KR102267067B1 - Fluorescent probes compound for the detection of diethylcyanophosphonate and use thereof - Google Patents

Abstract

The present invention relates to a fluorescent probe compound for detection of diethyl cyanophosphonate and uses thereof, wherein the fluorescent probe compound has high sensitivity to diethyl cyanophosphonate and a fast reaction time to be environmentally used, and in particular, the fluorescent probe compound can be usefully used to check the presence or absence of nerve agents in the environment and all related research fields in that it is possible to monitor the detection of diethyl cyanophosphonate in real time under a given environmental condition.

Description

Fluorescent probes compound for the detection of diethylcyanophosphonate and use thereof

The present invention relates to a fluorescent probe compound for detecting diethylcyanophosphonate and uses thereof.

O(P=O)R₂OR₃)인 사린(GB), 소만(GD), 타분(GA)는 극도로 낮은 농도에서도 높은 독성을 나타내기 때문에 인체유해물질로 알려져 있다. 이러한 이유로 인해, 신경작용제를 선택적이고 민감하게 감지할 수 있는 여러 분석 기술이 개발되고 있으며 이를 이용한 방어체계, 해독 및 안전관리법들이 꾸준히 개발되고 있다(Chem. Soc. Rev. 2013, 42, 9251; Chem. Soc. Rev. 2008, 37, 470; Chem. Soc. Rev. 2017, 46, 3357). Nerve agents are a kind of organic compound that disrupts the balance of the central nervous system by blocking acetylcholinesterase (AChE), an enzyme that can control the neurotransmitter acetylcholine). Among them, the G-series organophosphonate derivatives (R)

Sarin (GB), Soman (GD), and Tabun (GA), which are O(P=O)R₂OR₃), are known to be harmful to the human body because they show high toxicity even at extremely low concentrations. For this reason, various analytical techniques that can selectively and sensitively detect nerve agents have been developed, and defense systems, detoxification and safety management methods using them have been continuously developed (Chem. Soc. Rev. 2013, 42, 9251; Chem. Soc. Rev. 2008, 37, 470; Chem. Soc. Rev. 2017, 46, 3357).

As a representative analysis method of a nerve agent, equipment-based analysis techniques such as mass spectrometry, ion mobility spectrometry, and photonic crystal are known. However, in the case of the existing analysis method, there is a problem in that it is difficult to carry as a real-time detection method in the field due to low sensitivity and selectivity, complicated instrument usage, additional pre-processing, expensive analysis cost, and large volume.

Accordingly, the development of a technique for detecting a nerve agent using a fluorescent probe as a method capable of remarkably improving this problem is being actively developed (Chem. Soc. Rev. 2015, 115, 7893; Chem. Soc. Rev. (2015, 115, 7944).

Fluorescent probe systems known to be able to specifically detect diethylcyanophosphonate (DNCP) are (i) pyridine, alkyl-alcohol by diethylthionophosphonate. Fluorescence change due to phosphorylation of functional groups such as alcohol), phenolic-alcohol, and amine, (ii) hydroxy-imine functional group by diethylthionophosphonate It has a chemical reaction-based mechanism of action, such as a change in fluorescence according to the reaction of being replaced with a nitrile.

However, a fluorescent probe capable of real-time detection in the environment while having high sensitivity and selectivity to diethylcyanophosphonate has not yet been developed despite its importance.

Accordingly, the present inventors have developed a fluorescent probe compound that is easy to synthesize, interacts with diethylcyanophosphonate, which is highly toxic to the human body, emits fluorescence, and can specifically detect only the target material. . This fluorescent probe compound quantitatively shows an increase in fluorescence intensity according to the amount of the target material, and has selectivity to distinguish and detect diethylcyanophosphonate from other known nerve agents. In addition, the present invention was completed by revealing that the developed fluorescent probe compound can be used as a detection kit that can be utilized in real time in the field due to a fast reaction time with a target material.

