JP2024060941A - Method and kit for labeling sugar chain - Google Patents

Abstract

To provide a technique to obtain analysis results equivalent to those obtained when sodium cyanoborohydride is used as a reducing agent, even when highly toxic sodium cyanoborohydride is not used as a reducing agent, in labeling sugar chains.SOLUTION: A method for labeling sugar chains by reductive amination reaction includes step (A) of bringing the sugar chains into contact with a labeling reaction solution containing a labeling compound having an amino group, a reducing agent, and an additive with a pKa of 5 to 12 for conjugated acid, and obtaining labeled sugar chains. A kit for labeling sugar chains includes a labeled compound having an amino group, a reducing agent, and an additive with a pKa of 5 to 12 for conjugated acids.SELECTED DRAWING: None

Description

The present invention relates to a method and kit for labeling glycans.

Biopolymers such as glycans, glycoproteins, glycopeptides, peptides, oligopeptides, proteins, nucleic acids, and lipids play important roles in the fields of biotechnology, including medicine, cell engineering, and organ engineering, and elucidating the mechanisms by which these substances control biological reactions will lead to advances in the field of biotechnology.

Glycosylation is a general term for molecules in which monosaccharides such as glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine, N-acetylgalactosamine, and sialic acid and their derivatives are linked in a chain form via glycosidic bonds. Among biopolymers, glycans are extremely diverse, and it is becoming clear that they are deeply involved in various functions of naturally occurring organisms, such as intercellular signaling and regulating protein functions and interactions.

Glycosylation often occurs in the body as complex carbohydrates bound to proteins, lipids, etc. Examples of biopolymers with glycans include proteoglycans in the cell walls of plant cells that contribute to cell stabilization; glycolipids that affect cell differentiation, proliferation, adhesion, and migration; and glycoproteins that are involved in cell-cell interactions and cell recognition. The mechanisms by which the glycans contained in these biopolymers control sophisticated and precise biological reactions while acting as substitutes for, supporting, amplifying, regulating, or inhibiting the functions of the biopolymers are gradually becoming clear.

If the relationship between cell differentiation and proliferation, cell adhesion, immunity, and cell canceration and glycans can be clarified, it is expected that new developments will be achieved by closely linking glycotechnology, medicine, cell engineering, and organ engineering.

For example, in glycoprotein drugs, the glycans often play an important role in the expression of biological activity. Therefore, evaluation of glycans is extremely important as a parameter for quality control of glycoprotein drugs. In particular, it has been reported that the glycan structure of antibody drugs affects antibody-dependent cellular cytotoxicity (ADCC activity), and glycan structure analysis is becoming increasingly important.

Under these circumstances, there is a demand for methods that can analyze glycan structures quickly, easily, and accurately, and glycan analysis is being carried out using a wide variety of methods, including high-performance liquid chromatography (HPLC), nuclear magnetic resonance, capillary electrophoresis (CE), mass spectrometry (MS), and lectin arrays.

To analyze glycans using these various methods, it is necessary to first separate and purify the glycans from the proteins, peptides, lipids, nucleic acids, etc. contained in the biological sample. Purifying and labeling glycans takes time and effort, so it is difficult to prepare a large number of different samples at once.

As a technique for solving this problem, for example, Patent Document 1 describes a method for preparing a glycan sample using polymer particles that have functional groups for capturing glycans.

International Publication No. 2008/018170

In order to label glycans, the glycans must be brought into contact with a labeling compound and reduced with a reducing agent. Sodium cyanoborohydride is often used as the reducing agent. However, because sodium cyanoborohydride is a highly toxic compound, it is desirable to use a safer reducing agent.

However, the inventors of the present application found that in conventional glycan labeling methods, changing the reducing agent from sodium cyanoborohydride to a safer reducing agent can result in fluctuations in the peak pattern during glycan analysis by HPLC or the like.

The present invention aims to provide a technology that can obtain analytical results equivalent to those obtained when sodium cyanoborohydride is used as a reducing agent in labeling glycans, even when sodium cyanoborohydride is not used as a reducing agent.

