JP5672892B2 - Copolymer for immobilizing ligand and method for immobilizing ligand with the copolymer - Google Patents

Description

  The present invention relates to a copolymer for immobilizing a ligand and a method for immobilizing a ligand using the copolymer.

  In recent years, attempts have been widely made in various fields to increase the added value by modifying the surface of a material. In particular, a technique for immobilizing a ligand that exhibits a specific interaction with a specific target substance on the surface of a material is rapidly progressing in the medical, environmental, and bio fields. In particular, regarding sugar chain immobilization technology, it has become clear that sugar chains bound to proteins and lipids are involved in important processes such as canceration and immune reactions. Many attempts have been made to apply them to cell culture equipment. For example, Patent Document 1 discloses a verotoxin detection sensor in which a sugar chain derivative containing galactose at a terminal is used as a ligand and this is immobilized on a substrate surface.

  By the way, in order to effectively exert the function as a ligand in a biosensing device or the like, it is desirable to improve the interaction between the ligand and the target substance. Generally, in order to improve the interaction between a ligand and a target substance, it is known that integration of the ligand on the material surface is effective. For this purpose, it is necessary to immobilize the ligand on the material surface stably and with high density.

  As an immobilization method for integrating a ligand on a material surface, for example, there is a method in which a sugar chain as a ligand is two-dimensionally immobilized by a self-assembled monolayer of alkanethiol (hereinafter referred to as SAM). (Refer nonpatent literature 1). According to this, since SAM is formed on the material surface, the ligand can be immobilized at high density. Patent Document 2 discloses a method of polymerizing a monomer having an oligosaccharide that can serve as a ligand in the side chain by a reversible addition-fragmentation chain transfer polymerization reaction (hereinafter referred to as RAFT). According to this method, sugar chains can be efficiently accumulated on the material surface.

JP 2002-22745 A JP-T-2008-515996

Munrika Satjapipat, Raymond Sanedrin, and Feimeng Zhou; Langmuir; 2001; 17; p. 7637-7644

  However, according to the methods described in Non-Patent Document 1 and Patent Document 2, the ligand can be integrated on the surface of the material, but a high interaction has not been achieved.

  The present invention has been made in view of the problems as described above, and can immobilize a ligand stably on the surface of a carrier as a material, and can enhance the molecular recognition action of the ligand. It is an object of the present invention to provide a copolymer and a method for immobilizing a ligand using the copolymer. Another object of the present invention is to provide a cell culture method using a culture substrate on which the above-mentioned ligand immobilization copolymer is immobilized.

  As a result of repeated investigations to solve the above problems, the present inventors have found that, in order to improve the interaction between the ligand and the target substance, it is effective to increase the mobility of the ligand in addition to the integration of the ligand. I found out. As a result of further earnest research, it was found that a specific copolymer can be immobilized on the surface of the material in a brush shape, whereby the integration of ligands and the improvement of mobility can be realized, and the present invention is completed. It came to. Specifically, the following are provided.

  (1) A copolymer for immobilizing a ligand on the surface of a carrier having a reactive group (a), the reactive group (b) capable of being chemically bonded to the reactive group (a) Having a segment containing a structural unit derived from the monomer (A) at one end of the chain of the copolymer, and a segment containing a structural unit derived from the monomer (B) having the ligand of the chain of the copolymer A structural unit derived from the monomer (A) between the segments at both ends, and having a segment containing a structural unit derived from the monomer (B) at both ends of the copolymer chain. A copolymer having a segment comprising

  (2) Having a segment composed of a structural unit derived from the monomer (A) at one end of the chain of the copolymer and a segment composed of a structural unit derived from the monomer (B) of the chain of the copolymer The copolymer according to (1), which is provided at the other end.

  (3) The copolymer according to (1) or (2), which is formed by living radical polymerization.

  (4) The copolymer according to any one of (1) to (3) above, wherein either one of the reactive group (a) and the reactive group (b) is an azide group and the other is an alkynyl group.

(5) The copolymer according to any one of (1) to (4), wherein the monomer (A) is represented by the general formula (I).
(In the formula, R ^1a represents a hydrogen atom or a methyl group, R ^2a represents —O— or —NH—, X represents a spacer, and Y represents a reactive group (b).)

(6) The copolymer according to any one of (1) to (5), wherein the monomer (B) is represented by the general formula (II).
(In the formula, R ^1b represents a hydrogen atom or a methyl group, R ^2b represents —O— or —NH—, X represents a spacer, and Z represents a ligand.)

  (7) The copolymer according to any one of (1) to (6), wherein the ligand is at least one selected from the group consisting of sugars, sugar chains, and amino acids.

  (8) A carrier on which a ligand is immobilized by the copolymer according to any one of (1) to (7) above.

  (9) a ligand comprising a step of reacting the reactive group (b) of the copolymer according to any one of (1) to (7) above with the reactive group (a) on the surface of the carrier Immobilization method.

  (10) A cell culture method using a culture substrate on which the copolymer according to any one of (1) to (7) above is immobilized.

  (11) The cell culture method according to (10), wherein the cells are cultured in a serum-free medium.

The copolymer of the present invention is a copolymer comprising a segment containing a structural unit derived from the monomer (A) having a reactive group (b) that can be chemically bonded to the reactive group (a) on the surface of the carrier. and having at one end of the chain of the segment containing a constituent unit derived from the monomer (B) having a ligand than that Yusuke to the other end of the chain of the copolymer, fixed to the brush to the surface of the carrier Can be When the copolymer of the present invention is immobilized in a brush shape, the mobility of the ligand is improved, so that the interaction between the ligand and the target substance is enhanced and the molecular recognition action of the ligand is enhanced.

It is a figure which shows the reaction scheme of the copolymer which concerns on the 1st Embodiment of this invention by a reversible addition cleavage chain transfer polymerization reaction. (A) Modification of sugar chain by copolymer according to first embodiment of the present invention, (B) Schematic diagram showing modification of sugar chain by a self-assembled monolayer of alkanethiol. Evaluation of the copolymer of the present invention: Specific binding of RCA (A), non-specific binding of BSA / specific binding of RCA (B) (Poly-Lac (OAc) ₇ -Ab-Alkyne- It is a figure which shows A). Evaluation of the copolymer of the present invention: Specific binding of RCA (A), non-specific binding of BSA / specific binding of RCA (B) (Poly- [Lac (OAc) ₇ -A-co-HEA ] -B-Alkyne-A). Evaluation of copolymer of the present invention: Photograph showing rat liver parenchymal cell (hepatocyte) adhesion (Poly- [Lac (OAc) ₇ -A-co-HEA] -b-Alkyne-A). Evaluation of the copolymer of the present invention: It is a diagram showing the difference in the amount of albumin produced depending on the presence or absence of an additive factor. Evaluation of the copolymer of the present invention: It is a figure showing the difference in albumin production by the presence or absence of epidermal growth factor (EGF). Evaluation of the copolymer of the present invention: Photographs showing adhesion of primary hepatocytes on a gelatin-modified substrate ((A) with ASF, (B) without ASF). Evaluation of copolymer of the present invention: Photographs showing adhesion of primary hepatocytes on a lactose glycopolymer substrate ((A) with ASF, (B) without ASF).

  Hereinafter, specific embodiments to which the present invention is applied will be described in detail.

<First Embodiment>
The copolymer according to the first embodiment of the present invention is a copolymer for immobilizing a ligand on the surface of a carrier having a reactive group (a), and has a chemical structure with the reactive group (a). A structural unit derived from the monomer (B) having a ligand having a segment containing a structural unit derived from the monomer (A) having a reactive group (b) capable of being bound thereto at one end of the chain of the copolymer At the other end of the chain of the copolymer. In the copolymer of the present invention, it is important that the structural unit derived from the monomer (A) and the structural unit derived from the monomer (B) are not mixed and exist in different segments. In addition, you may have another monomer between the segment containing the structural unit derived from a monomer (A), and the segment containing the structural unit derived from a monomer (B). The copolymer of the present invention has a chain structure, and the structure may be either a linear polymer or a branched polymer.