An object of the present invention is ortho-hydroxy (hydroxy)-benzaldehyde (benzaldehyde) diethylcyanophosphonate (DNCP, diethylcyanophosphonate) analog of the nerve agent Tabun (Tabun, GA) using a naphthalene-based compound To provide a fluorescent probe compound for selectively detecting the and diethylcyanophosphonate detection kit comprising the same.

Another object of the present invention is to provide a method for detecting diethylcyanophosphonate using the fluorescent probe compound.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

The present invention provides a fluorescent probe compound for detecting diethylcyanophosphonate represented by the following formula (1):

[Formula 1]

In one embodiment of the present invention, the fluorescent probe compound is characterized in that it contains an ortho-hydroxy-benzaldehyde structure in the compound.

In another embodiment of the present invention, the fluorescent probe compound is an excited-state intramolecular proton transition (Excited-State Intramolecular) in the structure of ortho-hydroxy-benzaldehyde through a nucleophilic substitution reaction with diethylcyanophosphonate. It is characterized in that it exhibits fluorescence when the quenching effect by Proton Transfer is broken.

In addition, the present invention provides a kit for detecting diethylcyanophosphonate comprising the fluorescent probe compound.

In addition, the present invention provides a method for detecting diethylcyanophosphonate using the fluorescent probe compound.

In one embodiment of the present invention, the detection method is characterized in that it comprises the step of adding a sample containing diethylcyanophosphonate to the fluorescent probe compound, and irradiating UV light.

The fluorescent probe compound of the present invention can provide a fluorescent signal by selectively detecting diethylcyanophosphonate, which is an analog of the nerve agent Tabun (Tabun, GA). In addition, an analyte in an arbitrary environment containing diethylcyanophosphonate may be collected and analyzed in real time using the fluorescent probe compound.

In particular, the fluorescent probe compound of the present invention is easy to synthesize, and proton transfer in an excited state molecule due to the structural characteristics of an ortho-hydroxy-benzaldehyde compound having a bipolar naphthalene structure ( ESIPT, Excited-State Intramolecular Proton Transfer). Diethylcyanophosphonate, a nerve agent, inhibits proton transfer in an excited state in the structure of the compound and turns on the fluorescence of the molecular probe compound, which is a G-series nerve agent with a similar structure. It overcomes the problem of conventional fluorescent probes with low selectivity in that they show a specific increase in fluorescence without their interference.

In addition, the fluorescent probe compound of the present invention can detect diethyl cyanophosphonate even in environmental conditions such as soil, and in the form of a portable detection kit, it is possible to detect diethyl cyanophosphonate in a short time in situ. By providing a new method for detection, it can be usefully used in various fields such as actual military use (war), as a counter-terrorism prevention tool, and whether or not it is distributed in the environment.

1 shows the absorption and fluorescence graphs of the compound (DMHN1) according to the present invention measured under given solvent conditions.
2 shows the results of measuring the mass of the product after the reaction was completed using a high resolution mass spectra (JMS-700 spctrometer, JEOL, Tokyo, Japan).
3 shows the reaction mechanism of the compound (DMHN1) according to the present invention to diethylcyanophosphonate.
4 shows the results of confirming whether the fluorescence increases according to the concentration of diethylcyanophosphonate when the compound (DMHN1) according to the present invention is reacted with diethylcyanophosphonate.
5 shows the correlation analysis results of the fluorescence intensity of the compound (DMHN1) according to the present invention with respect to the concentration (0 to 1 mM) of diethylcyanophosphonate in the maximum fluorescence wavelength band.
6 shows the results of correlation analysis of the fluorescence intensity of the compound (DMHN1) according to the present invention to diethylcyanophosphonate (0 to 10 μM) at a low concentration.
7 is a result of confirming the selectivity of the compound (DMHN1) according to the present invention to diethylcyanophosphonate through a change in fluorescence.
8 is a result of analyzing the increase in fluorescence intensity based on the maximum fluorescence wavelength (486 nm) when the reaction of the compound (DMHN1) according to the present invention and diethyl cyanophosphonate proceeded for 3 minutes.
9 is a photograph showing the result of confirming whether diethylcyanophosphonate was detected under soil conditions of the compound (DMHN1) according to the present invention.
10 is a graph showing the change in fluorescence intensity according to the reaction time of the compound (DMHN1) according to the present invention with diethylcyanophosphonate under soil conditions.
11 is a photograph showing the change in fluorescence intensity over time of the compound (DMHN1) according to the present invention according to the type of each soil containing diethylcyanophosphonate.
12 is a graph showing the change in fluorescence intensity over time of the compound (DMHN1) according to the present invention according to the type of each soil containing diethylcyanophosphonate.
13 shows a real-time in situ detection process of diethylcyanophosphonate using the compound (DMHN1) according to the present invention.
14 shows the results of real-time detection of diethylcyanophosphonate by manufacturing the compound (DMHN1) according to the present invention with a field detection kit.

