JP2021038373A - Polymer of 1-acryloylimidazolidin-2-one compound, and cell culture material based on the same - Google Patents

Abstract

To provide a copolymer expected for applications as a cell culture material.SOLUTION: The copolymer comprises a hydrophobic block chain and a block chain having a repeating unit represented by the general formula (1) in the figure. (In the formula, R1 represents a hydrogen atom, C1-4 alkyl group, benzyl group, or oxyalkyl group; R2 represents a hydrogen atom or C1-4 alkyl group; and R3 represents a hydrogen atom or methyl group.)SELECTED DRAWING: None

Description

The present invention relates to a copolymer that is expected to be applied as a material for cell culture.

In recent years, the development of regenerative medicine technology has progressed, and it is possible to induce differentiation into mesenchymal stem cells, which have the ability to differentiate into osteoblasts, adipocytes, muscle cells, chondrocytes, etc., and all tissues and organs that make up humans. There is a demand for a technique for safely and efficiently culturing a large amount of artificial pluripotent stem cells (iPS cells) and the like. As such a material for cell culture, a culture bed in which a polymer of N-isopropylacrylamide (NIPAAM) is immobilized on the surface of a container has been developed. In the case of the NIPAAM homopolymer, the lower limit critical solution temperature (LCST) is 32 ° C. near the body temperature. For example, at the cell culture temperature (about 36 ° C.), the surface of the culture bed on which the NIPAAM polymer is immobilized is hydrophobic and the cells Adhesion and proliferation occur, and when the culture bed is controlled to a temperature lower than LCST, the surface of the culture bed becomes hydrophilic and the cultured cells are easily peeled off, enabling cell recovery with less damage to the cells (Non-Patent Documents). 1).

As a culture bed on which the polymer of NIPAAM is immobilized, a culture bed in which NIPAAM is graft-polymerized by electron beam irradiation (UpCell manufactured by CellSeed) is commercially available, but a large amount is required because a large-scale electron beam irradiation device is required. The problem is that it is not suitable for production. Further, in Patent Document 1, a large-scale device such as electron beam irradiation is required by using a base material whose surface is coated with a block copolymer having a structure in which a water-insoluble polymer segment and a temperature-responsive polymer segment are bonded. It has been shown that the temperature-responsive polymer can be immobilized on the surface of the substrate without using it. However, the block copolymer disclosed in Patent Document 1 has insufficient water resistance, and has a problem that elution occurs from the surface of the base material and a problem that cell exfoliation property is insufficient. Further, when the amount of the block copolymer immobilized on the surface of the substrate is large, the cell exfoliation property is good, but the cell adhesion is poor. On the contrary, when the amount of immobilization is small, the cell adhesion is good, but the cell exfoliation property is inferior, and the amount of the polymer of NIPAAM immobilized on the surface of the substrate greatly affects the performance of the cell culture bed.

The culture bed on which the NIPAAM polymer is immobilized in this way has room for improvement in terms of stable cell culture and recovery efficiency, and is a temperature-responsive polymer material that replaces the NIPAAM polymer, and further applies this. There is a demand for block copolymers.

Patent No. 5846584

Polymer Papers, Vol. 75 (No. 2), Paragraphs 174-186 (2018)

An object of the present invention is to provide a copolymer suitable as a material for cell culture.

As a result of diligent studies to solve the above problems, the present inventors have completed the present invention. That is, one aspect of the present invention is the following general formula (1).

(In the formula, R ¹ is a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, a benzyl group, or the following general formula (2).

(In the formula, p represents an integer of 2 to 4, and R ⁴ represents a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, a 2-methoxyethyl group or an acetyl group.). R ² represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. R ³ represents a hydrogen atom or a methyl group. ) Is a copolymer comprising a block chain having a repeating unit and a hydrophobic block chain.

Further, in another aspect of the present invention, the hydrophobic block chain has the following general formula (3).

(Represented in the formula, R ⁵ is an alkyl group having 1 to 6 carbon atoms, a cyclohexyl group, a benzyl group, or an .R ⁶ is a hydrogen atom or a methyl group and phenylethyl group. Provided that when R ⁶ is a hydrogen atom , R ⁵ is a copolymer which is a block chain having a repeating unit represented by a methyl group, an ethyl group, a cyclohexyl group or a benzyl group).

Further, another aspect of the present invention is a coating agent containing the above-mentioned copolymer.

Further, another aspect of the present invention is a cell culture material obtained by applying the above-mentioned coating agent to a substrate.

Further, another aspect of the present invention is a cell culture method using the above-mentioned cell culture material.

The present invention will be described in more detail below.

^{Examples of the alkyl group having 1 to 4 carbon atoms of R 1 in} the general formula (1) include a methyl group, an ethyl group, a propyl group, an isopropyl group, a cyclopropyl group, a butyl group, an isobutyl group, a sec-butyl group, and a tert-. Butyl group and cyclobutyl group can be mentioned. Alkyl group having 1 to 4 carbon atoms R ² is the same.

The same applies to the alkyl group having 1 to ⁴ carbon atoms of R4 in the general formula (2).

The chemical structure of the block chain having the repeating unit represented by the general formula (1) constituting the copolymer (hereinafter, may be referred to as imidazolidinone block chain) is the imidazolidinone monomer (4) used in the polymerization. Although it is naturally determined by the chemical structure of the above, the combination is not particularly limited as long as it is within the range of the above-mentioned substituents.

(In the formula, R ¹ , R ² and R ³ have the same meanings as described above.)
1-Acryloyluimidazolidine-2-one, 1-acryloyl-3-methylimidazolidine-2-one, 1- in terms of controlling the temperature responsiveness of imidazolidinone block chains and cell adhesion / detachability Acryloyl-3-ethylimidazolidine-2-one, 1-acryloyl-3-propylimidazolidine-2-one, 1-acryloyl-3-isopropylimidazolidine-2-one, 1-acryloyl-3-cyclopropylimidazolidine -2-one, 1-acryloyl-3-benzylimidazolidine-2-one, 1-acryloyl-3- (2-hydroxyethyl) imidazolidine-2-one, 1-acryloyl-3- (2-methoxyethyl) Imidazolidine-2-one, 1-acryloyl-3- (3-methoxypropyl) imidazolidine-2-one, 1-acryloyl-3- (4-methoxybutyl) imidazolidine-2-one, 1-acryloyl-3 -(2-ethoxyethyl) imidazolidine-2-one, 1-acryloyl-3- (2-propyloxyethyl) imidazolidine-2-one, 1-acryloyl-3- [2- (2-methoxyethoxy) ethyl ] Block chains obtained by polymerizing imidazolidine-2-one and 1-acryloyl-4,4-dimethylimidazolidine-2-one alone or in combination of two or more are preferable, among which 1-acryloy midazolidine-2-one, 1-acryloyl-3-methylimidazolidine-2-one, 1-acryloyl-3-ethylimidazolidine-2-one, 1-acryloyl-3-isopropylimidazolidine-2-one, 1-acryloyl-3-benzylimidazole Obtained by polymerizing lysine-2-one, 1-acryloyl-3- (2-methoxyethyl) imidazolidine-2-one, 1-acryloyl-4,4-dimethylimidazolidine-2-one alone or in combination of two or more. Block chains are particularly preferred.

The imidazolidinone block chain may contain a repeating unit represented by the general formula (1), but in addition to the imidazolidine monomer, hydrophobicity, hydrophilicity, solubility, film-forming property, heat resistance, and film Acrylate, methyl acrylate, ethyl acrylate, propyl acrylate, butyl acrylate, phenyl acrylate, benzyl acrylate, 2-hydroxyethyl acrylate, 2 -Methoxyethyl acrylate, 2- (N, N-dimethylamino) ethyl acrylate, methacrylic acid, methyl methacrylate, ethyl methacrylate, propyl methacrylate, butyl methacrylate, phenyl methacrylate, benzyl methacrylate, 2-phenylethyl methacrylate, 2-hydroxyethyl methacrylate , 2-methoxyethyl methacrylate, 2-methacryloyloxyethyl phosphorylcholine, acrylamide, N, N-dimethylacrylamide, N, N-diethylacrylamide, N-isopropylacrylamide, N-tert-butylacrylamide, N-phenylacrylamide, N-hydroxy Methyl acrylamide, N-hydroxyethyl acrylamide, N-butoxymethyl acrylamide, N- (2-hydroxyethyl) acrylamide, N'- [3- (N, N-dimethylamino) propyl] acrylamide, acryloyl morpholine, N-methylmethacryl Amid, N, N-dimethylmethacrylate, N-isopropylmethacrylate, N-tert-butylmethacrylate, N-phenylmethacrylate, N-methoxymethylmethacrylate, vinyl acetate, styrene, chloromethylstyrene, 2-vinylpyridine , Acrylonitrile and other small amounts of vinyl-based monomers may coexist. The amount of the vinyl-based monomer added is preferably 10% by weight or less, more preferably 5% by weight or less, based on the imidazolidine monomer, so as not to significantly impair the properties derived from the imidazolidine monomer.

A hydrophobic block chain refers to a polymer composed of only the hydrophobic block chain that does not show solubility in water or has extremely low solubility, and refers to polyethylene, polypropylene, polybutadiene, polyacrylonitrile, polyvinyl ether, polystyrene, etc. Examples thereof include poly (meth) acrylic acid ester, poly (meth) acrylamide, polyester, polycarbonate, polyamide, polyimide, polyurethane, polysulfone, polyphenylene oxide, polysiloxane, polysilarylenesiloxane, and derivatives thereof. Among them, from the viewpoint of film formation property and cell adhesion, a hydrophobic block chain having at least one of the melting point and the glass transition temperature of the polymer composed of only the hydrophobic block chain is 15 ° C. or higher is preferable.

As the hydrophobic block chain, block chain having a repeating unit represented by formula (3) a copolymer preferable in the structure control is easy in terms of the general formula (3) having 1 to 6 carbon atoms for R ⁵ in As the alkyl group, methyl group, ethyl group, propyl group, isopropyl group, cyclopropyl group, butyl group, isobutyl group, sec-butyl group, tert-butyl group, cyclobutyl group, pentyl group, neopentyl group, isopentyl group, tert -Pentyl group, 1-methylbutyl group, 1-ethylpropyl group, cyclobutylmethyl group, cyclopentyl group, hexyl group, 1-methylpentyl group, 4-methylpentyl group, 1-ethylbutyl group, 2-ethylbutyl group, cyclohexyl group Specific examples include methyl acrylate, ethyl acrylate, cyclohexyl acrylate, benzyl acrylate, methyl methacrylate, ethyl methacrylate, and propyl methacrylate in terms of controlling solubility, film-forming property, and cell adhesiveness / exfoliation. , Isopropyl methacrylate, butyl methacrylate, isobutyl methacrylate, sec-butyl methacrylate, tert-butyl methacrylate, cyclohexyl methacrylate, benzyl methacrylate, and 2-phenylethyl methacrylate are preferably polymerized alone or in combination of two or more.

The form of the copolymer may be any form such as a linear block copolymer, a branched block copolymer, a star-shaped block copolymer, and a graft copolymer.

The molecular weight of the copolymer is not particularly limited, and the copolymer can be used depending on the measuring method such as weight average molecular weight, number average molecular weight, and viscosity average molecular weight. The weight average molecular weight (Mw) is preferably 1,000 to 1,000,000, and more preferably 5,000 to 500,000 from the viewpoint of controlling the properties of the polymer and processability. The molecular weight distribution (PD) represented by Mw / Mn is not particularly limited, but is preferably in the range of about 1 to 10, and more preferably in the range of 1 to 3 from the viewpoint of polymer uniformity. .. As a method for calculating the molecular weight, known methods such as a size exclusion chromatography (SEC) method, a viscosity method, and a light scattering method in which a standard sample such as polystyrene or polyethylene glycol is converted as a reference can be mentioned. When graft-polymerized on the surface of the substrate, it is difficult to directly determine the molecular weight of the polymer. For example, the number average molecular weight (Mn) of the graft chain can be estimated from the density of reaction points on the surface of the substrate and the polymerization conversion rate. it can.

