KR101473457B1 - Dichlorofluorescein chloroacetate-based composition for detecting azide ion, and detecting method of azide ion using the same - Google Patents

Abstract

The present invention relates to a suture thread, which pulls, support and lifts skin. Specifically, in the present invention, provided are a dichlorofluorescein chloroacetate-based composition for detecting azide ions and a detecting method of the azide ions using colormetric signals and or fluorescence signals of the composition. More specifically, the composition for detecting azide ions comprises a fluorescein acetate derivative compound denoted by chemical formula 1.

Description

TECHNICAL FIELD [0001] The present invention relates to a dichlorofluorescein chloroacetate-based composition for detecting an azide ion, and a method for detecting an azide ion using the same. BACKGROUND OF THE INVENTION 1. Field of the Invention [0002]

The present invention relates to a dichlorofluoroethylene chloroacetate-based composition for azidated ion detection, and a method for detecting azidated ions using the azidated ion detection composition.

Signaling of anions is of great interest because it plays an important role in various chemical, biological, and environmental processes. Detection methods for the signaling of chemical, biological, and industrially important anions such as fluoride and cyanide are continuously being developed. However, the development of a detection method for conveniently detecting azide ions has not been developed so far. BACKGROUND OF THE INVENTION Sodium azide has been widely used industrially for the manufacture of biocides, explosive detonators, radical scavengers, and anti-corrosion solutions, as well as in automotive safety bags It is also used as a propellant. The sodium azide breaks down into heat and produces nitrogen gas, which quickly expands the airbag. The substitution and addition reaction of an azide ion or a hydrazoic acid is carried out in the presence of an organic azide for the preparation of a heterocyclic compound such as imidazole, oxazole, 1,2,3-triazone, and tetrazole And derivatives thereof. Recently, click chemistry, such as acetylenes catalyzed by dipolar cycloadditions of azides and sources of monovalent copper ions, has been used in the field of bioorthogonal ligation I am receiving great attention.

However, azidized ions are toxic and potentially fatal to humans, and are compared to cyanide with an LD ₅₀ of about 27 mg / kg in rats. The azide ions reduce oxygen consumption by somatic cells and cause harmful effects on the lungs, heart, and brain. Even a small amount of azide may cause headache, eye inflammation, mucosal inflammation, dyspnea, ≪ / RTI > The development of methods for measuring azide ions in environmental, industrial, and biological systems is required because the process of azide evaporation and the treatment of azides increase the likelihood of human exposure and environmental contamination.

Typically, the analysis of azide ions is performed in a chemical laboratory by redox titrimetry, argentometry, and potentiometric methods. The measurement of the azide ion is mostly based on a colorimetric reaction, electron paramagnetic resonance, gas chromatography-mass spectrometry, ion chromatography, and liquid chromatography. Recently, Cu ^{2 +} complex and India reel-fluorescent sensor based on naphthalene (indolyl-naphthalene) was successfully devised. However, development of a convenient colorimetric and fluorescence signaling system is required for the detection of industrially important toxicity.

Reaction-based signaling for optical signaling of metal ions, anions, and some neutral compounds in analytes has received great attention due to the particular characteristics of the reactions involved as well as the cumulative signaling effects. Hydrolysis of ester functionality has been used for the development of detection methods for signaling important target materials. Ester hydrolysis is promoted by the number of metal ions and anions, and is used for the detection of various important target substances. 2-hydroxypyridine, a bisacetylated BODIPY derivative of bis-acetylated BODIPY, and an ester of picolinate of 7-hydroxycoumarin. Cu ^{2 +} -mediated ) Copper (II) -elective chemodosimeters that induce hydrolysis are representative examples. Other compositions for detection of important target materials include reactive disulfide-containing fluororesin esters for hydrogen sulphide, resorufin levulinates for sulfites and fluorescein for BODIPY fluorophore, perborate and Acetate derivatives of resorufin, and chloroacetate derivatives of fluolecein to hydrazine.

Japanese Patent Application Laid-Open No. 2007-064945 discloses a method for detecting azide ion by electrochemical analysis as a method for detecting azide ion. However, optical signaling of azide ions by hydrolysis of fluorene acetate derivative compounds is not known.

The present invention provides a dichlorofluoroethylene chloroacetate-based composition for azidated ion detection and a method for detecting azidated ions using a colorimetric signal and / or a fluorescent signal of the azidated ion detection composition.

However, the problems to be solved by the present invention are not limited to the above-mentioned problems, and other problems not mentioned can be clearly understood by those skilled in the art from the following description.

According to a first aspect of the present invention, there is provided a composition for detecting azide ions, comprising a fluorescein acetate derivative compound represented by the following formula

 [Chemical Formula 1]

In Formula 1,

X ₁ and X ₂ are each independently Cl,

X ₃ and X ₄ are each independently selected from the group consisting of F, Cl, Br, I, and combinations thereof.

The second aspect of the present invention provides a method for detecting azide ions using a colorimetric signal and / or a fluorescence signal of a composition for detecting azide ion according to the first aspect of the present application.

According to one embodiment of the present invention, a colorimetric signal and / or a fluorescent signal is expressed by hydrolysis of the fluorescein acetate derivative compound contained in the composition for detecting azide ion. In addition, the detection of the azide ion may be selectively and rapidly performed by adding an anion-blocking agent to prevent the reaction of the anion with the composition for detecting the azide ion in the presence of other anions.

