JP6065202B2 - Method for producing polymer particles having hydrazide group / oxylamino group - Google Patents

Description

  The present disclosure relates to a method for producing polymer particles having a hydrazide group / oxylamino group.

  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, after a raw material containing a carboxylic acid ester monomer containing a polymerizable group is polymerized in the presence of a crosslinking agent to obtain a carboxylic acid ester-containing polymer, the carboxylic acid-containing polymer particles are added at a concentration of 10 volume percent or more. Disclosed is a process comprising treating with a hydrazine solution.

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

  The present disclosure provides a method for producing polymer particles having a hydrazide group or an oxylamino group, which can improve the yield of the polymer particles as compared with the methods disclosed in Patent Documents 1 and 2.

  In one aspect, the present disclosure provides (A1) a carboxylic acid ester monomer containing a polymerizable group, or (A2) a hydrazyl group or an oxyl protected with a polymerizable group and a protecting group [P] in a water-in-oil emulsion. The present invention relates to a method for producing polymer particles having a hydrazide group or an oxylamino group, comprising polymerizing a monomer containing an amino group and (B) a crosslinking agent to obtain (C) a polymer particle.

  According to the present disclosure, in one embodiment, the yield of polymer particles having a hydrazide group or an oxylamino group can be improved.

FIG. 1 is a graph showing an example of a result of detecting a labeled sugar chain using three types of beads including the beads of the present disclosure.

  In one aspect, the present disclosure provides a method for producing polymer particles including polymerizing a carboxylic acid ester monomer or a monomer containing a hydrazyl group or an oxylamino group derivative, wherein the polymerization is performed by a reverse phase suspension polymerization reaction. , Based on the knowledge that the yield can be significantly improved.

  That is, in one aspect, the present disclosure provides (A1) a carboxylic acid ester monomer containing a polymerizable group or (A2) a hydrazyl group protected with a polymerizable group and a protecting group [P] in a water-in-oil emulsion. Alternatively, a method for producing polymer particles having a hydrazide group or an oxylamino group, which comprises polymerizing a monomer containing an oxylamino group and (B) a crosslinking agent to obtain a polymer particle (C) (hereinafter referred to as “this disclosure”) It is also referred to as a “manufacturing method”.

［式（Ｉ）中、Ｒ₁は、−Ｏ−、−Ｓ−、−ＮＨ−、−ＣＯ−、及び−ＣＯＮＨ−からなる群から選択される少なくとも一つで中断されてもよい炭素数１〜２０の炭化水素鎖を示し、Ｒ₂は、Ｈ、ＣＨ₃又は炭素数２〜５の炭化水素鎖を示す。］ [Carboxylic acid ester monomer containing polymerizable group (A1)]
The carboxylic acid ester monomer (A1) containing a polymerizable group used in the production method of the present disclosure (hereinafter, also referred to as “monomer (A1)”) is not limited, and in one or more embodiments, the following general formula (I The monomer represented by this is mentioned.

[In Formula (I), R ₁ may be interrupted with at least one selected from the group consisting of —O—, —S—, —NH—, —CO—, and —CONH—. Represents a hydrocarbon chain having ˜20, and R ₂ represents H, CH ₃ or a hydrocarbon chain having 2 to 5 carbon atoms. ]

In the formula (I), R ₁ may include the followings in one or more embodiments. In the following formula, a, b, and d represent an integer of 1 to 5, and c represents an integer of 1 to 10.

Examples of the compound of the general formula (I) include those represented by the following formula (1) in one or a plurality of non-limiting embodiments.

  The content of the monomer (A1) in the water-in-oil emulsion before the polymerization reaction or the solution for forming the emulsion is not limited. In one or more embodiments, the content is 5 to 50 mmol, 5 to 5 30 mmol or 10-20 mmol. Note that the monomer (A1) 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.

  When performing the manufacturing method of this indication using a monomer (A1), the manufacturing method of this indication performs the process of providing a hydrazide group to the polymer particle (C) obtained by polymerization reaction in one or some embodiment. Including that. (C) The process which provides a polymer particle with a hydrazide group is performed by processing the polymer particle (C) obtained by polymerization reaction with a hydrazine solution in one or some embodiment. In one or more non-limiting embodiments, the hydrazine concentration in the hydrazine solution is 10 volume percent or more, 30 volume percent or more, 80 volume percent or more, 90 volume percent or more, or 98 volume percent or more.