  In addition, when the copolymer of the present invention is synthesized by a reversible addition-fragmentation chain transfer polymerization reaction (hereinafter referred to as RAFT), a copolymer having a structure derived from a chain transfer agent is obtained. The end in the invention means the end of the chain of the copolymer excluding the structure derived from the chain transfer agent. Further, when the copolymer of the present invention is a branched polymer, the structural unit derived from the monomer (B) may be at the end of the trunk polymer, or may be in the branched polymer portion branched from the end of the trunk polymer. May be.

[Monomer (A)]
The monomer (A) of the present invention has a reactive group (b) that can be chemically bonded to the reactive group (a) on the surface of the carrier. The reactive group (b) is not particularly limited as long as it can be chemically bonded to the reactive group (a), but preferably one of the reactive group (a) and the reactive group (b) is an azide group. The other is an alkynyl group, or one is an amino group or a thiol group and the other is a carboxyl group, a carboxylic acid activated by an N-hydroxysuccinimidyl ester, an isocyanate group or a thioisocyanate group. is there. The copolymer of the present invention is immobilized on the carrier by chemically bonding the reactive group (b) with the reactive group (a) on the surface of the carrier.

  The monomer (A) of the present invention is a polymerizable monomer and needs to have a polymerizable group in its structure, but the kind thereof is not particularly limited. For example, vinyl group, allyl group, styryl group, methacryloyl It may be a group, an acryloyl group, or the like. The monomer (B) can be polymerized through this polymerizable group.

In the copolymer of the present invention, the monomer (A) is preferably represented by the following general formula (I).

Here, R ^1a is a hydrogen atom or a methyl group, and R ^2a is —O— or —NH—. X is a spacer, and Y is a reactive group (b).

  In the monomer (A), the spacer is not particularly limited as long as it does not adversely affect the chemical bond between the reactive group (a) and the reactive group (b). An oligoalkyleneoxy group, an alkyl group, an alkylene group, etc. are mentioned, Preferably it is an oligoethyleneoxy group or alkylene group of 1-20 repeat units. The alkylene group may be linear or branched. Although carbon number of an alkylene group is not specifically limited, Preferably it is C1-C8. If the ethyleneoxy group having 1 to 50 repeating units or the C1 to C8 alkylene group is used, the mobility of the copolymer is improved, and the molecular recognition action of the ligand is also improved.

  The copolymer according to the first embodiment of the present invention has a segment containing a structural unit derived from the monomer (A) at one end of the chain of the copolymer. The segment may contain other monomers other than the monomer (B). Examples of other monomers include (meth) acrylamide, methylol (meth) acrylamide, methoxymethyl (meth) acrylamide, ethoxymethyl (meth) acrylamide, propoxymethyl (meth) acrylamide, butoxymethoxymethyl (meth) acrylamide, N- Methylol (meth) acrylamide, N-hydroxymethyl (meth) acrylamide, 2-hydroxyethyl (meth) acrylamide, 2-hydroxypropyl (meth) acrylamide, 2-hydroxybutyl (meth) acrylamide, (meth) acrylic acid, fumaric acid , Maleic acid, maleic anhydride, itaconic acid, itaconic anhydride, citraconic acid, citraconic anhydride, crotonic acid, methyl (meth) acrylate, ethyl (meth) acrylate, butyl (meth ) Acrylate, 2-ethylhexyl (meth) acrylate, cyclohexyl (meth) acrylate, 2-hydroxyethyl (meth) acrylate, 2-hydroxypropyl (meth) acrylate, 2-hydroxybutyl (meth) acrylate, 2-phenoxy-2- Hydroxypropyl (meth) acrylate, 2- (meth) acryloyloxy-2-hydroxypropyl phthalate, glycerin mono (meth) acrylate, tetrahydrofurfuryl (meth) acrylate, dimethylamino (meth) acrylate, glycidyl (meth) acrylate, etc. Can be mentioned. These monomers may be used alone or in combination of two or more.

  The proportion of the structural unit derived from the monomer (A) in the segment is not particularly limited, but is preferably 5 mol% or more, more preferably 30 mol% or more, and even more preferably 100 mol%. The higher the proportion of the structural unit derived from the monomer (A), the higher the proportion of the reactive group (b). Therefore, the reaction probability with the reactive group (a) on the surface of the carrier increases. In the copolymer according to the first embodiment of the present invention, it is important to have a segment containing a structural unit derived from the monomer (A) at the end of the copolymer chain, The segment need not have a structural unit derived from the monomer (A) at the end. Therefore, a segment including the above-mentioned other monomer between the end of the copolymer chain and the structural unit derived from the monomer (A) is also within the scope of the present invention.

[Monomer (B)]
The monomer (B) of the present invention is characterized by having a ligand.

  As a ligand, for example, molecular recognition showing a specific interaction with a specific target substance such as a sugar chain, sugar, glycoprotein, glycolipid, protein, antigen, antibody, amino acid, oligo DNA, oligo RNA, etc. An element is mentioned. By binding these ligands, various functions can be added to the copolymer.

  The monomer (B) of the present invention is a polymerizable monomer and needs to have a polymerizable group in its structure, but the type is not particularly limited, and examples thereof include a vinyl group, an allyl group, a styryl group, and methacryloyl. It may be a group, an acryloyl group, or the like. It is possible to polymerize with the monomer (A) or the like via this polymerizable group.

In the copolymer of the present invention, the monomer (B) is preferably represented by the following general formula (II).

Here, R ^1b is a hydrogen atom or a methyl group, and R ^2b is —O— or —NH—. X is a spacer, and Z is a ligand.

  In the monomer (B), the spacer is not particularly limited as long as the above-described ligand can be introduced, and examples thereof include an oligoalkyleneoxy group having 1 to 200 repeating units, an alkyl group, and an alkylene group, preferably repeating. It is an oligoethyleneoxy group or alkylene group having 1 to 50 units. The alkylene group may be linear or branched. Although carbon number of an alkylene group is not specifically limited, Preferably it is C1-C8. An ethyleneoxy group having 1 to 50 repeating units or a C1 to C8 alkylene group is preferable because the mobility of the copolymer becomes good. In the copolymer of the present invention, the above-described ligand is bound through this spacer.

  The copolymer according to the first embodiment of the present invention has a segment including a structural unit derived from the monomer (B) at the other end of the chain of the copolymer. The segment may contain the above-mentioned other monomers other than the monomer (A). The proportion of the structural unit derived from the monomer (B) in the segment is not particularly limited, but is preferably 20 mol% or more, more preferably 50 mol% or more, and even more preferably 80 mol% or more. The higher the proportion of the structural unit derived from the monomer (B) is, the higher the proportion of the ligand is, and the molecular recognition action of the ligand is improved. However, depending on the ligand, the other monomer may be included in the molecule. The recognition effect may be enhanced. In the copolymer according to the first embodiment of the present invention, it is important to have a segment containing a structural unit derived from the monomer (B) at the end of the copolymer chain, The segment need not have a structural unit derived from the monomer (B) at the end. Therefore, a segment containing the above-mentioned other monomer between the end of the copolymer chain and the structural unit derived from the monomer (B) is also within the scope of the present invention.

[Synthesis Method of Copolymer]
The polymerization method of the copolymer of the present invention is not particularly limited, and a conventionally known method can be used, but a living radical polymerization method such as RAFT or atom transfer radical polymerization (hereinafter referred to as ATRP) is preferable. The living radical polymerization method is preferable because the molecular weight and molecular weight distribution of the copolymer to be synthesized can be controlled. In the following, a segment composed of a structural unit derived from the monomer (A) is provided at one end of the chain of the copolymer, and a segment composed of a structural unit derived from the monomer (B) is added to the chain of the copolymer. A method for synthesizing the copolymer of the present invention at the end is illustrated. The polymerization method is a living radical polymerization method.

  In the case of RAFT, first, the monomer (B), the chain transfer agent, and the polymerization initiator are dissolved in a predetermined solvent, and oxygen in the reaction vessel including dissolved oxygen is completely removed, and then the polymerization initiator is cleaved. A macro chain transfer agent in which a chain transfer agent is introduced at the terminal of a polymer (hereinafter referred to as B block) obtained by polymerizing the monomer (B) by heating at a temperature of 100 ° C. or lower for 1 to 500 hours. Synthesize. Next, the macro chain transfer agent and the monomer (A) are dissolved in a predetermined solvent, a polymerization initiator is added, and the mixture is heated at a temperature not lower than 200 ° C. for 1 to 500 hours. Thus, the copolymer of the present invention in which the B block and the polymer obtained by polymerizing the monomer A (hereinafter referred to as A block) are coupled in series is synthesized. A schematic of the reaction scheme is shown in FIG.

