JP2012201653A - Sugar chain library, method for forming the same, and sugar chain array immobilizing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a polymer compound for bioassay, which immobilizes a sugar chain array using a conveniently formed sugar chain library without coating an adsorption inhibitor, and to provide various sugar chain array substrates using the polymer compound for bioassay.SOLUTION: The sugar chain array is formed by building a sugar chain library with a sugar chain which is obtained by: capturing a sugar chain or a sugar derivative with a primary amino group of a substrate for sugar chain purification, having the primary amino group to purify the sugar chain; recovering the sugar chain or the sugar derivative; and purifying the sugar chain or the sugar derivative, and immobilizing the sugar chain of the sugar chain library in a shape of an array by coating a substrate surface with the polymer compound for capturing sugar.

Description

The present invention relates to a sugar chain library, a method for producing the same, and a sugar chain array on which sugar chains of the sugar chain library are immobilized.

In the field of biochemistry, in recent years, sugar chain molecules have attracted attention as the third chain following nucleic acids and proteins. In particular, research on cell differentiation, canceration, immune reaction, fertilization, etc. has been conducted, and attempts to create new medicines and medical materials are continuing.
In addition, sugar chains are pathogenous foreign factor receptors such as many toxins, viruses and bacteria, and are also attracting attention as markers for cancer. There are ongoing attempts to create.

However, while the importance of research on sugar chains is recognized, the progress of research is significantly delayed compared to nucleic acids and proteins that are the first and second chains due to their complex structure and diversity.
In order to promote this research, various methods for purifying sugar chains have been developed. On the other hand, sugar chains are often confirmed to function as ligands for cell receptors rather than to function alone, and thus various sugar chains are immobilized for analysis of receptors for sugar chains. Substrates for this purpose have been developed.

Attempts have been made to create a library of various sugar chains using the above-mentioned base material, which is generally produced from complex sugars such as sugars, sugar chains, glycopeptides, glycoproteins and glycolipids derived from living organisms. The

In addition, as described in Patent Document 1, a method of synthesizing a sugar-binding protein by modifying a sugar chain recognition site of a cargo receptor has been developed, and a number of synthetic sugar chains have been produced. The sugar chain sequence increased dramatically, and the kind as a sugar chain library could be secured.

Furthermore, Patent Document 1 describes a method for producing a sugar chain array using the sugar chain library described above, wherein a trifunctional spacer is used to attach a first functional group to a sugar chain, A method is shown in which a sugar chain array is prepared by binding two functional groups to a solid phase carrier and the third functional group to a chromophore.
However, the sugar chain library prepared by this method has many impurities, and it is difficult to immediately prepare a sugar chain array.

Patent Document 2 describes a method of immobilizing a sugar chain to a substrate via a spacer, and it is shown that nonspecific adsorption is suppressed by using a hydrophilic compound for the spacer. Yes. Patent Document 2 describes a blocking operation using 3% bovine serum albumin in Examples in order to prevent non-specific adsorption after sugar chain fixation.
However, it is clear that bovine serum albumin already has a sugar chain and has a physiological function, and it is difficult to distinguish it from the target sugar chain.

Patent Document 3 describes a method of immobilizing a plurality of glycoconjugates on a base material via a spacer to form a library. By using a hydrophilic compound for the spacer, nonspecific adsorption is suppressed. It has been shown.
However, in the present invention, it is necessary to introduce a halogenated acetyl group into the solid phase carrier, and when the material is glass, it is relatively easy, but it is a method with poor versatility. In addition, as described above, sugar chains are also used in research on many receptors for toxins, viruses, bacteria, and the like. Therefore, incineration is preferable for disposal, and it is disadvantageous that the material is glass.

International Publication WO2004 / 016789 Special table 2007-527539 Special Table 2002-537562

The present invention provides a polymer compound for bioassay that can be used to easily prepare a sugar chain library and immobilize a sugar chain array using the sugar chain library without coating an adsorption inhibitor. It is an object to provide various sugar chain array substrates.

（Ｒは−Ｏ−、−Ｓ−、−ＮＨ−、−ＣＯ−、−ＣＯＮＨ−が挿入されてもよい炭素数１〜２０の炭化水素鎖を示す。）
（７）前記糖鎖精製用基材が、ビーズまたはプレート状の形態である（１）ないし（６）のいずれか１項に記載の糖鎖ライブラリー作製方法。
（８）前記糖鎖精製用基材が、下記一般式〔２〕の構造を有する架橋ポリマー構造を有するポリマーマトリックスで構成される粒子である（１）ないし（７）のいずれか１項に記載の糖鎖ライブラリー作製方法。

（Ｒ１，Ｒ２は−Ｏ−，−Ｓ−，−ＮＨ−，−ＣＯ−，−ＣＯＮＨ−が挿入されてもよい炭素数１〜２０の炭化水素鎖，Ｒ３，Ｒ４，Ｒ５はＨ，ＣＨ３，または炭素数２〜５の炭化水素鎖を示す。ｍ，ｎはモノマーユニット数を示す。）
（９）（１）ないし（８）のいずれか１項に記載の糖鎖ライブラリー作製方法で作製したことを特徴とする糖鎖ライブラリー。
（１０）前記糖鎖ライブラリーの糖鎖を基板の表面に固定化したことを特徴とする糖鎖アレイ。
（１１）前記糖鎖アレイが、下記一般式〔３〕で表される糖類固定用高分子化合物を基板表面に塗布し、高分子化合物の固定化層を介して前記糖鎖が、固定化した（１０）記載の糖鎖アレイ

