JP6818521B2 - Method for producing polymer - Google Patents

Description

The present invention relates to a method for producing a polymer capable of highly sensitive and specific quantitative detection of 8-oxo-2'-deoxyguanosine (hereinafter, also referred to as "8OHdG") in an aqueous sample solution. Is. 8OHdG has a tautomer and is represented by the following structural formula.

8OHdG is attracting attention as an oxidative stress marker because it directly reflects the amount of active oxygen generated by environmental factors and metabolic activity in the living body. Therefore, accurately measuring the abundance of 8OHdG in vivo and in urine is extremely significant in studying mutations, aging, and many diseases. In general, the current measurement technology can analyze various chemical substances even if it is below the ppt (1/1 trillion) level by selecting and combining highly sophisticated separation technology, concentration technology, and analysis method. It is possible. In the case of such a trace level of analysis, it is usually necessary to go through steps such as optimum separation, concentration, qualitative analysis, and quantitative analysis according to the detection target. Inevitably, accurately measuring the abundance of 8OHdG at the ppt level requires a great deal of labor, a lot of time, and a high analysis cost. However, since the amount of active oxygen generated is variable, it is expected to develop a method for rapid measurement in various measurement environments.

In general, it is known that a resin having a molecular recognition function can be manufactured by a molecular template method and used as a chemical sensor to quickly measure a target chemical substance in various environments at a relatively low cost (for example). Non-Patent Document 1). However, a method using such a chemical sensor is said to have many problems regarding specific selectivity (hereinafter referred to as specificity) for a target chemical substance. For example, a resin having a molecular recognition function has been studied for detecting the abundance of 8OHdG, but such a resin also detects uric acid which is abundantly present in a living body. Therefore, the specificity for 8OHdG is insufficient (for example, Non-Patent Document 2), and a study using an apparatus (for example, Quartz Crystal Microbalance) that is difficult to measure quickly in various measurement environments has been conducted (Non-Patent Document 3). It's only done.

Chem. Soc. Rev. , 2011, 40, 2922-2942 Analytica Chimica Acta 640 (2009) 82-86 Biosensors and Bioelectronics 86 (2016) 225-234

Therefore, an object of the present invention is to provide a method for producing a polymer capable of detecting 8OHdG with high sensitivity and high specificity.

前記式（１）中、Ｒ^１は炭素数１から１８の脂肪族炭化水素基または、下記式（２）で表わされる構造であり、

上記式（２）中、Ｒ^２およびＲ^３はそれぞれ独立に、ヒドロキシル基または炭素数１から１６のアルキルカルボニルオキシ基であり、ただしＲ^２およびＲ^３は同時にヒドロキシル基ではなく、＊は前記式（１）中のＲ^１が結合するＮの位置を示す。 In order to solve the above problems, the method for producing a polymer of the present invention is a method for producing a polymer, which comprises carrying out a polymerization reaction in the presence of a compound represented by the following formula (1).

In the above formula (1), R ¹ is an aliphatic hydrocarbon group having 1 to 18 carbon atoms or a structure represented by the following formula (2).

In the above formula (2), R ² and R ³ are independently hydroxyl groups or alkylcarbonyloxy groups having 1 to 16 carbon atoms, whereas R ² and R ³ are not hydroxyl groups at the same time, and * is the above formula. (1) Indicates the position of N to which R ¹ is bound.

According to the present invention, it is possible to provide a method for producing a polymer and a copolymer having a high sensitivity and a specific repeating unit with respect to a low molecular weight compound, particularly 8OHdG.
The present invention also makes it possible to provide a device used for quantitative detection of 8 OHdG with high sensitivity.

前記式（１）中、Ｒ^１は炭素数１から１８の脂肪族炭化水素基または、下記式（２）で表わされる構造であり、

上記式（２）中、Ｒ^２およびＲ^３はそれぞれ独立に、ヒドロキシル基、または炭素数１から１６のアルキルカルボニルオキシ基であり、ただしＲ^２およびＲ^３は同時にヒドロキシル基ではなく、＊は式（１）中のＲ^１が結合するＮの位置を示す。前記炭素数１から１６のアルキルカルボニルオキシ基は、炭素数１から１６のアルキル基がカルボニルオキシ基を介して結合する基を表す。
なお、本明細書では、脂肪族炭化水素基の炭素数の規定は、置換基、枝分かれ構造などに含まれるすべての炭素の数を意味する。 The method for producing a polymer according to the embodiment of the present invention is characterized in that the polymerization reaction is carried out in the presence of a compound represented by the following formula (1).

In the above formula (1), R ¹ is an aliphatic hydrocarbon group having 1 to 18 carbon atoms or a structure represented by the following formula (2).

In the above formula (2), R ² and R ³ are independently hydroxyl groups or alkylcarbonyloxy groups having 1 to 16 carbon atoms, whereas R ² and R ³ are not hydroxyl groups at the same time, and * is the formula. (1) Indicates the position of N to which R ¹ is bound. The alkylcarbonyloxy group having 1 to 16 carbon atoms represents a group to which an alkyl group having 1 to 16 carbon atoms is bonded via a carbonyloxy group.
In addition, in this specification, the definition of the carbon number of an aliphatic hydrocarbon group means the number of all carbons contained in a substituent, a branched structure and the like.