  In the case of ATRP, first, the monomer (B), the alkyl halide initiator, the metallizing agent, and the ligand are dissolved in a predetermined solvent and reacted to form an alkyl halide initiator at the end of the B block. Synthesize the introduced macrohalogenated alkyl initiator. Next, the macrohalogenated alkyl initiator and the monomer (A) are dissolved in a predetermined solvent, and the metalating agent and the ligand are added, followed by heating at room temperature to 200 ° C. for 1 to 500 hours. Thus, the copolymer of the present invention in which the B block and the A block are connected in series is synthesized.

  The solvent used for the polymerization reaction is not particularly limited, but for example, water, methanol, ethanol, propanol, t-butanol, benzene, toluene, dimethylformamide, tetrahydrofuran, chloroform, 1,4-dioxane, dimethyl sulfoxide or a mixture thereof. Liquid.

  The polymerization initiator is not particularly limited. For example, 2,2′-azobisisobutyronitrile (AIBN), 2,2′-azobis (2-methylbutyronitrile), diisopropyl peroxycarbonate, t-butyl Peroxy-2-ethylhexanoate, t-butylperoxypivalate, t-butylperoxydiisobutyrate, benzoyl peroxide, lauroyl peroxide, ammonium persulfate, potassium persulfate, and the like can be used. The suitable usage-amount of a polymerization initiator is 0.001-1 mass% with respect to a monomer, and 1-33 mass% with respect to a chain transfer agent.

  The chain transfer agent used in RAFT is not particularly limited, and examples thereof include butylbenzyl trithiocarbonate, cumyl dithiobenzoate, 4-cyanopentanoic acid dithiobenzoate, acetic acid dithiobenzoate, butanoic acid dithiobenzoate, and 4-toluic acid. And dithiobenzoate.

  Although it does not specifically limit as an alkyl halide initiator used for ATRP, For example, 2-bromoisobutyryl bromide, 2-chloroisobutyryl chloride, bromoacetyl bromide, bromoacetyl chloride, benzyl bromide etc. are mentioned.

  Although it does not specifically limit as a transition metal complex, Complexes, such as monovalent copper and bivalent ruthenium, can be used.

  The weight average molecular weight (measured by GPC using polystyrene as a standard substance) of the copolymer of the present invention is preferably 2000 to 100,000, and more preferably 2000 to 20000. By setting it as the said range, it can fix stably with respect to the surface of a support | carrier.

  The copolymer of the present invention has a segment containing a structural unit derived from the monomer (A) at one end of the chain of the copolymer and is derived from the monomer (B) at the other end of the chain of the copolymer. If it has a segment containing a structural unit, it has other monomers between the segment containing the structural unit derived from the monomer (A) and the segment containing the structural unit derived from the monomer (B). Also good.

  In the copolymer of the present invention, the proportion of the structural unit derived from the monomer (A) in the copolymer is not particularly limited, but is preferably 5 to 80 mol%, more preferably 5 to 20 mol%. . If it is the said range, the copolymer of this invention can be fix | immobilized in the shape of a brush with respect to the surface of a support | carrier. Moreover, the ratio for which the structural unit derived from the monomer (B) in the copolymer of this invention accounts is although it does not specifically limit, Preferably it is 20-80 mol%, More preferably, it is 50-80 mol%. If it is the said range, the molecule | numerator recognition effect of a ligand is favorable.

The copolymer of the present invention is a copolymer comprising a segment containing a structural unit derived from the monomer (A) having a reactive group (b) that can be chemically bonded to the reactive group (a) on the surface of the carrier. And a segment containing a structural unit derived from the ligand-containing monomer (B) at the other end of the copolymer chain. FIG. 2A shows a modification of a sugar chain by the copolymer of the present invention having a segment composed of a structural unit derived from the monomer (A) and a segment composed of a structural unit derived from the monomer (B) at both ends. It is a schematic diagram which shows. FIG. 2B is a schematic diagram showing modification of a sugar chain by a self-assembled monolayer of alkanethiol (hereinafter referred to as SAM), which is a conventional method. Since the copolymer of the present invention has a plurality of reactive groups (b) as shown in FIG. 2 (A), the reaction probability with the reactive groups (a) on the surface of the carrier is increased. In addition, since the copolymer is fixed in a multipoint manner, the copolymer can be fixed in the shape of a brush extended from the surface of the carrier, and the mobility of the copolymer is improved. Improves. Furthermore, since the ligand is integrated at one end, the interaction between the ligand and the target substance is enhanced by improving the mobility of the copolymer having the ligand and integrating the ligand. The molecular recognition effect of the ligand is also expected to increase.
In contrast, in the conventional method in which the ligand is immobilized two-dimensionally by SAM of alkanethiol, SAM is formed on the material surface as shown in FIG. However, due to the interaction between molecules, the mobility is reduced.

[Immobilization of copolymer]
The copolymer of the present invention immobilizes the ligand on the surface of the carrier by reacting the reactive group (b) of the copolymer with the reactive group (a) on the surface of the carrier. By this reaction, the reactive group (b) and the reactive group (a) are chemically bonded to each other, so that they can be stably immobilized on the surface of the carrier. The reactive group (a) is not particularly limited as long as it can be chemically bonded to the reactive group (b). Preferably, either the reactive group (a) or the reactive group (b) is an azide group. The other is an alkynyl group, one is an amino group, the other is a carboxyl group, one is an amino group or a thiol group, the other is a carboxyl group, active in N-hydroxysuccinimidyl ester It is preferably a carboxylic acid, isocyanate group or thioisocyanate group.

  When either one of the reactive group (a) and the reactive group (b) is an azide group and the other is an alkynyl group, the reaction between the reactive group (a) and the reactive group (b) It is preferable to carry out by reaction. In the click reaction, an azide group and an alkynyl group are reacted in a solvent so that a triazole ring is formed. By adding a catalyst such as copper and a reducing agent such as ascorbate to the reaction mixture, a triazole ring can be formed at room temperature even in an aqueous solvent. In addition to the fact that the click reaction proceeds even in a water-mixed solvent, other coupling reactions even under conditions where the reaction rate tends to be low in ordinary organic chemical reactions such as polymer end reactions on solid surfaces. It is also preferable in that the reaction proceeds with higher efficiency than the above.

  The type and form of the material constituting the carrier to be immobilized are not particularly limited. The carrier may be solid or semi-solid. Examples of the material include glass, silicon, plastic, ceramics, and metal, and can be used according to the purpose. Examples of the metal include gold, silver, iron, copper, and platinum. Examples of the form include a plate shape, a film shape, a fiber shape, a particle shape, and a gel shape.

  The method of modifying the reactive group (a) on the surface of the carrier is not particularly limited. For example, when the carrier is gold, a bond between gold and a thiol group is used, or the carrier is a hydroxyl group such as glass. In the case of having silane, a silane coupling agent can be used.

  The copolymer of the present invention can be stably immobilized on the surface of the carrier as described above. Therefore, for example, a copolymer in which a sugar chain or an antibody specifically recognizing a specific cell, virus, bacterium, toxin, peptide, or the like is bound as a ligand is used as a diagnostic sensing probe or active targeting drug delivery system. It can be suitably used for (active / target-oriented DDS).

[Cell culture method]
According to the culture substrate whose surface is modified with the copolymer of the present invention to which a ligand is bound, cell culture becomes possible. In particular, when a ligand that specifically binds to a specific cell is used, it can be used for selective culture of the cell. For example, by using lactose as a ligand that is recognized only by hepatocytes, hepatocytes and other non-parenchymal cells (sinusoidal endothelial cells, stellate cells, Kupffer cells, etc.) that constitute the liver Only hepatocytes can be easily separated from the mixture. In addition, according to the culture substrate whose surface is modified with the copolymer of the present invention in which lactose is bound as a ligand, lactose and cells adhere well via an asialoglycoprotein receptor, so even in a serum-free medium Cells can be cultured.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited to these Examples.

