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

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

Technical Field

[0001] The present invention relates to a separating agent carrier, a column, and a separation purification and measurement method capable of adsorbing and separating biopolymers having a cis-diol structure in a biological sample, such as glycated proteins, ribonucleic acids (RNAs), and saccharides, by liquid chromatography.

Background Art

[0002] Boronic acid affinity chromatography is used for the adsorption and separation of biopolymers having a cis-diol structure, such as glycated proteins, RNAs, and saccharides (Non-Patent Documents 1, 2, and 3). Among these, it is utilized in diabetes diagnosis because it can separate glycated hemoglobin that reflects the average blood glucose level during the past one to two months. In particular, in the diagnosis of diabetes in patients with hemoglobin abnormalities (abnormal hemoglobinosis, thalassemia), which is not easy with the currently widely used analysis using cation exchange chromatography, the boronic acid affinity method is used because glycated hemoglobin can be separated without being affected.

[0003] In the separation using the boronic acid affinity method, the boronic acid ester formation reaction between phenylboronic acid immobilized on the surface of the separating agent and the cis-diol of the sample is utilized. That is, in separation, analysis, and purification including diabetes diagnosis using high performance liquid chromatography (HPLC), first, while passing the first eluent, a boronic acid ester is formed by the reaction between the boronic acid site and the glycated protein, and only samples having no cis-diol structure, such as non-glycated proteins, are eluted. Next, a second eluent having a cis-diol such as sorbitol is passed, and samples having a cis-diol structure, such as glycated proteins, are eluted by a boronic acid ester exchange reaction. At this time, a basic eluent with a pH of 8 or higher is used to promote the formation and exchange reaction of the boronic acid ester (Patent Documents 1 and 2). At this time, it is known that non-glycated proteins may be non-specifically adsorbed when an eluent with a lower pH is used for separation (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 the cis-diol moiety. For example, a packing material supporting phenylboronic acid, in which a nitrogen or oxygen atom capable of coordinating with the boronic acid moiety is positioned at the ortho position of the boron atom on the benzene ring, can retain the cis-diol even at low pH. However, the boronic acid that can be used in this method is not directly available and must be synthesized through inefficient multi-step reactions (Non-Patent Literature 4).

[0005] In response to this, a filler was developed in which a coordinating functional group is positioned near phenylboronic acid supported on the filler surface. While this method is characterized by the use of readily available raw materials, the pH required for retaining the cis-diol is higher compared to the case where both boronic acid and a coordinating functional group are present on the benzene ring (Non-Patent Literature 5).

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

[0007] For the reasons stated above, columns using boronic acid-supported packing materials that can retain cis-diol moieties at low pH are not practical and are rarely used for liquid chromatography separation, analysis, and purification of biomacromolecules such as glycated proteins and RNA. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Japanese Patent Application Publication No. 5-5731 [Patent Document 2] Japanese Patent Publication No. 2002-139481 [Patent Document 3] Japanese Patent Publication No. 2007-284425 [Non-patent literature]

[0009] [Non-Patent Document 1] YuCai Li, Eva Linne Larsson,J. Chromatography A.2001,909,137-145. [Non-Patent Document 2] A. Gabriela Gomes, Ana. M. Azevedo,J. Chromatography A.2010,1217,2262-2266. [Non-Patent Document 3] Serap Senel, Colloids and Surfaces A.2003,219,17-23. [Non-Patent Document 4] H.Li, Y.Liu, J.Liu, Z.Liu,Chem.Commun.2011,47,8169-8171. [Non-Patent Document 5] H.Li,Z.Liu,Trends Anal.Chem.2012,37,148-161 [Non-Patent Document 6] D.Li,et al,J.Chromatogr.A,2014,1339,103-109 [Overview of the project] [Problems that the invention aims to solve]

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

[0011] As a result of diligent research, the inventors have discovered a separation agent carrier for boronic acid affinity chromatography that can retain and efficiently elute biomacromolecules having a cis-diol structure using a neutral to weakly acidic eluent, thereby completing the present invention.

