JPS63267437A - Adsorption carrier for chromatography - Google Patents

Abstract

PURPOSE:To increase the separation and refining capability by forming an adsorption carrier for chromatography with porous copolymer containing D- alanyl-D-alanine which is covalent-bonded, through a bonding group, to an epoxy group. CONSTITUTION:A1 glycidylmonovinylester or glycidylmonovinylester and B1 alkyleneglycoldivinylester are polymerized through aqueous suspension in the presence of water-insoluble organic diluent. Subsequently, porous copolymer containing D-alanyl-D-alanine which is covalent-bonded, through a bonding group, to an epoxy group based on component A of a gelatinized copolymer wherein of component A is crosslinked by component B. This copolymer is used to manufacture an adsorption carrier for chromatography. A preferable ratio of components A and B is 10-90mol.% of the former to 90-10mol.% of the latter.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an adsorption carrier for chromatography that can be used in various chromatography including affinity chromatography.

[Conventional technology]

As a type of chromatography technology, affinity chromatography is a method of separating and purifying biological substances by utilizing the affinity of pairs of substances that specifically interact with each other. It is useful for identifying and purifying certain chemical structures on molecules.

An adsorption carrier for affinity chromatography (AffinityDal) is an active support obtained by bonding a binding group (substrate) to an insoluble carrier (matrix), and a ligand is bonded to one of the above groups of the active support. The adsorption operation is performed by selecting a combination of substances that interact with this ligand.

For example, Streptomyces orientalis (St
Vancomycin (vancomycin) produced from Reptomyces orientalis
and Nocardia 0 Lurida (Nocardia 1url
ristocetin (ristoee) produced from
tin) is an antibiotic that is mainly effective against Durham-positive bacteria, but its action against bacteria is to inhibit the biosynthesis of cell wall peptidoglycan, which is caused by vancomycin and ristocetin adsorbing to the cell wall and containing D-alanyl-D-alanine at the C-terminus. [Journal of Bacteriology (J, Bact), Vol. 102, 476]
Page (1970)]. Therefore, by using an affinity adsorbent with D-alanyl-D-alani as a ligand, it is possible to analyze and separate and purify vancomycin antibiotics, and it is known to be useful for research and production. .

For example, in separation, purification, and analysis by affinity chromatography in which D-alanyl-D-alanine is used as a ligand and an antibiotic such as vancomycin is used as the adsorption target substance, the activity that is the main component of the adsorption carrier for affinity chromatography is The desired properties of the support include low non-specific adsorption, high porosity, easy binding of ligands and large immobilization capacity, chemical stability, vocal range, salt concentration, and temperature. These characteristics include sufficient stability and no volume change under a wide range of conditions, sufficient mechanical strength and stability, and good flow properties, and resistance to biological contamination.

Cellulose, dextran, polyacrylamide, agarose, etc., which have been conventionally used as base materials for adsorption carriers for affinity chromatography, do not necessarily have these desired properties. In particular, it has serious drawbacks such as poor flow characteristics and poor separation characteristics because it is a so-called soft gel lacking in hardness, and also has a short life.

Furthermore, although the silica beads that have been used in recent years are satisfactory in terms of hardness, they cannot be used under alkaline conditions, which poses the problem of placing significant restrictions on the selection of separation conditions and elution/washing conditions. Ta.

[Problem that the invention seeks to solve]

An object of the present invention is to overcome the drawbacks of the conventional adsorption carriers for chromatography and to provide an adsorbent i-form for chromatography that has all the above-mentioned properties desired in an adsorption carrier for affinity chromatography. be.

[Means for solving problems]

The present invention provides an adsorption carrier for chromatography that can achieve the above objects.

That is, the present invention comprises (A) glycysol monovinyl ester or glycidyl monovinyl ether and (B) alkylene glycol divinyl ester as main components;
A delta copolymer whose component is crosslinked with component (B) (hereinafter referred to as
A porous copolymer containing D-alanyl-D-alanine covalently bonded to an epoxy group via a bonding group based on the component (A) of the delta-shaped copolymer according to the present invention. The present invention relates to an adsorption carrier for chromatography characterized by the following.

