KR100688424B1 - Chemical Substrate with Dual Functions - Google Patents

Abstract

The present invention relates to a method for producing a bifunctional substrate compound, a method of producing an electric bifunctional substrate compound, and an electric bifunctional substrate compound which show a change in color and fluorescence by an enzymatic reaction. Since the bifunctional matrix compound of the present invention can lower the error occurrence rate that may occur during various analytical operations using the enzyme reaction, it may be widely used in the development of more effective analytical methods.

Bifunctional Substance Compounds, Colors, Fluorescence, Enzymes

Description

Chemical Substrate with Dual Functions             

Figure 1a is a graph showing the change in absorption spectrum of visible light over time when the reaction of alkaline phosphatase and 1,4-DPAQ.

Figure 1b is a graph showing the change in fluorescence spectrum over time when the reaction of alkaline phosphatase and 1,4-DPAQ.

Figure 2a is a graph showing the change in absorption spectrum of visible light over time when the reaction of alkaline phosphatase and 1-DPAQ.

Figure 2b is a graph showing the change in fluorescence spectrum over time when the reaction of alkaline phosphatase and 1-DPAQ.

The present invention relates to a bifunctional matrix compound. More specifically, the present invention is a method for measuring the activity of the enzyme using a bifunctional substrate compound, a method for producing an electric bifunctional substrate compound and a bifunctional substrate compound exhibiting a change in color and fluorescence by an enzymatic reaction. It is about.

Substrate compounds (or sensor compounds) in which compounds are decomposed by the activity of enzymes and exhibit changes in absorption or emission spectra are used in various applications as in an enzyme linked immunosorbent assay (ELISA) using antibody-enzyme coupling. E.g. The ELISA immobilizes the primary antibody that specifically recognizes the target substance on a solid substrate, reacts the sample containing the target substance, and then the secondary antibody that specifically recognizes the primary antibody bound to the target substance of the electrical sample. Since the end of the secondary antibody has an enzyme bound to it, when a specific substrate compound capable of reacting with the enzyme is treated on the sample reacted with the secondary antibody, the substrate compound reacts with the enzyme to develop or The signal is generated by fluorescence, and the degree of the generated signal is measured to measure the presence or content of the target substance present in the sample.

The substrate compound used in this way could only generate one signal. For example, 4-nitrophenyl phosphate, the most commonly used substrate compound in ELISA, reacts with alkaline phosphatase to produce a color signal, and another substrate compound, florose di Phosphate (fluorescein diphosphate) reacts with alkaline phosphatase to produce a fluorescence signal. However, it has been pointed out that the substrate compound exhibiting only one signal has a high probability of error. That is, when the enzyme and the substrate compound are reacted, an error may occur about 10% in the reaction result even under the same reaction conditions. Therefore, a method of confirming the reaction result using a different substrate compound is sometimes used. have. If a substrate compound capable of generating two signals is used, it is expected that the two signals can be identified and the probability of error occurrence can be reduced, and research to develop them has been actively conducted. There is no situation.

Thus, there is a constant need to develop substrate compounds capable of generating two signals.

Thus, the present inventors have diligently researched to develop a substrate compound capable of generating two signals, and in the case of using hydroxyanthraquinone and hydroxynaphthacequinone compound in which intramolecular hydrogen bonds are blocked, one enzymatic reaction It was confirmed that the color signal and the fluorescence signal can be detected, thereby reducing the probability of a reaction error, thereby completing the present invention.

After all, the main object of the present invention is to provide a bifunctional matrix compound capable of generating two signals by reacting with an enzyme.

It is another object of the present invention to provide a method for preparing an electric dual function matrix compound.                         

It is still another object of the present invention to provide a method for measuring enzymatic activity using an electric bifunctional matrix compound.

The bifunctional matrix compound of the present invention is represented by the following general formula (I) or a salt thereof:

In the above formula,

와

(이때, R₃는 C_1-4의 알킬 또는 페닐기R is

Wow

Where R ₃ is a C _1-4 alkyl or phenyl group

1 or 2 substituents equal to 1, 4, 5 or

Substituted at position 8; And,

R ₁ and R ₂ are both hydrogen or together form a benzene ring.

