A ratiometric fluorescent probe for detecting hydrazine and a preparation method and application thereof

Abstract

The application belongs to the technical field of synthetic chemistry and water quality detection, and provides a ratiometric fluorescent probe for detecting hydrazine, a preparation method and application thereof. The fluorescent probe DCN-Cl provided by the application exhibits excellent fluorescence performance and light stability, and can detect N2H4 through a ratio signal. The DCN-Cl realizes ratiometric detection of the concentration change of exogenous N2H4 in mitochondria and reproducible detection of N2H4 in water samples. The probe can be widely applied to detection of drug in-vivo metabolism and detection of water pollutants.

Description

Technical Field

[0001] This invention belongs to the fields of synthetic chemistry and water quality detection technology, specifically relating to a fluorescent probe for detecting hydrazine and its application. Background Technology

[0002] Hydrazine (N₂H₄) is a colorless, transparent liquid with a faint ammonia odor. Due to its strong alkalinity and reducing properties, it is used industrially in the synthesis of high-energy propellants, rocket diazo fuels, and breaker agents; in the pharmaceutical industry, it is used to produce anti-tuberculosis and anti-diabetic drugs; and in the pesticide industry, it is used to produce herbicides, insecticides, and plant growth regulators. However, the widespread use of N₂H₄ generates a large amount of waste that is harmful to the environment and human health, and these pollutants spread through soil and water sources. The metabolic process of some anti-tuberculosis drugs (such as isoniazid) can produce endogenous N₂H₄ in organisms, which may damage the liver, kidneys, and nervous system. To further study the toxicity of N₂H₄ and detect the hydrazine content in the environment, the development of convenient and efficient detection tools is of great significance. Fluorescence analysis, due to its low cost, ease of operation, and real-time detection capabilities, is considered a powerful technical tool in many fields, especially suitable for labeling small molecules in organelles and the microenvironment. Summary of the Invention

[0003] To address the problems in the prior art, this invention provides a fluorescent probe for detecting hydrazine, which can be used to evaluate and study physiological and pathological processes related to the toxicity of N2H4; and achieves reproducible detection of N2H4 in water samples through ratio signals.

[0004] To achieve the above objectives, the present invention adopts the following technical solution.

[0005] A ratiometric fluorescent probe for detecting hydrazine, abbreviated as DCN-Cl, has the chemical structural formula shown in formula (I): Formula (I).

[0006] The preparation method of the above ratiometric fluorescent probe includes the following steps: (1) A mixed solution of 6-(dimethylamino)-2-naphthaldehyde and 4-pyridineacetonitrile was reacted under the catalysis of pyrrolidine. After the reaction was completed, solid-liquid separation and purification were performed to obtain the intermediate product: ; (2) The intermediate product and the mixed solution of 1,4-dichlorobenzyl were reacted under a protective atmosphere. After the reaction was completed, solid-liquid separation and purification were performed to obtain the fluorescent probe.

[0007] In steps (1) and (2), the reaction temperature is 90℃.

[0008] The application of the above-mentioned fluorescent probe in the detection of hydrazine in solutions, cells or organisms.

[0009] The fluorescent probes described above can also be used to prepare reagents for the detection of hydrazine.

[0010] The mechanism of this invention is as follows: This invention uses 6-(dimethylamino)-2-naphthal and cyanopyridine derivatives as fluorescent reporter molecules. A response site for N2H4 is established through a C=C conjugated structure, and quaternary ammonium groups and benzyl chloride are selected as mitochondrial targeting and immobilization groups, respectively, to achieve probe targeting within the mitochondria.

[0011] The present invention has the following advantages: The fluorescent probe DCN-Cl provided by this invention exhibits excellent fluorescence performance and photostability, and can detect N2H4 through ratio signals. DCN-Cl enables the ratio detection of changes in mitochondrial exogenous N2H4 concentration and the reproducible detection of N2H4 in water samples. This probe can be widely used in the detection of drug metabolism in vivo and the detection of water pollutants. Attached Figure Description