The adsorption carrier for chromatography of the present invention will be explained below.

The delta copolymer according to the present invention is disclosed in Japanese Patent Application Laid-Open No. 60-1042.
As described in Publication No. 56 etc., for example, (A)
Glycidyl monovinyl ester or glycidyl monovinyl ether,! = (B) It can be prepared by aqueous suspension polymerization of alkylene glycol divinyl ester in the presence of a water-insoluble organic diluent to obtain a porous del.

Since the components (A) and (B) are sparingly soluble in water, they can be copolymerized by an aqueous suspension polymerization method, and the aqueous suspension polymerization can be carried out by a simple oil-in-water suspension method. Copolymerization is carried out in the presence of an organic diluent, and due to the presence of this organic diluent, the alkylene glycol divinyl ester component (B) acts as the main crosslinking component, and the glycidyl monovinyl ester component (A) or Most of the epoxy groups in the glycidyl monovinyl ether component remain in the produced copolymer without ring opening, and are also useful for adjusting the pore size of the produced porous del.

As the component (A), glycidyl ester of monovinylcarbic acid having 3 to 12 carbon atoms or 3 to 12 carbon atoms
Glycidyl ether of monovinyl alcohol is used. Among these, those having a small number of carbon atoms are particularly preferably used, and examples thereof include glycidyl methacrylate, glycidyl acrylate, allyl glycidyl ether, and the like.

On the other hand, as the component (B) which acts as a crosslinking component, an alkylene glycol having 2 or 3 carbon atoms or an ester of the polyalkylene glycol with acrylic acid or methacrylic acid is preferably used.

Examples include alkylene glycol divinyl esters such as ethylene glycol diacrylate, ethylene glycol dimethacrylate, propylene glycol diacrylate, propylene glycol diacrylate, and polyalkylene glycol novinyl esters such as polyethylene glycol dimethacrylate and polypropylene glycol dimethacrylate. .

The ratio of component (A) to component (B) is 10 to 90 moles for the former and 90 to 10 mole for the latter. Preferably f'
t (A) component 40-80 mole, (B) component 60-2
It is 0 moles. Up to half of component (A) can be replaced by a comonomer, ie, a lower vinyl ester such as methyl methacrylate, methyl acrylate, or vinyl acetate. Of course, the greater the proportion of component (A), the greater the content of epoxy groups in the copolymer. The higher the proportion of component (B), the higher the degree of crosslinking of the copolymer, and therefore the denser the network structure, the lower the degree of swelling, and the harder the copolymer becomes. If the ratio of both components is outside the above range, it will not be possible to obtain a dull copolymer that fully satisfies the desired characteristics and properties.

The organic diluent used may be one that is inert to the polymerization reaction, insoluble or sparingly soluble in water, but capable of dissolving the raw material monomer. The amount used is at least 30% by volume based on the content of monomer components and organic diluent, preferably Fi40~
The larger the volume dose (80 volume), the more suppressed is the opening of the epoxy group during the polymerization process. Generally speaking, use (
About 60 to 95 epoxy groups contained in the charged amount of component A) can be easily made to remain in the produced polymer.

It is believed that the remainder is hydrolyzed during the polymerization process and a portion is used for crosslinking.

As mentioned above, the content of epoxy groups in the product colander can be adjusted by the amount of component (A) used, i.e., the ratio to component (B), or by partial substitution with comonomers, and also by the amount of organic diluent. The amount of epoxy oxygen, expressed as epoxy oxygen, is preferably present in the resulting polymer at a maximum of about 10% by weight.

As mentioned above, the use of organic diluents during aqueous suspension polymerization also helps control the pore size of the resulting porous coll. When an organic diluent with low swelling property is used for the resulting gel,
Phase separation occurs within the suspended particles during the polymerization process, and as a result, a gel having a so-called giant pore structure with large pores can be obtained. On the other hand, when a highly swelling diluent is used, the polymerization proceeds with the product in a swollen state during the polymerization process, and phase separation is unlikely to occur, resulting in a polymer with relatively small pores formed by swelling. . The degree of swelling can be determined based on the solubility/grammeter of the organic diluent.