（式中Ｒ１、Ｒ２、Ｒ３は水素原子またはメチル基を、Ｒ４は疎水性基を示す。Ｘは炭素数１〜１０のアルキレンオキシ基を示し、ｐは１〜２０の整数を示す。ｐが２以上２０以下の整数である場合、繰り返されるＸは、同一であっても、または異なっていてもよい。Ｙはアルキレングリコール残基を含むスペーサーであり、Ｚは酸素原子またはＮＨである。Ｍｅはメチル基である。ｌ、ｍ、ｎは自然数である。）
（１２）前記疎水性基Ｒ４が環状アルキル基であることを特徴とすることを特徴とする（１１）記載の糖類アレイ。
（１３）前記環状アルキル基がシクロヘキシル基であることを特徴とする（１２）記載の糖類アレイ。
（１４）前記基板の材質がプラスチックである（１０）ないし（１３）のいずれか１項に記載の糖鎖アレイ。
（１５）前記プラスチックが飽和環状ポリオレフィンまたはポリスチレンを含むものである（１４）記載の糖鎖アレイ。
Such an object is achieved by the present invention described in the following (1) to (15).
(1) Capture a sugar chain by bringing a purification material containing a sugar chain into contact with a sugar chain purification substrate having a primary amino group,
Collect the entire base material,
A method for preparing a sugar chain library, wherein the sugar chain library is divided for each sugar chain group having predetermined characteristics.
(2) The method for preparing a sugar chain library according to (1), wherein the sugar chains collected together with the base material are released from the base material and fractionated by liquid chromatography.
(3) The method for preparing a sugar chain library according to (1) or (2), wherein the purified raw material is an organic compound, an enzyme compound, or a biological substance.
(4) The sugar chain to be separated is a sugar chain having two or more sugars, or a compound containing the same, sugar amino acid, glycopeptide, glycoprotein, glycolipid, glycosaminoglycan, proteoglycan, glycosylphosphatidylinositol, peptidoglycan, lipo The method for preparing a sugar chain library according to any one of (1) to (3), which is selected from polysaccharides and derivatives thereof.
(5) The method for preparing a sugar chain library according to any one of (1) to (4), wherein the primary amino group is an oxylamino group and / or a hydrazide group.
(6) The sugar chain library according to any one of (1) to (5), wherein the sugar chain purification base material includes a polymer compound for sugar chain capture represented by the following general formula [1]: Manufacturing method.

(R represents a hydrocarbon chain having 1 to 20 carbon atoms into which —O—, —S—, —NH—, —CO—, and —CONH— may be inserted.)
(7) The method for preparing a sugar chain library according to any one of (1) to (6), wherein the sugar chain purification substrate is in the form of beads or plates.
(8) The sugar chain purification substrate according to any one of (1) to (7), wherein the sugar chain purification base material is a particle composed of a polymer matrix having a crosslinked polymer structure having the structure of the following general formula [2]. Of preparing a sugar chain library.

(R1 and R2 are hydrocarbon chains having 1 to 20 carbon atoms into which —O—, —S—, —NH—, —CO—, and —CONH— may be inserted; R3, R4, and R5 are H, CH3, Or a hydrocarbon chain having 2 to 5 carbon atoms, m and n represent the number of monomer units.)
(9) A sugar chain library produced by the method of producing a sugar chain library according to any one of (1) to (8).
(10) A sugar chain array, wherein sugar chains of the sugar chain library are immobilized on the surface of a substrate.
(11) The sugar chain array was applied to a substrate surface with a polymer compound for immobilizing a saccharide represented by the following general formula [3], and the sugar chain was immobilized via a polymer compound immobilization layer. (10) The sugar chain array according to

(Wherein R1, R2 and R3 represent a hydrogen atom or a methyl group, R4 represents a hydrophobic group, X represents an alkyleneoxy group having 1 to 10 carbon atoms, and p represents an integer of 1 to 20. p is an integer. When X is an integer of 2 or more and 20 or less, the repeated Xs may be the same or different, Y is a spacer containing an alkylene glycol residue, and Z is an oxygen atom or NH. Is a methyl group, and l, m, and n are natural numbers.)
(12) The saccharide array according to (11), wherein the hydrophobic group R4 is a cyclic alkyl group.
(13) The saccharide array according to (12), wherein the cyclic alkyl group is a cyclohexyl group.
(14) The sugar chain array according to any one of (10) to (13), wherein a material of the substrate is plastic.
(15) The sugar chain array according to (14), wherein the plastic contains saturated cyclic polyolefin or polystyrene.

According to the present invention, a sugar chain can be easily purified from a contaminated state like a biological material, a purified sugar chain library can be constructed, and a sugar chain array using the sugar chain library is prepared. Is possible.

HPLC chart of IgG free sugar chain

The sugar chain library described in the present invention is constituted by collecting, purifying and separating only sugar chains from sugar chain resources which are purification raw materials such as various animal cells and serum glycoproteins. In order to construct a library, a technology for mass production based on various conditions for purification and separation of sugar chains is required.

(Description of sugar chain library production method)
First, the method for preparing a sugar chain library comprises the following steps.
(1) By bringing the purified raw material into contact with a sugar chain purification substrate having a primary amino group, a chemical bond is formed between the reducing end of the sugar chain and the primary amino group, and only the sugar chain is formed on the substrate. To capture.
(2) The substrate that has captured the sugar chain can easily remove impurities other than the sugar chain in the purified raw material by washing, and collects only the sugar chain together with the substrate.
(3) The sugar chain collected together with the base material is released from the base material and divided into sugar chain groups having predetermined characteristics to prepare a sugar chain library.

The sugar chain in the present invention is a narrowly-defined sugar chain of two or more sugars, or a compound containing the same, for example, sugar amino acid, glycopeptide, glycoprotein, glycolipid, glycosaminoglycan, proteoglycan, glycosylphosphatidylinositol, peptidoglycan, Lipopolysaccharides and their derivatives are shown.

(About refined raw materials)
Next, each step will be described in detail.
First, the purification raw material is an organic compound, an enzyme compound or a biological substance, and is often a mixture containing sugar chains. In the case of an organic compound, a reaction catalyst and a reaction intermediate are included, and in the case of an enzyme compound, a synthetic enzyme is included. In addition, in the case of biologically derived substances, it contains many contaminants other than substances containing sugar chains, such as tissue-derived, animal cell-derived or body fluid-derived substances such as blood, or roughly purified serum glycoproteins. It is a mixture.

A method for capturing only sugar chains from the purified raw material will be described.
A sugar chain is the only substance in the living body that generates an aldehyde group at the terminal by reduction treatment. By bringing the sugar chain into contact with a sugar chain purification substrate having a primary amino group on the surface, the sugar chain can be immobilized on the surface of the sugar chain purification substrate by chemical bonding.
The contact method is carried out by placing the sugar chain purification base material in the solution of the mixture containing the reducing end sugar chain, or by placing the mixture solution in a container made of the sugar chain purification base material and bringing it into contact with the aldehyde group. The primary amino group is reacted with each other to form a chemical bond, and the sugar chain is covalently bonded to the sugar chain-trapping substrate.

(About the substrate for sugar chain purification)
The sugar chain purification substrate is a substrate having on its surface a reactive primary amino group for capturing a sugar chain, and preferably has an oxylamino group or a hydrazide group as the primary amino. This is preferable because it can react with and bind to the aldehyde group which is the sugar chain reducing end even in the absence of an enzyme or a coupling reagent.

