JPWO2004072649A1 - Reagent for superoxide anion analysis - Google Patents

Abstract

Eliminates the problems of the conventional technology, and features high detection sensitivity, light emission in the long wavelength range, reduced influence on light emission by components such as proteins, lipids and sugar chains, improved water solubility, and analysis with high accuracy It is an object of the present invention to provide a reagent for analyzing a superoxide anion for detecting a superoxide anion having a salt, a method for producing the same, and a method for analyzing a superoxide anion using the same. The above object can be achieved by a compound in which a fluorescein molecule and an imidazo [1,2-a] pyrazin-3 (7H) -one molecule are covalently bonded to a cyclodextrin molecule.

Description

  The present invention relates to the analysis of superoxide anions. More specifically, the present invention contains, as an active ingredient, a compound in which a luminescent reagent (for example, a fluorescein molecule and an imidazo [1,2-a] pyrazin-3 (7H) -one molecule) is covalently bonded to a cyclodextrin molecule. The present invention relates to a reagent for analyzing superoxide anion, a method for producing the same, and a method for analyzing superoxide anion using the same.

ＣＬＡの最大発光波長は約３８０−４１０ｎｍであり、ＭＣＬＡの最大発光波長は約４２０−４６０ｎｍであり、ＦＣＬＡの最大発光波長は約５２０−５４０ｎｍである。生体のスーパーオキシドアニオン分析では他の成分の影響が少ない方が望ましいことから、より長波長域での発光が望まれる。このため、長波長域の発光を示すＦＣＬＡがよいとされる。また、ＣＬＡ、ＭＣＬＡ、およびＦＣＬＡのうち、発光強度はＭＣＬＡがもっとも強く、生体のスーパーオキシドアニオン分析では発光強度が高い方が望ましいことから、発光強度の点ではＭＣＬＡがよいとされる。従って、ＭＣＬＡとＦＣＬＡの両化合物の良い特性を兼ね備えた、すなわち、高い発光強度を有し、かつ発光波長が長波長領域であるという特性を有した発光分析試薬の創製が望まれる。更に好ましくは、ＭＣＬＡおよびＦＣＬＡよりも発光強度が高く、しかも、さらなる長波長領域（例えば、オレンジ色や赤色の発光）での発光を示す発光試薬が望まれる。また、ＣＬＡ、ＭＣＬＡ、ＦＣＬＡの発光は、生体中のタンパク質、脂質や糖鎖等の成分による影響を受けやすいため、スーパーオキシドアニオンの分析精度が低いと言う問題がある。また、ＣＬＡ、ＭＣＬＡ、ＦＣＬＡは水溶性が低いために、被検体系への注入が困難であり、加えて細胞表面等への吸着が生じ易く有効に作用できずに発光性能が低下するという問題がある。
本発明は、以上のとおりの事情に鑑みてなされたものであり、その目的は、従来技術の問題点を解消し、高い検出感度、長波長領域での発光、タンパク質・脂質や糖鎖等の成分による発光に対する影響の低減化、水溶性の向上、高い精度での分析等の特性を有するスーパーオキシドアニオンを検出するためのスーパーオキシドアニオン分析用試薬とその製造法およびそれを用いたスーパーオキシドアニオンの分析方法を提供することである。Superoxide anion is a kind of active oxygen and has physiological activity such as detoxification activity in living body. On the other hand, excessive superoxide anion destroys normal sites, leading to canceration and aging. In vivo analysis of superoxide anions having such functions is important, and colorimetric methods, fluorescent methods, and ESR methods have been used so far. However, these methods are not preferable analysis methods because they are analyzed by destroying a specimen in addition to low sensitivity. On the other hand, the luminescence method that has been developed in recent years has a high sensitivity and can analyze a specimen nondestructively.
As the luminescent reagent used in the luminescence method, 2-methyl-6-phenylimidazo [1,2-a] pyrazin-3 (7H) -one (CLA: Compound A in the figure below), 2-methyl-6- (4- Methoxyphenyl) imidazo [1,2-a] pyrazin-3 (7H) -one (MCLA: lower compound B), 3,7-dihydro-6- [4- [2- [N ′ (5-fluoresceinyl) thioureido ] -Ethoxy] phenyl] -2-methylimidazo [1,2-a] pyrazin-3 (7H) -one (FCLA: Compound C shown below) is known (for example, Tokyo Chemical Industry Co., Ltd./Tokyo Chemical Sales Co., Ltd.) Company).