Examples of the method for producing the copolymer include the following methods.
(Method 1) A method for producing a copolymer of the present invention by synthesizing an imidazolidinone block chain and a hydrophobic block chain, respectively, and then reacting the imidazolidinone block chain with the hydrophobic block chain. (Method 2) A method for producing the copolymer of the present invention by synthesizing an imidazolidinone block chain or a hydrophobic block chain by using living polymerization or the like, and then synthesizing the other block chain from this terminal. 3) A method for producing the copolymer of the present invention by introducing a polymerizable functional group at the end of the imidazolidinone block chain and polymerizing the polymerizable functional group (Method 4) From the surface of the polymer solid as the base material. A method for producing a copolymer of the present invention by synthesizing an imidazolidinone block chain by surface-initiated polymerization of an imidazolidinone monomer using electromagnetic waves such as ultraviolet light or radiation such as electron beam or gamma ray. Among them, Method 2 is preferably used because the structure of the copolymer can be precisely controlled and it can be easily applied to a coating agent described later.

When producing a block copolymer using Method 2, known living radical polymerization methods can be used, for example, iniferter polymerization method, nitroxide-mediated radical polymerization method, atom transfer radical polymerization method, reversible addition-. A rupture chain transfer (RAFT) polymerization method or the like can be used. For details of these polymerization methods, see "Radical Polymerization Handbook" published by NTS Co., Ltd., p. See 161 to 225 (2010). In particular, RAFT polymerization is preferably used because there is no residue of heavy metals and the like and the molecular weight can be easily controlled.

The manufacturing method using the RAFT polymerization method can be roughly divided into two manufacturing steps. In the first step, a polymer in which a RAFT agent residue is bonded to the terminal of the polymer A obtained by RAFT polymerization of the monomer A in the coexistence of a radical polymerization initiator and a RAFT agent (this is called a macro RAFT agent of the polymer A) is obtained. This is the manufacturing process. In the second step, RAFT polymerization is performed in the coexistence of the macro RAFT agent produced in the first step and the radical polymerization initiator, and the polymer B obtained by polymerizing the monomer B is bonded to one end of the polymer A. This is a step of producing a block copolymer (diblock copolymer). In the present invention, a monomer composed of a single or two or more types of imidazolidinone monomers and a vinyl-based monomer which may be added as needed corresponds to a monomer A or B, and a single or two or more types of (meth) acrylics. A monomer composed of an acid alkyl ester corresponds to monomer B or A.

A RAFT agent residue is bound to the end of this diblock copolymer, which acts as a macro RAFT agent. By using this macro RAFT agent instead of the RAFT agent in the first step, for example, Polymer A-Polymer B -A triblock copolymer in which each chain is linked, such as polymer A, can be produced. By repeating the process in the same manner, a multi-block copolymer in which arbitrary chains are connected can be produced.

The degree of polymerization can be controlled by the ratio [M] / [I ·] of the molar amount [M] of the monomer and the molar amount [I ·] of the radical species generated from the radical polymerization initiator, and the x and y of the block copolymer can be controlled. It suffices to charge more than the amount of the monomer satisfying the value, and the conversion rate of the monomer may be controlled so as to satisfy the values of x and y. The amount of RAFT agent used [RAFT] may be equal to or greater than [I ·], and generally [RAFT] / [I ·] is adjusted to be 1 to 10, preferably 1.5 to 5. .. The conversion rate of the monomer can be arbitrarily controlled by the type of RAFT agent, the value of [RAFT] / [I ·], the presence or absence or type of solvent, the initial concentration of the monomer, the viscosity of the solution, the polymerization temperature, the polymerization time and the like.

Examples of the radical polymerization initiator used in the first step and the second step include inorganic peroxides such as potassium persulfate and ammonium persulfate, and N, N, N', N'-tetramethyl called polymerization accelerators. Rapid polymerization at low temperatures is possible when used in combination with amine compounds such as ethylenediamine and N, N-dimethylparatoluidine. Further, as a radical initiator, organic peroxides such as dilauroyl peroxide, benzoyl peroxide, di-t-butyl peroxide, t-butyl hydroperoxide, and cumene hydroperoxide, and 2,2'-azobis (4-methoxy-2, 4-Dimethylvaleronitrile), 2,2'-azobis (2,4-dimethylvaleronitrile), 2,2'-azobisisobutyronitrile, 2,2'-azobis (2-methylbutyronitrile), 1,1'-azobis (cyclohexane-1-carbonitrile), 2,2'-azobis [2- (2-imidazolin-2-yl) propane] dihydrochloride, 2,2'-azobis [2- (2) -Imidazolin-2-yl) propane] disulfate dihydrate, 2,2'-azobis [2- (2-imidazolin-2-yl) propane], 2,2'-azobis (2-methylpropionamidine) ) Dihydrochloride, 2,2'-azobis [N- (2-carboxyethyl) -2-methylpropionamidine] n hydrate, 2,2'-azobis [2-methyl-N- (2-hydroxyethyl) ) Propionamide], 2,2'-azobis [N- (2-propenyl) -2-methylpropionamide], 2,2'-azobis (N-butyl-2-methylpropionamide), dimethyl 2,2' Azobisisobutyate, 4,4'-azobis (4-cyanopentanoic acid) and other azo compounds can be exemplified. Above all, an azo compound is preferably used from the viewpoint of suppressing side reactions.

The RAFT agent used in the first step is not particularly limited as long as the monomer A can be controlledly polymerized and the monomer B can be controlledly polymerized using the macro-RAFT agent of the polymer A, and a known RAFT agent used in the RAFT polymerization of a non-conjugated monomer. Can be used. Generally, dithiocarbamic acid ester compounds, dithiocarboxylic acid ester compounds, trithiocarbonate ester compounds and the like are known, and more specifically, 4-[(2-carboxyethyl sulfanylthiocarbonyl) sulfanyl-4-cyanopentanoic acid, 2 -{[(2-carboxyethyl) sulfanylthiocarbonyl] sulfanyl} propanoic acid, 4-chloro-3,5-dimethylpyrazole-1-dithiocarboxylic acid 2'-cyanobutane-2'-yl, 3,5-dimethylpyrazole -1-Dithiocarboxylic acid 2'-cyanobutane-2'-yl, 4-cyano-4-[(dodecylsulfanylthiocarbonyl) sulfanyl] pentanoic acid, 2-cyano-2-[(dodecylsulfanylthiocarbonyl) sulfanyl] propane , 3,5-Dimethylpyrazole-1-dithiocarboxylic acid cyanomethyl, 4-cyano-4-[(thiobenzoyl) sulfanyl] pentanoic acid, trithiocarbonate S, S-dibenzyl, 2-[(dodecylsulfanylthiocarbonyl) sulfanylpropane Examples of acids, 4-cyano-4-[(dodecylsulfanylthiocarbonyl) sulfanyl] methylpentanoate, (dodecylsulfanylthiocarbonyl) sulfanyl acetonitrile, 2-methyl-2-[(dodecylsulfanylthiocarbonyl) sulfanyl] propanoic acid, etc. However, the selection of the RAFT agent may differ depending on the structure and combination of the monomers and the order of further polymerization. For the selection of the RAFT agent, for example, publicly known documents such as Macromolecules, Vol. 45, pp. 5321-5342 (2012) may be referred to.

A solvent can be used in the first step and the second step to allow the polymerization reaction to proceed smoothly. Available solvents include, for example, water, methanol, ethanol, propanol, isopropyl alcohol, butanol, isobutanol, hexanol, 2-methoxyethanol, 2-butoxyethanol, ethylene glycol, pentane, cyclopentane, hexane, cyclohexane, heptane, Octane, benzene, toluene, xylene, chlorobenzene, dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane, acetone, methylethylketone, diethyl ether, cyclopentylmethylether, tetrahydrofuran, dioxane, 1,2-dimethoxyethane, ethyl acetate, acetate Propyl, butyl acetate, acetonitrile, dimethylsulfoxide, N, N-dimethylformamide (DMF), N, N-dimethylacetamide (DMAc), ammonia, trimethylamine, diethylamine, triethylamine, diisopropylamine, butylamine, dibutylamine, tributylamine, pyridine , Picolin and the like can be used, but the present invention is not limited thereto. In addition, two or more of these solvents can be mixed and used. In the polymerization reaction, the polymerization can be easily controlled by lowering the oxygen concentration in the reaction vessel to the extent that the radical reaction is not hindered, such as under a vacuum or an inert gas atmosphere. The temperature of the polymerization reaction usually proceeds smoothly within the range of 0 ° C. to 150 ° C.

A part of the imidazolidinone monomer (4) can be produced by allowing acrylic acid chloride to act on the corresponding imidazolidinone derivative (5b) in the presence of base1 as shown in the following formula.

(In the formula, R ^1b is a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, a benzyl group, or the following general formula (2b).

(In the formula, p represents the same meaning as described above. R ^4b represents an alkyl group having 1 to 4 carbon atoms or a 2-methoxyethyl group.) Represents an oxyalkyl group represented by. R ² and R ³ have the same meanings as described above. )
At this time, a tertiary amine, a pyridine derivative, or the like can be used as base 1, specifically, trimethylamine, triethylamine, tripropylamine, tributylamine, triisobutylamine, diethylmethylamine, ethyldipropylamine, ethyldiisopropyl. Amine, pyridine, α-picoline, β-picoline, γ-picoline, 4- (N, N-dimethylamino) pyridine, quinoline and the like can be used alone or in combination of two or more. Among these, triethylamine is preferable in terms of good yield and low cost. The reaction proceeds smoothly when a solvent is used, for example, n-pentane, cyclopentane, n-hexane, cyclohexane, n-heptane, n-octane, benzene, toluene, xylene, chlorobenzene, dichloromethane, chloroform, carbon tetrachloride, etc. 1,2-Dichloroethane, acetone, methyl ethyl ketone, diethyl ether, cyclopentyl methyl ether, tetrahydrofuran, dioxane, acetonitrile, dimethyl sulfoxide, N, N-dimethylformamide, N, N-dimethylacetamide, etc. are used alone or in combination of two or more. Can be done. The reaction is usually carried out at a temperature of 100 ° C. or lower, and is preferably carried out at 50 ° C. or lower in terms of suppressing decomposition of the product.

After completion of the reaction, the imidazolidinone monomer (4) or (4b) can be produced by combining conventional purification methods such as extraction, filtration, column chromatography, recrystallization, reprecipitation and distillation.

The imidazolidinone derivative (5b) as a raw material can be easily obtained by adding the corresponding diamine derivative (6b) and urea and heating to about 100 to 200 ° C.

(In the formula, R ^1b , R ² and R ³ have the same meanings as described above.)
Further, the imidazolidinone derivative (5c) is shown by ^{X-R 1c} after allowing 1 equivalent of base2 to act on imidazolidine-2-one as an anionizing agent to extract hydrogen on the N atom to anionize it. It can be produced by reacting with an alkylating agent.

(In the formula, R ^1c represents an alkyl group having 1 to 4 carbon atoms, a benzyl group or an oxyalkyl group represented by the general formula (2b). X represents a chlorine atom, a bromine atom, an iodine atom or a tosyl group.)
At this time, examples of base2 include sodium hydride, potassium hydride, phenyllithium, lithium diisopropylamide, n-butyllithium, and the like. When a solvent is used in the above reaction, the reaction proceeds smoothly, for example, hexane, cyclohexane, toluene, diethyl ether, tetrahydrofuran (THF), 1,4-dioxane, cyclopentyl methyl ether (CPME), N, N-dimethylformamide (DMF). ), N, N-dimethylacetamide and the like can be used alone or in combination of two or more.