  In this example, first, a molecule for modifying the reactive group (a) on the surface of the carrier was produced through steps 1 and 2. The reactive group (a) corresponds to the reactive group (b) of monomer A.

[Manufacture of molecules for carrier surface modification]
<Step 1: Synthesis of 3-azidopropyltriethoxysilane>
According to a known method (Eur. J. Org. Chem., 2006, 2934-2941), from 3.0 g (12.5 mmol) of 3-chloropropyltriethoxysilane represented by the formula (1), the formula (2) 3-azidopropyltriethoxysilane represented by the formula (yield: 2.12 g (8.60 mmol), yield: 69%) was obtained. The reaction scheme is shown below.

<Step 2: Synthesis of 1-azidoundecane-11-thiol>
With reference to a known method (Langmuir., 2004, 20 (4), 1051-1053), 1-bromoundecan-11-ol represented by formula (3) is converted to 1- Azidoundecan-11-thiol was synthesized.
Specifically, 2.51 g (10.0 mmol) of 1-bromoundecane-11ol represented by the formula (3) and 1.95 g (30.0 mmol) of sodium azide are suspended in 45 ml of DMF under an argon atmosphere. The mixture was stirred at 70 ° C. for 3 hours. Excess sodium azide was removed by filtration, and then the reaction solution was concentrated under reduced pressure. After adding isopropyl ether to the residue and washing twice with water, the organic layer was dehydrated with anhydrous magnesium sulfate and concentrated under reduced pressure to obtain 1-azidoundecanol represented by the formula (4) (yield: 2). .13 g (10.0 mmol), yield: 100%).
Next, in a 60 ml dehydrated THF solution of 2.07 g (9.70 mol) of 1-azidoundecan-11-thiol represented by the formula (4) and 2.29 g (20.0 mmol) of methanesulfonyl chloride under an argon atmosphere, A solution of 2.02 g (20.0 mmol) of triethylamine in 20 ml of dehydrated THF was added dropwise and stirred at room temperature for 3 hours. The reaction solution was filtered to remove the generated salt, and then concentrated under reduced pressure. Isopropyl ether is added to the residue, and the mixture is washed with 1M hydrochloric acid, water, and a NaHCO ₃ aqueous solution. The organic layer is dehydrated with anhydrous magnesium sulfate and concentrated under reduced pressure, and 1-azidoundecane-11-methyl represented by the formula (5) The sulfonate was obtained (yield: 2.70 g (9.27 mmol), yield: 96%).
Subsequently, 1.70 g (9.27 mmol) of 1-azidoundecane-11-methylsulfonate represented by the formula (5) and 2.10 g (18.6 mmol) of potassium thioacetate were dissolved in 80 ml of methanol, and an argon atmosphere was obtained. Under reflux for 2.5 hours. The reaction solution was concentrated under reduced pressure, water was added to the residue, and the mixture was extracted with diethyl ether. The organic layer was further washed with water, dehydrated with anhydrous magnesium sulfate, and concentrated under reduced pressure to obtain 1-azidoundecane-11-thioacetate represented by the formula (6). (Yield: 2.27 g (8.36 mmol), yield: 90%)
Finally, 1.27 g (8.36 mmol) of 1-azidoundecane-11-thioacetate represented by the formula (6) was dissolved in 150 ml of methanol, 8 ml of concentrated hydrochloric acid was added, and the mixture was heated under an argon atmosphere for 5 hours. Refluxed. After adding water to the reaction solution, methanol was removed under reduced pressure and extracted with diethyl ether. The organic layer was further washed with NaHCO ₃ aqueous solution, water and brine, then dehydrated with anhydrous magnesium sulfate and concentrated under reduced pressure to obtain 1-azidoundecan-11-thiol represented by formula (7) (yield: 1 .92 g (8.38 mmol), yield: 100%). The reaction scheme is shown below.

  Through the three steps, butylbenzyl trithiocarbonate, which is a reversible addition-cleavage chain transfer agent (hereinafter referred to as chain transfer agent) used in the present invention, was produced.

[Production of chain transfer agent]
<Step 3: Synthesis of butylbenzyltrithiocarbonate>
The butylbenzyl trithiocarbonate represented by the formula (10) was synthesized from the benzyl mercaptan represented by the formula (8) with reference to known literature (Journal of Chemical Society of Japan, 1987, 7, 1408-1413).
Specifically, 1.24 g (10.0 mmol) of benzyl mercaptan represented by Formula (8) and 1.52 g (20.20) of 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU). 0 mmol) was dissolved in 10 ml of dehydrated benzene, 1.52 g (20.0 mmol) of carbon disulfide was slowly added dropwise under an argon atmosphere, and the mixture was stirred at room temperature for 30 minutes. To this reaction solution, a solution of 1.37 g (10.0 mmol) of 1-bromobutane in 5 ml of dehydrated benzene was added dropwise and stirred at room temperature for 18 hours. The reaction solution was diluted with benzene and filtered through celite. The filtrate was washed with 1M hydrochloric acid, water and brine, then dehydrated over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain butylbenzyl trithiocarbonate represented by the formula (10) (yield: 2.48 g (9 .67 mmol), yield: 97%). The reaction scheme is shown below.

  The monomer (A) of this invention was manufactured through the process of 4.

[Production of Monomer (A)]
<Step 4: Synthesis of acrylic acid-4-pentynyl ester>
Acrylic acid-4-pentynyl ester represented by the formula (13) was synthesized from 4-pentyn-1-ol represented by the formula (11) and acrylic acid chloride.
Specifically, 2.83 g (40.0 mmol) of 4-pentyn-1-ol represented by the formula (11) and 4.45 g (44.0 mmol) of triethylamine are dissolved in 40 ml of dehydrated THF and cooled in an ice bath. Then, 40 ml of dehydrated THF in 3.98 g (44.0 mmol) of acrylic acid chloride represented by Table (12) was added dropwise. Thereafter, the ice bath was removed and the mixture was stirred at room temperature for 5 hours. The reaction solution was filtered to remove the generated salt, and then concentrated under reduced pressure. Isopropyl ether was added to the residue, which was washed with an aqueous NaHCO ₃ solution, an aqueous ammonium chloride solution and brine, then dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a crude product. The obtained crude product was purified by silica gel column chromatography (ethyl acetate: n-hexane = 1: 10) to obtain acrylic acid-4-pentynyl ester represented by the formula (13) (yield: 1.90 g (13.8 mmol), yield: 35%). The reaction scheme is shown below.

  The monomer (B) of this invention was manufactured through the process of 5 or 6.

[Production of Monomer (B)]
<Step 5: Synthesis of β-ethyl acrylate-D-lactose-heptaacetate>
With reference to a known method (Biomacromolecule, 2004, 5 (1), 224-231), β-rich D-lactose-octaacetate represented by formula (14) and hydroxy represented by formula (15) Β-ethyl acrylate-D-lactose-heptaacetate represented by the formula (16) was synthesized from ethyl acrylate.
Specifically, the β-form rich D-lactose-octaacetate 10.0 g (14.7 mmol) represented by the formula (14) and the acrylic acid hydroxyethyl ester 2.06 g represented by the formula (15) ( 17.7 mmol) was dissolved in dehydrated dichloromethane under an argon atmosphere, and 6 ml of boron trifluoride-diethyl ether complex (46-49%) was added dropwise while cooling in an ice bath. After stirring for 2 hours, the ice bath was removed and the mixture was further stirred at room temperature for 18 hours. The reaction solution was diluted with chloroform, washed with NaHCO ₃ aqueous solution and water, and the organic layer was dehydrated with anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a crude product. The obtained crude product was purified by silica gel column chromatography (acetone: dichloromethane = 1: 30 → 1: 25), and then reprecipitated with methanol / water, and β-acryl represented by formula (16). Acid ethyloxy-D-lactose-heptaacetate was obtained (yield: 5.36 g (7.30 mmol), yield: 50%). The reaction scheme is shown below.

