KR102294475B1 - Fluorescent probe compound for the detection of malononitrile and use thereof - Google Patents

Abstract

The present invention relates to a fluorescent probe capable of detecting malononitrile using a compound containing a structure of 6-dimethylamino-3-hydroxy-2-naphthaaldehyde and a use thereof. The fluorescent probe according to the present invention can be obtained through a simple synthesis method, and is characterized by high specificity to malononitrile. In addition, since it has a high sensitivity (6.6 ppb) to malononitrile and a rapid reaction time, the range of environmental applications using the compound is wide. In addition, applicability in the in vivo environment is important in that the substance with high toxicity to the human body is decomposed into toxic substances again through the metabolic process in the body. Since it has the strength that fluorescence can be turned on, it is expected to be valuable in all related applied research fields.

Description

Fluorescent probe compound for the detection of malononitrile and use thereof

The present invention relates to a fluorescent probe compound for detection of malononitrile and uses thereof.

Malononitrile (NCCH₂CN), also called propenedinitrile, is mainly used as an intermediate in synthetic reactions based on the condensation reaction of aldehydes or ketones (Toxicol. Mech. Methods). , 2003, 13, 241.; Pham. Res., 1995, 12, 380.; J. Heterocycl. Chem., 2007, 44, 419, Synth. Commun, 2002, 32, 3475.). Among them, Knoevenagel condensation of malononitrile is used in pharmaceutical core structures, heterocycle systems, and solvatochromic dyes. It has industrial value because it can be However, malononitrile is known to be more toxic than hydrogen cyanide (HCN), which is highly toxic in water. In addition, malononitrile has the potential to be converted to hydrogen cyanide in the metabolic process in animal tissues, as well as block respiration of brain, kidney and liver, and aerobic glycolysis. ) function, so it is necessary to develop a system that can specifically detect it.

Until now, a ^{number of fluorescent probes known to detect cyanide ions (CN −} ) have been reported, but despite their importance, probes that selectively detect only malononitrile remain a very challenging task. nothing has happened In particular, considering that the existing technology for detecting malononitrile is mainly known only for mass-based spectrometric analysis, and the low biological and environmental applicability of the technology, it can be used simply and quickly. The development of possible fluorescent probes has industrial value in that all these shortcomings can be ameliorated.

Accordingly, the present inventors have found that the process of synthesis is easy, toxic to the human body, and interacts with malononitrile, which can produce toxic substances even in the metabolic process in the body, to emit fluorescence, and only the target substance is specific A fluorescent probe that can detect This fluorescent probe quantitatively shows fluorescence changes according to the concentration of the target material, and its utility is expected to cross fields because it has malononitrile selectivity beyond metal ions and biomolecules that can cause interference in and out of the body. do. In addition, the developed fluorescent probe shows the possibility of real-time detection in the environment and industrial sites with a fast reaction time (within 5 minutes) with the target material, and furthermore, by revealing that it can be used in in vivo conditions, the present invention has been completed did.

An object of the present invention is to provide a fluorescent probe compound for selectively detecting malononitrile, a toxic substance, using a 6-dimethylamino-3-hydroxy-2-naphthaaldehyde compound having a bipolar naphthalene structure.

Another object of the present invention is to provide a malononitrile detection kit comprising the fluorescent probe compound.

Another object of the present invention is to provide a method for detecting malononitrile 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 malononitrile represented by the compound of Formula 1 below.

[Formula 1]

In one embodiment of the present invention, the compound is 6-dimethylamino-3-hydroxy-2-naphthaaldehyde (6-dimethylamon-3) proceeded by Knoevenagel condensation with malononitrile. -Hydoxy-2-naphthaldehyde) terminates the excited-state intramolecular proton transfer (ESIPT) through intramolecular cyclization to produce iminobenzo[g]coumarin and exhibits red fluorescence.

In addition, the present invention provides a kit for detecting malononitrile containing the fluorescent probe compound.

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

In one embodiment of the present invention, the detection method is characterized in that it is performed under the conditions of an aqueous solution.

In addition, the present invention provides a cell or tissue imaging method for measuring the fluorescence generated by reaction with malononitrile by treating the cell or tissue with the fluorescent probe compound.