The compound represented by the formula (1) has tautomers represented by the following formulas (1'), formula (1 ″), and formula (1 ′ ″). In, the compound represented by the formula (1) is assumed to include all of these tautomers.

In the embodiment of the present invention, among preferable specific examples of the compound represented by the formula (1), those in which R ¹ is an aliphatic hydrocarbon group having 1 to 18 carbon atoms are shown below, but are limited to the following examples. It is not something that is done.

In the preferred specific example of the compound represented by the formula (1) in the embodiment of the present invention, a specific example of the structure represented by the preferred formula (2) of the compound in which R ¹ is the structure represented by the formula (2) is as follows. However, the present invention is not limited to the following examples.

In the present specification, when R ^{1 of the} compound represented by the formula (1) represents a compound having a structure represented by the formula (2), the number assigned to the structure represented by the above formula (2) is assigned. , Also used as the number of the compound represented by the formula (1). For example, the compound represented by the formula (2-1) means a compound represented by the formula (1) in which R ¹ has a structure represented by the formula (2-1).

Usually, when a polymer having a molecular recognition function is produced by the molecular template method, the substance to be detected is used as a so-called template substance. However, the inventors of the present invention have stated that when a polymer is simply prepared using 8OHdG as a template substance and acrylic acid or acrylamide, the obtained polymer does not have sufficient sensitivity to 8OHdG. Is finding out. Further, the aliphatic hydrocarbon group number of carbon atoms in the aliphatic hydrocarbon group esterified to the deoxyribose of 8OHdG is represented by R ¹ directly bonded to a 8-hydroxy guanine a component or if 8OHdG above 17 Our study has also shown that when the number of carbon atoms is 19 or more, the obtained polymer does not have sufficient specificity for 8OHdG. In view of the above, although the details are unknown, it is considered that the size and shape of the pores formed by the template substance play an important role in the specificity of 8OHdG recognition. That is, the inventors of the present invention have found that if the size and shape of the pores formed by the aliphatic hydrocarbon group of the template substance are inappropriate, sufficient sensitivity and specificity cannot be obtained.

式（３）、式（４）、および式（５）中、Ａ^１はそれぞれ独立に、水素原子またはメチル基であり、Ｒ^４およびＲ^５はそれぞれ独立に、水素原子、または飽和もしくは不飽和の炭素数１から１２の脂肪族炭化水素基である。 The polymer according to the embodiment of the present invention is synthesized by polymerizing a single type of monomer as long as it is a polymer produced by carrying out a polymerization reaction in the presence of the compound represented by the formula (1). It may be either a so-called homopolymer or a so-called copolymer synthesized by polymerization of a plurality of types of monomers. However, from the viewpoint of specificity for 8OHdG, a polymer containing a repeating unit derived from at least one of the monomers represented by the formulas (3), (4) and (5) is preferable. Here, the polymer containing a repeating unit derived from a monomer means a polymer in which the monomer is contained as a repeating unit by a polymerization reaction.

In formulas (3), (4), and (5), A ¹ is independently a hydrogen atom or a methyl group, and R ⁴ and R ⁵ are independently hydrogen atoms, or saturated or unsaturated, respectively. It is an aliphatic hydrocarbon group having 1 to 12 carbon atoms.

Ａ^１はそれぞれ独立に、水素原子またはメチル基であり、Ａ^２はそれぞれ独立に、式（９）、式（１０）、式（１１）、または式（１２）で表わされる構造であり、Ａ^３は炭素数１から１８の脂肪族炭化水素基であり、

式（９）、式（１０）、式（１１）、および式（１２）中、Ｒ^６およびＲ^１６はそれぞれ独立に、水素原子、またはニトロ基もしくはハロゲン原子で置換されていてもよい飽和もしくは不飽和の脂肪族炭化水素基であり、Ｒ^７〜Ｒ^１５はそれぞれ独立に、水素原子、ニトロ基、ハロゲン原子、またはニトロ基もしくはハロゲン原子で置換されていてもよい飽和もしくは不飽和の脂肪族炭化水素基であり、Ｒ^７およびＲ^８、またはＲ^９、Ｒ^１０およびＲ^１１は、互いに結合し環中に不飽和結合を有していてもよい環状構造を形成してもよく、＊は式（６）、式（７）、および式（８）中のＮの位置を示す。 However, polymers containing only the monomer-derived repeating units represented by these formulas (3), (4), and (5) may still have low specificity for 8OHdG. Therefore, the polymer according to the embodiment of the present invention is a polymer containing a repeating unit derived from at least one of the monomers represented by the formulas (6), (7), and (8). Is more preferable. The repeating units derived from the monomers represented by the formulas (6), (7), and (8) perform molecular recognition by forming hydrogen bonds with respect to three hydrogen bond sites of 8OHdG. , The specificity for 8OHdG is increased. Hereinafter, the repeating unit derived from the monomer represented by the formulas (6), (7), and (8) is referred to as a molecular recognition unit. On the other hand, the repeating unit derived from the monomer represented by the formulas (3), (4) and (5) is called a low recognition unit.

A ¹ is an independent hydrogen atom or a methyl group, and A ² is an independent structure represented by the formula (9), the formula (10), the formula (11), or the formula (12). Reference numeral ³ is an aliphatic hydrocarbon group having 1 to 18 carbon atoms.