[0012] In a separation agent carrier supported by pyridylboronic acid, which can retain saccharified components even under neutral to weakly acidic conditions, we investigated a surface structure that efficiently retains cis-diol components and elutes them through boronic acid transesterification. As a result, we found that good results were obtained when a coordinating functional group, which normally lowers the pH of the eluent necessary for boronic acid ester formation between boronic acid and cis-diol components, was placed near the boronic acid. Specifically, compared to a separation agent carrier with only pyridylboronic acid, a separation agent carrier with pyridylboronic acid and a coordinating functional group efficiently retains cis-diol components and elutes them using boronic acid transesterification without changing the pH necessary for retaining cis-diol components, resulting in a better chromatogram shape. This led to the completion of the present invention.

[0013] That is, a separation agent carrier for boronic acid affinity chromatography has a pyridylboronic acid derivative as a binding site with a biopolymer having a cis-diol structure on the surface of, for example, silica gel or a crosslinked polymer, and a functional group having an amino group or a hydroxyl group at its terminus as a site for promoting boronic acid ester formation and exchange is covalently bonded in the vicinity thereof, and thus a separation agent carrier for boronic acid affinity chromatography has been developed.

[0014] Hereinafter, the present invention will be described in detail.

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

[0016] [1-2] The separation agent 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 a poly(ethyleneoxy)ethyl group having an amino group or a hydroxyl group at its terminus are covalently bonded to the surface of silica gel or a crosslinked polymer.

[0017] [Chemical formula]

[0018] (In formula (I), 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. R and R' are each independently a hydrogen atom or an alkyl group, and may form a ring together. A is an alkyl group having 2 to 10 carbon atoms or a poly(ethyleneoxy)ethyl group.). Note that the substitution position of the boronic acid site and the binding site (X) on the pyridine ring of the pyridylboronic acid derivative is not limited.

[0019] [1-3] The alkyl group or poly(ethyleneoxy)ethyl group having an amino group or a hydroxyl group at the terminal, which is the promoting site for boronic acid ester formation and exchange described above, is preferably 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 site.

[0020] [1-4] The separation agent carrier of the present invention can be filled into a tube having a cavity inside and used as a column for liquid chromatography. By liquid chromatography using the column, a biopolymer having a cis-diol group can be separated from a biological sample. In particular, glycated hemoglobin can be analyzed from a blood sample.

[0021] [1-5] The separation agent carrier of the present invention can be used for forming a covalent bond such as an epoxide site introduced into silica gel or a crosslinkable polymer, and can be produced by the reaction between a site for forming a covalent bond and a site for forming a covalent bond such as an amino group of a promoter for ester formation and exchange with a pyridylboronic acid derivative.

[0022] Further, the present application includes the inventions of the following aspects. [2-1] (1) A functional group containing a pyridylboronic acid derivative represented by the following general formula (II):

Chemical formula

Chemical formula

[0023] The separation agent carrier of the present invention can be used as a liquid chromatography column by packing it into a column tube. Analysis using this column allows for the separation of components with and without cis-diol moieties, even when using neutral to weakly acidic eluents. For example, it can be used for separating glycated proteins from non-glycated proteins, separating RNA from contaminants, and separating and / or removing RNA from samples containing deoxyribonucleic acid (DNA) and ribonucleic acid. It is particularly useful for separating hemoglobin from glycated hemoglobin. Furthermore, [Brief explanation of the drawing]

