of excessive aldehyde almost 100% amino and aldehyde reaction.Therefore, do not detect unreacted amino in the surface-modifying agent of the present invention.Therefore, adopt surface modification of the present invention, can only import phosphorylcholine and not be mixed with unreacted amino at body surface.In view of the above, compare with the method for carrying out the reaction of 2 steps on solid phase, it is high to obtain biocompatibility, the powder that protein adsorption is few.
[preparation method of modified powder]
In the methanol solution 20mL of the 0.3mmol/mL concentration of formula (5) or (6) compound, add distilled water 20mL, add the powder that carries out surface modification.The quality of powder must be adjusted according to its specific surface area.For example when being 100m
2During the powder of/g, its addition is to be suitable about 10g.With the 80 ℃ of backflows in oil bath of this powder dispersion liquid, with powder filter, use methanol cleaning after 5 hours, 80 ℃ of drying under reduced pressure 3 hours, obtain modified powder thus.
Used powder has no particular limits, and is meant that according to purposes average particle diameter be arbitrary objects about 0.01～10 μ m or 0.01～1000 μ m.As concrete powder, can enumerate for example inorganic powder (for example talcum powder, kaolin, mica, sericite, white mica, phlogopite, synthetic mica, rubellan, biotite, vermiculite, magnesiumcarbonate, lime carbonate, pure aluminium silicate, barium silicate, Calucium Silicate powder, Magnesium Silicate q-agent, strontium silicate, wolframic acid metal-salt, magnesium, silicon, zeolite, barium sulfate, calcined calcium sulfate hemihydrate (plaster of Paris), calcium phosphate, fluorapatite, hydroxyapatite, ceramics powder, metallic soap (for example Zinc tetradecanoate, calcium palmitate, aluminum stearate), boron nitride, cerium oxide etc.); Organic dust (for example polyamide resin powder (nylon powder), polyethylene powders, polymethylmethacrylate powder, benzo guanamine toner, polytetrafluorethylepowder powder, poly-methyl silsesquioxane powder, silicone elastomer powder, cellulose powder etc.); Inorganic white pigment (for example titanium dioxide, zinc oxide etc.); Inorganic red series pigments (for example ferric oxide (red iron oxide), iron titanate etc.); Inorganic brown series pigments (for example gamma-iron oxide etc.); Inorganic yellow series pigments (for example Zh 1, loess etc.); Inorganic black series pigments (for example black iron oxide, low order titanium oxide (low order acidifying チ Application) etc.); Inorganic violet pigment (for example manganese violet, cobalt violet etc.); Inorganic green series pigments (for example chromic oxide, chromium hydroxide, cobalt titanate etc.); Inorganic blue series pigments (for example ultramarine, Prussian green grass or young crops etc.); Pearl pigment (for example mica of the talcum powder of the pearl white of the mica of titanium oxide dressing, titanium oxide dressing, titanium oxide dressing, painted titanium oxide dressing, pearl white, guanin etc.); Metallic powdery pigment (for example aluminium powder, copper powder etc.); Pigment dyestuffs such as zirconium, barium or aluminium color lake (for example red No. 201, red No. 202, red No. 204, red No. 205, red No. 220, red No. 226, red No. 228, red No. 405, orange No. 203, orange No. 204, yellow No. 205, pigment dyestuffs such as yellow No. 401 and blue No. 404, red No. 3, red No. 104, red No. 106, red No. 227, red No. 230, red No. 401, red No. 505, orange No. 205, yellow No. 4, yellow No. 5, yellow No. 202, yellow No. 203, green No. 3 and blue No. 1 etc.); Natural pigment (for example chlorophyll, β-Hu Luobusu etc.) etc.
By above-mentioned preparation method, can obtain containing the powder of the wetting ability phosphorylcholine of any amount easily.In addition, at powder is under the situation of synthetic polymer, as its hydrophilic segment, also can contain carboxylic acid group, hydroxyl, primary～uncle amino, sulfonic group, phosphate, polyoxyethylene groups, ammonium, amido, carboxybetaine, carbohydrate etc., the function that can design powder by the kind and the content of these materials.In addition, as its hydrophobic part, also can contain hydrophobic groups such as heterocyclic aromatic series, perfluoroalkyl, poly-alkylsiloxane such as the cyclic alkyls such as straight or branched alkyl, cholesterol of carbonatoms 2～22, the alkyl that contains unsaturated link(age) such as oleyl, hydro carbons aromatic series, pyridine ring, imidazoles, thiazole, indoles, can select, design according to the purposes of powder with headed by phenyl ring, naphthalene nucleus, the pyrene.The combining form of the hydrophobic group of synthetic polymer powder can pass through ester, ether, acid amides, urethane, urea bond etc., directly is attached on the main polymer chain, also can combine with main chain by spacer.As the kind of spacer, can enumerate hydrophilic polyoxyethylene, hydrophobic polyoxytrimethylene, straight chained alkyl (carbonatoms 2～22) etc.
Modified powder of the present invention is the good powder of wetting ability and moisture retention.As having biocompatible powder, can be applied to makeup, medical material, chromatographic grade weighting agent, coating etc. widely in the purposes.
[the chromatographic grade weighting agent that comprises the modified support of handling with surface-modifying agent]
By surface-modifying agent of the present invention, thereby the carrier surface modification easily can be prepared the chromatographic grade weighting agent of phosphorylcholine with aequum.
Particularly, by the hydroxyl that exists on the carrier surface and the Si-OCH in formula (5) and (6) compound
3Dehydration reaction, phosphorylcholine is imported to carrier surface.
In methanol solution (0.3mmol/mL) 20mL of formula (5) and (6) compound, add distilled water 20mL, add median size 5 μ m, average fine pore 300 dusts, specific surface area 100m
2The spherical high-purity silica gel 4g of/g.With this powder dispersion liquid in oil bath in 80 ℃ of backflows, with powder filter,,, can easily obtain directly having on the surface powder of phosphorylcholine thus after 5 hours 80 ℃ of drying under reduced pressure 3 hours with methanol cleaning.
Reaction solvent can also make protic solvents such as water, ethanol, 2-propyl alcohol except the water-methanol mixed solvent, or non-protonic solvent such as methyl-sulphoxide, dimethyl formamide, toluene, ether, and these can be used alone or in combination.