In formulas (9), (10), (11), and (12), R ⁶ and R ¹⁶ are each independently saturated or optionally substituted with a hydrogen atom or a nitro or halogen atom. Unsaturated aliphatic hydrocarbon groups, each of R ^{7 to} R ¹⁵ is a saturated or unsaturated aliphatic hydrocarbon which may be independently substituted with a hydrogen atom, a nitro group, a halogen atom, or a nitro group or a halogen atom. It is a hydrocarbon group, and R ⁷ and R ⁸ or R ⁹ , R ¹⁰ and R ¹¹ may be bonded to each other to form a cyclic structure which may have an unsaturated bond in the ring. The positions of N in the formulas (6), (7), and (8) are shown.

式（１３）、式（１４）、および式（１５）中、Ｒ^１７およびＲ^２７はそれぞれ独立に、水素原子、または飽和もしくは不飽和の脂肪族炭化水素基であり、Ｒ^１８〜Ｒ^２６はそれぞれ独立に、水素原子、ニトロ基、ハロゲン原子、または飽和もしくは不飽和の脂肪族炭化水素基であり、＊は式（６）、式（７）、および式（８）中のＮの位置を示す。 The polymer according to the embodiment of the present invention, molecular recognition units, the formula (6), equation (7), and A ² monomers in the formula (8), equation (13), wherein A polymer which is a repeating unit derived from a monomer having a structure represented by (14) or (15) is more preferable.

In formulas (13), (14), and (15), R ¹⁷ and R ²⁷ are independently hydrogen atoms or saturated or unsaturated aliphatic hydrocarbon groups, and R ^{18 to} R ²⁶ are Each is independently a hydrogen atom, a nitro group, a halogen atom, or a saturated or unsaturated aliphatic hydrocarbon group, where * indicates the position of N in the formulas (6), (7), and (8). Shown.

式（１６）、式（１７）、および式（１８）中、Ａ^１はそれぞれ独立に、水素原子またはメチル基であり、Ｒ^１７およびＲ^２７はそれぞれ独立に、水素原子、または飽和もしくは不飽和の脂肪族炭化水素基であり、Ｒ^１８〜Ｒ^２６はそれぞれ独立に、水素原子、ニトロ基、ハロゲン原子、または飽和もしくは不飽和の脂肪族炭化水素基である。 A more preferable polymer as the polymer according to the embodiment of the present invention is a polymer whose molecular recognition unit is a repeating unit derived from a monomer represented by the formulas (16), (17), and (18). Is.

In formulas (16), (17), and (18), A ¹ is independently a hydrogen atom or a methyl group, and R ¹⁷ and R ²⁷ are independently hydrogen atoms, or saturated or unsaturated, respectively. R ^{18 to} R ²⁶ are independently hydrogen atoms, nitro groups, halogen atoms, or saturated or unsaturated aliphatic hydrocarbon groups.

In view of the sensitivity of the polymer according to the embodiment of the present invention to 8OHdG, in the method for producing the polymer according to the embodiment of the present invention, the monomer constituting the molecular recognition unit and the monomer constituting the low recognition unit The molar ratio with and from is preferably 1: 100 to 70:30, and more preferably 10:90 to 50:50.

The polymer according to the embodiment of the present invention has a repeating unit derived from a monomer represented by the formulas (6) and (7) as a molecular recognition unit, or the formulas (6), (7) and By having the repeating unit derived from the monomer represented by the formula (8), both high sensitivity and specificity can be realized. The details are unknown, but it is expected that the molecular recognition units will complement each other and form hydrogen bonds with different parts of 8OHdG. The molar ratio of the monomer-derived repeating unit represented by the formula (6) to the monomer-derived repeating unit represented by the formula (7) in the polymer according to the embodiment of the present invention is 1: 1. 1 to 1: 4 is preferable, and the closer to 1: 2, the higher the sensitivity and specificity of the polymer according to the embodiment of the present invention to 8OHdG. Further, the molar ratio of the monomer-derived repeating unit represented by the formula (6), the monomer-derived repeating unit represented by the formula (7), and the monomer-derived repeating unit represented by the formula (8). Is preferably 1: 1: 1 to 1: 4: 1, and the closer it is to 1: 1: 1, the higher the sensitivity and specificity of the polymer according to the embodiment of the present invention to 8OHdG.

式（１９）中、Ａ^１はそれぞれ独立に、水素原子またはメチル基であり、Ｒ^２８は、炭素数１から２０の直鎖または環状の脂肪族炭化水素基、置換基を有してもよい芳香族炭化水素基、または置換基を有してもよいヘテロ芳香族炭化水素基である。 The polymer according to the embodiment of the present invention may be a polymer in which the above-mentioned monomer having one polymerizable functional group is polymerized in a chain, and is a polyfunctional single amount having two or more polymerizable functional groups. It may be a crosslinked polymer obtained by polymerizing the body. By having the crosslinks, the polymer according to the embodiment of the present invention has improved specificity for 8OHdG. It is considered that this is because the structure of the molecular recognition portion is fixed to suppress the elimination of 8OHdG once captured, but the details are unknown. The polyfunctional monomer may have any structure, but is preferably a bifunctional monomer, and more preferably a monomer represented by the formula (19). When the polyfunctional monomer is a monomer represented by the formula (19), the repeating unit derived from the monomer represented by the formula (19) is the molecular recognition unit, and thus according to the embodiment of the present invention. The specificity of the polymer for 8 OHdG is further improved.