式１中のＲは−Ｏ−，−Ｓ−、−ＮＨ−、−ＣＯ−、−ＣＯＮＨ−が挿入されてもよい炭素数１〜２０の炭化水素を示す。
前記担体は、下記一般式〔２〕の構造を有する架橋ポリマー構造を有するポリマーマトリックスで構成される粒子であることが好ましい。

（Ｒ１，Ｒ２は−Ｏ−，−Ｓ−，−ＮＨ−，−ＣＯ−，−ＣＯＮＨ−が挿入されてもよい炭素数１〜２０の炭化水素鎖，Ｒ３，Ｒ４，Ｒ５はＨ，ＣＨ３，または炭素数２〜５の炭化水素鎖を示す。ｍ，ｎはモノマーユニット数を示す。）
前記、一般式〔２〕の担体は、式〔１〕のＲ−ＮＨ−ＮＨ２の構造を含むため、担体が式〔１〕の構造体そのものとなる。 The sugar chain purification substrate preferably has a structure represented by the following general formula [1].

R in Formula 1 represents a hydrocarbon having 1 to 20 carbon atoms into which —O—, —S—, —NH—, —CO—, and —CONH— may be inserted.
The carrier is preferably particles composed of a polymer matrix having a crosslinked polymer structure having the structure of the following general formula [2].

(R1 and R2 are hydrocarbon chains having 1 to 20 carbon atoms into which —O—, —S—, —NH—, —CO—, and —CONH— may be inserted; R3, R4, and R5 are H, CH3, Or a hydrocarbon chain having 2 to 5 carbon atoms, m and n represent the number of monomer units.)
Since the carrier of the general formula [2] includes the R—NH—NH 2 structure of the formula [1], the carrier is the structure of the formula [1] itself.

In addition, the carrier is preferably a carrier insoluble in an aqueous solution or an organic solvent, and the material is not particularly limited, but glass or a resin excellent in organic solvent resistance such as silicon, polystyrene, ethylene, and the like. -Maleic anhydride copolymer, polymethyl methacrylate and the like can be selected.

The form of the sugar chain purification substrate is not particularly limited, but is preferably a bead or plate form. In order to prepare a sugar chain library, it is possible to process a large number of samples at the same time. In this case, continuous processing is possible by using a column packed with beads. Moreover, if it is a multiwell plate, it is possible to process many samples simultaneously. As the multiwell plate, multiwell plates such as 6, 12, 24, 48, 96, and 384 wells can be appropriately used.

Other than the carrier of the formula [2], an inorganic substance can be used as beads. The carrier can be in the form of particles, and examples thereof include silica particles, alumina particles, glass particles, and metal particles. In addition, as the organic polymer substance, a polysaccharide gel typified by agarose or sepharose, or a polymer obtained by polymerizing a polymer of a vinyl compound can be used.

Moreover, it is preferable that the shape when it is set as a particle is a sphere, and it is a polymer particle having an average particle diameter of 0.1 μm to 500 μm. The average particle diameter in this case is obtained by measuring the diameter of each particle observed in the optical microscope field of view. It is considered that the carrier particles having a particle size in such a range can be easily collected by centrifugation, a filter, etc., and have a sufficient surface area, so that the reaction efficiency with sugar chains is high. When the particle size is significantly larger than the above range, the reaction efficiency with the sugar chain may be lowered due to the small surface area. In addition, when the particle size is significantly smaller than the above range, it may be difficult to collect particles particularly by a filter. Furthermore, when the particles are packed in a column and used, if the particle size is too small, the pressure loss at the time of liquid passage may increase.

(About sugar chain capture)
One specific example of the conditions for the coupling reaction between the terminal reduced sugar chain and the primary amino group on the sugar chain purification substrate is that the pH is 4 to 7, the reaction temperature is 25 to 90 ° C, preferably 60 to 85 ° C, more preferably 75-80 degreeC and reaction time are 1 to 16 hours. The most preferred conditions are pH 4.5 to 5.5, reaction temperature 80 ° C., and reaction time 1 hour.
When the pH is less than 4 or exceeds 7, the dissociation reaction of the capture sugar chain occurs at the same time, and the capture efficiency decreases. When the reaction temperature is less than 25 ° C., the reaction efficiency may be remarkably deteriorated, and sugar chains cannot be sufficiently captured.
When the temperature exceeds 90 ° C., the compound itself is adversely affected, and when the base material is plastic, deformation or melting may occur depending on the type.
When the reaction time is shorter than 1 hour, a sufficient binding reaction may not be obtained, and the sugar chain cannot be captured sufficiently. Moreover, the reaction over 16 hours has no effect of only taking time because no further capture of sugar chains is observed.

(About sugar chain recovery)
Here, as the solution used for the cleaning liquid, an aqueous solution containing a surfactant, alcohols such as methanol and ethanol; water, an aqueous buffer, and the like are used. Here, when an aqueous solution is used for washing, the pH of the aqueous solution is preferably near neutral, and the pH is 4 to 10, more preferably 6 to 8.
The substrate obtained by capturing the sugar chain in the previous period can easily remove impurities other than the sugar chain in the purified raw material by washing, and only the sugar chain can be recovered together with the substrate.
As a washing method, in the case of beads, the beads can be washed by immersing them in a washing solution and repeating the exchange of the washing solution.
Moreover, in the case of a plate, it can wash | clean simply by dispensing a washing | cleaning liquid in each well and repeating suction removal.

The surfactant is not particularly limited, but a buffer solution capable of controlling pH, such as a phosphate buffer solution or a Tris-hydrochloric acid buffer solution, is preferable. Various anionic surfactants, cationic surfactants, and neutral surfactants can be used, and although not particularly limited, Triton X, various Tweens, and sodium dodecyl sulfate are preferably used. The pH of the cleaning liquid is preferably 4-7. This is because when the pH is lower than 4 or higher than pH 7, a sugar chain elimination reaction occurs depending on the pH.

  In the case where the sugar chain capture reaction is performed in a continuous manner, this washing treatment may be performed continuously from the sugar chain capture reaction by passing a washing solution through the column. When a multiplate is used, substances other than the sugar chain-trapping substance may be removed by filtration or centrifugation.

  The washing step may not be performed depending on the state of the initial biological sample, for example, the degree of mixing of substances other than sugar chains.

(Glycan separation and purification)
As the next step, it will be described that the sugar chains recovered together with the base material are released from the base material and divided into sugar chain groups having predetermined characteristics.
Examples of the predetermined properties include, but are not limited to, a molecular weight of a sugar chain, a sugar chain binding mode, and a binding molecule other than the presence or absence of a specific sugar chain.

As described above, the sugar chain recovered together with the base material can be released from the base material, and the free sugar chain can be used as a sugar chain library. This release operation will be described below.