The fluorescent probe compound of the present invention can provide a fluorescent signal by selectively detecting malononitrile, a toxic substance. In addition, an analyte in an arbitrary environment containing malononitrile may be collected using the fluorescent probe compound and analyzed in real time.

In particular, the fluorescent probe compound of the present invention is easy to synthesize and has a proton transfer phenomenon in an excited state molecule due to the structural characteristics of naphthalene having a bipolar nature. By this phenomenon, fluorescence is generated specifically with malononitrile, thereby overcoming the problem of the conventional fluorescent probe for detecting malononitrile having low selectivity.

In addition, the fluorescent probe of the present invention can detect malononitrile even in aquatic environmental conditions and can be manufactured in the form of a portable detection kit, so it is a novel method that can detect malononitrile in a short time in the field. It provides a method that can be usefully used in various fields.

1 is a result of measuring the absorbance of MalP-1 according to the present invention under specific solvent conditions.
2 is a result of measuring the fluorescence intensity of MalP-1 according to the present invention under specific solvent conditions.
3 is a result of measuring absorption and fluorescence properties when MalP-1 according to the present invention detects malononitrile.
4 shows the mechanism of malP-1 sensing reaction of malononitrile according to the present invention.
5 is a result of measuring the absorption and fluorescence spectra of MalP-1 according to the present invention reacted with malononitrile at various concentrations.
6 is a linear correlation graph showing the average fluorescence intensity of MalP-1 according to the present invention for an analyte according to each concentration.
7 is a result of confirming the selective detection ability for malononitrile of MalP-1 according to the present invention through fluorescence change.
FIG. 8 is a result of analyzing the malononitrile sensing characteristics of MalP-1 according to the present invention under each pH condition (pH 4-9).
9 is a photograph confirming whether malononitrile was detected using MalP-1 according to the present invention in a sample collected in an aquatic environment.
10 is a result of measuring the change in fluorescence of MalP-1 with respect to malononitrile according to the present invention in a sample taken in an aquatic environment.
11 is a photograph of observing a change in fluorescence through serial dilution of malononitrile contained in an aqueous solution sample.
12 is a graph showing the maximum fluorescence wavelength of 610 nm as a result of fluorescence measurement of the aqueous solution sample of FIG. 11
13 shows a method for synthesizing tear gas containing malononitrile, which is a substance to be detected.
14 is a result of confirming whether or not the synthesized tear gas is generated using thin layer chromatography (TLC).
15 is a result of confirming whether a synthesized tear gas is generated using a hydrogen nuclear magnetic resonance device.
16 shows the fluorescence change of MalP-1 according to the concentration of tear gas.
17 is an imaging result for confirming whether MalP-1 according to the present invention is suitable for in vivo application.
18 is an experimental result confirming whether MalP-1 according to the present invention has cytotoxicity in vivo.

The present invention provides a fluorescent probe compound for detecting malononitrile represented by the compound of Formula 1 below.

[Formula 1]

In one embodiment of the present invention, the compound is an excited state molecule through a cyclization reaction in the molecule of 6-dimethylamino-3-hydroxy-2-naphthaaldehyde, which is carried out by the condensation reaction between malononitrile and Knovenagel. It terminates the internal proton transfer, produces iminobenzo[g]coumarin, and can exhibit red fluorescence.

In one embodiment of the present invention, the detection mechanism of malononitrile of the compound of Formula 1 developed in the present invention was investigated (Example 4), and it was confirmed that malononitrile could be selectively detected (Example 4). Examples 5 and 6)

In addition, the present invention provides a kit for detecting malononitrile containing the fluorescent probe compound.

In one embodiment of the present invention, it was confirmed that the compound of Formula 1 developed in the present invention can be utilized as a kit for detecting malononitrile that can be used in the field (Example 7).

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

In one embodiment of the present invention, the detection method is performed on a liquid sample.

In one embodiment of the present invention, it was confirmed that the compound of Formula 1 developed in the present invention can effectively detect malononitrile even in an aqueous environment (Example 7).

In addition, the present invention provides a cell or tissue imaging method for measuring the fluorescence generated by reaction with malononitrile by treating the cell or tissue with the fluorescent probe compound.

In one embodiment of the present invention, it was confirmed that the compound of Formula 1 developed in the present invention can be effectively utilized for imaging of cells or tissues (Example 9).

