WO2022244890A1 - ボロン酸アフィニティクロマトグラフィー用分離剤担体、カラム及びそれを用いた測定方法 - Google Patents

ボロン酸アフィニティクロマトグラフィー用分離剤担体、カラム及びそれを用いた測定方法 Download PDF

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WO2022244890A1
WO2022244890A1 PCT/JP2022/021144 JP2022021144W WO2022244890A1 WO 2022244890 A1 WO2022244890 A1 WO 2022244890A1 JP 2022021144 W JP2022021144 W JP 2022021144W WO 2022244890 A1 WO2022244890 A1 WO 2022244890A1
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group
liquid chromatography
separating agent
agent carrier
column
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French (fr)
Japanese (ja)
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友啓 目黒
和昭 村中
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Tosoh Corp
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Tosoh Corp
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/282—Porous sorbents
    • B01J20/283—Porous sorbents based on silica
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/282—Porous sorbents
    • B01J20/285—Porous sorbents based on polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention provides a separating agent carrier, a column, and separation and purification using the same that can adsorb and separate biopolymers having a cis-diol structure in a biological sample, such as glycated proteins, ribonucleic acids (RNA), and sugars, by liquid chromatography. , regarding the method of measurement.
  • Non-Patent Documents 1, 2, 3 Boronic acid affinity chromatography is used for adsorption separation of biopolymers having a cis-diol structure, such as glycated proteins, RNA, and sugars.
  • glycated hemoglobin which reflects the average blood sugar level over the past one to two months, can be separated, it is utilized in diabetes diagnosis.
  • glycated hemoglobin when diagnosing diabetes in patients with hemoglobinopathy (dyshemoglobinopathy, thalassemia), which is not easy to analyze using cation exchange chromatography, which is currently widely used, glycated hemoglobin can be separated without being affected by it.
  • the boronic acid affinity method is utilized from.
  • Separation using the boronic acid affinity method utilizes a boronic ester formation reaction between the phenylboronic acid immobilized on the surface of the separating agent and the cis-diol of the sample. That is, in separation, analysis, and purification including diagnosis of diabetes using high-performance liquid chromatography (HPLC), the boronic acid moiety reacts with the glycated protein to form a boronate ester while passing the first eluent. to elute only samples without cis-diol structures, eg, non-glycated proteins.
  • HPLC high-performance liquid chromatography
  • a second eluent having a cis-diol such as sorbitol is passed through to elute a sample having a cis-diol structure, eg glycated protein, by boronic acid transesterification.
  • a basic eluent having a pH of 8 or more is used in order to promote the formation and exchange reaction of the boronate ester (Patent Documents 1 and 2).
  • Patent Document 3 it is known that when an eluent with a pH lower than this is used for separation, non-glycated proteins may be non-specifically adsorbed.
  • Non-Patent Document 6 a separating agent carrying boronic acid in which the benzene ring portion of phenylboronic acid is replaced with a more electron-deficient heteroaromatic ring has been developed (Non-Patent Document 6).
  • boronic acid affinity chromatography capable of efficiently retaining and eluting glycated proteins, RNA, and polysaccharides, which are biopolymers having a cis-diol structure, under neutral to weakly acidic conditions without requiring basic conditions.
  • a separation agent carrier for graphics is provided. More specifically, the present invention provides a technique for separating a biopolymer having a cis-diol structure from a sample containing a biopolymer having a cis-diol structure using an eluent having a pH of 4.5 to 8.
  • the present inventors have found a separating agent carrier for boronic acid affinity chromatography that is capable of retaining and efficiently eluting biopolymers having a cis-diol structure using a neutral to weakly acidic eluent. , completed the present invention.
  • a separating agent carrier incorporating pyridylboronic acid and a coordinating functional group did not change the pH necessary to retain the cis-diol component. Furthermore, it was found that the retention of the cis-diol component and the elution of the cis-diol component using boronic acid ester exchange can be efficiently performed, so that the shape of the obtained chromatogram is improved, and the present invention has been completed. rice field.