In addition, do not exist on the carrier surface under the situation of hydroxyl, following method is effective: with the compound dissolution of formula (5) and (6) in volatile solvent, with this solution coat on material surface, dry solvent then.As concrete example,, it directly is coated on the material in right amount according to the specific surface area of material with the methanol solution (0.3mmol/mL) of formula (5) and (6) compound.In 10 ℃～250 ℃ temperature range, make methanol gasifying then.At this moment, the Si-OCH in formula (5) and (6) compound
3Between dehydration reaction takes place, generate the Si-O-Si key, thereby can be covered material surface.This dehydration reaction is known.The film that when methyl alcohol volatilizees, forms like this and since with nearly all material surface on the hydroxyl that exists of trace produce throughout and combine, the therefore surface modification that can have good stability.This law to there not being the material of hydroxyl, also is the surface-modification method of very imitating to the material that hydroxyl is arranged not only.
With at first to carrier surface import amino, will contain the method that the compound of the aldehyde body that the oxidative decomposition by Glycerophosphorylcholine obtains imports then and compare, using the maximum difference of the method for surface-modifying agent of the present invention is to have or not unreacted amino on body surface.
That is, using under the situation of surface-modifying agent of the present invention, can on material surface, only import phosphorylcholine and be not mixed with unreacted amino.At first amino is being imported under the situation of powder surface, second step import phosphorylcholine reaction since the hydroformylation thing of the Glycerophosphorylcholine in the liquid phase must with the amino reaction of solid phase surface, thereby the three-dimensional etc. of the steric barrier that causes owing to the three-dimensional arrangement of rate of diffusion or solid phase surface, phosphorylcholine self makes reactivity low.Can import phosphorylcholine having only on about 30% the amino.Although residual amino can block on degree ground by combining with other low molecular compound, the wetting ability of keeping body surface is very difficult, and can not block fully.
In addition, under the situation of residual a lot of amino on the body surface,, mainly be that acidic protein shows obviously very strong electricity interaction because amino has strong basicity, it nearly all produces absorption.During as the chromatographic grade weighting agent, become the reason that this recovery of protein rate reduces or the peak significantly trails.And absorption of proteins causes its sex change, when the biocompatible material, becomes the reason of inflammation etc., thereby bad.
On the contrary, surface-modifying agent of the present invention with respect to the aldehyde derivatives with the excessive phosphorylcholine of amino organic silane compound interpolation, reacts the two when synthetic in liquid phase.The reactivity of known amino and aldehyde radical is very high, generally almost 100% amino and aldehyde reaction under the situation of adding excessive aldehyde.
Therefore, in surface-modifying agent of the present invention, do not detect unreacted amino.Therefore, use surface modification of the present invention, can only import phosphorylcholine and not be mixed with unreacted amino at body surface.In view of the above, compare, can obtain the powder that biocompatibility is fabulous, protein adsorption is few with the method for on solid phase, carrying out the reaction of 2 steps.
In addition, known to by spherical powder being scattered in when carrying out surface modification in the liquid phase, the powder of weak strength exist stir in the phenomenon of powder disintegration.In this law, since can one step and directly phosphorylcholine being imported on the powder in short time, therefore compare with the methods of on solid phase, carrying out the reaction of 2 steps, the churning time of powder saved 1/4 or below, go for the powder of structure, material widely.Particularly, even fine pore is surpassed powder a little less than such physical strength of 1000 dusts, can not damage powder shape yet and carry out surface modification.
The carrier that uses among the present invention comprises inorganic porous body, porous organic polymer resins such as silicon-dioxide, silica gel, gac, zeolite, aluminum oxide, clay mineral.The preferred powder of carrier.Preferred ball-type or breaking type porous silica gel.The median size of spherical porous silica gel is 1～200 μ m, preferred 1～10 μ m, and the pore average diameter of spherical porous silica gel is 10～2000 dusts, preferred 80～1000 dusts, specific surface area is 0.01～800m
2/ g, preferred 80～600m
2/ g.
Chromatographic grade weighting agent of the present invention is used for GFC when using post, and the absorption of protein or polypeptide is few, and the performance high score is from ability.
That is, be good column packing aspect the absorption inhibition of protein and polypeptide.Therefore, go for utilizing the pattern (GFC pattern) of the differential liberation protein and the polypeptide of molecular weight.
Therefore and chromatographic grade weighting agent of the present invention is because phosphorylcholine has double charge, is not only based on the difference of molecular weight analyte, and has more high score based on faint charge differences that sample had from the column packing of ability.Importing the example with doubly charged functional group for the absorption of arrestin matter does not like this also have, and the chromatographic grade weighting agent for preparing with surface treatment agent of the present invention is the new GFC column packing that does not have so far.Utilization has this feature of this electric charge, not only can according to the difference of molecular weight and its with electric charge come isolated protein or polypeptide, and show, by changing the pH or the salt concn of moving phase, also can control the interaction strength of weighting agent surface and protein or polypeptide.Therefore, pH or salt concn optimization by making moving phase can keep target protein matter or polypeptide freely.
The GFC pattern with its separation, purifying, therefore aspect the separation or medical use of unknown sample, expects that also the high score of column packing of the present invention plays a role from ability owing to can not making protein or enzyme deactivation.
Chromatographic grade weighting agent of the present invention, particularly, as the few high score of the absorption of protein and polypeptide from the ability column packing, proteinic separation in human serum for example, the perhaps separation of the polypeptide that in the sample of tryptic digestion protein gained, contains, perhaps based on the separation of the activity rating of the agnoprotein matter that contains in the biology, divide that to get the aspect be good.