［式（１）中、Ｒ^１，Ｒ^２は、それぞれ独立に、１又は複数の－ＣＨ_２－が、－Ｏ－，－Ｓ－，－ＮＨ－，－ＣＯ－又は－ＣＯＮＨ－に置換されることにより中断されていてもよい炭素数１以上２０以下の炭化水素鎖を表し、Ｒ^３，Ｒ^４，Ｒ^５は、それぞれ独立に、Ｈ，ＣＨ_３又は炭素数２以上５以下の炭化水素鎖を表し、ｍ：ｎ＝９９：１～７０：３０である。］
［８］アミノ基を有する標識化合物、還元剤、及び、共役酸のｐＫａが５～１２である添加剤を含む、糖鎖を標識するためのキット。 The present invention includes the following aspects.
[1] A method for labeling a glycan by a reductive amination reaction, comprising the step (A) of contacting the glycan with a labeling reaction solution containing a labeling compound having an amino group, a reducing agent, and an additive whose conjugate acid has a pKa of 5 to 12, to obtain the labeled glycan.
[2] The method according to [1], wherein the additive is an organic amine.
[3] The method according to [2], wherein the organic amine is dimethylamine, triethylamine, methylamine or trimethylamine.
[4] The method according to any one of [1] to [3], wherein the concentration of the additive in the labeling reaction solution is 0.001 to 3.0 M.
[5] The method according to any one of [1] to [4], wherein the pH of the labeling reaction solution is 2 to 9.
[6] The method according to any one of [1] to [5], further comprising a step (A') of capturing the sugar chain released from the biomolecule on a carrier having a hydrazide group on the surface thereof, prior to the step (A).
[7] The method according to [6], wherein the support having a hydrazide group on the surface thereof is a support made of a compound having a crosslinked polymer structure represented by the following formula (1):

[In formula (1), R ¹ and R ² each independently represent a hydrocarbon chain having 1 to 20 carbon atoms which may be interrupted by replacing one or more -CH ₂ - with -O-, -S-, -NH-, -CO- or -CONH-, R ³ , R ⁴ and R ⁵ each independently represent H, CH ₃ or a hydrocarbon chain having 2 to 5 carbon atoms, and m:n is 99:1 to 70:30.]
[8] A kit for labeling a sugar chain, comprising a labeling compound having an amino group, a reducing agent, and an additive whose conjugate acid has a pKa of 5 to 12.

The present invention provides a technology that can obtain analytical results equivalent to those obtained when sodium cyanoborohydride is used as a reducing agent in labeling glycans, even when the highly toxic sodium cyanoborohydride is not used as a reducing agent.

FIG. 1 is an LC-MS chromatogram showing the results of Experimental Example 1. FIG. 2 is an LC-MS chromatogram showing the results of Experimental Example 2. FIG. 3 is an LC-MS chromatogram showing the results of Experimental Example 3. FIG. 4 is an LC-MS chromatogram showing the results of Experimental Example 4.

[Method of labeling glycans]
In one embodiment, the present invention provides a method for labeling a glycan by a reductive amination reaction, comprising a step (A) of contacting the glycan with a labeling reaction solution containing a labeling compound having an amino group, a reducing agent, and an additive whose conjugate acid has a pKa of 5 to 12, to obtain the labeled glycan.

As described later in the Examples, the method of this embodiment makes it possible to obtain analytical results equivalent to those obtained when sodium cyanoborohydride is used as a reducing agent in labeling glycans, even when highly toxic sodium cyanoborohydride is not used as the reducing agent.

In the method of this embodiment, a glycan is contacted with a labeling reaction solution containing a labeling compound having an amino group, a reducing agent, and an additive whose conjugate acid has a pKa of 5 to 12, to obtain a labeled glycan. Methods for labeling glycans include reductive amination and exchange reaction, and the method of this embodiment uses reductive amination.

In the reductive amination method, glycans are labeled with a labeling compound having an amino group. The labeling reaction solution may contain an organic solvent or a mixed solvent containing water and an organic solvent. For example, dimethyl sulfoxide (DMSO) is preferably used as the organic solvent.

When the labeling reaction solution contains a mixed solvent containing water and an organic solvent, it is preferable that the result obtained when measuring the pH is weakly acidic to basic, regardless of the ratio of the organic solvent. More specifically, the pH of the labeling reaction solution is preferably 2 to 9, more preferably 2 to 8, and even more preferably 2 to 7.

The reaction temperature for labeling the glycan is preferably 4°C or higher and 90°C or lower, more preferably 25°C or higher and 80°C or lower, and even more preferably 30°C or higher and 70°C or lower.

