JPH01107150A - Adsorbent carrier for chromatography - Google Patents

Abstract

PURPOSE:To enable use of an adsorbent carrier for a packing agent in affinity- chromatography, by composing the adsorbent carrier by glycidyl monovinyl ester of glycidyl monovinyl ether (A), and alkyleneglycol divinyl ester as main components (B). CONSTITUTION:An adsorbent carrier comprises a gelled copolymer modified with hydroxyl group which is obtained by bonding a compound having hydroxyl group with epoxy group based on component (A) of a gelled copolymer in which component (A) is cross-linked with component (B). Moreover, the adsorbent carrier comprises a porous copolymer containing phenylalanin or its ester bonded to the gelled copolymer modified with hydroxyl group through a bonding group by covalent bonding. The above copolymerization is carried out in the existence of organic diluent. Because of the presence of this diluent, component (B) works as the main cross-linking component and accordingly most of the epoxy group in component (A) is remained in the produced copolymer, without ring-opening. At the same time, the diameter of a porosity of the produced porous gel can be adjusted. Thus, the adsorbent carrier is utilizable as a packing agent for affinity-chromatography.

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.

Affinity chromatography, one of the conventional chromatography techniques, uses the affinity of pairs of substances that specifically interact with each other to separate and purify biological substances. It is useful in identifying and purifying a property, ie, a specific chemical structure on a molecule.

An adsorption carrier for affinity chromatography (affinity gel) is, for example, an active support obtained by bonding a binding group (spacer) to an insoluble carrier (matrix), and a ligand is bonded to the spacer. An adsorption operation is performed by selecting a combination of substances that interact with each other.

Phenylalanine, one of the ligands, is one of the essential amino acids and is a precursor of alkaloids, flavonoids, etc. in plants, and is converted to tyrosine by phenylalanine monooxygenase in animals. On the other hand, alkaliphilic proteases such as subtilisin are known to recognize phenylalanine residues in proteins. Taking advantage of these properties, affinity chromatography using phenylalanine as a ligand is considered to provide a particularly useful means for separating and purifying and analyzing enzyme-based substances involved in these conversions.

For example, in separation, purification, and analysis by affinity chromatography, in which phenylalanine or its ester is used as a ligand and a protein with affinity for these is used as the adsorption target substance, the active support that is the main component of the adsorption carrier for affinity chromatography is used. The desired properties for the body include low nonspecific adsorption, high porosity, easy binding of ligands and large immobilization capacity, chemical stability, pi range, salt concentration, and temperature. These characteristics include being sufficiently stable and not changing in volume under a wide range of conditions, having sufficient mechanical strength and stability, and having good flow properties, and being resistant to biological contamination.

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

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.

In addition, adsorption carriers using styrene polymers or acrylate polymers as base materials have recently been developed, but these have not been satisfactory due to the problem of nonspecific adsorption.

Problems to be Solved by the Invention The object of the present invention is to overcome the drawbacks of the conventional adsorption carriers for chromatography, and to provide a talomatography material that has all the above-mentioned properties desired in an adsorption carrier for affinity chromatography. An object of the present invention is to provide an adsorption carrier for

Means for Solving the Problems The present invention provides an adsorption carrier for chromatography that can achieve the above objects. - That is, the present invention provides (A)
A gel copolymer containing glycidyl monovinyl ester or glycidyl monovinyl ether and (B) alkylene glycol divinyl ester as the main components, and in which the component (A) is crosslinked with the component (B) (hereinafter referred to as the gel copolymer according to the present invention). A hydroxyl-modified gel-like copolymer in which a compound having a hydroxyl group is bonded to an epoxy group based on the component (A) (referred to as "coalescence"), which is further covalently bonded to the hydroxyl-modified gel-like copolymer via a bonding group. The present invention relates to an adsorption carrier for chromatography characterized by being made of a porous copolymer containing bound phenylalanine or its ester.

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

The gel-like copolymer according to the present invention is disclosed in Japanese Patent Application Laid-Open No. 80-1042.
As described in Publication No. 56 etc., for example, (A)
It can be prepared by aqueous suspension polymerization of glycidyl monovinyl ester or glycidyl monovinyl ether and (B) alkylene glycol divinyl ester in the presence of a water-insoluble organic diluent to obtain a porous gel. Turbid polymerization can be carried out using a simple oil-in-water suspension system. 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 resulting copolymer without ring opening, and are also useful for adjusting the pore size of the resulting porous gel.