1A and 1B show the structure of a fluorescein acetate derivative contained in the composition for azidation detection in one embodiment of the present invention.
FIG. 2 is a graph showing the change in fluorescence intensity of a compound prepared according to one embodiment of the invention at about 532 nm in the presence of azide ions, versus time, in one embodiment of the invention.
3 is a graph showing the absorbance change ( A / A ₀ ) at about 513 nm in the presence of various anions of a compound made according to one embodiment of the present application, wherein the illustration shows the ultraviolet- Is an ultraviolet-visible (hereinafter also referred to as "UV-vis") absorption spectrum.
Figure 4 is a graph showing the change in fluorescence intensity (I / I ₀₎ at about 532 nm in the presence of various anions of the compound according to an embodiment of the present application, sapdo the fluorescence emission of said compound in the presence of various anions Lt; / RTI >
Figure 5 is a partial ¹ H nuclear magnetic resonance (hereinafter also referred to as 'NMR') spectrum of the compound under the presence and absence of azide ion of the compound prepared according to one embodiment of the present application.
Figure 6 is a UV-vis absorption spectral change of a compound prepared according to one embodiment of the present application and 2 ', 7 ' -dichlorofluorescein under the presence and absence of azide ion in one embodiment of the invention.
FIG. 7 is a fluorescence emission spectral change of the compound and 2 ', 7'-dichlorofluorescein produced according to one embodiment of the invention under the presence and absence of azide ion in one embodiment of the invention.
Figure 8 is a graph depicting the inhibition of sulphide interference of compounds made according to one embodiment of the invention in the signaling of azide ions by TPEN-Hg ^{2 +} in one embodiment of the invention.
FIG. 9 is a graph showing the fluorescence intensity of azide ion by a compound prepared according to one embodiment of the present invention in the presence of various anions in one embodiment of the present invention. FIG.
10 is a graph showing changes in fluorescence emission spectrum of a compound prepared according to one embodiment of the present application, and an illustration showing a change in fluorescence intensity of the compound at about 532 nm.
11 is a graph depicting the concentration-dependence of azide ion signaling by a compound made according to one embodiment of the present application.
Figure 12 is a graph showing signaling of azide ion and sulphide ion over time by the compound prepared according to one embodiment of the present application.
13 is a graph showing fluorescence signaling of azide ions by distilled water, tap water, and a sample prepared according to one embodiment of the present invention in a sample of virtual wastewater in one embodiment of the present invention.
Figure 14 is a partial ¹ H NMR spectrum of a compound prepared according to one embodiment herein in CDCl ₃ , in one embodiment of the invention.
15 is a partial ¹³ C NMR spectrum of a compound prepared according to one embodiment herein in CDCl ₃ , in one embodiment of the invention.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily carry out the present invention. It should be understood, however, that the present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. In the drawings, the same reference numbers are used throughout the specification to refer to the same or like parts.

Throughout this specification, when a part is referred to as being "connected" to another part, it is not limited to a case where it is "directly connected" but also includes the case where it is "electrically connected" do.

Throughout this specification, when a member is " on " another member, it includes not only when the member is in contact with the other member, but also when there is another member between the two members.

Throughout this specification, when an element is referred to as " including " an element, it is understood that the element may include other elements as well, without departing from the other elements unless specifically stated otherwise. The terms " about ", " substantially ", etc. used to the extent that they are used throughout the specification are intended to be taken to mean the approximation of the manufacturing and material tolerances inherent in the stated sense, Accurate or absolute numbers are used to help prevent unauthorized exploitation by unauthorized intruders of the referenced disclosure. The word " step (or step) " or " step " used to the extent that it is used throughout the specification does not mean " step for.

Throughout this specification, the term " combination (s) thereof " included in the expression of the machine form means a mixture or combination of one or more elements selected from the group consisting of the constituents described in the expression of the form of a marker, Quot; means at least one selected from the group consisting of the above-mentioned elements.

Throughout this specification, the description of "A and / or B" means "A or B, or A and B".

Throughout this specification, the term "azide ion" refers to N ₃ ^- ion, which causes rapid substitution reaction with other materials, so that it is easy to generate various kinds of compounds, exhibits instability sensitive to small impacts, But it may not be so limited.

Hereinafter, embodiments and examples of the present invention will be described in detail with reference to the accompanying drawings. However, the present invention is not limited to these embodiments and examples and drawings.

According to a first aspect of the present invention, there is provided a composition for detecting azide ions, comprising a fluorescein acetate derivative compound represented by the following formula

[Chemical Formula 1]

In Formula 1,

X ₁ and X ₂ are each independently Cl and X ₃ and X ₄ are each independently selected from the group consisting of F, Cl, Br, I, and combinations thereof.

According to one embodiment of the present invention, the composition for detecting an azide ion may further include a mixed solution of a buffer solution and an organic solvent, but the present invention is not limited thereto.