The labeling compound having an amino group preferably has ultraviolet-visible absorption characteristics or fluorescent characteristics. Specific examples of the labeling compound include 8-aminopyrene-1,3,6-trisulfonate, 8-aminonaphthalene-1,3,6-trisulfonate, 7-amino-1,3-naphthalenedisulfonic acid, 2-amino-9(10H)-acridone, 5-aminofluorescein, dansylethylenediamine, 2-aminopyridine, 7-amino-4-methylcoumarin, 2-aminobenzamide, 2-aminobenzoic acid, 3-aminobenzoic acid, 7-amino-1-naphthol, 3-(acetylamino)-6-aminoacridine, 2-amino-6-cyanoethylpyridine, ethyl p-aminobenzoate, p-aminobenzonitrile, 7-aminonaphthalene-1,3-disulfonic acid, and procainamide. Of these, 2-aminobenzamide is preferred.

The concentration of the labeling compound in the labeling reaction solution is preferably 1 mM or more and 10 M or less, more preferably 10 mM or more and 10 M or less, and even more preferably 100 mM or more and 3 M or less.

In the method of this embodiment, a boron-based reducing agent that is safer than sodium cyanoborohydride, which has been used conventionally, is used as a reducing agent in the glycan labeling reaction. Examples of such reducing agents include picoline borane and dimethylamine borane.

The concentration of the reducing agent in the labeling reaction solution is preferably 1 mM or more and 10 M or less, more preferably 10 mM or more and 10 M or less, and even more preferably 100 mM or more and 2 M or less.

The reaction time for labeling the glycan is preferably from 10 minutes to 24 hours, more preferably from 10 minutes to 8 hours, and even more preferably from 10 minutes to 3 hours.

As described later in the Examples, the inventors of the present application found that when the reducing agent for labeling glycans is changed from sodium cyanoborohydride to picoline borane, the peak pattern may vary during glycan analysis by HPLC or the like. From the results of mass spectrometry, it was believed that the peak pattern variation was caused by the generation of glycans having a lactone structure (lactone body) as a by-product.

The inventors further demonstrated that by adding an additive whose conjugate acid has a pKa of 5 to 12 to the labeling reaction solution that is brought into contact with the glycan, the lactone structure of the glycan can be eliminated, and even when picoline borane is used as the reducing agent, analytical results equivalent to those obtained when sodium cyanoborohydride is used as the reducing agent can be obtained.

Additives whose conjugate acids have a pKa of 5 to 12 include organic amines. Specific organic amines include dimethylamine, triethylamine, methylamine, and trimethylamine.

The concentration of the additive in the labeling reaction solution is preferably 0.001 to 3.0 M. The lower limit of the concentration of the additive in the labeling reaction solution may be 0.005 M or more, 0.01 M or more, or 0.05 M or more. The upper limit of the concentration of the additive in the labeling reaction solution may be 2 M or less, 1 M or less, or 0.5 M or less. These lower and upper limits can be combined in any combination.

As described later in the Examples, by using an additive with a conjugate acid pKa of 5 to 12 at the above concentrations, it is possible to obtain analytical results equivalent to those obtained when sodium cyanoborohydride is used as a reducing agent, even when sodium cyanoborohydride is not used as a reducing agent.

In the method of this embodiment, the glycan to be labeled may be a glycan contained in a sample derived from a living organism (biological sample). Examples of biological samples include whole blood, serum, plasma, urine, saliva, cells, tissues, viruses, and plant tissues. In addition, purified glycoproteins or unpurified glycoproteins may be used as the biological sample. The biological sample may be pretreated by methods such as delipidation, desalting, protein fractionation, and heat denaturation.

By using a glycan releasing means, glycans can be released from glycan-containing biomolecules (e.g., glycoproteins) in a biological sample. As a glycan releasing means, methods such as glycosidase treatment using N-glycosidase or O-glycosidase, hydrazinolysis, and β-elimination by alkali treatment can be used. When analyzing N-glycans, a method using N-glycosidase is preferred. Prior to glycosidase treatment, protease treatment using trypsin, chymotrypsin, etc. may be performed.

In the method of this embodiment, the glycan to be labeled may be captured using a carrier having a hydrazide group on its surface. That is, the method of this embodiment may further include a step (A') of capturing the glycan released from the biomolecule on a carrier having a hydrazide group on its surface prior to step (A).

In this case, the method of this embodiment includes a step (A') of capturing the glycan released from the biomolecule on a carrier having a hydrazide group on the surface, and a step (A) of contacting the glycan with a labeling reaction solution containing a labeling compound having an amino group, a reducing agent, and an additive whose conjugate acid has a pKa of 5 to 12 to obtain the labeled glycan.

In a solution such as an aqueous solution, glycans are in equilibrium between cyclic hemiacetal type and non-cyclic aldehyde type. The hydrazide group reacts with the aldehyde group of the aldehyde type glycan to form a specific and stable bond. As a result, the glycan can be captured on the carrier.