［式（ＩＩ）及び（ＩＩＩ）中、Ｒ₁は、−Ｏ−、−Ｓ−、−ＮＨ−、−ＣＯ−、及び−ＣＯＮＨ−からなる群から選択される少なくとも一つで中断されてもよい炭素数１〜２０の炭化水素鎖を示し、Ｒ₂は、Ｈ、ＣＨ₃又は炭素数２〜５の炭化水素鎖を示し、［Ｐ］は、保護基を示す。］ [Monomer (A2) containing a polymerizable group and a hydrazyl group or oxylamino group protected with a protective group [P]]
The monomer (A2) (hereinafter also referred to as “monomer (A2)”) containing a polymerizable group and a hydrazyl group or oxylamino group protected with a protective group [P] used in the production method of the present disclosure is a polymerizable group. And a hydrazyl group or an oxylamino group protected with a protecting group [P]. As a monomer (A2), the monomer represented by the following general formula (II) or (III) is mentioned in one or some embodiment which is not limited.

[In the formulas (II) and (III), R ₁ may be interrupted with at least one selected from the group consisting of —O—, —S—, —NH—, —CO—, and —CONH—. shows good hydrocarbon chain having a carbon number of 1 to 20, R ₂ is H, CH ₃ or a hydrocarbon chain having 2 to 5 carbon atoms, [P] represents a protective group. ]

In the formula (II), R ₁ may include the following in one or more embodiments. In the following formula, a, b, and d represent an integer of 1 to 5, and c represents an integer of 1 to 10.

In the formula (III), R ₁ may include the following in one or more embodiments. In the following formula, a, b, and d represent an integer of 1 to 5, and c represents an integer of 1 to 10.

Examples of the compounds of the general formulas (II) and (III) include those represented by the following formulas (2) and (3), respectively, in one or a plurality of non-limiting embodiments.

  The content of the monomer (A2) in the water-in-oil emulsion before the polymerization reaction or the solution for forming the emulsion is not limited. In one or more embodiments, the content is 5 to 50 mmol, 5 to 5 30 mmol or 10-20 mmol.

  Examples of the protecting group [P] include, but are not limited to, 9-fluorenylmethyloxycarbonyl group (Fmoc), tert-butoxycarbonyl group (Boc), benzyloxycarbonyl group (Cbz), 2, 2,2-trichloroethoxycarbonyl group (Troc), allyloxycarbonyl group (Alloc), 4-methoxytrityl group (Mmt), amino group, alkyl group having 3 to 20 carbon atoms, 9-fluoreneacetyl group, 1- Fluorenecarboxylic acid group, 9-fluorenecarboxylic acid group, 9-fluorenone-1-carboxylic acid group, benzyloxycarbonyl group, xanthyl group (Xan), trityl group (Trt), 4-methyltrityl group (Mtt), 4- Methoxy 2,3,6-trimethyl-benzenesulfonyl group (Mtr), mesithi 2-sulfonyl group (Mts), 4,4-dimethoxybenzohydryl group (Mbh), tosyl group (Tos), 2,2,5,7,8-pentamethylchroman-6-sulfonyl group (Pmc) 4-methylbenzyl group (MeBzl), 4-methoxybenzyl group (MeOBzl), benzyloxy group (BzlO), benzyl group (Bzl), benzoyl group (Bz), 3-nitro-2-pyridinesulfenyl group (Npys) ), 1- (4,4-dimethyl-2,6-dioxocyclohexylidene) ethyl group (Dde), 2,6-dichlorobenzyl group (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl Group (2-Cl-Z), 2-bromobenzyloxycarbonyl group (2-Br-Z), benzyloxymethyl group (Bom), cyclohexyloxy Si group (cHxO), t-butoxymethyl group (Bum), t-butoxy group (tBuO), t-butyl group (tBu), acetyl group (Ac), trifluoroacetyl group (TFA) o-bromobenzyloxycarbonyl Group, t-butyldimethylsilyl group, 2-chlorobenzyl (Cl-z) group, cyclohexyl group, cyclopentyl group, isopropyl group, pivalyl group, tetrahydropyran-2-yl group, trimethylsilyl group and the like.