[0012] Figure 1 It is the fluorescent probe DCN-Cl 1 H NMR spectrum; Figure 2 These are the ultraviolet absorption spectra of DCN-Cl reacting with different concentrations of N2H4; Figure 3 The emission wavelength scans are performed under excitation at 550 nm (a) and 365 nm (b) after DCN-Cl reacts with different concentrations of N2H4. Figure 4 It is the ratio of the fluorescence intensity of different concentrations of N2H4 detected by DCN-Cl (I 460 / I 700 ); Figure 5 It is the ratio of the fluorescence intensity of N2H4 detected by DCN-Cl (I 460 / I 700 (Changes over time) Figure 6 It is the ratio of the fluorescence intensity of DCN-Cl to different analytes (I 460 / I 700 ); Figure 7 The images show fluorescence imaging and related channel intensities of HeLa cells stained with DCN-Cl and MTG; where a is the bright field image, b is the blue channel, c is the green channel, d is the deep red channel, e is the superposition of a and b, f is the deep red channel, g is the green channel, h is the superposition of f and g, i is the distribution of red and green fluorescence intensity in the area indicated by the arrow in h, and j is the co-location scatter plot of green and deep red channels. The scale bar is 20 μm. Figure 8 This is a fluorescence imaging image (scale bar 20 μm) of the blue and near-infrared channels of DCN-Cl after exogenous addition of N2H4 to living cells, along with the fluorescence intensity and the intensity ratio of the two channels. Detailed Implementation

[0013] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.

[0014] Example 1 Synthesis of fluorescent probe DCN-Cl The synthetic route of the fluorescent probe DCN-Cl is as follows: .

[0015] Specifically, the steps are as follows: (1) 6-(dimethylamino)-2-naphthaldehyde (199.3 mg, 1 mmol) and 4-pyridineacetonitrile (118.1 mg, 1 mmol) were dissolved in an appropriate amount of anhydrous ethanol, 4 drops of pyrrolidine were added, and the mixture was stirred at 90 °C for 14 h. After cooling to room temperature, the mixture was filtered. The crude product was purified by column chromatography (DCM:MeOH = 200:1 v / v) to remove the solvent and obtain an orange-red powder, namely compound 1. (2) Compound 1 (149.6 mg, 0.50 mmol) and 1,4-dichlorobenzyl (612.7 mg, 3.50 mmol) were dissolved in acetonitrile; the reaction was carried out at 90 °C for 5 h under a nitrogen atmosphere; after the reaction was completed, the mixture was cooled to room temperature, filtered under reduced pressure, and the filter cake was washed three times with petroleum ether and dried to obtain probe DCN-Cl. 1 H NMR spectrum as follows Figure 1 .

[0016] Example 2: Response of fluorescent probe to detection substance The DCN-Cl synthesized in Example 1 was prepared as a stock solution and then diluted with PBS buffer containing 50% ethanol to prepare a working solution.

[0017] 1. Ultraviolet absorption spectrum DCN-Cl was reacted with N2H4 at final concentrations ranging from 0 to 200 μM at a final concentration of 10 μM, and the results were analyzed by UV-Vis absorption spectroscopy. Figure 2 As shown: without N2H4, DCN-Cl exhibits an absorption peak centered at 550 nm; after the addition of N2H4, the intensity of the maximum absorption peak at 550 nm decreases, while the intensity of the absorption peak at 350 nm increases.

[0018] 2. Fluorescence emission spectrum DCN-Cl was reacted with N2H4 at final concentrations ranging from 0 to 200 μM at a final concentration of 10 μM. Fluorescence emission spectra were then analyzed using 550 nm as the excitation wavelength. Figure 3 As shown in Figure a: the pure probe DCN-Cl has no obvious emission peak at 460 nm; after adding N2H4, the emission peak at 460 nm gradually increases with the increase of concentration. Under the same conditions, fluorescence emission spectroscopy was scanned using 365 nm as the excitation wavelength, such as... Figure 3 As shown in b: the pure probe DCN-Cl shows a relatively obvious emission peak at 700 nm; after adding N2H4, the emission peak at 700 nm gradually weakens with increasing concentration.

[0019] The ratio of N2H4 concentration to fluorescence intensity at 460 nm and 700 nm (I 460 / I 700 ) Create a diagram, such as Figure 4 As shown above, the results indicate that the fluorescent probe DCN-Cl can effectively achieve fluorescence ratio detection of N2H4.

[0020] Example 3: Response kinetics of fluorescent probe to detection substance DCN-Cl was reacted with N2H4 at a final concentration of 200 μM at a final concentration of 10 μM. Excitation wavelengths of 365 nm and 550 nm were used, and fluorescence intensities at 460 nm and 700 nm were detected immediately, every 30 s for 10 min. Results are as follows: Figure 5 As shown: with the increase of reaction time, I 460 / I 700 The value also increased accordingly, and in the absence of a responder, the fluorescence intensity showed good stability during this period, which confirmed that the change in fluorescence was activated by N2H4.