The present invention provides a fluorescent probe compound for detecting diethylcyanophosphonate (DNCP, diethylcyanophosphonate) represented by the following formula (1).

[Formula 1]

The fluorescent probe compound of the present invention is characterized in that it includes an ortho-hydroxy-benzaldehyde structure.

In addition, the fluorescent probe compound is an ortho-hydroxy-benzaldehyde through a nucleophilic substitution reaction with diethylcyanophosphonate by proton transfer (ESIPT, Excited-State Intramolecular Proton Transfer) in the structure of benzaldehyde. Fluorescence may be exhibited when the quenching effect is broken.

In one embodiment of the present invention, the detection mechanism of DMHN1 of the compound of Formula 1 developed in the present invention was investigated, which is ortho through nucleophilic substitutions between DMHN1 and diethylcyanophosphonate. It was confirmed that the quenching effect caused by the excited-state intramolecular proton transfer (ESIPT) in the structure of -hydroxy-benzaldehyde was broken while exhibiting fluorescence (Example 3).

In one embodiment of the present invention, the fluorescence characteristics were checked using the compound of Formula 1 developed in the present invention as a fluorescent probe, and through this, the compound of Formula 1 of the present invention was converted to diethylcyanophosphonate. It was confirmed that the containing sample selectively exhibited fluorescence turn-on (Example 5).

In addition, the present invention provides a kit for detecting diethylcyanophosphonate comprising the fluorescent probe compound.

In one embodiment of the present invention, by adding a soil sample to the compound of Formula 1 developed in the present invention to confirm that diethylcyanophosphonate contained in the soil sample can be detected, a real-time on-site detection kit was confirmed to be applicable (Examples 6 and 7).

In addition, the present invention provides a method for detecting diethylcyanophosphonate using the fluorescent probe compound.

The detection method of the present invention may include adding a sample containing diethylcyanophosphonate to the fluorescent probe compound and irradiating UV light, but is not limited thereto.

In one embodiment of the present invention, it was confirmed that the method for detecting diethylcyanophosphonate of the compound of Formula 1 is effective (Example 7).

Therefore, since the compound of Formula 1 according to the present invention has high sensitivity and fast reaction time to diethylcyanophosphonate, it is possible to use the compound in an environment, and in particular, under given environmental conditions, diethylcyanophosphonate in real time In that the detection of annophosphonate can be monitored, it can be usefully used for checking the presence or absence of a nerve agent in the environment and all related research fields.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

Example 1. Synthesis and structural analysis of fluorescent probe compound (DMHN1)

The present inventors paid attention to that naphtol terminates the proton transfer in the excited state molecule in the fluorescent probe through a nucleophilic substitution reaction with diethylcyano. In order to develop a fluorescent probe capable of bringing about a change in optical properties, a compound of Formula 1 was synthesized according to Scheme 1 below.

[Scheme 1]

(1) Synthesis of 7- (dimethylamino) naphthalen-2-ol (7- (dimethylamino) naphthalen-2-ol) (compound No. 1 in Scheme 1)

7-(dimethylamino)naphthalen-2-ol; The synthesis of 7-(dimethylamino)naphthalen-2-ol was performed.