The monomer (A2) is a compound of the following formula (4) in one or more embodiments that are not limited.

  When the production method of the present disclosure is performed using the monomer (A2), the production method of the present disclosure includes, in one or more embodiments, (C) the protective group [P] of the polymer particle obtained by the polymerization reaction is converted to an acid. Including deprotection by treatment. Deprotection can be performed by a known method according to the type of the protecting group.

[Crosslinking agent (B)]
As a crosslinking agent (B), it is a polyfunctional compound, Comprising: The compound which copolymerizes with a carboxylic acid ester monomer can be used suitably, In one or some embodiment which is not limited,
(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.

  The crosslinking agent (B) is ethylene glycol di (meth) acrylate, propylene glycol di (meth) acrylate, or N, N′-methylenebis (meth) acrylamide in one or more non-limiting embodiments.

  The content of (B) the crosslinking agent in the water-in-oil emulsion before the polymerization reaction or the solution for forming the emulsion is not limited, and in one or more embodiments, the monomer (A1 or A2) is 1 -30 mol%, 3-20 mol%, or 3-15 mol%.

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

[Reverse phase suspension polymerization reaction]
In the present disclosure, “polymerization in a water-in-oil emulsion” refers to reverse phase suspension polymerization. In the present disclosure, reverse phase suspension polymerization refers to in one or a plurality of embodiments, an aqueous phase containing a water-soluble monomer, another monomer or a crosslinking agent as necessary, a polymerization initiator, and the like is not allowed to polymerize. This is a method for carrying out water-in-oil type suspension polymerization by dispersing and suspending in an oil phase containing an active hydrophobic organic solvent. Therefore, in one or a plurality of embodiments of the production method of the present disclosure, the organic solvent is protected with (A1) a carboxylic acid ester monomer containing a polymerizable group or (A2) a polymerizable group and a protecting group [P]. A step of dispersing a mixture containing a monomer containing a hydrazyl group or an oxylamino group, (B) a crosslinking agent, water, and a polymerization initiator to form a water-in-oil emulsion.

[Organic solvent]
The organic solvent that can be used in the production method of the present disclosure is, in one or more non-limiting embodiments, a hydrophobic organic solvent that is inert to polymerization. The organic solvent that can be used in the production method of the present disclosure includes (A1) a carboxylic acid ester monomer containing a polymerizable group or (A2) a polymerizable group and a hydrazyl group or oxylamino group protected with a protective group [P]. Since it is used for dispersing a monomer-containing mixture, (B) a crosslinking agent, water, and a mixture containing a polymerization initiator, in one or a plurality of non-limiting embodiments, it can also be referred to as an organic dispersion medium. 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.

  The amount of the organic solvent used is not particularly limited and can be appropriately set according to the amount of the monomer (A1 / A2). The usage-amount of the said organic solvent with respect to 100 mass parts of said monomers (A1 and / or A2) is 500-5000 mass parts or 500-3000 mass parts in one or some embodiment which is not limited.

[Polymerization initiator]
The polymerization initiator that can be used in the production method of the present disclosure is water-soluble in one or a plurality of non-limiting embodiments. 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.

  The usage-amount of the said polymerization initiator can be suitably set according to the conditions of reverse phase suspension polymerization. The amount of the polymerization disclosure agent used is not limited, and in one or more embodiments, 0.005 to 20 parts by mass, 0.01 to 10 parts by mass with respect to 100 parts by mass of the monomer (A1 and / or A2), It is 0.02-10 mass parts, or 0.05-5 mass parts. The method for adding the polymerization initiator is not particularly limited. In one or more embodiments that are not limited, the polymerization initiator may be added to the organic solvent separately from the monomer (A1 and / or A2), and the aqueous solution of the monomer (A1 and / or A2) may be added. It may be added in advance to prepare a polymerization initiator-containing liquid, which may be added to the organic solvent.

[Dispersant]
In one or a plurality of embodiments of the production method of the present disclosure that are not limited, it is preferable to contain a dispersant in the organic solvent when performing reverse phase suspension polymerization. Examples of the dispersant include oil-soluble surfactants having an HLB of 3 to 10, or 3 to 9, in one or more embodiments that are not limited. 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 the reverse phase suspension polymerization. The amount of the dispersant used is not limited, and in one or a plurality of embodiments, 0.1 to 20 parts by mass, 0.1 to 15 parts by mass with respect to 100 parts by mass of the water monomer (A1 and / or A2), Or it is 0.1-10 mass parts.