The basis of the releasing operation is that the sugar chain can be cut out from the base material by subjecting the sugar chain collected together with the base material to a heating reaction in an acidic solvent in the presence of a reducing agent.
A specific method is shown below.

The solvent for the reaction is preferably a free reaction 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 organic solvent, the water content is preferably 0.1% to 90%, more preferably 0.1% to 80%, and even more preferably 0.1% to 50%. . An aqueous buffer may be contained instead of water. The concentration of the buffer before mixing 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 reaction solution is preferably 2-9, more preferably 2-7, and even more preferably 2-6. The acid used is preferably, for example, acetic acid, formic acid, trifluoroacetic acid, hydrochloric acid, citric acid, phosphoric acid, sulfuric acid, more preferably acetic acid, formic acid, trifluoroacetic acid, phosphoric acid, and more preferably acetic acid, trifluoroacetic acid. .

As the reducing agent, sodium borohydride can be suitably used.
Here, if a compound containing an aminooxy group or a hydrazide group is used as the compound containing an amino group, it is not always necessary to use a reducing agent.

The reaction temperature is preferably 4 to 90 ° C, preferably 25 to 90 ° C, more preferably 40 to 90 ° C. The reaction time is 10 minutes to 24 hours, preferably 10 minutes to 8 hours, more preferably 10 minutes to 3 hours. The reaction is preferably carried out in an open system to completely evaporate the solvent.
Since the sugar chain cleaving reaction can be performed in the vicinity of pH 2 from acidic to neutral, compared to the conventional cleaving reaction in the presence of a strong acid such as cleaving by 10% trifluoroacetic acid treatment, It is possible to suppress the occurrence of sugar chain hydrolysis such as elimination of sialic acid residues.

In addition, when the sugar chain collected together with the base material is released and the molecular weight fractionation is performed by a method such as high performance liquid chromatography, it is necessary to fluorescently label the sugar chain. The sugar chain release and fluorescent labeling are described below.

The fluorescent labeling method is a method in which a sugar chain is labeled with a fluorophore or a chlorophore at the same time as a fluorophore or chlorophore containing an amino group is contacted to release the sugar chain. The details of the reaction solvent, reducing agent, reaction temperature, and time are as described above, but it can be achieved by adding a compound containing an amino group, for example, 10 equivalents or more of a sugar chain.

The use of the reducing agent is a chlorophore or fluorophore containing an amino group, specifically, wherein the chlorophore or fluorophore is selected from the group consisting of substances containing the following hydrazide groups or substances containing an aminooxy group: This is more preferable.
(Substances containing hydrazide groups)
5-Dimethylaminonaphthalene-1-sulfonyl hydrazine (Dansylhydrazine); 2-hydrazinopyridine; 9-fluorenylmethyl carbazate (Fmoc hydrazine); benzylhydrazine; 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s -indacene-3-propionoc acid, hydrazide; 2- (6,8-difluoro-7-hydroxy-4-methylcoumarin) acetohydrazide; 7-diethylaminocoumarin-3-carboxylic acid, hydrazide (DCCH); phenylhydrazine; 1-Naphthaleneacethydrazide; 2 -hydrazinobenzoic acid; phenylacetic hydrazide;
(Substances containing aminooxy groups) O-benzylhydroxylamine; O-phenylhydroxylamine; O- (2,3,4,5,6-pentafluorobenzyl) hydroxylamine; O- (4-nitrobenzyl) hydroxylamine; 2-aminooxypyridine; 2-aminooxymethylpyridine; 4-[(aminooxyacetyl) amino] benzoic acid methyl ester; 4-[(aminooxyacetyl) amino] benzoic acid ethyl ester; 4-[(aminooxyacetyl) amino] benzoic acid n-butyl ester

In the case of this method, it is necessary to further remove the label after the molecular weight fractionation of the sugar chain using the label.
The method of removing the label can be performed by the same method as separating the sugar chain from the substrate.

(Sugar chain library storage method)
As described above, sugar chains are collected in a solution state and prepared as a sugar chain library. This solution-like sugar chain library may be used immediately or may be stored until needed, depending on the purpose. The storage method is preferably transferred to a closed container such as a commercially available tube with a lid as it is and stored frozen. More preferably, the method is freeze-dried and then stored frozen. The temperature for frozen storage is preferably −20 ° C. or lower, more preferably −80 ° C.

(Overview of the entire sugar chain array)
Next, a method for producing a sugar chain array using the sugar chain library of the present invention will be described.
A sugar chain array is a general term for a sugar chain immobilized on an array substrate. In the present invention, a polymer compound that mediates the binding of the sugar chain to the substrate is coated on the array substrate. Thereafter, the sugar chain is immobilized on the array substrate using a sugar chain-binding functional group provided in the polymer compound.

(Glycan library array)
Specifically, the above polymer compound includes at least a unit A for retaining hydrophilicity, a unit B having a hydrophobic group for capturing saccharides, and a unit having a primary amino group for capturing saccharides. It is a polymer compound composed of C. This polymer compound has a property of immobilizing a physiologically active substance and a property of suppressing physical adsorption (non-specific enemy adsorption) of the detection target to the array substrate. That is, the primary amino group plays a role of immobilizing sugars, sugar chains, glycopeptides, glycolipids, or physiologically active substances having these, and a unit for maintaining hydrophilicity is physically applied to the substrate to be detected. It also has the property of suppressing adsorption (non-specific adsorption). The hydrophobic unit B is involved in the adsorption of the polymer compound to a plastic array substrate or a glass array substrate.

  As such a polymer compound for immobilization, those represented by the following formula are preferably used. The hydrophilic unit A contained in the polymer compound is typified by a phosphorylcholine group, and the structure is not particularly limited. However, in the following general formula [3], the unit A is represented by the left structural unit. As described above, a structure in which a (meth) acrylic residue and a phosphorylcholine group are bonded via a chain of an alkyleneoxy group X having 1 to 10 carbon atoms is most preferable. Of these, X is most preferably an ethyleneoxy group. The repeating number of the alkyleneoxy group in the formula is an integer of 1 to 20, and when the repeating number is 2 or more and 20 or less, the carbon number of the repeating alkyleneoxy group may be the same or different. Although l is naturally a natural number, it may be expressed as a composition ratio of each component.