Therefore, since the compound of Formula 1 according to the present invention has high sensitivity and fast reaction time to malononitrile, it can be used in an environment, and in particular, it is possible to monitor the detection of malononitrile in real time under given environmental conditions. It can be usefully used in related research fields.

In addition, since it exhibits selective fluorescence properties with respect to malononitrile even in cells or tissues, it can be usefully used for imaging related cells or tissues.

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 MalP-1

The present inventors have tried to develop a fluorescent probe that can selectively detect toxic malononitrile, and 6-dimethylamino-3-hydroxy-2-naphthaldehyde (6-dimethylamino-3-hydroxy-2-naphthaldehyde) ) (MalP-1 in Scheme 1 below) detects malononitrile through Knovenagel condensation reaction and intramolecular cyclization can occur in aqueous solution conditions, the fluorescent probe of the present invention (MalP-1) was developed.

6-dimethylamino-3-hydroxy-2-naphthaaldehyde can be synthesized according to Scheme 1 below.

[Scheme 1]

.

(1) Synthesis of 7-(dimethylamino)naphthalen-2-ol (7-(dimethylamino)naphthalen-2-ol)

The present inventors performed the synthesis of 7-(dimethylamino)naphthalen-2-ol) (compound 2 in Scheme 1 above). In Scheme 1, the starting material for the synthesis, a sealed glass container containing compound 1 (1 g, 6.243 mmol, Sigma-aldrich) and sodium metabisulfite (Na ₂ S ₂ O ₅ , 2.374 g, 12.486 mmol) ( dimethylamine solution (40% in H ₂ O, 3.95 mL, 31.215 mmol) and water (H ₂ O, 2.67 mL) were added to the sealed-tube) blocked the entrance. The mixture was stirred on a silicone oil container at 150° C. for about 4 hours. To terminate the reaction, first lower the temperature of the reactant to room temperature (25 ℃), open the container, put ethyl acetate (ethyl acetate, 100 mL), water (100 mL), and saturated brine (30 mL), and use a separatory funnel to extract the organic layer. The organic layer was dried over anhydrous sodium sulfate (Na ₂ SO ₄ , 1 g), and concentrated using an aspirator. The pale brown solid compound thus obtained was separated using column chromatography using silica gel (eluent: 20% EtOAc/Hexane), and a white solid compound (compound 2, 795.7 mg, 68.07 in Scheme 1 above) %) was obtained. When the product is analyzed by thin layer chromatography (TLC, thin layer chromatography, silica 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 (CDCl ₃ , 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) 7- (methoxymethoxy) - N, N - dimethyl naphthalene-2-amine Synthesis of (7- (methoxymethoxy) -N, N -dimethylnaphthalen-2-amine)

The present inventors performed the synthesis of 7-(methoxymethoxy) -N,N -dimethylnaphthalen-2-amine (compound 3 in Scheme 1 above). Sodium hydride (sodium hydride, NaH, sodium hydride, 235 mg, 5.875 mmol) is added to a dimethylformamide (DMF, N,N-dimethylformamide, 5 mL) solvent, an argon balloon is inserted, and saturated brine and The temperature was lowered to -15 °C using ice. In this mixed solution, compound 2 (1 g, 5.34 mmol) in Scheme 1 was dissolved in DMF (5 mL), and then slowly added dropwise for about 5 minutes while maintaining the temperature. At this time, hydrogen gas (H ₂ gas) is generated, which is discharged to the outdoors after passing through a silicon oil trap. After stirring for 1 hour at the same temperature, _{it was confirmed that gas bubbles (H 2} gas) were no longer 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). The extracted organic layer was dried with water using anhydrous sodium sulfate (10 g). The dried ethyl acetate organic layer was concentrated using a suction device. The pale brown liquid compound thus obtained was separated by column chromatography using silica gel (eluent: 5% EtOAc/Hexane), and the compound as a white solid (compound 3 in Scheme 1, 1336.1 mg, 87.3%) was obtained. got it _{The product has a developing value of R f} = 0.45 (20% EtOAc/Hexane - one run) when analyzed by thin layer chromatography.