In formula (19), A ¹ is independently a hydrogen atom or a methyl group, and R ²⁸ may have a linear or cyclic aliphatic hydrocarbon group or substituent having 1 to 20 carbon atoms. It is an aromatic hydrocarbon group or a heteroaromatic hydrocarbon group which may have a substituent.

式（１９−１）および式（１９−２）中、Ａ^１はそれぞれ独立に、水素原子またはメチル基であり、Ａ^４はそれぞれ独立に、窒素原子または水素原子が一つ結合した炭素原子であり、Ｒ^２９およびＲ^３０はそれぞれ独立に、炭素数１から２０のアルコキシ基、アルコキシカルボニル基、アルキルカルボニルオキシ基、アルキルアミノカルボニル基、アルキルカルボニルアミノ基、アルキルチオ基、またはアルキルスルホニル基である。これらの基は、それぞれ炭素数１から２０のアルキル基が、酸素原子、オキシカルボニル基、カルボニルオキシ基、アミノカルボニル基、カルボニルアミノ基、硫黄原子、またはスルホニル基を介して結合する基を表す。 Further, when the monomer represented by the formula (19) is a monomer represented by the structural formula (19-1) or the formula (19-2), 8OHdG of the polymer according to the embodiment of the present invention is used. The specificity for it is further improved.

In formulas (19-1) and (19-2), A ¹ is an independent hydrogen atom or methyl group, and A ⁴ is an independent carbon atom with one nitrogen atom or one hydrogen atom bonded. Yes, R ²⁹ and R ³⁰ are independently alkoxy groups, alkoxycarbonyl groups, alkylcarbonyloxy groups, alkylaminocarbonyl groups, alkylcarbonylamino groups, alkylthio groups, or alkylsulfonyl groups having 1 to 20 carbon atoms, respectively. Each of these groups represents a group in which an alkyl group having 1 to 20 carbon atoms is bonded via an oxygen atom, an oxycarbonyl group, a carbonyloxy group, an aminocarbonyl group, a carbonylamino group, a sulfur atom, or a sulfonyl group.

In the method for producing a polymer according to the embodiment of the present invention, the molar ratio of the monomer represented by the formula (19) to the other monomers is preferably 1:99 to 50:50. In order to increase the polymerization rate of the polymer according to the embodiment of the present invention and obtain higher specificity for 8OHdG, the monomer represented by the formula (19) in the method for producing the polymer according to the embodiment of the present invention. The molar ratio of the polymer to the other monomer is more preferably 1:90 to 30:70.

In the embodiment of the present invention, the hydrocarbon group in the above formula means a chemical structure capable of containing at least a carbon atom and a hydrogen atom, and may be substituted with a nitro group, a halogen atom and the like. When limited to an aliphatic hydrocarbon group, it means an acyclic or cyclic, non-aromatic hydrocarbon group, and may have a linear or branched structure. The aliphatic hydrocarbon group in the embodiment of the present invention forms a linear, branched, or alicyclic structure with at least one or more carbon atoms, and the carbon atom bonds are unsaturated even if they are saturated bonds. It means a group that may be either of bonds, for example, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a cycloalkenyl group, a cycloalkynyl group, an alkylene group, an alkenylene group, an alkynylene group, a cycloalkylene group, a cycloalkenylene group, Cycloalkynylene groups and the like can be mentioned.

The hydrocarbon group shall also include an aromatic hydrocarbon group unless otherwise specified as an aliphatic hydrocarbon group. Further, when limited to an aromatic hydrocarbon group, the above aliphatic hydrocarbon group is not included. Specific examples of the aromatic hydrocarbon group in the embodiment of the present invention include a phenyl group, a naphthyl group, an anthracenyl group, a phenanthrenyl group, a biphenyl group and a corresponding divalent group. Specific examples of the heteroaromatic hydrocarbon group include a pyridyl group, an imidazolyl group, a pyrazolyl group, a pyridinyl group, a thienyl group, a thiazolyl group, and a divalent group corresponding to these groups.

In the method for producing a polymer according to the embodiment of the present invention, the polymerization reaction may be any reaction and is not limited. The polymerization initiator used in the polymerization reaction may be a photopolymerization initiator or a thermal polymerization initiator, but in the thermal polymerization initiator, the polymerization temperature at the start of the reaction is equal to or higher than the glass transition temperature of the polymer. A photopolymerization initiator is preferable.