[with the strainer of surface-modifying agent processing]
When protein that contains in to biological sample etc. is analyzed, must remove pre-treatment from the impurity of biology.Particularly, in measuring blood during proteinic concentration,, must adopt in advance and filter or hemocyte or thrombocyte etc. are removed in centrifugation etc. in order to obtain being dissolved with proteinic serum.In the filtration procedure of blood etc., there is the problem that causes the reduction of proteinic quantitative property owing to used strainer adsorbed proteins.In addition, make its double-deck container that passes through punctulate film, in the concentrated or purposes such as desalination, solvent exchange of protein example, be widely used with centrifugal force.For example can enumerate the centrifugal Off イ of Ultrafree-MC Le one ユ ニ Star ト (ProductName) (Japanese ミ リ Port ア Co., Ltd.).This Off イ Le one ユ ニ Star ト is the sample before concentrating of packing on top, and the bottom is set to finish filtration towards the outside of centrifuge separator by the centrifugal force that applies about 5000G.Even the filtering membrane of such utensil, also exist the recovery of protein rate to drop to situation near 50%, there is not good problem.
By compound strainer is carried out surface modification, can obtain the few strainer of protein adsorption with formula (5) and (6).Because the Si-OCH in formula (5) and (6)
3Combine with the hydroxyl generation dehydration reaction of filter surfaces, can import protein adsorption easily and suppress the good phosphorylcholine of ability.Owing to all have the hydroxyl of autoxidisable substance on nearly all surface of the strainer of materials such as used metal, glass, glass fibre, therefore to material is all effective widely.
In addition, under the situation of the material that does not have hydroxyl, following method is effective: the compound dissolution of formula (5) and (6) in volatile solvent, is immersed in strainer or other materials in this solution, and dry solvent is cleaned then.Particularly, the methanol solution (0.3mmol/mL) of formula (5) and (6) compound according to the specific surface area of material direct impregnation material in right amount, is made methanol gasifying then in 10 ℃～250 ℃ temperature range.At this moment, the Si-OCH in formula (5) and (6) compound
3Between dehydration reaction takes place, generate the Si-O-Si key, thereby can be covered material surface.This dehydration reaction is known.The film that forms like this when methyl alcohol volatilizees combines owing to forming throughout with the hydroxyl of trace existence on nearly all material surface, therefore can compare tough surface modification.This law to there not being the material of hydroxyl, also is the surface-modification method of very imitating to the material that hydroxyl is arranged not only.
When by phosphorylcholine the object that has minute aperture as strainer being carried out surface modification, it is difficult being covered with known polymkeric substance with phosphorylcholine.Major cause is the polymer plugging minute aperture, reduces the ability as strainer.By use formula (5) and (6) so low molecule, can make the carrier surface modification and do not damage the micro hole construction characteristic of carrier.And, since with carrier surface be not physical adsorption and can form chemical bond, very good aspect weather resistance.
[testing utensil] with the glass that surface-modifying agent is handled
Glass experiment utensil be meant preserve container, metering with utensil, cuvette (cell), fractionating tube (fractionation tip), sample classification (sample fractionation) with experiment utensils such as syringes.Under situation about handling, can obtain the few experiment utensil of protein adsorption with surface-modifying agent of the present invention.
Embodiment
Below be that the basis further describes the present invention with embodiment.The present invention is not subjected to the restriction of these embodiment.
Synthesis example 1 contains the aldehyde cpd of phosphorylcholine
(450mg) is dissolved among the distilled water 15mL with L-α-Glycerophosphorylcholine, cools off in ice-water bath.Add sodium periodate (750mg) (Wako Pure Chemical Industries, Ltd.), stirred 5 hours.With the reaction solution concentrating under reduced pressure, drying under reduced pressure is used the methanol extraction object.Structure is shown in following compound (8).The 1H NMR collection of illustrative plates of formula (8) compound as shown in Figure 1, the Mass collection of illustrative plates as shown in figure 18.
The preparation of embodiment 1 formula (5) and (6) compound
The compound 7.5g of synthesis example 1 is dissolved among the dehydration methyl alcohol 30mL, will replaces with drying nitrogen in the container.In the methanol solution of compound 1, add 3-TSL 8330 (Shin-Etsu Chemial Co., Ltd) 3.6g then.This mixture solution is in stirring at room after 5 hours, ice-cold, add sodium cyanoborohydride (Wako Pure Chemical Industries, Ltd.) 2.5g, return to room temperature, and stirred 16 hours.In reaction vessel, continue to feed drying nitrogen during this period.Behind the filtering-depositing, obtain desired substance, i.e. the methanol solution of following formula (10) and formula (11) compound.
The preparation of embodiment 2 modified powders
In containing embodiment 1, add distilled water 35mL in the methanol solution of the formula (10) of preparation and (11) compound, further add median size 5 μ m, average fine pore 30nm, specific surface area 140m
2The silica gel 14g of/g.This powder dispersion soln was refluxed 5 hours at 80 ℃.Clean with methyl alcohol 100mL filtration after refluxing, obtain desired substance.
According to above step, the ultimate analysis value of the modified powder that the surface-modifying agent of usefulness embodiment 1 was handled is as shown in table 1.C% in the table or N% represent the quality % of carbon contained in the powder or nitrogen element.From this value as can be known, be 5.08 with the carbon of the powder after the surface-modifying agent processing of embodiment 1 and the ratio (C/N) of nitrogen-atoms number.Because it is not the C/N after the combination of the position, methoxyl group position of the surface-modifying agent of formula (10) and (11) is 5, destroyed so show that surface-modifying agent has been directed in the powder.
Table 1
In addition, the 13C-CPMAS collection of illustrative plates of this silica gel and 13C-PSTMAS collection of illustrative plates are as shown in Figure 2.So-called PSTMAS collection of illustrative plates is the method that selectivity obtains the collection of illustrative plates of free-moving molecular chain, is the method for widespread use in the parsing of the modification chain of powder surface.Observe the collection of illustrative plates that the carbon by the choline base produces among Fig. 2 at the 54.2ppm place.
On the other hand, in the 31P-CPMAS of identical silica gel shown in Figure 3 collection of illustrative plates since with the NaH that measures as object
2PO
4Detect the peak on the chemical displacement value much at one, therefore can confirm the existence of phosphate.Owing to the existence of having confirmed the choline base according to Fig. 2, confirmed the existence of phosphate again according to Fig. 3, therefore think phosphorylcholine to be imported to carrier silica gel surface.
