JPH11341995A - New chemiluminescent reagent and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new chemiluminescent reagent capable of detecting a peroxidase in high sensitivity by reacting an N,N'-disubstituted-9,9'-bisacridinium salt in the presence of an N,N-disubstituted carboxylic acid amide under light irradiation. SOLUTION: The objective chemiluminescent reagent enabling the highly sensitive determination of various substances by peroxidase labeling is produced by reacting an N,N'disubstituted-9,9'-bisacridinium salt of formula I [R<1> and R<2> are each an alkyl or a (halogenated) aryl; R<3> to R<6> are each H, an alkyl, an aryl, an alkoxy, an aryloxy or a halogen; X is an n-valent anion; (n) is 1 or 2] (e.g. lucigenin) in the presence of an N,N-disubstituted carboxylic acid amide compound of formula II [R1 is H, a 1-10C alkyl, a 2-10C alkenyl or a (substituted) 6-20C aryl; R2 is methyl or ethyl; R3 is a 1-10C alkyl, a 2-10C alkenyl, a (Substituted) 6-20C aryl or the like] (e.g. N,N-dimethylacetamide) under light irradiation.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a chemiluminescent reagent capable of performing controlled chemiluminescence, which is used for detection and quantification of various substances by chemiluminescence analysis.

[0002]

2. Description of the Related Art Lucigenin (N, N'-dimethyl-9,9'-bisacridinium dinitrate), which has been used for a long time as a chemiluminescent reagent, exhibits a faint light in an alkaline aqueous solution. It is known that light emission is strong when hydrogen oxide is added, and it is used for various trace analysis. However, lucigenin emits light quantitatively in the presence of hydrogen peroxide and alkali, so it can be used for the quantitative determination of hydrogen peroxide. Chemiluminescent enzyme immunoassay (CLEIA) has a problem that it is difficult to use. As a means for solving this problem, chemiluminescence depending on the concentration of the substance to be measured is achieved by using glucose oxidase as a labeling enzyme and causing hydrogen peroxide generated by an oxidation reaction of glucose to act on lucigenin in the presence of an alkali. And the like.

[0003] However, in CLEIA using glucose oxidase as a labeling enzyme, preparation of reagents and operation of a luminescence system are complicated. CLE using peroxidase as a labeling enzyme, which has excellent stability and is relatively easy to handle
Luminol is used as a chemiluminescent reagent that can be used for IA, but it is not always sufficient in terms of sensitivity even if a luminescence promoter is used in combination.

[0004] Therefore, there has been a demand for the development of a chemiluminescent reagent which can be used for CLEIA using peroxidase as a labeling enzyme, which can easily prepare the reagent and operate the luminescence system and can perform high-sensitivity measurement.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel chemiluminescent reagent which emits chemiluminescence depending on the number of moles of peroxidase using hydrogen peroxide as a substrate, and a method for producing the same.

[0006]

Means for Solving the Problems Accordingly, the present inventors have:
As a result of intensive studies to solve the above-mentioned problems, N, N'-disubstituted-9,9'-bisdiacridinium salts such as lucigenin were converted to N, N-disubstituted carboxylic acid amide compounds. Under specific alkaline pH conditions, the chemiluminescence reagent obtained by light irradiation does not react with hydrogen peroxide, and in the presence of hydrogen peroxide and peroxidase, the chemiluminescence depends on the number of moles of peroxidase. And found the present invention based on these findings.

Accordingly, a first aspect of the present invention is to convert N, N'-disubstituted-9,9'-bisacridinium salts under light irradiation in the presence of an N, N-disubstituted carboxylic acid amide compound. The present invention relates to a chemiluminescent reagent characterized by containing a chemiluminescent substance obtained by the reaction.