Specifically, compound 1 (1 g, 6.243 mmol, Sigma-aldrich, D116408) and sodium metabisulfite (Na ₂ S ₂ O ₅ , 2.374 g, 12.486 mmol) in Scheme 1 as a starting material for synthesis In a sealed-tube, dimethylamine solution (40 % in H ₂ O, 3.95 mL, 31.215 mmol) and water (H ₂ O, 2.67 mL) were added, and then a lid, Teflon and insulating tape were added. was used to block the entrance to the container. The mixture was stirred at 150° C. for 4 hours using a silicone oil container. To terminate the reaction, first lower the temperature of the reactant to room temperature (25 ℃), open the container, put ethyl acetate (ethylacetate, 100 mL), water (100 mL), and saturated brine (30 mL), and use a separatory funnel to add the organic layer was extracted. Water present in the organic layer of ethyl acetate obtained through extraction was dried over anhydrous sodium sulfate (Na ₂ SO ₄ , 1 g), and concentrated using an evaporator. The pale brown solid compound thus obtained was separated using column chromatography using silica gel (eluent: 20 % EtOAc/Hexane) to form a white solid compound No. 2 in Scheme 1 (710 mg, 60 %) got When the resulting compound is analyzed by thin layer chromatography (TLC, thin layer chromatography, silical gel 60F-254 glass plate, Merck), _{it has a developing value of R f} = 0.25 (20 % EtOAc/Hexane - one run).

¹ H NMR (CDCl ₃ , 300 MHz, 293K): δ 7.66-7.59 (m, 2H), 7.05-7.02 (m, 1H), 6.96-6.95 (d, 1H), 6.85-6.82 (m, 1H), 6.78 -6.77 (d, 1H), 5.10 (s, 1H), 3.05 (s, 6H).

¹³ C NMR (CDCl3, 75 MHz, 293K): δ153.88, 149.17, 136.24, 129.39, 128.61, 122.44, 114.22, 113.80, 108.02, 105.32, 40.91.

HRMS: m/z calcd. for C ₁₂ H ₁₃ NO 187.0997 found 187.0999.

(2) Synthesis of 7-(methoxymethoxy)-N,N -dimethylnaphthalen-2-amine (7-(methoxymethoxy) -N,N- dimethyl naphthalen-2-amine)

The synthesis of 7-(methoxymethoxy) -N,N -dimethylnaphthalen-2-amine (compound No. 2 in Scheme 1 above) was performed.

Specifically, sodium hydride (NaH, sodium hydride, 235 mg, 5.875 mmol) was added to a dimethylformamide (DMF, N,N-dimethylformamide, 5 mL) solvent, and an argon balloon was inserted, followed by saturated brine. and ice was used to lower the temperature to -15 °C. Compound No. 2 (1 g, 5.34 mmol) was dissolved in DMF (5 mL) in this mixed solution, and then slowly added dropwise for about 5 minutes while maintaining the temperature. In this process, hydrogen gas (H ₂ gas) is generated, which is discharged outdoors through a silicon oil trap. After stirring at the same temperature for 1 hour, it was confirmed that no more _{droplets (H 2 gas) were generated in the trap.} Then, chloromethyl methyl ether (chloromethyl methyl ether, 0.4 mL, 5.34 mmol) was slowly added dropwise for about 5 minutes. When the injection was completed, the temperature was changed to room temperature (25° C.), and the mixture was stirred for 6 hours. After 6 hours, water (100 mL) was added, and extraction was performed using ethyl acetate (EtOAc, 200 mL). Water present in the organic layer of ethyl acetate obtained through extraction was dried over anhydrous sodium sulfate (10 g), and the dried organic layer of ethyl acetate was concentrated using a suction device. The light brown liquid compound thus obtained was separated by column chromatography using silica gel (eluent: 5 % EtOAc/Hexane), and a white solid compound (compound No. 3 in Scheme 1) (988 mg, 80%) got Analysis of the resulting compound by thin layer chromatography _{has a developing value of R f} =0.45 (20% EtOAc/Hexane - one run).

