Separating agent carrier for boronate affinity chromatography, column, and measurement method employing same

Abstract

The present invention provides: a separating agent carrier for liquid chromatography that is characterized by having, on surfaces of particles of silica gel or a crosslinked polymer, a functional group that contains a pyridylboronic acid derivative serving as a bonding site with a biopolymer having a cis-diol structure, as well as a functional group that has, at an end thereof, an amino group or a hydroxyl group serving as an acceleration site for boronate ester formation and exchange; a column filled with the separating agent carrier; and a method employing the column for measuring, separating, purifying and / or removing the biopolymer.

Description

Separation carrier for boronic acid affinity chromatography, column, and measurement method using the same

[0001] The present invention relates to a separation carrier and column capable of adsorbing and separating biopolymers having a cis-diol structure in a biological sample, such as glycated proteins, ribonucleic acid (RNA), and sugars, by liquid chromatography, and to separation, purification, and measurement methods using the same.

[0002] Boronic acid affinity chromatography is used for the adsorption and separation of biopolymers with cis-diol structures, such as glycated proteins, RNA, and sugars (Non-Patent Documents 1, 2, and 3). It is particularly useful in diagnosing diabetes because it can separate glycated hemoglobin, which reflects the average blood glucose level over the past one to two months. In particular, the boronic acid affinity method is used to diagnose diabetes in patients with hemoglobinopathies (hemoglobinopathies and thalassemias), which are difficult to diagnose using currently widely used cation exchange chromatography, because it can separate glycated hemoglobin without being affected by the disease.

[0003] Separation using the boronic acid affinity method utilizes a boronate ester formation reaction between phenylboronic acid immobilized on the surface of a separation material and cis-diols in the sample. Specifically, in separation, analysis, and purification, including diabetes diagnosis using high-performance liquid chromatography (HPLC), a first eluent is passed through the separation material, forming a boronate ester through a reaction between the boronic acid moiety and glycated proteins, thereby eluting only samples lacking cis-diol structures, such as non-glycated proteins. Next, a second eluent containing a cis-diol, such as sorbitol, is passed through the separation material, and a boronate ester exchange reaction occurs to elute samples possessing cis-diol structures, such as glycated proteins. To promote the formation of boronate esters and the exchange reaction, a basic eluent with a pH of 8 or higher is used (see Patent Documents 1 and 2). It is known that the use of a lower pH eluent for separation can result in non-specific adsorption of non-glycated proteins (see Patent Document 3).

[0004] In response to this, various attempts have been made to lower the pH of the eluent used in the reaction between boronic acid and cis-diol moieties. For example, a packing material carrying phenylboronic acid, in which a nitrogen atom or oxygen atom capable of coordinating with the boronic acid moiety is positioned ortho to the boron atom on the benzene ring, can retain cis-diols even at low pH. However, the boronic acid usable in this method cannot be obtained directly and is synthesized via an inefficient multi-step reaction (Non-Patent Document 4).

[0005] In response to this, a packing material was developed in which a coordinating functional group was located near the phenylboronic acid supported on the packing surface. This method is characterized by the use of readily available raw materials, but the pH required to retain cis-diols is higher than when both boronic acid and a coordinating functional group are present on the benzene ring (Non-Patent Document 5).

[0006] In response to this, separation agents loaded with boronic acids in which the benzene ring moiety of phenylboronic acid has been replaced with a more electron-deficient heteroaromatic ring have been developed (Non-Patent Document 6). For example, it has been reported that a packing material in which pyridylboronic acid, in which one of the carbon atoms of the benzene ring has been replaced with a nitrogen atom, has been modified on the carrier surface via the nitrogen atom of the pyridine ring, can retain cis-diols even at low pH (pH 5-6). However, when using these boronic acids capable of retaining cis-diol moieties at low pH, the bond between the boronic acid and the cis-diol moiety is strong, so hydrolysis of the boronate ester under strongly acidic conditions is utilized to elute the cis-diol components. In this case, when elution is performed using the above-described boronate transesterification using d-sorbitol under neutral to weakly acidic conditions, there is a concern that the slowness of the transesterification will result in broadening of the peak shape of the chromatogram. Therefore, it may not be suitable for the separation, analysis, or purification of glycated proteins, such as glycated hemoglobin and RNA, which are biopolymers that denature or decompose under strongly acidic conditions.

[0007] For these reasons, columns using packing materials carrying boronic acids capable of retaining cis-diol moieties at low pH are not practical and are rarely used for liquid chromatography separation, analysis, and purification of samples of biopolymers such as glycosylated proteins and RNA.