  The reverse phase suspension polymerization reaction comprises (A1) a carboxylic acid ester monomer containing a polymerizable group or (A2) a monomer containing a polymerizable group and a hydrazyl group or oxylamino group protected with a protective group [P], (B ) Polymerization is performed while suspending a water-in-oil emulsion in which a mixture containing a crosslinking agent, 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. Polymerization reaction conditions are 50-90 degreeC or 60-80 degreeC, 0.5 to 24 hours, or 1 to 20 hours in one or some embodiment which is not limited.

[Polymer particles (C)]
(A1) When the said reverse phase suspension polymerization is performed using the carboxylic acid ester monomer containing a polymeric group, the polymer particle which has carboxylic acid ester as (C) polymer particle can be obtained. Thus, in another aspect, the present disclosure relates to the “polymer particles having a carboxylic acid ester” and / or a method for producing the same.

  The polymer particle which has a hydrazide group can be manufactured by the process of providing a hydrazide group to the obtained (C) polymer particle. (C) The step of imparting a hydrazide group to the polymer particles can be performed as described above.

  (A2) When the reverse phase suspension polymerization is carried out using a monomer containing a polymerizable group and containing a hydrazyl group protected with a protecting group [P], (C) the polymer particle is protected with the protecting group [P]. It is possible to obtain polymer particles having a modified hydrazyl group. Thus, in another aspect, the present disclosure relates to the “polymer particles having a hydrazyl group protected with a protecting group [P]” and / or a method for producing the same.

  By deprotecting the obtained polymer particles (C), polymer particles having a hydrazide group can be produced. (C) The step of deprotecting the polymer particles can be performed as described above.

  (A2) When the reverse phase suspension polymerization is performed using a monomer containing a polymerizable group and containing an oxylamino group protected with a protecting group [P], (C) As a polymer particle, the protecting group [P] Polymer particles having protected oxylamino groups can be obtained. Thus, in another aspect, the present disclosure relates to the “polymer particle having an oxylamino group protected with a protecting group [P]” and / or a method for producing the same.

  By deprotecting the obtained polymer particles (C), polymer particles having an oxylamino group can be produced. (C) The step of deprotecting the polymer particles can be performed as described above.

[Polymer particles having hydrazide group or oxylamino group]
The polymer particles having a hydrazide group or an oxylamino group that can be produced by the production method of the present disclosure have the following structure in one or more embodiments.
-(Hydrazide group-containing compound component) m- (crosslinking agent component) n-, or-(oxylamino group-containing compound component) m- (crosslinking agent component) n-

［式（ＩＶ）中、Ｒ₁は、−Ｏ−、−Ｓ−、−ＮＨ−、−ＣＯ−、−ＣＯＮＨ−で中断されてもよい炭素数１〜２０の炭化水素鎖を示し、Ｒ₃、Ｒ₄、Ｒ₅は、Ｈ、ＣＨ₃又は炭素数２〜５の炭化水素鎖を示し、ｍ、ｎはモノマーユニット数を示す。］ Examples of the polymer particles having a hydrazide group that can be produced by the production method of the present disclosure include those having a crosslinked polymer structure represented by the following general formula (IV) in one or a plurality of non-limiting embodiments.

[In the formula (IV), R ₁ represents a hydrocarbon chain having 1 to 20 carbon atoms which may be interrupted by —O—, —S—, —NH—, —CO— or —CONH—, and R ₃ , R ₄ and R ₅ represent H, CH ₃ or a hydrocarbon chain having 2 to 5 carbon atoms, and m and n represent the number of monomer units. ]

In the formula (IV), R ₁ may include the following in one or more embodiments that are not limited. In the following formula, a, b, and d represent an integer of 1 to 5, and c represents an integer of 1 to 10.

In the formula (IV), R ₂ may include the following in one or more embodiments that are not limited. In the following formulae, e and f represent an integer of 1 to 5, and g represents an integer of 1 to 10.

（ｍ、ｎはモノマーユニット数を示す。） Examples of the polymer particles having a hydrazide group that can be produced by the production method of the present disclosure include those having a crosslinked polymer structure represented by the following formula in one or a plurality of non-limiting embodiments.

