JP2014210845A - Polymer particle to be used for producing sugar chain capturing particle, and sugar chain capturing particle - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a polymer particle to be used for producing a sugar chain capturing particle, which can allow a method for producing the sugar chain capturing particle to be simplified by reducing the number of reaction steps to be carried out in order to obtain the sugar chain capturing particle, and to provide the sugar chain capturing particle.SOLUTION: The polymer particle to be used for producing the sugar chain capturing particle is obtained by polymerizing a monomer having a carboxyl group and a crosslinkable monomer. It is preferable that the polymer particle is obtained by polymerizing 70-99.99 mol% of the monomer having the carboxyl group and 0.01-30 mol% of the crosslinkable monomer.

Description

  The present disclosure relates to polymer particles and sugar chain-capturing particles used in the production of sugar-chain-capturing particles.

  Patent Document 1 discloses a support having a functional group that specifically reacts with an aldehyde group of a sugar chain, and polymer particles to which this support is applied. Specifically, the production of polymer particles from a monomer having an oxylamino group or a derivative thereof by a suspension polymerization reaction is disclosed.

  Patent Document 2 discloses a sugar chain affinity bead containing a hydrazide group or an aminooxy group and a method for producing the same. Specifically, a raw material containing a carboxylic acid ester monomer containing a polymerizable group is polymerized in the presence of a crosslinkable monomer to obtain a carboxylic acid ester-containing polymer, and then 10% by volume of the carboxylic acid ester-containing polymer particles. Disclosed is a process comprising treating with a hydrazine solution of the above concentration.

  However, many of the monomers used in these patent documents are not commercially available, and in order to obtain sugar chain capturing particles, many reaction steps are required including the monomer synthesis step. There was a problem that the manufacturing method was complicated.

International Publication No. 2005/097844 International Publication No. 2008/018170

  The object of the present invention is a polymer particle used in the production of sugar chain-trapping particles, and simplifies the method of producing sugar chain-trapping particles by reducing the number of reaction steps to obtain the sugar chain-trapping particles. It is an object of the present invention to provide polymer particles and sugar chain-capturing particles that can be used.

Such an object is achieved by the present invention described in the following (1) to (9): (1) polymer particles used for the production of sugar chain-trapping particles, and a monomer having a carboxyl group; Polymer particles obtained by polymerizing a crosslinkable monomer.
(2) The polymer particle is obtained by polymerizing a monomer having a carboxyl group of 70 mol% or more and 99.99 mol% or less and a crosslinkable monomer 0.01 mol% or more and 30 mol% or less. The polymer particles according to (1).
(3) The monomer (1) having the carboxyl group is represented by the following general formula (I):
Or the polymer particle as described in (2).
[In Formula (I), R ₁ represents 0 to 0 carbon atoms that may be interrupted with at least one selected from the group consisting of —O—, —S—, —NH—, —CO—, and —CONH—. 30 represents a hydrocarbon chain, and R ₂ represents H, CH ₃ or a hydrocarbon chain having 2 to 5 carbon atoms.]
(4) The polymer particle according to any one of (1) to (3), wherein the monomer having a carboxyl group contains 1 or less nitrogen atoms.
(5) The polymer particle according to any one of (1) to (4), wherein the monomer having a carboxyl group is represented by the following general formula (II).
[In formula (II), R ₂ represents H, CH ₃ or a hydrocarbon chain having 2 to 5 carbon atoms, and n represents an integer of 0 to 30. ]
(6) A sugar chain-trapping particle in which the polymer particle according to any one of (1) to (5) is reacted with hydrazine, and a hydrazide group is introduced into the polymer particle.
(7) The sugar chain-trapping particles according to the above (6), wherein the sugar chain-trapping particles have a hydrazide group content of 0.01 μmol / mg or more and 10 μmol / mg or less with respect to the entire sugar chain-trapping particles. .

  According to the present invention, it is a polymer particle used in the production of sugar chain capturing particles, and the method for producing the sugar chain capturing particles is simplified by reducing the number of reaction steps for obtaining the sugar chain capturing particles. It is possible to provide polymer particles and sugar chain-capturing particles that can be used.

  The polymer particles according to the present invention are used for producing sugar chain-trapping particles and are obtained by polymerizing a monomer having a carboxyl group and a crosslinkable monomer.