[0008] Japanese Patent Laid-Open No. 5-5731 Japanese Patent Laid-Open No. 2002-139481 Japanese Patent Laid-Open No. 2007-284425

[0009] YuCai Li, Eva Linne Larsson, J. Chromatography A. 2001, 909, 137-145. A. Gabriela Gomes, Ana. M. Azevedo, J. Chromatography A. 2010, 1217, 2262-2266. Serap Senel, Colloids and Surfaces A. 2003, 219, 17-23. H. Li, Y. Liu, J. Liu, Z. Liu, Chem. Commun. 2011, 47, 8169-8171. H. Li, Z. Liu, Trends Anal. Chem. 2012, 37, 148-161D. Li, et al, J. Chromatogr. A, 2014, 1339, 103-109

[0010] The present invention provides a separating support for boronic acid affinity chromatography that can efficiently retain and elute biopolymers having a cis-diol structure, such as glycosylated proteins, RNA, and polysaccharides, under neutral to weakly acidic conditions without requiring basic conditions. More specifically, the present invention provides a method for separating biopolymers having a cis-diol structure from a sample containing the biopolymers using an eluent having a pH of 4.5 to 8.

[0011] As a result of extensive research, the present inventors have discovered a separating material carrier for boronic acid affinity chromatography that can retain and efficiently elute biopolymers having a cis-diol structure using a neutral to weakly acidic eluent, and have completed the present invention.

[0012] The present inventors investigated a surface structure capable of efficiently retaining cis-diol components and eluting the cis-diol components by boronate ester exchange in a separating carrier loaded with pyridylboronic acid, which can retain glycated components even under neutral to weakly acidic conditions. As a result, good results were obtained when a coordinating functional group, which normally lowers the pH of the eluent required for forming a boronate ester between the boronic acid and the cis-diol component, was placed near the boronic acid. Specifically, compared to a separating carrier loaded with only pyridylboronic acid, a separating carrier loaded with pyridylboronic acid and a coordinating functional group was found to be capable of efficiently retaining cis-diol components and eluting the cis-diol components by boronate ester exchange without changing the pH required for retaining the cis-diol components, thereby resulting in a better chromatogram shape. This finding led to the completion of the present invention.

[0013] Specifically, we have developed a separation carrier for boronic acid affinity chromatography, which is characterized in that a pyridylboronic acid derivative is present on the surface of a separation carrier, such as silica gel or a crosslinked polymer, as a binding site for a biopolymer having a cis-diol structure, and a functional group having an amino group or a hydroxyl group at its terminus is covalently bonded to the vicinity of the pyridylboronic acid derivative as a site for promoting boronate ester formation and exchange.

[0014] The present invention will be described in detail below.

[0015] [1-1] The separating material carrier for boronic acid affinity chromatography of the present invention is characterized in that a pyridylboronic acid derivative and an alkyl group or poly(ethyleneoxy)ethyl group having an amino group or a hydroxyl group at its terminal are covalently bonded to the surface of silica gel or a crosslinked polymer.

[0016] [1-2] The separating material carrier for boronic acid affinity chromatography described above is characterized in that a pyridylboronic acid derivative represented by the following general formula (I) and an alkyl group or poly(ethyleneoxy)ethyl group having an amino group or a hydroxyl group at its terminal are covalently bonded to the surface of silica gel or a crosslinked polymer:

[0017]

[0018] (In formula (I), X is -NH-, -O-, -CH(OH)-NH-, or -CH(OH)-O-. X' is -NH-, -O-, -CH(OH)-NH-, or -CH(OH)-O-. X" is an amino group or a hydroxyl group. R and R' each independently represent a hydrogen atom or an alkyl group, and may combine to form a ring. A is an alkyl group having 2 to 10 carbon atoms or a poly(ethyleneoxy)ethyl group.) In addition, with respect to pyridylboronic acid derivatives, the substitution positions of the boronic acid moiety and the bonding moiety (X) on the pyridine ring do not matter.

[0019] [1-3] The aforementioned alkyl group or poly(ethyleneoxy)ethyl group having an amino group or hydroxyl group at its terminal, which is the moiety that promotes boronate ester formation and exchange, preferably accounts for 1 to 200 mol %, for example, 4 mol % to 185 mol %, 4 mol % to 100 mol %, 4 mol % to 80 mol %, or 4.5 mol % to 75 mol %, of the pyridylboronic acid moiety.

