A fluorescent probe for detecting hydrogen peroxide and viscosity, and a preparation method and application thereof

Abstract

The application belongs to the technical field of synthetic chemistry, and provides a fluorescent probe for detecting hydrogen peroxide and viscosity, and a preparation method and application thereof. The fluorescent probe can be used for detecting H2O2 content and / or viscosity in a solution, cells or organisms. The fluorescent probe can be accurately positioned in mitochondria, and is fixed in the mitochondria, and is not affected by the mitochondrial membrane potential, which has important significance for the related research of hydrogen peroxide and viscosity in mitochondria. The synthesis method of the fluorescent reagent has the characteristics of simple process and low cost.

Description

Technical Field

[0001] This invention belongs to the field of synthetic chemistry technology, specifically relating to a fluorescent probe for detecting hydrogen peroxide and viscosity and its application. Background Technology

[0002] Hydrogen peroxide (H2O2) is a key reactive oxygen species produced during mitochondrial metabolism, playing a crucial role in signal transduction, immune defense, and metabolic regulation. As the core site of cellular energy metabolism, mitochondria continuously produce H2O2 through the electron transport chain and superoxide dismutase (SOD), participating in signal transduction, immune responses, and redox homeostasis regulation. However, when the mitochondrial antioxidant system (such as catalase and glutathione peroxidase) malfunctions, the abnormal accumulation of H2O2 within mitochondria triggers oxidative stress, leading to mitochondrial dysfunction and DNA damage, ultimately inducing various related diseases.

[0003] Viscosity, as a key microenvironmental parameter, plays a crucial regulatory role in physiological functions, such as internal transport of substances and enzyme activity. Abnormal changes in viscosity within the mitochondrial microenvironment exacerbate oxidative stress damage and lead to gene mutations; this process is closely related to inflammatory responses, neurological diseases, and even the development of malignant tumors. Studies have shown that excessive H2O2 not only induces mitochondrial structural damage but can also interfere with metabolite transport and enzymatic reactions by altering the viscosity of the mitochondrial matrix, further aggravating disease responses. Therefore, developing methods for real-time monitoring of H2O2 and viscosity changes within mitochondria is of great significance for revealing the interrelationships between H2O2, viscosity, and mitochondrial-related diseases.

[0004] Fluorescence imaging technology boasts advantages such as high sensitivity, excellent spatiotemporal resolution, and non-invasiveness. In recent years, this method has attracted widespread attention from researchers and clinicians, becoming a core technique for analyzing subcellular organelle microenvironment parameters and bioactive molecules. Recently, several fluorescent probes for detecting H2O2 and viscosity have been reported. However, these probes can only be used to detect specific analytes individually and cannot simultaneously image H2O2 and viscosity, greatly limiting in-depth research on the interaction between H2O2 and viscosity. Therefore, there is an urgent need to develop a small organic molecule probe capable of simultaneously detecting H2O2 and viscosity in mitochondria. Summary of the Invention

[0005] To address the problems in the prior art, this invention provides a fluorescent probe that can be used to simultaneously detect H2O2 and viscosity in mitochondria, with fast response speed and strong anti-interference ability.

[0006] Another object of the present invention is to provide an application of the above-mentioned fluorescent probe in detecting H2O2 and viscosity in mitochondria.

[0007] To achieve the above objectives, the present invention adopts the following technical solution.

[0008] A fluorescent probe for detecting H2O2 and viscosity, abbreviated as BKI, has the chemical structural formula shown in formula (I): Formula (I).

[0009] The preparation method of the above-mentioned fluorescent probe includes the following steps: (1) 4-methylquinoline and 1-iodododecane were reacted in a solvent to purify the intermediate. ; (2) Intermediate The product was obtained by reacting and purifying 4-formylphenylboronic acid pinacol ester in a solvent.

[0010] In step (1), the solvent is acetonitrile; the reaction temperature is 80°C.

[0011] In step (1), the purification step is as follows: the solvent is removed from the reactants, and the eluent is dichloromethane / methanol at a volume ratio of 20:1, and the product is purified by silica gel chromatography.

[0012] In step (2), the solvent is ethanol; the reaction temperature is 80°C.

[0013] In step (2), the purification step is as follows: after the reactants are cooled to room temperature, solid-liquid separation is performed; the solid is purified by silica gel chromatography using dichloromethane / methanol at a volume ratio of 10:1 as the eluent.