（式中Ｒ１、Ｒ２、Ｒ３は水素原子またはメチル基を、Ｒ４は疎水性基を示す。Ｘは炭素数１〜１０のアルキレンオキシ基を示し、ｐは１〜２０の整数を示す。ｐが２以上２０以下の整数である場合、繰り返されるＸは、同一であっても、または異なっていてもよい。Ｙはアルキレングリコール残基を含むスペーサーであり、Ｚは酸素原子またはＮＨである。Ｍｅはメチル基である。ｌ、ｍ、ｎは自然数である。）

(Wherein R1, R2 and R3 represent a hydrogen atom or a methyl group, R4 represents a hydrophobic group, X represents an alkyleneoxy group having 1 to 10 carbon atoms, and p represents an integer of 1 to 20. p is an integer. When X is an integer of 2 or more and 20 or less, the repeated Xs may be the same or different, Y is a spacer containing an alkylene glycol residue, and Z is an oxygen atom or NH. Is a methyl group, and l, m, and n are natural numbers.)

The composition ratio of the unit A having a phosphorylcholine group contained in the polymer compound of the present invention (ratio of l to the sum of l, m, n) is preferably 5 to 98 mol% with respect to all units of the polymer. Is 10 to 80 mol%, most preferably 10 to 80%. When the composition ratio is below the lower limit, the hydrophilicity becomes weak and non-specific adsorption increases. On the other hand, when the upper limit is exceeded, water solubility increases, and the polymer compound may be eluted during the assay.

The composition ratio of the unit B having a hydrophobic group contained in the polymer compound of the present invention (ratio of m to the sum of l, m, and n) is preferably 10 to 90 mol% with respect to all units of the polymer. Preferably it is 10-80 mol%, Most preferably, it is 20-80%. If the upper limit is exceeded, non-specific adsorption may increase.

The unit C having a primary amino group contained in the polymer compound of the present invention is not particularly limited in structure, but as represented by the right structural unit in the general formula [3], ( A structure via a spacer Y containing a (meth) acrylic residue and an oxylamino residue is preferred. In the case of an oxylamino group, Z represents an oxygen atom, and in the case of a hydrazide group, Z represents NH. n is originally a natural number, but may be expressed as a composition ratio of each component. The structure of the spacer Y containing an alkylene glycol residue is not particularly limited, but is preferably the following general formula [4] or [5], and more preferably [2].

(式中ｑは１〜２０の整数)

(Where q is an integer from 1 to 20)

(式中ｒは１〜２０の整数)

(Wherein r is an integer of 1 to 20)

The composition ratio of the component of unit C having a primary amino acid (ratio of n to the sum of l, m, n) is preferably 1 to 94 mol%, more preferably 2 to 90 mol%, most preferably with respect to all units of the polymer. Preferably, it is 20 to 40%. When the composition value falls below the lower limit, it is impossible to immobilize a sufficient amount of sugar, sugar chain, and / or physiologically active substance having these. Moreover, when it exceeds an upper limit, nonspecific adsorption | suction increases.

The method for synthesizing the polymer compound of the present invention is not particularly limited. From the viewpoint of ease of synthesis, at least a monomer having a phosphorylcholine group as unit A, a monomer having a hydrophobic group as unit B, and a primary unit as unit C A production method including a step of radical copolymerizing a monomer in which an amino group is previously protected with a protecting group, and a step of removing the protecting group from the polymer compound obtained by the step is preferred. Alternatively, a step of radical copolymerizing a monomer having at least a phosphorylcholine group, a monomer having a hydrophobic group, and a monomer having a functional group capable of introducing a primary amino group, and the polymer compound obtained by the step having a primary amino group The manufacturing method including the step of introducing is preferable.

The monomer having a phosphorylcholine group as the unit A is not particularly limited in structure. For example, 2- (meth) acryloyloxyethyl phosphorylcholine, 2- (meth) acryloyloxyethoxyethyl phosphorylcholine, 6- (meth) acryloyloxy Hexyl phosphorylcholine, 10- (meth) acryloyloxyethoxynonyl phosphorylcholine, 2- (meth) acryloyloxypropyl phosphorylcholine and the like can be mentioned, and 2-methacryloyloxyethyl phosphorylcholine is preferable from the viewpoint of availability.

Specific examples of the monomer having a hydrophobic group as unit B include n-butyl (meth) acrylate, iso-butyl (meth) acrylate, sec-butyl (meth) acrylate, t-butyl (meth) acrylate, n-neopentyl (meth) acrylate, iso-neopentyl (meth) acrylate, sec-neopentyl (meth) acrylate, neopentyl (meth) acrylate, cyclohexyl (meth) acrylate, n-hexyl (meth) acrylate, iso-hexyl (meth) Acrylate, heptyl (meth) acrylate, n-octyl (meth) acrylate, iso-octyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, n-nonyl (meth) acrylate, iso-nonyl (meth) acrylate, n- Decyl (Me ) Acrylate, is o-decyl (meth) acrylate, n-dodecyl (meth) acrylate, iso-dodecyl (meth) acrylate, n-tridecyl (meth) acrylate, iso-tridecyl (meth) acrylate, n-tetradecyl (meth) Acrylate, iso-tetradecyl (meth) acrylate, n-pentadecyl (meth) acrylate, iso-pentadecyl (meth) acrylate, n-hexadecyl (meth) acrylate, iso-hexadecyl (meth) acrylate, n-octadecyl (meth) acrylate, iso-octadecyl (meth) acrylate, isobonyl (meth) acrylate, etc. are mentioned. Of these, cyclohexyl (meth) acrylate, n-butyl methacrylate, n-dodecyl methacrylate, and n-octyl methacrylate are most preferable.

The monomer in which the primary amino group is previously protected with a protecting group is not particularly limited in structure, but the following general formula [6] (wherein R3 is a hydrogen atom or a methyl group, Y is a spacer containing an alkylene glycol residue, Z Is a structure in which a (meth) acrylic group and an oxylamino group or a hydrazide group are interposed via a spacer Y containing an alkylene glycol residue, as represented by: oxygen atom or N, H, W represents a protecting group. It is preferable that

The protecting group W 1 is not limited as long as it can protect an amino group, and can be arbitrarily used, for example, from an acid anhydride. Of these, t-butoxycarbonyl group (Boc group), benzyloxycarbonyl group (Z group, Cbz group), 9-fluorenylmethoxycarbonyl group (Fmoc group) and the like are preferably used.

Specific examples of the monomer include those represented by the following formula [7].

Deprotection can be performed under general conditions using trifluoroacetic acid, hydrochloric acid, or anhydrous hydrogen fluoride.

On the other hand, the method for introducing a primary amino group after polymerizing a polymer compound is not limited at all, but at least a monomer having a phosphorylcholine group, a monomer having a hydrophobic group, and a monomer having an alkoxy group are radicalized. A method of producing a hydrazide group by reacting an alkoxy group introduced into the polymer compound with hydrazine after copolymerization is preferred. As the alkoxy group, a methoxy group, an ethoxy group, a propoxy group, a t-butoxy group and the like are preferable.