Specific examples of the polymerization initiator include 2,2'-azobis (isobutyronitrile), 2,2'-azobis (2,4-dimethylvaleronitrile), and 2,2'-azobis (2-methyl). Butyronitrile), 4,4'-azobis (4-cyanovaleric acid), 2,2'-azobis (2-methylpropionamidine) dihydrochloride, 2,2'-azobis [2- (2-imidazolin-) 2-Il) Propane] Dihydrochloride, benzoyl peroxide, tert-butyl hydroperoxide, cumene hydroperoxide, di-tert-butyl peroxide, 2,2'-azobis (2-methylpropionic acid) dimethyl, dicumyl peroxide, Acetphenone, p-anisyl, benzyl, benzoin, benzophenone, 4,4'-bis (diethylamino) benzophenone, 4,4'-bis (dimethylamino) benzophenone, benzoin methyl ether, benzoin isopropyl ether, benzoin isobutyl ether, benzoin ethyl ether , 4-Benzoyl benzoic acid, 2,2'-bis (2-chlorophenyl) -4,4', 5,5'-tetraphenyl-1,2'-biimidazole, methyl 2-benzoyl benzoate, 2-( 1,3-Benzodiol-5-yl) -4,6-bis (trichloromethyl) -1,3,5-triazine, 2-benzyl-2- (dimethylamino) -4'-morpholinobtyrophenone, 4 , 4'-Dichlorobenzophenone, 2,2-diethoxyacetophenone, 2,2-dimethoxy-2-phenylacetophenone, 2,4-diethylthioxanthene-9-one, 2,2-diethoxyacetophenone, 2,2- Dimethoxy-2-phenylacetophenone, diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide, 1,4-dibenzoylbenzene, 2-ethylanthraquinone, 1-hydroxycyclohexylphenylketone, 2-hydroxy-2-methylpropio Phenone, phenyl (2,4,6-trimethylbenzoyl) lithium phosphinate, 2-methyl-4'-(methylthio) -2-morpholinopropiophenonne, 2-isonitrosopropiophenone, 2-phenyl-2- Examples thereof include (p-toluenesulfonyloxy) acetophenonne, phenylbis (2,4,6-trimethylbenzoyl) phosphine oxide, and the like, but the initiator in the embodiment of the present invention is not limited to the above compounds.

In the method for producing a polymer according to the embodiment of the present invention, the polymerization reaction is preferably carried out without a solvent when the monomer, the template substance and the polymerization initiator are compatible with each other. Further, when the monomer, the template substance and the polymerization initiator cannot be reacted without being compatible with each other, it is preferable to use a reaction solvent. Therefore, the reaction solvent may be one that dissolves or disperses the monomer, the template substance and the polymerization initiator and does not inhibit the reaction. Specific examples of the reaction solvent include toluene, heptane, hexane, chloroform, tetrahydrofuran, dioxane, dimethylformamide, dimethyl sulfoxide, isopropyl alcohol, ethanol, methanol, water and a mixed solvent thereof. When water is used as the reaction solvent, it may be an aqueous solution of a salt such as NaCl, a cushioning material, an acid, a base or the like, and the pH of the aqueous solution can be adjusted using these.

After the completion of the polymerization reaction, the solvent for removing the template substance incorporated in the polymer (hereinafter, also referred to as “cleaning solvent”) is the template substance, the remaining monomer, and the start of polymerization without dissolving the resin. Any substance that can dissolve and remove the agent or the like will do. Specific examples of the cleaning solvent include toluene, heptane, hexane, chloroform, tetrahydrofuran, dioxane, dimethylformamide, dimethyl sulfoxide, isopropyl alcohol, ethanol, methanol, water and a mixed solvent thereof. When water is used as the cleaning solvent, it may be an aqueous solution of a salt such as NaCl, a buffer material, an acid, a base or the like, and the pH of the aqueous solution can be adjusted using these.

Further, the method for producing the polymer according to the embodiment of the present invention may be so-called random polymerization or block polymerization. Random polymerization is preferable from the viewpoint of cost.

Preferred specific examples of the monomer used in the method for producing the polymer of the embodiment of the present invention are shown below, but the present invention is not limited to the following specific examples. A ² in the structures represented by the formulas (6-1) to (8-2) is the structure represented by 9-1-X to 15-12-X, and the * part is the structure represented by the formula (6-1). The position of connection with N in the structure represented by the formula (8-2) is shown. In addition, the numbers of the compounds shown as specific examples, corresponds to the number of A ^2. When the main chain portion has a structure represented by the formula (6-1), the compound number is the one in which X of the number indicating A ² is 1, and the main chain portion is represented by the formula (6-2). In the case of, the compound number in which X of the number indicating A ² is 2. When the main chain portion has a structure represented by the formula (7-1), the compound number is the one in which X of the number indicating A ² is 3, and the main chain portion is represented by the formula (7-2). In the case of the structure, the compound number is the one in which X of the number indicating A ² is 4. Similarly, when the main chain portion has a structure represented by the formula (8-1), the compound number in which X of the number indicating A ² is 5 is used as the compound number, and the main chain portion is represented by the formula (8-2). In the case of the represented structure, the compound number is the one in which X of the number indicating A ² is 6. For example, compound 9-1-1 is a compound in which the main chain portion has a structure represented by the formula (6-1) and A ² is a molecular recognition portion represented by 9-1-X.

Preferred specific examples of the polyfunctional monomer used in the method for producing a polymer of the embodiment of the present invention are shown below, but the present invention is not limited to the following specific examples.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples as long as the gist of the present invention is not exceeded.

