Fluorescent probes for detecting hydrogen sulfide and viscosity / polarity targeting mitochondria and applications thereof

Abstract

The application belongs to the technical field of organic small-molecule fluorescent probes, and particularly relates to a near-infrared fluorescent probe for simultaneously detecting hydrogen sulfide and viscosity / polarity while targeting mitochondria and application thereof. The near-infrared fluorescent probe MQA-DNP provided by the application has specific response performance to hydrogen sulfide and viscosity / polarity, and can be used as a detection reagent to simultaneously detect changes in hydrogen sulfide and viscosity / polarity in an inflammatory cell model, a fatty liver tissue model and a tumor model, and the detection means is simple and sensitive.

Description

Technical Field

[0001] This invention belongs to the field of organic small molecule fluorescent probe technology, specifically relating to a near-infrared fluorescent probe that targets mitochondria and simultaneously detects hydrogen sulfide and viscosity / polarity, and its application. Background Technology

[0002] Hydrogen sulfide (H2S), following carbon monoxide (CO) and nitric oxide (NO), is the third endogenous gaseous neurotransmitter found in living organisms. In mammals, it is produced in situ in the cytoplasm and mitochondria through enzymatic reactions between cystathionine β-synthetase, cystathionine γ-lyase, and 3-mercaptopyruvate thiotransferase and cystathionine, involving cysteine, homocysteine, and cystathionine. Studies have shown that endogenous H2S participates in numerous physiological processes, primarily related to vasodilation, insulin secretion, cell protection, and nerve conduction. Disorders in H2S levels often lead to diseases such as cirrhosis, myocardial damage, diabetes, tumors, and Alzheimer's disease. Therefore, monitoring H2S levels in biological systems is essential.

[0003] Intracellular microenvironment parameters (including viscosity, polarity, and pH) are closely related to the normal functioning of various physiological activities, such as cellular metabolism, enzyme activity, energy transport, signal transduction, and biomolecule / protein interactions. Abnormal levels of viscosity and polarity in mitochondria can lead to cellular homeostasis imbalance, metabolic disorders, and even various diseases such as inflammation, atherosclerosis, cardiovascular metabolic diseases, and cancer. Therefore, developing sensitive and reliable methods for detecting mitochondrial H2S, viscosity, and polarity levels is of great significance for understanding the etiology and treatment of related diseases.

[0004] Organic small molecule fluorescent probes possess advantages such as non-invasive analysis, rapid response, high sensitivity, high selectivity, and good real-time performance, leading to their widespread application in fields like bioimaging. To date, numerous fluorescent probes have been developed for the detection of H2S, viscosity, and polarity. However, many biomolecules frequently interact and change simultaneously within the complex cellular environment, making multifunctional probes with multiple reaction sites capable of emitting different fluorescence to identify multiple analytes a promising tool. Furthermore, many physiological / pathological processes often involve anomalous fluctuations in microenvironmental parameters and bioactive molecule levels simultaneously. In various disease models, the changes in H2S, viscosity, and polarity levels remain unclear. Therefore, developing a near-infrared fluorescent probe with mitochondrial targeting capabilities that can simultaneously detect H2S, viscosity, and polarity for the visual diagnosis of multiple diseases is essential. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a near-infrared fluorescent probe (MQA-DNP) with mitochondrial targeting capability and the ability to simultaneously detect H2S and viscosity / polarity. It combines quinoline cations with the negative membrane potential within mitochondria via electrostatic interaction, allowing the probe to target the mitochondria. Simultaneously, it utilizes 2,4-dinitrophenyl (DNP) ether as a mitochondrial target. - The recognition unit triggers the release of the intermediate MQA, resulting in a significant enhancement in fluorescence intensity. N,N-dimethylamino is used as the viscosity-sensitive unit. Due to its distorted intramolecular charge transfer (TICT) state, it can rotate freely under low viscosity conditions, leading to enhanced nonradiative transitions and fluorescence quenching. In high viscosity environments, free rotation is restricted, causing fluorescence recovery. Simultaneously, the D-π-A structure, possessing excited-state intramolecular charge transfer (ESICT) properties, makes the probe highly sensitive to polarity changes. Using the multifunctional fluorescent probe MQA-DNP, we revealed overexpression of H2S, increased viscosity levels, and decreased polarity in inflammation, non-alcoholic fatty liver disease, and tumor models, demonstrating its potential for multi-marker disease diagnosis.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A fluorescent probe MQA-DNP targeting mitochondria for detecting hydrogen sulfide and viscosity / polarity has the following structural formula:

[0008]

[0009] A fluorescent probe MQA-DNP targeting mitochondria for detecting hydrogen sulfide and viscosity / polarity is prepared according to the following reaction formula:

[0010]

[0011] A method for preparing a fluorescent probe MQA-DNP targeting mitochondria for detecting hydrogen sulfide and viscosity / polarity, the specific steps of which are as follows:

[0012] Step 1: Add 4-hydroxybenzaldehyde and potassium carbonate to anhydrous N,N-dimethylformamide, stir at room temperature, add 1-bromo-2,4-dinitrobenzene, continue stirring under nitrogen, filter, remove solvent under reduced pressure, purify by silica gel column, and collect the yellow solid 4-(2,4-dinitrophenoxy)benzaldehyde, i.e., compound 1.