The maximum emission wavelength of CLA is about 380-410 nm, the maximum emission wavelength of MCLA is about 420-460 nm, and the maximum emission wavelength of FCLA is about 520-540 nm. In the superoxide anion analysis of a living body, it is desirable that the influence of other components is small, and therefore light emission in a longer wavelength region is desired. For this reason, the FCLA showing light emission in the long wavelength region is considered good. In addition, among CLA, MCLA, and FCLA, MCLA has the strongest emission intensity, and it is desirable that the emission intensity is high in the superoxide anion analysis of a living body. Therefore, it is desired to create a luminescence analysis reagent having the good characteristics of both the MCLA and FCLA compounds, that is, having a high emission intensity and a characteristic that the emission wavelength is in the long wavelength region. More preferably, a luminescent reagent that has higher luminescence intensity than MCLA and FCLA and that emits light in a longer wavelength region (for example, orange or red light emission) is desired. In addition, the light emission of CLA, MCLA, and FCLA is easily affected by components such as proteins, lipids, and sugar chains in the living body, so that there is a problem that the analysis accuracy of the superoxide anion is low. In addition, since CLA, MCLA, and FCLA are low in water solubility, it is difficult to inject them into the subject system, and in addition, they are easily adsorbed on the cell surface, etc., and cannot effectively function, resulting in a decrease in luminous performance. There is.
The present invention has been made in view of the circumstances as described above, and its purpose is to solve the problems of the prior art, such as high detection sensitivity, light emission in a long wavelength region, protein / lipid, sugar chain, etc. Reagent for superoxide anion analysis for detecting superoxide anion having characteristics such as reduction of luminescence by components, improvement of water solubility, analysis with high accuracy, and its production method, and superoxide anion using the same It is to provide an analysis method.

As a result of diligent research, the inventor of the present invention has solved the above-mentioned problems. The first invention is the compounds (13) and (14) shown in FIG. 1 (where M is an integer of 5 to 14, n Is an integer of 0 to (M-1), R _{1 to} R ₃ , and R _{5 to} R ₁₃ are H or C _{1 to} C ₂₀ alkyl group, alkenyl group, alkynyl group, phenyl group, hydroxyl group, alkyl hydroxyl group Group, benzyl group, aryl group, halogen group, nitro group, cyano group, sulfonyl group, carbonyl group, amino group, naphthyl group, indolyl group, and X and Y are integers of 0 to 20, R ₄ is a phenyl oxide ring having 0 to 4 substitution degrees) as an active ingredient. This compound contains both a molecule that reacts with superoxide and a molecule that emits light upon receiving the energy generated in one cyclodextrin molecule, and it can analyze the superoxide anion well. it can.
Cyclodextrin is a non-reducing cyclic oligosaccharide in which D-glucopyranose is linked by α1 → 4, and is composed of 6 molecules, 7 molecules, and 8 molecules of glucopyranose, α-, β-, γ-, respectively. It is called cyclodextrin. As the cyclodextrin of the present invention, in addition to these α to γ-cyclodextrins, cyclic oligosaccharides in which 9 to 15 molecules of glucopyranose are bonded can be used. Preferably, 6 to 6 molecules of glucopyranose are used. Eight molecules of α-, β-, and γ-cyclodextrin are preferable, and γ-cyclodextrin is more preferable.
The second invention is characterized in that it contains at least a compound in which a fluorescein molecule and an imidazo [1,2-a] pyrazin-3 (7H) -one molecule are covalently bonded to a cyclodextrin molecule as an active ingredient. The superoxide anion analysis reagent is represented by the formulas (15) and (16) in FIG. 2 (where M is an integer of 5 to 7, and n is an integer of 0 to (M-1)). ) Is preferably a compound in which a fluorescein molecule and an imidazo [1,2-a] pyrazin-3 (7H) -one molecule are both covalently bonded to a cyclodextrin molecule represented by Among these, preferably, when M = 7, that is, a compound in which a fluorescein molecule and an imidazo [1,2-a] pyrazin-3 (7H) -one molecule are both covalently bonded to a γ-cyclodextrin molecule as an active ingredient It is a reagent for analyzing superoxide anion.
The third invention provides a method for producing compounds (15) and (16) in which a fluorescein molecule and an imidazo [1,2-a] pyrazin-3 (7H) -one molecule are covalently bonded to a cyclodextrin molecule. To do.
The fourth invention provides a method for analyzing superoxide anion, characterized in that the luminescence intensity is measured after contacting the reagent for analyzing superoxide anion with a sample solution.
According to the present invention, there are provided a superoxide anion analysis reagent for easily and accurately analyzing a superoxide anion, a production method thereof, and an analysis method. The reagent for analyzing superoxide anion of the present invention is water-soluble and reacts with the superoxide anion to show high emission intensity. In addition, since the maximum emission wavelength is about 530 nm, the influence of other components is small and efficient analysis of the superoxide anion becomes possible.