According to one embodiment of the invention, the buffer solution may include, but is not limited to, selected from the group consisting of Tris buffer solution, phosphate buffer solution, acetate buffer solution, HEPES buffer solution, and combinations thereof . The acidity of the buffer solution may be, for example, from about pH 7 to about pH 9, but is not limited thereto. For example, the acidity of the buffer solution may range from about pH 7 to about pH 9, from about pH 7.5 to about pH 9, from about pH 8 to about pH 9, from about pH 8.5 to about pH 9, from about pH 7 to about pH 8.5, a pH of about 7.5 to about pH 8.5, a pH of about 8 to about pH 8.5, a pH of about 7 to about pH 8, a pH of about 7.5 to about pH 8, or a pH of about 7 to about pH 7.5, .

According to one embodiment of the present invention, the organic solvent may be selected from the group consisting of acetonitrile, tetrahydrofuran, dimethyl sulfoxide, methanol, dioxane, acetone, and combinations thereof. .

The second aspect of the present invention provides a method for detecting azide ions using a colorimetric signal and / or a fluorescence signal of a composition for detecting azide ion according to the first aspect of the present application.

According to one embodiment of the present invention, the colorimetric signal and / or the fluorescent signal may be generated by adding an anion and an azide ion to the composition for detecting azide ion, but the present invention is not limited thereto.

According to one embodiment, the anion is selected from the group consisting of F ^- , Cl ^- , Br ^- , I ^- , HPO ₄ ^{2 -} , P ₂ O ₇ ^{4 -} , SO ₄ ^{2 -} , SO ₃ ^{2 -} , NO ₃ ^- _But are not limited to, anions selected from the group consisting of ₃ ^- , OAc ^- , ClO ₄ ^- , S ^{2 -} , and combinations thereof.

According to one embodiment of the present invention, tetrakis- (2-pyridylmethyl) ethylenediamine-Hg ^{2 +} [tetrakis (2-pyridylmethyl) ethylenediamine) is used as an anion masking agent for preventing the reaction between the composition for detecting azide ion and the anion - (2-pyridylmethyl) ethylenediamine-Hg ^{2 +} ] (hereinafter also referred to as 'TPEN-Hg ^{2 +} '). For example, sulfide ions in the anion can react with fluorescein dinitrobenzenesulfonate and TPEN-Hg ^{2 +} is added as an anion-blocking agent to prevent the reaction. Ion and the TPEN-Hg ^{2 +} may form a stable HgS complex to prevent the reaction between the sulfide ion and the composition for detecting an azide ion, but the present invention is not limited thereto.

According to an embodiment of the present invention, the fluorescein acetate derivative compound contained in the composition for detecting azide ion may be hydrolyzed by the azide ion to generate the colorimetric signal and / or fluorescence signal However, the present invention is not limited thereto.

Hereinafter, embodiments of the present invention will be described in detail. However, the present invention is not limited thereto.

[ Example ]

Fluorescein, 2 ', 7'-dichlorofluorescein, acetyl chloride, chloroacetyl chloride, sodium azide, and sodium sulfide were purchased from Aldrich Chemical Co. and used without further purification. N, N, N ', N'-tetrakis (2-pyridylmethyl) ethylenediamine, TPEN] was purchased from TCI. All solvents were purchased from Aldrich Chemical Co. as " spectroscopic grade ". ¹ H NMR (600 MHz) and ¹³ C NMR (150 MHz) spectra were obtained on a Varian VNS NMR spectrometer and referenced to the residual solvent signal. The UV-vis absorption spectra were recorded on a Jasco V-550 spectrophotometer equipped with a Peltier temperature controller. Fluorescence emission spectra were measured with a PTI QuantaMaster steady-state spectrophotometer. Column chromatography was performed using silica gel (240 mesh). The compositions for detecting azide ions according to this example and Comparative Examples 1 to 3, including fluororesin acetate derivative compounds, were prepared by reacting acetyl chloride and chloroacetyl chloride with fluorescein and 2 ', 7'- Was prepared by the reaction of dichlorofluorescein

< Example > 2 ', 7'- Dichlorofluorescein Chloro  Acetate (hereinafter referred to as &quot; DCF - Chloro  Acetate &quot;) &lt; / RTI &gt;

As shown in FIG. 1A, a compound represented by the following formula 2 was obtained by the reaction of 2 ', 7'-dichlorofluorescein (DCF) with chloroacetyl chloride.

Specifically, chloroacetyl chloride (0.24 mL, 3.0 mmol) was added to a suspension of DCF (0.40 g, 1.0 mmol) and cesium carbonate (0.98 g, 3.0 mmol) in dimethylformamide (hereinafter also referred to as "DMF" mmol), the reaction mixture was stirred at room temperature for 1 hour, diluted with water, and extracted with dichloromethane. The organic phase was separated and washed with 0.1 N NaOH solution and water, then evaporated to give a white solid residue. The product was purified by column chromatography (silica gel, CH ₂ Cl ₂ ).

(2)

< Comparative Example  1 > 2 &apos;, 7 & Dichlorofluorescein  Acetyl chloride (hereinafter referred to as &quot; DCF -Acetyl chloride &quot;) &lt; / RTI &gt;

As shown in FIG. 1A, by reacting 2 ', 7'-dichlorofluorescein (hereinafter also referred to as "DCF") with acetyl chloride, a compound represented by the following general formula (3) .