[0008] A second aspect of the present invention is to provide an N, N'-disubstituted-9,9'-bisacridinium salt under light irradiation in the presence of an N, N-disubstituted carboxylic acid amide compound. The present invention relates to a method for producing a chemiluminescent reagent characterized by reacting.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail. N, used for producing the chemiluminescent reagent of the present invention,
N′-disubstituted-9,9′-bisacridinium salts are
The following general formula (1)

[0010]

Embedded image Can be represented by

In the general formula (1), R ¹ and R ² are
Each is selected from the group consisting of an alkyl group, an aryl group, and a halogenated aryl group, and may be the same or different from each other. Alkyl, aryl and halogenated aryl groups have 1 to 14 carbon atoms, and preferred alkyl groups have 1 to 10 carbon atoms. For example, a linear or branched alkyl group such as a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, and a decyl group can be exemplified. The aryl group preferably has 6 to 20 carbon atoms, and includes a phenyl group, a tolyl group, a xylyl group, and the like, and may be further substituted with an alkyl group. As the aryl group, a phenyl group is particularly preferred. Examples of the halogenated aryl group include a halogenated phenyl group, a halogenated tolyl group, and a halogenated xylyl group, and a chlorophenyl group is particularly preferable.

In the general formula (1), R ³ , R ⁴ , R ⁵
And R ⁶ are each selected from the group consisting of a hydrogen atom, an alkyl group, an aryl group, an alkoxy group, an aryloxy group and a halogen atom, and may be the same or different. These hydrocarbon groups include those having 1 to 2 carbon atoms.
0, preferably 1 to 10. Examples thereof include a linear or branched alkyl group having 1 to 20 carbon atoms, an alkoxy group, an aryl group having 6 to 20 carbon atoms, and an aryloxy group. The aryl group and the aryloxy group are substituted with an alkyl group. It may be something.

In the general formula (1), X ^n- is an n-valent anion, and n is 1 or 2. The anion is not particularly limited and may be a nitrate ion (NO
₃ ^-), halide ions (e.g., chloride ion, fluoride ion, bromide ion, etc.), (meth) phosphate ion (PO ₃ ^- can be mentioned) and the like. Among these anions, nitrate ion is particularly preferred.

Specific examples of N, N'-disubstituted-9,9'-bisacridinium salts include N, N'-dimethyl-
9,9′-bisacridinium salt, N, N′-diethyl-9,9′-bisacridinium salt, N, N′-dipropyl-9,9′-bisacridinium salt, N, N '-Diisopropyl-9,9'-bisacridinium salt, N,
N′-dibutyl-9,9′-bisacridinium salt,
N, N'-diisobutyl-9,9'-bisacridinium salt, N, N-diphenyl-9,9'-bisacridinium salt, N, N'-di-m-chlorophenyl-9,9 '
-Bisacridinium dinitrate (lucigenin) and the like. In particular, N, N'-dimethyl-
9,9'-Bisacridinium dinitrate (lucigenin) is preferred. The N, N′-disubstituted carboxylic acid amide compound used for producing the chemiluminescent reagent of the present invention is
The following general formula (2)

[0015]

Embedded image Can be represented by

In the general formula (2), R ₁ is selected from the group consisting of a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, and an aryl group having 6 to 20 carbon atoms. The aryl group may be substituted with a group selected from the group consisting of an alkyl group, a halogen group, a nitro group, a hydroxyl group, an amino group and the like. R ₂ is selected from the group consisting of a methyl group and an ethyl group; R ₃ is a group consisting of an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, and an aryl group having 6 to 20 carbon atoms. The selected aryl group may be substituted with a group selected from the group consisting of an alkyl group, a halogen group, a nitro group, a hydroxyl group, an amino group, and the like. R ₁ and R ₃ may be bonded to each other to form a ring together with the carbon atom of the carbonyl group and the nitrogen atom of the amide group to which they are bonded.

Specific examples of the N, N-disubstituted carboxylic acid amide compound include N, N'-dimethylformamide,
N, N'-dimethylacetamide, N, N'-dimethylacrylamide, N, N'-dimethylpropionamide, N, N'-dimethylbenzamide, N-methyl-2
-Pyrrolidone and the like can be mentioned without limitation.