,

,

,

및

로 구성된 그룹으로부터 선택되는 1종의 화합물이다. 이때, 상기 식에서, R은

와

(이때, R₃는 C_1-4의 알킬기 또는 페닐기이다) 중 1개 또는 서로 같은 2개의 치환기이다.Preferably, the bifunctional matrix compound of the present invention

,

,

,

And

It is 1 type of compound chosen from the group which consists of. Where R is

Wow

(Wherein, R ₃ is an alkyl group or a phenyl group of C _1-4 ) or one or two substituents the same.

On the other hand, the bifunctional matrix compound represented by the general formula (I) or a salt thereof of the present invention can be prepared by the following two methods, depending on the substituent of R:

인 경우:(i) R is

If:

The bifunctional matrix compound represented by general formula (I) or a salt thereof may contain a compound of the general formula (II), wherein -OH is substituted with one or two at positions 1, 4, 5 or 8 Dissolving in a solvent and reacting with diethyl chlorophosphate in the presence of a strong base to obtain a compound of formula (III); And dissolving the compound of the general formula (III) obtained in an organic solvent, and reacting the compound obtained by reacting with bromotrimethylsilane with H ₂ O, hydroxide of an alkali metal, aniline or cyclhexylamine. Are manufactured.

Where

R ₁ and R ₂ are both hydrogen or together form a benzene ring.

At this time, the organic solvent of the first process is not particularly limited thereto, but it is preferable to use pyridine, and the strong base of the first process is not particularly limited thereto, but it is preferable to use NaH, and the organic solvent of the second process is particularly Although not limited, it is preferable to use chloroform, and the hydroxide of alkali metal is not particularly limited thereto, but it is preferable to use NaOH.

인 경우(이때, R₃는 C_1-4의 알킬 또는 페닐기이다):(Ii) R is

Where R ₃ is an alkyl or phenyl group of C _1-4 :

The bifunctional matrix compound represented by general formula (I) or a salt thereof may contain a compound of the general formula (II), wherein -OH is substituted with one or two at positions 1, 4, 5 or 8 It is prepared by the process of dissolving in a solvent and reacting with a compound of the general formula (IV).

Where

R ₁ and R ₂ are both hydrogen or together form a benzene ring; And,

R ₃ is a C _1-4 alkyl or phenyl group

In this case, the organic solvent is not particularly limited thereto, but pyridine is preferably used.

The present inventors have conducted research on substrate compounds that bring about a change in color and fluorescence by enzymatic reaction. As a result, these properties can be obtained by using hydroxyanthraquinone and hydroxynaphthacequinone derivatives having intramolecular hydrogen bonding. Confirmed that it can. In other words, hydroxyanthraquinone and hydroxynaphthacequinone which have intramolecular hydrogen bonds have the maximum absorption wavelength in the strong fluorescence and visible light range, but when the intramolecular hydrogen bonds are blocked, the fluorescence disappears and the maximum absorption wavelength is also in the ultraviolet range. As a result, the color signal and the fluorescence signal are not generated as a result. However, it was confirmed that, by the enzymatic reaction, if a temporarily blocked intramolecular hydrogen bond is formed again, a color signal and a fluorescence signal may be generated.

Accordingly, the method for measuring enzymatic activity using the compound represented by the general formula (I) of the present invention comprises one enzyme selected from the group consisting of alkaline phosphatase, phosphodiesterase and chymotrypsin and general Reacting the compound represented by formula (I); And measuring a color signal and a fluorescence signal from the electric reactant, wherein the reaction conditions of the compound represented by the general formula (I) and the enzyme are not particularly limited thereto, but are completely dissolved in an aqueous solution. It can react with the enzyme or in the form of a suspension solution (suspension) and the reaction, the measurement method of the color signal is not particularly limited, but is preferably measured in the wavelength range of 300 to 550nm, The measuring method of is not particularly limited thereto, but is preferably measured in the wavelength range of 500 to 650 nm.

Since the bifunctional matrix compound of the present invention can lower the error occurrence rate that may occur during various analytical operations using the enzyme reaction, it may be widely used in the development of more effective analytical methods.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1 Preparation of Bifunctional Substrate Compound

Example 1-1 Preparation of 1,4-DPAQ (1,4-diphosphorylanthraquinone)

1.0 g (4.16 mmol) of quinizarin was dissolved in 100 mL of pyridine, 400 mg (16.65 mmol) of sodium hydride was added thereto, stirred at room temperature for 10 minutes, and then diethylchloro Phosphate (diethylchlorophosphate) 2.4mL (16.65mmol) was added and reacted by stirring at room temperature for 3 hours. The semi-applied solution was added to 200 mL of hexane and the precipitate was filtered off, and the filtered solution was removed in vacuo, then dissolved in 100 mL of diethyl ether, washed with a sufficient amount of water, and then the organic solvent layer was received. Water remaining with magnesium sulfate was removed, filtered to remove magnesium sulfate, and the solvent was removed under vacuum. The residue was purified by column (silica gel, ethyl acetate: hexane = 6: 4 (v / v)) to yield 1.53 g (72%) of 1,4-DEPAQ.