[0024] [Figure 1] This figure shows an example of a chromatogram obtained by measuring a blood sample in Example 1. [Figure 2] This figure shows an example of a chromatogram obtained by measuring a blood sample in Example 2. [Figure 3] This figure shows an example of a chromatogram obtained by measuring a blood sample in Example 3. [Figure 4] This figure shows an example of a chromatogram obtained by measuring a blood sample in Example 4. [Figure 5] This figure shows an example of a chromatogram obtained by measuring a blood sample in Example 5. [Figure 6] This is a plot of glycated hemoglobin % in Example 6, where quantitative analysis was conducted. [Figure 7] This figure shows an example of a chromatogram obtained by adsorption separation of crude purified RNA in Example 7. [Figure 8] This figure shows an example of a chromatogram obtained by adsorbing and removing RNA from a DNA sample in Example 8. [Figure 9] This figure shows an example of a chromatogram obtained by adsorption separation of polysaccharides in Example 9. [Figure 10] This figure shows an example of a chromatogram obtained by measuring a blood sample in Comparative Example 1. [Figure 11] This figure shows an example of a chromatogram obtained by measuring a blood sample in Comparative Example 2. [Figure 12] This figure shows an example of a chromatogram obtained by measuring a blood sample in Comparative Example 3. [Figure 13] This figure shows an example of a chromatogram obtained by measuring a blood sample in Comparative Example 4. [Modes for carrying out the invention]

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

[0026] In one embodiment, the present invention provides a separation agent carrier for liquid affinity chromatography. (1) Functional groups containing pyridylboronic acid derivatives represented by the following general formula (II): [ka] [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 boronic acid protecting group, and R and R' may form a ring together. * indicates the binding site with the particle. ]; and (2) Functional group represented by the following general formula (III): [ka] [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 in X''. * indicates the particle binding site. Toga, The present invention provides a separation agent carrier for liquid chromatography, characterized by being covalently bonded to the surface of silica gel or cross-linked polymer particles.

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

[0028] In the above-mentioned 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 separation agent support, and these functional groups may be bonded at any position on the pyridine ring. Examples of functional groups that can be used for surface modification of pyridylboronic acid and the separation agent support include hydroxyl groups, hydroxymethyl groups, amino groups, aminomethyl groups, carboxyl groups, and formyl groups. 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 boronic acid ester substituted with a protecting group such as a pinacol ester to form a ring, or other known boronic acid protecting groups, such as diaminonaphthaleneamide, MIDA ester, trifluoroborate salt, tecol ester, neopentyl glycol ester, pinanediol ester, biscyclohexyldiol ester, or MPMP ester. 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 can 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, and 6-carboxypyridine-3-boronic acid. In particular, 2-aminopyridine-5-boronic acid pinacol ester is preferred.

[0029] The functional group represented by the following general formula (III), which is the site that promotes boronic acid ester formation and exchange: [ka] [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 in X''. * indicates the particle binding site. The second surface modifier for introducing the above-mentioned nitrogen or oxygen atom only needs to have a linking site at the end that is not substituted with the above-mentioned nitrogen or oxygen atom, which can be used for surface modification with the separation agent carrier (particles). This linking site only needs to be usable for a covalent bond formation reaction. Examples of such functional groups include hydroxyl groups, amino groups, carboxyl groups, and formyl groups, but there are no particular restrictions. Specific examples of the second surface modifier include 1,3-propanediol, propylene glycol, dipropylene glycol, tripylene glycol, 1,4-butanediol, glycerol, polyglycerol, aminoethanol, aminopropanol, aminobutanol, and 2-(2-(2-aminoethoxy)ethoxy)acetic acid, but it is desirable that it has an alkyl group with 2 to 6 carbon atoms or a poly(ethyleneoxy)ethyl group.

[0030] The two surface modifiers described above are bonded to a separation agent composed of known silica gel or a crosslinked polymer. In this case, a known separation agent having a functional group for linking with the modifier on its surface can be used. Examples of functional groups for linking include epoxy groups, amino groups, hydroxyl groups, carboxyl groups, and formyl groups, but there are no particular restrictions. The two surface modifiers described above may be reacted simultaneously or separately with silica gel or crosslinked polymer particles having a functional group for linking on their surface. It is preferable to react the first surface modifier with the second 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] The separation agent used for modification, such as silica gel or cross-linked polymer, has no restrictions on particle size if it is in the case of particles, but it is preferable that the average particle size is 1 to 200 μm, with spherical particles having an average particle size of 1 to 10 μm for analytical applications and spherical particles with an average particle size of 10 to 200 μm for preparative applications. Furthermore, for preparative applications, it is preferable that the particles be porous with pores in order to adsorb a large amount of sample at once, and the size of the pores is preferably on average 10 to 500 nm to match the sample.