[0020] [1-4] The separating medium carrier of the present invention can be packed into a hollow tube and used as a liquid chromatography column. Liquid chromatography using this column enables the separation of biopolymers having cis-diol groups from biological samples. In particular, glycated hemoglobin can be analyzed from blood samples.

[0021] [1-5] The separating support of the present invention can be produced by reacting a moiety available for covalent bond formation, such as an epoxide moiety, introduced into silica gel or a crosslinkable polymer with a covalent bond-forming moiety, such as an amino group, of a promoter of ester formation and exchange with a pyridylboronic acid derivative.

[0022] The present application also includes the following aspects of the invention: [2-1] (1) A functional group containing a pyridylboronic acid derivative represented by the following general formula (II): [In 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 protecting group for boronic acid, and R and R' may be bonded together to form a ring, and * indicates a bonding site with the particle.]; and (2) a functional group represented by the following general formula (III): [In formula (III), X' represents -NH-, -O-, -CH(OH)-NH-, -CH(OH)-O-, or -CH(OH)-; A represents an alkyl group having 1 to 10 carbon atoms or a poly(ethyleneoxy)ethyl group, which is substituted at X" with an amino group or a hydroxyl group; and * represents a particle bonding site.] A separating material for liquid chromatography, characterized in that these are covalently bonded to the surface of silica gel or crosslinked polymer particles. [2-2] The separating material for liquid chromatography according to item 2-1, wherein the crosslinked polymer is a crosslinked polysaccharide. [2-3] The separating material for liquid chromatography according to item 2-1 or 2-2, wherein the crosslinked polymer is a copolymer of at least one monomer selected from the group consisting of a (meth)acrylic acid ester having a hydroxyl group or an amino group on the side chain, a (meth)acrylamide having a hydroxyl group or an amino group on the side chain, a vinylpyridine, and a vinylimidazole, and a polyfunctional unsaturated monomer. [2-4] The liquid chromatography separating support according to any one of Items 2-1 to 2-3, wherein the particles are spherical particles having an average particle size of 1 to 200 μm. [2-5] The liquid chromatography separating support according to Item 2-4, wherein the particles are porous particles having pores with an average size of 10 to 500 nm. [2-6] A method for producing a liquid chromatography separating support according to any one of Items 2-1 to 2-5, comprising simultaneously or separately reacting silica gel or crosslinked polymer particles having linking functional groups on their surfaces with a first surface modifier for introducing the functional group (1) and a second surface modifier for introducing the functional group (2), to obtain the liquid chromatography separating support according to any one of Items 2-1 to 2-5. [2-7] The method according to Item 2-6, wherein the second surface modifier is reacted in an amount of 1 to 200 mol % relative to the first surface modifier. [2-8] The method according to Item 2-6 or 2-7, wherein the linking functional group is an epoxy group, an amino group, a hydroxyl group, a carboxyl group, or a formyl group. [2-9] The method according to Item 2-6 or 2-7, wherein the linking functional group is an epoxy group.[2-10] A liquid chromatography separation carrier obtained by the method according to any one of Items 2-6 to 2-9. [2-11] A liquid chromatography column packed with the liquid chromatography separation carrier 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 liquid chromatography column according to Item 2-11. [2-13] The measurement method according to Item 2-12, wherein the biopolymer having a cis-diol structure is a glycosylated protein, ribonucleic acid, or polysaccharide. [2-14] The measurement method according to Item 2-13, wherein the glycosylated protein is glycosylated 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 from a sample containing deoxyribonucleic acid and ribonucleic acid, using the liquid chromatography column according to Item 2-11.

[0023] The separating material carrier of the present invention can be packed into a column tube and used as a column for liquid chromatography. In an analysis using this column, even when a neutral to weakly acidic eluent is used, components having a cis-diol moiety and components not having the same can be separated. For example, it can be used to separate glycated proteins from non-glycated proteins, to separate RNA from contaminants, and to separate and / or remove RNA from samples containing deoxyribonucleic acid (DNA) and ribonucleic acid, and is particularly useful for separating hemoglobin from glycated hemoglobin. In addition,