<Step 6: Synthesis of β-acrylamidylethyloxy-D-lactose-heptaacetate>
According to a known method (Aust. J. Chem., 1998, 51, 31-35), from 1-aminoethanol represented by formula (17) and acrylic acid chloride represented by formula (18), the formula 2-hydroxyethylacrylamide represented by (19) was synthesized. Next, with reference to a known method (Biomacromolecule, 2004, 5 (1), 224-231), a β-rich D-lactose-octaacetate represented by formula (14) and a formula (19) Β-acrylamidylethyloxy-D-lactose-heptaacetate represented by the formula (20) was synthesized from 2-hydroxyethylacrylamide.
Specifically, 30 ml (0.50 mol) of 1-aminoethanol represented by the formula (17) is dissolved in 300 ml of dehydrated dichloromethane, and 20 ml of acrylic acid chloride represented by the formula (18) is cooled in an ice bath ( 0.25 mol) of 100 ml of dehydrated dichloromethane was added dropwise. After stirring for 3 hours while cooling in an ice bath as it is, the formed salt was removed by filtration, and the filtrate was concentrated under reduced pressure to synthesize 2-hydroxyethylacrylamide represented by the formula (19) (yield: 15 g (0 .13 mol), yield: 52%).
Next, 10.0 g (14.7 mmol) of β-rich D-lactose-octaacetate represented by the formula (14) and 2.03 g (17.7 mmol) of 2-hydroxyethylacrylamide represented by the formula (19) ) Was dissolved in dehydrated dichloromethane under an argon atmosphere, and 6 ml of boron trifluoride-diethyl ether complex (46-49%) was added dropwise while cooling in an ice bath. After stirring for 2 hours, the ice bath was removed and the mixture was further stirred at room temperature for 18 hours. The reaction solution was diluted with chloroform, washed with NaHCO ₃ aqueous solution and water, and the organic layer was dehydrated with anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a crude product. The resulting crude product was purified by silica gel column chromatography (ethyl acetate: n-hexane = 1: 1 → ethyl acetate), and β-ethyl acrylate-D-lactose-hepta represented by the formula (20) Acetate was obtained (yield: 6.22 g (8.48 mmol), yield: 58%). The reaction scheme is shown below.

  The sugar chain polymer was produced through the steps 7, 8, 9, or 10.

[Manufacture of sugar chain polymer: homopolymer type (1)]
<Step 7: Synthesis of Poly-Lac (OAc) ₇ -A>
1.47 g (2.0 mmol) of β-ethyl acrylate-D-lactose-heptaacetate represented by the formula (16) and 26 mg (0.10 mmol) of butylbenzyl trithiocarbonate represented by the formula (10) Then, 1.6 mg (10 μmol) of AIBN was dissolved in 10 ml of dehydrated DMF, freeze degassed three times with a Schlenk tube, and then heated and stirred at 70 ° C. for 4 days in an argon atmosphere. The reaction solution is added dropwise to isopropyl ether for reprecipitation, and the resulting precipitate is dissolved in a small amount of chloroform, and added dropwise to isopropanol / isopropyl ether for purification by reprecipitation, which is represented by the formula (21). Poly-Lac (OAc) ₇ -A was obtained (yield: 445 mg, recovery rate: 30%). The reaction scheme is shown below.
¹ HNMR (CDCl ₃ ) of a crude product obtained by concentrating a part of the reaction solution under reduced pressure was measured, and the monomer conversion calculated from the integration ratio was 43%. The number average molecular weight (Mn _cal ) of the purified polymer measured by GPC (polystyrene standard) is 3842, the molecular weight distribution (Mw / Mn) is 1.20, and the average degree of polymerization (n _cal ) calculated from Mn _cal is It was 4.9. In addition, since the molecular weight distribution is narrow, this polymerization reaction is considered to have progressed by the RAFT mechanism. Therefore, the theoretical average molecular weight (Mn _th ) calculated from the monomer conversion was 6574, and the average degree of polymerization (n _th ) was 8.6.

[Production of sugar chain polymer: homopolymer type (2)]
<Step 8: Synthesis of Poly-Lac (OAc) ₇ -AA>
1.10 g (1.5 mmol) of β-acrylamidylethyloxy-D-lactose-heptaacetate represented by the formula (20) and 13 mg of butylbenzyl trithiocarbonate represented by the formula (10) (0. 050 mmol) and 1.6 mg (10 μmol) of AIBN were dissolved in 8 ml of dehydrated DMF, freeze-deaerated three times with a Schlenk tube, and then heated and stirred at 70 ° C. for 4 days in an argon atmosphere. The reaction solution is added dropwise to isopropyl ether for reprecipitation, and the resulting precipitate is dissolved in a small amount of chloroform and added dropwise to isopropanol / isopropyl ether for purification by reprecipitation, which is represented by the formula (22). Poly-Lac (OAc) ₇ -AA was obtained (yield: 555 mg, recovery: 50%). The reaction scheme is shown below.
In addition, it measured by the method similar to the process 7, and the conversion rate of the calculated monomer was 60%, Mn _cal was 4785, Mw / Mn was 1.20, n _cal was 6.2, Mn _th was 13463, n _th Was 18.

[Production of sugar chain polymer: copolymer type (1)]
<Step 9: Synthesis of Poly-Lac (OAc) ₇ -A-co-HEA>
1.47 g (2.0 mmol) of β-ethyl acrylate-D-lactose-heptaacetate represented by the formula (16), 232 mg (2.0 mol) of a hydroxyethyl acrylate represented by the formula (15), , 26 mg (0.10 mol) of butylbenzyltrithiocarbonate represented by the formula (10) and 3.3 mg (20 μmol) of AIBN were dissolved in 10 ml of dehydrated DMF, and freeze degassing was performed three times with a Schlenk tube. Thereafter, the mixture was heated and stirred at 70 ° C. for 4 days under an argon atmosphere. The reaction solution is added dropwise to isopropyl ether for reprecipitation, and the resulting precipitate is dissolved in a small amount of chloroform, and then added dropwise to isopropanol / isopropyl ether for purification by reprecipitation, which is represented by the formula (23). Poly-Lac (OAc) ₇ -A-co-HEA was obtained (yield: 1.04 g, recovery: 53%). The reaction scheme is shown below.
In addition, it measured by the method similar to the process 7, and the conversion rate of the calculated monomer was 70% (as 2 types of whole monomers), Mn _cal was 5186, and Mw / Mn was 1.28.

[Production of sugar chain polymer: Copolymer type (2)]
<Step 10: Synthesis of Poly-Lac (OAc) ₇ -AA-co-HEAA>
1.46 g (2.0 mmol) of β-acrylamidylethyloxy-D-lactose-heptaacetate represented by the formula (20) and 230 mg (2.0 mmol) of 2-hydroxyethylacrylamide represented by the formula (19) ), 26 mg (0.10 mmol) of butylbenzyltrithiocarbonate represented by the formula (10) and 1.6 mg (10 μmol) of AIBN are dissolved in 10 ml of dehydrated DMF, and freeze degassing three times with a Schlenk tube. Thereafter, the mixture was heated and stirred at 70 ° C. for 4 days under an argon atmosphere. The reaction solution is added dropwise to isopropyl ether for reprecipitation, and the resulting precipitate is dissolved in a small amount of chloroform, and purified by reprecipitation by adding dropwise to isopropanol / isopropyl ether, which is represented by the formula (24). Poly-Lac (OAc) ₇ -AA-co-HEAA was obtained (yield: 770 mg, recovery: 39%). The reaction scheme is shown below.
In addition, it measured by the method similar to the process 7, and the conversion rate of the calculated monomer was 45% (as 2 types of whole monomers), Mn _cal was 4930, and Mw / Mn was 1.18.

  The copolymer of this invention was manufactured through the process of 11, 12, 13 or 14.