Specific examples of the monomer having an alkoxy group are those represented by the following formula [8].

As the synthetic solvent for the polymer compound of the present invention, any solvent may be used as long as each monomer can be dissolved. For example, alcohols such as methanol, ethanol, isopropanol, n-butanol, t-butyl alcohol, and n-pentanol are used. , Benzene, toluene, tetrahydrofuran, dioxane, dichloromethane, chloroform, cyclohexanone, N, N-dimethylformamide, dimethyl sulfoxide, methyl acetate, ethyl acetate, butyl acetate, methyl ethyl ketone, methyl butyl ketone, ethylene glycol monoethyl ether, ethylene glycol monomethyl ether And ethylene glycol monobutyl ether. These solvents are used alone or in combination of two or more.

The polymerization initiator may be any ordinary radical initiator, such as 2,2′-azobisisobutylnitrile (hereinafter referred to as “AIBN”), 1,1′-azobis (cyclohexane-1-carbonitrile), and the like. Examples thereof include organic peroxides such as azo compounds, benzoyl peroxide, and lauryl peroxide.

The molecular weight of the polymer compound of the present invention is preferably 5,000 or more and more preferably 10,000 or more because the polymer compound and unreacted monomer can be easily separated and purified. The number average molecular weight is calculated by high performance liquid chromatography measurement.

Deprotection can be performed under general conditions by using the above-described trifluoroacetic acid, hydrochloric acid, and anhydrous hydrogen fluoride, but the timing of deprotection is as follows.
Usually, the polymerization is completed and the polymer compound is generally prepared, and the polymer compound of the present invention is generally obtained. To obtain the polymer compound of the present invention, the polymer compound is obtained by deprotection after the completion of the polymerization. be able to.

By covering the surface of the substrate with the polymer compound, it is possible to easily impart the property of suppressing nonspecific adsorption of the physiologically active substance and the property of immobilizing the physiologically active substance.
In this case, it is possible to coat a polymer compound having a primary amino group which has been deprotected, but in some cases, coating a polymer material having a highly reactive primary amino group in a solution may be It is conceivable that the primary amino group reacts and inactivates during the work.
Therefore, it is desirable to purify the polymer compound immediately before deprotection, coat the surface of the substrate, and then perform a deprotection reaction to form a state in which a primary amino group is present on the surface of the substrate.

For coating the surface of the array substrate with a polymer compound, for example, a polymer solution in which a polymer compound is dissolved in an organic solvent so as to have a concentration of 0.05 to 50% by weight is prepared, and a known method such as immersion or spraying is used. After coating on the surface of the substrate, drying is performed at room temperature or under heating.

Examples of organic solvents include ethanol, methanol, isopropanol, n-butanol, t-butyl alcohol, n-pentanol, cyclohexanol, and other alcohols, benzene, toluene, tetrahydrofuran, dioxane, dichloromethane, chloroform, acetone, methyl acetate, ethyl acetate, Examples thereof include butyl acetate, methyl ethyl ketone, methyl butyl ketone, ethylene glycol monoethyl ether, ethylene glycol monomethyl ether, ethylene glycol monobutyl ether, and cyclohexanone. These solvents are used alone or in combination of two or more. Of these, alcohols such as ethanol, methanol, isopropanol, n-butanol, t-butyl alcohol, and n-pentanolcyclohexanol are preferable because they do not denature the plastic substrate and can be easily dried.

The array substrate used in the present invention is preferably a slide-shaped substrate, 6, 12, 24, 48, 96, 384 hole plate, container, or microfluidic substrate. For example, a plastic substrate, a glass substrate, a substrate having a metal vapor deposition film, and the like can be mentioned. A slide-shaped substrate is most suitable. Specific examples of the plastic substrate include substrates made of polystyrene, cyclic polyolefin polymer, cycloolefin polymer, polypropylene, polyethylene, polysulfone, polyimide, polycarbonate, and polymethyl methacrylate.

In particular, a cyclic polyolefin polymer and a cycloolefin polymer are useful as a base material used for fluorescence observation. In the case of using the base material, if the hydrophobic group of the polymer compound is a cyclohexyl group, the interaction with the base material is good, and a polymer material having the same composition ratio is used as another base material (for example, polystyrene). Compared with the case of application to a glass substrate), the adsorption amount is high and the background value is low.

Further, when the polymer compound is applied to an array substrate made of a cyclic polyolefin polymer, it cannot be applied to a polymer compound having 100 mol% of aminooxy monomer. On the other hand, it can be applied to polystyrene polymer, but nonspecific adsorption of impurities is not suppressed and is not versatile.

The base material coated with the polymer compound can immobilize the sugar chain group in the sugar chain library and can be suitably used for bioassay applications.
The sugar chain group includes two or more sugar chains or a compound containing the same, sugar amino acid, glycopeptide, glycoprotein, glycolipid, glycosaminoglycan, proteoglycan, glycosylphosphatidylinositol, peptidoglycan, lipopolysaccharide, and their Derivatives and the like. As a method for immobilizing these sugar chain groups on a base material, a method of spotting a solution in which a physiologically active substance is dissolved using a spotter, or dispensing a solution in which a sugar chain group is dissolved into a container or the like. There are ways to fix it.

The method of immobilizing the sugar chain group in the sugar chain library on the surface of the substrate can be immobilized by the same reaction as the reaction between the terminal reduced sugar chain and the primary amino group on the aforementioned sugar chain purification substrate. That is, the terminal reduced sugar chain of the sugar chain in the sugar chain library and the primary amino group on the array substrate have a pH of 4 to 7, a reaction temperature of 25 to 90 ° C, preferably 60 to 85 ° C, more preferably Is reacted at 75 to 80 ° C. and reaction time of 1 to 16 hours. The most preferred conditions are pH 4.5 to 5.5, reaction temperature 80 ° C., and reaction time 1 hour.

When the lyophilized and stored sugar chain library is used, various buffer materials are preferably used as a solution for dissolving the sugar chain group in the sugar chain library. Although not particularly limited, for example, sodium carbonate, sodium bicarbonate, potassium phosphate, dipotassium hydrogen phosphate, Tris hydrochloride buffer, Tris acetate buffer, PBS buffer, sodium citrate, sodium acetate, HEPES (N-2- Hydroxyethylperazine-N′-ethanesulfonic acid) buffer, MOPS (3- (N-morpholino) propanesulfonic acid) buffer, and the like are used.