The component (A) is glycidyl ester of monovinylcarboxylic 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, preferably used is an alkylene glycol having 2 or 3 carbon atoms, a hydroxyl-substituted alkylene glycol having 3 carbon atoms, or an ester of the polyalkylene glycol with acrylic acid or methacrylic acid. Examples include alkylene glycol or hydroxyl substituted alkylene glycol divinyl esters such as ethylene glycol diacrylate, ethylene glycol dimethacrylate, propylene glycol diacrylate, propylene glycol dimethacrylate, glycerol diacrylate, glycerol dimethacrylate and polyethylene glycol dimethacrylate, polypropylene glycol dimethacrylate. Examples include polyalkylene glycol divinyl esters such as.

The ratio of component (A) to component (B) is 10 to 90 mol% for the former and 90 to 10 mol% for the latter. Preferably (A
) component 40 to 80 mol%, component (B) 60 to 20 mol%
It is.

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.

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 40-8% by volume.
0% by volume. Examples of organic diluents that can be suitably used in the present invention include cyclohexanone, chlorobenzene,
These include benzene, toluene, n-propyl acetate, n-butyl acetate, n-octane, and the like.

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 not particularly limited, but may be used in an amount up to about 10 times the volume.

Bonding of a compound having a hydroxyl group to the gel-like copolymer according to the present invention can be carried out by utilizing the reactivity of the epoxy group based on the component (A) of the gel-like copolymer according to the present invention. .

Examples of compounds having a hydroxyl group include polyols such as glycerin, erythritol, pentaerythritol, inositol, xylitol, annitol, and sorbitol, epoxy compounds such as glycidol, glyceraldehyde, erythrose, xylose, arabinose,
Compounds with aldehyde groups such as libons, glucose, galactose, annose, fructose, etc.
Examples include amino alcohols such as trimethylol aminomethane, glucamine, glucosamine, galactosamine, fucosamine, and the like.

When bonding these hydroxyl group-containing compounds to the gel-like copolymer according to the present invention, it can be carried out in an appropriate solvent using an appropriate catalyst or reaction reagent, if necessary. As the catalyst, for example, acids such as hydrochloric acid and sulfuric acid, and alkalis such as sodium hydroxide, potassium hydroxide, sodium carbonate, and potassium carbonate are mainly used. In particular, for compounds having aldehyde groups, the epoxy groups of the gel copolymer according to the present invention are ring-opened using diamines such as ammonia or propanediamine, and then a reducing agent such as sodium cyanoborohydride is used. can be combined in the presence of

Further, in the case of a compound having an epoxy group, the ring can be opened with water after bonding to form a hydroxyl group. Water, dimethylformamide, dioxane, etc. are usually used as a bonding solvent, but they can also be used as a buffer solution or inorganic salts can be added, if necessary.

The compound having a gel copolymer and a hydroxyl group according to the present invention has, for example, an ff1lik ratio of 1:0.03 to 1, preferably 0605 to 0.5, a reaction temperature of 0°C to 150°C, preferably room temperature to 80°C, The binding can be carried out under conditions where the reaction time is 1 to 72 hours, preferably 2 to 12 hours.

Regarding the gel-like copolymer according to the present invention bound to a compound having a hydroxyl group (hereinafter referred to as the hydroxyl-modified gel-like copolymer according to the present invention), in order to further introduce a bonding group, the bonding group may be phenylalanine. However, there is no particular restriction on the structure of the bonding group other than having the above-mentioned functional group. Examples of the functional group capable of covalently bonding with the phenylalanine or its ester include an epoxy group, an amino group, a hydrazino group, a carboxyl group, a formyl group, and a thiol group. In this case, the bonding group can be easily introduced using a known method, and
It is not necessarily necessary to introduce it in one reaction step, but it 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, by bonding a compound having a glycidyl group to the hydroxyl group of the hydroxyl group-modified gel copolymer according to the present invention,
Introducing a bonding group having an epoxy group based on a glycidyl group (hereinafter referred to as the epoxy-modified gel copolymer according to the present invention). Compounds having a glycidyl group that are bonded to a hydroxyl group include shrimp chlorohydrin, shrimp Shrimp halohydrins such as bromohydrin, diglycidyl ethers such as ethylene glycol diglycidyl ether, ■,4-butanediol diglycidyl ether, polyethylene glycol diglycidyl ether, l,3-butadiene diepoxide, 1,7-octa Examples include alkyl diene diepoxides such as diene diepoxide.