Examples of organic diluents that can be suitably used in the present invention include:
Listed in descending order of swelling with respect to the produced dal:
Cyclohexanone, chlorobenzene, benzene, toluene, ronorovir acetate, n-butyl acetate,
n-octane, etc.

The aqueous suspension polymerization can be carried out in the presence of a free-radical generating catalyst according to conventional methods known per se.

The amount of the aqueous phase is approximately the same volume or more based on the amount of the organic phase, and is used in an amount of about 10 times the volume, although there is no particular restriction.

The introduction of the bonding group into the glue-like copolymer according to the present invention includes:
This can be carried out by utilizing the reactivity of the epoxy group based on the component (A) of the glue-like copolymer according to the present invention, but in this case, the bonding group can be covalently bonded to D-alanyl-D-alanine. Although it is necessary to have a functional group, there is no particular restriction on the structure of the bonding group other than having the functional group.

Examples of the functional group capable of covalently bonding with the D-alanyl-D-alanine include an epoxy group, a carboxyl group, and a formyl group. In this case, the bonding group can be easily introduced using a known method, and it does not necessarily have to be introduced in one reaction step, but can also be introduced in two or more reaction steps. . A specific example of introducing a bonding group will be described below.

(1) Introduction of a bonding group having an epoxy group as a functional group For example, the epoxy group based on the component (4) of the gel-like copolymer according to the present invention is generated by ring-opening modification with a hydroxyl group-containing compound such as water. A compound having a glycidyl group is bonded to a hydroxyl group to introduce a bonding group having an epoxy group based on a glycidyl group. - (Hereinafter, a polymer into which a glycidyl group has been introduced in this way will be referred to as a modified gel copolymer according to the present invention.) Ring-opening modification is carried out in the presence of an acid or alkali,
For example, when carrying out in the presence of an acid, these copolymers "f" are prepared in water using sulfuric acid or hydrochloric acid as a catalyst at 70°C to 70°C.
This is done by heating to 100° C. for about 2 hours. The concentration of acid is preferably 0.1N to 0.5N. Ring-opening modification is performed almost quantitatively.

Compounds having a glycidyl group that are bonded to the hydroxyl group produced by ring-opening modification include epihalohydrins such as epichlorohydrin and epibromohydrin, ethylene glycol diglycinole ether,
Diglycidyl ethers such as 1.4-butanediol tuclycidyl ether and polyethylene glycol diglycidyl ether, 1,3-butadiene diepoxide,
Examples include alkyl diene diepoxides such as 1,7-octazidine diepoxide.

In order to react with a compound having a glycityl group, for example, a ring-opening modified delta copolymer is reacted with epichlorohydrin in an aqueous solution of an inorganic base such as sodium hydroxide or potassium carbonate, or a hydrophilic copolymer is reacted with epichlorohydrin. Hydroxide containing sodium borohydride in the polymer) Reaction with ethylene glycol or 1,4-butanediol tuclycidyl ether, or polyethylene glycol diglycidyl ether in an aqueous solution of an inorganic base such as sodium borohydride or potassium carbonate, etc. It will be done as follows. Repeating unit of polyethylene glycol diglycidyl ether used -C
It is preferable to use one having a repeating number of 2H40- of 1 to 10.

(11) Introduction of a bonding group having a carboxyl group as a functional group For example, as a method for introducing a bonding group having a carboxyl group, an epoxy group based on component (A) of the dull copolymer according to the present invention or an epoxy group based on the component (A) of the dull copolymer according to the present invention The general formula (1) (wherein R' represents a hydrogen atom, an amine group, an ω-aminoalkyl group, or an ω-cargoxyalkyl group) is added to the epoxy group based on the glycidyl group of the modified glycidyl copolymer. When the amine compound represented by R' is reacted with an amine compound other than the ω-carbyxyalkyl group, alkylenecarbic acid anhydride is further reacted with the epoxy group or glycidyl group. , general formula (11) (wherein R represents an ω-carbyxyalkyl group, an ω-cargexyalkanoyl group, an ω-cargexyalkanoylamino group, or an ω-carbaxyalkanoylaminoalkyl group.) There is a method of introducing a bonding group represented by

In this case, the reaction can be carried out according to the following reaction formula.

represents one epoxy group or glycidyl group in the gel-like copolymer according to the invention or the modified rcopolymer according to the invention. In the formula, m is 1 or 2, and n1plq and r are each an integer of 1 or more (more preferably 1 to 12).