(M and n represent the number of monomer units.)

[Usage]
In one or a plurality of embodiments, polymer particles having a hydrazide group or an oxylamino group that can be produced by the production method of the present disclosure can be used for sugar chain capture. 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.

［式（Ｉ）中、Ｒ₁は、−Ｏ−、−Ｓ−、−ＮＨ−、−ＣＯ−、及び−ＣＯＮＨ−からなる群から選択される少なくとも一つで中断されてもよい炭素数１〜２０の炭化水素鎖を示し、Ｒ₂は、Ｈ、ＣＨ₃又は炭素数２〜５の炭化水素鎖を示す。］
［６］ （Ａ１）重合性基を含むカルボン酸エステルモノマーが、下記式（１）で表される、［１］から［５］のいずれかに記載の製造方法。

［７］ （Ａ２）重合性基と保護基［Ｐ］で保護されたヒドラジル基又はオキシルアミノ基とを含むモノマーが、下記一般式（ＩＩ）又は（ＩＩＩ）で表される、［１］から［４］のいずれかに記載の製造方法。

［式（ＩＩ）及び（ＩＩＩ）中、Ｒ₁は、−Ｏ−、−Ｓ−、−ＮＨ−、−ＣＯ−、及び−ＣＯＮＨ−からなる群から選択される少なくとも一つで中断されてもよい炭素数１〜２０の炭化水素鎖を示し、Ｒ₂は、Ｈ、ＣＨ₃又は炭素数２〜５の炭化水素鎖を示し、［Ｐ］は、保護基を示す。］
［８］ （Ａ２）重合性基と保護基［Ｐ］で保護されたヒドラジル基又はオキシルアミノ基とを含むモノマーが、下記式（２）又は（３）で表される、［１］から［４］及び［７］のいずれかに記載の製造方法。

［９］ 架橋剤が、ポリオールのジ又はトリ（メタ）アクリル酸エステル、ポリオールのジ又はトリ不飽和酸エステル、ビスアクリルアミド、ポリイソシアネートと（メタ）アクリル酸ヒドロキシエステルを反応させて得られるジ（メタ）アクリル酸カルバミルエステル、多価アリル化合物、及びこれらの組み合わせからなる群から選択される、［１］から［８］のいずれかに記載の製造方法。
［１０］ 油中水滴型エマルションの有機溶媒が、脂肪族炭化水素、脂環族炭化水素、芳香族炭化水素、脂肪族アルコール、脂肪族ケトン、及び脂肪族エステル類、並びにこれらの組み合わせからなる群から選択される、［１］から［９］のいずれかに記載の製造方法。
［１１］ 油中水滴型エマルションの有機溶媒が、ｎ−ヘキサン、シクロヘキサン、ｎ−ヘプタン、及びこれらの組み合わせからなる群から選択される、［１０］記載の製造方法。
［１２］ 前記有機溶媒が、さらに、分散安定剤としてＨＬＢ３〜１０のソルビタン脂肪酸エステルを含む、［１］から［１１］のいずれかに記載の製造方法。
［１３］ 前記分散安定剤が、ソルビタンモノステアレート、ソルビタンモノラウレート、ソルビタン、及びモノオレートからなる群から選択される、［１２］記載の製造方法。
［１４］ 重合反応条件が、５０〜９０℃、０．５〜２４時間である、［１］から［１３］のいずれかに記載の製造方法。 The present disclosure further relates to one or more of the following embodiments.
[1] In a water-in-oil emulsion, (A1) a carboxylic acid ester monomer containing a polymerizable group, or (A2) a hydrazyl group or an oxylamino group protected with a polymerizable group and a protecting group [P] A method for producing polymer particles having a hydrazide group or an oxylamino group, comprising polymerizing a monomer and (B) a crosslinking agent to obtain (C) polymer particles.
[2] The monomer according to [1], wherein the monomer is (A1) a carboxylic acid ester monomer containing a polymerizable group, and the (C) polymer particles are further treated with a hydrazine solution having a concentration of 10 volume percent or more. Production method.
[3] The monomer is a monomer containing (A2) a polymerizable group and a hydrazyl group or oxylamino group protected with a protecting group [P], and (C) the protecting group [P] of the polymer particle is converted to an acid. The production method according to [1], comprising deprotecting by treatment.
[4] (A1) a carboxylic acid ester monomer containing a polymerizable group or (A2) a monomer containing a polymerizable group and a hydrazyl group or oxylamino group protected with a protective group [P] in an organic solvent, (B) The production method according to any one of [1] to [3], comprising a step of dispersing a mixture containing a crosslinking agent, water, and a polymerization initiator to form a water-in-oil emulsion.
[5] (A1) The production method according to any one of [1] to [4], wherein the carboxylic acid ester monomer containing a polymerizable group is represented by the following general formula (I).

