KR100969958B1 - Fluorescein derivative, preparation method thereof and detection method of anion using the same - Google Patents

Abstract

The present invention provides a fluorescein derivative that effectively recognizes anions, in particular adenosine 5'-triphosphate (ATP) and pyrophosphate (PPi), by binding to metal ions at pH 7.4, a method for preparing the same and in vivo using the same Provided are negative ion detection methods. Specifically, the fluorescein derivative of the present invention exhibits a selective significant fluorescence amplification and color change effects with respect to ATP and PPi in combination with the manganese ion (Mn ^{2 +)} in a 100% aqueous solution, signaling in vivo And sensors for ATP and PPi detection, which play an important role in energy storage.

Fluorescein, Adenosine Triphosphate, Pyrophosphate, Fluorescence

Description

Fluorescein derivative, preparation method thereof and anion detection method using the same {FLUORESCEIN DERIVATIVE, PREPARATION METHOD THEREOF AND DETECTION METHOD OF ANION USING THE SAME}

The invention fluorescein derivative, and more particularly, the manganese ions (Mn ^{2 +)} and binding to adenosine triphosphate (ATP) and fatigue optionally fluorescence amplification for the phosphoric acid (PPi) and the color having the anion selectivity in combination with a metal ion The present invention relates to a fluorescein derivative having a changing effect, a method for preparing the same, and a method for detecting adenosine triphosphate and pyrophosphate using the same.

The design and research of new sensors for key substances and ions in vivo has been actively conducted. Recent understanding and research on supramolecule chemistry has shown great potential in the design of host compounds that can selectively bind ions or other types of guest compounds. In addition, it has been a great help in the study of the development of fluorescent chemosensor, which makes it easier to observe the selective binding with guest compounds using fluorescence change.

Fluorescence is a photochemical phenomenon that occurs when photons with a specific wavelength (excitation wavelength) collide with an indicator molecule, and electrons excite at a high energy level as a result of the collision. Among the various analytical methods, fluorescence has the advantage of being able to observe signals even at 10 ^-9 M concentrations due to their excellent sensitivity.

Recently, studies on fluorescence chemical sensors for cations, anions and neutral organic molecules using these properties have been published.

Negative ions play an important role in a wide range of chemical and biological processes. Thus, the anion-fluorescence sensor based on induced changes are considered very interesting due to gender high detection limits and a simple operation (Martinez-Manez R and Sancanon F , Chem Rev 2003, 103, 4419..). In particular, phosphate ions and their derivatives play an important role in signal transmission and energy storage in biological systems. For example, adenosine triphosphate (ATP) is known as a general energy transporter in biological systems and is known as a significant target for the design of molecular receptors. On the other hand, pyrophosphate (PPi) is a biologically important target as a product of ATP hydrolysis under cellular conditions. Detection of PPi release has been studied as a real-time DNA sequencing method (Ronaghi M et al . , Anal . Biochem . 1996 , 242 , 84), and recently PPi measurement has also been used in cancer studies ( Xu S et al . , Anal . Biochem . 2001 , 299 , 188).