[0021] Example 4: Selectivity of fluorescent probes for different analytes DCN-Cl reacted with various analytes, including N2H4, and the fluorescence intensity at 460 nm and 700 nm was measured. Ig was then calculated. 460 / I 700 Value. Result as follows Figure 6 As shown: A significant change in fluorescence intensity ratio was only observed with the addition of N2H4, while the change was observed with the addition of 200 μM of other detectants (Ag). + Al 3+ Ba 2+ ,Br - Co 2- CO3 2- Cu 2+, Cys, Fe2(SO4)3, GSH, H2O2, HSO3 - I - K + Mg 2+ NaS, Ni 2+ Pd 2+ SO2, Vitamin C, Zn 2+ The ratio of N2H4 to GLU remained almost unchanged. These results confirm that DCN-Cl exhibits superior selectivity for N2H4 compared to other analytes.

[0022] Example 5: Co-localization of mitochondria by fluorescent probes and commercial probes HeLa cells were counterstained using the fluorescent probe DCN-Cl obtained in Example 1 and the commercial mitochondrial probe Mito-Tracker Green (MTG): DCN-Cl working solution was added to the culture medium of HeLa cells to a final concentration of 10 µM. The cells were incubated for 30 min under normal culture conditions. The culture medium containing DCN-Cl was aspirated, and the cells were washed with PBS before imaging using a fluorescence confocal microscope. Then, an appropriate amount of MTG was added and the cells were incubated for 2 min under normal culture conditions. The culture medium containing MTG was aspirated, and the cells were washed with PBS before imaging using a fluorescence confocal microscope.

[0023] The results are as follows Figure 7 As shown in the figure, ae is the image after the first imaging, and fj is the image after the second imaging. As can be seen from the figure, the deep red channel (λ...) ex =561 nm, λ em At 663-738 nm, a distinct red fluorescence was observed in the DCN-Cl stained region, while the green channel (λ) showed a different fluorescence. ex =488 nm, λ em At a wavelength of 500-550 nm, the MTG-stained region exhibited significant green fluorescence, and the two colors overlapped perfectly when merged, with a Pearson coefficient as high as 0.89. These results indicate that the probe DCN-Cl can specifically anchor in the mitochondria of living cells.

[0024] Example 6: Imaging applications of fluorescent probes in living cells When DCN-Cl working solution was added to the culture medium of HeLa cells to a final concentration of 10 µM and incubated for 30 min under normal culture conditions, a clear fluorescence signal in the deep red channel was observed. After incubation in 100 µM and 200 µM exogenous N2H4 for 30 min respectively, the fluorescence intensity of the deep red channel signal decreased significantly, while the fluorescence signal of the blue channel increased.

[0025] Experiments for detecting exogenous N2H4 in living cells. (Example) Figure 5As shown, after HeLa cells were pretreated with probe DCN-Cl (10 μM) for 30 min, the NIR channel fluorescence signal was significant; then, after HeLa cells were incubated with exogenous N2H4 for 30 min, the NIR channel fluorescence intensity decreased significantly, while the blue channel fluorescence signal increased, and the ratio of the two fluorescence intensities changed significantly. Figure 8 This result indicates that the probe DCN-Cl can effectively detect exogenous N2H4 in living cells.

[0026] Example 7 Detection of hydrazine in water samples using fluorescent probes Five natural surface water samples (rivers, lakes, and reservoirs) from different sources were collected. A stock solution of 10 mM N₂H₄ was prepared using the different water samples as solvents. Then, a certain amount of 10 μM N₂H₄ was added to the DCN-Cl solution (final concentration 10 μM). The reproducibility rate was calculated using the following formula: .

[0027] Table 1. Addition and detection of N2H4 in water samples The results are shown in Table 1: the reproducibility rate in different water samples was 101.1-103.5%, which indicates that the probe DCN-Cl has high reliability in analyzing environmental samples.

Claims

1. A ratiometric fluorescent probe for detecting hydrazine, the chemical structure of which is shown in formula (I): Formula (I).

2. A method for preparing a ratiometric fluorescent probe as described in claim 1, characterized in that, Includes the following steps: (1) A mixed solution of 6-(dimethylamino)-2-naphthaldehyde and 4-pyridineacetonitrile was reacted under the catalysis of pyrrolidine. After the reaction was completed, solid-liquid separation and purification were performed to obtain the intermediate product: ; (2) The intermediate product and a mixed solution of 1,4-dichlorobenzyl were reacted under a protective atmosphere, and the mixture was then separated into solid and liquid components and purified.

3. The preparation method according to claim 2, characterized in that, In steps (1) and (2), the reaction temperature is 90℃.

4. The application of a ratiometric fluorescent probe as described in claim 1 in the detection of hydrazine in solution, cells or organisms.

5. The application of the ratiometric fluorescent probe as described in claim 1 in the preparation of reagents for detecting hydrazine.

6. A reagent and kit comprising the ratiometric fluorescent probe as described in claim 1.

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