After dissolving 1.00 g (1.95 mmol) of 1,4-DEPAQ obtained in 4 mL of chloroform, 1.01 mL (7.80 mmol) of bromotrimethylsilane was slowly added to the mixed solution, and the mixture was kept at room temperature for 1 hour. After stirring, the solvent was removed under vacuum. The residue was again dissolved in water, washed with diethyl ether, 0.71 mL (7.80 mmol) of aniline was added to the aqueous layer, the mixture was stirred well, and then the solvent was removed under vacuum. The residue was recrystallized from a mixed solvent of methanol and acetone (methanol: acetone = 1: 4 (v / v)) to give 0.37 g (33%) of a yellow product 1,4-DPOAQ (1,4-diphosphorylanthraquinone). .

m.p. 124-125 ° C .;

¹ H NMR (300 MHz, DMSO): δ = 6.56 (t, 2H), 6.63 (d, 4H), 7.04 (t, 4H), 7.72 (s, 2H), 7.86 (m, 2H), 8.03 (m , 2H);

¹³ C NMR (75 MHz, DMSO): δ = 115.67, 118.02, 125.14, 126.02, 129.00, 131.91, 133.79, 140.47, 145.43, 147.14, 181.34;

³¹ P NMR (75 MHz, DMSO): δ = -5.52

Example 1-2 Preparation of 1,5-DPAQ (1,5-diphosphorylanthraquinone)

1.0 g (4.16 mmol) of anthrarufin was dissolved in 100 mL of pyridine, 400 mg (16.65 mmol) of sodium hydride were added and stirred at room temperature for 10 minutes, and 2.4 mL (16.65 mmol) of diethylchlorophosphate was added to the mixed solution. ) Was added and reacted by stirring at room temperature for 3 hours. The reaction solution was added to 200 mL of hexane and the precipitate was filtered off to remove the solvent. The filtered solution was removed in vacuo, then dissolved in 100 mL of diethyl ether, washed with a sufficient amount of water. Water remaining as sulfate was removed, filtered to remove magnesium sulfate, and the solvent was removed in vacuo. The residue was purified by column (silica gel, ethyl acetate: hexane = 6: 4 (v / v)) to yield 1.53 g (72%) of 1,5-DEPAQ.

After dissolving 1.00 g (1.95 mmol) of 1,5-DEPAQ obtained in 4 mL of chloroform, 1.01 mL (7.80 mmol) of bromotrimethylsilane was slowly added to the mixed solution, which was then stirred at room temperature for 1 hour. The solvent was removed under vacuum. The residue was dissolved in water again, washed with diethyl ether, 0.71 mL (7.80 mmol) of aniline was added to the aqueous layer, the mixture was stirred well, and the solvent was removed under vacuum. The residue was recrystallized from a mixed solvent of methanol and acetone (methanol: acetone = 1: 4 (v / v)) to give 0.37 g (33%) of a yellow product 1,5-DPOAQ.

¹ H NMR (300 MHz, DMSO): δ = 6.57 (t, 2H), 6.64 (d, 4H), 7.03 (t, 4H), 7.77 (d, 2H), 7.87 (m, 2H), 7.90 (m , 2H)

Example 1-3 Preparation of 1,8-DPAQ (1,8-diphosphorylanthraquinone)

1.0 g (4.16 mmol) of 1,8-dihydroxyanthraquinone (1,8-dihydroxyanthraquinone) was dissolved in 100 mL of pyridine, followed by addition of 400 mg (16.65 mmol) of sodium hydride at room temperature for 10 minutes. The mixture was stirred, and 2.4 mL (16.65 mmol) of diethylchlorophosphate was added to the mixed solution, followed by reacting for 3 hours at room temperature. The reaction solution was added to 200 mL of hexane and the precipitate was filtered off, and the filtered solution was removed in vacuo, then dissolved in 100 mL of diethyl ether, washed with a sufficient amount of water, and then the organic solvent layer was taken up with magnesium sulfate. The remaining water was removed, filtered to remove magnesium sulphate and the solvent was removed in vacuo to afford a residue. The residue was purified by column (silica gel, ethyl acetate: hexane = 6: 4 (v / v)) to yield 1.53 g (72%) of 1,8-DEPAQ.