In the imidazolidinone monomer (4), when R ¹ is a 2-hydroxyethyl group or 2-acetoxyethyl group and both R ² and R ³ are hydrogen atoms, the synthesis method is as shown in the following formula, for example. It can be synthesized by a route. That is, urea is added to 2- (2-aminoethylamino) ethanol and heated to about 100 to 200 ° C. to give 1- (2-hydroxyethyl) imidazolidine-2-one, and then treated with acetic anhydride. Obtains 1- (2-acetoxyethyl) imidazolidine-2-one. By allowing acryloyl chloride to act on this in the presence of base1, 1-acryloyl-3- (2-acetoxyethyl) imidazolidine-2-one is obtained. 1- (2-Hydroxyethyl) imidazolidine-2-one can be obtained by subjecting this to a hydrolysis reaction in the presence of base3.

At this time, a weak base such as an amine or a pyridine derivative, sodium hydrogencarbonate, or sodium carbonate can be used as the base3, and specifically, the amine derivative is ammonia, butylamine, dimethylamine, diethylamine, trimethylamine, triethylamine, or tri. Examples thereof include propylamine, tributylamine, triisobutylamine, diethylmethylamine, ethyldipropylamine, ethyldiisopropylamine, etc. Specific examples of the pyridine derivative include pyridine, α-picoline, β-picolin, and γ-picolin. , 4- (N, N-dimethylamino) pyridine, quinoline and the like. base3 can be used alone or in combination of two or more of these weak bases. Among these, ammonia, trimethylamine, triethylamine, sodium hydrogencarbonate, and sodium carbonate are preferable in terms of good yield and low cost. The reaction proceeds smoothly when a solvent is used, for example, n-pentane, cyclopentane, n-hexane, cyclohexane, n-heptane, n-octane, benzene, toluene, xylene, chlorobenzene, dichloromethane, chloroform, carbon tetrachloride, etc. 1,2-Dichloroethane, acetone, methyl ethyl ketone, diethyl ether, cyclopentyl methyl ether, tetrahydrofuran, dioxane, acetonitrile, dimethyl sulfoxide, N, N-dimethylformamide, N, N-dimethylacetamide, methanol, ethanol, propanol, isopropyl alcohol, butanol Etc. can be used alone or in combination of two or more. The reaction is usually carried out at a temperature of 60 ° C. or lower, and is preferably carried out at 30 ° C. or lower in terms of suppressing decomposition of the product. After completion of the reaction, 1-acryloyl-3- (2-acetoxyethyl) imidazolidine-2-one and 1- (2-hydroxyethyl) imidazolidine-2-one were extracted, filtered, column chromatographed, and recrystallized, respectively. , Can be produced by combining conventional purification methods such as reprecipitation.

A coating agent can be prepared by dissolving the above-mentioned copolymer in a solvent. The solvent used for the coating agent is not particularly limited, but water, methanol, ethanol, propanol, isopropyl alcohol, butanol, hexanol, pentane, cyclopentane, hexane, cyclohexane, heptane, octane, benzene, toluene, xylene, chloroform, Dichloromethane, chloroform, carbon tetrachloride, 1,2-dichloroethane, acetone, methyl ethyl ketone, diethyl ether, cyclopentyl methyl ether, tetrahydrofuran, dioxane, acetonitrile, dimethyl sulfoxide, DMF, N, N-dimethylacetamide, N-methylpyrrolidin-2- On, ethylene glycol, 2-methoxyethanol, 2-butoxyethanol and the like can be used, but are not limited thereto. Further, two or more kinds of these solvents can be mixed and used.

The concentration of the coating agent is not particularly limited as long as it does not interfere with the coating, but a suitable cell culture material can be obtained by adjusting the concentration of the copolymer to 0.01 to 20% by weight. ..

The above-mentioned cell culture material can be prepared by treating the surface of a carrier having various shapes with a coating agent. The term "cell culture equipment" in the present specification is synonymous with a material for cell culture. The material of the carrier is not particularly limited, but for example, a styrene resin such as polystyrene, a polyolefin resin such as polypropylene, a polyurethane resin, a polycarbonate, a polyethylene terephthalate, a polysulfone resin, a fluorine resin, and a large number such as cellulose. Natural saccharides Polypolymers, inorganic materials such as glass and ceramics, and metal materials such as stainless steel and titanium can be used. The shape of the carrier is also not particularly limited, and for example, the shape of a general cell culture equipment such as a petri dish, a plate or a flask, a spherical shape, a bag shape, a sponge-like porous shape, or a fibrous shape such as a non-woven fabric or a woven fabric is used. be able to. Further, the method for treating the coating agent is not particularly limited, and a known method can be used. For example, a dip method, a spray method, a casting method, a spin coating method, an inkjet printing method, or the like can be used. Further, annealing may be performed with heat or a solvent. As an example, when performing thermal annealing, the treatment may be performed at 40 to 100 ° C. for several minutes to several hours. The film thickness of the copolymer is not particularly limited as long as it can function as a material for cell culture, and is, for example, 1 nm to 1000 nm.

The cells to be cultured using the cell culture material are not particularly limited as long as they can adhere to the surface of the cell culture substrate before being stimulated by a temperature drop. For example, bone marrow-derived mesenchymal stem cells of mammals such as humans, adipose tissue-derived mesenchymal stem cells of mammals such as humans, fibroblasts of mammals such as humans, Chinese hamster ovary-derived cells (CHO cells), mice. In addition to various cultured cell lines such as connective tissue L929 cells, human fetal kidney-derived cells HEK293 cells, and human cervical cancer-derived HeLa cells, for example, epithelial cells and endothelial cells constituting each tissue and organ in the living body, contractility, etc. Skeletal muscle cells, smooth muscle cells, myocardial cells, neuronal cells that compose the nervous system, glial cells, fibroblasts, hepatic parenchymal cells involved in biological metabolism, non-hepatic parenchymal cells and fat cells, cells with differentiation ability As a result, stem cells existing in various tissues, cells induced to differentiate from them, and the like can be used. In addition to these, cells (living cells) contained in blood, lymph, cerebrospinal fluid, sputum, urine or stool, and microorganisms, viruses, protozoans and the like existing in the body or environment can be exemplified.

The medium used for culturing can be used without particular limitation. When mesenchymal stem cells are used as an example, Dulbecco Vogt's modified Eagle's minimum essential medium (10 vol% FBS / DMEM) containing 10% fetal bovine serum can be used. In addition to serum and basal medium, the medium may contain an antibiotic, an extracellular matrix such as fibronectin or laminin.

Cell culture on the cell culture material can be performed at a temperature equal to or higher than LCST of the imidazolidinone block chain. Since general cells are vulnerable to high temperatures, the temperature is preferably 45 ° C. or lower, more preferably 37 ° C. to 40 ° C.

The cells cultured on the cell culture material can be separated from the culture equipment and recovered by cooling to a temperature lower than LCST of the imidazolidinone block chain. The cooling method is not particularly limited, and the medium may be replaced with a cooled medium, or may be cooled in a cold place.

According to the present invention, properties such as hydrophilicity, hydrophobicity, and temperature responsiveness can be easily controlled, and application as a biocompatible material, a material for cell culture, and a temperature responsive polymer is expected.

Hereinafter, the present invention will be described in more detail with reference to Reference Examples and Examples, but the present invention is not limited thereto.

The chemical structure and copolymerization composition ratio of the product were determined from the results of ^{1 1 H-NMR measurement using AVANCE III-400 manufactured by Bruker-Biospin.}

The molecular weight of the obtained polymer was determined from the SEC results. The SEC system is GL-7400 manufactured by GL Science (detector: GL-7456, column (4): TSKgel Super H5000, H4000 × 2, H2000, column temperature: 40 ° C., developing solvent: 0.01 M LiCl DMF solution , Standard polystyrene conversion) was used.

A 1 wt% aqueous solution (dispersion liquid) of the polymer was filled in an optical cell installed in a constant temperature bath, and the transmitted light intensity of light at 650 nm when the temperature was changed was measured using an illuminometer. The temperature at which the temperature change of the transmitted light intensity was maximized was defined as the lower limit critical solution temperature (LCST).

Reference example-1
Synthesis of 1-acryloyl imidazolidine-2-one

In an argon atmosphere, 2.2 mL (27 mmol) of acryloyl chloride was added to a solution of 2.23 g (25.9 mmol) of imidazolidine-2-one and 7.0 mL (50 mmol) of triethylamine in chloroform (60 mL) cooled in an ice bath. 20 mL) The solution was added dropwise, and the mixture was stirred for 4 hours while returning the temperature to room temperature. The reaction solution was concentrated under reduced pressure, THF was added to the residue, and the precipitate was removed. The filtrate is concentrated and purified by silica gel column chromatography (developing solution: hexane / ethyl acetate = 1/2) to obtain 1.18 g of 1-acryloylmethylimidazolidine-2-one as a colorless solid (yield: 32). 6.6%) was obtained.
^{1 1} H-NMR (400 MHz, CDCl ₃ , ppm), δ: 3.51 to 3.55 (2H, m), 4.01 to 4.05 (2H, m), 5.11 (1H, brs), 5.80 (1H, dd, J = 2.0, 10.4Hz), 6.49 (1H, dd, J = 2.0, 17.1Hz), 7.59 (1H, dd, J = 10. 4,17.1Hz).
Reference example-2
Synthesis of 1-acryloyl-3-methylimidazolidine-2-one

13.0 mL (149 mmol) of N-methylethylenediamine, 9.01 g (150 mmol) of urea and 10 mL of ethylene glycol were added, and the mixture was stirred at 130 ° C. for 8 hours. Ethylene glycol was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (developing solution: chloroform / methanol = 10/1) to obtain 10.6 g of 1-methylimidazolidine-2-one as a colorless solid (developing solution: chloroform / methanol = 10/1). Yield: 71.1%) was obtained.
^{1 1} H-NMR (400 MHz, CDCl ₃ , ppm), δ: 2.79 (3H, s), 3.41 (2H, t, J = 2.6Hz), 3.42 (2H, J = 2.6Hz) ), 5.10 (1H, brs).
Acryloyl chloride 1.5 mL (18 mmol) in a solution of 1.84 g (18.4 mmol) of 1-methylimidazolidine-2-one and 5.1 mL (37 mmol) of triethylamine cooled in an ice bath under an argon atmosphere in chloroform (62 mL). Chloroform (12 mL) solution was added dropwise, and the mixture was stirred for 12 hours while returning the temperature to room temperature. The reaction solution is concentrated and purified by silica gel column chromatography (developing solution: hexane / ethyl acetate = 2/1) to obtain 1.54 g of 1-acryloyl-3-methylimidazolidine-2-one (yield) as a colorless solid. Rate: 54.3%) was obtained.
^{1 1} H-NMR (400 MHz, CDCl ₃ , ppm), δ: 2.90 (3H, s), 3.45 (2H, t, J = 8.0 Hz), 3.91 (2H, t, J = 8) .0Hz), 5.78 (1H, dd, J = 2.0, 10.5Hz), 6.48 (1H, dd, J = 2.0, 17.1Hz) 7.64 (1H, dd, J) = 10.5, 17.1 Hz).
Reference example-3
Synthesis of 1-acryloyl-3-ethylimidazolidine-2-one