<Synthesis example>
<Synthesis of polymer (1)>
Acrylic acid (manufactured by Kishida Chemical Co., Ltd.) as a monomer, 1-hydroxycyclohexylphenyl ketone (manufactured by Tokyo Kasei Co., Ltd.) as a polymerization initiator, and a compound represented by the formula (2-1) as a template substance (manufactured by Sanyu Chemical Co., Ltd.) Was mixed at a molar ratio of 10: 1:10. Then, the polymerization reaction was carried out by irradiating with ultraviolet rays for 8 hours with Handy Cure Love (manufactured by Sen Special Light Source Co., Ltd.) under a nitrogen stream. Then, the obtained polymer was stirred and washed in methanol and ion-exchanged water at room temperature for 8 hours each. The washing solvent was removed by vacuum filtration, and then vacuum drying was performed to obtain the desired polymer (1).

<Synthesis of polymer (2)>
The target polymer (2) was obtained in the same manner as in the synthesis of the polymer (1) except that the monomer was replaced with acrylamide (manufactured by Kishida Chemical Co., Ltd.).

<Synthesis of polymer (3)>
Acrylic acid (manufactured by Kishida Chemical Co., Ltd.) and Viscort # 802 (manufactured by Osaka Organic Chemical Co., Ltd.) are used as the monomers, 1-hydroxycyclohexylphenylketone (manufactured by Tokyo Kasei Co., Ltd.) is used as the polymerization initiator, and the formula (2- The compound represented by 1) (manufactured by Sanyu Chemical Co., Ltd.) was mixed at a molar ratio of 10: 100: 1:10. Then, the polymerization reaction was carried out by irradiating with ultraviolet rays for 8 hours with Handy Cure Love (manufactured by Sen Special Light Source Co., Ltd.) under a nitrogen stream. Then, the obtained polymer was stirred and washed in chloroform, methanol, and ion-exchanged water at room temperature for 8 hours each. The washing solvent was removed by vacuum filtration, and then vacuum dried to obtain the desired polymer (3).

<Synthesis of polymer (4)>
The target polymer (4) was obtained in the same manner as in the synthesis of the polymer (3) except that the monomer was replaced with acrylamide (manufactured by Kishida Chemical Co., Ltd.).

<Synthesis of polymer (5)>
Compounds represented by the formula (13-6-1) (manufactured by Sanyu Chemical Co., Ltd.), compounds represented by the formula (15-6-1) (manufactured by Sanyu Chemical Co., Ltd.), Viscort # 802 (Osaka Organic Chemical Co., Ltd.) 1-Hydroxycyclohexylphenylketone (manufactured by Tokyo Kasei Co., Ltd.) was used as a polymerization initiator, and a compound represented by the formula (2-1) was used as a template substance (manufactured by Sanyu Chemical Co., Ltd.). The polymerization initiator and the template compound were mixed at a molar ratio of 20:10:10: 100: 1:10. Then, the polymerization reaction was carried out by irradiating with ultraviolet rays for 8 hours with Handy Cure Love (manufactured by Sen Special Light Source Co., Ltd.) under a nitrogen stream. Then, the obtained polymer was stirred and washed in chloroform, methanol, and ion-exchanged water at room temperature for 8 hours each. The washing solvent was removed by vacuum filtration, and then vacuum drying was performed to obtain the desired polymer (5).

<Synthesis of polymer (6)>
The monomer is represented by the formula (13-6-1) (manufactured by Sanyu Chemical Co., Ltd.), the compound represented by the formula (14-1-1) (manufactured by Sanyu Chemical Co., Ltd.), and the formula (15-6-1). The compound represented by (manufactured by Sanyu Chemical Co., Ltd.) and Viscort # 802 (manufactured by Osaka Organic Chemical Co., Ltd.) are used, and the respective monomers, polymerization initiators and template substances are 10:10:10:10:10:100: 1:10. The target polymer (6) was obtained in the same manner as in the synthesis of the polymer (5) except that it was used in terms of molar ratio.

<Synthesis of polymer (7)>
The target polymer (7) was obtained in the same manner as in the synthesis of the polymer (2) except that the monomer was a compound represented by the formula (19-1-8) (manufactured by Sanyu Chemical Co., Ltd.).

<Synthesis of polymer (8)>
The target polymer (8) is obtained in the same manner as in the synthesis of the polymer (5) except that the template substance is a compound represented by the formula (2-6) (manufactured by Sanyu Chemical Co., Ltd.).

<Synthesis of polymer (9)>
The target polymer (9) is obtained in the same manner as in the synthesis of the polymer (6) except that the template substance is a compound represented by the formula (2-6) (manufactured by Sanyu Chemical Co., Ltd.).

<Synthesis of polymer (10)>
The target polymer (10) is obtained in the same manner as in the synthesis of the polymer (7) except that the template substance is a compound represented by the formula (2-6) (manufactured by Sanyu Chemical Co., Ltd.).

<Synthesis of polymer (11)>
The target polymer (11) was obtained in the same manner as in the synthesis of the polymer (5) except that the template substance was a compound represented by the formula (2-10) (manufactured by Sanyu Chemical Co., Ltd.).

<Synthesis of polymer (12)>
The target polymer (12) is obtained in the same manner as in the synthesis of the polymer (6) except that the template substance is a compound represented by the formula (2-10) (manufactured by Sanyu Chemical Co., Ltd.).

<Synthesis of polymer (13)>
The target polymer (13) is obtained in the same manner as in the synthesis of the polymer (7) except that the template substance is a compound represented by the formula (2-10) (manufactured by Sanyu Chemical Co., Ltd.).

