WO2022244890A1

WO2022244890A1 - Separating agent carrier for boronate affinity chromatography, column, and measurement method employing same

Info

Publication number: WO2022244890A1
Application number: PCT/JP2022/021144
Authority: WO
Inventors: 友啓目黒; 和昭村中
Original assignee: 東ソー株式会社
Priority date: 2021-05-21
Filing date: 2022-05-23
Publication date: 2022-11-24
Also published as: JPWO2022244890A1

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

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

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.

Boronic acid affinity chromatography is used for adsorption separation of biopolymers having a cis-diol structure, such as glycated proteins, RNA, and sugars (

Non-Patent Documents

1, 2, 3). In particular, since 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. In particular, 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. Next, 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. At this time, 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). At this time, 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 (Patent Document 3).

In contrast, various attempts have been made to lower the pH of the eluent used for the reaction between the boronic acid and the cis-diol site. For example, phenylboronic acid-loaded packings in which a nitrogen or oxygen atom capable of coordinating a boronic acid moiety is placed ortho to the boron atom on the benzene ring can retain cis-diols even at low pH. However, boronic acids that can be used in this method cannot be obtained directly and are synthesized through inefficient multi-step reactions (Non-Patent Document 4).

In contrast, a filler was developed in which a coordinating functional group was placed near the phenylboronic acid supported on the surface of the filler. This method is characterized by the use of easily available raw materials, but compared to the case of having both a boronic acid and a coordinating functional group on the benzene ring, the pH required to retain the cis-diol is higher ( Non-Patent Document 5).

On the other hand, 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). For example, a filler in which pyridylboronic acid, in which one of the carbon atoms in the benzene ring is replaced with a nitrogen atom, is modified on the carrier surface via the nitrogen atom in the pyridine ring retains cis-diol even at low pH (pH 5-6). reportedly possible. However, when these boronic acids capable of retaining the cis-diol moiety at low pH are used, the bond between the boronic acid and the cis-diol moiety is strong, so the cis-diol component can be eluted under strongly acidic conditions. hydrolysis of is utilized. At this time, when elution is performed using boronic acid transesterification using d-sorbitol described above under neutral to weakly acidic conditions, there is concern that the peak shape of the chromatogram may become broad due to the slow transesterification. be done. Therefore, it may not be suitable for separation, analysis, and purification of glycated proteins such as glycated hemoglobin, RNA, etc., which are biopolymers that are denatured or degraded under strongly acidic conditions.

From the above, it is not practical to use a column with a packing material loaded with boronic acid that can retain cis-diol moieties at low pH. , is rarely used for analysis and purification.

JP-A-5-5731 JP-A-2002-139481 JP 2007-284425 A

In the present invention, 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.

As a result of intensive studies, 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.

Investigating a surface structure that can efficiently retain a cis-diol component and elute the cis-diol component by boronic acid transesterification in a separation agent carrier carrying pyridylboronic acid that can retain glycated components even under neutral to weakly acidic conditions. did. As a result, good results were obtained when a coordinating functional group was placed near the boronic acid to lower the pH of the eluent normally required for boronate ester formation between the boronic acid and the cis-diol component. . Specifically, compared to a separating agent carrier incorporating only pyridylboronic acid, 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.

That is, the surface of 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. We have developed 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 present invention will be described in detail below.

[1-1] 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

[1-2] 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.

(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. A is an alkyl group having 2 to 10 carbon atoms or a poly(ethyleneoxy)ethyl group.) In the pyridylboronic acid derivative, the boronic acid site and the bonding site (X) on the pyridine ring can be substituted at any position.

[1-3] 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 %.

[1-4] 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. In particular, glycated hemoglobin can be analyzed from a blood sample.