26.0 mL (248 mmol) of N-ethylethylenediamine, 14.4 g (240 mmol) of urea and 18 mL of ethylene glycol were added, and the mixture was stirred at 130 ° C. for 6 hours. Ethylene glycol was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (developing solution: chloroform / methanol = 10/1) to obtain 13.2 g (13.2 g) of 1-ethylimidazolidine-2-one as a colorless solid. Yield: 48%) was obtained.
^{1 1} H-NMR (400 MHz, CDCl ₃ , ppm), δ: 1.12 (3H, t, J = 3.3 Hz), 3.24 (2H, q, J = 3.3 Hz), 3.42 (4H) , S), 5.38 (1H, brs).
Under an argon atmosphere, 7.2 mL (88 mmol) of chloroform (60 mL) of acryloyl chloride in a solution of 9.14 g (80.1 mmol) of 1-ethylimidazolidine-2-one and 16.7 mL (120 mmol) of triethylamine in chloroform (180 mL). The solution was added dropwise and stirred for 4 hours. The reaction solution was concentrated under reduced pressure, THF was added to the residue, and the precipitate was removed. The filtrate is concentrated and purified by silica gel column chromatography (developing solution: hexane / ethyl acetate = 1/1) to obtain 10.8 g of 1-acryloyl-3-ethylimidazolidine-2-one (10.8 g) as a pale yellow liquid. Yield: 79.9%) was obtained.
^{1 1} H-NMR (400 MHz, CDCl ₃ , ppm), δ: 1.18 (3H, t), 3.36 (2H, q, J = 6.6 Hz), 3.45 (2H, t, J = 8 Hz) ), 3.91 (2H, t, J = 8Hz), 5.78 (1H, dd, J = 2.0, 10.5Hz), 6.47 (1H, dd, J = 2.0, 17. 1Hz), 7.64 (1H, dd, J = 10.5, 17.1Hz).
Reference example-4
Synthesis of 1-acryloyl-3-isopropylimidazolidine-2-one

10 mL (81 mmol) of N-isopropylethylenediamine, 4.9 g (81 mmol) of urea and 5.4 mL of ethylene glycol were added, and the mixture was stirred at 130 ° C. for 1 hour and further at 180 ° C. for 4 hours. Ethylene glycol was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (developing solution: chloroform / methanol = 10/1) to obtain 7.7 g of 1-isopropylimidazolidine-2-one as a colorless solid (developing solution: chloroform / methanol = 10/1). Yield: 73%) was obtained.
^{1 1} H-NMR (400 MHz, CDCl ₃ , ppm), δ: 1.13 (6H, d, J = 6.8 Hz), 3.39 (4H, m), 4.14 (1H, sep, J = 6) 0.8Hz), 5.27 (1H, brs).
Acryloyl chloride 7.7 mL (94 mmol) in a solution of 10.5 g (82.1 mmol) of 1-isopropylimidazolidine-2-one and triethylamine 17.0 mL (122 mmol) in chloroform (170 mL) cooled in an ice bath under an argon atmosphere. Chloroform (60 mL) solution was added dropwise, and the mixture was stirred for 4 hours. The reaction solution was concentrated under reduced pressure, THF was added to the residue, and the precipitate was removed. The filtrate is concentrated and purified by silica gel column chromatography (developing solution: hexane / ethyl acetate = 1/1) to obtain 12.0 g of 1-acryloyl-3-isopropylimidazolidine-2-one (yield) as a colorless solid. Rate: 80.0%) was obtained.
^{1 1} H-NMR (400 MHz, CDCl ₃ , ppm), δ: 1.19 (6H, d, J = 6.8 Hz), 3.38 to 3.42 (2 H, m), 3.88 to 3.92 (2H, m), 4.24 (1H, sep, J = 6.8Hz), 5.77 (1H, dd, J = 2.0, 10.5Hz), 6.47 (1H, dd, J = 2.0, 17.1 Hz), 7.64 (1H, dd, J = 10.5, 17.1 Hz).
Reference example-5
Synthesis of 1-acryloryl-3- (2-methoxyethyl) imidazolidine-2-one

10 mL (0.11 mol) of 2-chloroethyl methyl ether was added dropwise to 37 mL (0.55 mol) of ethylenediamine at room temperature, and the mixture was stirred under reflux for 13 hours. Saturated saline and diethyl ether were added to the reaction solution to extract the organic layer. After concentration, the product was distilled and purified under reduced pressure to obtain 8.79 g (yield: 67.6%) of N- (2-methoxyethyl) ethylenediamine as a colorless liquid.
^{1 1} H-NMR (400 MHz, CDCl ₃ , ppm), δ: 1.36 (3H, bs), 2.69 (2H, t, J = 6.0 Hz), 2.78 to 2.81 (4H, m) ), 3.37 (3H, s), 3.50 (2H, t, J = 6.0Hz).
8.79 g (74.4 mmol) of N- (2-methoxyethyl) ethylenediamine, 4.45 g (74.4 mmol) of urea and 5.0 mL of ethylene glycol were added, and the mixture was stirred at 130 ° C. for 1 hour and at 180 ° C. for 4 hours. Ethylene glycol was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (developing solution: chloroform / methanol = 1/1) to form a colorless solid 1- (2-methoxyethyl) imidazolidine-2-. On 10.4 g (yield: 96.5%) was obtained.
^{1 1} H-NMR (400 MHz, CDCl ₃ , ppm), δ: 3.36-3.43 (7 H, m), 3.51 to 3.57 (4 H, m), 4.90 (1 H, bs).
Acryloyl chloride 2 in a solution of 1.95 g (34.3 mmol) of 1- (2-methoxyethyl) imidazolidine-2-one and 9.5 mL (69 mmol) of triethylamine in chloroform (115 mL) cooled in an ice bath under an argon atmosphere. A solution of .8 mL (34 mmol) of chloroform (23 mL) was added dropwise, and the mixture was stirred for 14 hours while returning to room temperature. The reaction solution was put into a saturated aqueous sodium hydrogen carbonate solution, extracted with chloroform and concentrated. The residue is purified by alumina column chromatography (developing solution: hexane / ethyl acetate = 3/1) and then recrystallized from a hexane solution to purify the colorless solid 1-acryloyl-3- (2-methoxyethyl) imidazolidine. 4.27 g of -2-one (yield: 62.8%) was obtained.
^{1 1} H-NMR (400 MHz, CDCl ₃ , ppm), δ: 3.36 (3H, s), 3.46 to 3.49 (2H, m), 3.55 to 3.56 (4H, m), 3.90 (2H, t, J = 8.2Hz), 5.79 (1H, dd, J = 2.0, 10.4Hz), 6.48 (1H, dd, J = 2.0, 17. 1Hz), 7.64 (1H, dd, J = 10.4, 17.1Hz).
Reference example-6
Synthesis of 1-acryloyl-4,4-dimethylimidazolidine-2-one

Under argon, 8.82 g (100 mmol) of 1,2-diamino-2-methylpropane and 6.06 g (100 mmol) of urea were dissolved in 6 mL of ethylene glycol, stirred at 130 ° C. for 1 hour, and then at 150 ° C. for 1 hour. The mixture was heated and stirred at 180 ° C. for 3 hours. After distilling off ethylene glycol under reduced pressure, the residue was purified by silica gel column chromatography (chloroform / methanol = 10/1) to obtain 9.53 g of 4,4-dimethylimidazolidine-2-one as a colorless solid. (83.5 mmol) (yield: 83.4%) was obtained. ^{1 1} H-NMR (400 MHz, CDCl ₃ , ppm): δ 1.32 (6H, s), 3.25 (2H, s), 4.90 (1H, brs), 4.96 (1H, brs).
Under argon, 5.74 g (50.1 mmol) of 4,4-dimethylimidazolidine-2-one and 14.0 mL (100 mmol) of triethylamine are added to 50 mL of a chloroform solution, and 6.00 mL (73.6 mmol) of acryloyl chloride is added to 50 mL of a chloroform solution. Was slowly added dropwise under an ice bath and stirred for 4 hours. After the reaction, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate / hexane = 1/1) to purify the colorless solid 1-acryloyl-4,4-dimethylimidazolidine-2. -On 1.57 g (9.36 mmol) (yield: 46.7%) was obtained.
^{1 1} H-NMR (400 MHz, CDCl ₃ , ppm): δ 1.37 (6H, s), 3.73 (2H, s), 5.11 (1H, brs), 5.80 (1H, dd, J) = 2.0, 10.4Hz), 6.49 (1H, dd, J = 2.0, 17.1Hz), 7.59 (1H, dd, J = 10.4, 17.1Hz).
Reference example-7
Synthesis of polybutyl methacrylate by RAFT polymerization-1 (PBMA (1))
Butyl methacrylate 5.68 g (40.0 mmol), 4-cyano-4-[(thiobenzoyl) sulfanyl] pentanoic acid 55.6 mg (199 μmol) and 2,2'-azobis (isobutyronitrile) (AIBN) 13 .1 mg (79.7 μmol) was dissolved in 2 mL of DMF. After freezing and degassing this DMF solution, the tube was sealed and stirred at 70 ° C. for 18 hours. The reaction solution was diluted with THF and purified by precipitation in an excess amount of methanol to obtain 5.62 g (yield: 98.8%) of butyl polymethacrylate (PBMA (1)) as a pink solid. As a result of SEC measurement, Mn was 18,700, Mw was 22,900, and PD was 1.23.

Reference example-8
Synthesis of polybutyl methacrylate by RAFT polymerization-2 (PBMA (2))
5.68 g (40.0 mmol) of butyl methacrylate, 111.6 mg (399 μmol) of 4-cyano-4-[(thiobenzoyl) sulfanyl] pentanoic acid and 13.1 mg (79.8 μ mmol) of AIBN were dissolved in 2 mL of DMF. After freezing and degassing this DMF solution, the tube was sealed and stirred at 70 ° C. for 18 hours. The reaction solution was diluted with THF and purified by precipitation in an excess amount of methanol to obtain 5.69 g (yield: 99.9%) of butyl polymethacrylate (PBMA (2)) as a pink solid. As a result of SEC measurement, Mn was 8,300, Mw was 10,700, and PD was 1.28.

Reference example-9
Synthesis of polybutyl methacrylate by RAFT polymerization-3 (PBMA (3))
5.71 g (40.2 mmol) of butyl methacrylate, 55.6 mg (199 μmol) of 4-cyano-4-[(thiobenzoyl) sulfanyl] pentanoic acid and 13.1 mg (84.7 μmol) of AIBN were dissolved in 2 mL of DMF. After freezing and degassing this DMF solution, the tube was sealed and stirred at 70 ° C. for 18 hours. The reaction solution was diluted with THF and purified by precipitation in an excess amount of methanol to obtain 5.12 g (yield: 89.5%) of butyl polymethacrylate (PBMA (3)) as a pink solid. As a result of SEC measurement, Mn was 18,500, Mw was 22,400, and PD was 1.22.

Reference example-10
Synthesis of polybenzyl polymethacrylate by RAFT polymerization-1 (PBnMA (1))
Benzyl methacrylate 6.80 mL (40.1 mmol), 4-cyano-4-[(thiobenzoyl) sulfanyl] pentanoic acid 55.6 mg (199 μmol) and AIBN 13.2 mg (80.6 μmol) were dissolved in 2 mL of DMF. After freezing and degassing this DMF solution, the tube was sealed and stirred at 70 ° C. for 18 hours. The reaction solution was diluted with THF and purified by precipitation in an excess amount of methanol to obtain 7.01 g (yield: 99.2%) of benzyl polymethacrylate (PBnMA (1)) as a pink solid. As a result of SEC measurement, Mn was 24,800, Mw was 30,600, and PD was 1.23.

Reference example-11
Synthesis of polybenzyl polymethacrylate by RAFT polymerization-2 (PBnMA (2))
Benzyl methacrylate 3.40 mL (20.0 mmol), 4-cyano-4-[(thiobenzoyl) sulfanyl] pentanoic acid 20.5 mg (73.5 μmol) and AIBN 4.95 mg (30.1 μmol) were dissolved in 1 mL of DMF. After freezing and degassing this DMF solution, the tube was sealed and stirred at 70 ° C. for 18 hours. The reaction solution was diluted with THF and purified by precipitation in an excess amount of methanol to obtain 3.44 g (yield: 97.5%) of benzyl polymethacrylate (PBnMA (2)) as a pink solid. As a result of SEC measurement, Mn was 31,000, Mw was 37,500, and PD was 1.20.