A compound of the reaction formula (D), general formula 〇)') or general formula (F) is added to the epoxy group of the gel copolymer according to the present invention or the glycidyl group of the modified gel copolymer according to the present invention. The reaction can be carried out in the presence of a solvent, but if these compounds are liquid under the reaction conditions, a solvent may not be used.

As a solvent, water is usually used, but other solvents include n-hexane, benzene, carbonized xylenato-1, ethers such as tetrahydrofuran and dioxa 7, chloroform, chlorinated hydrocarbons such as chlorobenzenato, ethanol and methylseron. In addition to alcohols such as alcohol, ketones such as acetone and methyl isobutyl ketone, dimethylformamide and dimethyl sulfoxide are also used, and these solvents may be used alone or in a mixed solvent system. Although no particular catalyst may be used, hydroxides or carbonates of alkali or alkaline earth metals such as sodium hydroxide and potassium carbonate may also be used.

Examples of the compound of general formula (Iy) include ethylenediamine,
1,3-diaminoglonogne, 1,4-diaminobutane, 1,5-diaminopentane, 1,6-diamithexane, 1,7-diaminohebutane, 1,8-diaminoctane, 1,9-diaminononane , 1,10-diaminodecane, 1,12-diaminododecane, and other diaminoalkanes.

Examples of the compound of general formula (F) include aminocaproic acids such as glycine, β-alanine, 4-aminobutyric acid, 6-aminocaproic acid, and 8-amitsukazolylic acid.

The reaction conditions are not necessarily limited, but it is generally preferable to select the following conditions to carry out the reaction.

Gel copolymer according to the present invention or modified polymer according to the present invention
Polymer t(a) of the cylindrical copolymer, formula (D), and general formula (D')
or the ratio of the weight (b) of the compound of general formula (F) 2a:b=
1:0.3-10 More preferably, a:b=1:0.3-3 Reaction degree 20-150°C, more preferably room temperature-100
℃.

Reaction time: 1 to 60 hours, more preferably 1 to 30 hours.

The reaction pressure is normal pressure to 10 atm, preferably normal pressure.

There are no special requirements for post-treatment after the reaction, and it can be carried out as appropriate by commonly used methods such as door-to-door cleaning and washing.

The chromatography carrier according to the present invention obtained above is prepared by using the compound of formula (D) or general formula (D'), and then, if necessary, an active support group as already shown in the general formula. The terminal amide 7 group can be converted into an active support group having a cargexyl group by reacting with an acid anhydride of general formula (E). Water is usually used as a solvent for the reaction, but ethers such as tetrahydrofuran and dioxane, carbic acids such as acetic acid, and pyridino may also be used. In addition, although it is not necessary to use a catalyst, the addition of an acid such as hydrochloric acid or sulfuric acid, or an alkali such as hydroxide or potassium carbonate can cause the reaction solution to become -
can also be adjusted.

In the reaction formula, general formula (E) or general formula (E) alkylene carbic anhydrides include succinic anhydride, geltaric anhydride, adipic anhydride, pimelic anhydride, speric anhydride, and azelaic anhydride. Examples include cepatic acid anhydride, cepatic acid anhydride, and the like.

Although there are no particular restrictions on the reaction conditions, it is generally preferable to select the following conditions to carry out the reaction.