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 C ₁₄ 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 present inventors performed the synthesis of 6-(dimethylamino)-3-(methoxymethoxy)-2-naphthaldehyde (compound 4 in Scheme 1 above). After putting compound 3 (2.2606 g, 9.774 mmol) in a 100 mL round-bottom flask, an argon balloon was inserted. Then, ethyl ether (ehthyl 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 the 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, extraction was performed using ethyl acetate (300 mL), saturated brine (100 mL) and water (200 mL). The organic layer obtained through extraction was dried with water over anhydrous sodium sulfate (15 g), and concentrated using a suction device. The obtained solid compound was separated by column chromatography using silica gel (eluent: 20% EtOAc/Hexane) to obtain a light yellow solid compound (compound 4, 1.27 g, 50% in Scheme 1 above). _{The product has a developing value of R f} = 0.25 (20% EtOAc/Hexane - one run) when analyzed by thin layer chromatography.

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)

We performed the synthesis of 6-(dimethylamino)-3-hydroxy-2-naphthaldehyde (MalP-1 in Scheme 1 above). Compound 4 (195 mg, 0.75 mmol), isopropyl alcohol, 10 mL), and 5 M HCl (5 mL) were added to a 25 mL round-bottom flask, followed by stirring at 60 °C for 3 hours. 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). The organic layer (EtOAc) obtained through extraction was dried with anhydrous sodium sulfate (3 g) with water present in the organic layer, and then concentrated using an aspirator. The yellow solid compound thus obtained was separated by column chromatography using silica gel (eluent: 20% EtOAc/Hexane) to obtain yellow MalP-1 (113 mg, 70%). _{The product has an evolution value of R f} = 0.35 (20% EtOAc/Hexane - one run) when analyzed by thin layer chromatography.

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 (CDCl ₃ , 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 developed in the present invention was named MalP-1.

Example 2. Confirmation of absorption and fluorescence properties of MalP-1

The present inventors measured the absorbance and fluorescence intensity of MalP-1 under the following solvent conditions. The results are shown in FIGS. 1 and 2 .

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. When measuring each absorption and fluorescence spectrum, a standard quartz cell (interanl volume = 0.1 cm, Hellma Analytics, Jena, Germany) having a thickness of 1 cm was used in a glass cuvette.

MalP-1 used for the measurement was measured within 5 minutes at room temperature by diluting a stock solution (10 mM) dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 10 uM in the indicated solvent. .

1 and 2 show the absorption spectrum (FIG. 1) and the fluorescence spectrum (FIG. 2) of MalP-1 under the condition that the solvent is deionized water (99.9%) and piperidine (0.1%), respectively. Under the corresponding conditions, the absorption spectrum (FIG. 1) of MalP-1 showed the highest absorption value at 390 nm, and MalP-1 showed no fluorescence in the fluorescence spectrum (FIG. 2).

Example 3. Confirmation of MalP-1 detection of malononitrile

The present inventors confirmed whether MalP-1 detected malononitrile. Absorption and fluorescence spectra were measured before and after MalP-1 (10 μM) detected malononitrile in a given solvent condition (99.9% distilled water, 0.1% piperidine). The results are shown in FIG. 3 .

In FIG. 3 , the absorption spectrum is measured on the left, and the fluorescence spectrum is measured on the right. As shown in FIG. 3 , it was confirmed that the red fluorescence was turned on when MalP-1 was reacted with malononitrile (100 μM) for 5 minutes at room temperature (see FIG. 3 ). picture inside the graph on the right).

From this result, it can be seen that MalP-1 can react immediately to malononitrile and shows a clear fluorescence change before and after the reaction.

Example 4. MalP-1 Malononitrile Sensing Response Mechanism

The present inventors measured the mass of the material produced with a high resolution mass spectra (JMS-700 spctrometer, JEOL, Tokyo, Japan) in order to confirm the reaction product generated when MalP-1 detects malononitrile. measured.

Figure 4 (a) is the mechanism of the reaction to whether toxic malononitrile is converted to hydrogen cyanide in the tissue metabolism of animals, Figure 4 (b) is MalP-1 malonite It is a schematic diagram showing a simple mechanism that fluorescence is turned on when it meets a reel.

Example 5. Confirmation of MalP-1 detection sensitivity to malononitrile

The present inventors evaluated the sensing properties and sensitivity of MalP-1 to malononitrile in a given solvent condition (99.9% distilled water, 0.1% piperidine). In order to evaluate the detection sensitivity of MalP-1 to malononitrile, fluorescence spectra of MalP-1 reacted with various concentrations of malononitrile were measured, and the results are shown in FIG. 5 .