The light beam used for producing the chemiluminescent reagent of the present invention has a wavelength range of about 290 to 800.
An ultraviolet-visible part in the range of nm is used, especially about 400-8.
Visible light in the range of 00 nm is desirable. As these light sources, high-pressure mercury lamps, low-pressure mercury lamps, germicidal lamps, fluorescent lamps and incandescent lamps can be used without limitation, but incandescent lamps are particularly preferably used. The chemiluminescent reagent obtained by this light irradiation has a very high luminescence in a short time when manufactured at the same raw material lucigenin concentration and used in the same amount, compared to a chemiluminescent reagent manufactured under natural light without using a light source. Light irradiation plays an important role in the production of a chemiluminescent compound because it shows strength and that if this reaction is performed under light shielding, a chemiluminescent reagent having a peroxidase concentration-dependent property cannot be obtained. Is recognized. N, N'-
Disubstituted-9,9'-bisacridinium salts have a pH of 8
In the vicinity, neither hydrogen peroxide nor hydrogen peroxide in the presence of peroxidase causes a remarkable luminescence reaction.
This is probably because the N'-disubstituted-9,9'bisacridinium cation forms a stable salt with a counter ion, particularly a nitrate ion. However, by performing light irradiation in the presence of a highly polar compound such as an N, N-disubstituted carboxylic acid amide compound, a reaction with the N, N′-disubstituted-9,9′-bisacridinium cation can be prevented. The charge transfer from the anion is promoted, and the salt is changed from a highly ionic salt to a charge transfer complex having a radical property. This complex is stabilized by coordination or solvation of the N, N'-disubstituted carboxylic acid amide compound. Since the stabilized radical compound reacts with active oxygen generated by enzymatic decomposition of hydrogen peroxide and emits light through a dioxetane structure in an excited state, it is thought that luminescence intensity depending on the peroxidase concentration can be obtained. Can be

In the production of the chemiluminescent reagent of the present invention, N, N'-disubstitution-9,
The molar ratio of the N, N-disubstituted carboxylic acid amide compound to the 9'-bisacridinium salt is desirably in excess, but the molar ratio of the N, N'-disubstituted-9,9'-bisacridinium salt to the N, N'-disubstituted-9,9'-bisacridinium salt is preferable. On the other hand, the N, N-disubstituted carboxylic acid amide compound can be used in a molar ratio of 10,000 to 10,000 times, and in this case, the same and / or different N, N-
Disubstituted carboxylic acid amide compounds and / or other solvents can also be used as reaction solvents.

The reaction temperature varies depending on the presence or absence and type of the solvent used, but is generally in the range of -10 to + 150 ° C, preferably 0 to 120 ° C, and particularly preferably 20 to 90 ° C. The reaction time can be 1 minute to 24 hours, preferably 10 minutes to 15 hours, particularly preferably 30 minutes to 10 hours.

The chemiluminescent reagent of the present invention emits light by reacting with hydrogen peroxide and peroxidase under basic conditions, and this reaction can be used for detecting hydrogen peroxide and quantifying peroxidase. By using peroxidase as a labeling substance, it becomes possible to further use it for quantification of various substances.

The novel chemiluminescent reagent of the present invention has a pH of 7.5.
Under 条件 下 13 basic conditions, light is emitted in an amount dependent on the concentration of peroxidase in the presence of excess hydrogen peroxide. It is recognized that this luminescence is enhanced by a luminescence enhancer such as a phenolic compound. Such phenolic compounds include p-hydroxycinnamic acid, p-phenylphenol, p- (4-chlorophenyl) phenol, p- (4-bromophenyl) phenol, p- (4
-Iodophenyl) phenol, p-iodophenol, p-bromophenol, p-chlorophenol, 6
-Hydroxybenzothiazole, 2-naphthol, firefly luciferin and the like.

When performing chemiluminescence analysis using the novel chemiluminescent reagent of the present invention, 10 ^{-8 to} 1M, preferably 1 to ⁸ M
It is used at a concentration ranging from 0 ^-6 to 10 ^-2 M, and the amount used is preferably from 10 to 500 µl, particularly preferably from 50 to 300 µl. The amount of the luminescence enhancer is 0.01 to 100 times the molar amount of the chemiluminescent reagent, preferably 0.1 to 100 times.
It is used in a molar concentration of 10-fold, and its concentration is preferably in the range of 10 ^-6 to 1 M, particularly 10 ^-4 to 10 ^-2 M. In addition, the amount of hydrogen peroxide used in the chemiluminescence reaction needs to be used in a sufficient excess amount with respect to the chemiluminescence reagent,
It is preferable to use the compound in an amount of 30,000 to 10,000 times, especially 10 to 1,000 times, the mole of the chemiluminescent reagent.

In the case of quantifying various substances by labeling an antibody, nucleic acid or the like with peroxidase as a labeling substance, horseradish peroxidase (HRP) is preferably used as the peroxidase, although it is not particularly limited. .