After dissolving 1.00 g (1.95 mmol) of 1,8-DEPAQ obtained in 4 mL of chloroform, 1.01 mL (7.80 mmol) of bromotrimethylsilane was slowly added to the mixed solution and stirred at room temperature for 1 hour. After removal, the solvent was removed under vacuum. The residue was again dissolved in water, washed with diethyl ether, and then 0.71 mL (7.80 mmol) of aniline was added to the aqueous layer, followed by gentle shaking, followed by removal of the solvent under vacuum. The residue was recrystallized from a mixed solvent of methanol and acetone (methanol: acetone = 1: 4 (v / v)) to prepare 0.37 g (33%) of a yellow product 1,8-DPOAQ.

¹ H NMR (300 MHz, DMSO): δ = 6.82 (m, 6H), 7.16 (t, 2H), 7.52 (d, 2H), 7.75 (t, 2H), 7.86 (d, 2H)

Example 1-4 Preparation of 1-DPAQ (1-diphosphorylanthraquinone)

1.0 g (4.46 mmol) of 1-hydroxyanthraquinone (1-HAQ) was dissolved in 100 mL of pyridine, 214 mg (8.92 mmol) of sodium hydride were added thereto, and the mixture was stirred at room temperature for 10 minutes, and then Diethylchlorophosphate 1.29mL (9.92mmol) was added to the mixed solution, followed by reaction at room temperature for 3 hours. The reaction solution was added to 200 mL of hexane and the precipitate was filtered off to remove the solvent. The filtered solution was removed in vacuo, dissolved in 100 mL of diethyl ether, washed with a sufficient amount of water, and then the organic solvent layer was taken up with magnesium Water remaining as sulfate was removed, filtered to remove magnesium sulfate, and the solvent was removed in vacuo. The residue was purified by column (silica gel, ethyl acetate: hexane = 4: 6 (v / v)) to yield 1.50 g (93%) of 1-DEPAQ.

After dissolving 1.50 g (4.16 mmol) of 1-DEPAQ obtained in 4 mL of chloroform, 1.08 mL (8.32 mmol) of bromotrimethylsilane was slowly added to the electric mixed solution, stirred at room temperature for 1 hour, and then under vacuum. Solvent was removed. The residue was again dissolved in water, washed with diethyl ether, 0.76 mL (8.32 mmol) of aniline was added to the aqueous layer, shaken appropriately, and the solvent was removed under vacuum. The residue was recrystallized from a mixed solvent of methanol and acetone (methanol: acetone = 1: 5 (v / v)) to prepare 0.48 g (29%) of yellow product 1-DPOAQ.

m.p. 145-146 ° C;

¹ H NMR (300 MHz, DMSO): δ = 6.51 (t, 1H), 6.59 (d, 2H), 7.01 (t, 2H), 7.87 (m, 4H), 8.00 (d, 1H), 8.15 (t , 2H);

¹³ C NMR (75 MHz, DMSO): δ = 116.64, 119.27, 122.12, 123.58, 123.66, 126.22, 126.70, 127.98, 129.10, 132.06, 133.76, 134.45, 134.75, 143.83, 151.87, 151.95, 180.84, 182.4.

³¹ P NMR (75 MHz, DMSO): δ = -5.42.

Example 1-5 Preparation of 6,11-DPNQ (diphosphoryl naphthacenequinone)

1.0 g (3.44 mmol) of 6,11-dihydroxy-5,12-naphthacequinone was dissolved in 20 mL of pyridine, 330 mg (13.78 mmol) of sodium hydride were added, stirred at room temperature for 30 minutes, and then 2 mL (13.78 mmol) of diethylchlorophosphate was added to the mixed solution, followed by reaction at room temperature for 4 hours. The reaction solution was added to 200 mL of hexane and filtered to give a precipitate. The obtained precipitate was dissolved in 100 mL of ethyl acetate, washed with a sufficient amount of water, received an organic solvent layer to remove water remaining with magnesium sulfate, filtered to remove magnesium sulfate, and the solvent was removed under vacuum. The residue was purified by column (silica gel, ethyl acetate: hexane = 4: 6 (v / v)) to yield 1.2 g (62%) of 6,11-DEPNQ.