[0032] As the crosslinked polymers mentioned above, crosslinked polysaccharide particles using cellulose or agarose, (meth)acrylic acid esters having hydroxyl or amino groups in their side chains, (meth)acrylamide monomers having hydroxyl or amino groups in their side chains, vinylpyridines, and copolymer particles of vinylimidazole and polyfunctional unsaturated monomers can be used. In particular, particles using (meth)acrylic acid esters and (meth)acrylamide monomers are preferred because the amount of hydroxyl and amino groups on the particle surface can be easily controlled. Examples of monomers having alcohol-based hydroxyl groups include (meth)acrylic acid esters, such as monomers in which the ester portion 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 containing an amino group include vinylpyridines, vinylimidazoles, N-(2-aminoethyl)acrylamide, and 2-(dimethylamino)ethyl (meth)acrylate. Examples of polyfunctional unsaturated monomers include (poly)ethylene glycol-di(meth)acrylate, (poly)glycerol-poly(meth)acrylate, (poly)propylene glycol-poly(meth)acrylate, and divinylbenzene.

[0033] Crosslinked polymers having hydroxyl groups can be used by partially introducing functional groups necessary for introducing pyridylboronic acid, such as epoxy groups or carboxylic acids.

[0034] Furthermore, a method using a crosslinked polymer with an epoxy group-containing (meth)acrylic acid monomer, such as glycidyl methacrylate, as the monomer is preferable because a hydroxyl group or an amino group and a pyridylboronic acid compound can be simultaneously introduced to the epoxy groups of the particles, or by reacting the remaining epoxy with water, a diol compound, or an amine after introducing the pyridylboronic acid compound, thereby introducing a hydroxyl group or an amino group, respectively.

[0035] Furthermore, 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 a crosslinked polymer, the introduction of pyridylboronic acid and the introduction of hydroxyl groups through the reaction of epoxy groups with water can be easily achieved by reacting an aminopyridylboronic acid compound in an alkaline aqueous solution.

[0036] There are no restrictions on the method of bonding the two surface modifiers described above to the separation agent support, and known bond formation reactions can be used. For example, using the linking sites described above, covalent bonds can be formed via epoxide ring-opening reactions, amide bond formation reactions, carbamate formation reactions, imine formation reactions, etc., but there are no restrictions on the method of bond formation.

[0037] A column tube made of metal, resin, glass, or the like with a hollow interior may be used, and a column filled with the separation agent carrier can be used as a liquid chromatography column, for example, for HPLC measurements.

[0038] When performing HPLC measurements, a column packed with the separation agent carrier can be used. Two or more eluents can be used, including an eluent that does not contain components having a cis-diol moiety and an eluent that contains cis-diol moieties such as galactose, fructose, and sorbitol.

[0039] It is preferable to use a step gradient, linear gradient, or similar method for creating gradients.

[0040] In one embodiment, by using the liquid chromatography column of the present invention, a measurement method for analyzing a sample containing a biomacromolecule having a cis-diol structure (e.g., glycated protein (e.g., glycated hemoglobin), ribonucleic acid, or polysaccharides) can be provided. In another embodiment, by using the liquid chromatography column of the present invention, a method for adsorbing and / or purifying ribonucleic acid can be provided. In yet another embodiment, by using the liquid chromatography column of the present invention, a method for separating and / or removing ribonucleic acid from a sample containing deoxyribonucleic acid and ribonucleic acid can also be provided.

[0041] By using the liquid chromatography column of the present invention, even when using neutral to weakly acidic eluents, which have previously been difficult to separate, the broadening of the peak shape of the chromatogram of biopolymers having cis-diol moieties is suppressed, and it becomes possible to efficiently separate biopolymers having cis-diol moieties from components that do not have cis-diol moieties. [Examples]

[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 measurements were performed using a column packed with a separation agent carrier on 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 with epoxide moieties on its surface.