[0024] FIG. 1 is a diagram showing an example of a chromatogram obtained by measuring a blood sample in Example 1. FIG. 2 is a diagram showing an example of a chromatogram obtained by measuring a blood sample in Example 2. FIG. 3 is a diagram showing an example of a chromatogram obtained by measuring a blood sample in Example 3. FIG. 4 is a diagram showing an example of a chromatogram obtained by measuring a blood sample in Example 5. FIG. 5 is a diagram showing an example of a chromatogram obtained by measuring a blood sample in Example 6. FIG. 6 is a diagram showing an example of a chromatogram obtained by measuring a blood sample in Example 6. FIG. 7 is a diagram showing an example of a chromatogram obtained by measuring a blood sample in Example 7. FIG. 8 is a diagram showing an example of a chromatogram obtained by measuring a blood sample in Example 8. FIG. 9 is a diagram showing an example of a chromatogram obtained by measuring a blood sample in Example 9. FIG. 10 is a diagram showing an example of a chromatogram obtained by measuring a blood sample in Comparative Example 1. FIG. 11 is a diagram showing an example of a chromatogram obtained by measuring a blood sample in Comparative Example 2. FIG. 12 is a diagram showing an example of a chromatogram obtained by measuring a blood sample in Comparative Example 3. FIG. 13 is a diagram showing an example of a chromatogram obtained by measuring a blood sample in Comparative Example 4.

[0025] The present invention will be described in detail below. However, the present invention can be embodied in various forms and is not limited to the following embodiments and examples.

[0026] In one embodiment, the present invention provides a separation support for liquid affinity chromatography, comprising: (1) a functional group containing a pyridylboronic acid derivative represented by the following general formula (II): [In 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 protecting group for boronic acid, and R and R' may be bonded together to form a ring, and * indicates a bonding site with the particle.]; and (2) a functional group represented by the following general formula (III): [In formula (III), X' represents -NH-, -O-, -CH(OH)-NH-, -CH(OH)-O-, or -CH(OH)-; A represents an alkyl group having 1 to 10 carbon atoms or a poly(ethyleneoxy)ethyl group, which is substituted with an amino group or a hydroxyl group at X"; and * represents a particle bonding site.] is covalently bonded to the surface of a silica gel or crosslinked polymer particle.

[0027] In one embodiment, the present invention provides a method for producing the above-mentioned separation carrier for liquid chromatography, which may include simultaneously or separately reacting silica gel or crosslinked polymer particles having linking functional groups on their surfaces with a first surface modifier for introducing the functional group (1) and a second surface modifier for introducing the functional group (2), to obtain a separation carrier for liquid chromatography.

[0028] In the pyridylboronic acid derivative, it is sufficient that the pyridine ring has a boronic acid moiety and a linking moiety used for surface modification of the separating agent carrier, and these functional groups may be bonded to any position on the pyridine ring.Furthermore, functional groups that can be used for surface modification of pyridylboronic acid and the separating agent carrier include a hydroxyl group, a hydroxymethyl group, an amino group, an aminomethyl group, a carboxyl group, a formyl group, etc.Furthermore, 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 boronate ester substituted with a protecting group such as pinacol ester to form a ring, or other known boronic acid protecting groups, such as diaminonaphthaleneamide, MIDA ester, trifluoroborate salt, tetracohol ester, neopentyl glycol ester, pinanediol ester, biscyclohexyldiol ester, or MPMP ester.Pyridylboronic acid may be in any form, such as a hydrate, hydrochloride, or sulfate. The first surface modifying agent for introducing a functional group containing a pyridylboronic acid derivative may be any substance that satisfies the above conditions, and 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-carboxypyridine-3-boronic acid, etc. 2-aminopyridine-5-boronic acid pinacol ester is particularly preferred.

[0029] The functional group represented by the following general formula (III), which is the site for promoting the formation and exchange of the boronic acid ester: [In formula (III), X' represents -NH-, -O-, -CH(OH)-NH-, -CH(OH)-O-, or -CH(OH)-; A represents an alkyl group having 1 to 10 carbon atoms or a poly(ethyleneoxy)ethyl group, which is substituted with an amino group or a hydroxyl group at X"; and * represents a particle bonding site.] The second surface modifier for introducing the second surface modifier may have a linking moiety at the end not substituted with the nitrogen atom or oxygen atom, which is used for surface modification with the separating agent carrier (particle). This linking moiety may be used in a covalent bond formation reaction. Examples of such functional groups include, but are not limited to, hydroxyl groups, amino groups, carboxy groups, and formyl groups. 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, and 2-(2-(2-aminoethoxy)ethoxy)acetic acid, although it is desirable for the second surface modifier to have an alkyl group having 2 to 6 carbon atoms or a poly(ethyleneoxy)ethyl group.