  • a separating agent carrier such as silica gel or a crosslinked polymer has a pyridylboronic acid derivative as a binding site with a biopolymer having a cis-diol structure, and promotion of boronate ester formation and exchange in the vicinity of this derivative.
  • a separating agent carrier for boronic acid affinity chromatography characterized in that a functional group having an amino group or a hydroxyl group as a terminal is covalently bonded as a site.
  • the separating agent carrier for boronic acid affinity chromatography of the present invention comprises a pyridylboronic acid derivative and an alkyl group or poly(ethyleneoxy)ethyl group having an amino group or a hydroxyl group at its end, which is composed of silica gel or a crosslinked polymer. characterized by being covalently bonded to the surface of
  • the separating agent carrier for boronic acid affinity chromatography comprises a pyridylboronic acid derivative represented by the following general formula (I) and an alkyl group or poly(ethyleneoxy ) ethyl groups are covalently bonded to the surface of the silica gel or crosslinked polymer.
  • X is -NH- or -O- or -CH (OH) -NH- or -CH (OH) -O-.
  • X' is -NH- or -O- or -CH (OH)--NH-- or --CH(OH)--O--.
  • X′′ is an amino group or a hydroxyl group.
  • A is an alkyl group having 2 to 10 carbon atoms or a poly(ethyleneoxy)ethyl group.
  • the alkyl group or poly(ethyleneoxy)ethyl group having an amino group or a hydroxyl group at its terminal, which is a site promoting boronic acid ester formation and exchange, is 1 to 200 mol% of the pyridyl boronic acid site, For example, it is preferably 4 mol % to 185 mol %, 4 mol % to 100 mol %, 4 mol % to 80 mol %, or 4.5 mol % to 75 mol %.
  • the separating agent carrier of the present invention can be used as a column for liquid chromatography by packing it into a tube having a hollow inside.
  • a biopolymer having a cis-diol group can be separated from a biological sample by liquid chromatography using the column.
  • glycated hemoglobin can be analyzed from a blood sample.
  • the separating agent carrier of the present invention comprises a site that can be used for covalent bond formation, such as an epoxide site introduced into a silica gel or a crosslinkable polymer, and an amino group that promotes ester formation and exchange with a pyridyl boronic acid derivative. can be produced by reaction with a covalent bond forming site of
  • X is -NH-, -O-, -CH(OH)-NH-, or -CH(OH)-O-
  • R and R' are each independently a hydrogen atom, an alkyl group, or a protective group for boronic acid, and R and R' may be combined to form a ring; * indicates binding sites with particles.
  • the separating agent carrier for liquid chromatography according to item 2-1 wherein the crosslinked polymer is a crosslinked polysaccharide.
  • the crosslinked polymer is a crosslinked polysaccharide.
  • [2-3] from the group consisting of a (meth)acrylic acid ester in which the crosslinked polymer has a hydroxyl group or an amino group in a side chain, a (meth)acrylamide having a hydroxyl group or an amino group in the side chain, vinylpyridine, and vinylimidazole;
  • the separating agent carrier for liquid chromatography according to item 2-1 or 2-2 which is a copolymer of at least one selected monomer and a polyfunctional unsaturated monomer.
  • the separating agent carrier for liquid chromatography according to any one of 2-1 to 2-3, wherein the particles are spherical particles having an average particle diameter of 1 to 200 ⁇ m.
  • [2-11] A column for liquid chromatography packed with the separating agent carrier for liquid chromatography according to any one of items 2-1 to 2-5 and 2-10.
  • [2-12] A measurement method for analyzing a sample containing a biopolymer having a cis-diol structure, using the column for liquid chromatography according to item 2-11.
  • [2-13] The measurement method according to item 2-12, wherein the biopolymer having a cis-diol structure is glycated protein, ribonucleic acid or polysaccharide.
  • the glycated protein is glycated hemoglobin.