The compound or carrier having a hydrazide group "-C(=O)-NHNH ₂ " is not particularly limited as long as the sugar chain to be contacted can interact with the hydrazide group, and may be in any form, such as a low molecular weight compound, a solid phase carrier, a polymer particle, a micelle, a liposome, or a soluble polymer.

Of these, solid particles or gel particles can be suitably used as the polymer particles. If such polymer particles are used, the polymer particles can be easily collected by centrifugal separation, filtration, or other means after capturing sugar chains on the polymer particles.

It is also possible to pack the polymer particles into a column. Packing the polymer particles into a column is particularly important from the viewpoint of continuous operation. By using a filter plate (e.g., Millipore's MultiScreen Solvinert Filter Plate) as a reaction vessel, it becomes possible to process multiple samples simultaneously. In this case, the throughput of glycan purification is significantly improved compared to conventional purification methods using columns, such as gel filtration. In addition, if magnetic beads are used as the polymer particles, the beads can be accumulated on the vessel wall surface, etc., using magnetic force, making it easy to wash the beads.

The shape of the polymer particles is not particularly limited, but a spherical shape or a similar shape is preferred. When the polymer particles are spherical, the average particle size is preferably 0.05 μm or more and 1000 μm or less, more preferably 0.05 μm or more and 200 μm or less, even more preferably 0.1 μm or more and 200 μm or less, and particularly preferably 0.1 μm or more and 100 μm or less.

When the average particle size of the polymer particles is equal to or greater than the lower limit, good liquid permeability can be maintained when the polymer particles are packed into a column, and there is no need to apply high pressure. In addition, the polymer particles can be easily recovered by centrifugation or filtration. In addition, when the average particle size of the polymer particles is equal to or less than the upper limit, the contact area between the polymer particles and the sample solution can be secured, and the efficiency of glycan capture can be improved. In addition, the polymer particles can be easily sucked up using a pipette, etc.

An example of a carrier having a hydrazide group on its surface is a carrier made of a compound having a crosslinked polymer structure represented by the following formula (1):

In formula (1), R ¹ and R ² each independently represent a hydrocarbon chain having from 1 to 20 carbon atoms which may be interrupted by replacing one or more -CH ₂ - with -O-, -S-, -NH-, -CO- or -CONH-, and R ³ , R ⁴ and R ⁵ each independently represent H, CH ₃ or a hydrocarbon chain having from 2 to 5 carbon atoms. Additionally, m and n represent the number of monomer units, and the ratio of m to n is m:n=99:1 to 70:30.

As a carrier (polymer particles) made of the compound represented by the above formula (1), a commercially available carrier (BlotGlyco (registered trademark), product number "BS-45407", manufactured by Sumitomo Bakelite Co., Ltd.) can be suitably used.

In formula (2), m and n represent the number of monomer units, and the ratio of m to n is m:n = 99:1 to 70:30.

The pH of the reaction system when capturing glycans on a carrier having hydrazide groups on its surface is preferably 2 to 9, more preferably 2 to 7, and even more preferably 2 to 6. Various buffer solutions can be used to adjust the pH.

In addition, a mixed solvent of an acid and an organic solvent can also be used as the solvent for capturing the glycans. When a mixed solvent of an acid and an organic solvent is used, the acid is not particularly limited, and examples thereof include acetic acid, formic acid, trifluoroacetic acid, hydrochloric acid, citric acid, phosphoric acid, and sulfuric acid. Among these, acetic acid, formic acid, trifluoroacetic acid, and phosphoric acid are preferred, and acetic acid and trifluoroacetic acid are more preferred.

The organic solvent is not particularly limited as long as it dissolves the glycan and the above-mentioned acid. Since the capture efficiency often increases when the glycan is dried during capture, an organic solvent with a relatively low boiling point, such as acetonitrile, is preferably used.

The mixing ratio (volume ratio) of the acid to the organic solvent is preferably 0.1:99.9 to 10:90, more preferably 0.5:99.5 to 5:95, and even more preferably 1:99 to 5:95.

The temperature during glycan capture is preferably 4°C or higher and 90°C or lower, more preferably 30°C or higher and 90°C or lower, even more preferably 30°C or higher and 80°C or lower, and particularly preferably 40°C or higher and 80°C or lower. The reaction time for capturing glycans can be set appropriately. Glycans may be captured by filling a column with a carrier having hydrazide groups on its surface and passing a sample solution containing glycans through the column.