[0030] The two types of surface modifiers are bonded to a separating agent composed of a known silica gel, crosslinked polymer, or the like. In this case, known separating agents having functional groups on their surface for linking to the modifiers can be used. Examples of linking functional groups include, but are not limited to, epoxy groups, amino groups, hydroxyl groups, carboxyl groups, and formyl groups. The two types of surface modifiers may be reacted simultaneously or separately with silica gel or crosslinked polymer particles having linking functional groups on their surfaces. It is preferable to react the second surface modifier with the first surface modifier in an amount of 1 to 200 mol%, for example, 4 mol% to 185 mol%, 4 mol% to 100 mol%, 4 mol% to 80 mol%, or 4.5 mol% to 75 mol%.

[0031] When the separating agent used for modification, which is composed of silica gel, crosslinked polymer, or the like, is a particle, there is no limitation on the particle size, but the average particle size is 1 to 200 μm, and for analytical purposes, it is preferably spherical particles with an average particle size of 1 to 10 μm, and for preparative purposes, it is preferably spherical particles with an average particle size of 10 to 200 μm. Furthermore, for preparative purposes, porous particles having pores are preferred in order to adsorb a large amount of sample at one time, and the pore size is preferably an average of 10 to 500 nm, depending on the sample.

[0032] Examples of crosslinked polymers that can be used include crosslinked polysaccharide particles using cellulose or agarose, (meth)acrylic acid esters having hydroxyl or amino groups on their side chains, (meth)acrylamide monomers having hydroxyl or amino groups on their side chains, vinylpyridines, and copolymer particles of vinylimidazole and a polyfunctional unsaturated monomer. Particles using (meth)acrylic acid esters or (meth)acrylic acid amide monomers are particularly preferred because the amount of hydroxyl and amino groups on the particle surface can be easily controlled. Examples of monomers having an alcohol-based hydroxyl group include (meth)acrylic acid esters, monomers whose ester moiety is glycerol, 2-hydroxyethyl, diethylene glycol, triethylene glycol, propylene glycol, dipropylene glycol, or tripropylene glycol, as well as N-(2-hydroxyethyl)methacrylamide and N-(2-hydroxypropyl)methacrylamide. Examples of monomers having an amino group include vinylpyridines, vinylimidazole, N-(2-aminoethyl)acrylamide, 2-(dimethylamino)ethyl (meth)acrylate, etc. Examples of polyfunctional unsaturated monomers include (poly)ethylene glycol-di(meth)acrylate, (poly)glycerol-poly(meth)acrylate, (poly)propylene glycol-poly(meth)acrylate, divinylbenzene, etc.

[0033] A crosslinked polymer having a hydroxyl group can be used by partially introducing functional groups, such as epoxy groups and carboxylic acids, which are necessary for introducing pyridylboronic acid.

[0034] Furthermore, a method using a crosslinked polymer using a (meth)acrylic acid monomer having an epoxy group, such as glycidyl methacrylate, as a monomer is preferred because it is possible to simultaneously introduce a hydroxyl group or an amino group and a pyridylboronic acid compound into the epoxy groups of the particles, respectively, or to introduce a hydroxyl group or an amino group by reacting the remaining epoxy after introducing the pyridylboronic acid compound with water, a diol compound, or an amine, respectively.

[0035] Alternatively, commercially available hydrophilic vinyl polymer particles having epoxy groups can also be used. For example, when TOYOPEARL AF-Epoxy-650 manufactured by Tosoh Corporation is used as the crosslinked polymer, introduction of pyridylboronic acid and introduction of hydroxyl groups due to the reaction of the epoxy groups with water can be easily achieved by reacting an aminopyridylboronic acid compound in an alkaline aqueous solution.

[0036] There is no limitation on the method for bonding the two types of surface modifiers to the separating agent carrier, and known bond-forming reactions can be used. For example, using the linking moiety described above, a covalent bond can be formed via an epoxide ring-opening reaction, an amide bond-forming reaction, a carbamate-forming reaction, an imine-forming reaction, or the like, but the bond-forming method is not limited.

[0037] The column tube may be a hollow tube made of metal, resin, glass, etc., and the column packed with the separating medium carrier can be used as a column for liquid chromatography, for example, for HPLC measurement.

[0038] When performing HPLC measurement, a column packed with the separating agent carrier can be used. As the eluent, two or more types of eluents can be used: an eluent that does not contain components having cis-diol moieties, and an eluent that contains cis-diol moieties such as galactose, fructose, and sorbitol.

[0039] The gradient is preferably a step gradient, a linear gradient, or the like.