By using the monomer having a carboxyl group for the polymerization, a carboxyl group is introduced into the polymer particle. The introduced carboxyl group easily reacts with hydrazine, which will be described later, to form a hydrazide group. Therefore, the hydrazide group can be easily introduced into the polymer particle, and the sugar chain capturing particles can be easily produced. it can.
Moreover, by using the crosslinkable monomer for the polymerization, a crosslinked structure is formed in the polymer particles, and high solvent resistance and mechanical strength can be imparted to the polymer particles.
That is, by using a monomer having a carboxyl group for the polymerization and a crosslinkable monomer, polymer particles capable of easily producing sugar chain-trapping particles having high solvent resistance and mechanical strength are obtained. Can be obtained.

[Monomer having a carboxyl group]
Here, examples of the monomer having a carboxyl group (hereinafter, also referred to as “monomer (A)”) include monomers represented by the following general formula (I) in one or a plurality of non-limiting embodiments. It is done.
[In Formula (I), R ₁ represents 0 to 0 carbon atoms that may be interrupted with at least one selected from the group consisting of —O—, —S—, —NH—, —CO—, and —CONH—. 30 represents a hydrocarbon chain, and R ₂ represents H, CH ₃ or a hydrocarbon chain having 2 to 5 carbon atoms.]

The number of nitrogen atoms contained in the monomer (A) is preferably 1 or less. As a result, the number of nitrogen atoms in the polymer particles obtained and the sugar chain-capturing particles produced from the polymer particles is reduced, and the surface charge of the particles is kept low. Electrostatic and non-specific adsorption of proteins other than chains on the particle surface can be suppressed.
In addition, it is more preferable that the monomer (A) does not contain a nitrogen atom. Thereby, the said effect can be exhibited more notably.

Furthermore, as a compound of general formula (I), what is represented by following formula (II) is mentioned in one or some embodiment which is not limited.
[In formula (II), R ₂ represents H, CH ₃ or a hydrocarbon chain having 2 to 5 carbon atoms, and n represents an integer of 0 to 30. ]

In addition, as a compound of the said general formula (II), what is represented by following formula (III) and (IV) from an availability is preferable.

  In addition, the carboxyl group of the monomer (A) used in the present invention may be a carboxyl group as it is, or may be ionized, or may be neutralized into a salt.

[Crosslinking monomer]
Here, as the crosslinkable monomer (hereinafter, also referred to as “monomer (B)”), a polyfunctional compound that can be copolymerized with the monomer (A) can be suitably used. In one or more non-limiting embodiments,
(1) Di or tri (meth) acrylic acid esters of polyols, for example, where the polyol is ethylene glycol, propylene glycol, trimethylolpropane, glycerin, polyoxyethylene glycol, polyoxypropylene glycol, polyglycerin, etc.
(2) In (1), the unsaturated acid is other than (meth) acrylic acid, such as maleic acid, fumaric acid,
(3) Bisacrylamides such as N, N′-methylenebisacrylamide
(4) Di- or tri (meth) acrylic acid esters obtained by reacting polyepoxide and (meth) acrylic acid,
(5) Di (meth) acrylic acid carbamyl esters obtained by reacting polyisocyanate with (meth) acrylic acid hydroxy ester, for example, polyisocyanate is tolylene diisocyanate, hexamethylene diisocyanate and / or the like, and / or
(6) Polyvalent allyl compounds such as allylated starch, allylated cellulose, diallyl phthalate, tetraallyloxyethane, pentaerythritol triallyl ether, trimethylolpropane triallyl ether, diethylene glycol diallyl ether, triallyl trimellitate, etc. Is mentioned.

In addition, the combination of the monomer (A) and the monomer (B) is not limited. In one or a plurality of embodiments, the monomer (A) is any one of (II), (III), (IV), methacrylic acid, and acrylic acid. The monomer (B) is preferably any of ethylene glycol di (meth) acrylate, propylene glycol di (meth) acrylate, and N, N′-methylenebisacrylamide, and the monomer (A) is the above ( When it is either III) or (IV), it is more preferable that the monomer (B) is any one of ethylene glycol di (meth) acrylates.