Then, the carrier bound to the glycans (capturing the glycans) is washed. This makes it possible to remove substances other than the glycans (non-specifically adsorbed substances) from the substances captured by the carrier.

Methods for removing substances other than glycans include washing with an aqueous guanidine solution, which is a chaotropic agent capable of dissociating hydrophobic interactions, or washing with pure water or a water-soluble buffer solution (e.g., phosphate buffer, Tris buffer, etc.).

The washing conditions are preferably a temperature of 4°C or higher and 40°C or lower, and a washing time of 10 seconds or higher and 30 minutes or lower. The washing method includes immersing the carrier in a washing solution and repeatedly replacing the washing solution.

Specifically, the carrier is placed in a centrifuge tube or other tube, a washing solution is added, the tube is shaken, the carrier is precipitated by centrifugation, and the supernatant is removed. This process is repeated to wash the carrier. The carrier can be precipitated by natural settling or centrifugation. It is preferable to perform the washing process three to six times. When magnetic beads are used as the carrier, centrifugation is not required, making the process simple.

It is also possible to use a filter tube, which is a tube-shaped container with a filter attached to the bottom, the filter having a pore size that allows liquid to pass through but does not allow the carrier to pass through. By using the carrier in a filter tube, the washing liquid used for washing can be removed through the filter, and the above-mentioned process of removing the supernatant after centrifugation is no longer necessary, improving workability.

In addition, various types of multi-well plates with filters attached to the bottom of 6- to 384-well plates are commercially available, and using these plates makes it possible to achieve high throughput. In particular, 96-well multi-well plates are ideal for achieving high throughput, as solution dispensing devices, suction and removal systems, and plate transport systems have been developed.

After the glycan capture reaction, a washing solution may be passed through the column to perform a washing process continuously from the glycan capture reaction. In addition, when a multi-well plate is used, substances other than the carrier that captured the glycan may be removed by a filtration operation or a centrifugation operation.

Excess hydrazide groups on a carrier having hydrazide groups on its surface can be capped using, for example, acetic anhydride.

When step (A') is performed, it is preferable to release the glycan captured on the carrier having hydrazide groups on the surface after step (A') and before step (A). The release of the glycan can be performed by acid treatment. The acid treatment can be performed by contacting the carrier on which the glycan has been captured with a mixed solvent of an acid and an organic solvent, or a mixed solvent of an acid, water, and an organic solvent.

In the case of a mixed solvent of acid, water, and an organic solvent, the proportion of water (volume ratio) is preferably 0.1% or more and 90% or less, more preferably 0.1% or more and 80% or less, and even more preferably 0.1% or more and 50% or less.

An aqueous buffer solution may be used instead of water. The concentration of the buffering agent in the aqueous buffer solution is preferably 0.1 mM to 1 M, more preferably 0.1 mM to 500 mM, and even more preferably 1 mM to 100 mM.

The pH of the mixed solvent of acid and organic solvent, or the mixed solvent of acid, water and organic solvent, is preferably 2 to 9, more preferably 2 to 7, and even more preferably 2 to 6. By carrying out the glycan release reaction at a weakly acidic to near-neutral pH, hydrolysis of the glycan, such as the elimination of sialic acid residues, can be suppressed, compared to the glycan release reaction by treatment with, for example, 10% trifluoroacetic acid, which is a strong acid.

Examples of the acid in the mixed solvent of an acid and an organic solvent, or the mixed solvent of an acid, water, and an organic solvent, include acetic acid, formic acid, trifluoroacetic acid, hydrochloric acid, citric acid, phosphoric acid, and sulfuric acid. Among these, acetic acid, formic acid, trifluoroacetic acid, and phosphoric acid are preferred, and acetic acid and trifluoroacetic acid are more preferred.

The temperature of the acid treatment is preferably 4°C or more and 90°C or less, more preferably 25°C or more and 90°C or less, and even more preferably 40°C or more and 90°C or less. The time of the acid treatment is preferably 10 minutes or more and 24 hours or less, more preferably 10 minutes or more and 8 hours or less, and even more preferably 10 minutes or more and 3 hours or less.

From the viewpoint of efficiently releasing the glycans, it is preferable to carry out the acid treatment in an open system so that the solvent is completely evaporated.

In the method of this embodiment, the reaction product after step (A) contains unreacted labeled compound, reducing agent, salts, etc. in addition to the labeled glycan. Therefore, it is preferable to remove these substances before analyzing the labeled glycan.