ATP based on fluorescence changes (Jose DA et al . , Org . Lett . 2007 , 9, 1979; Kanekiyo Y et al . , Chem . Commun . 2004, 1006; Kwon, JY et al . , J. Am . Chem . Soc . 2004 , 126 , 8892; McCleskey SC et al . , J. Am . Chem . Soc . 2003 , 125 , 1114; Sancenon F et al . , Helv. Chim . Acta 2002 , 85 , 1505; Ojida A et al., Tetrahedron Lett . 2002 , 43 , 6193; Sancenon, F et al . , Angew . Chem . Int . Ed . 2001 , 40 , 2640; Schneider, SE et al . , J. Am . Chem . Soc . 2000 , 122 , 542) and PPi (Singh NJ et al . , Org . Lett . 2007 , 9, 485; Kim SK et al., Tetrahedron 2006 , 62, 6065; Gunnlaugsson T et al. , Org . Biomol . Chem. 2005 , 3, 48; Aldakov D and Anzenbacher P Jr., Chem . Comm . 2003 , 1394; Gunnlaugsson T et al . , Org . Lett . 2002 , 4, 2449; Anzenbacher P et al . , J. Am . Chem . Soc. 2000 , 122, 9350; Nishizawa S et al . , J. Am . Chem . Soc . 1999 , 121, 9463; Lee HN et al . , Org . Lett . 2007 , 9, 243; Jang YJ et al . , J. Org . Chem . 2005 , 70, 9603; Cho HK et al . Chem Commun . 2005 , 1690; Lee DH et al . , Angew . Chem ., Int . Ed . 2004 , 43, 4777; Aldakov D and Anzenbacher P Jr., J. Am . Chem . Soc . 2004 , 126, 4752; Mizukami S et al . J. Am . Chem . Soc . 2002 , 124, 3920; Vance DH and Czarnik AW, J. Am. Chem . Soc . 1994 , 116, 9397) has been a major goal of many research groups.

However, although several types of fluorescent chemical sensors capable of selectively detecting anions have been reported as described above, only a few fluorescent chemical sensors recognize anions in an aqueous solution similar to the in vivo environment (Jang YJ et al. , J. Org . Chem. 2005 , 70, 9603; Lee DH et al . , Angew . Chem ., Int . Ed . Engl . 2004 , 43 , 4777; Fabbrizzi L et al ., Angew . Chem ., Int . Ed . Engl . 2002 , 41 , 3811; Mizukami S et al . , J. Am . Chem . Soc . 2002 , 124 , 3920).

The present inventors have synthesized novel fluorescein derivatives while trying to study the fluorescence chemical sensor that recognizes anion in a similar aqueous solution and in vivo environment, and the derivative is combined with a manganese ion (Mn ^{2 +)} fluorescent represents a quenching effect and the color change effect, the fluorescein derivative-complex of manganese ions (Mn ^{2 +)} confirms the selective fluorescence amplification and indicate a color change effect on the pyrophosphate (PPi) and adenosine triphosphate (ATP) The present invention was completed.

Accordingly, the present invention seeks to provide a fluorescein derivative which exhibits a fluorescent amplification reaction by reacting with ATP and PPi in the presence of manganese ions.

The present invention also provides an intermediate compound of the fluorescein derivative.

In addition, the present invention is to provide a method for producing the fluorescein derivative.

In addition, the present invention is to provide a method for detecting PPi and ATP using the fluorescein derivative.

In order to achieve the above object, the present invention provides a fluorescein derivative represented by the following formula (3).

The present invention also provides an intermediate compound of the fluorescein derivative represented by the following formulas (1) and (2).

In another aspect, the present invention provides a method for producing a fluorescein derivative represented by the formula (3).

Method for producing the fluorescein derivative,

1) N- (2-aminoethyl) -N-methylcarbamic acid tertiary butyl ester [ N- (2-aminoethyl) -N -methylcarbamic acid t -butyl ester] and 2-pyridylmethyl bromide bromide) to obtain [2- (bis-2-pyridylmethylamino) ethyl-N-methyl] carbamic acid tert-butyl ester as a compound of Formula 1;

2) [2- (bis-2-pyridylmethylamino) ethyl-N-methyl] carbamic acid tert-butyl ester obtained in step 1) was treated with an acid, specifically trifluoroacetic acid, Obtaining a compound N'-methyl-N, N-bis-2-pyridylmethylethane-1,2-diamine; And

3) reacting N'-methyl-N, N-bis-2-pyridylmethylethane-1,2-diamine with formaldehyde and 2 ', 7'-dichlorofluorescein obtained in step 2) It characterized by comprising a step of obtaining a compound of.

More specifically, in step 3), 2 ', 7 and iminium ions of the reaction product of N'-methyl-N, N-bis-2-pyridylmethylethane-1,2-diamine and formaldehyde The '-dichlorofluorescein can be iminomethylated via a condensation reaction, ie the Mannich reaction, to give a compound of formula 3.