The ratio of the monomer (A) and the monomer (B) used in the polymerization reaction is such that when the monomer (A) is 70 mol% or more and 99.99 mol% or less, the monomer (B) is 0.01 mol% or more, It is preferably 30 mol% or less, and more preferably 1 mol% or more and 20 mol% or less of the monomer (B) when the monomer (A) is 80 mol% or more and 99 mol% or less.
The said effect can be exhibited more notably because the ratio of the monomer (A) and monomer (B) used for the said polymerization reaction exists in the said range.

  In addition, the polymerization method according to the present invention includes suspension polymerization, reverse phase suspension polymerization, and the like in one or more embodiments that are not limited.

[Suspension polymerization reaction]
Here, suspension polymerization refers to, in one or a plurality of embodiments, a water-insoluble monomer or a monomer that is hardly soluble in water, if necessary, another monomer or a crosslinkable monomer, a polymerization initiator, and the like. The oil phase containing is dispersed and suspended in an aqueous phase inert to the polymerization to carry out oil-in-water suspension polymerization. Accordingly, in the present invention, in one or a plurality of embodiments, an oil-in-water emulsion is formed by dispersing a mixture containing at least the monomer (A), the crosslinkable monomer, and the polymerization initiator in the aqueous phase. .
On the other hand, reverse phase suspension polymerization refers to polymerization of an aqueous phase containing a water-soluble monomer, if necessary, another monomer or a crosslinkable monomer, and a polymerization initiator in one or more embodiments. This is a method in which water-in-oil type suspension polymerization is carried out by dispersing and suspending in an oil phase containing an inert hydrophobic organic solvent. Therefore, in the present invention, in one or a plurality of embodiments, a water-in-oil emulsion is formed by dispersing a mixture containing at least the monomer (A), the crosslinkable monomer, and the polymerization initiator in an organic solvent. .

  The content of the monomer (A) in the oil-in-water emulsion or the water-in-oil emulsion before the polymerization reaction or the dispersion medium for forming the emulsion is not limited. In one or more embodiments, the solvent is 100 mL. 1 mmol or more, 80 mmol or less, 5 mmol or more, 50 mmol or less, or 5 mmol or more and 30 mmol or less. Note that the monomer (A) used in the production method of the present disclosure may be one type or a plurality of types in one or a plurality of embodiments.

  In addition, the content of the (B) crosslinkable monomer in the oil-in-water emulsion or the water-in-oil emulsion before the polymerization reaction or the solution for forming the emulsion is not limited in one or a plurality of embodiments. The amount is preferably 0.01 mmol or more and 30 mol% or less, more preferably 0.1 mmol or more and 20 mol% or less, and most preferably 0.1 mmol or more and 10 mol% or less with respect to all monomers.

  In addition, the addition timing to the polymerization reaction system of a crosslinkable monomer (B) is not specifically limited, In one or some embodiment, it can add at any time before superposition | polymerization before superposition | polymerization.

[Water phase]
The aqueous phase that can be used for normal suspension polymerization is an aqueous solution that is inert to polymerization in one or a plurality of non-limiting embodiments. You may add well-known dispersing agents, such as polyvinyl alcohol, as needed.
[Organic solvent]
Furthermore, the organic solvent that can be used for reverse phase suspension polymerization is a hydrophobic organic solvent that is inert to the polymerization, in one or more non-limiting embodiments. The organic solvent that can be used in the production method of the present disclosure is, in one or more non-limiting embodiments, aliphatic hydrocarbons, alicyclic hydrocarbons, aromatic hydrocarbons, aliphatic alcohols, aliphatic ketones, and aliphatics. Examples include esters. Examples of the aliphatic hydrocarbon include, but are not limited to, aliphatic hydrocarbons having 5 or more carbon atoms such as n-pentane, n-hexane, and n-heptane. Examples of the alicyclic hydrocarbon include, but are not limited to, alicyclic hydrocarbons having 5 or more carbon atoms such as cyclopentane, methylcyclopentane, cyclohexane, and methylcyclohexane. The aromatic hydrocarbon includes, in one or more non-limiting embodiments, benzene and benzene derivatives such as toluene and xylene. Examples of the aliphatic alcohol include, but are not limited to, aliphatic alcohols having 4 or more carbon atoms or 4 to 6 carbon atoms such as n-butyl alcohol and n-amyl alcohol. Examples of the aliphatic ketone include, but are not limited to, aliphatic ketones having 4 or more carbon atoms or 4 to 6 carbon atoms such as methyl ethyl ketone. Examples of the aliphatic esters include, but are not limited to, aliphatic esters having 4 or more carbon atoms or 4 to 6 carbon atoms such as ethyl acetate. The organic solvent that can be used in the production method of the present disclosure may be one kind or a mixed solvent of two or more kinds in one or a plurality of non-limiting embodiments. In one or more embodiments that are not limited, the organic solvent that can be used in the production method of the present disclosure may be only one kind of each of the organic solvents described above, or may be a mixture of two or more kinds.