[0040] In one embodiment, by using the liquid chromatography column of the present invention, it is possible to provide a measurement method for analyzing a sample containing a biopolymer having a cis-diol structure (for example, a glycated protein (for example, glycated hemoglobin), ribonucleic acid, or polysaccharide). In another embodiment, by using the liquid chromatography column of the present invention, it is possible to provide a method for adsorbing and / or purifying ribonucleic acid. In another embodiment, by using the liquid chromatography column of the present invention, it is possible to provide a method for separating and / or removing ribonucleic acid from a sample containing deoxyribonucleic acid and ribonucleic acid.

[0041] By using the liquid chromatography column of the present invention, broadening of the peak shape of a chromatogram of a biopolymer having a cis-diol moiety is suppressed, even when a neutral to weakly acidic eluent is used, which has previously been difficult to separate, and it has become possible to efficiently separate a biopolymer having a cis-diol moiety from a component not having a cis-diol moiety.

[0042] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. (Example 1) HPLC measurement was carried out using a column packed with a separating agent carrier in which a 2-aminopyridine-5-boronic acid derivative was supported on the surface of a silica gel carrier.

[0043] 30 g of spherical silica gel (7 μm) and 15 g of 3-glycidyloxypropyltrimethoxysilane were stirred in 30 g of phosphate buffer (pH 7.0) at 100° C. After 24 hours, the reaction solution was separated to obtain silica gel having epoxide moieties on its surface.

[0044] 10 g of the resulting silica gel particles having epoxide moieties, 400 mg of 2-aminopyridine-5-boronic acid pinacol ester (Mw: 220.08), and 5.5 mg of aminoethanol (Mw: 61.08) were stirred in 40 g of phosphate buffer (pH 7.0) at 40° C. After 24 hours, the reaction solution was separated to obtain silica gel having pyridylboronic acid and moieties for promoting boronic acid ester formation and exchange on its surface.

[0045] The obtained separating agent carrier was packed into a column with an inner diameter of 4.6 mm and a length of 3.5 cm, and blood components were separated under the following conditions. Eluent Solution A: 100 mM phosphate buffer (pH 6.5) Solution B: 100 mM phosphoric acid, 100 mM d-sorbitol mixed solution (pH 6.5) Flow rate: 1.0 mL / min Monitor wavelength: 415 nm Step gradient: Eluent Solution A (0-1.0 min), Eluent Solution B (1.0-4.0 min) When the column of Example 1 was used, non-glycated components and glycated components could be separated, as shown in Figure 1, and the peak shapes were good. (Example 2) The silica gel having epoxide moieties on its 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 reaction solution was separated to obtain silica gel having, on its surface, pyridylboronic acid and moieties promoting boronic acid ester formation and exchange.

[0046] The obtained separating medium carrier was packed into the same column as that used in Example 1 above, and separation was carried out under the same conditions.

[0047] When the column of Example 2 was used, the non-glycated and glycated components could be separated, as shown in Figure 2, and the peak shapes were good. (Example 3) The silica gel having epoxide moieties on its 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 reaction solution was separated to obtain silica gel having pyridylboronic acid and moieties promoting boronic acid ester formation and exchange on its surface.

[0048] The obtained separating medium carrier was packed into the same column as that used in Example 1 above, and separation was carried out under the same conditions.

[0049] When the column of Example 3 was used, the non-glycated and glycated components could be separated, as shown in Figure 3, and the peak shapes were good. (Example 4) The silica gel having epoxide moieties on its 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 reaction solution was separated to obtain silica gel having pyridylboronic acid and moieties promoting boronic acid ester formation and exchange on its surface.

[0050] The obtained separating medium carrier was packed into the same column as that used in Example 1 above, and separation was carried out under the same conditions.

[0051] When the column of Example 4 was used, the non-glycated and glycated components could be separated, as shown in Figure 4, and the peak shapes were good. (Example 5) The silica gel having epoxide moieties on its 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 stirred in 40 g of phosphate buffer (pH 7.0) at 40°C. After 24 hours, the reaction solution was separated to obtain silica gel having pyridylboronic acid and moieties promoting boronic acid ester formation and exchange on its surface.

[0052] The obtained separating medium carrier was packed into the same column as that used in Example 1 above, and separation was carried out under the same conditions.

[0053] When the column of Example 5 was used, the non-glycated components and the glycated components could be separated with good peak shapes, as shown in Figure 5. (Example 6) In order to investigate the quantitative determination of glycated hemoglobin, HPLC measurements were carried out on samples with different amounts of glycated hemoglobin using the column prepared in Example 2.