In detail, the preparation method is represented by the following Scheme 1.

The present invention also provides a method for detecting pyrophosphoric acid (PPi) and / or adenosine triphosphate (ATP) using the compound of Formula 3. The detection method comprises the steps of mixing a sample of the composite of the fluorescein derivative and manganese ion (Mn ^{+ 2),} to detect a pyrophosphate (PPi) and / or adenosine triphosphate (ATP); Irradiating the mixture with light of a predetermined wavelength; And confirming the fluorescence amplification effect.

Among these, as an example of a method for detecting PPi and / or ATP in a sample,

comprising the steps of: (a) each of preparing a buffer solution that the dissolution of derivative and manganese ion (Mn ^{+ 2)} fluorescein of the present invention;

(b) mixing the fluorescein derivative with a buffer solution in which the manganese ions are dissolved to induce the formation of a fluorescein derivative-manganese ion complex in which the manganese ions are bonded to the fluorescein derivative;

(c) inducing a reaction between the complex and PPi and / or ATP by adding and mixing a sample to detect PPi and / or ATP to a buffer solution in which the fluorescein derivative-manganese ion complex is formed;

(d) irradiating the mixture with an excitation wavelength of 505 nm; And

(e) detecting a fluorescence amplification signal by the excitation wavelength, and detecting PPi and / or ATP, wherein the presence of pyrophosphate reacts with the complex. The detection method of the invention is not limited to the above method.

The sample according to the invention is characterized in that the sample is an extracorporeal secretion selected from the group comprising blood, saliva and urine.

In addition, the compounds of formula (3) according to the invention, for metal ions, such as from pH 7.4 Ag ^+, Co ^{2 +,} Cu ^{2 +,} Hg ^{2 +,} stand for the addition of Mn ^{2 +,} Ni ^{2 +} Since it exhibits chelation enhanced fluorescence quenching (CHEQ) effect, it can be used as a fluorescence chemical sensor for the metal.

Compounds of formula 3 according to the invention, combined with a manganese ion (Mn ^{2 +)} at pH 7.4 and fluorescence quenching, and shows a change in color, consisting of the coupling compound of formula (3) - manganese ion (Mn ^{2 +)} complex anion No significant changes were shown for the addition of, for example, ADP, AMP, HSO ₄ ⁻ , CH ₃ COO ⁻ , H ₂ PO ₄ ⁻ , but a large fluorescence amplification effect selective for the addition of PPi and / or ATP and Because of the color change, the compound of formula 3 according to the present invention can be used as a fluorescent chemical sensor selective for PPi and / or ATP.

Derivatives (Fluorescein derivatives) fluorescein of the present invention, manganese ions in the presence of (Mn ^{2 +),} it represents the optionally high fluorescence amplification effect for PPi and / or ATP, in the signal transmission and energy storage in the body It can be usefully used as a fluorescent chemical sensor for the detection of PPi and / or ATP, which plays an important role.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention.

However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited thereto.

Example 1 Compound of Formula 1 [Intermediate Compound of Fluorescein Derivative]

Butyl ester [N- (2-aminoethyl) -N -methylcarbamic acid t -butyl ester] (2 g, 11.48 mmol) - N- (2- Amino-ethyl) -N- methyl-carbamic acid t in 125ml of absolute ethanol , A mixture of sodium carbonate (5.37 g, 50.62 mmol) and 2-pyridylmethyl bromide (6.39 g, 25.25 mmol) was added and refluxed under an argon gas atmosphere for 6 hours, and then the solvent was evaporated. I was. The residue was dissolved in an 80 mL aqueous layer containing 10% sodium hydroxide and extracted three times with 100 mL of dichloromethane. The combined organic layers were trapped with potassium carbonate to evaporate the solvent. The residue was chromatographed on aluminum oxide (dichloromethane: methanol = 98: 2, volume / volume ratio) to [2- (bis-2-pyridylmethylamino) ethyl-N-methyl] carbamic acid tert-butyl ester ( 3.2 g of compound of formula 1, yield 78%) was obtained.