Reference example-12
Synthesis of Polybenzyl Polymethacrylate by RAFT Polymerization-3 (PBnMA (3))
Benzyl methacrylate 3.40 mL (20.0 mmol), 4-cyano-4-[(thiobenzoyl) sulfanyl] pentanoic acid 42.0 mg (150 μmol) and AIBN 9.53 mg (58.0 μmol) were dissolved in 1 mL of DMF. After freezing and degassing this DMF solution, the tube was sealed and stirred at 70 ° C. for 18 hours. The reaction solution was diluted with THF and purified by precipitation in an excess amount of methanol to obtain 3.70 g (yield: 99.1%) of benzyl polymethacrylate (PBnMA (3)) as a pink solid. As a result of SEC measurement, Mn was 15,600, Mw was 19,200, and PD was 1.23.

Reference example-13
Synthesis of Polybenzyl Polymethacrylate by RAFT Polymerization-4 (PBnMA (4))
Benzyl methacrylate 3.40 mL (20.0 mmol), 4-cyano-4-[(thiobenzoyl) sulfanyl] pentanoic acid 83.5 mg (300 μmol) and AIBN 19.3 mg (118 μmol) were dissolved in 1 mL of DMF. After freezing and degassing this DMF solution, the tube was sealed and stirred at 70 ° C. for 18 hours. The reaction solution was diluted with THF and purified by precipitation in an excess amount of methanol to obtain 3.47 g (yield: 98.4%) of benzyl polymethacrylate (PBnMA (4)) as a pink solid. As a result of SEC measurement, Mn was 6,150, Mw was 8,360, and PD was 1.36.

Reference example-14
Poly by RAFT polymerization (Synthesis of butyl-ran-benzyl methacrylate-1 (P (BMA-ran-BnMA) (1)))

Butyl methacrylate 3.20 mL (20.1 mmol), benzyl methacrylate 3.4
0 mL (20.0 mol), 4-cyano-4-[(thiobenzoyl) sulfanyl] pentanoic acid 56.7 mg (203 μmol) and AIBN 13.4 mg (82.8 μmol) were dissolved in 2 mL of DMF. After freezing and degassing this DMF solution, the tube was sealed and stirred at 70 ° C. for 18 hours. 6. Dilute the reaction solution with THF and precipitate and purify it in an excess amount of methanol to obtain a pink solid poly (butyl methacrylate-ran-benzyl methacrylate) (P (BMA-ran-BnMA) (1)). 34 g (yield: 99.4%) was obtained. As a result of SEC measurement, Mn was 22,200, Mw was 27,000, and PD was 1.22. ^{1 From the} integrated value in the 1 H-NMR spectrum, it was p / q = 51/49 (%).

Reference example-15
Synthesis of benzyl polyacrylate by RAFT polymerization-1 (PBnA (1))
Benzyl acrylate 3.10 mL (20.2 mmol), 4-cyano-4-[(thiobenzoyl) sulfanyl] pentanoic acid 27.8 mg (99.5 μmol) and AIBN 6.71 mg (40.9 μmol) were dissolved in 1 mL of DMF. After freezing and degassing this DMF solution, the tube was sealed and stirred at 70 ° C. for 18 hours. The reaction solution was diluted with THF and purified by precipitation in an excess amount of methanol to obtain 2.17 g (yield: 66.3%) of benzyl polymethacrylate (PBnA (1)) as a pink solid. As a result of SEC measurement, Mn was 18,900, Mw was 25,400, and PD was 1.34.

Example-1
Synthesis of polybutylmethacrylate-block-poly [(1-acryloyl-3-isopropylimidazolidine-2-one) -ran- (1-acryloyl-3- (2-methoxyethyl) imidazolidine-2-one)] -1 (block copolymer (1))

1-acryloyl-3-isopropylimidazolidine-2-one 1.02 g (5.60 mmol) synthesized by the method of Reference Example-4, 1-acryloyl-3- (2-methoxy) synthesized by the method of Reference Example-5. Ethyl) imidazolidine-2-one 477 mg (2.41 mmol), PBMA (1) 1.14 g (7.99 mmol in terms of monomer unit) and 2,2'-azobis (4-methoxy-) synthesized in Reference Example-7. 5.02 mg (16.3 μmol) of 2,4-dimethylvaleronitrile (V-70) was dissolved in 6 mL of DMF. After freezing and degassing this DMF solution, the tube was sealed and stirred at 40 ° C. for 18 hours. The reaction solution was diluted with DMF and precipitated and purified in an excess amount of methanol / water (= 1/1) mixed solvent. The precipitate was collected, dried, and washed with diethyl ether to obtain 2.28 g (yield: 86.6%) of the light pink block copolymer (1). As a result of SEC measurement, Mn was 45,200, Mw was 80,100, and PD was 1.77. ^{1 From the} integrated values in the 1 H-NMR spectrum, x / y = 68/32 (%) and m / n = 52/48 (%).

Example-2
Synthesis of polybutyl methacrylate-block-poly [(1-acryloyl-3-isopropylimidazolidine-2-one) -ran- (1-acryloyl-3- (2-methoxyethyl) imidazolidine-2-one)] -2 (block copolymer (2))

1.53 g (8.25 mmol) of 1-acryloyl-3-isopropylimidazolidine-2-one synthesized by the method of Reference Example-4, 1-acryloyl-3- (2-methoxy) synthesized by the method of Reference Example-5. Ethyl) imidazolidine-2-one 714 mg (3.60 mmol), PBMA (2) 569 mg (4.19 mmol in terms of monomer unit) and V-70 10.5 mg (33.9 μmol) synthesized in Reference Example-8 in 6 mL of DMF. Dissolved in. After freezing and degassing this DMF solution, the tube was sealed and stirred at 40 ° C. for 18 hours. The reaction solution was diluted with DMF and precipitated and purified in an excess amount of methanol / water (= 1/1) mixed solvent. The precipitate was collected, dried, and washed with diethyl ether to obtain 2.51 g (yield: 89.2%) of the light pink block copolymer (2). As a result of SEC measurement, Mn was 45,200, Mw was 85,100, and PD was 1.88. ^{1 From the} integrated values in the 1 H-NMR spectrum, x / y = 68/32 (%) and m / n = 28/72 (%).

Example-3
Synthesis of polybutylmethacrylate-block-poly [(1-acryloyl-3-isopropylimidazolidine-2-one) -ran- (1-acryloyl-3- (2-methoxyethyl) imidazolidine-2-one)] -3 (block copolymer (3))

1-acryloyl-3-isopropylimidazolidine-2-one 1.02 g (5.60 mmol) synthesized by the method of Reference Example-4, 1-acryloyl-3- (2-methoxy) synthesized by the method of Reference Example-5. Ethyl) imidazolidine-2-one 476 mg (2.40 mmol), PBMA (2) 1.14 g (8.00 mmol in terms of monomer unit) and V-70 21.5 mg (69.6 μmol) synthesized in Reference Example-8. Was dissolved in 6 mL of DMF. After freezing and degassing this DMF solution, the tube was sealed and stirred at 40 ° C. for 18 hours. The reaction solution was diluted with DMF and precipitated and purified in an excess amount of methanol / water (= 1/1) mixed solvent. The precipitate was collected, dried, and washed with diethyl ether to obtain 2.05 g (yield: 77.7%) of the light pink block copolymer (3). As a result of SEC measurement, Mn was 21,500, Mw was 44,500, and PD was 1.53. ^{1 From the} integrated values in the 1 H-NMR spectrum, x / y = 68/32 (%) and m / n = 50/50 (%).

Example-4
Synthesis of polybutyl methacrylate-block-poly (1-acryloyl-4,4-dimethylimidazolidine-2-one) -1 (block copolymer (4))

2.02 g (12.0 mmol) of 1-acryloyl-4,4-dimethylimidazolidine-2-one synthesized by the method of Reference Example-6, and 568 mg of PBMA (1) synthesized by the method of Reference Example-7 (in terms of monomer unit). 4.00 mmol) and 4.74 mg (15.4 μmol) of V-70 were dissolved in 6 mL of DMF. After freezing and degassing this DMF solution, the tube was sealed and stirred at 40 ° C. for 18 hours. The reaction solution was diluted with DMF and precipitated and purified in an excess amount of methanol / water (= 1/1) mixed solvent. The precipitate was collected, dried, and washed with diethyl ether to obtain 2.29 g (yield: 88.4%) of the light pink block copolymer (4). As a result of SEC measurement, Mn was 69,000, Mw was 165,500, and PD was 2.40. ^{1 From the} integrated value in the 1 H-NMR spectrum, m / n = 30/70 (%).

Example-5
Synthesis of polybutyl methacrylate-block-poly (1-acryloyl-4,4-dimethylimidazolidine-2-one) -2 (block copolymer (5))

2.02 g (12.0 mmol) of 1-acryloyl-4,4-dimethylimidazolidine-2-one synthesized by the method of Reference Example-6, and 569 mg of PBMA (2) synthesized by the method of Reference Example-8 (monomer unit equivalent). 4.00 mmol) and 10.7 mg (34.7 μmol) of V-70 were dissolved in 6 mL of DMF. After freezing and degassing this DMF solution, the tube was sealed and stirred at 40 ° C. for 18 hours. The reaction solution was diluted with DMF and precipitated and purified in an excess amount of methanol / water (= 1/1) mixed solvent. The precipitate was collected, dried, and washed with diethyl ether to obtain 2.21 g (yield: 85.4%) of the light pink block copolymer (5). As a result of SEC measurement, Mn was 46,100, Mw was 77,500, and PD was 1.68. ^{1 From the} integrated value in the 1 H-NMR spectrum, m / n = 30/70 (%).

Example-6
Synthesis of polybutylmethacrylate-block-poly [(1-acryloyl-3-isopropylimidazolidine-2-one) -ran- (1-acryloyl-3- (2-methoxyethyl) imidazolidine-2-one)] -5 (block copolymer (6))

1.97 g (10.8 mmol) of 1-acryloyl-3-isopropylimidazolidine-2-one synthesized by the method of Reference Example-4, 1-acryloyl-3- (2-methoxy) synthesized by the method of Reference Example-5. 12 mL of DMF contains 238 mg (1.20 mmol) of imidazolidine-2-one (ethyl), 570 mg (4.01 mmol) of PBMA (3) synthesized in Reference Example-9, and 4.60 mg (14.9 μmol) of V-70. Dissolved in. After freezing and degassing this DMF solution, the tube was sealed and stirred at 40 ° C. for 18 hours. The reaction solution was diluted with DMF and precipitated and purified in an excess amount of methanol / water (= 1/1) mixed solvent. The precipitate was collected, dried, and washed with diethyl ether to obtain 1.88 g (yield: 67.7%) of the light pink block copolymer (6). As a result of SEC measurement, Mn was 78,300, Mw was 140,700, and PD was 1.80. ^{1 From the} integrated values in the 1 H-NMR spectrum, x / y = 89/11 (%) and m / n = 35/65 (%).

