JP2008180588A - Reagent and method for detecting acrolein and/or acrolein adduct - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a reagent capable of analyzing acrolein or an acrolein adduct with high sensitivity, and a method for detecting acrolein or the acrolein adduct using the reagent. <P>SOLUTION: The reagent for detecting acrolein or the acrolein adduct contains a compound having a compound having a structure represented by the formula (I) (wherein A is a group for forming a ring structure along with C1 and C2; X is a substituted or non-substituted aromatic hydrocarbon group and may form an A-containing ring or a condensed ring, R<SB>1</SB>and R<SB>2</SB>are each independently O, S, etc.). <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to reagents and methods for detecting acrolein and / or acrolein adducts.

  Currently, in most lifestyle-related diseases such as cancer, hypertension, arteriosclerosis, and diabetes, oxidative stress such as active oxygen and lipid peroxide is one of the major causes. Oxidative stress oxidizes important proteins and lipids that make up living organisms and damages DNA, thus inhibiting normal functions of the organism and accelerating aging, leading to lifestyle-related diseases such as those mentioned above . For this reason, in order to prevent diseases, it is necessary to stop oxidative stress to a minimum.

  As a marker for examining the magnitude of oxidative stress, there are 8-hydroxydeoxyguanosine (8-OHdG), acrolein, and improper substances thereof. Among them, acrolein is an α, β-unsaturated aldehyde generated by burning petroleum, coal, etc., tobacco smoke and exhaust gas, and heating of oils and fats, and is a substance having strong cell toxicity. Recently, it has been clarified that acrolein is produced by a lipid peroxidation reaction that is the most typical chemical reaction associated with oxidative stress. For example, a method of diagnosing stroke / asymptomatic cerebral infarction using the content of acrolein as an index has been reported (Patent Document 1). For this reason, it is important to establish a technique for analyzing acrolein or its adducts simply and quickly in order to make use of the results in the field of medical treatment and health care.

  As a method for quantifying acrolein or an acrolein adduct in a sample, an immunological technique using a monoclonal antibody is used. For the measurement, for example, an anti-acrolein antibody and an ELISA kit for measuring acrolein (ACR) (Nippon Yushi) Is commercially available. The advantage of this method is that multiple samples can be measured simultaneously and semi-quantitatively by immunoslot blotting, including simple immunoassays such as the enzyme antibody method. In addition, the localization of the oxidative stress occurrence site can be examined by immune tissue, cell staining and the like. On the other hand, there are problems that sufficient consideration is required for antigen preparation and epitope identification of the obtained antibody, and that the operation is complicated and it takes time to obtain the result (usually about 2 hours).

  On the other hand, an absorptiometric method (colorimetric analysis) is used as a conventional method for analyzing various chemical substances. In particular, the colorimetric analysis method is based on visual quantification, and does not require a large-scale apparatus or skilled technique, so that it can be linked to a simple analysis method that anyone can easily handle. Furthermore, not only analysis in solution (wet chemistry) but also analysis in solid phase media (dry chemistry) including test papers such as litmus paper, miniaturization of equipment, The analysis procedure can be greatly reduced, the analysis time can be shortened, and the analysis operation can be simplified.

  Therefore, a method and a means capable of detecting and quantifying acrolein or an acrolein adduct using a simple method such as a colorimetric analysis method are desired.

JP 2005-304476 A

  An object of the present invention is to develop a reagent capable of analyzing acrolein or an acrolein adduct with high sensitivity and to provide a method for easily and rapidly detecting acrolein or an acrolein adduct.

  As a result of intensive studies to solve the above-mentioned problems, the present inventor succeeded in synthesizing a dye compound showing a color change by reaction with acrolein or an acrolein adduct, and using this dye compound, high sensitivity and The inventors have found that acrolein or an acrolein adduct can be easily detected, and have completed the present invention.