  • [2-15] A method for adsorbing and/or purifying ribonucleic acid using the liquid chromatography column according to item 2-11.
  • [2-16] A method for separating and/or removing ribonucleic acid in a sample containing deoxyribonucleic acid and ribonucleic acid using the liquid chromatography column according to item 2-11.
  • the separating agent carrier of the present invention can be used as a column for liquid chromatography by filling a column tube.
  • a neutral to weakly acidic eluent is used, components having cis-diol sites and those not having cis-diol sites can be separated.
  • it can be used for separation of glycated proteins and non-glycated proteins, separation of RNA and contaminants, separation and/or removal of RNA in a sample containing deoxyribonucleic acid (DNA) and ribonucleic acid, particularly hemoglobin and glycated hemoglobin. is useful for the separation of again,
  • FIG. 4 is a diagram showing an example of a chromatogram obtained by measuring a blood sample in Example 1;
  • FIG. 10 is a diagram showing an example of a chromatogram obtained by measuring a blood sample in Example 2;
  • FIG. 10 is a diagram showing an example of a chromatogram obtained by measuring a blood sample in Example 3;
  • FIG. 10 is a diagram showing an example of a chromatogram obtained by measuring a blood sample in Example 4;
  • FIG. 10 is a diagram showing an example of a chromatogram obtained by measuring a blood sample in Example 5;
  • FIG. 10 is a plot diagram of saccharified hemoglobin % in Example 6 in which quantification was investigated.
  • FIG. 10 is a plot diagram of saccharified hemoglobin % in Example 6 in which quantification was investigated.
  • FIG. 10 is a diagram showing an example of a chromatogram obtained by adsorbing and separating crudely purified RNA in Example 7.
  • FIG. 10 is a diagram showing an example of a chromatogram of adsorption removal of RNA in a DNA sample in Example 8.
  • FIG. 10 is a diagram showing an example of a chromatogram obtained by adsorption separation of polysaccharides in Example 9.
  • FIG. 4 is a diagram showing an example of a chromatogram obtained by measuring a blood sample in Comparative Example 1.
  • FIG. FIG. 10 is a diagram showing an example of a chromatogram obtained by measuring a blood sample in Comparative Example 2;
  • FIG. 10 is a diagram showing an example of a chromatogram obtained by measuring a blood sample in Comparative Example 3;
  • FIG. 10 is a diagram showing an example of a chromatogram obtained by measuring a blood sample in Comparative Example 4;
  • the present invention provides a separating agent carrier for liquid affinity chromatography, (1) a functional group containing a pyridylboronic acid derivative represented by the following general formula (II): [in the formula (II), X is -NH-, -O-, -CH(OH)-NH-, or -CH(OH)-O-, R and R' are each independently a hydrogen atom, an alkyl group, or a protective group for boronic acid, and R and R' may be combined to form a ring; * indicates binding sites with particles.
  • a functional group containing a pyridylboronic acid derivative represented by the following general formula (II): [in the formula (II), X is -NH-, -O-, -CH(OH)-NH-, or -CH(OH)-O-, R and R' are each independently a hydrogen atom, an alkyl group, or a protective group for boronic acid, and R and R' may be combined to form
  • X' is -NH-, -O-, -CH(OH)-NH-, -CH(OH)-O- or -CH(OH)-
  • A is an alkyl group having 1 to 10 carbon atoms or a poly(ethyleneoxy)ethyl group
  • X′′ is substituted with an amino group or a hydroxyl group
  • * indicates the binding site of the particle.
  • a separating agent carrier for liquid chromatography characterized by being covalently bonded to the surface of silica gel or crosslinked polymer particles.
  • the present invention also provides a method for producing the separating agent carrier for liquid chromatography, the method comprising: A first surface modifier for introducing the functional group of (1) and a second surface modifier for introducing the functional group of (2) into the silica gel or crosslinked polymer particles having a functional group for linking on the surface. It may include reacting two surface modifiers simultaneously or separately to obtain a separating agent carrier for liquid chromatography.