  There are no particular limitations on the amounts of the aqueous phase and the organic solvent used, and they can be appropriately set according to the amount of the monomer (A). The amount of the aqueous phase or organic solvent used with respect to 100 parts by mass of the monomer (A) is not limited, and is preferably 500 parts by mass or more and 5000 parts by mass or less, more preferably 500 parts by mass or more and 3000 parts by mass. The following is more preferable.

[Polymerization initiator]
Moreover, the polymerization initiator which can be used for suspension polymerization is oil-soluble in one or some embodiment which is not limited. Examples of the oil-soluble polymerization initiator used in the present invention include organic peroxides such as benzoyl peroxide, lauroyl peroxide, t-butyl peroxy-2-ethyl hexanate, methyl ethyl ketone peroxide, azobisisobutyl nitrile, ( Examples thereof include azo compounds such as 1-phenylethyl) azodi-phenylmethane and 2,2-azobis (4-methoxy-2,4-dimethylvaleronitrile). These polymerization initiators may be used alone or in combination of two or more.
Moreover, the polymerization initiator which can be used for reverse phase suspension polymerization is water-soluble in one or some embodiment which is not limited. Examples of the polymerization initiator include, but are not limited to, a water-soluble radical polymerization initiator in one or more embodiments. In one or a plurality of non-limiting embodiments, the polymerization initiator includes (1) hydrogen peroxide, (2) persulfates such as sodium persulfate, potassium persulfate, and ammonium persulfate, and (3) potassium perchlorate. And perchlorates such as sodium perchlorate, (4) halogenates such as potassium chlorate and potassium bromate, (5) t-butyl hydroperoxide, cumene hydroperoxide, and dialkyl peroxide (di- peroxides such as t-butyl peroxide and t-butyl cumyl peroxide), (6) ketone peroxides such as methyl ethyl ketone peroxide and methyl isobutyl ketone peroxide, (7) t-butyl peroxyacetate, t-butyl Alkyl pars such as peroxyisobutyrate and t-butyl peroxypivalate Carboxy ester, and (8) azobisisobutyronitrile and 2,2'-azobis (N, N'-dimethylene isobutyl amidine) azo compounds such as dihydrochloride, and the like.

  Furthermore, the usage-amount of the said polymerization initiator can be suitably set according to the conditions of suspension polymerization and reverse phase suspension polymerization. In one or more embodiments in which the polymerization disclosure agent is not limited, 0.005 parts by mass or more, 20 parts by mass or less, 0.01 parts by mass or more, and 10 parts by mass with respect to 100 parts by mass of the monomer (A). It is not more than parts by mass, not less than 0.02 parts by mass and not more than 10 parts by mass, or not less than 0.05 parts by mass and not more than 5 parts by mass. The method for adding the polymerization initiator is not particularly limited.