[In Formula (I), R ₁ may be interrupted with at least one selected from the group consisting of —O—, —S—, —NH—, —CO—, and —CONH—. Represents a hydrocarbon chain having ˜20, and R ₂ represents H, CH ₃ or a hydrocarbon chain having 2 to 5 carbon atoms. ]
[6] The production method according to any one of [1] to [5], wherein the carboxylic acid ester monomer containing a polymerizable group (A1) is represented by the following formula (1).

[7] (A2) A monomer containing a polymerizable group and a protective group [P] -protected hydrazyl group or oxylamino group is represented by the following general formula (II) or (III): [4] The production method according to any one of [4].

[In the formulas (II) and (III), R ₁ may be interrupted with at least one selected from the group consisting of —O—, —S—, —NH—, —CO—, and —CONH—. shows good hydrocarbon chain having a carbon number of 1 to 20, R ₂ is H, CH ₃ or a hydrocarbon chain having 2 to 5 carbon atoms, [P] represents a protective group. ]
[8] (A2) A monomer containing a polymerizable group and a protective group [P] -protected hydrazyl group or oxylamino group is represented by the following formula (2) or (3): 4] and the production method according to any one of [7].

[9] A di- or tri- (meth) acrylic acid ester of a polyol, a di- or tri-unsaturated acid ester of a polyol, bisacrylamide, a polyisocyanate and a (meth) acrylic acid hydroxy ester obtained by reacting a di ( The production method according to any one of [1] to [8], which is selected from the group consisting of a methacrylic acid carbamyl ester, a polyvalent allyl compound, and a combination thereof.
[10] The organic solvent of the water-in-oil emulsion is a group consisting of aliphatic hydrocarbons, alicyclic hydrocarbons, aromatic hydrocarbons, aliphatic alcohols, aliphatic ketones, aliphatic esters, and combinations thereof. The production method according to any one of [1] to [9], selected from:
[11] The production method according to [10], wherein the organic solvent of the water-in-oil emulsion is selected from the group consisting of n-hexane, cyclohexane, n-heptane, and combinations thereof.
[12] The production method according to any one of [1] to [11], wherein the organic solvent further contains a sorbitan fatty acid ester of HLB 3 to 10 as a dispersion stabilizer.
[13] The production method according to [12], wherein the dispersion stabilizer is selected from the group consisting of sorbitan monostearate, sorbitan monolaurate, sorbitan, and monooleate.
[14] The production method according to any one of [1] to [13], wherein the polymerization reaction conditions are 50 to 90 ° C. and 0.5 to 24 hours.

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

[Example]
[Synthesis of Carboxylic Acid Ester Monomer (A1) Containing Polymerizable Group]
Compound (e), which is a carboxylic acid ester monomer containing a polymerizable group, was synthesized according to the following scheme.

  A solution of methacrylic anhydride (MAH: 5 g, 0.03 mol) dissolved in 100 mL of chloroform was dissolved in 25 g of (ethylenedioxy) bis (ethylamine) (EDBEA: 25 g, 0.17 mol) in 100 mL of chloroform. The solution was added dropwise on 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 an intermediate product (c). In a solution of 5 g (0.023 mol) of this intermediate product (c) in 100 mL of chloroform, 1.5 equivalents of monomethyl succinate and 1.5 equivalents of water-soluble carbodiimide compound (WSC), 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 carboxylic acid ester monomer (e) containing a polymerizable group. In addition, the obtained compound (e) was confirmed to be the target product by NMR, matrix-assisted laser ionization-time-of-flight mass spectrometer (MALDI-TOF-MS).