[0014] An application of the aforementioned fluorescent probe in detecting H2O2 and viscosity in solutions, cells, or organisms. The concentration of H2O2 and the viscosity of the system are detected by measuring changes in the red fluorescence intensity, green fluorescence intensity, or the ratio of red to green fluorescence intensity of the fluorescent probe before and after contact with the analyte.

[0015] A reagent or kit for preparing the above-mentioned fluorescent probe. The reagent or kit is used to detect the H2O2 content and / or viscosity in solutions, cells, or organisms.

[0016] The mechanism of the present invention is as follows Figure 1 As shown: This invention uses quinoline linked to a benzene ring as a fluorescent parent compound, and introduces a borate salt at one end of the benzene ring of this parent compound as an active site for the H2O2 reaction, thereby achieving selective detection of H2O2. A long alkyl chain (C10-C10) is introduced at the quinoline end. 12The probe mediates hydrophobic interactions with lipids on the mitochondrial membrane, ensuring its immobilization within the mitochondria. Simultaneously, the introduction of a cationic salt enables mitochondrial targeting driven by electrostatic interactions. This dual modification strategy ensures the probe accurately locates to the mitochondria for long-term anchoring. Furthermore, the double bond between the benzene ring and quinoline serves as a viscosity-responsive site for viscosity detection. The introduction of the quinoline salt also allows the probe to aggregate in the mitochondrial region, achieving both targeted and immobilized imaging of the mitochondria independently of mitochondrial membrane potential.

[0017] The present invention has the following advantages: The fluorescent probe described in this invention can be accurately located and immobilized within mitochondria, unaffected by mitochondrial membrane potential. This is of great significance for studies on hydrogen peroxide and viscosity within mitochondria. The synthesis method of this fluorescent reagent is simple and inexpensive. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the response mechanism of the probe BKI; Figure 2 This is the mass spectrum of the probe BKI; Figure 3 It is a probe BKI 1 H NMR spectrum; Figure 4 The fluorescence spectra of the probe BKI for recognizing different concentrations of hydrogen peroxide are shown, with an excitation wavelength of 405 nm. Figure 5 This is a selective fluorescence pattern of the probe BKI recognizing hydrogen peroxide, with an excitation wavelength of 405 nm and a probe concentration of 10 µM. Figure 6 The fluorescence spectrum of probe BKI in different pH buffers, with an excitation wavelength of 405 nm and a probe concentration of 10 µM. Figure 7 The fluorescence images of the probe BKI in different proportions of glycerol and water are shown. The excitation wavelength is 405 nm and the probe concentration is 10 µM. Figure 8 The fluorescence spectrum of the probe BKI in different solvents is shown. The excitation wavelength is 405 nm, and the probe concentration is 10 µM. Figure 9 The images show fluorescence imaging (a) of cells after co-incubation with probes BKI and MTDR and fluorescence imaging (b) of cells after co-incubation with BKI and CCCP for different times. Probe concentration: 10 µM, MTDR concentration: 0.2 µM, CCCP concentration: 10 µM. Figure 10This is a fluorescence imaging image of cells after co-incubation with probe BKI and different concentrations of hydrogen peroxide. Probe concentration: 10µM. Figure 11 This is a fluorescence imaging image of cells after co-incubation with probe BKI and nystatin or monensin. Probe concentration: 10 µM, nystatin or monensin concentration: 20 µM. Detailed Implementation

[0019] 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.

[0020] Example 1 Synthesis of fluorescent probes 1. Synthesis of intermediate (3) Methylquinoline (2 g, 14 mmol) and 1-iodododecane (6.22 g, 21 mmol) were dissolved in acetonitrile solution and heated to 80 °C for 24 hours. After the reaction was completed, the acetonitrile was removed by rotary evaporation. The compound (3) was purified by silica gel chromatography using dichloromethane / methanol (20:1, v / v) as the eluent.

[0021] 2. Synthesis of fluorescent probe (BKI) Compound (3) (0.23 g, 10 mmol) and compound (4) (0.53 g, 12 mmol) were dissolved in ethanol solution and heated to 80 °C for 2 hours. After the reaction solution cooled to room temperature, it was filtered to obtain the crude product. Subsequently, using dichloromethane / methanol (10:1, v / v) as the eluent, the crude product was purified by silica gel chromatography to obtain the fluorescent probe BKI. Its mass spectrometry and... 1 H NMR spectrum as follows Figure 2 and 3 As shown.

[0022] Example 2: Response of fluorescent probe BKI to different concentrations of hydrogen peroxide A 1 mM solution of the fluorescent probe BKI prepared in Example 1 in dimethyl sulfoxide (DMSO) was prepared as the test stock solution for later use.