  • the pyridine ring has a boronic acid site and a linking site used for surface modification of the separating agent carrier.
  • Functional groups that can be used to modify the surface of pyridylboronic acid and the separating agent carrier include hydroxyl group, hydroxymethyl group, amino group, aminomethyl group, carboxy group, formyl group and the like.
  • the form of the boron moiety may be a boronic acid in which the substituent other than the pyridine ring is a hydroxyl group, or a boronic acid ester substituted with a protecting group such as a pinacol ester to form a ring,
  • a protecting group such as a pinacol ester to form a ring
  • Other known boronic acid protecting groups such as diaminonaphthalenamide, MIDA ester, trifluoroborate salt, techol ester, neopentyl glycol ester, pinanediol ester, biscyclohexyldiol ester, or MPMP ester may also be used.
  • Pyridylboronic acid may be in any state such as hydrate, hydrochloride, or sulfate.
  • the first surface modifier for introducing a functional group containing a pyridylboronic acid derivative may be any substance that satisfies the above conditions. Specific examples include 6-hydroxypyridine-3-boronic acid pinacol ester, 6- (Hydroxymethyl)pyridine-3-boronic acid pinacol ester, 2-aminopyridine-5-boronic acid pinacol ester, 2-aminopyridine-4-boronic acid pinacol ester, 5-formylpyridine-5-boronic acid, 6-carboxy Examples include pyridine-3-boronic acid. 2-Aminopyridine-5-boronic acid pinacol ester is particularly preferred.
  • X' is -NH-, -O-, -CH(OH)-NH-, -CH(OH)-O- or -CH(OH)-
  • A is an alkyl group having 1 to 10 carbon atoms or a poly(ethyleneoxy)ethyl group
  • X′′ is substituted with an amino group or a hydroxyl group
  • * indicates the binding site of the particle.
  • the second surface modifier for introducing may have a linking site used for surface modification with the separating agent carrier (particles) at the terminal not substituted with the above nitrogen atom or oxygen atom.
  • this linking site can be used for the covalent bond formation reaction.
  • functional groups include a hydroxyl group, an amino group, a carboxyl group, and a formyl group, but are not particularly limited.
  • Specific examples of the second surface modifier include 1,3-propanediol, propylene glycol, dipropylene glycol, tripropylene glycol, 1,4-butanediol, glycerol, polyglycerol, aminoethanol, aminopropanol, aminobutanol, Examples thereof include 2-(2-(2-aminoethoxy)ethoxy)acetic acid, and it is desirable to have an alkyl group having 2 to 6 carbon atoms or a poly(ethyleneoxy)ethyl group.
  • the above two types of surface modifiers are bound to a separation agent composed of known silica gel, crosslinked polymer, or the like.
  • a known separating agent having a functional group for linking with a modifier on its surface can be used.
  • Functional groups for linking include epoxy group, amino group, hydroxyl group, carboxy group, formyl group and the like, but are not particularly limited.
  • the two types of surface modifiers described above may be reacted simultaneously or separately with the silica gel or crosslinked polymer particles having functional groups for linking on their surfaces.
  • 1 to 200 mol% of the second surface modifier relative to the first surface modifier such as 4 mol% to 185 mol%, 4 mol% to 100 mol%, 4 mol% to 80 mol%, or It is preferable to react in an amount of 4.5 mol % to 75 mol %.
  • the particle size is not limited, but the average particle size is 1 to 200 ⁇ m. Spherical particles having an average particle size of 10 to 200 ⁇ m are preferred for preparative applications. In preparative applications, porous particles having pores are preferable in order to adsorb a large amount of sample at once. is preferred.
  • crosslinked polymer examples include crosslinked polysaccharide particles using cellulose or agarose, (meth)acrylic acid esters having hydroxyl or amino groups in side chains, (meth)acrylamide monomers having hydroxyl or amino groups in side chains, and vinylpyridine.