[Dispersant]
In one or a plurality of embodiments of the production method of the present disclosure that are not limited, a dispersant may be added to the aqueous phase when performing normal suspension polymerization, and the organic solvent when performing reverse phase suspension polymerization. preferable. Examples of dispersants that can be used in ordinary suspension polymerization include (1) polymer dispersants and nonionic surfactants having an HLB value of 8 to 18 such as proteins (eg, gelatin); lecithin; Water-soluble rubber such as rubber and tragacanth rubber; sodium alginate; cellulose derivatives such as carboxymethylcellulose and ethoxycellulose; starch and derivatives thereof; polyoxyethylene alkyl ether, polyoxyethylene alkylphenyl ether, polyethylene glycol fatty acid ester, polyvinyl alcohol and the like; Sorbitan fatty acid esters such as sorbitan oleate, sorbitan stearate, sorbitan palmitate; cetyl alcohol, etc. (2) Higher alcohol sulfuric acid as ionic surfactant Anionic surfactants such as sodium stealth, sodium alkylbenzene sulfonate, sodium dialkyl ester succinate; cationic surfactants such as alkyl pyridinium chloride, alkyl dimethyl benzyl ammonium; hydroxyethyl imidazoline sulfate sodium, imidazoline sulfone Examples of amphoteric surfactants such as sodium phosphate and (3) inorganic compound dispersants include calcium phosphate, magnesium phosphate, calcium carbonate, magnesium carbonate, calcium hydroxide, magnesium hydroxide, and aluminum hydroxide. Such dispersants or surfactants can be used alone or in combination.
In one or more embodiments that are not limited, the dispersant for the reverse phase suspension polymerization includes an oil-soluble surfactant having an HLB of 3 to 10, or 3 to 9. In one or a plurality of embodiments, such an oil-soluble surfactant is a sorbitan fatty acid ester (sorbitan monostearate, sorbitan monolaurate, sorbitan, sorbitan monooleate, etc.), sucrose fatty acid ester (sucrose and stearin). Acid, palmitic acid, lauric acid, mono-, di- or triester with fatty acid such as oleic acid). The said dispersing agent may be used individually by 1 type, and may use 2 or more types together.

  The amount of the dispersant used is not particularly limited and can be appropriately selected according to the conditions of suspension polymerization and reverse phase suspension polymerization. The amount of the dispersant used is not limited, and in one or more embodiments, 0.1 parts by mass or more, 20 parts by mass or less, 0.1 parts by mass or more, 15 parts by mass with respect to 100 parts by mass of the monomer (A). Or 0.1 mass part or more and 10 mass parts or less.

In the polymerization of the present invention, a solvent may be appropriately added to the monomer droplets.
In normal suspension polymerization, an organic solvent may be appropriately added to the oil droplets in addition to the monomer (A), the crosslinkable monomer, and the initiator. The organic solvent preferably dissolves the monomer and is insoluble or hardly soluble in water as a dispersion medium. On the other hand, in reverse phase suspension polymerization, water may be appropriately added to the water droplets in addition to the monomer (A), the crosslinkable monomer, and the initiator. The amount of the organic solvent or water to be added is arbitrary as long as droplets can be produced in the dispersion medium.

The usual suspension polymerization reaction is carried out while suspending a water-in-oil emulsion in which a mixture containing the monomer (A), the crosslinkable monomer and the polymerization initiator is dispersed in the organic solvent. The formation of the water-in-oil suitable emulsion and the suspension may be simultaneous, or the formation of the water-in-oil suitable emulsion may be performed first. In addition, all the components may be mixed before the polymerization reaction, or a part or all of them may be added and mixed with stirring. The polymerization reaction conditions are not limited, and in one or more embodiments, the temperature is 50 ° C. or more and 90 ° C. or less, preferably 60 ° C. or more and 80 ° C. or less, and the time is 0.5 hours or more and 48 hours or less, preferably Is 1 hour or more and 24 hours or less.
In the reverse phase suspension polymerization reaction, polymerization is performed while suspending a water-in-oil emulsion in which a mixture containing the monomer (A), the crosslinkable monomer, water, and the polymerization initiator is dispersed in the organic solvent. The formation of the water-in-oil suitable emulsion and the suspension may be simultaneous, or the formation of the water-in-oil suitable emulsion may be performed first. In addition, all the components may be mixed before the polymerization reaction, or a part or all of them may be added and mixed with stirring. The polymerization reaction conditions are not limited, and in one or more embodiments, the temperature is 50 ° C. or more and 90 ° C. or less, preferably 60 ° C. or more and 80 ° C. or less, and the time is 0.5 hours or more and 48 hours or less, preferably Is 1 hour or more and 24 hours or less.

Here, by reacting the polymer particles of the present invention with hydrazine, it is possible to obtain sugar chain-trapping particles in which a carboxyl group and hydrazine in the polymer particles form a chemical bond and a hydrazide group is introduced.
The method of reacting the polymer particles and hydrazine is not particularly limited, but a method of reacting the polymer particles and the hydrazine compound using a dehydration condensing agent is simple and preferable.

  Examples of the dehydrating condensing agent include 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methyl Morpholinium chloride, dicyclohexylcarbodiimide, diphenylphosphoric acid azide "diphenylphosphoric acid azide or diisopropylcarbodiimide".