[0023] In the test solution, an appropriate amount of the fluorescent probe BKI was measured into a 2 mL volumetric flask and diluted to volume with a DMSO / PBS (V / V=1:5) mixture, with an appropriate amount of hydrogen peroxide added to one side. This resulted in a probe concentration of 10 μM in the test solution. The hydrogen peroxide concentrations used in the tests were 10 µM, 20 µM, 30 µM, 40 µM, 70 µM, 90 µM, and 100 µM, respectively. Fluorescence detection (λ) was then performed. ex = 405 nm).

[0024] like Figure 4 As shown, with the increase of hydrogen peroxide concentration, the fluorescence signal at 475 nm gradually weakens, while the fluorescence signal at 570 nm gradually strengthens. When the hydrogen peroxide concentration reaches 100 μM, the fluorescence intensity of the reaction system reaches saturation.

[0025] Example 3: Selectivity of the fluorescent probe BKI in recognizing hydrogen peroxide Prepare a 1 mM BKI solution in dimethyl sulfoxide (DMSO) as a test stock solution. Prepare 100 mM solutions of various ions, amino acids, or reactive oxygen species / reactive nitrogen species as backup. Add 20 μL of the probe stock solution, 50 equivalents of each ion solution, or 10 equivalents of each active substance solution to a 2 mL volumetric flask. Dilute to volume with a DMSO / PBS (V / V = 1:5) mixture. The concentration of the test ions is 500 μM, and the concentration of reactive oxygen species / nitrogen species is 200 μM. After mixing well, perform fluorescence detection (λ). ex =405 nm), and a bar chart of fluorescence intensity versus each ion was constructed.

[0026] like Figure 5 As shown: Other substances have almost no effect on the fluorescence of the probe BKI; only in the presence of hydrogen peroxide does the fluorescence signal ratio (F) increase. 570 / F 475 The significantly enhanced sensitivity indicates that the probe has good selectivity for hydrogen peroxide.

[0027] Example 4: Effect of different pH values ​​on the fluorescent probe BKI Prepare a 1 mM dimethyl sulfoxide (DMSO) test stock solution of the fluorescent probe BKI described in Example 1. Before testing, add 20 μL of the probe stock solution to a 2 mL volumetric flask, dilute to volume with a DMSO / PBS (V / V = 1:5) mixture at different pH, shake well, and then perform fluorescence detection (λ). ex = 405 nm), the fluorescence emission spectrum of the test solution was measured, and a bar chart of fluorescence intensity versus different pH values ​​was established.

[0028] The results are as follows Figure 6 The ratio of fluorescence signal of probe BKI in solutions with different pH values ​​(F) 570 / F 475 The value remained almost unchanged, indicating that the probe was not affected by pH.

[0029] Example 5: Response of fluorescent probe BKI to solutions of different viscosities Prepare a 1 mM test stock solution of dimethyl sulfoxide (DMSO) for the fluorescent probe BKI described in Example 1. Before testing, add 20 μL of the probe stock solution to a 2 mL volumetric flask. Using a water-glycerol binary mixture system, prepare mixed solutions with water volume fractions of 10%, 20%, 30%, 50%, 60%, and 70%, respectively, and bring them to a final volume. After shaking well, record the changes in fluorescence signal under 405 nm excitation.

[0030] The results are as follows Figure 7 As shown, the fluorescence signal of the BKI probe gradually increases with increasing glycerol content, indicating that the probe can be used to detect viscosity changes.

[0031] Example 6: Effect of solution polarity on the fluorescent probe BKI Prepare a 1 mM test stock solution of dimethyl sulfoxide (DMSO) of the fluorescent probe BKI described in Example 1. Before testing, add 20 μL of the probe stock solution to a 2 mL volumetric flask, dilute to volume with different solvents (acetone, N,N-dimethylformamide, dioxane, ethanol, dimethyl sulfoxide, ethyl acetate), shake well, and perform fluorescence testing under 405 nm excitation, recording the changes in fluorescence signal.

[0032] The results are as follows Figure 8 As shown, the fluorescence signal of the probe BKI remains basically unchanged in different solvents, except for the glycerol solution where the fluorescence signal is significantly enhanced, indicating that the probe is not affected by polarity and can selectively detect viscosity changes.