[0054] HPLC measurements were performed under 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. Specifically, Samples 1 and 5 were mixed at a ratio of 3:1 (No. 2), 1:1 (No. 3), and 1:3 (No. 4). As shown in Figure 6, the measurement results for the glycated hemoglobin percentage for the five samples were linear, demonstrating that the liquid chromatography separation support of the present invention exhibits sufficient quantitative performance. (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, Ltd.) 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 added to the separable flask and polymerized for 6 hours with stirring. The resulting reaction solution was filtered through a glass filter, washed with warm water and then with acetone, and further classified using a sieve to obtain crosslinked polymer particles having hydroxyl groups. 200 g (wet weight) of the resulting crosslinked polymer particles having hydroxyl groups was dispersed in 300 g of water in a 500 mL separable flask, and 50 g of epichlorohydrin was added and heated to 40°C. 40 g of 48% sodium hydroxide was gradually added to introduce epoxy groups.

[0055] The resulting particles were washed with water, acetone, and water, yielding epoxidized crosslinked polymer particles. 25 g (dry weight) of the crosslinked polymer particles were dispersed in 225 mL of ion-exchanged water, and 4.5 g of 2-aminopyridine-5-boronic acid pinacol ester was added and dissolved. 25 mL of 0.5 M aqueous sodium hydroxide solution was added, and the mixture was heated at 70°C for 2 hours. The reaction solution was filtered through a glass filter and further washed with three volumes of 80% aqueous ethanol, three volumes of ion-exchanged water, three volumes of 0.1 M aqueous hydrochloric acid, and three volumes of ion-exchanged water, yielding 2-aminopyridine-5-boronic acid-immobilized particles with hydroxyl groups bound to the substrate. The resulting particles were packed into an empty column with an inner diameter of 7.5 mm and a column length of 7.5 cm.

[0056] Using the obtained column, crude RNA was purified under the following conditions: Eluent A: 0.15 M sodium chloride, 0.1 M phosphate buffer (pH 6.0); Solution B: 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: 260 nm. The sample, Ribonucleic acid from yeast (Fujifilm Wako Pure Chemical Industries, Ltd., product number 185-00202), was dissolved in Eluent A to a concentration of 20 g / L, and 20 μL was injected into the column.

[0057] The resulting chromatogram (Figure 7) confirmed the elution of the fraction obtained by passing eluent B through the column, but not with the acidic eluent C. This indicates that adsorption is possible at neutral pH without the need for alkaline conditions for adsorption, and that elution does not require acidic conditions, enabling RNA purification under mild conditions. The absorbance of this fraction was measured, revealing that the A260 / A280 ratio, which was 1.69 before purification, increased to 2.05, indicating high purity. Furthermore, 120 μg of RNA components were recovered from 400 μg of crudely purified DNA. (Example 8) The elution behavior was confirmed in the same manner as in Example 7, except that the sample used was Merck DNA from Salmon testes D-1626, dissolved in eluent A to a concentration of 10 g / L. The resulting chromatogram (Figure 8) confirmed that DNA was eluted without being retained on the column, demonstrating that RNA, an impurity present in DNA, could be easily and effectively removed. Example 9: As a sample, Blue Dextran D5751 manufactured by Merck 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 monitoring wavelength was set to 620 nm. The obtained chromatogram ( FIG. 9 ) revealed that dextran was adsorbed at neutral pH and could be eluted under mild conditions without the need for acidic elution.

[0058] Comparative Example 1 For comparison, a column using an existing separation carrier was used. Boronate-5PW manufactured by Tosoh Corporation, which carried a phenylboronic acid derivative, was packed into the same column as that used in Example 1, and separation was carried out under the same conditions.

[0059] When the column of Comparative Example 1 was used, non-glycated components were not eluted due to non-specific adsorption when eluent A was passed through, as shown in the chromatogram in Figure 10, but were eluted together with glycated components when eluent B was passed through. (Comparative Example 2) The silica gel having epoxide moieties on its surface, prepared in Example 1, was used. 10 g of silica gel particles having epoxide moieties and 400 mg of 2-aminophenyl-4-boronic acid were stirred in 40 g of phosphate buffer (pH 7.0) at 40°C. After 24 hours, the reaction solution was separated to obtain silica gel having phenylboronic acid on its surface.

[0060] The obtained separating medium carrier was packed into the same column as that used in Example 1 above, and separation was carried out under the same conditions.