The pH of the solution for dissolving the sugar chain group in the sugar chain library is preferably 2 to 8 when dissolving sugar or a narrowly-defined sugar chain. When glycoprotein is dissolved, the pH is preferably 4-9. When the sugar nucleic acid is dissolved, the pH is preferably 2-8. When the glycolipid is dissolved, the pH is preferably 2-8.

The concentration of the sugar chain group in the sugar chain library is not particularly limited, but is preferably 0.0001 mg / ml to 10 mg / ml.

The temperature for immobilizing the sugar chain group in the sugar chain library is preferably from 0 ° C to 100 ° C.

EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example and a comparative example, this invention is not limited to this.

<Preparation of sugar chain library>
(Free sugar chain)
1 mg of IgG (derived from Bovine Serum) was subjected to reductive alkylation treatment, trypsinization, N-glycosidase (PNGaseF) treatment according to the protocol attached to the sugar chain purification kit BlotGlyco (BS-45603 manufactured by Sumitomo Bakelite), and sugar chains from IgG Was released. the detail is right below.

The following solution was added to a tube containing IgG (1 mg) and stirred until completely dissolved.
・ 1M ammonium bicarbonate aqueous solution (5μL)
・ Pure water (50μL)
120 mM dithiothreitol (DTT, Sigma, D9779) aqueous solution (5 μL)

The solution was allowed to stand at 60 ° C. for 30 minutes, 123 mM iodoacetamide (IAA, manufactured by Wako Pure Chemical Industries, Ltd., 093-02152) aqueous solution (10 μL) was added, and the mixture was allowed to stand at room temperature for 1 hour in the dark.
After standing, trypsin (400 units) was added. (Trypsin is dissolved in 1 mM hydrochloric acid at 40 units / μL, and 10 μL is added to the sample solution)
The reaction was allowed to stand for more than 1 hour in a 37 ° C. incubator. Then, it heated with the 90 degreeC heat block for 5 minutes, and the trypsin was deactivated. After the solution is cooled to 37 ° C., N-glycosidase (PNGase F, manufactured by Roche, 11-365-193-001) (5 μL = 5 units) is added and allowed to react overnight (12 hours or longer) in an incubator at 37 ° C. It was. After reaction, it heated at 90 degreeC for 5 minute (s), and PNGaseF was deactivated.

(Glycan purification)
According to the BlotGlyco protocol, 50 μL of a BlotGlyco bead dispersion liquid in which sugar chains corresponding to 200 μg of IgG were captured on beads was pipetted and injected into the bottom of a reaction tube attached to the kit. The reaction tube is centrifuged for several seconds with a table centrifuge (Chibitan, manufactured by Nihon Millipore), water is removed, sugar chain sample solution (20 μL) is added to the beads, and 2% acetic acid / acetonitrile (180 μL) is further added. Added. The reaction tube was inserted into an 80 ° C. heat block and heated for 1 hour to confirm that the solvent was completely evaporated and the beads were completely dried. After completion of the reaction, the reaction tube was inserted into a 2 mL Eppendorf tube, 2M guanidine solution (200 μL) was added to the reaction tube, and the mixture was centrifuged for several seconds with a tabletop centrifuge. After repeating this operation three times, the solution accumulated in the Eppendorf tube was removed. The reaction tube was washed three times with pure water (200 μL), followed by washing three times with 1% triethylamine / methanol (200 μL), and finally three times with methanol (200 μL).
Add 100 μL of 10% acetic anhydride / methanol solution to the beads in the reaction tube and react for 30 minutes at room temperature. After the reaction, the reaction tube was again inserted into a 2 mL Eppendorf tube and centrifuged to remove the acetic anhydride solution.
Further, the beads were washed with methanol (200 μL) three times, followed by washing with 10 mM hydrochloric acid (200 μL) three times, and finally with pure water (200 μL) three times.

(Sugar chain labeling)
Add 20 μL of 20 mM 2-aminobenzohydrazide (2-ABh, manufactured by Wako Pure Chemical Industries, Ltd., 574-92441) solution and 180 μL of 2% acetic acid / acenitrile solution, heat at 80 ° C. for 1 hour, and release sugar chain from BrotoGlyco. Labeling was performed.
Specifically:

Next, excess unreacted 2-ABh reagent was removed.
Acetonitrile (950 μL) is added to the collected sugar chain solution (about 50 μL), mixed, the cleanup column in the BlotGlyco kit is set in a centrifuge, 200 μL of pure water is added to the column, and the mixture is centrifuged for several seconds and filtered. .
200 μL of acetonitrile was added to the column, centrifuged and filtered. This was repeated 3 times to wash the cleanup column.
After discarding the waste solution, the whole amount of the sugar chain solution / acetonitrile solution was added to the cleanup column, and the solution was allowed to stand and let the solution pass by natural fall.
Acetonitrile (400 μL) was added, centrifuged and passed, and this was repeated three times.
400 μL of acetonitrile / pure water (95: 5, v / v) was added to the column, centrifuged and passed, and this was repeated three times.
The cleanup column was inserted into a new Eppendorf tube, and 50 μL of pure water was added and centrifuged to elute the labeled sugar chain.
Using the collected solution, fractionation was performed using HPLC.

(Preparation by HPLC)
The 2ABh-labeled sugar chain obtained above was fractionated by HPLC.
For fractionation, one fraction was collected every 2 minutes to obtain a total of 14 fractions.

The HPLC conditions are as follows.
Column: TSK-GEL Amide-80 (manufactured by Tosoh Corporation)
Solvent A: 50 mM formic acid aqueous solution (adjusted to pH 4.4 with aqueous ammonia)
Solvent B: Acrylonitrile Gradient: A 20% (0 min) → A 58% (158 min)
・ Flow rate: 0.4 mL / min
-Column temperature: 30 ° C
・ Device: Waters Delta600 (manufactured by Nippon Waters)

Each fraction obtained above was concentrated and dried.

(Removal of 2ABh from sugar chain)
The sugar chain obtained as described above is labeled with 2ABh. Although it may be preserved as it is, since the library can be used immediately by removing the label, the method for removing the label is described below.