  • Copolymer particles of vinylimidazole and polyfunctional unsaturated monomers can be used.
  • particles using a (meth)acrylic acid ester or a (meth)acrylic acid amide monomer are preferable because the amounts of hydroxyl groups and amino groups on the particle surface can be easily controlled.
  • Examples of monomers having an alcoholic hydroxyl group include (meth)acrylic acid esters, monomers in which the ester portion is glycerol, 2-hydroxyethyl, diethylene glycol, triethylene glycol, propylene glycol, dipropylene glycol, or tripropylene glycol. , N-(2-hydroxyethyl)methacrylamide, and N-(2-hydroxypropyl)methacrylamide.
  • Examples of monomers having an amino group include vinylpyridines, vinylimidazole, N-(2-aminoethyl)acrylamide, and 2-(dimethylamino)ethyl (meth)acrylate.
  • polyfunctional unsaturated monomers examples include (poly)ethylene glycol-di(meth)acrylate, (poly)glycerol-poly(meth)acrylate, (poly)propylene glycol-poly(meth)acrylate, Divinylbenzene and the like can be exemplified.
  • a crosslinked polymer having a hydroxyl group can be used by partially introducing a functional group necessary for introducing pyridylboronic acid, such as an epoxy group or a carboxylic acid.
  • a method using a crosslinked polymer using a (meth)acrylic acid monomer having an epoxy group, such as glycidyl methacrylate, as a monomer simultaneously attaches a hydroxyl group or an amino group and a pyridylboronic acid compound to the epoxy group of the particles, respectively.
  • a diol compound, or an amine by reacting the epoxy remaining after the introduction of the pyridylboronic acid compound with water, a diol compound, or an amine, hydroxyl groups and amino groups can be introduced, respectively, which is preferable.
  • hydrophilic vinyl polymer particles having an epoxy group can also be used. can easily achieve introduction of pyridylboronic acid and introduction of hydroxyl groups by reacting epoxy groups with water.
  • a covalent bond can be formed through an epoxide ring-opening reaction, an amide bond-forming reaction, a carbamate-forming reaction, an imine-forming reaction, or the like using the linking site described above, but the bond-forming method is not limited.
  • the column tube a tube made of metal, resin, glass, or the like having a hollow inside may be used, and the column filled with the separating agent carrier can be used as a column for liquid chromatography, for example, for HPLC measurement. .
  • a column filled with the separating agent carrier can be used.
  • the eluent two or more types of eluents can be used, ie, an eluent containing no component having a cis-diol moiety and an eluent containing a cis-diol moiety such as galactose, fructose and sorbitol.
  • liquid chromatography column of the present invention by using the liquid chromatography column of the present invention, a sample containing a biopolymer having a cis-diol structure (e.g., glycated protein (e.g., glycated hemoglobin), ribonucleic acid, polysaccharide, etc.) A measurement method to be analyzed can be provided.
  • the use of the liquid chromatography column of the present invention makes it possible to provide a method for adsorbing and/or purifying ribonucleic acid.
  • the use of the liquid chromatography column of the present invention also makes it possible to provide a method for separating and/or removing ribonucleic acid in a sample containing deoxyribonucleic acid and ribonucleic acid.
  • the column for liquid chromatography of the present invention By using the column for liquid chromatography of the present invention, even when using a neutral to weakly acidic eluent, which has been difficult to separate, the chromatogram peaks of biopolymers having cis-diol moieties can be obtained. Shape broadening was suppressed, and biopolymers having cis-diol sites and components not having cis-diol sites could be efficiently separated.
  • Example 1 HPLC measurement was performed using a column filled with a separating agent carrier having a 2-aminopyridine-5-boronic acid derivative supported on the surface of a silica gel carrier.