In addition, Fig. 2 observes the collection of illustrative plates that is produced by the ethyl in the phosphorylcholine near the collection of illustrative plates that observes 9ppm, the 23ppm by the carbon generation of the propyl group that exists near 60ppm, 69ppm between Siliciumatom and phosphorylcholine.As known from the above, the structure of formula (5) and (6) can not be destroyed and imports in the silica gel.In addition, the bound fraction of phosphorylcholine and propyl trimethoxy silicane mixes with the bound fraction of the secondary amine shown in the formula (5), the bound fraction of the amido linkage shown in the formula (6).
Shown the FT-IR collection of illustrative plates of synthetic modified powder in the present embodiment among Fig. 4.Can be at 1650cm
-1Near observe the peculiar absorption of amido linkage.
Embodiment 3: the chromatographic grade weighting agent
The modified powder that will prepare in embodiment 2 is filled in by conventional slurry process in the void column of internal diameter 4.6mm, long 250mm as carrier.The condition that obtains color atlas is as follows:
Moving phase: 50mmol/L phosphoric acid buffer+500mmol/L NaCl pH 6.8
Flow velocity: 100 μ L/min
Temperature: 25 ℃
Detect: UV 280nm
Calibration curve when using the post of embodiment 3 under this condition as shown in Figure 5.Calibration curve shown in Figure 5 is fabulous in the measurement range internal linear.Owing to absorption of proteins is few, so the weighting agent surface is minimum with protein interactions, as its result, carries out the GFC pattern separation of wash-out early of the big molecule of molecular weight as can be known.Secondly, the result of separation of human serum protein as shown in Figure 6 under these conditions.
What use as sample is with the コ Application セ one ラ N (ProductName) of 2 times of distilled water dilutings, injects 2 μ L.The such laboratory sample of human serum also is to separate with the GFC pattern of molecular weight order as can be known, and practicality is very high.
Comparative Examples 1
Next, commercially available chromatographic grade post (Shodex PROTEIN KW803, Showa Denko K. K's system) is directly used with commercially available state, attempt the protein in the separation of human serum sample (with 2 times of distilled water dilutings of コ Application セ one ラ N (ProductName)).This post of enumerating in the Comparative Examples has identical internal diameter and length with the post described in the embodiment 3.
It is the weighting agent that the hydrophilic radical Chemical bond is used to the lip-deep size exclusion pattern of porasil that this weighting agent is illustrated as.Average fine pore is 300 dusts, and median size 5 μ m are fit to compare with the weighting agent described in the embodiment 1.Be illustrated as and be suitable for molecular weight 10,000～hundreds thousand of proteinic separation.
Shodex PROTEIN KW803 with different with the weighting agent of surface-modifying agent preparation of the present invention, does not have charged functional group owing to use non-dissociating property hydrophilic radical.Therefore, think almost do not have and proteinic ionic interaction.
Similarly to Example 3, use to the 50mmol/L phosphoric acid buffer (by Na
2HPO
4And KH
2PO
4Preparation) moving phase that adds 500mmol/L sodium-chlor at flow velocity 100 μ L/min, under the condition that column temperature is 25 ℃, is attempted the protein in the separation of human serum sample (pure コ Application セ one ラ N (ProductName) is diluted 2 times with pure water).Detection is carried out at UV 280nm place.The result is shown in Fig. 7 (b).
In addition, for relatively, the weighting agent that the surface-modifying agent of the present invention that obtains in showing with embodiment 3 among Fig. 7 (a) prepares is under the same conditions with the color atlas of same sample acquisition.
In addition, the color atlas when figure (8) shows that the weighting agent for preparing with surface-modifying agent of the present invention uses with salt concn 150mM in (a); Show the color atlas when Shodex PROTEIN KW803 uses with salt concn 150mM among Fig. 8 (b).Among Fig. 8 except salt concn other conditions identical with Fig. 7.
In having used with the color atlas of the weighting agent of surface-modifying agent of the present invention preparation (Fig. 7 (a)), except being the gamma globulin and albumin of main protein, it is very big feature that the peak of Transferrins,iron complexes is confirmed to be the acromion shape in the human serum.The ownership at peak uses the commercially available product of isolating range protein to carry out.
On the contrary, using existing GFC to use under the situation of weighting agent (Fig. 7 (b)), albumin and Transferrins,iron complexes do not have separated fully.This is by identical with the elution time of the range protein of isolating commercially available product and confirm.
The molecular weight of albumin and Transferrins,iron complexes is about 69,000 and 75,000 respectively, and is closely similar.Can not separate with post with existing GFC and to resemble the albumin sample close with the such molecular weight of Transferrins,iron complexes.Use the weighting agent of surface-modifying agent preparation of the present invention as can be known, not only absorption of proteins is few, and owing to phosphorylcholine has double charge and has faint ionic interaction with protein, even the close protein of molecular weight also can separate with hydrophobic difference based on iso-electric point.
That is, chromatographic grade weighting agent of the present invention in the GFC pattern, not only can utilize the difference of molecular weight, and can be according to iso-electric point that protein had and the hydrophobic different sample separation of coming.
Weighting agent with surface-modifying agent preparation of the present invention has faint ion-exchange interaction, and this can confirm by the salt concn that reduces moving phase.
The salt concn of moving phase is among Fig. 7 (a) of 500mM, even use weighting agent with surface-modifying agent preparation of the present invention, Transferrins,iron complexes also is the degree of confirming as the acromion at albumin peak, the salt concn of moving phase is reduced among Fig. 8 (a) of 150mM, and Transferrins,iron complexes and albuminous peak reach baseline separation.
In addition, in Fig. 8 (a), can confirm the peak that a large amount of protein produce.It is reducing and grow along with the moving phase salt concn that the ion-exchange of usually, having an effect between the known material in weighting agent surface and moving phase interacts.This is meant that by reducing the moving phase salt concn material that interacted by ion-exchange will be kept more strongly.With the weighting agent of surface-modifying agent of the present invention preparation, by reducing the salt concn of moving phase, form especially to albuminous strong reservation, reach and the separating fully of Transferrins,iron complexes.
By under low salt concn, keeping electronegative albumin under image pattern 7, the such neutral pH of Fig. 8, infer that the weighting agent with surface-modifying agent preparation of the present invention has the anionresin pattern.