FIG. 1 is a chemical structural formula showing compounds (13) and (14) according to the present invention.
FIG. 2 is a chemical structural formula showing compounds (15) and (16) according to the present invention.
FIG. 3 illustrates the procedure for producing compounds (1) and (2) when M = 7 in compounds (15) and (16).

The inventor has so far reported the synthesis of a compound in which MCLA is covalently bonded to cyclodextrin and the luminescence reaction thereof (for example, Carbohydr. Res., 306, 177-187 (1998). Luminescence, 14, 303). -314 (1999), JP-A-10-77286).
The inventor has conducted extensive research based on these findings, and as a result, the cyclodextrin molecule reacts with a superoxide anion (for example, an imidazo [1,2-a] pyrazin-3 (7H) -one molecule, Or a derivative thereof) and a compound in which a molecule that receives light from the substance and emits light (for example, a fluorescein molecule or a derivative thereof) is covalently bonded, can be used to detect a superoxide anion in a specimen. The present invention has been found and led to the present invention.
The reagent for analyzing superoxide anion according to the present invention contains at least a compound in which an imidazo [1,2-a] pyrazin-3 (7H) -one molecule and a fluorescein molecule are covalently bonded to a cyclodextrin molecule. It is.
Specifically, a compound in which an imidazo [1,2-a] pyrazin-3 (7H) -one molecule and a fluorescein molecule are covalently bonded to the cyclodextrin molecule used in the reagent for analyzing superoxide anion of the present invention is shown in FIG. The formulas (1) and (2) are preferably exemplified.
The reagent for analyzing superoxide anion of the present invention is water-soluble, reacts with the superoxide anion, emits green light having a maximum emission wavelength of about 530 nm, and exhibits higher emission intensity than MCLA and FCLA (FCLA Is more preferable as an active ingredient because it is hardly affected by biological components such as proteins and lipids.
A compound in which a fluorescein molecule and an imidazo [1,2-a] pyrazin-3 (7H) -one molecule are both covalently bonded to a cyclodextrin molecule used as an active ingredient of a superoxide anion analysis reagent in the present invention is And provides a method for its manufacture. For example, there is the following method shown in FIG. Known obtained by alkaline treatment of mono-2-O-tosyl-γ-CD synthesized by a known method (for example, JP-A-10-77303, Biosci. Biotech. Biochem., 1998, 62, 1249-1252) The substance monomannoepoxy-γ-cyclodextrin (Bull. Chem. Soc. Jpn., 1990, 63, 1409-1414) is reacted with m-nitrobenzenesulfonyl chloride in pyridine to bond the nitrobenzenesulfonyl group to the 6-position hydroxyl group. New compounds 3 and 4 are obtained. Although the present compound has 8 isomers, these 8 isomers are not separated and the subsequent reaction proceeds as a mixture of 8 isomers, that is, a mixture of compounds 3 and 4. 