1.00 g (1.78 mmol) of 6,11-DEPNQ obtained above was dissolved in 4 mL of chloroform, and then 0.92 mL (7.11 mmol) of bromotrimethylsilane was slowly added to the electric mixed solution and stirred at room temperature for 1 hour 30 minutes. Afterwards, the solvent was removed under vacuum. The residue was dissolved in water again, washed with diethyl ether, 0.65 mL (7.11 mmol) of aniline was added to the aqueous layer, followed by gentle shaking, and then the solvent was removed under vacuum. The residue was recrystallized from acetone to give 0.30 g (26.5%) of the orange product 6,11-DPNQ.

¹ H NMR (300 MHz, DMSO): δ = 6.69 (m, 6H), 7.09 (t, 4H), 7.84 (dd, 2H), 7.90 (dd, 2H), 8.05 (dd, 2H), 8.45 (dd , 2H)

Example 1-6 Preparation of 1,4-DAAQ (1,4-diacetylanthraquinone)

5.0 g (21 mmol) of quinizarine was dissolved in 100 mL of pyridine, 5.98 mL of acetyl chloride was added, and the mixture was stirred at room temperature for 3 hours. 500 mL of cold water was added to the reaction solution, followed by filtration to obtain a precipitate. The precipitate was recrystallized (ethyl acetate: ethyl alcohol = 1: 3 (v / v)) to obtain 4.54 g (67%) of 1, 4-DAAQ was prepared.

¹ H-NMR (300 MHz, CDCl ₃ ); δ 2.49 (s, 6H), δ 7.43 (s, 2H) δ 7.77 (dd, 2H), δ 8.17 (dd, 2H)

Example 1-7 Preparation of 1,4-DBAQ (1,4-dibromoanthraquinone)

1.0 g (4.16 mmol) of quinizarine was dissolved in 30 mL of pyridine, 0.97 mL (8.39 mmol) of benzoyl chloride was added, and the mixture was stirred at room temperature for 3 hours. 250 mL of cold water was added to the reaction solution, followed by filtration to obtain a precipitate, which was recrystallized (ethyl acetate: ethyl alcohol = 1: 3 (v / v)) to prepare 1,4-DBAQ.

¹ H-NMR (300 MHz, CDCl ₃ ); δ 7.59 (m, 4H), δ 7.62 (s, 2H), δ 7.69 (m, 4H), δ 8.11 (d, 2H), δ 8.31 (d, 4H)

Example 2 Effect of Bifunctional Substrate Compound

Example 2-1 : Reaction effect of alkaline phosphatase and 1,4-DPAQ

1,4-DPAQ prepared in Example 1-1 was dissolved in 5 mL of Hepes (HEPES) buffer solution (5 mM, pH 8.0, 0.5 mM MgCl ₂ ) at a concentration of 0.1 mM and 40 mg / alkaline alkaline phosphatase. After adding to the final concentration of mL, the reaction was carried out at 25 ℃, and the amount of the reaction product over time (0, 10, 12, 15 and 20 minutes) was investigated. At this time, the amount of the reaction product was measured by adding 5 mL of chloroform to the sample, mixing, and analyzing the chloroform layer by using a visible light and a fluorescence spectrometer (see FIGS. 1A and 1B). FIG. 1A is a graph showing changes in absorption spectrum of visible light over time, FIG. 1B is a graph showing changes in fluorescence spectrum over time, and FIGS. 1A and 1B (a) show a reaction for 0 minutes. Represents one sample, (b) represents a sample reacted for 10 minutes, (c) represents a sample reacted for 12 minutes, (d) represents a sample reacted for 15 minutes, and (e) represents 20 minutes The reacted sample is shown. As shown in Figure 1a and 1b, as time passes, a new peak is increased, and this change occurs simultaneously in the visible and fluorescence region, it can be seen that one enzyme reaction can be confirmed by two methods Could.

Example 2-2 : Reaction effect of alkaline phosphatase and 1,5-DPAQ

1,5-DPAQ prepared in Example 1-2 was dissolved in diethanolamine (DEA) buffer (5 mM, pH 10.0, 0.5 mM MgCl ₂ ) at a concentration of 1.0 mM, and 0.1 mg of alkaline phosphatase. / mL was added to the final concentration, the reaction was carried out at 37 ℃, the amount of reaction product over time was measured using a fluorescence spectrometer, the color change of the reaction mixture was visually observed. As a result, it was found that the peak of about 490 nm gradually increased in the fluorescence spectrum, and the reaction mixture was changed from yellow to orange.

