JPS61110036A - Benzofuranyl phenylmaleimide derivative and assay of thiol compound using the same - Google Patents

Abstract

PURPOSE:To elevate the measuring accuracy of thiol compound, by arranging an analysis reagent composed of 2-benzophynyl maleimide derivative given by a specified chemical formula to enhance the fluorescent intensity along with a greater stokes shift. CONSTITUTION:A thiol compound is assayed using a benzofuronyl phenyl maleimide derivative as given by the formula (R is hydrogen or lower alkyl amine group). The derivative herein used, is, for example, 2-(6-dimethylamino) benzofuranyl phenyl maleimide. Fluorescent product from this compound and the thiol compound is stable, and remain unchanged at least for 24hr. With the exciting wavelength of 355nm and fluorescent wavelength 457nm, this enables a highly sensitive assay of 2pmol-2nmol/ml of glutathione and the coexistence of boron potassiumhydroxide also enables the assay of the disulfide of glutathione as well at a low concentration of 5pmol-1nmol/ml.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a benzofuranylphenylmaleimide derivative that reacts specifically with thiol compounds and exhibits extremely strong fluorescence intensity, and a method for quantifying thiol compounds using the same.

The most abundant thiol compound in the body is glutathione, which plays an important role in redox and detoxification in the body. Glutathione is concentrated in cells, especially in the liver, where it plays an important role in detoxification. On the other hand, the concentration in serum is extremely low at approximately 25, f, M. Therefore, highly sensitive analytical reagents are required to track the concentration of glutathione in serum and study the detoxification mechanism. .

When measuring thiol compounds in biological samples, proteins have their own fluorescence in the vicinity of 300-350 nm due to fluorescent amino acids such as tryptophan and tyrosine, so the fluorescence shown by the thiol compound quantitative reagent should be The wavelength is also preferably on the long wavelength side, and the difference between the excitation wavelength and the fluorescence wavelength, ie, the Stokes shift, is also required to be large. Furthermore, since thiol compounds are easily oxidized in the air to form disulfide bonds, it is desirable to use a reagent that easily reacts with these disulfide bonds.

Conventionally, it has been known that aromatic maleimide compounds become fluorescent due to addition reactions with thiol groups, and that the compounds themselves do not exhibit any fluorescence and the blank size is small. maleimide-based compounds have been devised as quantitative reagents for thiol compounds.

However, all of these compounds have advantages and disadvantages in terms of Stokes shift, reactivity with disulfides, fluorescence intensity, etc., and a thiol compound detection reagent that satisfies all aspects has not yet been found.

As a result of intensive research into fluorescent compounds that have extremely high fluorescence intensity, give a fluorescence spectrum over 300 nm, and have a large Stokes shift, the inventors discovered that 2-benzo compounds with a trans double bond fixed in the furan ring Furanylaniline has an extremely high fluorescent f-ion yield of 0.84;
It was also found that the Stokes shift was as large as 1100n, and the present invention was made based on this knowledge.

That is, the compound of the present invention is a novel 2-benzophenylmaleimide derivative represented by the general formula (1) (wherein R is hydrogen or a lower alkylamino group), such as 2-(6-dimethylamino)benzo Mention may be made of furanylphenylmaleimide.

All of the compounds of the present invention are new compounds that have not been described in any literature, and the fluorescent products produced with thiol compounds are stable and small (both remain constant for 24 hours.Excitation wavelength: 355 nm7)
．． At a fluorescence wavelength of 457 nm, 2 pmo of glutathione
Highly sensitive quantification of 1-2 nmol/ytl is possible. If potassium borohydride is present, the disulfide form of glutathione will also be reduced to 5 pmol to 1 nmol/
Low concentrations of xl can be quantified.

The compound of the present invention can be prepared, for example, by reacting a 2-benzofuranylaniline derivative represented by formula (1) (wherein R is as defined in general formula (1)) with maleic anhydride in chloroform, and further It can be synthesized by dehydrating it with acetic anhydride to form maleimide.

Next, the present invention will be explained in more detail with reference to Examples. Examples 1 and 2 relate to the method for producing the compound of the present invention.
Examples 3 and 4 relate to quantitative methods using them.

Example 1 1.22 F of 2-benzofuranylaniline maleic acid adduct was added to 20 tel of acetic anhydride and 0.03 F of anhydrous sodium acetate melted in advance, heated under reflux for 15 hours, and then cooled. Pour out the precipitated crystals. washing with water,
After drying, recrystallize from ethanol to obtain a pure product. m
, p205.5-251.5°C yield 45%, elemental analysis results are as follows.

0% N% N% Calculated value as Cl8H11NO3 74.43 3,83 4.84
Actual value 74.52 3.80 4.81
Example 2 2-(6-dimethylamino)benzofuranyl phenylmaleimide was produced by performing the same operation using 2-(6-dimethylamino)benzofuranylaniline instead of 2-benzofuranylaniline in Example 1. obtain.

Recrystallize from acetone to make a pure product. m, p, 2
The yield was 46% at 03-204°C, and the results of elemental analysis were as follows.

0% N% N% as C20H16N203 Calculated value 72.28 4.85 8.43 Actual value 72.15 4.81 8.28 Example 3 1% homogenate of Wistar male rat liver (0
, 02M, EDTA solution) was taken and the amount of glutathione was measured. Add 0.1 ml of 3% metaphosphoric acid, centrifuge at 4ooorpm for 15 minutes (4°C), and collect 0.2 ml of supernatant. Add 2M KOH5QAA' and further add 225 g' of 0.1 M boric acid-carbonate buffer (pH 8) to adjust the pH. Add 2.5xl of an acetonitrile solution (84M) of 2-(6-dimethylamino)benzofuranylphenylmaleimide and heat at 60°C.
Incubate for 30 minutes. Cool with tap water and measure the fluorescence intensity at an excitation wavelength of 355 nm and a fluorescence wavelength of 457 nm. Glutathione is 2 pmol-2 nmol 7m
l shows a good linear relationship, and the coefficient of variation is 2.44% (Q
, 2nmol/xi? , n=10), and is superior to conventional methods in both sensitivity and accuracy.

Example 4 The disulfide form of glutathione in liver homogenate was quantified. Add 2MKOH to 0.2ml of homogenate supernatant.
Add 50% KBH4250Al and 4mM N-ethylmaleimide, add 18% KBH4250Al, and incubate at 60°C.
Reduce to glutathione by heating for 0 minutes.

After that, glutathione is quantified according to the method shown in Example 3. Since the reagent of the present invention is stable and does not decompose even during hot reactions, it is not necessary to react with thiol at room temperature as in conventional methods, and rapid quantitative operations are possible.

A good linear relationship was obtained in the range of disulfide 5 pmol to lnmol/H1, and the accuracy was 2.37% with a coefficient of variation.
(0.2 nmol, n=5).

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Claims (2)

[Claims] (1) Benzofuranylphenylmaleimide derivative represented by the following general formula (I) ▲Mathematical formula, chemical formula, table, etc.▼(I) (R in the formula is hydrogen or a lower alkylamino group) (2) Using the benzofuranylphenylmaleimide derivative represented by the following general formula (I) ▲Mathematical formulas, chemical formulas, tables, etc.▼(I) (R in the formula is hydrogen or a lower alkylamino group) Quantitative analysis method for thiol compounds, characterized by quantifying thiol compounds