¹ H NMR (CDCl ₃ ) δ 8.52 (br s, 2H), 7.64 (m, 4H), 7.16 (br s, 2H), 3.85 (s, 4H), 3.38 (m, 2H), 2.93, (m, 5H), 1.72 (s, 3H), 1.47 (s, 6H);

¹³ C NMR (CDCl ₃ ) δ 159.4, 155.6, 149.3, 122.7, 121.9, 79.3, 60.4, 51.9, 46.7, 34.5, 27.9;

LRMS (FAB) m / z = 357.2 (M + H) ⁺ , calc. for C ₂₀ H ₂₉ N ₄ 0 ₂ = 357.2.

Example 2 Compounds of Formula 2 [Intermediate Compounds of Fluorescein Derivatives]

To a solution of compound (3 g, 8.42 mmol) prepared in Example 1 in 25 ml of dichloromethane (CH ₂ Cl ₂ ), 0.74 ml of trifluoroacetic acid was added dropwise at 0 ° C. Stirred at room temperature for 1 hour. The solvent was evaporated and the residue was dissolved in a 20 mL aqueous layer containing 10% sodium hydroxide (NaOH) and extracted four times with 25 mL of dichloromethane. The combined organic layers were watered with potassium carbonate, and the solvent was evaporated to give 2 g of N'-methyl-N, N-bis-2-pyridylmethylethane-1,2-diamine (Compound 2, yield 91%). Obtained.

¹ H NMR (CDCl ₃ ) δ 8.52 (d, 2H, J = 4.9 Hz), 7.63 (t, 2H, J = 13.5 Hz), 7.43 (d, 2H, J = 7.8 Hz), 7.15 (t, 2H, J = 4.9 Hz), 3.89 (s, 4H), 2.60 (m, 4H), 2.39 (s, 3H);

¹³ C NMR (CDCl ₃ ) δ 158.7, 148.9, 136.7, 123.2, 122.6, 59.8, 51.2, 47.8, 33.1;

LRMS (FAB) m / z = 257.2 (M + H) ⁺ , calc. for C ₁₅ H ₂₁ N ₄ = 257.2.

Example 3. Compound of Formula 3 [Fluorescein Derivative]

2,7-dichlorofluorescein (0.5 g, 1.25 mmol) and paraformaldehyde (0.11 g, 3.73 mmol) were combined in 20 mL of acetonitrile (CH ₃ CN). Compound (1 g, 3.99 mmol) was added with stirring at room temperature, followed by addition of 10 mL of water. The reaction mixture was refluxed for 24 hours, then the solvent was completely removed under vacuum and the residue was chromatographed using basic alumina with dichloromethane: methanol (95: 5, volume / volume ratio) as the developing solution. The solvent was evaporated to give a pale pink solid, 9- (o-carboxyphenyl) -2,7-dichloro-4,5-bis [N'-methyl-N, N-bis-2-pyridylmethylethane]- 0.25 g of 6-hydroxy-3-zantenone (compound of formula 3, yield: 21%) was obtained:

mp 65 ° C., dec .;

¹ H NMR (CDCl ₃ ) δ 8.50 (d, 4H, J = 4.9 Hz), 8.02 (d, 1H, J = 5.6 Hz), 7.61 (m, 10H), 7.14 (m, 5H), 6.62 (s, 2H), 3.91 (d, 4H, J = 3.8 Hz), 3.86 (s, 8H), 2.81 (m, 8H), 2.19 (s, 6H);

¹³ C NMR (CDCl ₃ ) δ 168.8, 158.7, 156.8, 148.9, 147.9, 136.6, 135.2, 130.2, 127.3, 125.5, 124.1, 123.6, 122.2, 117.3, 109.8, 109.1, 60.6, 54.7, 54.5, 50.9, 41.8;

HRMS (FAB) m / z = (M + H) ⁺ , calc. for C ₅₂ H ₅₁ Cl ₂ N ₈ O ₅ = 937.3359.