)의 조건에서 5분이내로 고정하여 측정하였다. MalP-1은 다이메틸 설폭사이드에 녹여서 사용하였으며, 최종으로 사용되는 용매조건에서 다이메틸 설폭사이드의 양이 동일하도록하여 10 uM의 농도조건에서 분석하였다.Specifically, in order to confirm the characteristic of fluorescence change according to the amount of malononitrile in MalP-1 and the minimum detectable concentration of malononitrile, a certain equivalent of malononitrile was used to determine the fluorescence change at high concentration and low concentration. The amount of increase in fluorescence was evaluated. The solvent used for the measurement was deionized water (99.9%) and piperidine (0.1%) used as a catalyst for the reaction, and the reaction was carried out at room temperature (25

) was fixed and measured within 5 minutes. MalP-1 was used by dissolving in dimethyl sulfoxide, and the amount of dimethyl sulfoxide was the same in the final solvent condition and analyzed at a concentration of 10 uM.

5, (a) and (b) are conditions in which the concentration range of malononitrile is 0-1 mM (0 uM, 10 uM, 50 uM, 100 uM, 300 uM, 500 uM, 700 uM, 1 mM). The graph of the increase in fluorescence when reacted with MalP-1 in , and the correlation between the concentration of malononitrile and the fluorescence intensity of MalP-1 in the maximum fluorescence wavelength band (610 nm) are respectively analyzed. The spectrum shows that the fluorescence of the produced material (IBC) increases in a linear correlation as the concentration of the target material, malononitrile, increases.

In addition, even when the concentration of malononitrile is low, in order to confirm the range of detection possible by MalP-1, three analytes according to each given concentration were determined and analyzed, and the average fluorescence intensity thereof was calculated as shown in FIG. 6 . It is shown as a linear correlation graph.

In the course of this experiment, the concentration of MalP-1 was fixed at 10 μM and analyzed, and all fluorescence intensities were reacted within 5 minutes at 25°C and measured with a fluorescence photometer.

As a result, as shown in FIG. 6 , it was confirmed that MalP-1 had a detectable sensitivity to malononitrile at a very low concentration of 6.6 ppb.

Example 6. MalP-1 malononitrile selectivity and characterization under pH conditions

The present inventors confirmed the selective detection ability of MalP-1 for malononitrile by changing the fluorescence before and after the reaction, and the results are shown in FIG. 7 .

Specifically, in order to confirm whether MalP-1 can selectively detect malononitrile, fluorescence of MalP-1 for each substance using various metal ions and representative biomolecules that can affect the reaction It was checked whether the characteristics were changed. The solvents used in the experiment were deionized water (99.9%) and piperidine (0.1%), and each metal ion and biomolecules were manufactured by Sigma Aldrich (USA), Alfa Aesar (USA), TCI (Japan). product was used. MalP-1 was dissolved in a dimethyl sulfoxide solution at a concentration of 10 mM, and the concentration of MalP-1 in the final solvent condition was 10 μM and the amount of dimethyl sulfoxide was controlled so that the amount of dimethyl sulfoxide was the same for each substance. The concentration of the compound was fixed at 10 μM, and the concentration of each analog was uniformly measured at 100 μM. This result is a summary of the fluorescence values at 611 nm showing the maximum fluorescence.

The types of metal ions and biomolecules indicated on the horizontal axis (A-X) of the graph of FIG. 7 are as follows:

(A) MalP-1;

(B) MalP-1 with malononitrile;

(C) MalP-1 with NaCN;

(D) MalP-1 with NaHSO ₃ ;

(E) MalP-1 with NaOH;

(F) MalP-1 with NaOAc;

(G) MalP-1 with NaCl;

(H) MalP-1 with NaCl(anion);

(I) MalP-1 with NaF;

(J) MalP-1 with NaI;

(K) MalP-1 with MgCl ₂ ;

(L) MalP-1 with KCl;

(M) MalP-1 with CaCl ₂ ;

(N) MalP-1 with FeCl ₃ ;

(O) MalP-1 with NiCl ₂ ;

(P) MalP-1 with CuCl ₂ ;

(Q) MalP-1 with ZnCl ₂ ;

(R) MalP-1 with CdCl ₂ ;

(S) MalP-1 with glutamine;

(T) MalP-1 with glutathione;

(U) MalP-1 with cysteine;

(V) MalP-1 with homocysteine;

(W) MalP-1 with lysine;

(X) MalP-1 with aspartic acid;

As shown in FIG. 7 , MalP-1 showed no fluorescence change in the reaction between each metal ion and biomolecules, but high fluorescence intensity was measured when reacted with malononitrile corresponding to B, and selectively malP-1 It was shown to detect only nitrile.