〔式中、
Ａは、炭素原子１及び炭素原子２と共に環状構造を形成する基であり、
Ｘは、置換若しくは非置換の芳香族炭化水素基、及び置換若しくは非置換の複素環基から選択される基であり、ここでＸはＡを含む環と縮合環を形成していてもよく、
Ｒ_１及びＲ_２は、それぞれ独立して、酸素原子、硫黄原子、二級アミン又は三級アミンであり、ここで、Ｒ_１及び／又はＲ_２が三級アミンである場合には、炭素数１〜１０のアルキル鎖を有するアミノ基である。〕
を有する化合物を含むことを特徴とするアクロレイン又はアクロレイン付加物検出用試薬である。 That is, the present invention provides a structure represented by the following formula (I):

[Where,
A is a group that forms a cyclic structure with carbon atom 1 and carbon atom 2;
X is a group selected from a substituted or unsubstituted aromatic hydrocarbon group and a substituted or unsubstituted heterocyclic group, wherein X may form a condensed ring with the ring containing A;
R ₁ and R ₂ are each independently an oxygen atom, a sulfur atom, a secondary amine, or a tertiary amine. Here, when R ₁ and / or R ₂ is a tertiary amine, the number of carbon atoms An amino group having 1 to 10 alkyl chains. ]
A reagent for detecting acrolein or an acrolein adduct, comprising a compound having

  In the detection reagent, A in the formula (I) is, for example, a substituted or unsubstituted aromatic hydrocarbon group or heterocyclic group.

  X in formula (I) is selected from the group consisting of substituted or unsubstituted benzene, naphthalene, anthracene, pyrene, biphenyl, coumarin, benzothiazole, fluorescein, rhodamine, azobenzene, pyridine, bipyridine, quinoline and phenanthroline. And a group of the compound.

  In the detection reagent, it is preferable that A and X in the formula (I) together form a naphthyl group.

In the detection reagent, _one of R ₁ and R ₂ in formula (I) is, for example, a sulfur atom and the other is a tertiary amine.

〔式中、Ｒ_３は、水素、又は炭素数１〜１０のアルキル基である。〕
であることが好ましい。また式（Ｉ）は、下記式（ＩＩＩ）： In the detection reagent, the formula (I) is, for example, the following formula (II):

[Wherein, R ₃ represents hydrogen or an alkyl group having 1 to 10 carbon atoms. ]
It is preferable that Formula (I) is represented by the following formula (III):

であることが特に好ましい。

It is particularly preferred that

  The present invention is also a method for detecting acrolein or an acrolein adduct in a test sample, wherein the test sample is brought into contact with the detection reagent to detect a change in the color of the detection reagent.

  In the above method, the color change can be detected by methods such as visual observation, absorptiometry, reflection spectrum analysis, and the like.

  According to the present invention, a detection reagent and a detection method for detecting acrolein or an acrolein adduct are provided. When such a detection reagent is used, it is possible to detect acrolein or an acrolein adduct with high sensitivity, in a short time, and without using a complicated instrument. Therefore, this detection reagent and this detection method are useful in the fields of biochemistry, medicine, and analytical chemistry.

  In the present invention, when a compound having the structure of formula (I) and acrolein or an acrolein adduct are brought into contact with each other, they form a Schiff base, so that a color change occurs due to the appearance of a new absorption band due to the extension of the conjugated system. Based on what happens. By measuring the change in absorption spectrum due to this color change, qualitative analysis and quantitative analysis of acrolein or an acrolein adduct can be performed.

  Below, the detection reagent which concerns on this invention, and the detection method of acrolein or an acrolein adduct using the same are demonstrated.

〔式中、
Ａは、炭素原子１及び炭素原子２と共に環状構造を形成する基であり、
Ｘは、置換若しくは非置換の芳香族炭化水素基、及び置換若しくは非置換の複素環基から選択される基であり、ここでＸはＡを含む環と縮合環を形成していてもよく、
Ｒ_１及びＲ_２は、それぞれ独立して、酸素原子、硫黄原子、二級アミン又は三級アミンであり、ここで、Ｒ_１及び／又はＲ_２が三級アミンである場合には、炭素数１〜１０のアルキル鎖を有するアミノ基である。〕
で表される構造を有する化合物を含むことを特徴とする。 1. Reagent for detection The acrolein or acrolein adduct detection reagent according to the present invention (hereinafter also referred to as the present detection reagent) has the general formula (I):

[Where,
A is a group that forms a cyclic structure with carbon atom 1 and carbon atom 2;
X is a group selected from a substituted or unsubstituted aromatic hydrocarbon group and a substituted or unsubstituted heterocyclic group, wherein X may form a condensed ring with the ring containing A;
R ₁ and R ₂ are each independently an oxygen atom, a sulfur atom, a secondary amine, or a tertiary amine. Here, when R ₁ and / or R ₂ is a tertiary amine, the number of carbon atoms An amino group having 1 to 10 alkyl chains. ]
It contains the compound which has the structure represented by these.