[Synthesis of polymer particles by reverse phase suspension polymerization reaction]
A sealable polymerization vessel was charged with 40 mL of 0.1% sorbitan monostearate-containing cyclohexane and purged with nitrogen. 2 g of the carboxylic acid ester monomer (e) (6 mmol), ethylene glycol dimethacrylate (EGDMA: 5 mol% based on methyl ester-containing monomer), 1.5 mL of pure water, and 0.0175 g of potassium peroxodisulfate (total A mixture consisting of 1 mol% with respect to the monomer was introduced into the polymerization vessel while purging with nitrogen, and dispersed in cyclohexane containing sorbitan monostearate. After the nitrogen purge was stopped and sealed, the reaction was carried out at 70 ° C. for 16 hours with stirring. The produced polymer particles were collected by centrifugation and washed with ethanol and water. The yield of polymer particles was 65%.

[Comparative Example: Synthesis of polymer particles by suspension polymerization reaction]
A sealable polymerization vessel was charged with 40 mL of a 0.1% aqueous polyvinyl alcohol solution and purged with nitrogen. 2 g of the carboxylic acid ester monomer (e) (6 mmol), ethylene glycol dimethacrylate (EGDMA: 5 mol% based on methyl ester-containing monomer), 1.5 mL of chloroform, and 0.011 g of azobisisobutyronitrile ( A mixture consisting of 1 mol% with respect to all monomers was introduced into the polymerization vessel while purging with nitrogen, and dispersed in an aqueous polyvinyl alcohol solution. After the nitrogen purge was stopped and sealed, the reaction was carried out at 70 ° C. for 16 hours with stirring. The produced polymer particles were collected by centrifugation and washed with methanol and water. The yield of polymer particles was 12%.

[Introduction of hydrazide group]
400 mg of polymer particles were placed in a container, 4 mL of hydrazine monohydrate (stock solution, 98% by volume or more) 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 polymer particles having a hydrazide group.

[Characteristic evaluation of polymer particles having hydrazide group]
Beads (X) made of polymer particles having hydrazide groups produced by the above-described reverse phase suspension polymerization, beads (Y) made of polymer particles having hydrazide groups produced by the above suspension polymerization, and commercially available beads containing hydrazide groups Using (Z), the amount of functional groups was quantified, and the sugar chain analysis of bovine serum-derived IgG was performed. As a bead (Z), a commercially available hydrazide group-containing bead was manufactured by Bio-Rad, and a trade name “Affigel-Hz” was used. Specifically, the amount of functional group and sugar chain analysis were performed under the following conditions.

(Quantification of functional group amount)
1 mg each of beads (X) and beads (Y), or 50 μL of beads (Z) dispersion (16.5 mg as the dry weight of beads (Z)) are placed in a container, and 1 μmol of N-acetyl-D-lactosamine (LacNAc) is added. Further, 180 μL of acetonitrile containing 2% acetic acid was added. This was heated at 80 ° C. for 45 minutes to react LacNAc with the hydrazide group on the beads. The beads were rinsed with pure water to recover unreacted sugar chains, which were quantified by MALDI-TOF-MS measurement (internal standard method) to determine the amount of LacNAc bound to the beads.

  Result: 0.83 μmol (830 nmol) of LacNAc was bound to 1 mg of beads (X). To the beads (Y), 0.85 μmol (850 nmol) of LacNAc was bound per mg. About 8 nmol of LacNAc was bound to 1 mg (dry weight) of beads (Z).

(Sugar chain analysis of bovine serum-derived IgG using hydrazide group-containing polymer beads)
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)
Add 20 μL of pre-treated biological sample suspension and 180 μL of 2% acetic acid / acetonitrile solution to a disposable column containing 5 mg of beads (X), (Y), or (Z) having hydrazide groups at 80 ° C. The reaction was carried out for 1 hour. 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, the beads were washed with a guanidine solution, pure water, methanol, and a triethylamine solution, 10% acetic anhydride / methanol was added, and the mixture was 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)
20 μL of pure water and 180 μL of a 2% acetic acid / acetonitrile solution were added to a disposable column containing beads (X), (Y), or (Z), 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 beads 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. Measurement was performed using an amino column (Shodex Asahipak NH2P-50) at an excitation wavelength of 330 nm and a fluorescence wavelength of 420 nm. As a result, a sugar chain labeled with 2AB was detected. FIG. 1 shows the total peak area obtained from the obtained HPLC chart. As shown in FIG. 1, the beads (X) and beads (Y) had the same size and were much larger than the beads (Z). From this, the capture amount of bovine serum-derived IgG sugar chains of the beads (X) of the present disclosure is equivalent to that of beads (Y) prepared by conventional suspension polymerization, and commercially available hydrazide group-containing beads (Z ) Was found to be more.