[Production of copolymer of the present invention: sugar chain polymer is homopolymer type (1)]
<Step 11: Synthesis of Poly-Lac (OAc) ₇ -Ab-Alkyne-A>
Poly-Lac (OAc) ₇ -A 384 mg (Mn _cal = 3842, Mn _th = 6574) represented by the formula (21) and 69 mg of acrylic acid-4-pentynyl ester represented by the formula (13) (0 .50 mol) and 5.0 mg (30 μmol) of AIBN were dissolved in 3 ml of dehydrated DMF, freeze-deaerated three times with a Schlenk tube, and then heated and stirred at 70 ° C. for 6 days in an argon atmosphere. The reaction solution was dropped into isopropyl ether and purified by reprecipitation to obtain Poly-Lac (OAc) ₇ -Ab-Alkyne-A represented by the formula (25) (yield: 341 mg, recovery rate: 75%). The reaction scheme is shown below.
¹ HNMR (CDCl ₃ ) of a crude product obtained by concentrating a part of the reaction solution under reduced pressure was measured, and the monomer conversion calculated from the integration ratio was 100%. Further, the number average molecular weight (Mn _cal ) of the purified polymer measured by GPC (polystyrene standard) was 3712, and the molecular weight distribution (Mw / Mn) was 1.19. In addition, since the molecular weight distribution is narrow, this polymerization reaction is considered to have progressed by the RAFT mechanism. Therefore, the theoretical average molecular weight (Mn _th ) calculated from Mn _th of the macro chain transfer agent and the conversion rate of the monomer was 7757, and the average degree of polymerization was n _th 8.6 and m _th 8.6. .

[Production of copolymer of the present invention: sugar chain polymer is homopolymer type (2)]
<Step 12: Synthesis of Poly-Lac (OAc) ₇ -AA-b-Alkyne-A>
Poly-Lac (OAc) ₇ -AA 540 mg (Mn _cal = 4785, Mn _th = 13463) represented by the formula (22) and 55 mg (0 of acrylic acid-4-pentynyl ester represented by the formula (13) .40 mol) and 3.3 mg (20 mmol) of AIBN were dissolved in 4 ml of dehydrated DMF, freeze-deaerated three times with a Schlenk tube, and then heated and stirred at 70 ° C. for 6 days in an argon atmosphere. The reaction solution was dropped into isopropyl ether and purified by reprecipitation to obtain Poly-Lac (OAc) ₇ -AA-b-Alkyne-A represented by the formula (26) (yield: 526 mg, recovery rate: 88%). The reaction scheme is shown below.
In addition, it measured by the method similar to the process 11, and the conversion rate of the calculated monomer was 100%, Mn _cal was 4452, Mw / Mn was 1.23, Mn _th was 14840, the average degree of polymerization was n _th 18; m _th was 10.

[Production of Copolymer of the Present Invention: Sugar Chain Polymer is Copolymer Type (1)]
<Step 13: Synthesis of Poly- [Lac (OAc) ₇ -A-co-HEA] -b-Alkyne-A>
Poly-Lac (OAc) ₇ -A-co-HEA 1.04 g (Mn _cal = 5186) represented by Formula (23) and 83 mg of acrylic acid-4-pentynyl ester represented by Formula (13) ( 0.60 mmol) and 4.9 mg (30 μmol) of AIBN were dissolved in 10 ml of dehydrated DMF, freeze-deaerated three times with a Schlenk tube, and then heated and stirred at 70 ° C. for 6 days in an argon atmosphere. The reaction solution was added dropwise to isopropyl ether and purified by reprecipitation to obtain Poly- [Lac (OAc) ₇ -A-co-HEA] -b-Alkyne-A represented by the formula (27) (yield) : 807 mg, recovery rate: 72%). The reaction scheme is shown below.
In addition, it measured by the method similar to the process 11, and the conversion rate of the calculated monomer was 100%, _Mncal was 3502, Mw / Mn was 1.32.

[Production of Copolymer of the Present Invention: Sugar Chain Polymer is Copolymer Type (2)]
<Step 14: Synthesis of Poly- [Lac (OAc) ₇ -AA-co-HEAA] -b-Alkyne-A>
742 mg (Mn _cal = 4930) of Poly-Lac (OAc) ₇ -AA-co-HEAA represented by formula (24) and 207 mg of acrylic acid-4-pentynyl ester represented by formula (13) (1. 50 mmol) and 4.9 mg (30 μmol) of AIBN were dissolved in 5 ml of dehydrated DMF, freeze-deaerated three times with a Schlenk tube, and then heated and stirred at 70 ° C. for 6 days in an argon atmosphere. The reaction solution was added dropwise to isopropyl ether and purified by reprecipitation to obtain Poly- [Lac (OAc) ₇ -AA-co-HEAA] -b-Alkyne-A represented by the formula (28) (yield) : 718 mg, recovery: 76%). The reaction scheme is shown below.
In addition, it measured by the method similar to the process 11, and the conversion rate of the calculated monomer was 78%, _Mncal was 5594, Mw / Mn was 1.23.

[Formation of sugar chain polymer brush on carrier surface]
A method for forming a sugar chain polymer brush on the surface of a carrier includes the first step of modifying the azide group on the surface of the carrier, the terminal alkyne portion of the synthesized copolymer of the present invention, and the azide modified on the surface of the carrier in the first step. The second step of performing a click reaction with a group and the third step of performing a deprotection reaction to remove the protecting group of the sugar chain. In addition, a 3rd process can be skipped by performing deprotection in the state of a copolymer. The degree of reaction progress in each step was confirmed by a change in the contact angle of water droplets.

(1) First step: Modification of azide group on carrier surface As a carrier for modifying an azide group, a glass substrate (slide glass) and a gold-deposited glass substrate were used. Such dirt such as organic matter adhering to the surface of the carrier may inhibit the promotion of the surface reaction. Therefore, after being treated with an ozone cleaner for 15 minutes, a carrier which was washed by pouring methanol and dried was used.

(1-1) Modification of glass substrate surface Modification of the azide group on the glass substrate surface was performed by a silane coupling reaction. As the silane coupling agent, 3-azidopropyltriethoxysilane represented by the formula (2) synthesized in Step 1 was used. A 1 vol% toluene solution of 3-azidopropyltriethoxysilane was prepared, thinly coated on the glass substrate surface with absorbent cotton, and heated and dried for 3 seconds with a hair dryer three times.

(1-2) Modification of gold-deposited glass substrate surface Modification of the azide group on the gold-deposited glass substrate surface was performed by a reaction of gold and a thiol group. Since alkanethiol is known to form a self-assembled monolayer (SAM) on the gold surface, 1 is represented by the formula (7) obtained by synthesizing a gold-deposited glass substrate in step 2. -It was immersed in a 0.2 vol% toluene solution of azidoundecane-11-thiol and allowed to stand for 18 hours.

(2) Second step: Click reaction Poly- [Lac (OAc) ₇ -AA- represented by formula (28) synthesized in step 14 on the support surface modified with the azide group in the first step. Co-HEAA] -b-Alkyne-A powder was sprinkled in a small amount (less than 1 mg per cm ² ), and then the click reaction solution was applied and allowed to stand for 18 hours. As the click reaction solution, a solution of 10 mg of copper (II) sulfate pentahydrate and 15 mg of sodium ascorbate dissolved in a THF / milli-Q water (20 ml / 5 ml) solution was used. After completion of the reaction, the support surface was washed thoroughly in order of acetone, methanol, and milli-Q water and dried.

(3) Third step: sugar chain deprotection reaction The sugar chain deprotection reaction was carried out with reference to a known method (Biomacromolecule, 2004, 5, 224-231). The carrier subjected to the click reaction in the second step was immersed in a 3 vol% DMSO solution of hydrazine monohydrate, and left in a sealed state for 5 hours. After completion of the reaction, the support surface was washed thoroughly in order of acetone, methanol, and milli-Q water and dried.

[Confirmation of reaction progress: measurement of contact angle]
Formation of the sugar chain polymer brush on the carrier surface was confirmed by measuring the contact angle on the carrier surface. This is because if the sugar chain polymer brush is formed on the surface of the carrier, the density of hydroxyl groups on the surface of the carrier will increase, and the hydrophilicity will increase.
About the glass substrate, the deprotection of the 3rd process was partially performed, and the contact angle of the part which is deprotected and the part which is not deprotected was compared. In addition, the contact angle calculated | required the average value of two glass substrates (measurement of four places per sheet).
About the gold vapor deposition glass substrate, the click reaction of the 2nd process was partially performed, and also the deprotection of the 3rd process was partially performed, and the contact angle after each process was compared. In addition, the contact angle calculated | required the average value of 1 gold | metal vapor deposition glass substrate (10 places measurement per sheet).
At room temperature (about 23 ° C.), 2 μl of ion-exchanged water (Milli Q water) was dropped on the surface of the carrier in air, and the contact angle between the surface of the carrier and the water droplets was measured. A surface tension meter (DropMaster-500 manufactured by Kyowa Interface Science Co., Ltd., using analysis software FAMAS) was used as the measuring device.