Specifically, acetyl chloride (0.21 mL, 3.0 mmol) was added to a suspension of DCF (0.40 g, 1.0 mmol) and cesium carbonate (0.98 g, 3.0 mmol) in DMF (10 mL) Stirred for a period of time, diluted with water, and extracted with dichloromethane. The organic phase was separated and washed with 0.1 N NaOH solution and water, then evaporated to give a white solid residue. The product was purified by column chromatography (silica gel, CH ₂ Cl ₂ ).

(3)

< Comparative Example  2> Fluororesin  Preparation of acetyl chloride

As shown in Fig. 1B, a compound represented by the following formula 4 was obtained by the reaction of fluororesin and acetyl chloride.

Specifically, acetyl chloride (0.21 mL, 3.0 mmol) was added to a suspension of fluorescein (0.33 g, 1.0 mmol) and cesium carbonate (0.98 g, 3.0 mmol) in DMF (10 mL) For 1 hour, diluted with water and extracted with dichloromethane. The organic phase was separated and washed with 0.1 N NaOH solution and water, then evaporated to give a white solid residue. The product was purified by column chromatography (silica gel, CH ₂ Cl ₂ ).

[Chemical Formula 4]

< Comparative Example  3> Fluororesin Chloro  Preparation of acetate

As shown in Fig. 1B, a compound represented by the following formula (5) was obtained by the reaction of fluorescein with chloroacetyl chloride.

Specifically, chloroacetyl chloride (0.24 mL, 3.0 mmol) was added to a suspension of fluorescein (0.33 g, 1.0 mmol) and cesium carbonate (0.98 g, 3.0 mmol) in DMF (10 mL) The mixture was stirred at room temperature for 1 hour, diluted with water and extracted with dichloromethane. The organic phase was separated and washed with 0.1 N NaOH solution and water, then evaporated to give a white solid residue. The product was purified by column chromatography (silica gel, CH ₂ Cl ₂ ).

[Chemical Formula 5]

< Example  And Comparative Example  1 to 3 Fluororesin  Acetate derivative stock  solution( stock solution ) And anion stock  Solution preparation>

A stock solution (5.0 × 10 ^-4 M) of the fluorescein acetate derivative compound obtained in this Example and Comparative Examples 1 to 3 was prepared in acetonitrile.

Sodium salts include N ₃ ^- , F ^- , Cl ^- , Br ^- , I ^- , HPO ₄ ^{2 -} , P ₂ O ₇ ^{4 -} , SO ₄ ^{2 -} , SO ₃ ^{2 -} , NO ₃ ^- , HCO Stock solutions (0.1 M) of ^{3 -} , OAc ^- , ClO ₄ ^- , and S ^{2 -} ions were prepared in distilled water.

[ Experimental Example ] Example  And Comparative Example  1 to 3 Fluororesin  Acetate derivative compound Azid  Detection of ions

< Signaling  Measurement of the behavior>

UV-vis and fluorescence signaling behaviors of the fluorescein acetate derivative compounds obtained in this Example and Comparative Examples 1 to 3 for azide ion and other anions were measured using a Tris buffer solution (pH 8.0) and acetonitrile (1: 9, v / v). The solution for the measurement was prepared by adding a stock solution (30 μL, 5.0 × 10 ^-4 M) of fluorene acetate derivative compound, 30 μL of Tris buffer solution (1.0 μM) and an azide ion or anion stock solution mu L, 0.1 M) were sequentially placed in a glass bottle. The prepared solution was diluted with water and acetonitrile in a volume ratio of 1: 9 (v / v) to give a final volume of 3.0 mL. The final concentrations of the fluorescein acetate derivative compound, the buffer solution, and the anion were 5.0 × 10 ^-6 M, 1.0 × 10 ^-2 M, and 5.0 × 10 ^-4 M, respectively. The excitation wavelength for fluorescence measurement was 480 nm.

<Shielding of sulphide ion interference>

TPEN-Hg ^{2 +} complex solution was added as an anion-blocking agent in order to eliminate the interference by sulfide ions. Each sample solution contained different anions and TPEN-Hg ^{2 +} stock solution (150 μL) and the fluorescein acetate derivative compound were added successively under the same measurement conditions. The TPEN-Hg ^{2 +} stock solution was prepared by dissolving Hg (ClO ₄ ) ₂ (43 mg, 10 mM) and TPEN (40 mg, 10 mM) in acetonitrile. The final concentrations of the fluorescein acetate derivative compounds, anions, buffer solutions, and TPEN-Hg ^{2 +} for signaling measurements were 5.0 × 10 ^-6 M, 5.0 × 10 ^-4 M, 1.0 × 10 ^-2 M , And 5.0 x 10 &lt;&quot; ⁴ &gt;

<Detection limit>

According to the IUPAC recommendation, the detection limit was calculated by the equation 3S _bl / m. Where S _bl is the standard deviation of the blank measurement (number of measurements = 15) and m is the calibration sensitivity defined as the slope of the calibration curve.