In addition, the malP-1 detection characteristics of MalP-1 were analyzed under each pH condition (pH 4-9), and the results are shown in FIG. 8 .

As shown in FIG. 8 , as a result of the experiment, it was possible to observe the fluorescence of iminobenzo[g]coumarin, a product of the reaction, under the in vivo pH conditions of pH 7.4 and alkaline (akaline) pH conditions. This was confirmed by the intramolecular cyclization of MalP-1.

These results indicate that MalP-1 can react specifically to malononitrile by avoiding interference with other substances. In addition, MalP-1 showed its reactivity under various pH conditions, including biological pH conditions, confirming the possibility that it could be utilized in vivo and in environmental conditions.

Example 7. Evaluation of the detection of malononitrile in aquatic samples using MalP-1

The present inventors evaluated the fluorescence change in the corresponding environment using a total of seven types of water in order to confirm whether malononitrile was detected using MalP-1 based on samples collected in an aquatic environment.

Specifically, the concentrations of MalP-1 (10 μM) and malononitrile (100 μM) were adjusted to a total volume of 500 μL using an aqueous sample as a solvent, and then reacted for about 3 minutes at room temperature. The aqueous solution samples collected were sea water, river water, lake water, tap water, and commercially sold drinking water, respectively, and the results before and after the reaction were UV (UV) water. The change in fluorescence was photographed under UV light, 365 nm) conditions, and is shown in FIG. 9 .

In addition, the fluorescence change photographed as described above was measured using a fluorescence device, and was summarized and graphed in FIG. 10 based on the maximum fluorescence wavelength of 610 nm. The types of aqueous solution samples indicated on the horizontal axis (A-G) of the graph of FIG. 10 are as follows:

(A) MalP-1 without/with malononitrile in deionized water;

(B) MalP-1 without/with malononitrile in sea water;

(C) MalP-1 without/with malononitrile in lake water;

(D) MalP-1 without/with malononitrile in river water;

(E) MalP-1 without/with malononitrile in tap water;

(F) MalP-1 without/with malononitrile in bottled water1;

(G) MalP-1 without/with malononitrile in bottled water2;

In addition, as shown in FIG. 11 , the aqueous solution sample corresponding to B-G is the result of photographing the fluorescence change measured by serial dilution of malononitrile at a concentration of 0-200 μM. 12 is a graph showing the maximum fluorescence wavelength of 610 nm as a result of fluorescence measurement of the aqueous solution sample of FIG. 11 .

These results show that MalP-1 can detect malononitrile sensitively and quickly in a short time even under the conditions of an aquatic environment, as shown in the photos and graphs of FIGS. 11 and 12, and thus, developed according to the present invention It can be confirmed that MalP-1 can be utilized within aquatic environmental conditions.

Example 8. Confirmation of detection of trace malononitrile in tear gas of MalP-1

The present inventors verified the applicability in actual military and industrial uses of malononitrile using MalP-1. Specifically, by synthesizing in large quantities, tear gas (CS riot gas), which is the most used for military training in each country, was synthesized through the method shown in FIG. 13 . The starting material used for the synthesis is 2-chlorobenzaldehyde, which reacts with a certain amount of malononitrile under catalyst conditions to produce tear gas. FIG. 14 shows whether or not pure tear gas was produced as a result of checking the tear gas obtained through synthesis under 254 nm conditions using thin layer chromatography (TLC).

15 is a result of confirming whether or not the synthesized tear gas is generated by using a hydrogen nuclear magnetic resonance apparatus, as in FIGS. 13 and 14 . As a trace amount of malononitrile remained in the results, it was assumed that the following characteristics would be shown even in the mass-synthesized tear gas in the actual process. In other words, if it is possible to detect residual malononitrile in tear gas from synthesis using this logic, it seems that the target material can be controlled for industrial and military uses.