[0061] As shown in Figure 11, when the column of Comparative Example 2 was used, similar to Comparative Example 1, non-glycated components were not eluted due to non-specific adsorption when eluent A was passed through, but were eluted together with glycated components when eluent B was passed through. (Comparative Example 3) The silica gel having epoxide moieties on its 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, the reaction solution was separated to obtain silica gel having pyridylboronic acid on its surface.

[0062] The obtained separating medium carrier was packed into the same column as that used in Example 1 above, and separation was carried out under the same conditions.

[0063] When the column of Comparative Example 3 was used, the non-glycated components and glycated components could be separated, as shown in Figure 12, but the peak for the glycated components was broad. (Comparative Example 4) Using a column packed with the gel prepared in Comparative Example 3, HPLC measurement was performed using Solution A, which has a higher pH than that of Example 1. Eluent Solution A: 100 mM phosphate buffer (pH 6.8) Solution B: 100 mM phosphoric acid, 100 mM d-sorbitol mixed solution (pH 6.5) Separation was performed under the same conditions as in Example 1, except for the eluent.

[0064] 13, when the pH of the eluent was increased, glycated hemoglobin was hardly retained and was eluted together with the non-glycated hemoglobin peak. This result shows that the pH of the eluent in Comparative Example 3 for the boronate ester bond between the gel prepared in Comparative Example 3 and glycated hemoglobin was sufficiently high, and that the coordinating functional groups introduced in Examples 1 to 3 did not lower the optimal pH for boronate ester formation.

Claims

1. (1) A functional group containing a pyridylboronic acid derivative represented by the following general formula (II): 【Chemistry 1】 [In formula (II), X is -NH-, -O-, -CH(OH)-NH-, or -CH(OH)-O-; R and R' each independently represent a hydrogen atom, an alkyl group, or a protecting group for boronic acid, and R and R' may be bonded together to form a ring; * indicates the binding site with the particle. (2) A functional group represented by the following general formula (III): 【Chemistry 2】 [In formula (III), 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, and is substituted with an amino group or a hydroxyl group at X″; * indicates particle binding site.] But, A separating material carrier for liquid chromatography, characterized in that it is covalently bonded to the surface of silica gel or crosslinked polymer particles.

2. 2. The liquid chromatography separation support according to claim 1, wherein the crosslinked polymer is a crosslinked polysaccharide.

3. 3. The liquid chromatography separation support according to claim 1 or 2, wherein the crosslinked polymer is a copolymer of at least one monomer selected from the group consisting of (meth)acrylic acid esters having a hydroxyl group or an amino group on a side chain, (meth)acrylamides having a hydroxyl group or an amino group on a side chain, vinylpyridines, and vinylimidazoles, and a polyfunctional unsaturated monomer.

4. 2. The separating material carrier for liquid chromatography according to claim 1, wherein the particles are spherical particles having an average particle size of 1 to 200 μm.

5. 5. The separating material carrier for liquid chromatography according to claim 4, wherein the particles are porous particles having pores with an average size of 10 to 500 nm.

6. A method for producing the liquid chromatography separation support according to claim 1, comprising the steps of: A method for obtaining the liquid chromatography separation carrier according to claim 1, comprising reacting a first surface modifier for introducing the functional group (1) and a second surface modifier for introducing the functional group (2) simultaneously or separately with silica gel or crosslinked polymer particles having a functional group for linking on the surface thereof. A method comprising:

7. The method according to claim 6, wherein the second surface modifier is reacted in an amount of 1 to 200 mol % relative to the first surface modifier.

8. The method according to claim 6 or 7, wherein the linking functional group is an epoxy group, an amino group, a hydroxyl group, a carboxyl group, or a formyl group.

9. The method according to claim 6 or 7, wherein the linking functional group is an epoxy group.

10. A separation carrier for liquid chromatography obtained by the method according to claim 6.

11. A liquid chromatography column packed with the separating material carrier for liquid chromatography according to claim 1.

12. A measurement method for analyzing a sample containing a biopolymer having a cis-diol structure, using the liquid chromatography column according to claim 11.

13. The method according to claim 12, wherein the biopolymer having a cis-diol structure is a glycosylated protein, a ribonucleic acid, or a polysaccharide.

14. The method according to claim 13, wherein the glycated protein is glycated hemoglobin.

15. A method for adsorbing and / or purifying ribonucleic acid, using the liquid chromatography column according to claim 11.

16. A method for separating and / or removing ribonucleic acid from a sample containing deoxyribonucleic acid and ribonucleic acid, using the liquid chromatography column according to claim 11.

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