  The present inventor selected a thiazolinone hydrazone group as a site that forms a Schiff base after reaction with acrolein or an acrolein adduct and promotes a color change. In addition, a naphthyl group was introduced into the thiazolinone hydrazone group so that the conjugated system was elongated and an absorption band was generated in the visible region. Therefore, when this detection reagent is mixed with acrolein or an acrolein adduct, the dye compound and acrolein or acrolein adduct react to form a Schiff base to form a covalent bond. At the same time, since the conjugated system extends, a new absorption band is generated in the visible region (near 650 nm), and the color changes from a colorless and transparent state to dark green to blue. For this reason, it is possible to detect a trace amount of acrolein or an acrolein adduct by measuring a change in the color of the reagent.

  Accordingly, in the above general formula (I), A may be any group as long as it is a group that forms a cyclic structure with the carbon atom 1 and the carbon atom 2. For example, A may be a group having 4 to 30 atoms in which a cyclic structure is formed by atoms selected from the group consisting of carbon atom 1 and carbon atom 2 and carbon atom, oxygen atom, sulfur atom and nitrogen atom. . More specifically, A is a substituted or unsubstituted aromatic hydrocarbon group, a substituted or unsubstituted heterocyclic group, or a linear or branched cycloalkyl group, cycloalkenyl group, cycloalkynyl group, cyclic Type oxyethylene chains, cyclodextrins, and the like. A may have a substituent in addition to the X group. Examples of such substituents include, but are not limited to, alkyl groups (methyl, ethyl, n-propyl, etc.), alkoxy groups (methoxy, ethoxy, phenoxy, etc.), halogen atoms (fluoro, chloro, bromo, etc.) ), Aralkyl groups (such as benzyl and phenethyl), aryl groups (such as phenyl, toluyl, biphenyl, naphthyl, and pyrenyl), amino groups, nitro groups, cyano groups, and morpholino groups. A may have one or more of the above substituents.

  In a preferred embodiment, A in Formula (I) is a substituted or unsubstituted aromatic hydrocarbon, a substituted or unsubstituted heterocyclic compound, and the like.

  In the formula (I), X is an arbitrary group selected from a substituted or unsubstituted aromatic hydrocarbon group and a substituted or unsubstituted heterocyclic group. The aromatic hydrocarbon group includes, for example, a phenyl group and a phenylene group, and is not specifically limited. However, phenol (1-5 valent), toluene, xylene, benzoic acid, salicylic acid, aniline, nitrobenzene. Aralkyl (benzyl group, phenethyl group, etc.) and the like. The aromatic hydrocarbon group may be a polycyclic aromatic group having 10 to 30 carbon atoms, preferably 10 to 18 carbon atoms, such as a biphenyl group, a terphenyl group, a naphthyl group, an acenaphthylenyl group, an anthryl group, A phenanthryl group, a pyrenyl group, a benzoanthryl group, a naphthacenyl group, a picenyl group, a fluorenyl group, and the like are included. Further, as the heterocyclic group, a 3-membered, 4-membered, 5-membered, 6-membered carbon atom having 1 to several hetero atoms (oxygen, nitrogen, sulfur, etc.) having 6 to 20, preferably 6 to 12 carbon atoms. Alternatively, a seven-membered heterocyclic group can be used, for example, pyrrolyl group, pyrrolylene group, pyridyl group, pyrimidinyl group, pyrazinyl group, pyridazinyl group, thienyl group, furyl group, furylene group, quinolyl group, quinoxalinyl group, naphthyridinyl group, Examples include quinazolinyl group, phenanthridinyl group, indolizinyl group, phenazinyl group, carbazolyl group, oxazolyl group, thiazolyl group, acridinyl group, phenazinyl group and the like.