  The concentration of the dehydrating condensing agent is not particularly limited, but is preferably 10 mg / mL or more and saturated concentration or less, more preferably 50 mg / mL or more and 300 mg / mL or less, and most preferably 100 mg / mL or more and 300 mg / mL or less. If the amount used is small, the reaction does not proceed sufficiently, and if the amount used is large, it is not economical.

  The reaction temperature during the dehydration condensation is preferably 0 ° C. or higher and 80 ° C. or lower, more preferably 10 ° C. or higher and 70 ° C. or lower, and particularly preferably 15 ° C. or higher and 50 ° C. or lower.

  Further, the dehydration condensation reaction is preferably performed in an aqueous solution. In that case, 3-7 are preferable and, as for pH of aqueous solution, 4-6 are more preferable.

  The hydrazide group content of the sugar chain capturing particles is preferably 0.01 μmol / mg or more and 10 μmol / mg or less, and preferably 1 μmol / mg or more and 5 μmol / mg or less with respect to the entire sugar chain capturing particles. It is more preferable that When the hydrazide group content of the sugar chain capturing particles is within the above range, the sugar chain capturing particles efficiently suppress sugar chains while suppressing non-specific adsorption of foreign substances other than sugar chains. Can be captured.

[Usage]
The polymer particles of the present invention can be used for producing sugar chain-trapping particles. The sugar chain capturing particles can be used for sugar chain capturing. For example, it can be used for capturing a sugar chain having a reducing end in a sample. In one or a plurality of embodiments, sugar chains captured by polymer particles can be purified by an enzyme such as PNGaseF or hydrazine decomposition. Alternatively, in one or a plurality of embodiments, the sugar chain captured by the polymer particles can label the labeling compound by a hydrazone-oxime exchange reaction or a hydrazone-hydrazone exchange reaction. This makes it possible to increase the detection sensitivity, increase the resolution, and / or facilitate the detection.

  EXAMPLES Hereinafter, although this invention is demonstrated based on a following example and a comparative example, this invention is not limited to this.

[Example 1]
[Synthesis of Particles Having Carboxyl Group Using Monomer (A)]
4.6 g (20 mmol) of mono-2- (methacryloyloxy) ethyl succinate (manufactured by Sigma Aldrich), 0.1 g (0.5 mmol) of ethylene glycol dimethacrylate and 5 mL of chloroform were mixed, and 66 mg of azobisisobutyronitrile ( 0.4 mmol) was added and completely dissolved to prepare a monomer solution. The monomer solution was added to 100 mL of a 0.26 wt% polyvinyl alcohol aqueous solution and bubbled with nitrogen gas for 20 minutes while stirring. The mixture was heated at 60 ° C. for 16 hours while continuing stirring and bubbling, and then washed with ethanol and pure water to obtain particles having a carboxyl group.
[Hydrazide conversion of particles]
A water-soluble carbodiimide (2.5 g) was dissolved in 10 mL of a 50 μL / mL hydrazine monohydrate solution (dissolved in a PBS buffer and adjusted to pH 5.8 with HCl). 0.5 g of the carboxyl group-containing particles was added to this solution and mixed by inverting at 37 ° C. for 2 hours. After washing with water, the particles were dispersed in 1N HCl. The supernatant was removed by centrifugation, and washing with water was repeated. Lyophilized particles (sugar chain-capturing particles) were obtained.
The number of reaction steps until obtaining the sugar chain capturing particles is 2, and the amount of hydrazide groups of the sugar chain capturing particles is generally determined by the trinitrobenzenesulfonic acid (TNBS) method (hereinafter referred to as TNBS) method used for quantification of amino groups and hydrazide groups. It was 2.5 μmol / mg as determined by “TNBS method”.
In addition, as a result of evaluating the obtained particles for sugar chain capture based on the following “Method for evaluating sugar chain capture function”, the total peak area (S) was 3.2 million.

[Example 2]
A sugar chain-capturing particle was produced in the same manner as in Example 1 except that the concentration of the hydrazine monohydrate solution at the time of particle hydrazide was changed to 10 μL / mL.
The number of reaction steps until obtaining the sugar chain capturing particles was 2, and the amount of hydrazide groups of the sugar chain capturing particles was determined by the TNBS method and found to be 1.2 μmol / mg.
In addition, as a result of evaluating the obtained particles for sugar chain capture based on the following “evaluation method of sugar chain capture function”, the total peak area (S) was 2.9 million.