<Time course measurement>

The elapsed time of signaling of the azide ions to the fluorescein acetate derivative compounds obtained in this Example and Comparative Examples 1 to 3 was measured by measuring the fluorescence intensity of the sample solution at 532 nm. In the 1: 9 (v / v) mixed solution of tris-buffer solution (pH 8.0) and acetonitrile, the fluorescein acetate derivative compound obtained in this Example and Comparative Examples 1 to 3, TPEN-Hg ² The concentrations of ⁺ , S ^{2 -} , and N ₃ ^- ions were 5.0 × 10 ^-6 M, 5.0 × 10 ^-4 M, 5.0 × 10 ^-4 M, and 5.0 × 10 ^-4 M, respectively.

<Competition test>

A stock solution of DCF-chloroacetate (30 μL), anion solution (15 μL), and N ₃ ^- solution (15 μL) obtained in this Example was placed in a glass bottle continuously to prepare a test solution. The final concentrations of DCF-chloroacetate, buffer solution, anion (A ^{n -} ), and azide ion in a 1: 9 (v / v) mixed solution of tris-buffer solution pH 8.0 and acetonitrile were 5.0 10 ^-6 M, 1.0 10 ^-2 M, 5.0 10 ^-4 M, and 5.0 10 ^-4 M, respectively.

< ^One H NMR  Spectrum>

Sodium azide (13.4 mg, 0.24 mmol) in D ₂ O solution (0.06 mL) was added to DCF-chloroacetate solution (3.3 mg, 0.006 mmol) in CD ₃ CN (0.54 mL). The mixed solution was stirred at room temperature for 10 minutes, and partial ¹ H NMR spectrum of DCF-chloroacetate and azide ion was obtained without any separation.

<Tap water and virtual In the water Azid  Analysis of ions>

A stock solution (0.1 M) of azide ion was prepared in distilled water, tap water, and virtual wastewater. The solution obtained by adding N ₃ ^- , a Tris buffer solution, and a stock solution of DCF-chloroacetate obtained in this example to a vial was diluted to 3.0 mL with acetonitrile and distilled water, tap water, or wastewater. The final concentrations of DCF-chloroacetate, Tris buffer solution (pH 8.0), and azide ion in a 1: 9 (v / v) mixed solution of tris-buffered distilled water, tap water or wastewater and acetonitrile were 5.0 x 10 ^-6 M, 1.0 x 10 ^-2 M, and 0 to 1.0 x 10 ^-4 M. All measurements were performed 5 times. The virtual wastewater was prepared according to the composition of the already known wastewater: [Na ⁺ ] = 2.39 × 10 ^-3 M, [K ⁺ ] = 2.81 × 10 ^-4 M, [Mg ^{2 +} ] = 2.88 × 10 ^-4 M, ^{[Ca 2 +] = 2.50 ×} 10 -4 M, [F -] = 1.60 × 10 -5 M, [Cl -] = 9.87 × 10 -4 M, [PO 4 3 -] = 1.05 × 10 -4 M , [SO ₄ ^2- ] = 2.08 × 10 ^-4 M, [NO ₃ ^- ] = 4.84 × 10 ^-4 M, [HCO ₃ ^- ] = 1.23 × 10 ^-3 M.

[result]

Fluorescein acetate derivative compounds according to this Example and Comparative Examples 1 to 3 were prepared by reacting fluororesin or 2 ', 7'-dichlorofluorescein (DCF) with acetyl chloride or chloroacetyl chloride 1a and 1b). The fluorescein acetate derivative compounds obtained in this Example and Comparative Examples 1 to 3 showed typical colorimetric and fluorescence-on type signaling behaviors of fluorescein dye towards azide ions in aqueous acetonitrile and tetrahydrofuran .

In this example, a fluorescein derivative containing a dichloro derivative was irradiated as a signaling handle, and the DCF derivative showed better signaling efficiency compared to the fluorescein derivative in terms of rate towards the azide ion and, I found out. FIG. 2 is a graph showing changes in fluorescence intensity of the compound prepared according to this example and Comparative Examples 1 to 3 at 532 nm in the presence of azide ions with respect to time according to this example, The concentration of the compound prepared according to this Example and Comparative Examples 1 to 3 was 5.0 × 10 ^-6 M in the mixed solution of the solution (pH 8.0) and acetonitrile (volume ratio 1: 9), and the concentration of the azide ion Was 5.0 × 10 ^-4 M, and the excitation wavelength (λ _ex ) was 480 nm. As shown in FIG. 2, the compounds containing the fluorescein derivatives obtained in Comparative Examples 2 and 3 exhibited very poor reaction toward azide ions. Therefore, a compound containing a DCF derivative according to this example and the comparative example 1 which exhibited more reactivity and absorbed a long wavelength was tested as a composition for detecting azidated ions. After systematic irradiation, it was found that the DCF-chloroacetate obtained in this example had sufficient stability of the DCF-chloroacetate itself as well as reactivity towards azidated ions. Based on these results, all signaling behavior towards azide ions was investigated using DCF-chloroacetate obtained in this example.