For glass substrates, the contact angle of the non-deprotected portion is 67.6 °, whereas the contact angle of the deprotected portion is 41.8 °, and the deprotected portion is about 26 °. A small value was shown.
For the gold-deposited glass substrate, the contact angle after the first step (after modification of the azide group) is 67.1 °, and the contact angle after the second step (after click reaction) is 65.8 °. On the other hand, the contact angle after the third step (after deprotection reaction) was 51.8 °, and the contact angle of the deprotected portion was the smallest value.
From the above results, it was suggested that the reaction in each step proceeded in both the glass substrate and the gold-deposited glass substrate, and a sugar chain polymer brush was formed on the surface.

[Evaluation of sugar chain polymer brush]
1. Evaluation by specific binding of lectin and nonspecific adsorption of protein (formation of sugar chain polymer brush on sensing surface of QCM sensor)
(1) First step: Modification of the azide group on the sensing surface A piranha cleaning solution (concentrated sulfuric acid: hydrogen peroxide solution = 3: on the sensing surface of the sensor chip of the QCM sensor (Initium, AFFINIX Q, 27 MHz): 1) was applied, allowed to stand for 10 minutes, washed with water, further rubbed with a 1% SDS solution on a cotton swab, then washed with water and dried.
Since the sensing surface is made of a gold thin film, the first step (modification of the azide group) was performed by a reaction between gold and a thiol group. The sensing surface was immersed in a 0.2 vol% toluene solution of 1-azidoundecane-11-thiol represented by the formula (7) synthesized in Step 2, and left for 2 hours.

(2) Second step: Click reaction Poly-Lac (OAc) ₇ − represented by the formula (25) synthesized in step 11 on the sensing surface of the QCM sensor in which the azide group was modified in the first step. A small amount (1 cm) of the powder of Ab-Alkyne-A or Poly- [Lac (OAc) ₇ -A-co-HEA] -b-Alkyne-A represented by the formula (27) synthesized in Step 13 after sprinkling less than ² per 1 mg), click reaction solution was applied and allowed to stand 18 hours.
For the click reaction solution, 10 mg of copper (II) sulfate pentahydrate and 15 mg of sodium ascorbate dissolved in t-BuOH / milli-Q water (1: 1) ⇒ (10 ml / 10 ml) solution were used. It was. After completion of the reaction, the sensing surface was washed thoroughly in the order of acetone, methanol and milli-Q water and dried.

(3) Third step: sugar chain deprotection reaction The sugar chain deprotection reaction was carried out with reference to a known method (Biomacromolecule, 2004, 5, 224-231). The 3 vol% DMSO solution of hydrazine monohydrate was applied to the sensing surface of the QCM sensor that was click-reacted in the second step, and left for 2 hours was repeated twice. After completion of the reaction, the sensing surface was washed thoroughly in the order of acetone, methanol and milli-Q water and dried.

(Confirmation of specific binding of lectins)
RCA ₁₂₀ (manufactured by Funakoshi), which is a lactose lectin, was used as the lectin. A QCM sensor was used as the measuring device. Into the glass cell attached to the QCM sensor, 8 ml of ion-exchanged water (Milli-Q water) degassed in advance is put, and a sensor chip with a sugar chain polymer brush formed on the surface by the above method is immersed in the cell. Stirring (850 rpm) was performed. After waiting in this state until the frequency became constant, 8 μl of RCA ₁₂₀ (5 mg / ml) was added, and the frequency change was recorded.

(Confirmation of non-specific protein adsorption)
BSA (manufactured by Sigma) was used as the protein. Into the glass cell attached to the QCM sensor, 8 ml of ion-exchanged water (Milli-Q water) degassed in advance is put, and a sensor chip with a sugar chain polymer brush formed on the surface by the above method is immersed in the cell. Stirring (850 rpm) was performed. After waiting in this state until the frequency became constant, 8 μl of BSA (5 mg / ml) was added, and the frequency change was recorded. Subsequently, after waiting until the frequency became constant, 8 μl of RCA ₁₂₀ (5 mg / ml) was added and the frequency change was recorded.

Evaluation results of the sugar chain polymer brush formed by immobilizing Poly-Lac (OAc) ₇ -Ab-Alkyne-A are shown in FIGS. 3 (A) and 3 (B), and Poly- [Lac (OAc) ₇ -A. -Co-HEA] Evaluation results of the sugar chain polymer brush formed by immobilizing -b-Alkyne-A are shown in FIGS. Both Poly-Lac (OAc) ₇ -Ab-Alkyne-A and Poly- [Lac (OAc) ₇ -A-co-HEA] -b-Alkyne-A showed almost no change in frequency in BSA. On the other hand, in RCA ₁₂₀ , a change in frequency of 1500 to 2000 Hz was observed. From this, it was confirmed that the sensing surface on which the copolymer of the present invention was immobilized and a sugar chain polymer brush was formed showed specific binding to lectin (RCA ₁₂₀ ) and hardly showed nonspecific adsorption of protein. It was done.

2. Evaluation by adhesion of rat primary hepatocytes (I)
(Formation of sugar chain polymer brush on culture substrate surface)
(1) First step: Modification of azide group on glass substrate surface Modification of the azide group on the glass substrate surface was performed by silane coupling reaction. As the silane coupling agent, 3-azidopropyltriethoxysilane represented by the formula (2) synthesized in Step 1 was used. A 1 vol% toluene solution of 3-azidopropyltriethoxysilane was prepared, thinly coated on the glass substrate surface with absorbent cotton, and heated and dried for 3 seconds with a hair dryer three times.

(2) Second step: Click reaction Poly- [Lac (OAc) ₇ -A represented by the formula (27) synthesized in step 13 on the glass substrate surface modified with the azide group in the first step. -Co-HEA] -b-Alkyne-A powder was sprinkled in a small amount (less than 1 mg per cm ² ), and then the click reaction solution was partially applied and left for 18 hours. In the click reaction solution, 10 mg of copper (II) sulfate pentahydrate and 15 mg of sodium ascorbate were dissolved in a t-BuOH / milli-Q water (1: 1) ⇒ (10 ml / 10 ml) solution. Was used. After completion of the reaction, the glass substrate surface was washed thoroughly in order of acetone, methanol, and milli-Q water and dried.

(3) Third step: sugar chain deprotection reaction The sugar chain deprotection reaction was carried out with reference to a known method (Biomacromolecule, 2004, 5, 224-231). The glass substrate subjected to the click reaction in the second step was immersed in a 3 vol% DMSO solution of hydrazine monohydrate and left in a sealed state for 5 hours. After completion of the reaction, the surface of the glass substrate was washed thoroughly in order of acetone, methanol and milli-Q water, dried, and used as a culture substrate.

(Confirmation of cell adhesion)
For evaluation by cell adhesion, rat primary liver parenchymal cells (hepatocytes), which are known to have a lactose recognition site on the cell surface, were used.
Hepatocytes were separated from a rat (7 weeks old, male) with a collagenase solution, and centrifuged (50 × g, 2 min) three times to purify the hepatocytes. The purified hepatocytes were seeded on the above culture substrate with 3.0 × 10 ⁶ cells and cultured. In order to avoid cell adhesion to the entire culture substrate, serum-free Williams'E medium (50 ml FBS, 10 ml penicillin-streptomycin) was used for culturing hepatocytes.

The culture results are shown in FIG. The arrow in the figure indicates the position where Poly- [Lac (OAc) ₇ -A-co-HEA] -b-Alkyne-A is immobilized on the culture substrate. It was confirmed that hepatocytes selectively and spherically adhered only to the site where the sugar chain polymer brush was formed by Poly- [Lac (OAc) ₇ -A-co-HEA] -b-Alkyne-A (FIG. Below the dotted line 5).