¹ H NMR (CDCl ₃ , 300 MHz, 293K): δ 7.75 (d, 1H), 7.72 (d, 1H), 7.40-7.39 (d, 1H), 7.15-7.08 (m, 2H), 6.98-6.97 (d) , 1H), 5.39 (s, 2H), 3.69 (s, 3H), 3.11 (s, 6H).

¹³ C NMR (CDCl ₃ , 75 MHz, 293K): δ 155.75, 149.16, 136.23, 129.11, 128.55, 123.00, 114.99, 114.58, 108.69, 105.96, 94.62, 56.07, 40.83.

HRMS: m/z calcd. for C1 ₄ H ₁₇ NO ₂ 231.1259 found 231.1262.

(3) Synthesis of 6-(dimethylamino)-3-(methoxymethoxy)-2-naphthaldehyde (6-(dimethylamino)-3-(methoxymethoxy)-2-naphthaldehyde)

The synthesis of 6-(dimethylamino)-3-(methoxymethoxy)-2-naphthaldehyde) (compound 3 in Scheme 1 above) was carried out.

Specifically, after putting compound 3 (2.2606 g, 9.774 mmol) in a 100 mL round-bottom flask, an argon balloon was inserted. Then, diethyl ether (Et ₂ O, 50 mL) was added, and the temperature was lowered to -20 °C using saturated brine and ice. After checking the temperature, tertiary butyl lithium (tert-BuLi, 8.6 mL, 14.66 mmol) was slowly added dropwise over about 30 minutes. At this time, the color gradually changed to dark brown, and when injection was completed, the mixture was stirred at the same temperature for 2 hours. After stirring for 2 hours, DMF (1.3 mL, 16.62 mmol) was slowly added dropwise over about 5 minutes. When the injection was completed, the mixture was stirred for 1 hour at the same temperature. At this time, the color gradually changed to bright yellow. After stirring for 1 hour, 4N HCl (10 mL) and primary distilled water (10 mL) were added and stirred for 10 minutes. After 10 minutes, the extraction process was performed using ethyl acetate (300 mL), saturated brine (100 mL), and water (200 mL). Water present in the organic layer of ethyl acetate obtained through extraction was dried over anhydrous sodium sulfate (15 g), and concentrated using an aspirator. The light yellow solid compound thus obtained was separated by column chromatography using silica gel (eluent: 20 % EtOAc/Hexane) to obtain a light yellow solid compound No. 4 in Scheme 1 (1.27 g, 50 %). . Analysis of the resulting compound by thin layer chromatography _{has a developing value of R f} = 0.25 (20% EtOAc/Hexane - one run).

¹ H NMR (CDCl ₃ , 300 MHz, 293K): δ 10.49(s, 1H), 8.25(s, 1H), 7.75-7.73(d, 1H), 7.29-7.24(d, 1H), 7.05-7.03(dd , 1H), 6.77-6.76 (d, 1H), 5.40 (s, 2H), 3.59 (s, 3H), 3.12 (s, 6H).

¹³ C NMR (CDCl ₃ , 75 MHz, 293K): δ 189.61, 155.99, 150.71, 139.74, 131.16, 130.89, 122.26, 121.29, 114.76, 107.66, 104.30, 94.75, 56.39, 40.32.

HRMS: m/z calcd. for C ₁₅ H ₁₇ NO ₃ 259.1208 found 259.1211.

(4) Synthesis of 6-(dimethylamino)-3-hydroxy-2-naphthaldehyde (6-(dimethylamino)-3-hydroxy-2-naphthaldehyde) (compound of formula 1)

The synthesis of 6-(dimethylamino)-3-hydroxy-2-naphthaldehyde was performed.