FIG. 3 is a graph showing the absorbance change ( A / A ₀ ) of DCF-chloroacetate according to this example at 513 nm in the presence of various anions, and the illustration shows the UV-absorbance of the DCF-chloroacetate in the presence of various anions. vis absorption spectrum of DCF-chloroacetate in the mixed solution of Tris buffer solution (pH 8.0) and acetonitrile (volume ratio 1: 9) was 5.0 × 10 ^-6 M and the concentration of anions was 5.0 × 10 ^-4 M, and the excitation wavelength (λ _ex ) was 480 nm. The DCF-chloroacetate showed a slight absorption above 400 nm due to the ring-closed lactone form of the DCF moiety (FIG. 3). In the presence of azide ions, a strong absorption band was observed at 513 nm with a shoulder at 480 nm. The absorbance improvement ( A / A ₀ ), as indicated by the ratio of absorbance at 513 nm, was 154 times in the presence and absence of azide ions. Incidentally, the solution turned from colorless to visually detectable greenish yellow. The sulphide ion known to react with fluorescein dinitrobenzenesulfonate also exhibited a remarkable reaction with an A / A ₀ value of 32. Fortunately, the reaction to the sulphide ion was Hg ^{2 +} of TPEN as a masking agent, (TPEN-Hg ²⁺ ), and the selectivity toward azide ions could be realized (see below). Other ions were 0.04 and HPO ₄ ² for on P ₂ O ₇ ^{4 - -} on for having an absorbance ratio (a / a ₀₎ of the range between 3.3 and relatively non-reactivity are shown.

The DCF-chloroacetate also showed significant fluorescence enhancement towards azide ions in a 10% aqueous acetonitrile solution (Figure 4). Figure 4 is a graph showing the change (I / I ₀₎ of the fluorescence intensity of DCF- chloroacetate according to the present embodiment in the presence, 532 nm of a variety of anionic, sapdo is the DCF- chloroacetate in the presence of various anions As the fluorescence emission spectrum, the concentration of DCF-chloroacetate in the mixed solution of Tris buffer solution (pH 8.0) and acetonitrile (1: 9 volume ratio) was 5.0 × 10 ^-6 M and the concentration of anions was 5.0 × 10 ^-4 M, and the excitation wavelength (λ _ex ) was 480 nm. Fluorescence emission of the DCF-chloroacetate was very weak at over 490 nm, due to the ring-closed form of the spirolactone moiety of O -functionalized fluorescein. In the presence of an azide ion, significant emission was seen from the DCF-chloroacetate at 532 nm. Fluorescence emission ratio at 532 nm (I / I ₀₎ is displayed by improving the ratio of the azide ion in the presence and absence of the addition is 245 times higher. Fluorescence of the solution changed from colorless to greenish yellow under UV lamp irradiation. Among other negative ions, sulfide ions also fluorescence emission ratio at 532 nm (I / I ₀₎ showed a remarkable 32-fold enhanced response. The other anions showed a small and almost constant reaction between 0.95 and 3.4 times Br ^- to pyrophosphate.

Selective degradation of the chloroacetate moiety by the azide ion resulted in signaling for the azide ion in DCF-chloroacetate (Scheme 1). The indicated conversion was confirmed by NMR, UV-vis, and fluorescence measurements. The ¹ H NMR spectrum of the DCF-chloroacetate measured in the presence of sodium azide (40 equiv.) Was identical to the partial ¹ H NMR spectrum of DCF in the presence of sodium azide (FIG. 5). Figure 5 shows a partial ¹ H NMR spectrum of DCF-chloroacetate according to this example, in the presence and absence of azide ion, at which time, in a mixed solution of D ₂ O and CD ₃ CN (1: 9 by volume) The concentrations of DCF-chloroacetate and DCF were 1.0 × 10 ^-2 M and the concentration of azide ion was 0.4 M. 5, resonance of DCF was observed at 7.05 ppm and 6.56 ppm, resonance of 1 ', 8'-hydrogen at 7.06 ppm of DCF-chloroacetate and 4', 5'-hydrogen at 7.04 ppm has disappeared. The UV-vis and fluorescence emission spectra of DCF-chloroacetate in the presence of 100 equivalents of azide ion were the same as the UV-vis and fluorescence emission spectra of DCF under the same measurement conditions (FIGS. 6 and 7). 6 and 7 show changes in UV-vis absorption spectrum and fluorescence emission spectrum of DCF-chloroacetate and DCF according to the present embodiment, respectively, in the presence and absence of azide ion, The concentration of DCF-chloroacetate and DCF in the mixed solution of the solution (pH 8.0) and acetonitrile (1: 9 by volume) was 5.0 × 10 ^-6 M and the concentration of the azide ion was 5.0 × 10 ^-4 M , And the excitation wavelength ( _ex ) was 480 nm.

 [Scheme 1]