[1-5] 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

Further, the present application includes inventions of the following aspects.
[2-1]
(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. ]; and (2) a functional group represented by the following general formula (III):

[In the 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 X″ is substituted with an amino group or a hydroxyl group,
* indicates the binding site of the particle. ]
and
A separating agent carrier for liquid chromatography, which is covalently bonded to the surface of silica gel or crosslinked polymer particles.
[2-2] The separating agent carrier for liquid chromatography according to item 2-1, wherein 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.
[2-4] 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 μm.
[2-5] The separating agent carrier for liquid chromatography 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 the separating agent carrier for liquid chromatography according to any one of items 2-1 to 2-5,
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. 2 simultaneously or separately to obtain the separating agent carrier for liquid chromatography 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 % with respect 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 carboxy 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 separating agent carrier for liquid chromatography obtained by the method according to any one of items 2-6 to 2-9.
[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.
[2-14] The measurement method according to item 2-13, wherein 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. In the analysis using this column, even when a neutral to weakly acidic eluent is used, components having cis-diol sites and those not having cis-diol sites can be separated. For example, 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 diagram showing an example of a chromatogram obtained by adsorbing and separating crudely purified RNA in Example 7. FIG. FIG. 10 is a diagram showing an example of a chromatogram of adsorption removal of RNA in a DNA sample in Example 8. FIG. 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 will be described in detail below. However, the present invention can be embodied in different forms, and is not limited to the embodiments and examples shown below.

In one embodiment, 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 (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 X″ is substituted with an amino group or a hydroxyl group,
* indicates the binding site of the particle. ]
and
Provided is a separating agent carrier for liquid chromatography characterized by being covalently bonded to the surface of silica gel or crosslinked polymer particles.

In one embodiment, 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.

In the pyridylboronic acid derivative described above, it is sufficient that the pyridine ring has a boronic acid site and a linking site used for surface modification of the separating agent carrier. may 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. 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, 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.

A functional group represented by the following general formula (III), which is a site promoting boronic acid ester formation and exchange:

[in the 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 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. It is sufficient that this linking site can be used for the covalent bond formation reaction. Examples of such 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. At this time, 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 %.

If the separating agent composed of silica gel or crosslinked polymer used for modification is a particle, the particle size is not limited, but the average particle size is 1 to 200 μm. Spherical particles having an average particle size of 10 to 200 μm 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.

Examples of the above-mentioned crosslinked polymer 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. In particular, 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. Examples of polyfunctional unsaturated monomers 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.

In addition, 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. Alternatively, 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.

In addition, commercially available 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.

There are no restrictions on the method of binding the above two surface modifiers to the separating agent carrier, and known bond forming reactions can be used. For example, 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.

As 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. .

When performing HPLC measurement, a column filled with the separating agent carrier can be used. As 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.

It is desirable to use a step gradient, linear gradient, etc. for the gradient.

In one embodiment, 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. In one embodiment, 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. In one embodiment, 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.

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.

EXAMPLES 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 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.

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, separation from the reaction solution was performed to obtain silica gel having epoxide sites on its surface.

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.

Using the obtained separating agent carrier packed in a column having an inner diameter of 4.6 mm and a length of 3.5 cm, blood components were separated under the following conditions.
Eluent A solution: 100 mM phosphate buffer (pH 6.5)
B solution: 100 mM phosphoric acid, 100 mM d-sorbitol mixed solution (pH 6.5)
Flow rate: 1.0 mL/min
Monitor wavelength: 415nm
Step gradient: eluent A solution (0-1.0 min), eluent B solution (1.0-4.0 min)
When the column of Example 1 was used, the non-saccharified component and the saccharified component could be separated as shown in FIG. 1, and the peak shape was good.
(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.

The obtained separating agent carrier was packed into a column similar to that used in Example 1 above, and separated under the same conditions.

When the column of Example 2 was used, the non-saccharified component and the saccharified component could be separated as shown in FIG. 2, and the peak shape was good.
(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.

When the column of Example 3 was used, the non-saccharified components and the saccharified components could be separated as shown in FIG. 3, and the peak shape was good.
(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.

When the column of Example 4 was used, the non-saccharified components and the saccharified components could be separated as shown in FIG. 4, and the peak shape was good.
(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.

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). As shown in 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 μm using a sieve to obtain crosslinked polymer particles having hydroxyl groups. In a 500 mL separable flask, 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.

Using the obtained column, crude 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
A sample, Ribonucleic acid from yeast (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., product number 185-00202), was dissolved in eluent A to a concentration of 20 g/L, and injected into a 20 μL column.