Specifically, after putting compound 4 (195 mg, 0.75 mmol), isopropyl alcohol, 10 mL), and 5M HCl (5 mL) in Scheme 1 in a 25 mL round-bottom flask, 3 hours at 60 ° C. was stirred. After 3 hours, after cooling the flask to room temperature, isopropyl alcohol was removed from the aspirator, and extraction was performed using ethyl acetate (100 mL) and water (100 mL). Water present in the organic layer of ethyl acetate obtained through extraction was dried over anhydrous sodium sulfate (3 g), and concentrated using a suction device. The yellow solid compound thus obtained was separated by column chromatography using silica gel (eluent: 20% EtOAc/Hexane) to obtain a yellow compound of Formula 1 (113 mg, 70%).

[Formula 1]

When the compound of Formula 1 was analyzed by thin layer chromatography, it had _{a developing value of R f} = 0.35 (20 % EtOAc/Hexane - one run).

¹ H NMR (CDCl ₃ , 300 MHz, 293K): δ 10.54(s, 1H), 9.89(s, 1H), 7.90(s, 1H), 7.70-7.67(d, 1H), 7.02-6.98(m, 2H), 6.66-6.65 (d, 1H), 3.13 (s, 6H).

¹³ C NMR (CDCl3, 75 MHz, 293K): δ 195.26, 156.83, 151.39, 140.62, 137.75, 130.87, 120.63, 119.03, 114.18, 108.73, 103.22, 40.26.

HRMS: m/z calcd. for C ₁₃ H ₁₃ NO ₂ 215.0946 found 259.0946.

At this time, the compound of Formula 1 developed in the present invention was named DMHN1.

Example 2. Confirmation of absorption and fluorescence properties of fluorescent probe compound (DMHN1)

The absorption and fluorescence graphs of DMHN1 were measured under the solvent conditions given below, and the results are shown in FIG. 1 .

An absorption spectrophotometer (UV/Vis spectrophotometer; Agilent Technologies Cary 8454, USA) was used to analyze the absorbance of the material, and a spectro-fluorophotometer (SHIMADZU CORP) was used to measure the fluorescence intensity. (RF-6000, Japan) was used. A standard quartz cell (interanl volume = 0.1 cm, Hellma Analytics, Jena, Germany) having a thickness of 1 cm on all sides was used as a glass cuvette used for measuring absorption and fluorescence spectra.

The graph of FIG. 1 shows the absorption and fluorescence graphs of DMHN1 under the conditions that the solvent is acetonitrile (acetonitrile, 99%) and triethylamine (1%), respectively, under the conditions DMHN1 is an absorption spectrum (FIG. 1). (a)) showed the highest absorption value at 390 nm. However, DMHN1 showed very low fluorescence in the fluorescence spectrum (FIG. 1 (b)).

Example 3. Mechanism of Diethylcyanophosphonate Reaction of Fluorescent Probe Compound (DMHN1)

In order to confirm the reaction mechanism of DMHN1 to diethylcyanophosphonate, the mass of the finished product was measured with a high resolution mass spectra (JMS-700 spctrometer, JEOL, Tokyo, Japan), The results are shown in FIG. 2 . In addition, FIG. 3 shows the reaction mechanism of DMHN1 to diethylcyanophosphonate.

Through these results, the detection mechanism of DMHN1 was identified, which is a proton transfer in the excited state molecule in the structure of ortho-hydroxy-benzaldehyde through nucleophilic substitutions between DMHN1 and diethylcyanophosphonate. It was confirmed that the quenching effect by (ESIPT, Excited-State Intramolecular Proton Transfer) is broken and shows fluorescence.

Example 4. Confirmation of sensitivity to detection of diethylcyanophosphonate of the fluorescent probe compound (DMHN1)

The diethylcyanophosphonate detection characteristics and sensitivity of DMHN1 in a given solvent condition were analyzed based on the observation of fluorescence changes, and the results are shown in FIGS. 4 to 6 .