The weight (a') of the aminated product of the kelp-like copolymer according to the present invention or the modified glue-like copolymer according to the present invention and the general formula (E
) or the weight of the compound of general formula (H) (ratio of b: a'
: b'-=1 : 0.3-10 more preferably, a' : b' = 1 : 0.3-3 reaction degree 20-150°C, more preferably room temperature-100°C, reaction time: 1 -60 hours, more preferably 1 to 30 hours, reaction pressure - normal pressure - 10 atm, more preferably normal pressure.

There are no special requirements for post-treatment after the reaction, and it can be carried out as appropriate by commonly used methods such as door-to-door cleaning and washing.

Another method for introducing a bonding group having a carpixyl group as a functional group is to introduce an amine calgenic acid having a hydroxyl group into an epoxy group based on the component (A) of the dull copolymer according to the present invention or a modified method according to the present invention. There is a method of binding by acting on the glycidyl groups of a glycidyl copolymer.

Examples of aminocardigenic acid having a hydroxyl group include serine,
homoserine, threonine/, 4-hydroxyzololine, 4
Examples include amino acid derivatives such as -amino-3-hydroxybutyric acid, N-tris(hydroxymethyl), and methylglycine, and amino sugar derivatives such as glucosaminic acid.

The bonding state is such that the epoxy group of the glycidyl copolymer according to the present invention or the tuclicidyl group of the modified glycidyl copolymer according to the present invention is bonded to the amino group of the amine calgenic acid having a hydroxyl group, so that the epoxy group or the glycidyl group The group opens to form a hydroxyl group and a carbyxyl residue having a hydroxyl group.

The action of the amine carbic acid having a hydroxyl group can be carried out in the presence of a solvent.

Water is usually used as a solvent, but other hydrocarbons such as rhohexane, benzene, and xylene, nitrogen compounds such as tetrahydrofuran and dioxane, chloroform, chlorinated hydrocarbons such as chloropenzene, ethanol, and methyl cellosolve are also used as a solvent. In addition to alcohols such as , ketones such as acetone and methyl isobutyl ketone, trimethylformamide and dimethyl sulfoxide are also used, and these may be used alone or in a mixed solvent system.

Although no particular catalyst may be used, hydroxides or carbonates of alkali or alkaline earth metals such as sodium hydroxide and potassium carbonate may also be used.

There are no particular limitations on the reaction conditions, and for example, the same conditions as in the case of reacting the compound of general formula (1) above can be used.

When a bonding group is introduced using an aminocarboxylic acid [- having such a hydroxyl group, non-specific adsorption originating from hydrophobicity based on a portion other than the base material, that is, a portion of the bonding group can also be eliminated.

(Mountain) Introduction of a bonding group having a formyl group as a functional group For example, the bonding group having an epoxy group as a functional group obtained by the above (1) can be easily hydrolyzed into 1
, 2-glycol, and this 1.2-
Glycol can be converted into a diformyl group by the action of periodic acid. There are no particular restrictions on the reaction conditions, and the reaction can be carried out according to a conventional method using, for example, water, ethanol, acetic acid, or a mixture thereof as a solvent at room temperature to 50° C. by reacting periodic acid or an inorganic salt thereof. .

When D-alanyl-D-alanine is covalently bonded to the bonding group, it can be carried out in an appropriate solvent using an appropriate catalyst or reaction reagent, if necessary depending on the functional group that the bonding group has. For example, as a catalyst, an acid or alkali such as hydrochloric acid, sodium carbonate or sodium bicarbonate is mainly used when the functional group is an epoxy group, and as a reaction reagent, for example, N-hydroxysuccinimide,
dicyclohexylcarbodiimide or 1-ethyl-3-
Examples include condensing agents such as (3-nomethylaminoglovir) carbodiimide used when the functional group is a carbyxyl group, reducing agents such as sodium cyanoborohydride when the functional group is a formyl group, etc. You can. Water is usually used as a solvent, but if necessary, it can also be used as a phosphoric acid or acetate buffer, or an inorganic salt such as sodium chloride can be added thereto.

The reaction conditions with D-alanyl-D-alanine are not necessarily limited, but it is generally appropriate to carry out the reaction within the following range.