Experimental Example  1. Observation of fluorescence change

1-1. Fluorescence Emission Spectrum Measurement of Compounds of Formula 3 in the Presence of Multiple Metal Ions

Compounds of Formula 3 and various metal ions (Ag ⁺ , Ca ^{2 +} , Cd ^{2 +} , Co ^{2 +} , Cs ⁺ , Cu ²⁺ , Hg ^{2 +} , K ⁺ , Li ⁺ , Mg ^{2 +} , Mn ^{2 +} , Na ⁺ , Ni ^{2 +} , Pb ^{2 +} and Zn ^{2 +} ) to determine the binding properties, the following experiment was performed.

Metal ions ^{(Ag +, Ca 2 +,} Cd 2 +, Co 2 +, Cs +, Cu 2 +, Hg 2 +, K +, Li +, Mg 2 +, Mn 2 +, Na +, Ni 2+, Pb ² stock solution (1mM) of a tetrabutyl ammonium salt with a ⁺ and Zn ^{+ 2)} was prepared by using the pH (20mM HEPES 7.4) buffer. The stock solution (1 mM) of the compound of Formula 3 prepared in Example 3 was prepared using double distilled demineralized water. The stock solutions were used on the day of preparation.

Test solution, the probe stock solution into a test tube containing a final concentration of 12 μM ㎕ to 3 (which is the compound of formula (III) stock solutions of the present invention), each of the metal ions (Ag ^+, Ca ^{+ 2,} Cd ^{+ 2,} Co ^2+, Cs ⁺ , Cu ^{2 +} , Hg ^{2 +} , K ⁺ , Li ⁺ , Mg ^{2 +} , Mn ^{2 +} , Na ⁺ , Ni ^{2 +} , Pb ^{2 +} and Zn ^{2 +} After addition of 24 μl, the solution was diluted to 4 ml with 20 mM HEPES (pH 7.4) buffer, and all fluorescence changes were performed using a fluorescence spectrophotometer (RF-5301 / PC Spcectrofluorophotometer, Shimadzu), and the excitation wavelength was 505. Nm, excitation and emission slit widths were measured at 1.5 nm and the results are shown in FIG. 1.

1, the emission spectrum of the compounds of the formula I according to the present invention, Ag ⁺ at pH 7.4, Co ^{2 +,} Cu ^{2 +,} Hg ^{2 +,} Mn ^{2 +,} eyes to the addition of Ni ^{2 +} It showed a significant chelation enhanced fluorescence quenching (CHEQ) effect.

1-2. Mercury ions (Hg ²⁺ )of  Fluorescence titration experiment of the compound of formula 3 in the presence of

In order to determine the binding properties between the compound of formula 3 and the mercury ion of the present invention, the following experiment was performed.

A stock solution of tetrabutylammonium salt with mercury ions (10 mM) was prepared using 20 mM HEPES (pH 7.4) buffer. In addition, the stock solution (1 mM) of the compound of Formula 3 prepared in Example 3 was prepared using deionized water distilled twice. The stock solutions were used on the day of preparation.

Test solution 1 contains 12 μl of the probe storage solution (compound of formula 3 of the present invention) into the test tube so that the final concentration is 3 μM, and 0.24-12 μl of the aliquot of the mercury ion storage solution to 0.1 to 5 equivalents. After addition, the solution was diluted to 4 ml with 20 mM HEPES (pH 7.4) buffer, and all fluorescence changes were measured using a fluorescence spectrophotometer, with an excitation wavelength of 505 nm and excitation and emission slit width of 1.5 nm. Measured as is shown in Figure 2 the results.