Specifically, in order to determine whether DMHN1 shows a fluorescence change according to the amount of diethyl cyanophosphonate and whether it is possible to detect a trace amount of diethylcyanophosphonate, a certain equivalent of diethylcyanophosphonate was used to check the degree of fluorescence change at high concentration and fluorescence change at low concentration. The solvent used for the measurement was acetonitrile (acetonitrile, 99%) and triethylamine (triethylamine, 1%), and the reaction time at room temperature (25 ℃) was fixed to less than 3 minutes and analyzed. DMHN1 was used by dissolving 10 mM in dimethyl sulfoxide (DMSO), and the amount of dimethyl sulfoxide was controlled to be the same in the final solvent condition.

Through the graph of FIG. 4 , it can be confirmed that the fluorescence of DMHN1 increases when the concentration of diethylcyanophosphonate (0 to 1 mM high concentration range) increases. 5 shows the correlation analysis results between the concentration of diethylcyanophosphonate and the fluorescence intensity of DMHN1 in the maximum fluorescence wavelength band. 4 and 5, it can be confirmed that the intensity of fluorescence linearly increases as the concentration of the target material (diethylcyanophosphonate) increases.

In order to check whether diethylcyanophosphonate (0 to 10 μM) can be detected at a low concentration, the number of analytes according to each concentration was increased by three, and a correlation graph was confirmed as an average value. In all experiments, the concentration of DMHN1 was fixed at 10 μM and analyzed, and all fluorescence intensities were measured with a fluorescence device by reacting at 25° C. for less than 3 minutes. As a result, as shown in FIG. 6, it was confirmed that the sensitivity of DMHN1 to diethylcyanophosphonate was 8.16 ppm.

Example 5. Confirmation of diethylcyanophosphonate selectivity of fluorescent probe compound (DMHN1)

The selectivity of DMHN1 to diethylcyanophosphonate was confirmed through fluorescence change, and the results are shown in FIGS. 7 and 8 .

Specifically, in order to determine whether DMHN1 selectively binds to diethyl cyanophosphonate, an acidic substance that can affect the reaction and a nerve agent having a function similar to that of diethylcyanophosphonate are used. Thus, it was confirmed whether the fluorescence of DMHN1 was changed for each material. The solvents used in the experiment were acetonitrile (99%) and triethylamine (1%), and each nerve agent analogue was manufactured by TCI (Tokyo chemical industry Co., Ltd). DMHN1 was dissolved in dimethyl sulfoxide solution at a concentration of 10 mM, and the concentration of DMHN1 in the final solvent condition was 10 μM, so that the amount of dimethyl sulfoxide was controlled so that the amount of dimethyl sulfoxide was the same for each substance.

The inner photo in FIG. 7 is a photo taken with a camera in a fluorescence device having a wavelength of 365 nm, showing that DMHN1 (left side of the photo) hardly emits fluorescence. However, DMHN1 was put in a mixed solvent of acetonitrile (99%) and triethylamine (1%) containing diethylcyanophosphonate (1 mM) and reacted at 25 ° C for about 3 minutes. It can be confirmed that strong blue fluorescence is turned on when

8 is a result of analyzing the increase in fluorescence intensity based on the maximum fluorescence wavelength (486 nm) when the reaction of DMHN1 and diethyl cyanophosphonate proceeded for 3 minutes. In FIG. 8 , the fluorescence intensity of the nerve agent analogs that did not react with DMHN1 were all clustered at the bottom of the graph, and the fluorescence increased in the case of the substance reacted with diethylcyanophosphonate (red line). The concentration of DMHN1 was fixed at 10 μM, and the concentration of each analogue was uniformly measured at 1 mM. The types of nerve agent analogs indicated on the horizontal axis of the graph shown in FIG. 8 are as follows.

(A) DMHN1 (compound of formula 1);

(B) DMHN1 with diethylcyanophosphonate;

(C) DMHN1 with diethyl chlorophosphate

(D) DMHN1 with dimethyl methylphosphate;

(E) DMHN1 with triphenylphosphate;

(F) DMHN1 with triethylphosphate;

(G) DMHN1 with acetic acid;

As a result, as shown in FIG. 8 , it was confirmed that DMHN1 selectively increased fluorescence only in the presence of diethylcyanophosphonate. Therefore, it can be seen that DMHN1 according to the present invention has selective reactivity only with diethyl thiaphosphonate.