6-O-nitrobenzenesulfonyl-γ-cyclodextrin (3 and 4) is reacted with sodium azide in DMF to introduce an azide group at the 6-position to synthesize novel compounds (5) and (6). Next, the azide group is reduced by catalytic hydrogenation to obtain a mixture of the new compound (7) and the compound (8). Next, the mixture of compound (7) and compound (8) is reacted with fluorescein isothiocyanate to obtain a mixture of novel compound (9) and compound (10). Subsequently, a mixture of the compound (9) and the compound (10) was reacted with aqueous ammonia to open the epoxy, and a mixture of the novel compound (11) and the compound (12) having an amino group introduced at the 3-position was obtained. obtain. Subsequently, the mixture of compound (11) and compound (12) is amide-bonded with MCLA-COOH to obtain the desired mixture of novel compound (1) and compound (2). Of course, it may be synthesized by other methods and is not limited by the above example.
Further, the reagent for analyzing superoxide anion of the present invention comprises a compound in which a fluorescein molecule and an imidazo [1,2-a] pyrazin-3 (7H) -one molecule are both covalently bound to the cyclodextrin molecule as described above as an active ingredient. In other words, other substances such as solvents, buffers, stabilizers, surfactants and the like may be contained.
And this invention also provides the method of detecting a superoxide anion using the reagent for a superoxide anion analysis as mentioned above. That is, the reagent for analyzing superoxide anion of the present invention is brought into contact with a sample solution by mixing, mixing, etc., and imidazo [1,2-a] pyrazin-3 (7H) -one molecule and fluorescein molecule are added to the cyclodextrin molecule. The superoxide anion can be easily analyzed by measuring the intensity of light emitted by the covalently bonded compound.
At this time, analysis conditions such as the amount of the superoxide anion analysis reagent to be added are not particularly limited. Specifically, the concentration of the superoxide anion analysis reagent may be a level that does not affect the analysis system. The superoxide anion analysis may be performed in a temperature range that does not affect the analyte. For example, in general, when a living body is a subject and analysis is performed in an aqueous solution, a temperature range of about 10 to 40 ° C. is preferably exemplified.
A fluorescein molecule and an imidazo [1,2-a] pyrazin-3 (7H) -one molecule are both covalently bonded to a cyclodextrin molecule which is an active ingredient of a superoxide anion analysis reagent used in the method for analyzing a superoxide anion of the present invention. These compounds are soluble in water, so that they can be dissolved in blood, serum, cell culture medium, etc., and the presence of proteins, lipids, and sugars does not affect the luminescence reaction. In addition, since the maximum emission wavelength is about 530 nm, the influence of other components is small. Furthermore, the emission intensity is very high compared to CLA, MCLA, and FCLA. Therefore, a compound in which a fluorescein molecule and an imidazo [1,2-a] pyrazin-3 (7H) -one molecule are covalently bonded to such a cyclodextrin molecule is analyzed by a conventional superoxide anion analysis such as CLA, MCLA, FCLA. This makes it possible to analyze the superoxide anion with higher accuracy than the reagents for use.
Hereinafter, examples will be shown, and the embodiments of the present invention will be described in more detail. Of course, the present invention is not limited to the following examples, and can be implemented in various modes within the scope not changing the gist thereof. In addition, the technical scope of the present invention extends to a so-called equivalent range.