The obtained sugar chain was treated with 0.5% TFA at 37 ° C. for 1 hour to liberate 2ABh, and then 2ABh was removed using a silica column. The resulting solution was concentrated and dried.
Acetonitrile was added to the collected sugar chain and mixed to 1000 μL.
The cleanup column in the BlotGlyco kit was set in a centrifuge, and 200 μL of pure water was added to the column, followed by centrifugation for several seconds and filtration.
200 μL of acetonitrile was added to the column, centrifuged and filtered. This was repeated 3 times to wash the cleanup column.
After discarding the waste solution, the whole amount of the sugar chain solution / acetonitrile solution was added to the cleanup column, and the solution was allowed to stand and let the solution pass by natural fall.
Acetonitrile (400 μL) was added, centrifuged and passed, and this was repeated three times.
400 μL of acetonitrile / pure water (95: 5, v / v) was added to the column, centrifuged and passed, and this was repeated three times.
The cleanup column was inserted into a new Eppendorf tube, and 50 μL of pure water was added and centrifuged to elute sugar chains.

<Production of sugar chain array>
Immobilization of sugar chains and evaluation of binding to lectins were carried out by the following methods.
(Production of array substrate)
For the production of the microarray, a glass substrate made of glass slide made of Sumitomo Bakelite and cyclic polyolefin resin was used.
2-methacryloyloxyethyl phosphorylcholine-cyclohexyl methacrylate-N- [2- [2- [2-t-butoxycarbonylaminooxyacetylamino] ethoxy] ethoxy] ethyl] -methacrylamide copolymer in 1.0 wt% ethanol solution The slide was immersed in and a polymer compound was applied.
After the coating, it was treated with 2M HCl at 37 ° C. for 2 hours to deprotect the t-butoxycarbonyl group (Boc group), and an amino group was produced on the substrate surface.

(Immobilization of sugar chains)
The IgG-derived sugar chain obtained by fractionation by HPLC was dissolved by adding 20 uL of the following solution, spotted on a substrate, and reacted at 80 ° C. for 1 hour to immobilize the sugar chain on the substrate.
Solution: 100 mM NaOAc buffer (pH 5.0) + 0.01% Triton X-100 (Wako Pure Chemicals, A16046), 0.01% PVA (degree of polymerization = 1500)

(Detection of fixed sugar chains)
Biotin-labeled ConA lectin (SIGMA, C2272), which is a biotin-labeled ConA lectin that binds to many of the IgG-derived sugar chains, was prepared at 5 ug / mL with the following solution.
Solution: 50 mM Tris.HCl (pH 7.5), 100 mM NaCl,
1 mM CaCl2, MnCl2, MgCl2, 0.05% Tween20

The solution was reacted on the substrate and reacted at room temperature for 2 hours.
After the reaction, it was washed with the cleaning agent.
Cy3-Streptavidin (GE Healthcare, PA43001) prepared at 2 ug / mL with the following solution was developed on a substrate and reacted at room temperature for 1 hour.
Solution: 50 mM Tris.HCl (pH 7.5), 100 mM NaCl,
1 mM CaCl2, MnCl2, MgCl2, 0.05% Tween20
After the reaction, washing with the washing solution. The measurement was performed by measuring the fluorescence intensity of Cy3 using an X-100 microarray reader, ScanArrayLite (manufactured by PerkinElmer). (Laser = 90, PMT = 65)

The results are shown in the table below.

According to the present invention, it is possible to easily prepare a sugar chain library, and also use a sugar chain capture substrate that suppresses nonspecific adsorption / binding of a detection target substance, sugar, narrowly defined sugar chain, glycopeptide, Various sugar chain arrays for bioassays capable of immobilizing glycoproteins, glycolipids, or sugar chains having these can be provided.

Claims (15)

The purification raw material containing a sugar chain is brought into contact with a sugar chain purification substrate having a primary amino group to capture the sugar chain,
Collect the entire base material,
A method for preparing a sugar chain library, wherein the sugar chain library is divided for each sugar chain group having predetermined characteristics. The method for preparing a sugar chain library according to claim 1, wherein the sugar chains recovered together with the base material are released from the base material and fractionated by liquid chromatography. The method for preparing a sugar chain library according to claim 1 or 2, wherein the purified raw material is an organic compound, an enzyme compound, or a biological substance. The sugar chain to be separated is a sugar chain of two or more sugars, or a compound containing the same, a sugar amino acid, a glycopeptide, a glycoprotein, a glycolipid, a glycosaminoglycan, a proteoglycan, a glycosylphosphatidylinositol, a peptidoglycan, a lipopolysaccharide, and The method for preparing a sugar chain library according to any one of claims 1 to 3, which is selected from those derivatives. The method for preparing a sugar chain library according to any one of claims 1 to 4, wherein the primary amino group is an oxylamino group and / or a hydrazide group. The method for preparing a sugar chain library according to any one of claims 1 to 5, wherein the sugar chain purification substrate contains a polymer compound for capturing a sugar chain represented by the following general formula [1].

(R represents a hydrocarbon chain having 1 to 20 carbon atoms into which —O—, —S—, —NH—, —CO—, and —CONH— may be inserted.) The method for preparing a sugar chain library according to any one of claims 1 to 6, wherein the sugar chain purification substrate is in the form of beads or plates. The sugar chain library according to any one of claims 1 to 7, wherein the sugar chain purification substrate is a particle composed of a polymer matrix having a crosslinked polymer structure having the structure of the following general formula [2]. Manufacturing method.

(R1 and R2 are hydrocarbon chains having 1 to 20 carbon atoms into which —O—, —S—, —NH—, —CO—, and —CONH— may be inserted; R3, R4, and R5 are H, CH3, Or a hydrocarbon chain having 2 to 5 carbon atoms, m and n represent the number of monomer units.) A sugar chain library produced by the method of producing a sugar chain library according to any one of claims 1 to 8. A sugar chain array, wherein sugar chains of the sugar chain library are immobilized on the surface of a substrate. 11. The sugar chain array is obtained by applying a saccharide-fixing polymer compound represented by the following general formula [3] to a substrate surface, and the sugar chains are immobilized via a polymer compound-immobilized layer. The sugar chain array described

(Wherein R1, R2 and R3 represent a hydrogen atom or a methyl group, R4 represents a hydrophobic group, X represents an alkyleneoxy group having 1 to 10 carbon atoms, and p represents an integer of 1 to 20. p is an integer. When X is an integer of 2 or more and 20 or less, the repeated Xs may be the same or different, Y is a spacer containing an alkylene glycol residue, and Z is an oxygen atom or NH. Is a methyl group, and l, m, and n are natural numbers.) The saccharide array according to claim 11, wherein the hydrophobic group R4 is a cyclic alkyl group. The saccharide array according to claim 12, wherein the cyclic alkyl group is a cyclohexyl group. The sugar chain array according to any one of claims 10 to 13, wherein a material of the substrate is plastic. The sugar chain array according to claim 14, wherein the plastic contains saturated cyclic polyolefin or polystyrene.

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