<Synthesis of polymer (14)>
The target polymer (14) is obtained in the same manner as in the synthesis of the polymer (5) except that the template substance is a compound represented by the formula (1-1) (manufactured by Sanyu Chemical Co., Ltd.).

<Synthesis of polymer (15)>
The target polymer (15) is obtained in the same manner as in the synthesis of the polymer (6) except that the template substance is a compound represented by the formula (1-1) (manufactured by Sanyu Chemical Co., Ltd.).

<Synthesis of polymer (16)>
The target polymer (16) is obtained in the same manner as in the synthesis of the polymer (7) except that the template substance is a compound represented by the formula (1-1) (manufactured by Sanyu Chemical Co., Ltd.).

<Synthesis of polymer (17)>
The target polymer (17) is obtained in the same manner as in the synthesis of the polymer (5) except that the template substance is a compound represented by the formula (1-7) (manufactured by Sanyu Chemical Co., Ltd.).

<Synthesis of polymer (18)>
The target polymer (18) is obtained in the same manner as in the synthesis of the polymer (6) except that the template substance is a compound represented by the formula (1-7) (manufactured by Sanyu Chemical Co., Ltd.).

<Synthesis of polymer (19)>
The target polymer (19) is obtained in the same manner as in the synthesis of the polymer (7) except that the template substance is a compound represented by the formula (1-7) (manufactured by Sanyu Chemical Co., Ltd.).

<Synthesis of polymer (20)>
The target polymer (20) is obtained in the same manner as in the synthesis of the polymer (5) except that the template substance is a compound represented by the formula (1-11) (manufactured by Sanyu Chemical Co., Ltd.).

<Synthesis of polymer (21)>
The target polymer (21) is obtained in the same manner as in the synthesis of the polymer (6) except that the template substance is a compound represented by the formula (1-11) (manufactured by Sanyu Chemical Co., Ltd.).

<Synthesis of polymer (22)>
The target polymer (22) is obtained in the same manner as in the synthesis of the polymer (7) except that the template substance is a compound represented by the formula (1-11) (manufactured by Sanyu Chemical Co., Ltd.).

<Synthesis of polymer (23)>
The target polymer (23) was obtained in the same manner as in the synthesis of the polymer (1) except that the template substance was 8OHdG (manufactured by Tokyo Kasei Co., Ltd.).

<Synthesis of polymer (24)>
The target polymer (24) was obtained in the same manner as in the synthesis of the polymer (2) except that the template substance was 8OHdG (manufactured by Tokyo Kasei Co., Ltd.).

<Synthesis of polymer (25)>
The target polymer (25) was obtained in the same manner as in the synthesis of the polymer (1) except that the template substance was the following compound 2-40 (manufactured by Sanyu Chemical Co., Ltd.).

<Synthesis of polymer (26)>
The target polymer (26) was obtained in the same manner as in the synthesis of the polymer (2) except that the template substance was compound 2-40 (manufactured by Sanyu Chemical Co., Ltd.) below.

<Synthesis of polymer (27)>
The target polymer (27) was obtained in the same manner as in the synthesis of the polymer (1) except that the monomer was replaced with styrene (manufactured by Kishida Chemical Co., Ltd.).

The method for calculating the sensitivity of the polymer to 8 OHdG in the examples of the present invention is shown below. For the measurement, Lambda Bio (manufactured by PerkinElmer) was used.
(Calculation of 8OHdG sensitivity)
The absorbance (abs _r ) at 300 nm of a methanol solution (10 μM) of 40 μl of 8 OHdG (manufactured by Wako Pure Chemical Industries, Ltd.) was measured.
40 μl of a 10 μM 8OHdG (manufactured by Wako Pure Chemical Industries, Ltd.) hexane solution was added to each of 4 mg of the polymers (1) to (26) produced above, and the mixture was stirred at 20 ° C. for 24 hours. The absorbance (abs _k ) of the supernatant component extracted by centrifugation at 10000 rpm was measured at 300 nm, and 1.0- (abs _k / abs _r ) was calculated and used as the sensitivity.
In the evaluation of the sensitivity, the following criteria were used in the examples of the present invention, with A to C being the allowable level and D being the unacceptable level.
A: Sensitivity is 0.90 or more B: Sensitivity is 0.75 or more and less than 0.90 C: Sensitivity is 0.50 or more and less than 0.75 D: Sensitivity is less than 0.50

Table 1 shows the sensitivity and evaluation results of the polymers obtained in the examples of the present invention.

The method for calculating the specificity of the polymer for 8OHdG in the examples of the present invention is shown below. For the measurement, Lambda Bio (manufactured by PerkinElmer) was used.
(Calculation of specificity) Sensitivity of the polymer to uric acid in the embodiment of the present invention (uric acid sensitivity) in the same manner as the calculation of the sensitivity of 8OHdG (8OHdG sensitivity) except that 8OHdG was replaced with uric acid (manufactured by Kishida Chemical Co., Ltd.). ) Was measured, and 8OHdG sensitivity / uric acid sensitivity was calculated and used as the specificity.
As the evaluation of specificity, the following criteria were used in the examples of the present invention, with A to C being the allowable level and D being the unacceptable level.
A: Specificity is 10.0 or more and B: Specificity is 5.0 or more and less than 10.0 C: Specificity is 1.1 or more and less than 5.0 D: Specificity is less than 1.1

The polymer produced by the production method of the present invention can detect oxidative stress in a living body with high sensitivity, relatively inexpensively, and easily by detecting 8OHdG.