  Examples of substituents include, but are not limited to, alkyl groups (methyl, ethyl, n-propyl, etc.), alkoxy groups (methoxy, ethoxy, phenoxy, etc.), halogen atoms (fluoro, chloro, bromo, etc.), aralkyl Examples include groups (benzyl, phenethyl, etc.), aryl groups (phenyl, toluyl, biphenyl, naphthyl, pyrenyl, etc.), amino groups, nitro groups, cyano groups, morpholino groups, and the like. X may have one or more of the above substituents.

  In the formula (I), the compound of the specific group corresponding to X is not limited, but is substituted or unsubstituted benzene, naphthalene, anthracene, pyrene, biphenyl, coumarin, benzothiazole, fluorescein, rhodamine Aromatic hydrocarbons and heterocyclic compounds such as azobenzene, pyridine, bipyridine, quinoline and phenanthroline are preferred. More preferably, it is a group having the structure shown below.

  In the above structure, R is independently of each other a hydrogen atom, a linear or branched alkyl group having 1 to 10 carbon atoms, a linear or branched ether having 1 to 10 carbon atoms, a phenyl group, Phenyl group in which a part of phenyl group is substituted with amino group, halogen, nitro group, amino group, cyano group, nitro group, carboxylic acid or its salt or ester or amide, sulfonic acid or its salt or ester or amide, thiol group , Hydroxyl groups or salts thereof, ketones, halogens, sugars and the like, but are not limited thereto. n is an integer of 1 to 4, and when n is 2 or more, each R may be the same as or different from each other.

  In a preferred embodiment, X in formula (I) is a substituted or unsubstituted aromatic hydrocarbon, a heterocyclic compound, or the like. In another preferred embodiment, X is a ring containing A and a condensed ring such as a naphthyl group, anthracenyl group, tetracenyl group, pyrenyl group, pyridyl group, biphenyl group, coumarin group, benzothiazole group, fluorescein group, rhodamine group. Azobenzene group, bipyridine group, quinoline group, phenanthroline group and the like. Furthermore, these functional groups may have a substituent. Examples of such substituents include, but are not limited to, alkyl groups (methyl, ethyl, n-propyl, etc.), alkoxy groups (methoxy, ethoxy, phenoxy, etc.), halogen atoms (fluoro, chloro, bromo, etc.) ), Aralkyl groups (such as benzyl and phenethyl), aryl groups (such as phenyl, toluyl, biphenyl, naphthyl, and pyrenyl), amino groups, nitro groups, cyano groups, and morpholino groups.

  In a particularly preferred embodiment, A and X in formula (I) together form a naphthyl group.

In Formula (I), R ₁ and R ₂ are each independently an oxygen atom, a sulfur atom, a secondary amine, or a tertiary amine. When R ₁ and / or R ₂ is a tertiary amine, it is preferably an amino group having an alkyl chain having 1 to 10 carbon atoms (for example, methyl, ethyl, n-propyl, etc.). R ₁ and R ₂ may be the same as or different from each other.

In a preferred embodiment, _one of R ₁ and R ₂ is a sulfur atom and the other is a tertiary amine. Particularly preferably, R ₁ and R ₂ are tertiary amines, one of which is a sulfur atom and the other of which has a methyl group.

〔式中、Ｒ_３は、水素、又は炭素数１〜１０のアルキル基である〕。 Specific examples of the compound having the structure represented by the formula (I) include compounds represented by the following formula (II):

[Wherein R ₃ is hydrogen or an alkyl group having 1 to 10 carbon atoms].

Particularly preferably, the compound having a structure represented by the formula (I) is a compound represented by the following formula (III):

  The compound represented by the formula (III) is colorless in a free state, but exhibits a dark green color when bound to FDP-lysine, which is one of acrolein and an acrolein adduct.