In order to act with a compound having a glycidyl group, for example, a hydroxyl group-modified gel copolymer is reacted with shrimp chlorohydrin in an aqueous solution of an inorganic base such as sodium hydroxide or potassium carbonate, or a compound containing sodium borohydride is used. This is carried out by reacting with ethylene glycol, 1,4-butanediol diglycidyl ether, or polyethylene glycol diglycidyl ether in an aqueous solution of an inorganic base such as sodium hydroxide or potassium carbonate. The repeating unit -C2H,O- of the polyethylene glycol diglycidyl ether used is 1
It is preferable to use 10 to 10.

(11) Introduction of a bonding group having an amino group, hydrazino group, carboxyl group, etc. as a functional group For example, as a method for introducing a bonding group having an amino group, hydrazino group, carboxyl group, etc. General formula (1) [
In the formula, R' represents a hydrogen atom, an amino group, an ω-aminoalkyl group or an ω-carboxyalkyl group. ] The above epoxy group is reacted with an amino compound represented by the above, or when an amino compound other than the ω-carboxyalkyl group is reacted with R' as the amino compound, an alkylenecarboxylic anhydride is further reacted with the epoxy group. General formula (
II) NHR [wherein R is a hydrogen atom, an amino group, an ω-aminoalkyl group, an ω-carboxyalkyl group, an ω-carboxyalkanoyl group, an ω-carboxyalkanoylamino group, or an ω
- represents a carboxyalkanoylaminoalkyl 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.

In addition, in the following reaction formula, (base+CH-CH2 represents \ 1 the epoxy-modified gel copolymer according to the present invention, -
CH-CH2 shall represent one \ 1 epoxy group in these copolymers. In the formula, m is 1 or 2
, n+1), q and r are each an integer of 1 or more (more preferably 1 to 12).

(The following is a blank space) / \ 0H 0 (E) / \ A compound of the above reaction formula (D), general formula (Do) or general formula (P) is added to the epoxy group of the epoxy-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 hydrocarbons such as n-hexane, benzene, and xylene, ethers such as tetrahydrofuran and dioxane, chlorinated hydrocarbons such as chloroform and chlorobenzene, alcohols such as ethanol and methyl cellosolve, and acetone In addition to ketones such as and methyl isobutyl ketone, dimethyl formamide 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.

Examples of the compound of general formula (Do) include ethylenediamine,
1.3-diaminopropane, ■,4-diaminobutane,
■, 5-diaminopencune, l,8-diaminohexane, 1,7-diaminohexane, l,8-diaminooctane, 1,9-diaminononane, 1,10-diaminodecane, 1,12-diaminododecane, etc. Examples include diaminoalkanes.

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

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

Weight (a) of the epoxy-modified gel copolymer according to the present invention
and the weight (b) of the compound of formula (D), general formula (Do) or general formula (12): a:b-1:0.3-1G More preferably, a:b-1:0. 3-3 Reaction temperature 20-150°C, more preferably room temperature-100°C
°C, reaction time = 1 to 60 hours, more preferably 1 to 30 hours, reaction pressure normal pressure to IOaLm, more preferably normal pressure.

There are no special requirements for post-treatment after the reaction;
Washing and the like are carried out as appropriate using commonly used methods.

The chromatography carrier according to the present invention obtained above can be prepared by adding the necessary active support group (if any) to the compound of formula (D) or general formula (Do). The terminal amino group of can be converted into an active supporting group having a carboxyl group by reacting with the acid anhydride of the general formula (E).Water is usually used as a solvent for the reaction, but other solvents such as tetrahydrofuran and dioxane can be used. Ethers such as ethers, carboxylic acids such as acetic acid, pyridine, etc. are also used.Although there is no need to use a catalyst, the reaction can be carried out by adding acids such as hydrochloric acid, sulfuric acid, or alkalis such as sodium hydroxide or potassium carbonate. pH of liquid
can also be adjusted.