Claims (12)

Method of manufacturing a row cormorants polymer polymerization reaction in the presence of a compound represented by the following formula (1).

(In the above formula (1), R ¹ is an aliphatic hydrocarbon group having 1 to 18 carbon atoms or a structure represented by the following formula (2).

In the above formula (2), R ² and R ³ are independently hydroxyl groups or alkylcarbonyloxy groups having 1 to 16 carbon atoms, whereas R ² and R ³ are not hydroxyl groups at the same time, and * is the above. The position of N to which R ¹ is bound in the formula (1) is shown. ) The R ² And the above R ³ The method for producing a polymer according to claim 1, wherein each of them is independently an alkylcarbonyloxy group having 1 to 16 carbon atoms. The polymer according to claim 1 or 2 , wherein the polymerization reaction polymerizes at least one of the monomers represented by the following formulas (3), (4) and (5). Production method.

(In the formulas (3), (4), and (5), A ¹ is independently a hydrogen atom or a methyl group, and R ⁴ and R ⁵ are independently hydrogen atoms, or saturated or unsaturated, respectively. It is an aliphatic hydrocarbon group having 1 to 12 carbon atoms. The invention according to claim 1 or 2 , wherein the polymerization reaction polymerizes at least one of the monomers represented by the following formulas (6), (7), and (8). Method for producing a polymer.

(In the formulas (6), (7), and (8), A ¹ is an independent hydrogen atom or a methyl group, and A ² is an independent formula (9), formula (10), and formula. (11), or a structure represented by the formula (12), ^{a 3} is an aliphatic hydrocarbon group having 1 to 18 carbon atoms,

In the formulas (9), (10), (11), and (12), R ⁶ and R ¹⁶ are each independently saturated or unsaturated which may be substituted with a hydrogen atom or a nitro group or a halogen atom. R ⁷ to R ¹⁵ are each independently substituted with a hydrogen atom, a nitro group, a halogen atom, or a nitro group or a halogen atom. Saturated or unsaturated aliphatic hydrocarbons. R ⁷ and R ⁸ or R ⁹ , R ¹⁰ and R ¹¹ which are groups may be bonded to each other to form a cyclic structure which may have an unsaturated bond in the ring, where * is the above formula. The positions of N in (6), (7), and (8) are shown. ) A ² in the monomers represented by the formulas (6), (7), and (8) has a structure represented by the following formula (13), formula (14), or formula (15). The method for producing a polymer according to claim 4 , wherein the polymer is produced.

(In the formulas (13), (14), and (15), R ¹⁷ and R ²⁷ are independently hydrogen atoms or saturated or unsaturated aliphatic hydrocarbon groups, and R ¹⁸ to R ²⁶ are Each is independently a hydrogen atom, a nitro group, a halogen atom, or a saturated or unsaturated aliphatic hydrocarbon group, and * indicates the position of N in the above formulas (6) to (8). The weight according to claim 1 or 2 , wherein the polymerization reaction polymerizes at least one of the monomers represented by the following formulas (16), (17), and (18). Manufacturing method of coalescence.

(In the formulas (16), (17) and (18), A ¹ is independently a hydrogen atom or a methyl group, and R ¹⁷ and R ²⁷ are independently hydrogen atoms or saturated or unsaturated, respectively. It is an aliphatic hydrocarbon group, and R ¹⁸ to R ²⁶ are independently hydrogen atoms, nitro groups, halogen atoms, or saturated or unsaturated aliphatic hydrocarbon groups.) The method for producing a polymer according to claim 4 or 5 , wherein the polymerization reaction polymerizes at least the monomers represented by the formulas (6) and (7). The method for producing a polymer according to claim 7 , wherein the polymerization reaction polymerizes at least the monomers represented by the formulas (6), (7), and (8). The method for producing a polymer according to any one of claims 1 to 8 , wherein the polymerization reaction polymerizes at least a polyfunctional monomer. The method for producing a polymer according to claim 9 , wherein the polyfunctional monomer is a monomer represented by the following formula (19).

(In the above formula (19), A ¹ is independently a hydrogen atom or a methyl group, and R ²⁸ has a linear or cyclic aliphatic hydrocarbon group having 1 to 20 carbon atoms and a substituent. It is also a good aromatic hydrocarbon group, or a heteroaromatic hydrocarbon group which may have a substituent.) The method for producing a polymer according to claim 10 , wherein the monomer represented by the formula (19) is selected from the group consisting of the following formulas (19-1) and (19-2). ..

(In the above formulas (19-1) and (19-2), A ¹ is an independent hydrogen atom or a methyl group, and A ⁴ is an independent carbon having one nitrogen atom or one hydrogen atom bonded thereto. Atoms, R ²⁹ and R ³⁰ are independently an alkoxy group, an alkoxycarbonyl group, an alkylcarbonyloxy group, an alkylaminocarbonyl group, an alkylcarbonylamino group, an alkylthio group, or an alkylsulfonyl group, respectively. is there.) The method for producing a polymer according to any one of claims 1 to 11, wherein the polymerization reaction is carried out using a photopolymerization initiator.