Therefore, use lactic acid, acetic acid, succsinic acid, propanedioic acid, 5 kinds of organic acids of Citric Acid subsequently, estimated the anion exchange capacity that weighting agent had with surface-modifying agent preparation of the present invention.Appreciation condition is as follows.The result as shown in Figure 9.
Post: 4.6 * 150mm
Moving phase: 50mmol/L phosphoric acid buffer (pH 6.8)
Flow velocity: 1000mL/min
Detect: UV 210nm
As shown in Figure 9, the weighting agent with surface-modifying agent preparation of the present invention can separate various organic acids according to the quantity of carboxylic acid.
Particularly, when each peak that is produced by 5 kinds of organic acids was belonged to, 2 kinds of organic acids with 1 carboxylic acid were separated by wash-out the earliest, are that 2 kinds of organic acids with 2 carboxylic acids are separated by wash-out then, are that the Citric Acid with three carboxylic acids is separated by wash-out at last.Owing to confirmed, has anion exchange capacity with regard to clear and definite this weighting agent along with the increase of carboxylic acid quantity keeps the trend that becomes big.Therefore we can say that the powder of handling with surface-modifying agent of the present invention also is very effective as anionresin with weighting agent.More than shown in ion exchangeable, be suitable for not making the intensity of protein adsorption, denaturation degrees, the good uniqueness of the rate of recovery is separated.
On the other hand, as the Shodex PROTEIN KW803 of general GFC, as shown in Fig. 8 (b), when the salt concn of 150mM, can not separate Transferrins,iron complexes and albumin with post.
With the weighting agent of surface-modifying agent of the present invention preparation, be GFC pattern (size exclusion pattern) that causes because of good function and the extremely weighting agent of uniqueness that has the ion-exchange pattern with the absorption of arrestin matter.Because absorption of proteins is few, therefore can keep separating under the condition of its enzymic activity state, protein purification not making protein denaturation.And, owing to not only have the GFC pattern also to be mixed with the ion-exchange pattern, be to control isolating epoch-making weighting agent therefore according to the salt concn or the pH of moving phase.
Embodiment 4 chromatographic grade weighting agents
(be used in have 2 nitrogen-atoms between Siliciumatom and the phosphorylcholine, R is that the represented compound of formula (4) p=1 carries out surface modification in the formula (1))
The compound 7.5g of synthesis example 1 is dissolved among the dehydration methyl alcohol 30mL, will replaces with drying nitrogen in the container.In the methanol solution of compound 1, add 3-(2-aminoethylamino propyl group) Trimethoxy silane (Shin-Etsu Chemial Co., Ltd) 3.6g then.
This mixing solutions is in stirring at room after 5 hours, ice-cold, add sodium cyanoborohydride 2.5g, return to room temperature, stirred 16 hours.In reaction vessel, continue to feed drying nitrogen during this period.
Behind the filtering-depositing, obtain the following formula (12) as desired substance, the methanol solution of (13) compound.
In the methanol solution that contains formula (12) and (13) compound, add distilled water 35mL, further add median size 5 μ m, average fine pore 30nm, specific surface area 140m
2The silica gel 14g of/g.This powder dispersion soln was refluxed 5 hours at 80 ℃.Clean with methyl alcohol 100mL filtration after refluxing, obtain desired substance.
The silica gel that demonstration will import formula (12) and (13) in Figure 10 (b) slurry process is routinely filled, the color atlas when injecting human serum sample (with コ Application セ one ラ N (ProductName) with 2 times of distilled water dilutings).
Color atlas when Figure 10 (a) is to use the powder for preparing among the embodiment 3.
Secondary amine between Siliciumatom and phosphorylcholine is Duoed than Figure 10 (a) among Figure 10 (b) of 1, and Transferrins,iron complexes separates further improvement with albuminous as can be known.This is considered to increase the alkalescence of modification group thus owing to inserted secondary amine between Siliciumatom and phosphorylcholine, is kept more strongly as the albumin of acidic protein.
Like this, surface-modifying agent of the present invention, suppress effect because of phosphorylcholine produces protein adsorption, and can give the interaction of so-called ion exchangeable, hydrophobicity, wetting ability, hydrogen associativity by changing the character of spacer between Siliciumatom and the phosphorylcholine.
＜be combined with the preparation of the pyrex fiber filter material of phosphorylcholine 〉
Add the methanol solution 1.0mL of distilled water 20g, the formula (10) that contains preparation among the embodiment 1 and (11) compound (approximately 0.4mmol) in the 100mL triangular flask, vibration mixes.After adding the pyrex fiber filter sheet (glass fiber filter paper グ レ one De GF/F, diameter 25mm Φ, per 1 about 0.70g) of 8 ワ Star ト マ Application ジ ヤ パ Application Co., Ltd. systems therein, refluxed 5 hours in 100 ℃ of heated and boiled.Behind the cool to room temperature, strainer is filtered, clean,, obtain directly having from the teeth outwards the pyrex fiber filter sheet of phosphorylcholine 80 ℃ of drying under reduced pressure 3 hours.
＜filtering material is to the mensuration of the inhibition effect of protein adsorption 〉
Bovine serum albumin (BSA) 10mg is dissolved in (1 of the PBS sheet of カ ラ バ イ オ society system is dissolved in the distilled water, and making total amount is 100mL in the 100mL phosphoric acid buffer.PH 7.4～7.5) as BSA solution.Get in the 30mL stopple coupon of 2.0g to 3 polypropylene system of BSA solution of preparation the pyrex fiber filter sheet of preparation among the dipping embodiment 6 in 1 stopple coupon wherein, the untreated pyrex fiber filter sheet of dipping in 1 stopple coupon in addition respectively.It was placed 24 hours in room temperature (25 ℃), respectively the BSA solution in 3 stopple coupons is carried out color development, photometric analysis of extinction by the Lowry method, thus the BSA adsorptive capacity of per 1 filter of quantitative analysis.
Pyrex fiber filter sheet of the present invention is as can be known compared with the product of being untreated, and the BSA adsorptive capacity reduces.
Table 2
| Sample | BSA adsorptive capacity (μ g/ sheet) |
| Untreated pyrex filter | 37.8 |
| The pyrex filter that PC handles | 7.8 |
By The above results as can be known, the present invention can provide the few filtering material of absorption of protein or polypeptide.