16 shows the fluorescence change of MalP-1 according to the concentration of tear gas. As the amount of tear gas increases, it can be seen that the amount of malononitrile contained therein also increases proportionally. Through this result, MalP-1 can detect harmful substances that can occur when exposed to tear gas and cyanide (cyanide) that can be produced incidentally therefrom and can detect toxic malononitrile in combination. that can be checked

Example 9. Intracellular malononitrile imaging method using MalP-1

The present inventors imaged malononitrile present in cells using MalP-1 to confirm the suitability and cytotoxicity of MalP-1 for in vivo application.

Specifically, it was determined whether the characteristic of fluorescence changes as MalP-1 reacts with selectivity to malononitrile in vivo, and MalP-1 treated malononitrile-treated HeLa cells (cervical cancer). cells), it was confirmed whether malononitrile could be detected and an imaging method therefor.

)조건에서 배양시켜준 다음, 말로노나이트릴(100 μM)을 처리하여 2시간 동안 추가로 배양시켜주었다. 그 결과를 도 17에 나타내었다.An experiment was conducted to determine whether MalP-1 could be imaged with specificity for malononitrile in cells. The present inventors used HeLa cells as a control. In addition, three comparative groups (i: MalP-1, ii: MalP-1 with malononitrile, iii: MalP-1 with malononitrile including 0.1% piperidine) were prepared for each condition. Attempted imaging. The concentration of MalP-1 used in the experiment was 30 μM, 37 °C, 5% carbon dioxide (CO2) for 2 hours.

) conditions, and then treated with malononitrile (100 μM) and further incubated for 2 hours. The results are shown in FIG. 17 .

As shown in FIG. 17 , although no fluorescence was observed in the group not containing malononitrile, it was confirmed that the red fluorescence was turned on in the group treated with malononitrile, and MalP-1 was also found in malono in vivo. It can be confirmed that it has selectivity with respect to nitrile. At this time, the excitation wavelength used for imaging is 499 nm, and the scale bar is 20 μm.

In addition, it was confirmed whether MalP-1 had cytotoxicity in vivo. The old results are shown in FIG. 18 .

Specifically, the cytotoxicity experiment was performed using the CCK-8 assay kit (dojindo molecular technologies, Inc., CatNO: CK04-13), and was performed according to the protocol provided by the company. After dispensing the same number of cells in a 96 well plate (SPL, 30096), the cells were cultured in an incubator (Thermo Scientific Forma Series3) maintained _{at 37 °C and 5% CO 2 for 24 hours.} Then, the culture medium was changed to a serum-free culture medium and incubated in an incubator for 30 minutes, then treated with MalP-1 at a given concentration. Cells treated with MalP-1 were further cultured in an incubator for about 2 hours. Next, the culture medium was removed and washed once with 1X PBS. Afterwards, the serum-free culture medium was dispensed in 90 μL to each well, and 10 μL of CCK-8 solution was treated in each well. Finally, the absorbance value was measured at 450 nm by culturing in an incubator at 37° C. and 5% CO _{2 conditions for 2 hours.} The absorbance value was measured with a microplate reader (Microplate reader, Multiskan FC, Thermo Fisher), and the percentage of cytotoxicity was calculated as (absorption value of sample *100)/(absorption value of control). The results are shown in FIG. 18 .

As shown in FIG. 18, as a result of confirming the cytotoxicity based on the concentration conditions corresponding to 10 μM, 25 μM, 50 μM, 100 μM, and 200 μM, it can be confirmed that even high concentrations corresponding to 100 μM exhibit low toxicity. .

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 malononitrile, comprising a compound of Formula 1 below:
[Formula 1]

The method of claim 1,
The compound is a cyclization reaction within a molecule of 6-dimethylamino-3-hydroxy-2-naphthaldehyde, which is carried out by a condensation reaction between malononitrile and Knovenagel. A fluorescent probe, characterized in that it terminates the proton transition in the excited state molecule through A kit for detecting malononitrile comprising a compound of Formula 1 below.
[Formula 1]

A method for detecting malononitrile using a compound of Formula 1 below.
[Formula 1]

5. The method of claim 4,
The detection method is malononitrile detection method, characterized in that it is carried out under the conditions of an aqueous solution. An imaging method of cells or tissues for measuring fluorescence generated by reaction with malononitrile by treating cells or tissues with a compound of Formula 1 below.
[Formula 1]

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