Buffered aqueous solution (pH 7) containing KCl (Nacalai Tesque) (0.2M), EDTA (SIGMA) (0.1 mM), 3-morpholinopropanesulfonic acid (MOPS, Nacalai Tesque) (20 mM) .2, 0.5 ml) at 25 ° C. with a hypoxanthine (Wako Pure Chemical Industries, Ltd.) aqueous solution (0.3 mM, 0.5 ml), a mixed aqueous solution of compound (1) and compound (2) (2.5 × 10 ^{− 5} M, 40 μl) and xanthine oxidase (manufactured by SIGMA) aqueous solution (0.37 unit / ml, 40 μl) were added, and the luminescence intensity was measured using Aloka luminescence reader BL201 (manufactured by Aloka). Luminescence showed the highest intensity immediately after addition of the xanthine oxidase aqueous solution. For comparison, experiments were conducted in the same manner using MCLA (manufactured by Tokyo Chemical Industry Co., Ltd.) and FCLA (manufactured by Tokyo Chemical Industry Co., Ltd.) instead of the mixture of compounds (1) and (2). The results of the relative light emission intensity obtained are shown in Table 1.

Hexadecyltrimethylammonium bromide (CTAB) was added to a buffered aqueous solution (pH 7.2, 0.5 ml) containing KCl (0.2 M), EDTA (0.1 mM) and 3-morpholinopropanesulfonic acid (MOPS) (20 mM) at 25 ° C. : Nacalai Tesque, Inc.) aqueous solution (0.5%, 50 μl), hypoxanthine aqueous solution (0.3 mM, 0.5 ml), mixed aqueous solution of compound (1) and compound (2) (2.5 × 10 ⁻⁵ M, 40 μl) and an aqueous xanthine oxidase solution (0.37 unit / ml, 40 μl) were added, and the luminescence intensity was measured using Aloka luminescence reader BL201. Luminescence showed the highest intensity immediately after addition of the xanthine oxidase aqueous solution. For comparison, MCLA and FCLA were similarly tested instead of the mixture of compounds (1) and (2). The results of the relative light emission intensity obtained are shown in Table 1.

以上の実施例１−３より、シクロデキストリン分子にフルオレセイン分子およびイミダゾ［１，２−ａ］ピラジン−３（７Ｈ）−オン分子が共に共有結合した化合物（１）および（２）の混合物は、ＭＣＬＡおよびＦＣＬＡよりも高い発光強度を示すことが明かとなった。また、ＣＴＡＢは、ＭＣＬＡやＦＣＬＡの発光強度を増強する効果があるが、シクロデキストリン分子にフルオレセイン分子およびイミダゾ［１，２−ａ］ピラジン−３（７Ｈ）−オン分子が共に共有結合した化合物（１）および（２）の混合物は、ＣＴＡＢの影響をあまり受けないことが確認された。また、ＢＳＡはＭＣＬＡやＦＣＬＡの発光強度を増減する効果があるが、シクロデキストリン分子にフルオレセイン分子およびイミダゾ［１，２−ａ］ピラジン−３（７Ｈ）−オン分子が共に共有結合した化合物（１）および（２）の混合物は、ＢＳＡの影響が小さいことが確認された。ＣＴＡＢ添加実験あるいはＢＳＡ添加実験の結果は、シクロデキストリン分子にフルオレセイン分子およびイミダゾ［１，２−ａ］ピラジン−３（７Ｈ）−オン分子が共に共有結合した化合物（１）および（２）の混合物は、タンパク質や脂質の影響を受けにくい発光をする発光試薬であることを示している。Buffered aqueous solution containing KCl (0.2 M), EDTA (0.1 mM), albumin protein (BSA: Wako Pure Chemical Industries, Ltd.) (0.1%), 3-morpholinopropanesulfonic acid (MOPS) (20 mM) (PH 7.2, 0.5 ml) at 25 ° C., hypoxanthine aqueous solution (0.3 mM, 0.5 ml), mixed aqueous solution of compound (1) and compound (2) (2.5 × 10 ⁻⁵ M, 40 μl) and An aqueous xanthine oxidase solution (0.37 unit / ml, 40 μl) was added, and the luminescence intensity was measured using Aloka luminescence reader BL201. Luminescence showed the highest intensity immediately after addition of the xanthine oxidase aqueous solution. For comparison, MCLA and FCLA were similarly tested instead of the mixture of compounds (1) and (2). The results of the relative light emission intensity obtained are shown in Table 1.

From the above Example 1-3, the mixture of the compounds (1) and (2) in which the fluorescein molecule and the imidazo [1,2-a] pyrazin-3 (7H) -one molecule are both covalently bonded to the cyclodextrin molecule, It was revealed that the emission intensity was higher than that of MCLA and FCLA. CTAB has the effect of enhancing the luminescence intensity of MCLA and FCLA, but a compound in which a fluorescein molecule and an imidazo [1,2-a] pyrazin-3 (7H) -one molecule are covalently bonded to a cyclodextrin molecule ( It was confirmed that the mixture of 1) and (2) was not significantly affected by CTAB. BSA has the effect of increasing or decreasing the luminescence intensity of MCLA or FCLA, but is a compound in which a fluorescein molecule and an imidazo [1,2-a] pyrazin-3 (7H) -one molecule are covalently bonded to a cyclodextrin molecule (1 ) And (2) were confirmed to be less affected by BSA. As a result of the CTAB addition experiment or BSA addition experiment, a mixture of compounds (1) and (2) in which a fluorescein molecule and an imidazo [1,2-a] pyrazin-3 (7H) -one molecule are covalently bonded together to a cyclodextrin molecule is obtained. Indicates a luminescent reagent that emits light that is not easily affected by proteins and lipids.