[Example 3]
Particles were produced in the same manner as in Example 1 except that the amount of mono-2- (methacryloyloxy) ethyl succinate was changed to 2.3 g (10 mmol) and the amount of ethylene glycol dimethacrylate was changed to 2 g (10 mmol). The hydrazide formation of the particles was performed in the same manner as in Example 1 to obtain sugar chain capturing particles. The number of reaction steps until obtaining the sugar chain capturing particles was 2, and the amount of hydrazide groups of the sugar chain capturing particles was 0.6 μmol / mg.
In addition, as a result of evaluating the obtained particles for sugar chain capture based on the following “Method for evaluating sugar chain capture function”, the total area (S) of the peak was 700,000.

Comparative example

Monomer synthesis and particle synthesis were performed based on the examples described in International Publication No. 2008/018170.
(1) Synthesis of Monomer Precursor A solution of methacrylic anhydride (MAH: 5 g, 0.03 mol) dissolved in 100 ml of chloroform was added to 25 g of (ethylenedioxy) bis (ethylamine) (EDBEA: 25 g, 0. 17 mol) was added dropwise to a solution of 100 ml of chloroform in an ice bath. Nitrogen was sealed therein and stirred overnight. The residue obtained by evaporating the solvent from the obtained reaction solution is applied to a silica gel column (developing solvent: mixed solvent of 90 parts of chloroform / 10 parts of methanol) to fractionate a predetermined fraction, and the solvent is evaporated from this fraction. To obtain a monomer precursor.

(2) Monomer synthesis To a solution of 5 g of compound monomer precursor (0.023 mol) dissolved in 100 ml of chloroform, 1.5 equivalents of monomethyl succinate and 1.5 equivalents of a water-soluble carbodiimide compound (WSC) Certain 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide was added and sealed. Nitrogen was sealed therein and stirred overnight. The residue obtained by evaporating the solvent from the obtained reaction solution is applied to a silica gel column (developing solvent: mixed solvent of 90 parts of chloroform / 10 parts of methanol) to fractionate a predetermined fraction, and the solvent is evaporated from this fraction. To obtain a monomer.

2. Synthesis of methyl ester-containing polymer particles A three-necked flask was charged with 25 ml of 5% polyvinyl alcohol (PVA) aqueous solution and purged with nitrogen. A mixture consisting of 1 g of monomer (2.6 mmol), ethylene glycol dimethacrylate (EGDMA: 5 mol% relative to monomer) and 1 ml of chloroform was introduced into the three-necked flask and stirred while maintaining at 60 ° C. And EGDMA were dispersed in an aqueous PVA solution. Subsequently, 3% by weight of azobisisobutyronitrile (AIBN) as a polymerization initiator was added to the monomer to initiate polymerization. After reacting at 60 ° C. for 16 hours, the produced polymer particles were recovered by centrifugation and washed with methanol and water.

3. Introduction of hydrazide group 400 mg of polymer particles were placed in a container, 4 ml of hydrazine monohydrate was added and stirred, and allowed to stand at room temperature for 2 hours. After the reaction, hydrazine monohydrate was removed, washed with methanol, and rinsed with 1M hydrochloric acid. Thereafter, it was further washed with pure water to obtain particles for capturing sugar chains. The number of reaction steps until obtaining the sugar chain capturing particles was 4, and the amount of hydrazide groups of the sugar chain capturing particles was determined by the TNBS method and found to be 3.0 μmol / mg.
In addition, as a result of evaluating the obtained particles for sugar chain capture based on the following “evaluation method of sugar chain capture function”, the total peak area (S) was 3.7 million.

<Evaluation method of sugar chain capture function>
(Sugar chain analysis of bovine serum-derived IgG using sugar chain capture particles)
1 Pretreatment of biological sample 1 mg of bovine serum-derived IgG was dissolved in 50 μL of 100 mM ammonium bicarbonate, 5 μL of 120 mM DTT (dithiothreitol) was added, and the mixture was reacted at 60 ° C. for 30 minutes. After completion of the reaction, 10 μL of 123 mM IAA (iodoacetamide) was added and allowed to react at room temperature for 1 hour in the dark. Subsequently, protease treatment was performed with 400 U of trypsin, and the protein portion was fragmented into peptides. The reaction solution was treated at 90 ° C. for 5 minutes, and then treated with 5 U glycosidase F to release sugar chains from the peptide, thereby obtaining a pretreated biological sample.