[0044] The obtained 10 g of 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, and silica gel having pyridylboronic acid and boronic acid ester formation and exchange promoting sites on its surface was obtained.

[0045] The obtained separation 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: Mixture of 100 mM phosphoric acid and 100 mM d-sorbitol (pH 6.5) Flow rate: 1.0mL / min Monitor wavelength: 415nm Step gradient: Eluent A solution (0-1.0min), Eluent B solution (1.0-4.0min) When using the column from Example 1, non-glycated and glycated components could be separated as shown in Figure 1, and the peak shape was good. (Example 2) 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 pyridylboronic acid and moieties that promote boronic acid ester formation and exchange on its surface.

[0046] The obtained separation agent carrier was packed into the same column as used in Example 1, and separation was performed under the same conditions.

[0047] When using the column from Example 2, non-glycated and glycated components could be separated as shown in Figure 2, and the peak shape was also good. (Example 3) 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 that promote boronic acid ester formation and exchange on its surface.

[0048] The obtained separation agent carrier was packed into the same column as used in Example 1, and separation was performed under the same conditions.

[0049] When using the column from Example 3, non-glycated and glycated components could be separated as shown in Figure 3, and the peak shape was also good. (Example 4) 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 that promote boronic acid ester formation and exchange on its surface.

[0050] The obtained separation agent carrier was packed into the same column as used in Example 1, and separation was performed under the same conditions.

[0051] When using the column from Example 4, non-glycated and glycated components could be separated as shown in Figure 4, and the peak shape was also good. (Example 5) 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 boronic acid ester formation and exchange promoting sites on its surface.

[0052] The obtained separation agent carrier was packed into the same column as used in Example 1, and separation was performed under the same conditions.

[0053] When using the column of Example 5, non-glycated and glycated components can be separated as shown in Figure 5, and the peak shape is also good. (Example 6) To investigate the quantitative properties of glycated hemoglobin, HPLC measurements were performed on samples with different levels of glycated hemoglobin using the column prepared in Example 2.

[0054] HPLC measurements were 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. Specifically, samples 1 and 5 were mixed in ratios of 3:1 (No. 2), 1:1 (No. 3), and 1:3 (No. 4). As shown in Figure 6, the measurement results of glycated hemoglobin % in the five samples showed linearity, indicating that the liquid chromatography separation agent carrier of the present invention exhibits sufficient quantitative accuracy. (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 heated to 60 degrees Celsius. 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 acetone, and further classified into particles with a size of 40 to 100 μm using a sieve to obtain crosslinked polymer particles with hydroxyl groups. 200 g (wet weight) of the obtained crosslinked polymer particles with hydroxyl groups were dispersed in 300 g of water in a 500 mL separable flask, and 50 g of epichlorohydrin was added and heated to 40 degrees Celsius. 40 g of 48% sodium hydroxide was gradually added to introduce epoxy groups.

[0055] The obtained particles were washed in the order of water, acetone, and water to obtain epoxidized crosslinked polymer particles. 25 g (dry weight) of the 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 and further washed in the order of 3 times the volume of 80% ethanol aqueous solution, 3 times the volume of deionized water, 3 times the volume of 0.1 M hydrochloric acid aqueous solution, and 3 times the volume of deionized water to obtain 2-aminopyridine-5-boronic acid immobilized particles having hydroxyl groups bound to the substrate. The obtained 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 Solution A: 0.15 M sodium chloride, 0.1 M phosphate buffer (pH 6.0) Solution B: 0.1M sorbitol, 0.15M sodium chloride, 0.1M phosphate buffer (pH 6.0) Solution C 20mM phosphate buffer (pH1.9) Step gradient:: Eluent A (0-16.6 minutes), Eluent B (16.6-33.1 minutes), Eluent C (33.1-49.7 minutes) Flow rate: 1.0mL / min Monitor wavelength: 260nm The sample, Ribonucleic acid from yeast (product code 185-00202) from Fujifilm Wako Pure Chemical Industries, was dissolved in eluent A to a concentration of 20 g / L and injected into a 20 μL column.