Compounds having the structure of formula (I) can be easily synthesized by those skilled in the art according to methods known in the art. For example, as shown in FIG. 2, the compound represented by the formula (II) is obtained by reacting compound 1 and compound 2 in the presence of triethylamine (TEA) and 2,3,4,5-tetrahydrofuran (THF). Then, after obtaining compound 3, the amine in compound 3 can be obtained by substituting with an arbitrary substituent (R ₃ ). For example, when a compound represented by the formula (III) is obtained, the amine in compound 3 may be substituted with a methyl group by reacting compound 3 with methyl iodide in the presence of potassium carbonate and a suitable solvent. it can. After the compound is synthesized, purification is performed using an operation such as recrystallization, reprecipitation, or column chromatography.

2. Detection method of acrolein and acrolein adducts When this detection reagent is brought into contact with acrolein or acrolein adducts, they form a Schiff base as shown in the reaction formulas of FIGS. 1A and 1B. A color change is caused by the appearance of a new absorption band. Therefore, acrolein or an acrolein adduct in a sample can be detected by bringing the detection reagent into contact with a sample that may contain acrolein or an acrolein adduct and detecting a color change. This detection reagent can react with acrolein or an acrolein adduct even in the presence of a competitor or inhibitor, and also reacts with a trace amount of acrolein or an acrolein adduct. Can be detected. In the present invention, “detection” of acrolein or an acrolein adduct not only detects the presence or absence of acrolein or an acrolein adduct in a sample, but also quantitatively detects acrolein or an acrolein adduct. Including.

Acrolein to be detected using this detection reagent is an unsaturated aldehyde having a molecular formula of C ₃ H ₄ O and a molecular weight of 56.06 (CAS registration number 107-02-8). The detection of acrolein is very important because it is a volatile, flammable liquid with a pungent odor and is toxic to the environment and organisms. An acrolein adduct is a general term for compounds having a piperidine structure formed by a reaction between two molecules of acrolein and a primary alkylamine. For example, FDP-lysine is an acrolein-added amino acid in which a lysine residue of a protein is added to acrolein. (N-formylpiperidinolysine). The structures of acrolein and FDP-lysine are shown below.

  The test sample is not particularly limited as long as it is suspected of containing acrolein or an acrolein adduct, and may be either a liquid sample or a solid sample. Specifically, for example, solutions that may contain acrolein or acrolein adducts, biological samples (urine, saliva, cerebrospinal fluid, etc.), environmental samples (industrial wastewater, sludge, air, exhaust gas, etc.) And food-derived samples.

  The detection reagent to be used may be used after being dissolved in an appropriate solvent or buffer solution (for example, MeOH: phosphate buffer solution) or fixed to an appropriate carrier (membrane, test paper, etc.). May be used.

  Detection of acrolein or an acrolein adduct typically involves contacting the sample with the detection reagent and detecting a change in the color of the detection reagent. In the present invention, “contact” means that acrolein or an acrolein adduct present in a sample and a dye compound in the detection reagent are brought into close proximity so as to bind, for example, a liquid sample and the main detection. Mixing with a reagent solution, infiltrating a liquid sample into a carrier (membrane, test paper, etc.) on which the detection reagent is fixed, applying the detection reagent to a solid sample, Operations such as immersing a solid sample in a reagent are included.

  Conditions for bringing the sample into contact with the detection reagent can be appropriately selected by those skilled in the art in consideration of the type and amount of the sample to be used, the contact form, and the like. For example, when the amount of acrolein or an acrolein adduct contained in a sample is estimated to be about 0 to 500 μM, 100 to 20000 μM, preferably 5000 μM of this detection reagent is used. The contact is performed at room temperature (about 20 to 30 ° C., preferably about 25 ° C.) for about 5 to 30 minutes, preferably about 10 to 20 minutes.

  After contacting the sample with the present detection reagent, a change in the color of the detection reagent is detected using methods known in the art. The change in color can be detected by visual observation, absorptiometry (using a spectrophotometer, a plate reader, etc.), reflection spectrum analysis, or the like. If a color change is observed, it indicates the presence of acrolein or an acrolein adduct in the sample.

  When quantifying acrolein or acrolein adduct in a sample, prepare a standard sample containing a known concentration of acrolein or acrolein adduct, react the standard sample with this detection reagent, It is preferable to prepare a calibration curve based on the change (for example, absorbance) and compare it with the color change of the present detection reagent when the test sample is used.