Filtering material of the present invention the separation of antibody, enzyme etc., concentrate or the filtration of biological substance widely such as blood purifications such as hemodialysis, blood filtration, analysis in useful.
Embodiment 6 vials
＜be combined with the making of the vial of phosphorylcholine 〉
Add the methanol solution 1.0mL of distilled water 20g, the formula (10) that contains preparation among the embodiment 1 and (11) compound (approximately 0.4mmol) in the 100mL triangular flask, vibration mixes.After adding the vial (the spiral vial of 12 * 32mm) of 10 Japanese ウ オ one one ズ Co., Ltd. systems therein, refluxed 5 hours 100 ℃ of heated and boiled.Behind the cool to room temperature,,, obtain directly having from the teeth outwards the vial of phosphorylcholine 80 ℃ of drying under reduced pressure 3 hours with the described bottle of methanol cleaning.
Like this, by surface-modifying agent of the present invention, can obtain the few glass experiment utensil of protein adsorption.Known usefulness can not arrestin matter during the common phial of absorption, and proteinic sample concentration reduced along with the time in the experimental implementation.Particularly, known when when preserving container and to high performance liquid chromatography, injects sample, although the volume of same amount, the phenomenon that peak area reduces are in time injected in existence at every turn.The vial of making in the present embodiment, since good aspect the protein adsorption inhibition, therefore can avoid such phenomenon.
In addition, thus known under the situation of the absorption that makes container inner wall adsorbed polymer arrestin matter, when containing organic solvent in the sample, the high molecular absorption of taking off can take place, thereby have the problem of weather resistance.With polymer carry out surface-coated, be to realize by the absorption that the hydrophobic interaction between the such base material of polymer and polypropylene container causes.When containing organic solvent in the sample solvent, the hydrophobic interaction between polymer and the base material weakens, and polymer is peeled off.On the other hand, surface-modifying agent of the present invention (silane coupling agent) owing to carry out finishing by Chemical bond, therefore can substantially not be subjected to the influence of solvent in the sample, can keep effect.
Embodiment 7 has the preparation of the phosphoryl choline derivative of carboxyl
In the 200mL flask, add Glycerophosphorylcholine 5g (19.4mmol), sodium periodate 17g (79.7mmol, 4.1 equivalent) (Wako Pure Chemical Industries, Ltd.), ruthenium trichloride (hydrate) 81mg (0.39mmol, 0.02mol equivalent) (Wako Pure Chemical Industries, Ltd.) and ion exchanged water 70g, acetonitrile 30g., filter after 2 hours in stirring at room, from filtrate, remove and desolvate.From the solids that obtains, use the methanol extraction object, obtain the phosphoryl choline derivative shown in the following formula (9) by removing methyl alcohol then with carboxyl.The 1H NMR collection of illustrative plates of formula (9) compound as shown in figure 16, Mass composes as shown in figure 17.
Embodiment 8 has between Siliciumatom and phosphorylcholine that R is the represented organic silane compound (silane coupling agent) of formula (3) L=2 in amido linkage, the formula (1)
Following formula (9) compound 3g (12.4mmol) is dissolved among the dehydration methyl alcohol 100mL, will replaces with drying nitrogen in the container.Add 3-TSL 8330 1.1g (6.2mmol), N-hydroxy-succinamide 1.4g (12.4mmol) and N-ethyl-N '-3-dimethylaminopropyl carbodiimide 2.4g (12.4mmol) then,-10 ℃ of reactions 16 hours, obtain containing the solution that has amido linkage in the spacer shown in the following formula (11), has the organic silane compound of phosphorylcholine endways.
In addition, replace the compound shown in the following formula (9), between phosphorylcholine and carboxyl, have the O-phosphorylcholine hydroxycaproic acid that carbonatoms is 5 saturated alkyl chain by using, can obtain with same step that R is the compound shown in formula (3) L=6 in the formula (1).
Surface-modifying agent of the present invention also can be fixed on phosphorylcholine on the base material without purifying.Yet, also can use-case method described as follows carry out purifying.
Purification process
The solution decompression that obtains is concentrated, it is dissolved in the distilled water.With this aqueous solution as sample.(size: 4.6mm i.d. * 250mm) (Shiseido) is connected in the HPLC device high performance liquid chromatography that will have hydrophobic interaction and a cation exchange capacity (CEC) with post-カ プ セ Le パ Star Network SCX UG80 S-5, the flow velocity that its phosphoric acid buffer with 0.2mmol/L (pH3.5) is divided with 1mL/ injects 10 μ L samples after flowing through and carrying out balance.Use the differential refractometer to obtain color atlas, can separate the purpose compound thus as detector.
Have amido linkage in the embodiment 9 usefulness spacers, have a modified powder that the organic silane compound of phosphorylcholine is handled endways
In containing embodiment 8, among the solution 30mL (0.25mmol/mL) of formula (11) compound of preparation, add distilled water 35mL, further add median size 5 μ m, average fine pore 30nm, specific surface area 140m
2The silica gel 14g of/g.This powder dispersion liquid was refluxed 5 hours at 80 ℃.Clean with methyl alcohol 100mL filtration after refluxing, obtain desired substance.The carbon content of the modified powder that the surface-modifying agent with embodiment 8 that obtains by above-mentioned steps was handled (carbon content before handling is 0.15%) is 2.49%.
[confirming the importing of PC base by the assay of phosphorus]
Phosphoric in the powder that has phosphorylcholine from the teeth outwards of preparation among the embodiment 9 has been carried out assay.In preparation method of the present invention, phosphoric is a phosphorylcholine inherent element, can confirm the phosphorylcholine that in fact imports by measuring the phosphoric amount that exists on the powder surface.
The assay of phosphoric is undertaken by molybdic acid color development method.Content assaying method below is described.
(1) powder of being measured is metered in the test tube of fully cleaning.
(2) in 1 powder, add 60% high chloro acid solution (Wako Pure Chemical Industries, Ltd.) 3mL.