As mentioned earlier, sulfide ions interfere with signaling of the azide by the DCF-chloroacetate. This example tested the possibility of improving azide ion selectivity in the presence of sulfide ions under measurement conditions by inhibiting interference from sulfide ions through the formation of stable metal ion-sulfide complexes. FIG. 8 is a graph showing the suppression of interference of sulfide ions of DCF-chloroacetate according to the present embodiment in the signaling of an azide ion by TPEN-Hg ^{2 +} , wherein a Tris buffer solution (pH 8.0) The concentration of DCF-chloroacetate was 5.0 × 10 ^-6 M, the concentration of anions was 5.0 × 10 ^-4 M, and the concentration of TPEN-Hg ^{2 +} was 5.0 (molar ratio) in acetonitrile × 10 ^-4 M, and the excitation wavelength (λ _ex ) was 480 nm. As shown in FIG. 8, it was found that the TPEN-Hg ^{2 +} complex effectively inhibited sulfide ion signaling without significantly affecting the azide ion signaling of the DCF-chloroacetate. Using this complex, the reaction of DCF-chloroacetate towards the sulfide ion was almost completely inhibited (see Example of FIG. 8 + TPEN-Hg ^{2 +} + S ^{2 -} ). The selectivity toward azide ion through the sulfide ion in the presence of TPEN-Hg ^{2 +} at the rate of reaction at 532 nm, I ( _{example + azide} ) / I ( _{example + sulfide} ) Significantly improved by double. In the presence of Hg ^2+-TPHE, the DCF- azide ion selective UV-vis signal by the painter chloroacetate was also observed, this is of 4.8 to 109 A at 513 nm _{(Example azide +)} / A _{( Lt; RTI ID = 0.0 & gt ; + sulphide)} &lt; / RTI &gt;

The efficient inhibition of sulphide ion interference in the azide ion signaling of DCF-chloroacetate is due to the formation of a highly stable HgS complex with TPEH-Hg ^{2 +} , which provides sulphide-shielded Hg ^{2 +} ions (Scheme 2). The dissociation constant K _d of TPEN-Hg ^{2 +} is 10 ^-25 M, while the K _sp of HgS is 2 × 10 ^-53 . K _sp of HgS is effectively separated from the sulfide ions in the analyte to form a Hg ^{2 +} ions and stable HgS complexes release is much smaller from the lower TPEN-Hg ^{2 +} complex stability than the K _d of TPEN-Hg ^{2 +} I could. In contrast, azidized ion signaling was not affected by the addition of TPEN-Hg ^{2 +} , because the Hg ^{2 +} ion is strongly bound and remains in a complexed state. This can be reasonably explained by the fact that the overall formation constant β ₂ of the Hg (N ₃ ) ₂ complex is 2.44 × 10 ¹⁴ M ^-2 , while the TPEN-Hg ^{2 +} K _d value is 10 ^-25 M.

 [Scheme 2]

Next, the influence of other coexisting anions on the signaling of azide ions by the DCF-chloroacetate was determined. FIG. 9 is a graph showing the fluorescence intensity of the azide ion by DCF-chloroacetate according to the present example in the presence of various anions, wherein the ratio of the Tris buffer solution (pH 8.0) and acetonitrile (volume ratio 1: ), The concentration of DCF-chloroacetate was 5.0 × 10 ^-6 M, the concentration of azide ion and anion was 5.0 × 10 ^-4 M, and the excitation wavelength (λ _ex ) was 480 nm. As shown in Figure 9, in the presence of common anions, the fluorescence of the DCF-chloroacetate-azide ion system was not significantly affected. The ratio of the fluorescence intensity of the DCF-chloroacetate-azide ion system at 532 nm, I _{( example + azide + anion)} / I _{( example + azide )} , in the presence and absence of other anions, Lt; RTI ID = 0.0 &gt; 0.97 &lt; / RTI &gt; This suggests that the DCF-chloroacetate may be useful for convenient detection of azide ions in common industrial and environmental systems.

To understand quantitative signaling behavior, fluorescence titration of azide ions and the DCF-chloroacetate was performed. Figure 10 shows a graph The fluorescence emission spectrum of DCF-chloroacetate according to the present embodiment as a function of the concentration is shown in the graph. The illustration shows the change in fluorescence intensity at 532 nm. At this time, in the mixed solution of Tris buffer solution (pH 8.0) and acetonitrile (volume ratio 1: 9), the concentration of DCF-chloroacetate was 5.0 × 10 ^-6 M and the concentration of azide ion was 0 to 4.0 × 10 ^-4 M, and the excitation wavelength (λ _ex ) was 480 nm. As shown in FIG. 10, the concentration-dependent fluorescence changes of the DCF-chloroacetate as a function of azide ion produced a well-defined calibration plot suitable for the quantification of azide ions. As shown in the illustration of FIG. 10, the fluorescence intensity of the DCF-chloroacetate steadily increased at 532 nm as the azide ion concentration increased to 2.0 x 10 &lt; ^-4 &gt; The detection limit calculated for three times the standard deviation of the background signal was 4.0 × 10 ^-7 M (17 ppb). The UV-vis titration of the azide ion and the DCF-chloroacetate showed a similar reaction up to 4.0 × 10 ^-4 M of the azide ion (FIG. 11). FIG. 11 is a graph showing the concentration of azide ion &lt; RTI ID = 0.0 &gt; As a graph showing the concentration-dependency of signaling, the concentration of DCF-chloroacetate in the mixed solution of Tris buffer solution (pH 8.0) and acetonitrile (1: 9 volume ratio) was 5.0 × 10 ^-6 M, The concentration of ions was 0 to 4.0 x 10 &lt; ^-4 &gt;