Example 6. Confirmation of detection of diethyl cyanophosphonate under soil conditions

In order to confirm whether diethyl cyanophosphonate was detected under soil conditions of DMHN1, the fluorescence change of DMHN1 in the corresponding environment was confirmed using three types of soil.

Specifically, 1 g of sand, clay, and field were added to an aluminum dish and then treated with 100 mM diethylcyanophosphonate dispersed in acetonitrile. Finally, each type of soil (1 g) treated with diethyl cyanophosphonate was added to a mixed solvent of acetonitrile (acetonitrile, 99%) and triethylamine (1%) at a concentration of 10 μM. After addition to DMHN1 (3 mL), the change in fluorescence was observed. The change in fluorescence was observed for the reactant at 25 °C for 60 minutes, and after 10 minutes of reaction with diethylcyanophosphonate, a picture was taken in a 365 nm fluorescence device. The results are shown in FIG. 9 .

In addition, as mentioned above, the change in fluorescence intensity according to the reaction time of diethylcyanophosphonate and DMHN1 was measured and shown in FIG. 10 . As shown in FIGS. 9 and 10 , it can be confirmed that DMHN1 exhibits a fluorescence change in the presence of diethylcyanophosphonate in the corresponding environment through the above experiment.

11 is a photograph showing the change in fluorescence intensity over time according to the type of each soil. In addition, FIG. 12 graphically shows the change in fluorescence intensity over time according to the type of each soil. 11 and 12, it can be confirmed that DMHN1 can rapidly detect diethylcyanophosphonate in a short time even under soil conditions.

Through the above results, it can be confirmed that DMHN1 according to this nickname can be utilized within environmental conditions.

Example 7. Application to diethylcyanophosphonate real-time in situ detection kit

The possibility of developing a real-time in-situ detection kit of diethyl cyanophosphonate using DMHN1 was verified by manufacturing a simplified kit.

The real-time in situ detection process of diethylcyanophosphonate is shown in FIG. 13 . Specifically, for the evaluation of portability, a commercially available ultraviolet flashlight (UV light, 365 nm, 3W) was prepared, and 10 μM DMHN1 was dissolved in cetonitrile (acetonitrile, 99%) and triethylamine. (triethylamine, 1 %) 1 mL of a mixed solvent was prepared in a high-resolution liquid chromatography vial (2 mL size). The kit consists of a vial containing DMHN1, a capillary tube (capillary), and an ultraviolet flashlight. The diethyl cyanophosphonate stock solution (about 20 μL) was collected through a capillary tube and inserted into a vial containing DMHN1. The experimental results are shown in FIG. 14 .

As shown in FIG. 14 , a change in fluorescence of DMHN1 can be confirmed in a short time within 30 seconds.

The description of the present invention stated above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (6)

A fluorescent probe for detecting diethylcyanophosphonate, comprising a compound of Formula 1 below:
[Formula 1]

The method of claim 1,
The compound is characterized in that it contains an ortho-hydroxy (hydroxy)-benzaldehyde structure in the compound, a fluorescent probe. ◈Claim 3 was abandoned when paying the registration fee.◈ The method of claim 1,
The compound is quenched by excited-state intramolecular proton transfer in the structure of ortho-hydroxy-benzaldehyde through a nucleophilic substitution reaction with diethylcyanophosphonate. A fluorescent probe, characterized in that it exhibits fluorescence when the effect is broken. A kit for detecting diethylcyanophosphonate comprising a compound of Formula 1 below:
[Formula 1]

A method for detecting diethylcyanophosphonate using a compound of Formula 1 below:
[Formula 1]

6. The method of claim 5,
The detection method is
A method of detecting diethyl cyanophosphos comprising adding a sample containing diethylcyanophosphonate to the compound of Formula 1 and irradiating UV light.

Images