  • silica gel particles having an epoxide moiety 10 g of the obtained silica gel particles having an epoxide moiety, 400 mg of 2-aminopyridine-5-boronic acid pinacol ester (Mw: 220.08) and 5.5 mg of aminoethanol (Mw: 61.08) were added to 40 g of It was stirred at 40° C. in a phosphate buffer (pH 7.0). After 24 hours, the silica gel was separated from the reaction solution to obtain a silica gel having pyridyl boronic acid and sites promoting boronic ester formation and exchange on the surface.
  • Example 2 Silica gel having epoxide sites on the surface prepared in Example 1 was used. 10 g of silica gel particles having epoxide moieties, 400 mg of 2-aminopyridine-5-boronic acid pinacol ester and 22 mg of aminoethanol were stirred in 40 g of phosphate buffer (pH 7.0) at 40°C. After 24 hours, the silica gel was separated from the reaction solution to obtain a silica gel having pyridyl boronic acid and sites promoting boronic ester formation and exchange on the surface.
  • phosphate buffer pH 7.0
  • the obtained separating agent carrier was packed into a column similar to that used in Example 1 above, and separated under the same conditions.
  • Example 3 Silica gel having epoxide sites on the surface prepared in Example 1 was used. 7 g of silica gel particles having epoxide moieties, 210 mg of 2-aminopyridine-5-boronic acid pinacol ester and 42 mg of aminoethanol were stirred in 40 g of phosphate buffer (pH 7.0) at 40°C. After 24 hours, the silica gel was separated from the reaction solution to obtain a silica gel having pyridyl boronic acid and sites promoting boronic ester formation and exchange on the surface.
  • the obtained separating agent carrier was packed into a column similar to that used in Example 1 above, and separated under the same conditions.
  • Example 4 Silica gel having epoxide sites on the surface prepared in Example 1 was used. 7 g of silica gel particles having epoxide moieties, 210 mg of 2-aminopyridine-5-boronic acid pinacol ester and 105 mg of aminoethanol were stirred in 40 g of phosphate buffer (pH 7.0) at 40°C. After 24 hours, the silica gel was separated from the reaction solution to obtain a silica gel having pyridyl boronic acid and sites promoting boronic ester formation and exchange on the surface.
  • phosphate buffer pH 7.0
  • the obtained separating agent carrier was packed into a column similar to that used in Example 1 above, and separated under the same conditions.
  • Example 5 Silica gel having epoxide sites on the surface prepared in Example 1 was used. 7 g of silica gel particles having epoxide moieties, 210 mg of 2-aminopyridine-5-boronic acid pinacol ester and 21 mg of 1,6-hexamethylenediamine (Mw: 116.20) were added to 40 g of phosphate buffer (pH 7.0). 0) and stirred at 40°C. After 24 hours, the silica gel was separated from the reaction solution to obtain a silica gel having pyridyl boronic acid and sites promoting boronic ester formation and exchange on the surface.
  • phosphate buffer pH 7.0
  • the obtained separating agent carrier was packed into a column similar to that used in Example 1 above, and separated under the same conditions.
  • Example 5 When the column of Example 5 is used, the non-saccharified components and the saccharified components can be separated as shown in FIG. 5, and the peak shape is good.
  • Example 6 In order to examine the quantification of glycated hemoglobin, the column prepared in Example 2 was used to perform HPLC measurement of samples with different amounts of glycated hemoglobin.
  • HPLC measurement was performed using the same conditions as in Example 1.
  • the five samples used for quantitative evaluation were prepared by mixing two samples with different amounts of glycated hemoglobin. That is, samples 1 and 5 were mixed at a ratio of 3:1 (No. 2), 1:1 (No. 3) and 1:3 (No. 4).
  • FIG. 6 the measurement results of glycated hemoglobin % in five kinds of samples show linearity, so that the separating agent carrier for liquid chromatography of the present invention exhibits sufficient quantification. It was revealed.