The basic buffer used in the chemiluminescence reaction includes Tris buffer, phosphate buffer, borate buffer,
Carbonate buffer, glycine-sodium hydroxide buffer and the like can be optionally used. The concentration of these buffers was 1 m
It is preferable to use in the range of M to 1M. In addition, additives such as a surfactant and a chelating agent can be optionally used during the reaction.

[0026]

EXAMPLES The present invention will be described below in detail with reference to examples, but the present invention is not limited to the following examples.

Example 1 1.5 mg of lucigenin was placed in a test tube, and 1 ml of N, N-dimethylacetamide was added thereto.
And dissolved in a water bath at a temperature of 30 ° C.
By irradiating a W copy lamp for 7 hours, a chemiluminescent reagent containing a chemiluminescent substance is obtained. [Measurement of peroxidase activity] This chemiluminescent reagent 50
Take 0.1 μl of this and add 0.1 M Tris-HCl buffer (pH 8.4)
After mixing with 2.95 ml, 10 mM p-
0.1 M Tris-HCl buffer of iodophenol (pH 8.4)
100 μl of the solution is added and mixed to prepare a chemiluminescent reagent solution. Also, a plurality of wells of a chemiluminescence measurement microplate were filled with 100 μl of 0.1 M Tris-HCl buffer (pH 8.4) solutions having various concentrations of horseradish peroxidase (HRP), and the solution was injected from a solution injection device. The chemiluminescent reagent solution and 100 μl of a 0.0017% by weight aqueous hydrogen peroxide solution were sequentially injected to cause a luminescence reaction, and the luminescence amount was measured using a luminometer (Luminous CT manufactured by Diatron Co., Ltd.).
As a result of integration for 0 to 5 seconds at −9000 D), luminescence intensities as shown in Table 1 were obtained, and a significant improvement in sensitivity and luminescence intensity was recognized as compared with Comparative Example 1 in which the reaction was performed without light irradiation. Was.

[0028]

[Table 1]

[Comparative Example 1] 1 mg of lucigenin was placed in a test tube, and 2 ml of N, N-dimethylacetamide was added and dissolved in the test tube.
By adding and mixing l, a chemiluminescent reagent containing a chemiluminescent substance is obtained. [Measurement of Peroxidase Activity] Immediately take 20 μl of this chemiluminescent reagent solution, add it to a plurality of chemiluminescence measurement wells, and then add 0.1 M Tris at different concentration levels of horseradish peroxidase (HRP) to each well. Hydrochloric acid buffer (pH 8.4) solution 200 μl and p-iodophenol 10 mM Tris hydrochloric acid buffer (pH 8.4) solution 20 μl
After adding l, 50 µl of a 0.0034% by weight aqueous hydrogen peroxide solution was added thereto, and the luminescence amount was integrated for 0 to 5 seconds by a luminometer (Diatron Co., Ltd., Luminous CT-9000D). Luminescence intensity was obtained.

[0030]

[Table 2]

Example 2 1.5 mg of lucigenin was placed in a test tube, and 1 ml of N, N-dimethylformamide was added thereto.
And dissolved in a water bath at a temperature of 30 ° C.
By irradiating a W copy lamp for 5 hours, a chemiluminescent reagent containing a chemiluminescent substance is obtained. [Measurement of peroxidase activity] This chemiluminescent reagent 50
Take 0.1 μl of this and add 0.1 M Tris-HCl buffer (pH 8.4)
After mixing with 2.95 ml, 10 mM p-
0.1 M Tris-HCl buffer of iodophenol (pH 8.4)
100 μl of the solution is added and mixed to prepare a chemiluminescent reagent solution. A plurality of wells of a chemiluminescence measurement microplate were filled with 100 μl of 0.1 M Tris-HCl buffer (pH 8.4) solutions having various concentrations of horseradish peroxidase (HRP), and the solution was injected from a solution injection device. The chemiluminescent reagent solution and 100 μl of a 0.0017% by weight aqueous hydrogen peroxide solution were sequentially injected to cause a luminescence reaction, and the luminescence amount was measured using a luminometer (Luminous CT manufactured by Diatron Co., Ltd.).
As a result of integration for 0 to 5 seconds at −9000 D), emission intensity as shown in Table 3 was obtained, and improvement was observed in emission intensity as compared with Comparative Example 2 in which the reaction was performed without irradiation with light.