[0057] The resulting chromatogram (Figure 7) confirmed the elution when eluted with eluent B, while no elution was observed with the acidic eluent C. This indicates that adsorption does not require alkalinity and occurs in a neutral environment, and furthermore, elution does not require acidity, allowing RNA purification to be performed under mild conditions. Measurement of the absorbance of this fraction showed that A260 / A280 increased from 1.69 before purification to 2.05, indicating high purity purification. In addition, 120 μg of RNA component was recovered from 400 μg of crude purification. (Example 8) The elution behavior was confirmed in the same manner as in Example 7, except that Merck's DNA from Salmon Testes D-1626 was dissolved in eluent A to a concentration of 10 g / L as the sample. From the obtained chromatogram (Figure 8), it was confirmed that the DNA was not retained in the column and was eluted, indicating that RNA, which is an impurity present in the DNA, can be removed simply and effectively. (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. From the obtained chromatogram (Figure 9), it was found that dextran was adsorbed in a neutral solution and could be eluted under mild conditions without the need for acidity.

[0058] (Comparative Example 1) For comparison, a column using an existing separation agent support, Boronate-5PW manufactured by Tosoh Corporation and supported with a phenylboronic acid derivative, was packed into the same column as in Example 1, and separation was performed under the same conditions.

[0059] When the column of Comparative Example 1 was used, as shown in the chromatogram in Figure 10, the non-saccharified components were not eluted because they were non-specifically adsorbed when eluent A was passed through, but they were eluted together with the saccharified components when eluent B was passed through. (Comparative Example 2) 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 separation agent carrier was packed into the same column as used in Example 1, and separation was performed under the same conditions.

[0061] As shown in Figure 11, when the column of Comparative Example 2 was used, similar to Comparative Example 1, the non-saccharified components were not eluted when eluent A was passed through because they were non-specifically adsorbed, but they were eluted together with the saccharified components when eluent B was passed through. (Comparative Example 3) 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 separation agent carrier was packed into the same column as used in Example 1, and separation was performed under the same conditions.

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

[0064] As shown in Figure 13, when the pH of the eluent was increased, glycated hemoglobin was hardly retained and was eluted along with the non-glycated hemoglobin peak. From these results, it can be seen that the pH of the eluent in Comparative Example 3 was sufficiently high for boronic acid ester bonding between the gel prepared in Comparative Example 3 and glycated hemoglobin, and that the coordinating functional groups introduced in Examples 1 to 3 did not lower the optimal pH for boronic acid ester formation.

Claims

1. (1) Functional groups containing pyridylboronic acid derivatives 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' are each independently a hydrogen atom, an alkyl group, or a boronic acid protecting group, and R and R' may form a ring together. * indicates the binding site with the particle. ]; and (2) 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 in X''. * indicates the particle binding site. Toga, A separation agent carrier for liquid chromatography, characterized by being covalently bonded to the surface of silica gel or cross-linked polymer particles.

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

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

4. The separation agent carrier for liquid chromatography according to claim 1, wherein the particles are spherical particles with an average particle diameter of 1 to 200 μm.

5. The separation agent 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 a separation agent carrier for liquid chromatography according to claim 1, A separation agent carrier for liquid chromatography according to claim 1 is obtained by simultaneously or separately reacting a first surface modifier for introducing the functional group (1) and a second surface modifier for introducing the functional group (2) with silica gel or crosslinked polymer particles having a functional group for linking on their surface. Methods that include...

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

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

10. A separation agent carrier for liquid chromatography obtained by the method described in claim 6.

11. A liquid chromatography column packed with the liquid chromatography separation agent carrier described in claim 1.

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

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

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

15. A method for adsorbing and / or purifying ribonucleic acid using a 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 described in claim 11.

Images