CTAB aqueous solution (0.5%) at 25 ° C. in a buffered aqueous solution (pH 7.2, 0.5 ml) containing KCl (0.2 M), EDTA (0.1 mM), 3-morpholinopropanesulfonic acid (MOPS) (20 mM). , 50 μl), hypoxanthine aqueous solution (0.3 mM, 0.5 ml), mixed aqueous solution of compound (1) and compound (2) (2.5 × 10 ⁻⁵ M, 40 μl) and xanthine oxidase aqueous solution (0.37 unit / ml, 40 μl) was added, and the emission spectrum was measured using a fluorescence spectrophotometer FP750DS (manufactured by JASCO Corporation) (excitation light was not used). The emission spectrum showed a maximum emission wavelength of about 530 nm.

Synthesis of Mixture of Mono-6-O-nitrobenzenesulfonyl-2,3-monomannoepoxy γ-CD (3) and (4) Next, a method for producing the compounds (1) and (2) will be described. In the following description, compound numbers are given by reference numerals in FIG.
Monomannoepoxy-γ-cyclodextrin (0.500 g, 0.000391 mol) was dissolved in pyridine (10.0 ml), m-nitrobenzenesulfonyl chloride (0.130 g, 0.000586 mol) was added, and 30 ° C. at −20 ° C. Stir for minutes. Pyridine was removed under reduced pressure, and ODS column chromatography (20 × 130 mm, 0% MeOH / H ₂ O → 20% MeOH / H ₂ O) was performed to obtain a mixture of compound (3) and (4) (0.204 g). Obtained in 35% yield. The mixture was measured by ¹ H NMR (DMSO ^D6 , 40 ° C.) and found to be δ3.2-3.9 (majority, H of _Y- cyclodextrin unit), 4.4-4.5 (2H, m, H— 6 ^* ), 4.7-5.0 (8H, m, H-1), 8.00 (1H, m, Ar-H), 8.39 (1H, m, Ar-H) and 8.61. The result of (2H, m, Ar-H) was obtained.

Synthesis of a mixture of mono-6-azido-2,3-monomannoepoxy _Y- CD (5) and (6) A mixture of compounds (3) and (4) (0.190 g, 0.000129 mol) was added to DMF ( 3.80 ml), sodium azide (0.0250 g, 0.000384 mol) was added, and the mixture was stirred at room temperature for 20 hours. DMF was removed under reduced pressure, and ODS column chromatography (20 × 130 mm, 0% MeOH / H ₂ O → 15% MeOH / H ₂ O) was performed to obtain a mixture of compounds (5) and (6) (0.165 g). Obtained in 97% yield. When the mixture was measured by ¹ H NMR (DMSO ^D6 , 50 ° C.), δ 3.3-3.8 (majority, H of _Y- cyclodextrin unit) and 4.9-5.0 (8H, m, H— The result of 1) was obtained.

Synthesis of a mixture of mono-6-N- (5-fluoresceinyl) thioureido-2,3-monomannoepoxy _Y- CD (9) and (10) A mixture of compounds (5) and (6) (0.154 g, 0.000118 mol) was dissolved in methanol: water = 1: 1 (5.00 ml), Pd—C (5 wt.% On activated carbon) (0.100 g) was added, and the mixture was stirred at room temperature for 15 hours in a hydrogen atmosphere. Pd-C was separated by filtration using Celite and membrane filter to obtain a mixture (0.172 g) of crude amino compounds (7) and (8). This crude amino compound (0.150 g, 0.000117 mol) was dissolved in pyridine (3.00 ml), FITC (0.0460 g, 0.000118 mol) was added, and the mixture was stirred at room temperature for 1 hour. Pyridine was removed under reduced pressure and ODS column chromatography (15 × 70 mm, 0% MeOH / H ₂ O (including 0.1% TFA) → 30% MeOH / H ₂ O (including 0.1% TFA)) was performed. As a result, a mixture (0.121 g) of the compounds (9) and (10) was obtained in a yield of 70%. When the mixture was measured by ¹ H NMR (DMSO ^D6 , 50 ° C.), δ3.3-3.9 (majority, H of _Y- cyclodextrin unit), 4.9-5.1 (br., H-1). ), 6.63 (4H, m, Ar-H), 6.75 (2H, s, Ar-H), 7.18 (1H, m, Ar-H), 7.81 (br., Ar-). H) and 8.34 (br., Ar-H).