Binding Fund Weight ratio of the glue-like copolymer or modified glue-like copolymer according to the present invention bound to D-alanyl-〇-alanine: 1:0.03 to 0.3, preferably 1:0.05 ~
0.2; Reaction temperature: 0°C to 80°C, preferably 4°C to 6°C
0°C; reaction time: 1 to 72 hours, preferably 2 to 12 hours.

There are no special requirements for post-treatment after the reaction, and it can be carried out as appropriate by commonly used methods such as door-to-door cleaning and washing.

〔Effect of the invention〕

The adsorption carrier for chromatography provided by the present invention uses a base having all the properties desired as an adsorption carrier for affinity chromatography, and connects D-alanyl-D- Since alanine is covalently bonded, the adsorption carrier of the present invention can not only be used as a packing material for affinity chromatography in the same way as conventional adsorption carriers using soft dulls, but also particularly suitable for pressure-resistant columns. The biggest advantage is that it can be filled with water and used even under pressure, which greatly shortens the working time and therefore greatly improves the efficiency of separation and purification. Needless to say, this is indispensable for applications such as high performance liquid chromatography and industrial separation and purification equipment.

In particular, for the glue-like copolymer which has many advantages as mentioned above, adsorption carriers to which 9D-alanyl-D-alanine is covalently bonded via the bonding group as mentioned above, for example. Compared to commonly used adsorption carriers for affinity chromatography using cyanogen bromide, the ligand does not fall off during use, and the length of the bonding group can be adjusted arbitrarily. The adsorption carrier for chromatography provided by the present invention is highly effective, as it has excellent adsorption ability for the target substance and less non-specific adsorption.

〔Example〕

Typical examples of the method for producing the adsorption carrier for chromatography of the present invention will be shown below and explained in more detail.

However, these are merely examples for explanation, and it goes without saying that the present invention is not limited thereto.

Example 1 An epoxy group-containing glycidyl copolymer obtained from glycidyl methacrylate and ethylene glycol dimethacrylate was ring-opened and modified with water, and further epoxy groups were introduced with 1,4-butanediol diglycidyl ether. To a suspension of 600 rn9 (epoxy group: 0.3 mmol per Ig of dry glue) in 4.5 ml of water was added 140 m9 of D-alanyl-D-alanine and mixed with a 4 molar aqueous sodium hydroxide solution (p)! The temperature was adjusted to 8.5 and shaken at room temperature for 1 day. The entire dal was collected and washed with a 1M aqueous sodium chloride solution and water. The adsorption carrier obtained in this way contains 0.00% of D-alanyl-D-alanine per dry glue II
It was confirmed that 6 mmol of unreacted D-alanyl + D-alanyl was supported by high performance liquid chromatography.

Example 2 The same procedure as in Example 1 was carried out using shrimp chlorhydroline in place of 1,4-buta-7-ol-glycidyl ether, and 9D-alanyl- An adsorption carrier carrying 0.12 mmol of D-alanine was obtained.

Example 3 An epoxy group-containing glu-like copolymer obtained from glycidyl methacrylate and ethylene glycol dimethacrylate was subjected to ring-opening modification of the epoxy group with water, and then the epoxy group was introduced with ethylene glycol diglycinol ether, and then further 4 - After thoroughly washing 450 m9 of glue into which a carboxyl group was introduced with aminobutyric acid with anhydrous dioxane, 74 m7 of anhydrous dioxane! In addition, 80 ml of N-hydroxysuccinimide and 144 ml of dicyclohexylcaledodiimide were added, and after shaking at room temperature for 2 hours, the glue was removed, and 720 ml of anhydrous dioxa, 5 ml of methanol,
It was quickly washed with 3 ml of cold water. This glue was added to a solution of D-alanyl-D-alanine 70rn9 in 0.1 molar sodium bicarbonate in 2rnl of water and shaken overnight at room temperature. The glue was then washed with a 1 molar aqueous sodium chloride solution and water. The adsorption carrier thus obtained contained 0.03 D-alanyl-D-alanine per dry dal II.
It was confirmed that mmol was supported.