(3) for fear of the gasification diffusion of liquid, prolong is installed on the test tube top 2, in 120 ℃ of heating 1 hour, then in 180 ℃ of heating 2 hours.In this operation, make the modification chain of powder surface all oxidized, especially, make phosphoric free with phosphoric acid.
(4) 3 solution is put into separating centrifuge (3000rpm, 5 minutes), supernatant liquor is transferred in the stopple coupon of 1mL.
(5) in 4 solution, add distilled water 1mL and Sodium orthomolybdate (Wako Pure Chemical Industries, Ltd.) the 500 μ L of 0.5M and the xitix 500 μ L of 0.5M.
(6) 5 solution was heated 5 minutes in 95 ℃ hot water bath, cool off with cold water then.
(7) the solution 200 μ L that color development in 6 is finished are added drop-wise in 96 orifice plates, measure the coloring intensity at 710nm place in this plate device.
(8) calibration curve to be obtained by the coloring intensity of the phosphoric acid solution of concentration known in advance carries out quantitatively the amount of phosphoric contained in the sample.The standardized solution of phosphoric acid can obtain from Wako Pure Chemical Industries, Ltd..
Consequently, the phosphoric of the modified powder that has phosphorylcholine from the teeth outwards of preparation is 0.13mmol/g among the embodiment 9
GelThat is, as can be known can be with 0.13mmol/g
GelPhosphorylcholine be fixed on the powder surface.
The FT-IR spectrum that shows synthetic modified powder in the present embodiment among Figure 11.
At 1650cm
-1Near can observe the peculiar absorption of amido linkage.
Have amido linkage in the embodiment 10 usefulness spacers, have a phase chromatography-use weighting agent that the surface-modifying agent (silane coupling agent) of phosphorylcholine was handled endways
Be used in that the synthetic modified powder is filled in by conventional slurry process in the void column of internal diameter 4.6mm, long 250mm as carrier among the embodiment 9.The condition that obtains color atlas is as follows:
Moving phase: 50mmol/L phosphoric acid buffer+500mmol/L NaCl pH 6.9
Flow velocity: 200 μ L/min
Temperature: 25 ℃
Detect: UV 280nm
At first, show that in Figure 12 injecting 2 μ L is mixed with alpha2 Macroglobulin 0.67mg/mL (molecular weight about 800,000, abbreviation α 2M), gamma globulin 1.3mg/mL (molecular weight about 160,000, abbreviation γ G), human serum albumin 1.7mg/mL (molecular weight about 70,000, the abbreviation HSA), N,O-Diacetylmuramidase 0.3mg/mL (molecular weight about 14,000, abbreviation LYZ), the color atlas the during aqueous sample of uridylic 0.017mg/mL (molecular weight 112, abbreviation U).α 2M, γ-G, HSA, LYZ from シ グ マ ア Le De リ Star チ ジ ヤ パ Application Co., Ltd. obtain, uridylic obtains from Na カ ラ イ テ ス Network Co., Ltd..5 peaks are confirmed clearly, with the elution time of commercially available single sample it are identified, the result is α 2M, γ G, HSA, LYZ, U according to wash-out order sooner or later successively.The small peak that observes outside 5 peaks is the impurity that contains in the commercially available standard model.By amido linkage phosphorylcholine is fixed on as can be known and makes absorption of proteins few on the silica gel, the weighting agent surface is minimum with protein interactions, the result with the big molecule of molecular weight early the GFC pattern of wash-out separate.
The amido linkage wetting ability is good.Consider the functional group of surface-coated wetting ability of preferred substance and nonionic from the viewpoint of arrestin matter absorption.Phosphorylcholine has the extremely good wetting ability of zwitter-ion inherent, because the equilibrium of zwitterionic charge balance is ionic in fact very faint, can be described as the good functional group in arrestin matter absorption aspect.Yet, phosphorylcholine is fixed to the hydrophobicity of the used spacer of material surface when very high, can bring out proteinic non-specific irreversible adsorption.Therefore, it is epochmaking having amido linkage on spacer, and it realizes more hydrophilic surface modification.The contained wetting ability of this spacer in absorption of proteins suppresses also the utmost point produce effect.
＜the Comparative Examples (with the comparison of prior art) compared with the surface-modifying agent of betaine structure 〉
Comparative Examples 2
About having the organosilicon alkanes surface-modifying agent of the betaine structure different with phosphorylcholine, the silane compound with sultaine shown in the formula (14) is disclosed in the Japanese kokai publication hei 5-222064 communique.In addition, the silane compound with carboxybetaine shown in the formula (15) is disclosed in Japanese kokai publication sho 63-295593 communique.
The silane compound of formula (14) and (15) and formula (10) and (11) compound of embodiment 1 are compared.
The preparation method of formula (14) and (15) compound carries out according to known document.
In formula (14) compound 2.0g, add methyl alcohol 20mL and distilled water 20mL, further add median size 5 μ m, average fine pore 30nm, specific surface area 140m
2The silica gel 5.0g of/g.This powder dispersion soln was refluxed 5 hours at 80 ℃, the compound shown in the formula (14) is fixed on the silica gel.Clean with methyl alcohol 50mL filtration after refluxing, obtain by the silica gel of compound surface modification shown in the formula (14).
About compound shown in the formula (15), also carry out same operation, obtain by the silica gel of compound surface modification shown in the formula (15).
Then, the surface modification silica gel that obtains is filled in the void column of internal diameter 4.6mm, long 250mm by conventional slurry process respectively.The condition that obtains color atlas is as follows:
Moving phase: 50mmol/L phosphoric acid buffer+500mmol/L NaCl
pH 6.9
Flow velocity: 200 μ L/min
Temperature: 25 ℃
Detect: UV 280nm
At first, in Figure 13, show toward what filled embodiment 3 and have on the surface that injection 2 μ L are mixed with alpha2 Macroglobulin 0.67mg/mL (molecular weight about 800 in the post of weighting agent of phosphorylcholine, 000, abbreviation α 2M), gamma globulin 1.3mg/mL (molecular weight about 160,000, abbreviation γ G), human serum albumin 1.7mg/mL (molecular weight about 70,000, abbreviation HSA), N,O-Diacetylmuramidase 0.3mg/mL (molecular weight about 14,000, abbreviation LYZ), the color atlas the during aqueous sample of uridylic 0.017mg/mL (molecular weight 112, abbreviation U).α 2M, γ-G, HSA, LYZ from シ グ マ ア Le De リ Star チ ジ ヤ パ Application Co., Ltd. obtain, uridylic obtains from Na カ ラ イ テ ス Network Co., Ltd..