Example 2-3 : Effect of Alkaline Phosphatase and 1,8-DPAQ

Instead of the 1,5-DPAQ prepared in Examples 1-2 above, the same method as in Example 2-2 was carried out, except that 1,8-DPAQ prepared in Examples 1-3 was used. The reaction product was measured using a fluorescence spectrometer, and the color change of the reaction mixture was visually observed. As a result, it was found that the peak of about 500 nm gradually increased in the fluorescence spectrum, and the reaction mixture was found to change from yellow to orange.

Example 2-4 : Reaction effect of alkaline phosphatase and 1-DPAQ

Instead of 1,4-DPAQ prepared in Example 1-1, except that 1-DPAQ prepared in Examples 1-4 was used, the time elapsed in the same manner as in Example 2-1 ( The amount of reaction product according to 0, 1, 5 and 10 minutes was investigated (see FIGS. 2A and 2B). Figure 2a is a graph showing the change in the absorption spectrum of visible light over time, Figure 2b is a graph showing the change in fluorescence spectrum over time, in Figures 2a and 2b (a) is a reaction for 0 minutes One sample is shown, (b) represents a sample reacted for 1 minute, (c) represents a sample reacted for 5 minutes, and (d) represents a sample reacted for 10 minutes. As shown in Fig. 2a and 2b, as time passes, a new peak is increased, and this change occurs simultaneously in the visible region and the fluorescence region, indicating that one enzymatic reaction can be confirmed in two ways. Could.

Example 2-5 : Reaction effect of alkaline phosphatase and 6,11-DPNQ

Instead of 1,5-DPAQ prepared in Examples 1-2, the same method as in Example 2-2 was carried out, except that 6,11-DPNQ prepared in Examples 1-5 was used. The reaction product was measured using a fluorescence spectrometer, and the color change of the reaction mixture was visually observed. As a result, as time passed, it was found that the peak of about 500 nm gradually increased in the fluorescence spectrum, and the reaction mixture was changed from yellow to orange.

Example 2-6 : Effect of chymotrypsin and 1,4-DAAQ

1,4-DAAQ prepared in Example 1-6 was dissolved in 5 mL of Hepes (HEPES) buffer at a concentration of 0.1 mM, and chymotrypsin was added to a final concentration of 40 mg / mL, followed by reaction at 25 ° C. The amount of the reaction product over time was analyzed by using a visible light and a fluorescence spectrometer. As a result, as time passed, the visible light spectrum was found to gradually increase the peak at about 480nm, the fluorescence spectrum was found to increase the peak at 530nm and 570nm.

As described and demonstrated in detail above, the present invention is to measure the enzyme activity by using a bifunctional substrate compound, a method of producing an electric bifunctional substrate compound and an electric bifunctional substrate compound exhibiting a change in color and fluorescence by an enzymatic reaction. Provide a way to. Since the bifunctional matrix compound of the present invention can lower the error occurrence rate that may occur during various analytical operations using the enzyme reaction, it may be widely used in the development of more effective analytical methods.

Claims (13)

delete delete delete delete delete delete delete delete delete (Iii) reacting a compound represented by the following general formula (I) with one enzyme selected from the group consisting of alkaline phosphatase, phosphodiesterase and chymotrypsin; And, (Ii) measuring enzyme activity using a compound represented by the following general formula (I), comprising measuring a color signal and a fluorescence signal from an electric reactant:

Where OR is applied to one or two sites selected from positions 1, 4, 5 and 8 Where R is

or

(this When R ₃ is a C _1-4 alkyl group or a phenyl group); And, R ₁ and R ₂ are both hydrogen or together form a benzene ring. The method of claim 10, The compound represented by the general formula (I) is characterized in that the reaction with the enzyme in the form of completely dissolved in an aqueous solution or in the form of a suspension (suspension) A method for measuring enzymatic activity using a compound represented by general formula (I). The method of claim 10, The color signal is measured in the wavelength range of 300 to 550 nm. A method for measuring enzymatic activity using a compound represented by general formula (I). The method of claim 10, The fluorescent signal is measured in the wavelength range of 500 to 650 nm A method for measuring enzymatic activity using a compound represented by general formula (I).

Images