In the reaction formula, the alkylenecarboxylic anhydride of general formula (E) or general formula (E゛) includes succinic anhydride, geltaric anhydride, adipic anhydride, pimelic anhydride, speric anhydride, and azelain. Examples include acid anhydrides and cepatic anhydride.

The reaction conditions are not necessarily limited, but the reaction can be carried out by selecting, for example, the same conditions as for the compounds of formula (D), general formula (Do) or general formula (F) above.

Another method for introducing a bonding group having a carboxyl group as a functional group is a method in which an aminocarboxylic acid having a hydroxyl group is made to act on the epoxy group of the epoxy-modified gel copolymer according to the present invention and bonded thereto.

Examples of aminocarboxylic acids having a hydroxyl group include serine, homoserine, threonine, 4-hydroxyproline, 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 epoxy-modified gel copolymer according to the present invention is bonded to the amino group of the aminocarboxylic acid having a hydroxyl group, and as a result, the epoxy group is ring-opened, and a hydroxyl group and a carboxyl residue having a hydroxyl group are formed. will be generated.

There are no particular restrictions on the solvent, catalyst, and reaction conditions in which the aminocarboxylic acid having a hydroxyl group is reacted; It can be made into a condition.

When a bonding group is introduced using an aminocarboxylic acid having such a hydroxyl group, nonspecific 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.

(iii) 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 in the above (i) can be easily hydrolyzed into 1
．． It can be modified to 2-glycol, and this 1.2-
Glycol can be converted into formyl 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. .

(1v) Introduction of a bonding group having a thiol group as a functional group For example, the bonding group having an epoxy group as a functional group obtained by the above (1) is prepared by treating the functional group with sodium thiosulfate and then adding hydrochloric acid. A thiol group can be introduced by, for example, allowing the reaction to occur.

There are no particular restrictions on the reaction conditions, and the reaction can be easily carried out according to a conventional method, for example, at room temperature in an aqueous solvent.

Examples of phenylalanine or ester thereof to be bonded to the bonding group include L-phenylalanine, D-phenylalanine, DL-phenylalanine, and lower alkyl esters thereof having 1 to 6 carbon atoms. When bonding to a group, for example, salts such as their hydrochloride and sodium salt, or those having a protecting group such as a carbobenzoxyl group or a butoxycarbonyl group can also be used.

When phenylalanine or its ester is covalently bonded to a 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 possessed by the bonding group.

For example, as catalysts, acids such as hydrochloric acid, sodium carbonate, or sodium bicarbonate, and alkalis are mainly used when the functional group is an epoxy group, and as reaction reagents, for example, N-hydroxysuccinimide and dicyclohexylcarbodiimide are used. When the functional group is a carboxyl group,
The condensing agent such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide has a carboxyl functional group,
In the case of an amino group or a hydrazino group, reducing agents such as sodium cyanoborohydride are used when the functional group is a formyl group, and so on. Water is usually used as a solvent, but if necessary, it can also be used as a phosphoric acid or acetate buffer, or inorganic salts such as sodium chloride or sodium sulfate can be added thereto.

The conditions for the reaction with phenylalanine or its ester are not necessarily limited, but it is generally appropriate to carry out the reaction within the following range.

Weight ratio of hydroxyl-modified gel copolymer with bonding groups to phenylalanine or its ester = 1: 0.0
3-0.3, preferably 1:0.05-0.2;
"Reaction temperature: 0°C to 100°C, preferably 4°C to 80°C
C; Reaction time: 1 to 72 hours, preferably 2 to 12 hours.

There are no special requirements for the post-treatment after the reaction, and it may be appropriately carried out by commonly used methods such as separate furnaces and washing.

Effects 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 as described above, and binds phenylalanine or its like via an appropriate bonding group to the base. Since the ester 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 adsorption carriers using conventional soft gels, but also particularly suitable for pressure-resistant columns. The biggest advantage is that it can be filled with water and used under pressure, which greatly reduces working time and greatly improves the efficiency of separation and purification. Needless to say, this is essential for applications such as high performance liquid chromatography and industrial separation and purification equipment.