FIG. 12 is a graph showing signaling of azide ion and sulfide ion with respect to time by DCF-chloroacetate according to the present embodiment, wherein Tris buffer solution (pH 8.0) and acetonitrile (1: ), The concentration of DCF-chloroacetate was 5.0 × 10 ^-6 M, the concentration of azide ion and sulfide ion was 5.0 × 10 ^-4 M, the concentration of TPEN-Hg ^{2 +} was 5.0 × 10 ^{-4 -4} M, and the excitation wavelength (? _Ex ) was 480 nm. Signaling of the azide ion by DCF-chloroacetate was completed within 10 minutes after preparation of the sample (FIG. 12). In addition, the signaling rate of the azide ion by DCF-chloroacetate was not affected by the addition of TPEN-Hg ^{2 +} complex. On the other hand, the reaction of the DCF-chloroacetate toward the sulfide ion gradually increased. However, in the presence of the TPEN-Hg ^{2 +} complex, the signaling of the sulfide was negligible until 30 minutes after sample preparation. Changes over time also confirmed the intrinsic stability of the DCF-chloroacetate towards hydrolysis under the measurement conditions.

Finally, the practical applicability of the DCF-chloroacetate in signaling of azide ions was tested using virtual waste water samples. Signaling was measured on solutions containing 20 equivalents of azide ion (1.0 x ^10-4 M) in distilled water, tap water, and virtual wastewater. Wastewater samples were prepared according to the existing paper procedure. 13 is a graph showing the fluorescence signaling of azide ions by DCF-chloroacetate according to this example in distilled water, tap water, and imaginary wastewater samples, wherein a 10% aqueous acetonitrile solution (pH 8.0, Solution, the concentration of DCF-chloroacetate was 5.0 × 10 ^-6 M, the concentration of azide ion was 0 to 1.0 × 10 ^-4 M, and the excitation wavelength λ _ex was 480 nm. As can be seen from Fig. 13, the azide ion signaling in tap water and virtual wastewater closely coincided with distilled water. All these observations indicated that the DCF-chloroacetate may be useful for convenient optical signaling of azide ions in chemical and industrial systems.

14 is a partial ¹ H NMR spectrum of DCF-chloroacetate in CDCl ₃ .

Figure 15 is a part of the ¹³ C NMR spectrum in CDCl ₃ DCF- chloroacetate.

A novel reaction-based dual signaling azide ion detection composition for the detection of azide ions has been developed. Selective degradation of dichlorofluorescein chloroacetate by azide ion was used for colorimetric and fluorescence signaling. Fluorescein derivatives also showed selective signaling to azide ions, but the signaling rate was very slow. Azide ion signaling by dichlorofluorescein chloroacetate was rapid and selective in the presence of other anions. In particular, interference from sulphide ions was easily inhibited by using TPEN-Hg ²⁺ complex as a masking agent, which did not affect the azide ion signaling of the compositions for detecting azide ions. Selective dual signaling of azide ions was possible in a mixed aqueous solution with a detection limit of 4.0 × 10 ^-7 M (17 ppb). The designed dichlorofluorescein chloroacetate can be useful as a convenient, visually detectable, optically signaling composition for the detection of azide ions in chemical and industrial systems.

It will be understood by those of ordinary skill in the art that the foregoing description of the embodiments is for illustrative purposes and that those skilled in the art can easily modify the invention without departing from the spirit or essential characteristics thereof. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present invention is defined by the appended claims rather than the detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

Claims (10)

1. A fluorene derivative represented by the following formula (1)
Wherein the fluorescein acetate derivative compound is hydrolyzed by an azide ion to generate a colorimetric signal and / or a fluorescence signal.
Compositions for detecting azide ions:
[Chemical Formula 1]

In Formula 1,
X ₁ and X ₂ are each independently Cl,
X ₃ and X ₄ are each independently selected from the group consisting of F, Cl, Br, I, and combinations thereof.
The method according to claim 1,
A composition for detecting an azide ion further comprising a mixed solution of a buffer solution and an organic solvent.
3. The method of claim 2,
Wherein the buffer solution comprises one selected from the group consisting of Tris buffer solution, phosphate buffer solution, acetate buffer solution, HEPES buffer solution, and combinations thereof.
3. The method of claim 2,
Wherein the acidity of the buffer solution is from pH 7 to pH 9.
3. The method of claim 2,
Wherein the organic solvent comprises one selected from the group consisting of acetonitrile, tetrahydrofuran, dimethylsulfoxide, methanol, dioxane, acetone, and combinations thereof.
A colorimetric signal and / or a fluorescence signal of the composition for detecting azide ion according to any one of claims 1 to 5,
Wherein the colorimetric signal and / or the fluorescence signal is obtained by adding azide ion to the composition for detecting azide ion, whereby the fluorescein acetate derivative compound contained in the composition for azide ion detection is azidated Wherein the colorimetric signal and / or the fluorescent signal is generated by hydrolysis by an ion.
A method for detecting azide ions.
The method according to claim 6,
And adding an anion to the composition for detecting azide ion.
8. The method of claim 7,
Wherein the anion is ^{F -, Cl -, Br -} , I -, HPO 4 2 -, P 2 O 7 4 -, SO 4 2 -, SO 3 2 -, NO 3 -, HCO 3 -, OAc -, ClO 4 ^- , S ^2- , and combinations thereof.
8. The method of claim 7,
Wherein the azide ion detection composition further comprises TPEN-Hg ^{2 +} as an anion-blocking agent for preventing reaction of the anion with the azidated ion detecting composition.
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