2 Sugar chain capture step (a), washing step (b)
20 μL of a pre-treated biological sample suspension and 180 μL of a 2% acetic acid / acetonitrile solution were added to the disposable column containing 5 mg of the sugar chain-capturing particles prepared in Examples 1 to 3 and Comparative Example. Reacted for hours. The reaction was carried out in an open system, and it was visually confirmed that the solvent was completely evaporated and the beads were dried. Subsequently, after washing the particles with a guanidine solution, pure water, methanol, and a triethylamine solution, 10% acetic anhydride / methanol was added and reacted at room temperature for 30 minutes to cap unreacted hydrazide groups. After capping, the beads were washed with pure water.

3 Sugar chain release step (c)
To the disposable column containing the particles, 20 μL of pure water and 180 μL of 2% acetic acid / acetonitrile solution were added and reacted at 70 ° C. for 1.5 hours. The reaction was carried out in an open system, and it was visually confirmed that the solvent was completely evaporated and the beads were dried.

4 Labeling step (d)
Solution prepared by dissolving in a 30% acetic acid / DMSO mixed solvent in a disposable column containing particles so that the final concentrations of 2-aminobenzomide (2-AB) and sodium cyanoborohydride are 0.35M and 1M, respectively. 50 μL was added and reacted at 60 ° C. for 2 hours.

  The sugar chain capturing step (a), the washing step (b), the sugar chain releasing step (c), and the labeling step (d) were performed in a disposable column which is the same container.

5 Excess reagent removal step 50 μL of the reaction solution is recovered, diluted 10-fold with acetonitrile, and then added to a column packed with silica gel (Iatrobeads, 6RS-8060, manufactured by Mitsubishi Chemical Yatron) to adsorb the labeled sugar chain to the silica gel. It was. After washing the column with acetonitrile / acetonitrile / water mixed solution (95: 5), the labeled sugar chain was recovered with 50 μL of pure water.

6 Detection of labeled sugar chain The obtained labeled sugar chain was measured by HPLC (Nexera, Shimadzu Corporation). Using an amino column (Shodex Asahipak NH2P-50), measurement was performed at an excitation wavelength of 330 nm and a fluorescence wavelength of 420 nm, and a sugar chain labeled with 2AB was detected.
In the HPLC chart obtained by the above method, the total area (S) of peaks derived from the sugar chain labeled with 2AB was determined. The total area (S) of the peak corresponds to the total sugar chain capture amount.

Claims (7)

Polymer particles used for the production of sugar chain capturing particles,
A polymer particle obtained by polymerizing a monomer having a carboxyl group and a crosslinkable monomer. The polymer particle is obtained by polymerizing 70 mol% or more and 99.99 mol% or less of a monomer having a carboxyl group and 0.01 mol% or more and 30 mol% or less of a crosslinkable monomer. The polymer particles described. The monomer having a carboxyl group is represented by the following general formula (I):
Polymer particles as described in 1.
[In Formula (I), R ₁ represents 0 to 0 carbon atoms that may be interrupted with at least one selected from the group consisting of —O—, —S—, —NH—, —CO—, and —CONH—. 30 represents a hydrocarbon chain, and R ₂ represents H, CH ₃ or a hydrocarbon chain having 2 to 5 carbon atoms.] The polymer particle according to any one of claims 1 to 3, wherein the monomer having a carboxyl group contains 1 or less nitrogen atoms. The polymer particle according to any one of claims 1 to 4, wherein the monomer having a carboxyl group is represented by the following general formula (II).
[In formula (II), R ₂ represents H, CH ₃ or a hydrocarbon chain having 2 to 5 carbon atoms, and n represents an integer of 0 to 30. ] A sugar chain-trapping particle obtained by reacting the polymer particle according to any one of claims 1 to 5 with hydrazine and introducing a hydrazide group into the polymer particle. The glycan-capturing particle according to claim 6, wherein a hydrazide group content of the glycan-trapping particle is 0.01 µmol / mg or more and 10 µmol / mg or less with respect to the entire glycan-trapping particle.