3. Effect on differentiation function of rat primary hepatocytes (I)
For the test, Rat liver parenchymal cells were used in the same manner as above. Hepatocytes were separated from a rat (7 weeks old, male) with a collagenase solution, and centrifuged (50 × g, 2 min) three times to purify the hepatocytes. A cell suspension prepared by adjusting the purified hepatocytes to 1 × 10 ⁵ cells / ml using serum-free Williams'E medium is seeded on a 12-well plate so that the cell suspension becomes 1 × 10 ⁵ cells / well. (2 ml / well) and cultured. As the substrate, a lactose glycopolymer (Poly- [Lac (OAc) ₇ -A-co-HEA] -b-Alkyne-A) substrate and a gelatin-modified substrate were used. Formation of a sugar chain polymer brush on the surface of a lactose glycopolymer substrate is performed as described in 2. above. The same method as in (1) to (3) was performed. After 6 hours of culturing, Williams'E medium (no additive factor, 10% FBS, 2% penicillin-streptomycin) and Williams'E medium (with additive factor, 10% FBS, 2% penicillin-streptomycin, ^10-6 M Insulin) , 10 ⁻⁷ Dexamethasone, 0.3 mg / ml L-glutamine).

On the first, second, third, and fourth days, the cell morphology was observed, and the MTT assay and albumin production were measured. In addition, the MTT assay followed a conventional method. The measurement of albumin production was performed by the following method.
(1) 1 ml of the supernatant of the medium was collected and stored at -80 ° C.
(2) A primary antibody (rabbit anti rat IgG) diluted 5000-fold was added to a 96-well plate in an amount of 100 μl, and allowed to stand for 1 hour.
(3) The solution in the well was sucked and washed 3 times with a washing solution (0.02% Tween 20-PBS solution).
(4) 200 μl of 0.1% BSA-PBS solution was added and allowed to stand for 30 minutes.
(5) The solution in the well was sucked and washed 3 times with a washing solution (0.02% Tween 20-PBS solution).
(6) 100 μl of sample solution was added and allowed to stand for 1 hour.
(7) The solution in the well was sucked and washed 3 times with a washing solution (0.02% Tween 20-PBS solution).
(8) 100 μl of secondary antibody (goat anti rat albumin IgG) was added and allowed to stand for 1 hour.
(9) The solution in the well was sucked and washed 3 times with a washing solution (0.02% Tween 20-PBS solution).
(10) 50 μl of a coloring reagent (SAT Blue) was added and reacted until the solution in the well turned blue.
(11) After the solution turned blue, 50 μl of a reaction stop solution (1 MH ₂ SO ₄ solution) was added.
(12) The amount of albumin produced was measured by measuring the absorbance at 450 nm with a microplate reader.

  The culture results are shown in FIG. The lactose glycopolymer substrate had higher albumin production than the gelatin modified substrate. This revealed that the lactose glycopolymer substrate activated and maintained the differentiation function of primary hepatocytes. This seems to be because, according to the lactose glycopolymer substrate, cells are adhered in a spherical shape via the asialoglycoprotein receptor.

4). Effect on differentiation function of rat primary hepatocytes (II)
In the test, 2. Rat liver parenchymal cells were used in the same manner as above. Hepatocytes were separated from a rat (7 weeks old, male) with a collagenase solution, and centrifuged (50 × g, 2 min) three times to purify the hepatocytes. A cell suspension prepared by adjusting the purified hepatocytes to 1 × 10 ⁵ cells / ml using serum-free Williams'E medium is seeded on a 12-well plate so that the cell suspension becomes 1 × 10 ⁵ cells / well. (2 ml / well) and cultured. As the substrate, a lactose glycopolymer (Poly- [Lac (OAc) ₇ -A-co-HEA] -b-Alkyne-A) substrate and a gelatin-modified substrate were used. Formation of a sugar chain polymer brush on the surface of a lactose glycopolymer substrate is performed as described in 2. above. The same method as in (1) to (3) was performed. After 6 hours of culture, Williams'E medium (no EGF, 10% FBS, 2% penicillin-streptomycin, ^10-6 M Insulin, ^10-7 Dexamethasone, 0.3 mg / ml L-glutamine) and Williams'E medium ( The medium was replaced with EGF, 10% FBS, 2% penicillin-streptomycin, ^10-6 M Insulin, ^10-7 Dexamethasone, 0.3 mg / ml L-glutamine, EGF 50 ng / ml).

  On the first, second, third, and fourth days, the cell morphology was observed, and the MTT assay and albumin production were measured. In addition, the MTT assay followed a conventional method. The measurement of albumin production was performed by the same method as described above.

  The culture results are shown in FIG. The lactose glycopolymer substrate had higher albumin production than the gelatin modified substrate. This revealed that the lactose glycopolymer substrate activated and maintained the differentiation function of primary hepatocytes. This seems to be because, according to the lactose glycopolymer substrate, cells are adhered in a spherical shape via the asialoglycoprotein receptor.

5. Evaluation of rat primary hepatocytes by adhesion (II)
For the test, Rat liver parenchymal cells were used in the same manner as above. Hepatocytes were separated from a rat (7 weeks old, male) with a collagenase solution, and centrifuged (50 × g, 2 min) three times to purify the hepatocytes. The purified hepatocytes were adjusted to 5 × 10 ⁴ cells / ml using serum-free Williams'E medium (2% penicillin-streptomycin), and asialofetin (ASF) was added at a concentration of 10 μg / ml. And preincubated at 4 ° C. for 30 minutes. Similarly, a cell suspension with an asialofetin concentration of 0 μg / ml was preincubated at 4 ° C. for 30 minutes. Thereafter, cells were seeded on a 12-well plate so as to be 1 × 10 ⁵ cells / well, and cultured at 37 ° C. for 6 hours.

  The culture results are shown in FIGS. As shown in FIG. 8, no adhesion inhibition was observed on the gelatin-modified substrate (ASF 10 μg / ml). In contrast, adhesion inhibition was observed in the lactose glycopolymer substrate (ASF 10 μg / ml) (FIG. 9). This suggests that the lactose glycopolymer substrate is a surface that can adhere to primary hepatocytes in a sugar chain-specific manner.

  From the above results, it is considered that the lactose glycopolymer has a brush structure, and thus hepatocyte adhesion specific to lactose could be easily achieved. And it is thought that the significant raise of albumin production ability was recognized by adding an addition factor and EGF.

Claims (7)

A copolymer having a chain structure for immobilizing a ligand on the surface of a carrier having a reactive group (a),
The monomer (A) having a reactive group (b) that can be chemically bonded to the reactive group (a) is polymerized alone, or the monomer (A) and another monomer are copolymerized. The segment (A) has one end of the main chain of the copolymer, and the monomer (B) having the ligand is polymerized alone, or the monomer (B) and another monomer are copolymerized. have a segment (B) to the other end of the main chain of the copolymer,
Either one of the reactive group (a) and the reactive group (b) is an azide group and the other is an alkynyl group, or the reactive group (a) and the reactive group (b) Either one is an amino group or a thiol group, and the other is a carboxyl group, a carboxylic acid activated with an N-hydroxysuccinimidyl ester, an isocyanate group, or a thioisocyanate group,
The proportion of the structural unit derived from the monomer (A) in the segment (A) is 5 mol% or more,
The proportion of the structural unit derived from the monomer (B) in the segment (B) is 20 mol% or more,
The proportion of the structural unit derived from the monomer (A) in the copolymer is 5 to 80 mol%,
The proportion of the structural unit derived from the monomer (B) in the copolymer is 20 to 80 mol%,
A copolymer in which the monomer (A) is represented by the following general formula (I) and the monomer (B) is represented by the following general formula (II) .
(In the formula, R ^1a represents a hydrogen atom or a methyl group, R ^2a represents —O— or —NH—, X represents a spacer, and Y represents a reactive group (b).)
(In the formula, R ^1b represents a hydrogen atom or a methyl group, R ^2b represents —O— or —NH—, X represents a spacer, and Z represents a ligand.) The copolymer according to claim 1, which is formed by living radical polymerization. The copolymer according to claim 1 or 2 , wherein the ligand is at least one selected from the group consisting of sugars, sugar chains, and amino acids. Carrier ligand by a copolymer according to claims 1 to 3 or is immobilized. A method for immobilizing a ligand, comprising a step of reacting the reactive group (b) of the copolymer according to any one of claims 1 to 3 with the reactive group (a) on the surface of the carrier. A cell culture method using a culture substrate on which the copolymer according to any one of claims 1 to 3 is immobilized. The cell culture method according to claim 6 , wherein the cells are cultured in a serum-free medium.