  The detection reagent and detection method for acrolein or an acrolein adduct according to the present invention can detect acrolein or an acrolein adduct with high sensitivity and in a short time without using a complicated instrument. In addition, since this detection reagent binds to acrolein or an acrolein adduct at a certain ratio, it can be easily quantified by preparing a calibration curve. Furthermore, since this detection reagent can bind acrolein or an acrolein adduct satisfactorily even in the presence of an interfering substance, it can detect acrolein or an acrolein adduct in any sample with high accuracy. Further, since the dye compound to be used can be easily synthesized, the cost and labor required for the reagent can be reduced. From the above, the present detection reagent and the present detection method are useful in the fields of biochemistry, medicine, and analytical chemistry.

  In this example, a dye compound of formula (III) was synthesized according to the following reaction formula and the scheme shown in FIG.

Specifically, compound 1 (0.2 g, 1.0 mmol), compound 2 (0.2 g, 0.9 mmol), triethylamine (0.1 mL, 0.9 mmol), and THF 40 mL were added to a 50 ml three-necked flask, and argon was added. The mixture was refluxed for 24 hours under an air stream. After the solvent was distilled off under reduced pressure, the residue was dissolved in chloroform and washed with water. After drying over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure. Purification by column chromatography (SiO ₂ , chloroform) gave a yellow solid (yield 55%).

Subsequently, as shown above, compound 3 (0.2 g, 1.0 mmol), methyl iodide (1.0 g, 10 mmol), potassium carbonate (0.1 g, 0.9 mmol), and acetone were added to a 50 ml three-necked flask. 40 mL was added and refluxed for 24 hours under an argon stream. After distilling off the solvent under reduced pressure, the residue was dissolved in chloroform and washed with water. After drying over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure. Purification by column chromatography (SiO ₂ , chloroform) gave a yellow solid (yield 80%).

  In this example, it was confirmed whether the dye compound synthesized in Example 1 responded to acrolein.

First, after mixing a dye solution and an acrolein solution having the following concentrations, an absorption spectrum was measured at 25 ° C.
[Dye] = 1 wt% (in MeOH: phosphate buffer (pH 5.0) = 1: 2 v / v)
[Acrolein] = 100 μM

  FIG. 3 shows absorption spectra of the dye before and after the addition of acrolein. The dye alone had absorption in the ultraviolet region, and did not have an absorption band in the visible region (longer wavelength side than 500 nm). On the other hand, when acrolein was added, a new absorption band was observed at 500 to 750 nm.

  FIG. 4 shows photographs of the dye solution before and after the addition of acrolein. The pigment alone was colorless, but it changed to dark green when the pigment and acrolein reacted.

  Subsequently, in order to observe the reaction rate between the dye and the acrolein, the dye solution and the acrolein solution were mixed, and the change with time in the absorbance at 650 nm was measured. The results are shown in FIG. It took about 10 minutes for the absorbance to become constant.

  Next, when the absorbance at 650 nm when the acrolein concentration was changed from 0 to 500 μM was plotted against the acrolein concentration, a good linear relationship as shown in FIG. 6 was obtained. Therefore, it was found that acrolein in the sample can be quantitatively detected using this detection reagent.

  In this example, it was confirmed whether the dye compound synthesized in Example 1 responded to FDP-lysine.

First, after mixing a dye solution having the following concentration and an FDP-lysine solution, an absorption spectrum was measured at 25 ° C.
[Dye] = 1 wt% (in MeOH: phosphate buffer (pH 5.0) = 1: 2 v / v)
[FDP-lysine] = 100 μM

  FIG. 7 shows absorption spectra of the dye before and after the addition of FDP-lysine. The dye alone had absorption in the ultraviolet region, and did not have an absorption band in the visible region (longer wavelength side than 500 nm). On the other hand, when FDP-lysine was added, a new absorption band was observed at 500 to 750 nm.

  In addition, with the dye alone, the color of the solution was colorless, but when the dye and FDP-lysine reacted, it changed to dark green (results not shown).