As shown in Figure 2, when the final concentration of the compound of formula 3 according to the invention at pH 7.4 is 3 μM, chelation enhanced fluorescence quenching as the amount of mercury ions added increases from 0.1 to 5 equivalents: CHEQ) effect. Through titration experiments with mercury ions, the binding constant of the compound of formula 3 and the mercury ion of the present invention was found to be 6.3 × 10 ⁷ M ^-2 .

In addition, when the final concentration of the compound of formula 3 according to the present invention at pH 7.4 is 3 μM, the fluorescence change and color change when 5 equivalents of mercury ions were added, which showed the largest chelate amplification fluorescence quenching effect in the above experiment. It is shown in Figure 3 by measuring.

As shown in FIG. 3, when 5 equivalents of mercury ions were added to the compound of Chemical Formula 3 of the present invention, the pale yellow solution was changed to pink, and a noticeable chelate amplification fluorescence quenching effect occurred.

1-3. Manganese ions ( Mg ^{2 +} ) Fluorescence titration experiment for the analysis of the binding of the compound of formula 3 to various anions

Investigate the binding properties between the manganese ions (Mg ^{2 +)} in the presence, the compound and various anions of formula (3) of the present invention (PPi, ATP, ADP, AMP, HSO ₄ ^-, CH ₃ COO ^{- -,} and H ₂ PO ₄₎ In order to perform the following experiment.

Tetrabutyl ammonium having a tetrabutyl ammonium salt stock solution (1mM) and manganese ion (Mn ^{2 +)} having the anion (PPi, ATP, ADP, AMP , HSO 4 -, CH 3 COO - - , and H ₂ PO ₄₎ Stock solution (10 mM) was prepared using 20 mM HEPES (pH 7.4) buffer. In addition, the stock solution (1 mM) of the compound of Formula 3 prepared in Example 3 was prepared using deionized water distilled twice. The stock solutions were used on the day of preparation.

Test solution, and 6 so that the probe stock solution Final concentration containing 12 ㎕ to 3 μM (Compound storage solution (III) of the invention), 2.5 equivalent of the Ally quart of the manganese ion (Mn ^{2 +)} stock solution into the test tube, After 24 μl of 24 μl aliquots of PPi or aliquots of each anion (ATP, ADP, AMP, HSO ₄ ⁻ , CH ₃ COO ^−, and H ₂ PO ₄ ⁻ ) were added to 10 equivalents, followed by 20 mM HEPES ( pH 7.4) was used by diluting the solution to 4 ml with buffer, and all fluorescence changes were measured using a fluorescence spectrophotometer, the excitation wavelength was measured at 505 nm, and the excitation and emission slit widths were at 1.5 nm. The results are shown in FIG. 4. It was.

As shown in Figure 4, at pH 7.4, in the presence of manganese ions (2.5 equivalents), when the final concentration of the compound of formula 3 according to the invention is 3 μM, the emission spectrum of the compound of formula 1 according to the invention is PPi And selective fluorescence amplification effect for the addition of 10 equivalents of ATP. On the other hand, no significant change was shown for the addition of 10 equivalents of other anions. Accordingly, the compounds of formula (3) according to the invention may form the manganese ions (Mg ^{2 +)} and the complex, and the complex is found to be very selectively coupled to PPi and ATP. In particular, it was shown to have excellent selectivity for PPi compared to H ₂ PO ₄ ⁻ in 100% aqueous solution.

Via fluorescence titration experiments, the binding constant of the compound 3 and PPi and ATP in the present invention are each 4.2 × 10 ⁴ M ^-1 and 3.5 × 10 ⁴ M ^- it was found to be ^1.

1-4. Manganese ions ( Mg ^{2 +} ) In the presence of PPi Fluorescence Titration Experiments for Binding Properties

In the presence of manganese ions, in order to determine the binding properties between the compound of Formula 3 and PPi of the present invention, the following experiment was performed.