Example-7
Synthesis of polybutyl butyl methacrylate-block-poly (1-acryloyl-3-isopropylimidazolidine-2-one) -1 (block copolymer (7))

2.19 g (12.0 mmol) of 1-acryloyl-3-isopropylimidazolidine-2-one synthesized by the method of Reference Example-4, and 570 mg of PBMA (3) synthesized by Reference Example-9 (monomer unit equivalent 4. 01 mmol) and 4.91 mg (15.9 μmol) of V-70 were dissolved in 12 mL of DMF. After freezing and degassing this DMF solution, the tube was sealed and stirred at 40 ° C. for 18 hours. The reaction solution was diluted with DMF and precipitated and purified in an excess amount of methanol / water (= 1/1) mixed solvent. The precipitate was collected, dried, and washed with diethyl ether to obtain 2.07 g (yield: 75.2%) of the light pink block copolymer (7). As a result of SEC measurement, Mn was 74,300, Mw was 132,200, and PD was 1.78. ^{1 From the} integrated value in the 1 H-NMR spectrum, m / n = 34/66 (%).

Example-8
Synthesis of polybenzyl methacrylate-block-poly [(1-acryloyl-3-isopropylimidazolidine-2-one) -ran- (1-acryloyl-3- (2-methoxyethyl) imidazolidine-2-one)] -1 (block copolymer (8))

1.53 g (8.40 mmol) of 1-acryloyl-3-isopropylimidazolidine-2-one synthesized by the method of Reference Example-4, 1-acryloyl-3- (2-methoxy) synthesized by the method of Reference Example-5. Ethyl) imidazolidine-2-one 715 mg (3.61 mmol), PBnMA (1) 704 mg (4.00 mmol in terms of monomer unit) and V-70 4.62 mg (15.0 μmol) synthesized in Reference Example-10 in 16 mL of DMF. Dissolved in. After freezing and degassing this DMF solution, the tube was sealed and stirred at 40 ° C. for 18 hours. The reaction solution was diluted with DMF and precipitated and purified in an excess amount of methanol / water (= 1/1) mixed solvent. The precipitate was collected, dried, and washed with diethyl ether to obtain 2.11 g (yield: 71.5%) of the light pink block copolymer (8). As a result of SEC measurement, Mn was 72,200, Mw was 187,600, and PD was 2.60. ^{1 From the} integrated values in the 1 H-NMR spectrum, x / y = 69/31 (%) and m / n = 33/67 (%).

Example-9
Synthesis of polybenzyl methacrylate-block-poly [(1-acryloyl-3-isopropylimidazolidine-2-one) -ran- (1-acryloyl-3- (2-methoxyethyl) imidazolidine-2-one)] -2 (Block copolymer (9))

1.53 g (8.40 mmol) of 1-acryloyl-3-isopropylimidazolidine-2-one synthesized by the method of Reference Example-4, 1-acryloyl-3- (2-methoxy) synthesized by the method of Reference Example-5. Ethyl) imidazolidine-2-one 715 mg (3.61 mmol), PBnMA (1) 704 mg (4.00 mmol in terms of monomer unit) and V-70 4.69 mg (15.2 μmol) synthesized in Reference Example-10 in 16 mL of DMF. Dissolved in. After freezing and degassing this solution, the tube was sealed and stirred at 40 ° C. for 18 hours. The reaction solution was diluted with DMF and precipitated and purified in an excess amount of methanol / water (= 1/3) mixed solvent. The precipitate was collected, dried, and washed with diethyl ether to obtain 1.99 g (yield: 67.4%) of the light pink block copolymer (9). As a result of SEC measurement, Mn was 79,300, Mw was 153,300, and PD was 1.93. ^{1 From the} integrated values in the 1 H-NMR spectrum, x / y = 68/32 (%) and m / n = 33/67 (%).

Example-10
Synthesis of polybenzyl methacrylate-block-poly [(1-acryloyl-3-isopropylimidazolidine-2-one) -ran- (1-acryloyl-3- (2-methoxyethyl) imidazolidine-2-one)] -3 (block copolymer (10))

1-33 g (7.28 mmol) of 1-acryloyl-3-isopropylimidazolidine-2-one synthesized by the method of Reference Example-4, 1-acryloyl-3- (2-methoxy) synthesized by the method of Reference Example-5. 2 618 mg (3.12 mmol) of imidazolidine-2-one (ethyl), 988 mg (5.61 mmol in terms of monomer unit) of PBnMA (2) synthesized in Reference Example-11, and 4.51 mg (14.6 μmol) of V-70. -Dissolved in 16 mL of methoxyethanol. After freezing and degassing this solution, the tube was sealed and stirred at 40 ° C. for 18 hours. The reaction solution was diluted with 2-methoxyethanol and precipitated and purified in an excess amount of water. The precipitate was collected, dried, and washed with diethyl ether to obtain 2.45 g (yield: 83.4%) of a light pink block copolymer (10). As a result of SEC measurement, Mn was 70,900, Mw was 127,300, and PD was 1.80. ^{1 From the} integrated values in the 1 H-NMR spectrum, x / y = 68/32 (%) and m / n = 39/61 (%).

Example-11
Synthesis of polybenzyl methacrylate-block-poly [(1-acryloyl-3-isopropylimidazolidine-2-one) -ran- (1-acryloyl-3- (2-methoxyethyl) imidazolidine-2-one)] -4 (block copolymer (11))

1.74 g (9.53 mmol) of 1-acryloyl-3-isopropylimidazolidine-2-one synthesized by the method of Reference Example-4, 1-acryloyl-3- (2-methoxy) synthesized by the method of Reference Example-5. 2 809 mg (4.08 mmol) of imidazolidine-2-one (ethyl), 424 mg (2.41 mmol in terms of monomer unit) of PBnMA (3) synthesized in Reference Example-12, and 4.22 mg (13.7 μmol) of V-70. -Dissolved in 18 mL of methoxyethanol. After freezing and degassing this solution, the tube was sealed and stirred at 40 ° C. for 18 hours. The reaction solution was diluted with 2-methoxyethanol and precipitated and purified in an excess amount of water. The precipitate was collected, dried, and washed with diethyl ether to obtain 1.91 g (yield: 64.3%) of a light pink block copolymer (11). As a result of SEC measurement, Mn was 82,400, Mw was 175,100, and PD was 2.13. ^{1 From the} integrated values in the 1 H-NMR spectrum, x / y = 67/33 (%) and m / n = 21/79 (%).

Example-12
Synthesis of polybenzyl methacrylate-block-poly [(1-acryloyl-3-isopropylimidazolidine-2-one) -ran- (1-acryloyl-3- (2-methoxyethyl) imidazolidine-2-one)] -5 (block copolymer (12))

1.89 g (10.4 mmol) of 1-acryloyl-3-isopropylimidazolidine-2-one synthesized by the method of Reference Example-4, 1-acryloyl-3- (2-methoxy) synthesized by the method of Reference Example-5. 2 880 mg (4.44 mmol) of imidazolidine-2-one (ethyl), 212 mg (1.20 mmol in terms of monomer unit) of PBnMA (4) synthesized in Reference Example-13, and 5.40 mg (17.5 μmol) of V-70. -Dissolved in 20 mL of methoxyethanol. After freezing and degassing this solution, the tube was sealed and stirred at 40 ° C. for 18 hours. The reaction solution was diluted with 2-methoxyethanol and precipitated and purified in an excess amount of warm water. The precipitate was collected, dried, and washed with diethyl ether to obtain 1.69 g (yield: 56.7%) of the light pink block copolymer (12). As a result of SEC measurement, Mn was 87,600, Mw was 152,900, and PD was 1.75. ^{1 From the} integrated values in the 1 H-NMR spectrum, x / y = 68/32 (%) and m / n = 11/89 (%).

Example-13
Synthesis of polybenzyl methacrylate-block-poly [(1-acryloyl-3-isopropylimidazolidine-2-one) -ran- (1-acryloyl-3-ethylimidazolidine-2-one)] (block copolymer (13) )))

1-acryloyl-3-isopropylimidazolidine-2-one 1.53 g (8.40 mmol) synthesized by the method of Reference Example-4, 1-acryloyl-3-ethylimidazolidine synthesized by the method of Reference Example-3- 2-On 613 mg (3.64 mmol), PBnMA (1) 705 mg (4.00 mmol in terms of monomer unit) and V-70 4.41 mg (14.3 μmol) synthesized in Reference Example-10 to 16 mL of 2-methoxyethanol. Dissolved. This 2-methoxyethanol solution was frozen and degassed, sealed, and stirred at 40 ° C. for 18 hours. 2. The light pink block copolymer (13) was obtained by pouring the reaction solution into an excess amount of methanol / water (= 1/3) mixed solvent, recovering the precipitate formed, drying the reaction solution, and washing with diethyl ether. 04 g (yield: 71.7%) was obtained. As a result of SEC measurement, Mn was 80,000, Mw was 152,500, and PD was 1.91. ^{1 From the} integrated values in the 1 H-NMR spectrum, x / y = 68/32 (%) and m / n = 33/67 (%).

Example-14
Poly (butyl methacrylate-ran-benzyl methacrylate) -block-poly [(1-acryloyl-3-isopropylimidazolidine-2-one) -ran- (1-acryloyl-3- (2-methoxyethyl) imidazolidine) -2-on)] synthesis-1 (block copolymer (14))

1.53 g (8.40 mmol) of 1-acryloyl-3-isopropylimidazolidine-2-one synthesized by the method of Reference Example-4, 1-acryloyl-3- (2-methoxy) synthesized by the method of Reference Example-5. Ethyl) imidazolidine-2-one 715 mg (3.61 mmol), P (BMA-ran-BnMA) synthesized in Reference Example-14 (1) 636 mg (4.00 mmol in terms of monomer unit) and V-70 4.51 mg. (14.6 μmol) was dissolved in 16 mL of DMF. After freezing and degassing this DMF solution, the tube was sealed and stirred at 40 ° C. for 18 hours. The reaction solution was diluted with DMF and precipitated and purified in an excess amount of methanol / water (= 1/1) mixed solvent. The precipitate was collected, dried, and washed with diethyl ether to obtain 2.13 g (yield: 73.8%) of the light pink block copolymer (14). As a result of SEC measurement, Mn was 70,500, Mw was 190,100, and PD was 2.70. ^{1 From the} integrated values in the 1 H-NMR spectrum, x / y = 68/32 (%), p / q = 51/49 (%), and m / n = 33/67 (%).

Example-15
Synthesis of benzyl-block-polypolyacrylate [(1-acryloyl-3-isopropylimidazolidine-2-one) -ran- (1-acryloyl-3- (2-methoxyethyl) imidazolidine-2-one)] (Block Copolymer (15))

1.53 g (8.40 mmol) of 1-acryloyl-3-isopropylimidazolidine-2-one synthesized by the method of Reference Example-4, 1-acryloyl-3- (2-methoxy) synthesized by the method of Reference Example-5. 2 715 mg (3.60 mmol) of imidazolidine-2-one (ethyl), 704 mg (4.00 mmol) of PBnA (1) synthesized in Reference Example-15, and 5.37 mg (17.4 μmol) of V-70. -Dissolved in 16 mL of methoxyethanol. This 2-methoxyethanol solution was frozen and degassed, sealed, and stirred at 40 ° C. for 18 hours. 2.03 g (yield: 70.0%) of light pink block copolymer (15) was obtained by pouring the reaction solution into an excess amount of water, collecting the precipitate formed, drying the reaction solution, and washing with diethyl ether. Obtained. As a result of SEC measurement, Mn was 67,400, Mw was 127,900, and PD was 1.90. ^{1 From the} integrated values in the 1 H-NMR spectrum, x / y = 68/32 (%) and m / n = 31/69 (%).

Reference example-16
Synthesis of poly (butyl-ran-benzyl methacrylate (P (BMA-ran-BnMA) (2)) by RAFT polymerization-2)

Butyl methacrylate 3.20 mL (20.1 mmol), benzyl methacrylate 3.40 mL (20.0 mmol) and 4-cyano-4-[(thiobenzoyl) sulfanyl] pentanoic acid 28.0 mg (100 μmol), AIBN 6.52 mg ( 40.2 μmol) was dissolved in 4 mL of DMF. After freezing and degassing this DMF solution, the tube was sealed and heated and stirred at 70 ° C. for 18 hours. 6. Dilute the reaction solution with THF and precipitate and purify it in an excess amount of methanol to obtain a pink solid poly (butyl methacrylate-ran-benzyl methacrylate) (P (BMA-ran-BnMA) (2)). 02 g (yield: 94.2%) was obtained. As a result of SEC measurement, Mn was 37,400, Mw was 48,400, and PD was 1.29. ^{1 From the} integrated value in the 1 H-NMR spectrum, it was p / q = 50/50 (%).