Mono-6-N- (5-fluoresceinyl) thioureido-mono-3-deoxy-3-[[6- (4-methoxyphenyl) imidazo [1,2-a] pyrazin-3 (7H) -one-2- Synthesis of (yl) propionyl] amino- _Y- CD (1) and (2) A mixture of compounds (9) and (10) (0.110 g, 0.0000659 mol) was dissolved in concentrated aqueous ammonia (2.20 ml), The reaction was carried out at 37 ° C. for 3 days. The reaction solution was concentrated to obtain a mixture (0.142 g) of crude amino compounds (11) and (12). This crude amino compound (0.130 g, 0.0000771 mol) dissolved in pyridine (2.60 ml) was dissolved in a known compound MCLA-COOH (Carbohydr. Res., 1998, 306, 177-187) (0.0250 g, 0.0000797 mol). ), WSC (manufactured by Nacalai Tesque Co., Ltd.) (0.0440 g, 0.000229 mol) was added, and the mixture was stirred at room temperature for 4 hours under an argon atmosphere. Pyridine was removed under reduced pressure and ODS column chromatography (20 × 90 mm, 0% MeOH / H ₂ O (including 0.1% TFA) →, 50% MeOH / H ₂ O (including 0.1% TFA)) Was done. Further, HPLC (COSMOSIL Code No. 379-76 Size 20 × 250 mm 5C-18-MS Type Waters flow rate 6.0 ml / min, 30% MeOH / H ₂ O (including 0.1% TFA) → 50% MeOH / H ₂ O (containing 0.1% TFA) (60 min)). The obtained compound was dissolved in a small amount of methanol, and a large amount of ethyl acetate was added to form a powder, whereby a mixture (0.0300 g) of compounds (1) and (2) was obtained in a yield of 25%. When the mixture was measured by ¹ H NMR (CD ₃ OD, 20 ° C.), δ2.7-3.2 (m, CH ₂ CH ₂ ), 3.3-4.1 (majority, H of _Y- cyclodextrin). unit), 4.6-5.1 (m, H-1), 6.6-6.8 (m, Ar-H), 7.08 (m, Ar-H), 7.6-7. 8 (m, Ar-H and H of pyridine) and 7.9-8.2 (m, Ar-H and H of pyridine) were obtained. Moreover, when it measured by UV (0.03M phosphate buffer, pH8.0), the result of (lambda) maxnm ((epsilon)): 492 (48000) was obtained.

Claims (5)

The following compounds (13) and (14):

(Where, M is an integer of 5 to 14, n is an integer of _{0~ (M-1), R} 1 ~R 3, and _R 5 _{to R 13} is H or an alkyl group _C 1 _{-C 20,} alkenyl groups , Alkynyl group, phenyl group, hydroxyl group, alkyl hydroxyl group, benzyl group, aryl group, halogen group, nitro group, cyano group, sulfonyl group, carbonyl group, amino group, naphthyl group, indolyl group , X and Y are integers of 0 to 20, and R ₄ is a phenyl ring having 0 to 4 substitution degrees) as an active ingredient. A reagent for analyzing a superoxide anion comprising at least a compound in which a fluorescein molecule and an imidazo [1,2-a] pyrazin-3 (7H) -one molecule are covalently bonded to a cyclodextrin molecule as an active ingredient. The following compounds (15) and (16):

(Wherein M is an integer of 5 to 14 and n is an integer of 0 to (M-1)), a fluorescein molecule and imidazo [1,2-a] pyrazine-3 (7H) A compound in which the on molecule is covalently bonded together. A method for producing a compound in which a fluorescein molecule and an imidazo [1,2-a] pyrazin-3 (7H) -one molecule are covalently bonded to a cyclodextrin molecule, comprising: (1) the hydroxyl group of monomannoepoxy-cyclodextrin Nitrobenzenesulfonyl group was bound to (2), an azide group was introduced into the 6-position of the resulting 6-O-nitrobenzenesulfonyl-cyclodextrin, (3) this azide group was reduced, and (4) fluorescein isothi After reacting with the isocyanate, (4) opening the epoxy, introducing an amino group at the 3-position, and (5) amide bonding the resulting product with MCLA-COOH to obtain the desired compound. A featured manufacturing method. A method for analyzing a superoxide anion, wherein the luminescence intensity is measured after contacting the compound according to claim 1 with a sample solution.

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