Stock solution (10 mM) of tetrabutyl ammonium salt with PPi was prepared using 20 mM HEPES pH 7.4 buffer. Further, in Example 3 a stock solution (1 mM) and manganese ions in the compound of formula (3) prepared in the storage solution of the tetrabutyl ammonium salt has a ^{(Mn 2 +) (10 mM} ) are prepared using deionized water by distillation in a double It was. The stock solutions were used on the day of preparation.

Test solutions are put 6㎕ such that 2.5 equivalent of probe stock solution Ally quart of a final concentration of 3 μM to contain 12 ㎕ (Compound stock solution of Formula 3 of the present invention), manganese ion (Mn ^{+ 2)} stock solution into the test tube, After forming the complex, add 2.4-168 μL of aliquots of the PPi stock solution to 1-70 equivalents. The solution was diluted to 4 ml with 20 mM HEPES (pH 7.4) buffer and all fluorescence changes were measured at excitation wavelength 505 nm and excitation and emission slit width 1.5 nm and the results are shown in FIG. 5.

As shown in FIG. 5, in the presence of manganese ions at pH 7.4, the compound of formula 3 (final concentration: 6 μM) of the present invention showed a gradual fluorescence amplification effect as the amount of PPi added increased from 1 to 70 equivalents. . Through titration experiments with PPi, the compound-manganese ions (Mn ^{2 +} ) complex of the present invention and the binding constant of PPi was found to be 4.2 × 10 ⁴ M ^-1 .

In addition, when the final concentration of the compound of formula 3 according to the present invention at pH 7.4 is 15 μM, the fluorescence change and the color change were measured when 10 and 50 equivalents of PPi were added, which showed a fluorescence amplification effect in the above experiment. 6 is shown.

As shown in FIG. 6, the compound of Formula 3 of the present invention at pH 7.4 changed the color of the solution to pink by the addition of manganese ions (2.5 equivalents), and a noticeable chelate fluorescence quenching effect occurred. However, when 10 and 50 equivalents of PPi were further added thereto, the color of the solution was recovered to pale yellow when only the compound of Formula 3 was present, and it was found that the amplification effect of quenched fluorescence occurred.

1 shows two equivalents of various metal ions [Ag ⁺ , Ca ^{2 +} , Cd ^{2 +} , Co ^{2 +} , Cs ⁺ , at pH 7.4 (20 mM HEPES buffer) in a compound of formula 3 (3 μM) according to the invention. is a diagram showing a change in fluorescence when the the ^{Cu 2 +, Hg 2 +,} K +, Li +, Mg 2+, Mn 2 +, Na +, Ni 2 +, Pb 2 + , and Zn ^{+ 2],}

2 is a diagram showing the fluorescence changes when the pH 7.4 (20mM HEPES buffer) at, by varying the concentration of Hg ^{2 +} in the compound of formula 3 (3 μM) in accordance with the present invention (0.1 to 5 eq.) Hayeoteul,

3 is a diagram showing the color and fluorescence changes when Hg ^{2 +} (5 equivalents) is added to the compound of formula 3 (3 μM) according to the present invention at pH 7.4 (20 mM HEPES buffer).

FIG. 4 shows 10 equivalents of PPi and anions [CH ₃ CO ₂ ⁻ , HSO ₄ ⁻ , in a compound of formula 3 (3 μM) in the presence of Mn ^{2 +} 2.5 equivalents at pH 7.4 (20 mM HEPES buffer) a diagram showing a change in fluorescence when the a], ^- H ₂ PO ₄

5 is a diagram showing the fluorescence change at the time when the the PPi (1 to 70 eq.) In pH 7.4 (20mM HEPES buffer) in, Mn ^{2 +} 2.5 equivalent of the compound of the presence of the, general formula (3) according to the invention (3 μM) ,

6 is a photograph showing the change in color and fluorescence when PPi is added to the compound of formula 3 (20 μM) according to the present invention at pH 7.4 (20 mM HEPES buffer) in the presence of Mn ^{2 +} 2.5 equivalents: ( a) Compound ( 1 ) of Formula 3; (b) Compound of Formula 3 ( 1 ) And Mn ^{2 +} 2.5 equivalents; (c) compound ( 1 ) of formula 3, Mn ^{2 +} 2.5 equivalents and 10 equivalents of PPi; (d) Compound 1 of formula 3, Mn ^{2 +} 2.5 equivalents and 50 equivalents of PPi.