Example-16
Poly (butyl methacrylate-ran-benzyl methacrylate) -block-poly [(1-acryloyl-3-isopropylimidazolidine-2-one) -ran- (1-acryloyl-3- (2-methoxyethyl) imidazolidine) -2-on)] synthesis-2 (block copolymer (16))

1-Acryloyl-3-isopropylimidazolidine-2-one 267 g (1.47 mmol), 1-acryloyl-3- (2-methoxyethyl) imidazolidine-2-one 125 mg (0.631 mmol), in Reference Example-16 1.62 g (10.2 mmol in terms of monomer unit) of the synthesized P (BMA-ran-BnMA) (2) and 7.44 mg (24.1 μmol) of V-70 were dissolved in 12 mL of 2-methokiethanol. This 2-methokiethanol solution was frozen and degassed, sealed, and stirred at 40 ° C. for 18 hours. The reaction solution was added to an excess amount of water to carry out precipitation purification. The precipitate was collected, dried, and washed with diethyl ether to obtain 1.77 g (yield: 88.2%) of a light pink block copolymer (16). As a result of SEC measurement, Mn was 44,600, Mw was 62,500, and PD was 1.40. ^{1 From the} integrated values in the 1 H-NMR spectrum, x / y = 75/25 (%), p / q = 50/50 (%), and m / n = 84/14 (%).

Reference example-17
Synthesis of poly (butyl-ran-benzyl methacrylate (P (BMA-ran-BnMA) (3)) by RAFT polymerization-3)

Butyl methacrylate 3.20 mL (20.1 mmol), benzyl methacrylate 3.40 mL (20.0 mmol) and 4-cyano-4-[(thiobenzoyl) sulfanyl] pentanoic acid 13.9 mg (49.6 μmol), AIBN 3. 49 mg (21.5 μmol) was dissolved in 4 mL of DMF. After freezing and degassing this DMF solution, the tube was sealed and heated and stirred at 70 ° C. for 18 hours. 6. Dilute the reaction solution with THF and precipitate and purify it in an excess amount of methanol to obtain a pink solid poly (butyl methacrylate-ran-benzyl methacrylate) (P (BMA-ran-BnMA) (3)). 25 g (yield: 97.9%) was obtained. As a result of SEC measurement, Mn was 66,100, Mw was 95,200, and PD was 1.44. ^{1 From the} integrated value in the 1 H-NMR spectrum, it was p / q = 50/50 (%).

Example-17
Poly (butyl methacrylate-ran-benzyl methacrylate) -block-poly [(1-acryloyl-3-isopropylimidazolidine-2-one) -ran- (1-acryloyl-3- (2-methoxyethyl) imidazolidine) -2-on)] synthesis-3 (block copolymer (17))

1-Acryloyl-3-isopropylimidazolidine-2-one 267 g (1.47 mmol), 1-acryloyl-3- (2-methoxyethyl) imidazolidine-2-one 125 mg (0.631 mmol), in Reference Example-17 1.62 g (10.2 mmol in terms of monomer unit) of the synthesized P (BMA-ran-BnMA) (3) and 3.74 mg (12.1 μmol) of V-70 were dissolved in 12 mL of 2-methokiethanol. This 2-methokiethanol solution was frozen and degassed, sealed, and stirred at 40 ° C. for 18 hours. The reaction solution was added to an excess amount of water to carry out precipitation purification. The precipitate was collected, dried, and washed with diethyl ether to obtain 1.45 g (yield: 71.9%) of the light pink block copolymer (17). As a result of SEC measurement, Mn was 75,400, Mw was 116,400, and PD was 1.54. ^{1 From the} integrated values in the 1 H-NMR spectrum, x / y = 77/23 (%), p / q = 50/50 (%), and m / n = 87/13 (%).

Reference example-18
Synthesis of polyethyl methacrylate (PetMA) by RAFT polymerization 4.57 g (40.0 mmol) of ethyl methacrylate, 44.7 mg (160 μmol) of 4-cyano-4-[(thiobenzoyl) sulfanyl] pentanoic acid and 10.4 mg of AIBN (AIBN 10.4 mg). 63.1 μmol) was dissolved in 2 mL of DMF. After freezing and degassing this DMF solution, the tube was sealed and stirred at 70 ° C. for 18 hours. The reaction solution was diluted with THF and purified by precipitation in an excess amount of methanol to obtain 3.69 g (yield: 65.6%) of benzyl polymethacrylate (PetMA) as a pink solid. As a result of SEC measurement, Mn was 17,300, Mw was 20,600, and PD was 1.19.

Example-18
Synthesis of polyethyl methacrylate-block-poly [(1-acryloyl-3-isopropylimidazolidine-2-one) -ran- (1-acryloyl-3- (2-methoxyethyl) imidazolidine-2-one)] -1 (block copolymer (18))

1-Acryloyl-3-isopropylimidazolidine-2-one 1.53 g (8.40 mmol), 1-acryloyl-3- (2-methoxyethyl) imidazolidine-2-one 714 mg (3.60 mmol), Reference Example- 571 mg of PETMA (5.00 mmol in terms of monomer unit) and 5.72 mg (18.5 μmol) of V-70 synthesized in No. 18 were dissolved in 16 mL of 2-methokiethanol. After freezing and degassing this solution, the tube was sealed and stirred at 40 ° C. for 18 hours. The reaction solution was added to an excess amount of water to carry out precipitation purification. The precipitate was collected, dried, and washed with diethyl ether to obtain 1.85 g (yield: 65.6%) of the light pink block copolymer (18). As a result of SEC measurement, Mn was 65,700, Mw was 109,600, and PD was 1.67. ^{1 From the} integrated values in the 1 H-NMR spectrum, x / y = 67/33 (%) and m / n = 41/59 (%).

Reference example-19
Polymethacrylic acid ethyl by RAFT polymerization ^(P i BuMA) Synthesis of -1
5.70 g (40.1 mmol) of isobutyl methacrylate, 44.7 mg (160 μmol) of 4-cyano-4- (thiobenzoylthio) pentanoic acid and 10.4 mg (63.0 μmol) of AIBN were dissolved in 2 mL of DMF. After freezing and degassing this DMF solution, the tube was sealed and stirred at 70 ° C. for 18 hours. The reaction solution was diluted with THF, by precipitation purification to an excess of methanol, pink solid poly isobutyl methacrylate and ^(P i BuMA) 4.46g ^(yield: 78.3%) was obtained. As a result of SEC measurement, Mn was 20,100, Mw was 23,400, and PD was 1.16.

Example-19
Synthesis of polyisobutyl methacrylate-block-poly [(1-acryloyl-3-isopropylimidazolidine-2-one) -ran- (1-acryloyl-3- (2-methoxyethyl) imidazolidine-2-one)] -1 (block copolymer (19))

1-Acryloyl-3-isopropylimidazolidine-2-one 1.53 g (8.40 mmol), 1-acryloyl-3- (2-methoxyethyl) imidazolidine-2-one 714 mg (3.60 mmol), Reference Example- It was dissolved and V-70 4.99mg of (16.2Myumol) 2-methoxide ethanol 16 mL (4.01 mmol in monomer unit equivalent) synthesized ^P i BuMA570mg 19. After freezing and degassing this solution, the tube was sealed and stirred at 40 ° C. for 18 hours. The reaction solution was added to an excess amount of water to carry out precipitation purification. The precipitate was collected, dried, and washed with diethyl ether to obtain 1.45 g (yield: 51.3%) of a light pink block copolymer (19). As a result of SEC measurement, Mn was 77,700, Mw was 132,700, and PD was 1.71. ^{1 From the} integrated values in the 1 H-NMR spectrum, x / y = 67/33 (%) and m / n = 33/67 (%).

Reference example-20
Synthesis of polybenzyl polymethacrylate (PBnMA (5)) by RAFT polymerization-5
Benzyl methacrylate 6.90 mL (40.7 mmol), 4-cyano-4- (thiobenzoylthio) pentanoic acid 56.7 mg (203 μmol) and AIBN 13.2 mg (80.9 μmol) were dissolved in 2 mL of DMF. After freezing and degassing this DMF solution, the tube was sealed and stirred at 70 ° C. for 18 hours. The reaction solution was diluted with THF and purified by precipitation in an excess amount of methanol to obtain 7.15 g (yield: 99.8%) of benzyl polymethacrylate (PBnMA) as a pink solid. As a result of SEC measurement, Mn was 21,300, Mw was 24,900, and PD was 1.17.

Example-20
Synthesis of polybenzyl methacrylate-block-poly (1-acryloyl-3-isopropylimidazolidine-2-one) (block copolymer (20))-1

1-acryloyl-3-isopropylimidazolidine-2-one 2.19 g (12.0 mmol), PBnMA (5) 706 mg (4.01 mmol in terms of monomer unit) and V-70 4.79 mg synthesized in Reference Example-20. (15.5 μmol) was dissolved in 16 mL of 2-methokiethanol. After freezing and degassing this solution, the tube was sealed and stirred at 40 ° C. for 18 hours. The reaction solution was added to an excess amount of water to carry out precipitation purification. The precipitate was collected, dried, and washed with diethyl ether to obtain 2.73 g (yield: 95.8%) of a light pink block copolymer (20). As a result of SEC measurement, Mn was 46,800, Mw was 85,900, and PD was 1.84. ^{1 From the} integrated value in the 1 H-NMR spectrum, m / n = 25/75 (%).

Example-21
Synthesis of polybenzyl methacrylate-block-poly [(1-acryloyl-3-isopropylimidazolidine-2-one) -ran- (1-acryloyl-3- (2-methoxyethyl) imidazolidine-2-one)] -6 (block copolymer (21))

1-Acryloyl-3-isopropylimidazolidine-2-one 1.97 g (10.8 mmol), 1-acryloyl-3- (2-methoxyethyl) imidazolidine-2-one 240 mg (1.21 mmol), Reference Example- 705 mg of PBnMA (5) synthesized in No. 20 (4.00 mmol in terms of monomer unit) and 4.81 mg (15.6 μmol) of V-70 were dissolved in 16 mL of 2-methokiethanol. After freezing and degassing this solution, the tube was sealed and stirred at 40 ° C. for 18 hours. The reaction solution was added to an excess amount of water to carry out precipitation purification. The precipitate was collected, dried, and washed with diethyl ether to obtain 2.54 g (yield: 87.1%) of the light pink block copolymer (21). As a result of SEC measurement, Mn was 52,500, Mw was 92,600, and PD was 1.76. ^{1 From the} integrated values in the 1 H-NMR spectrum, x / y = 88/12 (%) and m / n = 26/74 (%).

Example-22
(2-4) Synthesis of polybenzyl methacrylate-block-poly [(1-acryloyl-3-isopropylimidazolidine-2-one) -ran- (1-acryloyl midazolidine-2-one)]-1 (block) Copolymer (22))

1-acryloyl-3-isopropylimidazolidine-2-one 1.53 g (8.41 mmol), 1-acryloyl imidazolidine-2-one 505 mg (3.60 mmol), PBnMA (5) 706 mg synthesized in Reference Example-20 (4.01 mmol in terms of monomer unit) and 4.94 mg (16.0 μmol) of V-70 were dissolved in 16 mL of 2-methokiethanol. The solution was frozen and degassed, sealed, and stirred at 40 ° C. for 18 hours. The reaction solution was added to an excess amount of water to carry out precipitation purification. The precipitate was collected, dried, and washed with diethyl ether to obtain 2.33 g (yield: 84.9%) of a light pink block copolymer (JST-18806). As a result of SEC measurement, Mn was 53,700, Mw was 93,900, and PD was 1.75. ^{1 From the} integrated values in the 1 H-NMR spectrum, x / y = 70/30 (%) and m / n = 28/72 (%).