In particular, while gel copolymers have many advantages as described above, adsorption carriers to which phenylalanine or its ester is covalently bonded via a bonding group as described above, for example, Compared to adsorption carriers for affinity chromatography using cyanogen oxide, the ligand does not fall off during use, and the length of the bonding group can be adjusted arbitrarily, making it possible to adsorb the ligand to the target substance. The adsorption carrier for chromatography provided by the present invention is highly effective, as it has excellent performance and very little non-specific adsorption.

EXAMPLES Below, typical examples of the method for producing the adsorption carrier for chromatography of the present invention will be shown 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.

Reference Example 1 12 g of an epoxy group-containing gel copolymer obtained from glycidyl methacrylate and ethylene glycol dimethacrylate was added to 72 g of aqueous ammonia (25%),
The mixture was stirred for 12 hours while warming to ℃. Next, the gel was taken and washed with water, and it was confirmed by neutralization titration that 1.4 mmol of amino groups were supported per gram of dry gel. This gel was added to 36 g of a 0.2 mol dipotassium phosphate aqueous solution, and 7.92 g of glucose and 7.2 g of sodium cyanoborohydride were added.
Stirred at ℃ for 10 hours. After taking the gel, washing it with water and drying it, it was added to 18 g of acetic anhydride, and 18 g of sodium acetate was added.
was added and stirred at room temperature for 2 hours. Next, the gel was taken out and washed with water. The gel thus obtained (hydroxyl group-modified gel copolymer) was packed into a stainless steel column with an inner diameter of 4.8 ml and a length of 75 mm, and the retention time ratio of butanol and ethylene glycol was determined using a high performance liquid chromatograph. When I searched for it, I got the following result.

Reference Example 2 Analysis was conducted using α-chymotrypsinogen A and ovalbumin in place of butanol and ethylene glycol in Reference Example 1, and the following results were obtained.

Elution conditions Eluent ■: 0.1M sodium phosphate buffer (pH 7
,,0) Eluent ■: Addition of 1.8 mol ammonium sulfate to eluent ■ Linear gradient elution rate from eluent ■ to eluent ■ in 60 minutes: 1. Oml/min, Detector: Ultraviolet spectrophotometer (280 nm) Reference example 3 4 g of an epoxy group-containing gel copolymer obtained from glycidyl methacrylate and ethylene glycol dimethacrylate was added to 12 ml of dimethylformamide, and further 15 ml of glycerol and powder were added. 1.5 g of potassium hydroxide was added and stirred at 60° C. for 3 hours.

Next, the gel was removed and washed with water, and then packed into the same column as in Reference Example 1. When this was used in place of (column A) in Reference Example 2 and the same analysis as in Reference Example 2 was conducted, α-chymotrypsinogen A and ovalbumin were not retained.

Example] An epoxy group-containing gel copolymer obtained from glycidyl methacrylate and ethylene glycol dimethacrylate was modified with hydroxyl groups using ammonia water and glucose in the same manner as in Reference Example 1, and further epoxy groups were introduced with shrimp chlorohydrin. gel (0.3 per gram of epoxy box dry gel)
5 mmol) 2 g of 5% sodium carbonate aqueous solution 6 ml
330 mg of L-phenylalanine was added to the suspension and shaken at 60°C for 4 hours. The gel was drained and washed with water. The adsorption carrier obtained in this way is L per 1 g of dry gel.
- It was confirmed by analyzing unreacted L-phenylalanine by high performance liquid chromatography that 0.11 mmol of phenylalanine was supported.

Example 2 The epoxy group-introduced gel obtained in Example 1 was subjected to 1.
2 g of gel into which carboxyl groups were introduced by reacting with 3-diaminopropane and then with succinic anhydride (carboxyl group: 0.22 mmol per 1 g of dry gel) was added to 6 ml of dimethylformamide, and 230 o+g of L-phenylalanine and dicyclo 412 mg of hexylcarbodiimide was added and stirred at 10°C for 2 hours. By taking the gel and washing it with methanol and water, an absorption carrier supporting 0.070 mmol of L-phenylalanine per gram of dry gel was obtained.

Example 3 By carrying out the same operation as in Example 1 using ethylene glycol diglycidyl ether instead of epichlorohydrin and using D-phenylalanine instead of L-phenylalanine, the yield per 1 g of dry gel was A absorber carrying 0.085 mmol of D-phenylalanine was obtained.