Methods for removal include removal by solid-phase extraction using a glycan affinity carrier such as a silica column, removal by gel filtration, removal by ion exchange resin, etc. Any method may be used, but it is preferable that the pH of the solvent used is near neutral to prevent the release of sialic acid. As a result, it becomes possible to prepare a labeled glycan sample from which substances other than the labeled glycan have been removed.

In particular, it is preferable to adsorb a solution containing labeled glycans to a glycan affinity carrier, wash the carrier, and then elute the labeled glycans from the glycan affinity carrier.

In other words, it is preferable that the method of this embodiment further includes, after step (A), step (B) of adsorbing the labeled glycan to a glycan affinity carrier, washing the glycan while it is captured on the glycan affinity carrier, and then eluting the labeled glycan from the glycan affinity carrier.

Examples of carriers with affinity for sugar chains include carriers containing silica, which may consist of silica alone or may be a silica-organic hybrid type. There are also no particular limitations on the shape of the carrier, and the carrier may be particulate, plate-like, fibrous, porous, or the like. The silica may be silica having silanol groups, or may be surface-modified with aminopropyl groups, etc.

The sample solution used to adsorb the labeled glycans to the glycan affinity carrier is preferably a solution containing more than 95% by volume of organic solvent, more preferably a solution containing 96% or more by volume of organic solvent, even more preferably a solution containing 98% or more by volume of organic solvent, and particularly preferably a solution containing 99% or more by volume of organic solvent.

As the organic solvent, acetonitrile, hexane, ethyl acetate, methylene chloride, tetrahydrofuran, etc. can be used, but acetonitrile is particularly preferred.

After the labeled glycans are adsorbed to the glycan affinity carrier by the above operation, unnecessary substances other than the labeled glycans can be washed away by passing a washing solution through the carrier, and the labeled glycans can be purified. The washing solution preferably contains 90% or more by volume of an organic solvent and 10% or less by volume of water, and more preferably 95% or more by volume of an organic solvent and 5% or less by volume of water. As the organic solvent contained in the washing solution, acetonitrile, methanol, ethanol, 2-propanol, hexane, ethyl acetate, methylene chloride, tetrahydrofuran, etc. can be used, but acetonitrile is particularly preferred. Multiple types of washing solutions with different compositions may be used, and for example, after washing with 100% acetonitrile, further washing with a mixture of acetonitrile:water = 95:5 (v/v) may be used.

After the washing operation, an elution solution is added to the glycan affinity carrier to elute the labeled glycans. The elution solution preferably contains 10% or more by volume of water, more preferably 50% or more by volume of water, and water is particularly preferred. Components other than water contained in the elution solution include acetonitrile and organic solvents selected from alcohols such as methanol, ethanol, propanol, and butanol.

By the above steps, a labeled glycan can be obtained. The labeled glycan can be analyzed by known methods such as mass spectrometry (e.g., LC-MS, MALDI-TOF MS, etc.), chromatography (e.g., HPLC, HPAE-PAD, etc.), and electrophoresis (e.g., capillary electrophoresis, etc.). When analyzing the glycan, various databases (e.g., GlycoMod, Glycosuite, SimGlycan, etc.) can be referenced as necessary.

[kit]
In one embodiment, the present invention provides a kit for labeling a glycan, comprising a labeling compound having an amino group, a reducing agent, and an additive having a conjugate acid with a pKa of 5 to 12.

In the kit of this embodiment, the labeling compound having an amino group, the reducing agent, and the additive whose conjugate acid has a pKa of 5 to 12 are the same as those described above. The kit of this embodiment can be suitably used for carrying out the above-mentioned method for labeling glycans by reductive amination reaction.

The present invention will be described in more detail below with reference to examples and comparative examples, but the present invention is not limited to the following examples.

[Experimental Example 1]
(Glycosylation analysis of fetuin derived from calf serum 1)
The sugar chains of fetuin derived from calf serum were labeled with 2-aminobenzamide (2-AB) and analyzed by LC-MS. A labeling reaction solution containing sodium cyanoborohydride as a reducing agent was used. As described below, the labeled sugar chains were adsorbed onto a clean-up column and washed, and then eluted from the clean-up column with pure water and collected.

<Sample Pretreatment>
2 mg of fetuin derived from calf serum was dissolved in 200 μL of pure water, and then 20 μL of 120 mM dithiothreitol (DTT) and 20 μL of 1 M ammonium bicarbonate aqueous solution were added and reacted at 60° C. for 30 minutes. After the reaction was completed, 40 μL of 123 mM iodoacetamide (IAA) was added and reacted at room temperature for 1 hour under light shielding. The protein portion was then treated with protease using 400 U of trypsin to fragment the protein into peptide fragments. The reaction solution was then treated at 90° C. for 5 minutes, and then reacted with 5 U of N-glycosidase to cleave the N-glycan from the peptide.