Example-23
(2-5) Synthesis of polybenzyl methacrylate-block-poly [(1-acryloyl-3-isopropylimidazolidine-2-one) -ran- (1-acryloyl-3-methylimidazolidine-2-one)] -1 (block copolymer (23))

1-Acryloyl-3-isopropylimidazolidine-2-one 1.97 g (10.8 mmol), 1-acryloyl-3-methylimidazolidine-2-one 185 mg (1.20 mmol), PBnMA synthesized in Reference Example-20 (5) 706 mg (4.01 mmol in terms of monomer unit) and 4.95 mg (16.0 μmol) of V-70 were dissolved in 16 mL of 2-methokiethanol. After freezing and degassing this solution, the tube was sealed and stirred at 40 ° C. for 18 hours. The reaction solution was added to an excess amount of water to carry out precipitation purification. The precipitate was collected, dried, and washed with diethyl ether to obtain 2.62 g (yield: 91.6%) of the light pink block copolymer (23). As a result of SEC measurement, Mn was 48,500, Mw was 88,600, and PD was 1.83. ^{1 From the} integrated values in the 1 H-NMR spectrum, x / y = 89/11 (%) and m / n = 26/74 (%).

Example-24
Synthesis of polybenzyl methacrylate-block-poly [(1-acryloyl-3-ethylimidazolidine-2-one) -ran- (1-acryloyl-3-benzylimidazolidine-2-one)]-1 (block copolymer) (24))

1-Acryloyl-3-ethylimidazolidine-2-one 1.92 g (11.4 mmol), 1-acryloyl-3-benzylimidazolidine-2-one 138 mg (0.599 mmol), PBnMA synthesized in Reference Example-20 (5) 705 mg (4.00 mmol in terms of monomer unit) and 4.92 mg (16.0 μmol) of V-70 were dissolved in 16 mL of 2-methokiethanol. After freezing and degassing this solution, the tube was sealed and stirred at 40 ° C. for 18 hours. The reaction solution was added to an excess amount of water to carry out precipitation purification. The precipitate was collected, dried, and washed with diethyl ether to obtain 2.15 g (yield: 77.8%) of the light pink block copolymer (24). As a result of SEC measurement, Mn was 56,000, Mw was 100,800, and PD was 1.80. ^{1 From the} integrated values in the 1 H-NMR spectrum, x / y = 94/6 (%) and m / n = 28/72 (%).

Reference example-21
Synthesis of polybutyl methacrylate (PBMA (4)) by RAFT polymerization-4
Butyl methacrylate 6.40 mL (40.2 mmol), 4-cyano-4- (thiobenzoylthio) pentanoic acid 13.9 mg (50.0 μmol) and AIBN 3.67 mg (22.3 μmol) were dissolved in 2 mL of DMF. After freezing and degassing this DMF solution, the tube was sealed and stirred at 70 ° C. for 18 hours. The reaction solution was diluted with THF and purified by precipitation with an excess amount of methanol to obtain 5.61 g (yield: 98.1%) of butyl polymethacrylate (PBMA) as a pink solid. As a result of SEC measurement, Mn was 64,100, Mw was 86,900, and PD was 1.35.

Example-25
Synthesis of polybutylmethacrylate-block-poly [(1-acryloyl-3-isopropylimidazolidine-2-one) -ran- (1-acryloyl-3- (2-methoxyethyl) imidazolidine-2-one)] -6 (block copolymer (25))

1-Acryloyl-3-isopropylimidazolidine-2-one 762 mg (4.18 mmol), 1-acryloyl-3- (2-methoxyethyl) imidazolidine-2-one 92.0 mg (0.464 mmol), Reference Example- 1.71 mg (12.0 mmol in terms of monomer unit) of PBMA (4) synthesized in No. 21 and 5.52 mg (17.9 μmol) of V-70 were dissolved in 14 mL of DMF. After freezing and degassing this solution, the tube was sealed and stirred at 40 ° C. for 15 hours. The reaction solution was added to an excess amount of a mixed solvent of methanol / water (= 1/3) to carry out precipitation purification. The precipitate was collected, dried, and washed with diethyl ether to obtain 1.78 g (yield: 69.6%) of a light pink block copolymer (25). As a result of SEC measurement, Mn was 76,500, Mw was 131,600, and PD was 1.72. ^{1 From the} integrated values in the 1 H-NMR spectrum, x / y = 89/11 (%) and m / n = 84/16 (%).

Example-26
Synthesis of polybutylmethacrylate-block-poly [(1-acryloyl-3-isopropylimidazolidine-2-one) -ran- (1-acryloyl-3- (2-methoxyethyl) imidazolidine-2-one)] -7 (block copolymer (26))

1-Acryloyl-3-isopropylimidazolidine-2-one 1.97 g (10.8 mmol), 1-acryloyl-3- (2-methoxyethyl) imidazolidine-2-one 238 mg (1.20 mmol), Reference Example- 569 mg of PBMA (4) synthesized in No. 21 (4.00 mmol in terms of monomer unit) and 1.89 mg (6.13 μmol) of V-70 were dissolved in 20 mL of DMF. After freezing and degassing this solution, the tube was sealed and stirred at 40 ° C. for 15 hours. The reaction solution was added to an excess amount of a mixed solvent of methanol / water (= 1/3) to carry out precipitation purification. The precipitate was collected, dried, and washed with diethyl ether to obtain 1.12 g (yield: 40.4%) of the light pink block copolymer (26). As a result of SEC measurement, Mn was 151,200, Mw was 352,600, and PD was 2.33. ^{1 From the} integrated values in the 1 H-NMR spectrum, x / y = 88/12 (%) and m / n = 47/53 (%).

Example-27
Synthesis of polybutylmethacrylate-block-poly [(1-acryloyl-3-isopropylimidazolidine-2-one) -ran- (1-acryloyl-3- (2-methoxyethyl) imidazolidine-2-one)] -8 (block copolymer (27))

1-Acryloyl-3-isopropylimidazolidine-2-one 2.95 g (16.2 mmol), 1-acryloyl-3- (2-methoxyethyl) imidazolidine-2-one 357 mg (1.80 mmol), Reference Example- 567 mg of PBMA (4) synthesized in No. 21 (3.99 mmol in terms of monomer unit) and 1.99 mg of V-70 (6.45 μmol) were dissolved in 25 mL of DMF. After freezing and degassing this solution, the tube was sealed and stirred at 40 ° C. for 15 hours. The reaction solution was added to an excess amount of a mixed solvent of methanol / water (= 1/7) to carry out precipitation purification. The precipitate was collected, dried, and washed with diethyl ether to obtain 1.37 g (yield: 35.3%) of the light pink block copolymer (27). As a result of SEC measurement, Mn was 253,900, Mw was 735,300, and PD was 2.90. ^{1 From the} integrated values in the 1 H-NMR spectrum, x / y = 88/12 (%) and m / n = 39/61 (%).
(Evaluation of culture equipment coated with block copolymer)
Incubators using block copolymers (1) to (11), (13) to (15), and (25) to (27) were prepared and evaluated by the following procedure. Since the block copolymer (12) has a structure similar to that of the block copolymer (11), the evaluation is omitted. A dish not coated with block copolymer was also prepared as a culture device for comparison.
I Preparation of culture equipment [Preparation of surface treatment agent]
Isopropanol or 2-methoxyethanol was added to the block copolymer, and the mixture was completely dissolved by stirring, and then filtered through a filter made of regenerated cellulose (manufactured by Sartorius) having a pore size of 0.2 μm to prepare a surface treatment agent. The concentration of the block polymer was changed depending on the block polymer in order to make the coating film thickness approximately constant.
[Coat to dish 1]
1.0 mL of a surface treatment agent was added to a tissue culture dish (made of AGC technoglass, diameter 6 cm), and the dish was allowed to stand for 1 minute. Excess surface treatment agent was recovered with a Pasteur pipette, and then the surface of the dish was air-dried and heat-annealed at 60 ° C. for 60 minutes to prepare a culture equipment coated with a block copolymer.
[Coat to dish 2]
Add 100 μL of a surface treatment agent to the center of a tissue culture dish (made of AGC technoglass, diameter 6 cm), and spin coat the block copolymer using a spin coater under the conditions of a rotation speed of 2,000 rpm and a rotation time of 60 seconds. Coated culture equipment was prepared.

In any case of coating by any method, the film thickness was measured at 5 points from the center to the end of the culture equipment using a microspectroscopy (OPTM-A1 manufactured by Otsuka Electronics Co., Ltd.).
II Cell culture and exfoliation treatment [Culture step]
Human bone marrow-derived mesenchymal stem cells (Product Code: PT-2501, manufactured by Lonza Japan Co., Ltd.) were seeded in the ^{culture equipment at a cell density of 0.75 × 10 5 to} 1.0 × 10 ^{5 cells / dish.} The cells were cultured at 37 ° C. and a CO ₂ concentration of 5%. As the medium, Dulbecco Vogt's modified Eagle's minimum essential medium (DMEM: manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) containing 10 vol% of fetal bovine serum (manufactured by BioWest, Colombia) was used, and the culture period was 5 to 8 days.
[Cooling process]
After completion of the culturing step, the culture supernatant was gently removed, a new medium cooled to 4 ° C. was added, and the mixture was cooled at room temperature for 10 minutes or more.
[Cell exfoliation process]
After the completion of the cooling treatment step, the culture medium was pipeted with a volume of 1 mL using a 1 mL pipettor so as to apply the culture solution to the entire surface of the culture surface of the culture substrate 10 times, and the culture solution was collected together with the cells. The collected culture broth was centrifuged at 160 rcf, 25 ° C. for 5 minutes, the supernatant was removed, and 500 μL of new medium was added and suspended. From the obtained cell suspension, 10 μL was added to a slide for cell number measurement (manufactured by Thermo Fisher Scientific, trade name: Countess Cell Counting Chamber Slide), and an automatic cell counter (manufactured by Thermo Fisher Scientific, trade name: Center) was added. The number of cells was measured (number of recovered cells after cooling), and the cells remaining in the culture equipment after recovery of the culture solution were recovered by treating with trypsin at 37 ° C. for 10 minutes, and then the number of cells. Was measured and added to the number of recovered cells after cooling to obtain the total number of cells.

The results are shown in Table 1.

Claims (5)

The following general formula (1)
(In the formula, R ¹ is a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, a benzyl group, or the following general formula (2).
(In the formula, p represents an integer of 2 to 4, and R ⁴ represents a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, a 2-methoxyethyl group or an acetyl group.). R ² represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. R ³ represents a hydrogen atom or a methyl group. A copolymer comprising a block chain having a repeating unit represented by) and a hydrophobic block chain. The hydrophobic block chain has the following general formula (3).
(Represented in the formula, R ⁵ is an alkyl group having 1 to 6 carbon atoms, a cyclohexyl group, a benzyl group, or an .R ⁶ is a hydrogen atom or a methyl group and phenylethyl group. Provided that when R ⁶ is a hydrogen atom , R ⁵ is a methyl group, an ethyl group, a cyclohexyl group or a benzyl group.) the copolymer according to claim 1, characterized in that a block chain having repeating units represented by. A coating agent containing the copolymer according to claim 1 or 2. A material for cell culture obtained by applying the coating agent according to claim 3 to a substrate. A cell culture method according to claim 4, wherein the cell culture material is used.