[0033] Example 7 Localization of the fluorescent probe BKI in cells Fadu cells at appropriate densities were seeded into two sterile 35 mm imaging culture dishes and cultured in a CO2 incubator (37°C, 5% CO2). After cell attachment, the fluorescent probe BKI was added to both dishes to a final concentration of 10 μM, and the cells were cultured for another 30 minutes. The culture medium was then discarded, and the cells were washed three times with PBS buffer. One culture dish was then inoculated with the commercial mitochondrial dye Mito Tracker Deep Red (MTDR) to a final concentration of 0.2 μM and cultured for another 10 minutes before fluorescence imaging. The other culture dish was then inoculated with the mitochondrial membrane potential inhibitor 3-chlorophenylhydrazone carbonyl cyanide (CCCP) to a final concentration of 10 μM, and fluorescence imaging was performed at different time points.

[0034] like Figure 9 As shown, the green fluorescence of the probe overlaps well with the red fluorescence of the mitochondrial dye, indicating that the probe can be localized in the mitochondria. Figure 9a). Under the action of CCCP, the fluorescence signal of the probe in the mitochondria remained unchanged and remained in the mitochondria, indicating that the probe was not affected by the mitochondrial membrane potential and could be immobilized on the mitochondria. Figure 9 b).

[0035] Example 8: Detection of hydrogen peroxide in cells using the fluorescent probe BKI Fadu cells at an appropriate density were seeded into three sterile 35 mm imaging culture dishes and cultured in a CO2 incubator (37°C, 5% CO2). After cell attachment, the fluorescent probe BKI described in this invention was added to each of the three culture dishes to a final concentration of 10 μM. The dishes were then cultured for another 30 minutes, the culture medium was discarded, and the cells were washed three times with PBS buffer. Hydrogen peroxide was added to the other two culture dishes to final concentrations of 100 μM and 200 μM, respectively, and the dishes were cultured for another 30 minutes before fluorescence imaging experiments were performed.

[0036] The results are as follows Figure 10 As shown, with the increase of hydrogen peroxide concentration, the green and red fluorescence in the cells were significantly enhanced. The enhancement of green fluorescence was due to the increase in intracellular viscosity caused by excessive hydrogen peroxide. These results indicate that the probe can be used to detect changes in intracellular hydrogen peroxide.

[0037] Example 9: Detection of viscosity changes in cells using the fluorescent probe BKI Fadu cells at an appropriate density were seeded into three sterile 35 mm imaging culture dishes and cultured in a CO2 incubator (37°C, 5% CO2). After cell attachment, the fluorescent probe BKI described in this invention was added to each of the three culture dishes to a final concentration of 10 μM. The dishes were then cultured for another 30 minutes, the culture medium was discarded, and the cells were washed three times with PBS buffer. Nystatin (Nys) and monensin (Mon) were added to the other two culture dishes, respectively, to a final concentration of 10 μM. After culturing for another 30 minutes, fluorescence imaging experiments were performed.

[0038] The results are as follows Figure 11 As shown, under the stimulation of nystatin (Nys) and monensin (Mon), the green fluorescence in the cells was significantly enhanced, while the red fluorescence remained unchanged. This is consistent with the fluorescence test results of the probe in glycerol, indicating that the probe can be used to detect changes in intracellular viscosity.

Claims

1. A fluorescent probe for detecting H2O2 and viscosity, the chemical structure of which is shown in formula (I): Formula (I).

2. A method for preparing a fluorescent probe as described in claim 1, characterized in that, Includes the following steps: (1) 4-methylquinoline and 1-iodododecane were reacted in a solvent to purify the intermediate. ; (2) Intermediate The product was obtained by reacting and purifying 4-formylphenylboronic acid pinacol ester in a solvent.

3. The preparation method according to claim 2, characterized in that, In step (1), the solvent is acetonitrile; the reaction temperature is 80°C. In step (2), the solvent is ethanol; the reaction temperature is 80°C.

4. The preparation method according to claim 2, characterized in that, In step (1), the purification step is as follows: the solvent is removed from the reactants, and the eluent is dichloromethane / methanol at a volume ratio of 20:1, and the product is purified by silica gel chromatography. In step (2), the purification step is as follows: after the reactants are cooled to room temperature, solid-liquid separation is performed; the solid is purified by silica gel chromatography using dichloromethane / methanol at a volume ratio of 10:1 as the eluent.

5. The application of the fluorescent probe as described in claim 1 in detecting H2O2 and viscosity in solutions, cells or organisms.

6. A reagent or kit for preparing the fluorescent probe as described in claim 1.

Images