Claims (16)

Fluorescein derivative represented by the following formula (3). (3)

delete delete A method for producing the fluorescein derivative of claim 1, wherein the compound is prepared by adding formaldehyde and 2 ', 7'-dichlorofluorescein. [Formula 2]

The compound of claim 4, wherein 1) N- (2-aminoethyl) -N-methylcarbamic acid tertiary butyl ester [ N- (2-aminoethyl) -N -methylcarbamic acid t -butyl ester] and 2-pyridylmethyl bromide bromide) to obtain a compound of formula 1; And 2) The method of preparing a fluorescein derivative of claim 1, characterized in that it is prepared by adding an acid to the compound of formula 1 obtained in step 1). [Formula 1]

Mixing a sample for detecting pyrophosphate (PPi) with a complex of the fluorescein derivative of claim 1 and manganese ions (Mn ²⁺ ); Irradiating the mixture with light; And confirming the fluorescence amplification effect. The method of claim 6, wherein the method (a) fluorescein derivative and manganese ion according to claim 1 (Mn ^{+ 2)} a step of manufacturing each of the lysis buffer; (b) mixing the fluorescein derivative with a buffer solution in which the manganese ions are dissolved to induce the formation of a fluorescein derivative-manganese ion complex in which the manganese ions are bonded to the fluorescein derivative; (c) inducing a reaction between the complex and the pyrophosphate by adding and mixing a sample to detect pyrophosphate to the buffer solution in which the fluorescein derivative-manganese ion complex is formed; (d) irradiating the mixture with light of an excitation wavelength; And (e) detecting the fluorescence amplification signal by the light of the excitation wavelength, and detecting the presence of pyrophosphate reacting with the complex. The method of claim 6, wherein the sample is selected from an extracorporeal secretion consisting of blood, saliva, or urine. The method for detecting pyrophosphoric acid according to claim 7, wherein the excitation wavelength is 505 nm. Mixing a sample to detect adenosine triphosphate (ATP) in a complex of the fluorescein derivative of claim 1 and manganese ions (Mn ²⁺ ); Irradiating the mixture with light; And detecting the amplification effect of adenosine triphosphate. The method of claim 10, wherein the method is (a) preparing a buffer solution in which the fluorescein derivative of claim 1 and manganese ions (Mn ²⁺ ) are dissolved, respectively; (b) mixing the fluorescein derivative with a buffer solution in which the manganese ions are dissolved to induce the formation of a fluorescein derivative-manganese ion complex in which the manganese ions are bonded to the fluorescein derivative; (c) inducing a reaction between the complex and adenosine triphosphate by adding and mixing a sample to detect adenosine triphosphate to a buffer solution in which the fluorescein derivative-manganese ion complex is formed; (d) irradiating the mixture with light of an excitation wavelength; And (e) detecting the presence of adenosine triphosphate reacting with the complex by detecting a fluorescence amplification signal by light of the excitation wavelength, the adenosine triphosphate detection method. The method for detecting adenosine triphosphate according to claim 10, wherein the sample is selected from an extracorporeal secretion consisting of blood, saliva or urine. The method for detecting adenosine triphosphate according to claim 11, wherein the excitation wavelength is 505 nm. A fluorescent sensor comprising the compound of claim 1. The fluorescence sensor of claim 14, wherein the fluorescence sensor is complexed with manganese ions (Mg ²⁺ ) to selectively detect and bind pyrophosphate. The fluorescence sensor of claim 14, wherein the fluorescence sensor is complexed with manganese ions (Mg ²⁺ ) to selectively detect and bind adenosine triphosphate.

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