[0032]

[Table 3]

Comparative Example 2 1 mg of lucigenin was placed in a test tube, 2 ml of N, N-dimethylformamide was added and dissolved therein.
By adding and mixing l, a chemiluminescent reagent containing a chemiluminescent substance is obtained. [Measurement of peroxidase activity] Immediately add 20 µl of this chemiluminescent reagent to a plurality of wells for measuring chemiluminescence.
The wells were then filled with horseradish peroxidase (H
RP) at various concentrations of 0.1 M Tris-HCl buffer (p
H8.4) 200 μl of solution and 10 m of p-iodophenol
After adding 20 μl of M Tris-HCl buffer (pH 8.4) solution,
To this, 50 μl of a 0.0034% by weight aqueous hydrogen peroxide solution was added, and a luminometer (Luminous C manufactured by Diatron Co., Ltd.) was added.
T-9000D) as a result of integrating the light emission amount for 0 to 5 seconds,
The emission intensity as shown in Table 4 was obtained.

[0034]

[Table 4]

Example 3 1.5 mg of lucigenin was placed in a test tube, 1 ml of N-methyl-2-pyrrolidone was added and dissolved therein, and the solution was poured into a water bath at a temperature of 30 ° C. to give 250 W.
Irradiation with a copy lamp for 3 hours to obtain a chemiluminescent reagent containing a chemiluminescent substance. [Measurement of peroxidase activity] This chemiluminescent reagent 50
Take 0.1 μl of this and add 0.1 M Tris-HCl buffer (pH 8.4)
After mixing with 2.95 ml, 10 mM p-
0.1 M Tris-HCl buffer of iodophenol (pH 8.4)
100 μl of the solution is added and mixed to prepare a chemiluminescent reagent solution. Also, a plurality of wells of a chemiluminescence measurement microplate were filled with 100 μl of 0.1 M Tris-HCl buffer (pH 8.4) solutions having various concentrations of horseradish peroxidase (HRP), and the solution was injected from a solution injection device. The chemiluminescent reagent solution and 100 μl of a 0.0017% by weight aqueous hydrogen peroxide solution were sequentially injected to cause a luminescence reaction, and the luminescence amount was measured using a luminometer (Luminous CT manufactured by Diatron Co., Ltd.).
As a result of integration at −9000 D) for 0 to 5 seconds, emission intensity as shown in Table 5 was obtained, and improvement in emission intensity was recognized as compared with Comparative Example 3 in which the reaction was performed without irradiation with light.

[0036]

[Table 5]

[Comparative Example 3] 1 mg of lucigenin was placed in a test tube, 2 ml of N-methyl-2-pyrrolidone was added and dissolved therein. The mixture was allowed to stand at room temperature for 90 minutes, and 2 ml of pure water was added and mixed. Thus, a chemiluminescent reagent containing a chemiluminescent substance is obtained. [Measurement of peroxidase activity] Immediately, 20 μl of this chemiluminescent reagent solution was added to a plurality of chemiluminescence measurement wells, and then 0.1 M Tris-HCl buffer (HRP) was added to the wells at various concentrations. pH8.4) 200 μl of solution and p-iodophenol 1
After adding 20 μl of a 0 mM Tris-HCl buffer (pH 8.4) solution, 50 μl of a 0.0034 wt% aqueous hydrogen peroxide solution was added thereto.
1 was added, and the luminescence amount was integrated for 0 to 5 seconds with a luminometer (Luminous CT-9000D manufactured by Diatron), and the luminescence intensity as shown in Table 6 was obtained.