Example 4 Epoxy group-containing glue copolymer obtained from glycidyl methacrylate and ethylene glycol dimethacrylate 1. 4 ml of concentrated ammonia water was added to the OI, and the mixture was shaken at 40°C for 5 hours. FJ Dell! ! After washing with 2 L water and drying, add 1.0 g of succinic anhydride to 5 rnt of 0.1 N sodium chloride water.
The mixture was added to the solution at room temperature, and after adjusting the - to 6, it was shaken at room temperature for one day. 0.1N sodium bicarbonate,
It was washed successively with 0.1N hydrochloric acid and water and dried. This product weighs 1g (dry) and contains 0.0g of carboxyl group. It was confirmed by titration with 0.1 N sodium hydroxide that it contained 0.1 mmol. Next, to this glue 600rn9' was added D-alanyl-D-alanine 1641n9 to 0.1M sodium chloride LO ml while keeping it at 0°C to adjust - to 7.0, and then l-ethyl-3-(3-zo methylaminoglovir) cal-diimide hydrochloride 1651n9'i plus 3
Shake for hours. Then, after being left at 4° C. overnight, the glue was washed with 0.1N sodium chloride. The adsorption carrier obtained in this way is dried glue 1. ! D-alanyl-D- per i+
It was confirmed that 0.02 mmol of alanine was supported.

Example 5 By carrying out the same operation as in Example 4 using 1,3-diaminoglopane instead of concentrated ammonia water, D-alanyl-D-alanine' was obtained per gram of dry glue! z0.0
An adsorption carrier carrying 2 mmol was obtained.

Example 6 1.0 g of the epoxy group-introduced glue in Example 1
in acetic acid water (9:1) containing 1.0 g of sodium periodate.
) solution and shaken at room temperature for 2 hours. After washing the glass with water, it was added to 3 ml of an aqueous solution containing 0.03 mol acetic acid and 0.06 mol sodium acetate, and 0.0 ml of D-alanyl-D-alanine was added. Add IF and shake at room temperature for 8 hours. After cleaning the glass with a 1M aqueous sodium chloride solution and water, add 20m90 of sodium cyanoborohydride.
After adding a solution in 1 molar Tris-HCl buffer (pH 8,0) at 2 mA' and shaking for 1 hour at room temperature, the glue was washed several times with water and methanol again. It was confirmed that the adsorption carrier thus obtained supported 0.041 mmol of D-alanyl-D-alanine per 11 dry particles.

Test Example 1 The adsorption carrier obtained in Example 3 had an inner diameter of 4.6 mm and a length of 75 mm.
When an aqueous solution of vancomycin (manufactured by Sigma) was analyzed using a high performance liquid chromatography device, the results shown in FIG. 1 were obtained.

Analysis conditions Eluent: 0.2M aqueous acetic acid solution (hereinafter referred to as eluent A)
, elution rate: 0.5 m 11 min, detector: ultraviolet spectrophotometer (280 nm).

Test Example 2 When a similar test was conducted in Test Example 1 using pure water instead of eluent A, vancomycin was not eluted (FIG. 2). Then, when the eluent was switched from pure water to eluent A (as indicated by the arrow in FIG. 2), purified vancomycin was eluted.

Test Example 3 When a similar test was conducted in Test Example 1 using ristocetin (manufactured by Sigma) instead of vancomycin, the results shown in FIG. 3 were obtained.

Test Example 4 When a similar test was conducted in Test Example 2 using ristocetin instead of vancomycin, the results shown in FIG. 4 were obtained.

[Brief explanation of drawings]

1 to 4 are chromatogram diagrams showing separation adsorption or elution patterns using a high performance liquid chromatography column using the chromatography adsorption carrier according to the present invention.

Claims (1)

[Claims] Epoxy based on component (A) of a gel-like copolymer containing (A) glycidyl monovinyl ester or glycidyl monovinyl ether and (B) alkylene glycol divinyl ester as main components, and component (A) crosslinked with component (B). D covalently bonded to the group via a bonding group
- An adsorption carrier for chromatography, comprising a porous copolymer containing alanyl-D-alanine.