《Capturing and washing sugar chains》
A commercially available glycan purification and labeling kit (BlotGlyco (registered trademark), product number "BS-45407", manufactured by Sumitomo Bakelite Co., Ltd.) was used. The kit contained a carrier having hydrazide groups on the surface (BlotGlyco (registered trademark) beads).

First, 20 μL of a suspension of pretreated fetuin derived from calf serum and 180 μL of a 2% acetic acid/acetonitrile solution were added to a disposable column containing 5 mg of BlotGlyco (registered trademark) beads, and the reaction was allowed to proceed at 80°C for 1 hour. The reaction was carried out in an open system, and it was visually confirmed that the solvent had completely evaporated at the end of the reaction, and the contents of the column had dried up.

Then, the column was washed successively with a guanidine solution, pure water, methanol, and a 1% triethylamine-methanol solution, after which 10% acetic anhydride/methanol was added and reacted at room temperature for 30 minutes to cap any unreacted hydrazide groups. After capping, the column was washed with pure water.

<<Liberation of glycans>>
Next, 20 μL of pure water and 180 μL of a 2% acetic acid/acetonitrile solution were added to the column, and the reaction was carried out for 1.5 hours at 70° C. The reaction was carried out in an open system, and it was visually confirmed that the solvent had completely evaporated at the end of the reaction and the contents of the column had dried up.

<Glycan labeling>
Subsequently, 50 μL of a labeling reaction solution prepared by dissolving 2-AB and sodium cyanoborohydride in a 30% acetic acid/DMSO solution to final concentrations of 1 M and 350 mM, respectively, was added to the column, and the reaction was allowed to proceed at 50° C. for 1 hour.

Removal of excess reagents
The column was then centrifuged to recover 50 μL of the reaction solution containing the labeled glycan and unreacted 2-AB. The recovered reaction solution was then diluted 20-fold with acetonitrile and added to a clean-up column (silica gel carrier) included in a glycan purification labeling kit (BlotGlyco (registered trademark), product number "BS-45407", manufactured by Sumitomo Bakelite Co., Ltd.), and the labeled glycan was adsorbed onto the clean-up column.

The cleanup column was then washed twice with a washing solution. A mixed solution of acetonitrile and water (acetonitrile:water (volume ratio) = 95:5) was used as the washing solution. The labeled glycans were then eluted with 50 μL of pure water and collected.

Glycosylation Analysis
Subsequently, the labeled glycan was analyzed by LC-MS. The labeled glycan was applied to an amide column (ACQUITY UPLC® Glycan BEH Amide, 1.7 μm, 2.1×150 mm) and measured at an excitation wavelength of 330 nm and a fluorescence wavelength of 420 nm. FIG. 1 shows a chromatogram of LC-MS in which a glycan labeled with 2-AB was detected. In FIG. 1, the horizontal axis indicates the elution time, and the vertical axis indicates the signal intensity (relative value). The solid line indicates the peak of a glycan having a lactone form, and the dashed line indicates the peak of a glycan having a non-lactone form.

[Experimental Example 2]
(Glycosylation analysis of fetuin derived from calf serum 2)
The sugar chains of fetuin derived from calf serum were labeled with 2-AB and analyzed by LC-MS. The labeled sugar chains were analyzed by LC-MS in the same manner as in Experimental Example 1, except that a labeling reaction solution containing picoline borane instead of sodium cyanoborohydride was used as the reducing agent.

Specifically, the labeling reaction solution used was prepared by dissolving 2-AB and picoline borane in a mixed solvent of acetic acid, dimethyl sulfoxide (DMSO) and water (acetic acid:DMSO:water (volume ratio) = 3:1:2) to final concentrations of 2.6 M and 1.7 M, respectively.

Figure 2 shows an LC-MS chromatogram detecting glycans labeled with 2-AB. In Figure 2, the horizontal axis shows elution time, and the vertical axis shows signal intensity (relative value). The solid line shows the peak of a glycan having a lactone form, and the dashed line shows the peak of a glycan having a non-lactone form. As a result, the peak pattern of the glycan having a lactone form was different from the results of Experimental Example 1.