[0038]

[Table 6]

[0039]

The novel chemiluminescent reagent provided by the present invention can be easily produced in a relatively short period of time using inexpensive raw materials, and is capable of producing peroxidase in the presence of hydrogen peroxide and peroxidase. By utilizing the property of chemiluminescence depending on the concentration, a peroxidase enzyme can be detected with high sensitivity. Further, using an enzyme-labeled substance obtained by labeling an antigen, an antibody, a nucleic acid or the like with peroxidase as a labeling substance, using an enzyme immunoassay to detect an antibody, an antigen, or the like, a protein by Western blotting, a DNA and an RNA by Southern blotting and Northern blotting. And using an enzyme-labeled nucleic acid probe, the nucleic acid can be measured specifically and with high sensitivity.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Toshifumi Katsuragi 1-9-4 Horinouchi, Adachi-ku, Tokyo Dainichi Seika Industry Co., Ltd. Technology Research Center (72) Inventor Mio Sato 1-chome Horinouchi, Adachi-ku, Tokyo 9-4 Dainichi Seika Kogyo Co., Ltd. Technology Research Center

Claims (6)

[Claims] [Claim 1] The following general formula (1) (In the above general formula (1), R ¹ and R ² are selected from the group consisting of an alkyl group, an aryl group and a halogenated aryl group, and may be the same or different from each other, and R ³ , R ⁴ , R ⁵ And R ⁶ are selected from the group consisting of a hydrogen atom, an alkyl group, an aryl group, an alkoxy group, an aryloxy group and a halogen group, and may be the same or different from each other, X is an n-valent anion, and n is 1 or 2), N, N′-disubstituted-9,
The 9′-bisacridinium salt is converted to a compound represented by the following general formula (2): (In the general formula (2), R ₁ is selected from the group consisting of a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, and an aryl group having 6 to 20 carbon atoms. aryl group is an alkyl group, a halogen group, a nitro group, may be substituted with a group selected from the group consisting of a hydroxyl group and an amino group, R ₂ is selected from the group consisting of methyl and ethyl, R ₃ Is an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms and 6 carbon atoms.
Is selected from the group consisting of an aryl group to 20, the aryl group, alkyl group, halogen group, a nitro group, may be substituted with a group selected from the group consisting of hydroxyl and amino group, R ₁ and R ₃ may be bonded to each other to form a ring together with the carbon atom of the carbonyl group to which they are bonded and the nitrogen atom of the amide group. A chemiluminescent reagent comprising a chemiluminescent substance obtained by reacting under light irradiation in the presence of an N, N-disubstituted carboxylic acid amide compound represented by the formula (1). 2. The N, N′-disubstituted-9,9′-
Bisacridinium salts are N, N'-dimethyl-9,
The chemiluminescent reagent according to claim 1, which is 9'-bisacridinium dinitrate. 3. The N, N-disubstituted carboxylic acid amide compound is at least one compound selected from the group consisting of N, N-dimethylformamide, N, N-dimethylacetamide and N-methyl-2-pyrrolidone. The chemiluminescent reagent according to claim 1 or 2. 4. The chemiluminescent reagent according to claim 1, wherein the light source used for the light irradiation is a light source that emits visible light. 5. The following general formula (1): (In the above general formula (1), R ¹ and R ² are selected from the group consisting of an alkyl group, an aryl group and a halogenated aryl group, and may be the same or different from each other, and R ³ , R ⁴ , R ⁵ And R ⁶ are selected from the group consisting of a hydrogen atom, an alkyl group, an aryl group, an alkoxy group, an aryloxy group and a halogen group, and may be the same or different from each other, X is an n-valent anion, and n is 1 or 2), N, N′-disubstituted-9,
The 9′-bisacridinium salt is converted to a compound represented by the following general formula (2): (In the general formula (2), R ₁ is selected from the group consisting of a hydrogen atom, an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 2 to 10 carbon atoms, and an aryl group having 6 to 20 carbon atoms. The aryl group may be substituted with an alkyl group, a halogen group, a nitro group, a hydroxyl group, an amino group and the like.
₂ is selected from the group consisting of a methyl group and an ethyl group,
R ₃ is an alkyl group having 1 to 10 carbon atoms, 2 to 10 carbon atoms.
Selected from the group consisting of an alkenyl group and an aryl group having 6 to 20 carbon atoms, wherein the aryl group is substituted with a group selected from the group consisting of an alkyl group, a halogen group, a nitro group, a hydroxyl group and an amino group. R ₁ and R ₃
May be bonded to each other to form a ring together with the carbon atom of the carbonyl group to which they are bonded and the nitrogen atom of the amide group. A) reacting under light irradiation in the presence of an N, N-disubstituted carboxylic acid amide compound represented by the formula (1). 6. The method according to claim 5, wherein the light source used for the light irradiation is a light source that emits visible light.