Example 4 An epoxy group-containing gel copolymer obtained from glycidyl methacrylate and ethylene glycol dimethacrylate was hydroxyl-modified using aqueous ammonia and glucose in the same manner as in Reference Example 1, and further modified with 1,4-butanediol diglycidyl ether. 2 g of the gel obtained by introducing a glycidyl group with 4-aminoacetic acid was thoroughly washed with anhydrous dioxane, then added to 6 ml of anhydrous dioxane, and further added with N
- Add 230 mH of hydroxysuccinimide and 412 mg of dicyclohexylcarbodiimide and
After shaking for an hour, remove the gel and add 20ml of anhydrous dioxane.
1. It was quickly washed with 6 ml of methanol and 6 ml of cold water in that order. Add this gel to 330 ll1g of L-phenylalanine.
in 4 ml of a 0.1 molar aqueous sodium carbonate solution, shaken for 2 hours at room temperature, and then left overnight at 4°C. After the gel was taken out and washed with water, it was added to 4 ml of 1M Tris-HCl buffer (pH 8,0) and shaken at room temperature for 1 hour, and then the gel was taken out again and washed with water. The gel thus obtained contains L-phenylalanine in O, Oa per gram of dry gel.
It was confirmed that mmol was supported.

Example 5 530 mg of N-t-BOC-L-phenylalanine and 6 ml of N-hydroxysuccinimide 230 a+g
Using 412 mg of dicyclohexylcarbodiimide in dimethylformamide, the mixture is stirred and reacted at 10° C. for 2 hours. After the reaction, 2 g of the gel obtained by treating the glycidyl group-introduced gel obtained in Example 4 with ammonia (amino group: 0.15 mmol per 1 g of dry gel) was added to the reaction solution, and an additional 5 g of gel was added to the reaction solution. The reaction was allowed to shake overnight at °C. After taking the gel and washing it with methanol and water, the gel is treated with dioxane in which hydrogen chloride is dissolved and further washed with water, so that L-phenylalanine per gram of dry gel is reduced to 0.
An adsorption carrier carrying 0.12 mmol was obtained.

Example 6 The epoxy group-introduced gel obtained in Example 3 was treated with 4
- Gel obtained by the action of amino-3-hydroxyacetic acid (
Carboxyl group; 0.18 mmol per 1 g of dry gel)
2g in 6ml of dioxane, 380mg of L-phenylalanine methyl ester and 412.mg of dicyclohexylcarbodiimide. mg @ was added and stirred at 10°C for 2 hours. An adsorption carrier supporting 0.02 mmol of 1-phenylalanine methyl ester per gram of dry gel was obtained by taking one portion of the gel and washing it with methanol and water.

Test Example The adsorption carrier obtained in Example 3 was prepared with an inner diameter of 4.6 and a length of 75.
When the alkaliphilic protease was analyzed using a high-performance liquid chromatography device packed in a stainless steel column (mm), the results shown in the figure were obtained.

Elution conditions Eluent ■: 0.01M sodium phosphate buffer (p
l+8.0) Eluent ■: Added 0.5M sodium sulfate to 0.01M sodium borate-hydrochloric acid buffer (pH 9,0) Elution rate: 0.5 ml/min Detector: Ultraviolet spectrophotometer (280 nm) The figure shows that components that were not adsorbed with eluent ■ are eluted with eluent ■. Diagonal lines indicate sections where enzyme activity was confirmed.

BRIEF DESCRIPTION OF THE DRAWINGS The figure is a chromatogram showing a separation and elution pattern using a high performance liquid chromatography column using the chromatography adsorption carrier according to the present invention.

Claims (1)

[Claims] Based on the above (A) component of a gel-like copolymer containing (A) glycidyl monovinyl ester or glycidyl monovinyl ether and (B) alkylene glycol divinyl ester as the main components, and the above (A) component being crosslinked with the (B) component. A porous hydroxyl-modified gel copolymer in which a compound having a hydroxyl group is bonded to an epoxy group, and further contains phenylalanine or its ester covalently bonded to the hydroxyl-modified gel copolymer via a bonding group. An adsorption carrier for chromatography, characterized in that it consists of a copolymer of