[Experimental Example 3]
(Sugar chain analysis of fetuin derived from calf serum 3)
The sugar chains of fetuin derived from calf serum were labeled with 2-AB and analyzed by LC-MS. The labeled sugar chains were analyzed by LC-MS in the same manner as in Experimental Example 2, except that an additive with a conjugate acid pKa of 5 to 12 was added to the labeling reaction solution.

Specifically, the labeling reaction solution used was prepared by dissolving 2-AB, picoline borane, and dimethylamine in a mixed solvent of acetic acid, dimethyl sulfoxide (DMSO), and water (acetic acid:DMSO:water (volume ratio) = 3:1:2) to final concentrations of 2.6 M, 1.7 M, and 0.1 M, respectively.

Figure 3 shows an LC-MS chromatogram detecting glycans labeled with 2-AB. In Figure 3, the horizontal axis indicates elution time, and the vertical axis indicates signal intensity (relative value). The solid line indicates the peak of a glycan having a lactone form, and the dashed line indicates the peak of a glycan having a non-lactone form. As a result, it was revealed that the lactone form peak disappeared from the peak pattern of Experimental Example 3, and a pattern equivalent to that of Experimental Example 1 was obtained.

These results show that even when picoline borane, a safe reducing agent, is used, adding an additive to the labeling reaction solution can produce a peak pattern equivalent to that obtained when sodium cyanoborohydride, a conventional reducing agent, is used.

[Experimental Example 4]
(Glycosylation analysis of fetuin derived from calf serum 4)
The sugar chains of fetuin derived from calf serum were labeled with 2-AB and analyzed by LC-MS. The labeled sugar chains were analyzed by LC-MS in the same manner as in Experimental Example 2, except that an additive with a conjugate acid pKa of 5 to 12 was added to the labeling reaction solution.

Specifically, the labeling reaction solution was prepared by dissolving 2-AB, picoline borane, and triethylamine in a mixed solvent of acetic acid, dimethyl sulfoxide (DMSO), and water (acetic acid:DMSO:water (volume ratio) = 3:1:2) to final concentrations of 2.6 M, 1.7 M, and 0.1 M, respectively.

Figure 4 shows an LC-MS chromatogram detecting glycans labeled with 2-AB. In Figure 4, the horizontal axis indicates elution time, and the vertical axis indicates signal intensity (relative value). The solid line indicates the peak of a glycan having a lactone form, and the dashed line indicates the peak of a glycan having a non-lactone form. As a result, the peak pattern of Experimental Example 4 revealed that the lactone form peak was smaller.

This result further supports the idea that by adding an additive to the labeling reaction solution, it is possible to obtain a peak pattern equivalent to that obtained when using the conventional reducing agent, sodium cyanoborohydride, even when using picoline borane, a safe reducing agent.

The present invention provides a technology that can obtain analytical results equivalent to those obtained when sodium cyanoborohydride is used as a reducing agent in labeling glycans, even when the highly toxic sodium cyanoborohydride is not used as a reducing agent.

Claims (8)

A method for labeling a glycan by reductive amination reaction, comprising the steps of:
The method includes a step (A) of contacting the glycan with a labeling reaction solution containing a labeling compound having an amino group, a reducing agent, and an additive whose conjugate acid has a pKa of 5 to 12, to obtain the labeled glycan. The method of claim 1, wherein the additive is an organic amine. The method of claim 2, wherein the organic amine is dimethylamine, triethylamine, methylamine or trimethylamine. The method according to any one of claims 1 to 3, wherein the concentration of the additive in the labeling reaction solution is 0.001 to 3.0 M. The method according to any one of claims 1 to 3, wherein the pH of the labeling reaction solution is 2 to 9. The method according to any one of claims 1 to 3, further comprising a step (A') of capturing the glycan released from the biomolecule on a carrier having a hydrazide group on its surface prior to the step (A). The method according to claim 6 , wherein the support having a hydrazide group on the surface thereof is a support made of a compound having a crosslinked polymer structure represented by the following formula (1):

[In formula (1), R ¹ and R ² each independently represent a hydrocarbon chain having 1 to 20 carbon atoms which may be interrupted by replacing one or more -CH ₂ - with -O-, -S-, -NH-, -CO- or -CONH-, R ³ , R ⁴ and R ⁵ each independently represent H, CH ₃ or a hydrocarbon chain having 2 to 5 carbon atoms, and m:n is 99:1 to 70:30.] A kit for labeling glycans, comprising a labeling compound having an amino group, a reducing agent, and an additive whose conjugate acid has a pKa of 5 to 12.

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