  Subsequently, in order to observe the reaction rate of the dye and FDP-lysine, the dye solution and the FDP-lysine solution were mixed, and then the change with time in absorbance at 650 nm was measured. The results are shown in FIG. It took about 10 minutes for the absorbance to become constant.

  Next, when the absorbance at 650 nm when the concentration of FDP-lysine was changed from 0 to 500 μM was plotted against the FDP-lysine concentration, a good correlation as shown in FIG. 9 was obtained. Therefore, it was found that the acrolein adduct in the sample can be quantitatively detected using this detection reagent.

  In this example, it was confirmed how much the reaction between the dye compound synthesized in Example 1 and FDP-lysine was affected by the interfering substance.

After mixing the following dye solution, FDP-lysine solution, and interfering substances shown in Table 1 below, the absorption spectrum was measured at 25 ° C.
[Dye] = 1 wt% (in MeOH: phosphate buffer (pH 5.0) = 1: 2 v / v)
[FDP-lysine] = 100 μM
[Interfering substance] = 10, 50, 100 mM

  As a result, the results as shown in Table 1 above were obtained. From this result, it was found that this detection reagent can detect acrolein or an acrolein adduct with high accuracy even in the presence of an interfering substance.

  According to the present invention, a detection reagent and a detection method for detecting acrolein or an acrolein adduct are provided. When such a detection reagent is used, it is possible to detect acrolein or an acrolein adduct with high sensitivity, in a short time, and without using a complicated instrument. Therefore, this detection reagent and this detection method are useful in the fields of biochemistry, medicine, and analytical chemistry.

The reaction formula of the structure (A) of the dye compound in this detection reagent and an example (B) of the dye compound and acrolein or an acrolein adduct is shown. 1 shows a synthesis scheme of a dye compound of formula (III). The absorption spectrum of the pigment before and after the addition of acrolein is shown. It is a photograph of the dye solution before and after the addition of acrolein. The time change of the light absorbency in 650 nm after acrolein addition is shown. The relationship between the acrolein concentration and the absorbance at 650 nm is shown. The absorption spectrum of the pigment | dye before and after addition of FDP-lysine is shown. The time change of the light absorbency in 650 nm after FDP-lysine addition is shown. The relationship between FDP-lysine concentration and absorbance at 650 nm is shown.

Claims (9)

A reagent for detecting acrolein or an acrolein adduct comprising a compound having a structure represented by the following formula (I):

[Where,
A is a group that forms a cyclic structure with carbon atom 1 and carbon atom 2;
X is a group selected from a substituted or unsubstituted aromatic hydrocarbon group and a substituted or unsubstituted heterocyclic group, wherein X may form a condensed ring with the ring containing A;
R ₁ and R ₂ are each independently an oxygen atom, a sulfur atom, a secondary amine, or a tertiary amine. Here, when R ₁ and / or R ₂ is a tertiary amine, the number of carbon atoms An amino group having 1 to 10 alkyl chains. ]   The detection reagent according to claim 1, wherein A is a substituted or unsubstituted aromatic hydrocarbon group or heterocyclic group.   X is a substituted or unsubstituted group of a compound selected from the group consisting of benzene, naphthalene, anthracene, pyrene, biphenyl, coumarin, benzothiazole, fluorescein, rhodamine, azobenzene, pyridine, bipyridine, quinoline and phenanthroline, The detection reagent according to claim 1 or 2.   The detection reagent according to claim 1, wherein A and X together form a naphthyl group. The detection reagent according to any one of claims 1 to 4, wherein one of R ₁ and R ₂ is a sulfur atom, and the other is a tertiary amine. Formula (I) is represented by the following formula (II):

[Wherein, R ₃ represents hydrogen or an alkyl group having 1 to 10 carbon atoms. ]
The detection reagent according to any one of claims 1 to 5, wherein Formula (I) is represented by the following formula (III):

The detection reagent according to claim 6, wherein   Acrolein or acrolein addition in a test sample, wherein the test sample is brought into contact with the detection reagent according to any one of claims 1 to 7 to detect a change in the color of the detection reagent. How to detect things.   The method according to claim 8, wherein the color change is detected by a method selected from the group consisting of visual observation, absorptiometry, and reflection spectrum analysis.

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