Claims (13)

In a water-in-oil emulsion, (A1) a carboxylic acid ester monomer containing a polymerizable group, or (A2) a monomer containing a polymerizable group and a hydrazide group or oxylamino group protected with a protecting group [P]; (B) observed including that obtained by the (C) polymer particles by polymerizing and cross-linking agent,
If the monomer is a carboxylic acid ester monomer comprising (A1) a polymerizable group, further including a treatment with hydrazine solution of (C) a concentration of more than 10% by volume of polymer particles,
A method for producing polymer particles having a hydrazide group or an oxylamino group. If the monomer is (A2) Ru monomer der comprising a protected hydrazide group or Okishiruamino group with a polymerizable group and the protective group [P], further protecting groups [P] The acid of the (C) polymer particles The manufacturing method of Claim 1 including deprotecting by a process. (A1) a carboxylic acid ester monomer containing a polymerizable group or (A2) a monomer containing a polymerizable group and a hydrazide group or oxylamino group protected with a protecting group [P], (B) a crosslinking agent, The manufacturing method of Claim 1 or 2 including the process of disperse | distributing the mixture containing water and a polymerization initiator to form a water-in-oil emulsion. (A1) The manufacturing method in any one of Claim 1 to 3 with which the carboxylic acid ester monomer containing a polymeric group is represented by the following general formula (I).

[In Formula (I), R ₁ may be interrupted with at least one selected from the group consisting of —O—, —S—, —NH—, —CO—, and —CONH—. Represents a hydrocarbon chain having ˜20, and R ₂ represents H, CH ₃ or a hydrocarbon chain having 2 to 5 carbon atoms. ] (A1) The production method according to any one of claims 1 to 4 , wherein the carboxylic acid ester monomer containing a polymerizable group is represented by the following formula (1).

(A2) a monomer containing a polymerizable group and the protective group protected hydrazide group or Okishiruamino group [P] is represented by the following general formula (II) or (III), any of claims 1 to 3 The manufacturing method of crab.

[In the formulas (II) and (III), R ₁ represents —O—, —S—, —NH—, —CO—, and —
1 represents a hydrocarbon chain having 1 to 20 carbon atoms which may be interrupted by at least one selected from the group consisting of CONH—, and R ₂ represents H, CH ₃ or a hydrocarbon chain having 2 to 5 carbon atoms. , [P] represents a protecting group. ] (A2) monomers comprising a protected hydrazide group or Okishiruamino group with a polymerizable group and the protective group [P] is represented by the following formula (2) or (3), of claims 1 to 3 and 6 The manufacturing method in any one.

Di (meth) acryl obtained by reacting a di- or tri (meth) acrylic acid ester of a polyol, a di- or triunsaturated acid ester of a polyol, bisacrylamide, a polyisocyanate and a (meth) acrylic acid hydroxy ester The manufacturing method in any one of Claim 1 to 7 selected from the group which consists of acid carbamyl ester, a polyvalent allyl compound, and these combination. The organic solvent of the water-in-oil emulsion is selected from the group consisting of aliphatic hydrocarbons, alicyclic hydrocarbons, aromatic hydrocarbons, aliphatic alcohols, aliphatic ketones, aliphatic esters, and combinations thereof. The manufacturing method according to any one of claims 1 to 8 . The production method according to claim 9 , wherein the organic solvent of the water-in-oil emulsion is selected from the group consisting of n-hexane, cyclohexane, n-heptane, and combinations thereof. The manufacturing method in any one of Claim 1 to 10 in which the said organic solvent contains further the sorbitan fatty acid ester of HLB3-10 as a dispersion stabilizer. The production method according to claim 11 , wherein the dispersion stabilizer is selected from the group consisting of sorbitan monostearate, sorbitan monolaurate, and sorbitan monooleate. The production method according to any one of claims 1 to 12 , wherein the polymerization reaction conditions are 50 to 90 ° C and 0.5 to 24 hours.

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