  • Example 7 200 g of 2-hydroxyethyl methacrylate, 30 g of ethylene glycol dimethacrylate, 700 g of monochlorobenzene and 5 g of polymerization initiator V65 (manufactured by Fujifilm Wako Pure Chemical Industries) were mixed. Polyvinyl alcohol was dissolved at a concentration of 2% in a 3 L separable flask, and the temperature was adjusted to 60°C. The monomer mixture was put into a separable flask and polymerized for 6 hours while stirring.
  • the obtained reaction solution was filtered through a glass filter, washed with warm water and acetone in that order, and further classified into particle sizes of 40 to 100 ⁇ m using a sieve to obtain crosslinked polymer particles having hydroxyl groups.
  • 200 g (wet weight) of the obtained crosslinked polymer particles having hydroxyl groups were dispersed in 300 g of water, 50 g of epichlorohydrin was added, and 40 g of 48% sodium hydroxide heated to 40° C. was gradually added. , introduced an epoxy group.
  • the obtained particles were washed with water, acetone, and water in this order to obtain epoxidized crosslinked polymer particles.
  • 25 g (dry weight) of crosslinked polymer particles were dispersed in 225 mL of deionized water, and 4.5 g of 2-aminopyridine-5-boronic acid pinacol ester was added and dissolved.
  • 25 mL of 0.5 M sodium hydroxide aqueous solution was added, and the mixture was heated at 70° C. for 2 hours.
  • the reaction solution was filtered through a glass filter, washed with 3 times the amount of 80% ethanol aqueous solution, 3 times the amount of deionized water, 3 times the amount of 0.1 M hydrochloric acid aqueous solution, and 3 times the amount of ion exchanged water in this order.
  • 2-Aminopyridine-5-boronic acid immobilized particles having hydroxyl groups bound to the substrate were obtained. The obtained particles were packed into an empty column having an inner diameter of 7.5 mm and a column length of 7.5 cm.
  • RNA was purified under the following conditions.
  • Eluent A solution 0.15 M sodium chloride, 0.1 M phosphate buffer (pH 6.0)
  • B solution 0.1 M sorbitol, 0.15 M sodium chloride, 0.1 M phosphate buffer (pH 6.0)
  • Solution C 20 mM phosphate buffer (pH 1.9)
  • Step gradient :: eluent A (0-16.6 min), eluent B (16.6-33.1 min), eluent C (33.1-49.7 min)
  • Flow rate 1.0 mL/min Monitor wavelength: 260nm
  • Ribonucleic acid from yeast manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., product number 185-00202
  • Example 9 Blue Dextran D5751 manufactured by Merck Co. was dissolved in eluent A to a concentration of 50 g/L, and the elution behavior was confirmed in the same manner as in Example 7 except that the monitor wavelength was set to 620 nm. From the resulting chromatogram (Fig. 9), it was found that dextran was neutrally adsorbed and could be eluted under mild conditions without the need for acidity for elution.
  • the obtained separating agent carrier was packed into a column similar to that used in Example 1 above, and separated under the same conditions.
  • Comparative Example 3 Silica gel having epoxide sites on the surface prepared in Example 1 was used. 10 g of silica gel particles having epoxide moieties and 400 mg of 2-aminopyridine-5-boronic acid pinacol ester were stirred in 40 g of phosphate buffer (pH 7.0) at 40°C. After 24 hours, separation from the reaction solution was performed to obtain silica gel having pyridylboronic acid on its surface.
  • the obtained separating agent carrier was packed into a column similar to that used in Example 1 above, and separated under the same conditions.
  • Comparative Example 4 When the column of Comparative Example 3 was used, the non-saccharified components and the saccharified components could be separated as shown in FIG. 12, but the peak of the saccharified components was broad.
  • Comparative Example 4 Using the gel-filled column produced in Comparative Example 3, HPLC measurement was performed using liquid A having a pH higher than that of Example 1. Eluent A solution: 100 mM phosphate buffer (pH 6.8) B solution: 100 mM phosphoric acid, 100 mM d-sorbitol mixed solution (pH 6.5) Separation was carried out under the same conditions as in Example 1 except for the eluent.

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