5 peaks are confirmed clearly, by the elution time of commercially available single sample it are identified, the result is α 2M, γ G, HSA, LYZ, U according to wash-out order sooner or later successively.It is the previously described GFC pattern that begins wash-out from the big material of molecular weight.The small peak that observes outside 5 peaks is the impurity that contains in the commercially available standard model.
On the other hand, to usefulness be fixed with when injecting same sample in the post that the weighting agent of the sultaine shown in the formula (14) fills color atlas as shown in figure 14.In 5 kinds of materials, only observe peak, the especially N,O-Diacetylmuramidase of human serum albumin and urea, under this sample concentration, can not confirm wash-out.Between quaternary ammonium that constitutes sultaine and sulfonic acid, do not reach electric neutrality, discovery exists the sulfonic acid as strong acid to cause in the end cation exchange capacity (CEC), result are owing to and produce extremely strong ion-exchange between the N,O-Diacetylmuramidase of neutrality and positively charged and interact.
To usefulness be fixed with when injecting same sample in the post that the weighting agent of the carboxybetaine shown in the formula (15) fills color atlas as shown in figure 15.The peak of 5 kinds of materials is compared with the situation of the post that is fixed with sultaine, and wash-out is good.Sequentially eluting according to α 2M, gamma globulin, N,O-Diacetylmuramidase, urea, human serum albumin.Should be noted in the discussion above that under the neutral moving phase that electronegative human serum albumin is kept by characteristic ground, wash-out than low molecular urea also after.This is because do not reach electric neutrality between quaternary ammonium that constitutes carboxybetaine and carboxyl, therefore and neutral and have and produce extremely strong ion-exchange between the human serum albumin of negative charge and interact owing to found the reinforcing yin essence ion exchangeable of ionic strong relatively quaternary ammonium.
Like this, general known trimethyl-glycine and not exclusively be the ion neutral.The such betaine structure of sultaine of enumerating or carboxybetaine has good wetting ability, but because strong excessively ion exchangeable can bring out absorption of proteins.Wherein, the charge balance of phosphoric acid and quaternary ammonium is suitable in the known phosphorylcholine, is being good aspect the arrestin matter adsorption function of extremely good wetting ability of betaine structure inherent and nonionic.
We can say that by above the organosilicon alkanes surface-modifying agent (silane coupling agent) with phosphorylcholine of report example so far is being very effective aspect the material surface modification of purpose with adsorbed proteins not.The surface-modifying agent of the application of the invention can be implemented the surface modification that absorption of proteins is few, biocompatibility is good.
Industrial applicability
The present invention contains the new compound of phosphorylcholine, can be used as surface modifier. Surface modifier of the present invention is given object biocompatible, moisture retention and other multiple useful function. By surface modifier of the present invention can easily make modified powder with the phosphorylcholine modification, with this modified powder as the chromatographic grade filler of carrier, with the filter of this surface modifier modification, with the glass wares of this surface modifier modification.
Claims (8)
1. the compound that contains phosphorylcholine, with following formula (1) expression,
In the formula, m is 2～6, and n is 1～4,
X
1, X
2, X
3Be methoxyl group, oxyethyl group or halogen independently of one another, precondition is X
1, X
2, X
3In at the most 2 be in methyl, ethyl, propyl group, sec.-propyl, butyl, the isobutyl-any one,
R is any one in the structure shown in following formula (2)～(4), and wherein in following formula (2)～(4) structure, formula (1) compound represents with A-R-B,
A-(CH
2)
L-B
(2)
In formula (2)～(4), L represents 1～6, and P represents 1～3.
2. the described compound that contains phosphorylcholine of claim 1 is represented with following formula (5) or (6),
In the formula, m is 2～6, and n is 1～4, X
1, X
2, X
3Be methoxyl group, oxyethyl group or halogen independently of one another, precondition is X
1, X
2, X
3In at the most 2 be in methyl, ethyl, propyl group, sec.-propyl, butyl, the isobutyl-any one.
3. surface-modifying agent, it comprises claim 1 or the 2 described compounds that contain phosphorylcholine.
4. the preparation method of compound shown in the above-mentioned formula (6), it is characterized in that: have the phosphoryl choline derivative of carboxyl by synthesizing, synthesize the compound shown in the above-mentioned formula (6) with phosphoryl choline derivative by condensing agent with carboxyl by having amino organic silane compound with the reaction of sodium periodate and ruthenium trichloride oxidation Glycerophosphorylcholine.
5. the modified powder of handling with claim 3 described surface-modifying agent.
6. chromatographic grade weighting agent, it comprises the modified support of handling with the described surface-modifying agent of claim 3.
7. the strainer of handling with claim 3 described surface-modifying agent.
8. carried out surface-treated glass experiment utensil with the described surface-modifying agent of claim 3.
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| CN102399237A (en) * | 2010-09-08 | 2012-04-04 | 上海生物医学工程研究中心 | Surface treatment agent with quasi-phosphorylcholine structure, preparation method thereof, and application thereof |
| CN103739621A (en) * | 2010-09-08 | 2014-04-23 | 上海生物医学工程研究中心 | Application of surface treating agent with phosphorylcholine-like structure in material surface modification |
| JP6360482B2 (en) * | 2012-09-17 | 2018-07-18 | ダブリュー・アール・グレース・アンド・カンパニー−コーンＷ Ｒ Ｇｒａｃｅ ＆ Ｃｏ−Ｃｏｎｎ | Chromatographic carrier and device |
| EP2860183B1 (en) | 2013-03-25 | 2018-04-25 | Sumitomo Riko Company Limited | Reactive ionic liquid and ion-immobilized metal oxide particles produced using same, ion-immobilized elastomer, and transducer |
| TWI503157B (en) * | 2014-03-27 | 2015-10-11 | Nat University Of Kaohsuing | The method for surface modification of a filter material |
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