From the obtained chromatogram (Fig. 7), elution by eluent B was confirmed, and elution was not confirmed with eluent C, which is acidic. It was found that the purification of RNA can be carried out under mild conditions without the need of acidity for elution, and adsorption. When the absorbance of this fraction was measured, it was found that the A260/A280, which was 1.69 before purification, became 2.05, indicating that the purification can be performed with high purity. Also, 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 DNA from Salmon tests D-1626 manufactured by Merck Co., Ltd. was dissolved as a sample with the eluent A to a concentration of 10 g/L. From the obtained chromatogram (Fig. 8), it was confirmed that the DNA was eluted without being retained on the column, and it was found that RNA as an impurity present in the DNA could be easily and effectively removed.
(Example 9)
As a sample, 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.

(Comparative example 1)
As a column using an existing separating agent carrier for comparison, Boronate-5PW manufactured by Tosoh Corporation carrying a phenylboronic acid derivative was packed into a column similar to that used in Example 1 above, and under the same conditions. separation was performed.

When the column of Comparative Example 1 was used, as shown in the chromatogram in FIG. 10, the non-saccharified components were not eluted because they were non-specifically adsorbed when the eluent A was passed through, and were not eluted together with the saccharified components when the eluent B was passed. eluted.
(Comparative 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 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, separation from the reaction solution was performed to obtain silica gel having phenylboronic acid on its surface.

Even when the column of Comparative Example 2 is used as shown in FIG. 11, as in Comparative Example 1, the non-saccharified components are not eluted because they are non-specifically adsorbed when the eluent A is passed through, and the eluent B is not eluted. It was eluted together with the saccharified component during passage.
(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.

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.

As shown in FIG. 13, when the pH of the eluent was increased, almost no glycated hemoglobin was retained and eluted together with the non-glycated hemoglobin peak. From this result, the pH of the eluent of Comparative Example 3 in the boronic ester bond between the gel prepared in Comparative Example 3 and glycated hemoglobin was sufficiently high, and the coordinating functional group introduced in Examples 1 to 3 was boronic ester. It can be seen that the optimum pH for formation is not lowered.

Claims

(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. ]; and (2) a functional group represented by the following general formula (III):

[in the 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 X″ is substituted with an amino group or a hydroxyl group,
* indicates the binding site of the particle. ]
and
A separating agent carrier for liquid chromatography, which is covalently bonded to the surface of silica gel or crosslinked polymer particles.
The separating agent carrier for liquid chromatography according to claim 1, wherein the crosslinked polymer is a crosslinked polysaccharide.
The crosslinked polymer is at least one selected from the group consisting of a (meth)acrylic acid ester having a hydroxyl group or an amino group in a side chain, a (meth)acrylamide having a hydroxyl group or an amino group in a side chain, vinylpyridine, and vinylimidazole. 3. The separating agent carrier for liquid chromatography according to claim 1, which is a copolymer of the above monomers and a polyfunctional unsaturated monomer.
The separating agent carrier for liquid chromatography according to any one of claims 1 to 3, wherein the particles are spherical particles having an average particle diameter of 1 to 200 µm.
The separating agent carrier for liquid chromatography according to claim 4, wherein said particles are porous particles having pores with an average size of 10 to 500 nm.
A method for producing a separating agent carrier for liquid chromatography according to any one of claims 1 to 5,
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. A method comprising reacting two surface modifiers simultaneously or separately to obtain the separating agent carrier for liquid chromatography according to any one of claims 1 to 5.
The method according to claim 6, wherein the second surface modifier is reacted in an amount of 1 to 200 mol% with respect to the first surface modifier.
The method according to claim 6 or 7, wherein the linking functional group is an epoxy group, an amino group, a hydroxyl group, a carboxy group, or a formyl group.
The method according to claim 6 or 7, wherein the linking functional group is an epoxy group.
A separating agent carrier for liquid chromatography obtained by the method according to any one of claims 6 to 9.
A column for liquid chromatography packed with the separating agent carrier for liquid chromatography according to any one of claims 1 to 5 and 10.
A measurement method for analyzing a sample containing a biopolymer having a cis-diol structure using the liquid chromatography column according to claim 11.
The measurement method according to claim 12, wherein the biopolymer having a cis-diol structure is glycated protein, ribonucleic acid or polysaccharide.
The measurement method according to claim 13, wherein the glycated protein is glycated hemoglobin.
A method for adsorbing and/or purifying ribonucleic acid using the liquid chromatography column according to claim 11.
A method for separating and/or removing ribonucleic acid in a sample containing deoxyribonucleic acid and ribonucleic acid using the column for liquid chromatography according to claim 11.