[0013] Step 2: Dissolve compound 1 in methanol, cool to room temperature, add sodium borohydride, stir, then quench the reaction, extract the yellow crude product with dichloromethane, concentrate by rotary evaporation, and purify by silica gel column chromatography to obtain the yellow solid (4-(2,4-dinitrophenoxy)phenyl)methanol, i.e., compound 2.

[0014] Step 3: Dissolve compound 2 in dichloromethane, add phosphorus tribromide dropwise, stir, then quench the reaction, extract with ethyl acetate, wash with saturated sodium bicarbonate solution, dry on sodium sulfate, remove solvent under reduced pressure, and purify by silica gel column chromatography to obtain a yellow solid 1-(4-(bromomethyl)phenoxy)-2,4-dinitrobenzene, i.e., compound 3.

[0015] Step 4: Add 4-(dimethylamine)cinnamaldehyde and 4-methylquinoline to anhydrous N,N-dimethylformamide and p-toluenesulfonic acid solution, stir and reflux, cool and concentrate, then dissolve in dichloromethane, wash with deionized water and saturated sodium chloride solution, then dry with anhydrous sodium sulfate, evaporate the solvent, and obtain a yellowish-brown solid N,N-dimethyl-4-((1E,3E)-4-(quinoline-4-yl)but-1,3-dien-1-yl)aniline, i.e., compound MQA, by silica gel column chromatography;

[0016] Step 5: Add MQA and compound 3 to N,N-dimethylformamide, stir at room temperature, filter, and recrystallize in a dichloromethane / methanol mixed solvent to obtain a dark brown solid 4-(1E,3E)-4-(4-(dimethylamino)phenyl)but-1,3-dien-1-yl)-1-(4-(2,4-dinitrophenoxy)benzyl)quinoline-1-ammonium bromide, i.e., MQA-DNP.

[0017] The preparation processes of compounds 1, 2, 3 and MQA were based on existing technologies with similar structures, and the references are Sensor Actuat. B-Chem. 2022, 351, 130940; Anal. Chem. 2022, 94, 4594-4601.

[0018] Furthermore, in step 1, the molar ratio of 4-hydroxybenzaldehyde to potassium carbonate is 1:2.

[0019] Furthermore, in step 3, the molar ratio of compound 2 to phosphorus tribromide is 1:3.

[0020] Furthermore, in step 5, the molar ratio of MQA to compound 3 is 1:1.

[0021] Furthermore, in step 5, the volume ratio of the dichloromethane / methanol mixed solvent ranges from 50:1 to 20:1.

[0022] An application of a fluorescent probe MQA-DNP targeting mitochondria to simultaneously detect hydrogen sulfide, viscosity, and polarity.

[0023] Furthermore, the aforementioned fluorescent probe MQA-DNP is used to prepare an inflammation detection reagent.

[0024] Furthermore, the aforementioned fluorescent probe MQA-DNP was used to prepare a fatty liver detection reagent.

[0025] Furthermore, the aforementioned fluorescent probe MQA-DNP is used to prepare tumor detection reagents.

[0026] The mitochondrial-targeted near-infrared fluorescent probe provided by this invention has the characteristic of simultaneously detecting H2S and viscosity / polarity. The fluorescence intensity of the probe in the near-infrared channel (>714nm) gradually increases with the increase of environmental viscosity or the decrease of environmental polarity, and the fluorescence intensity of the probe at 634nm gradually increases with the increase of H2S concentration.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] (1) The near-infrared fluorescent probe for simultaneous detection of H2S and viscosity / polarity targeting mitochondria described in this invention forms a typical D-π-A molecular configuration by bridging N,N-dimethylamino (electron donor, D) and quinoline cation (electron acceptor, A) with divinyl bridging.

[0029] (2) The principle of the probe to recognize changes in H2S: H2S reacts with the 2,4-dinitrophenyl ether group of the probe, causing the intermediate MQA to be released and emit strong fluorescence, which is easy to identify.

[0030] (3) Detection of H2S changes by the probe: In a mixed system of acetonitrile (CH3CN) and phosphate buffered solution (PBS), the fluorescence at 634 nm significantly increased with increasing H2S concentration; the linear detection range for H2S was 0-1000 μM (R0). 2 =0.9909), and the detection limit is 0.295μM, meaning the probe can achieve a highly sensitive response to H2S.

[0031] (4) The probe has high selectivity and high sensitivity for H2S detection and is not affected by biological systems or other substances in the environment.

[0032] (5) The response principle of the probe to viscosity changes: N,N-dimethylamino is a viscosity-sensitive unit. Based on twisted intramolecular charge transfer (TICT), it rotates freely under low viscosity conditions, and the energy is dissipated in a non-radiative transition manner, resulting in fluorescence quenching; while in a high viscosity environment, free rotation is restricted, the rigid plane is enhanced, and fluorescence is restored.

[0033] (6) Detection of viscosity changes by the probe: In the water / glycerol system, when the solution viscosity coefficient η increases from 0.89 cP to 945 cP, the near-infrared fluorescence intensity at 740 nm is significantly enhanced. log F 740 It exhibits a linear relationship with logη in the range of 0.89–163.6 cP (R 2=0.9912), with a slope of 0.8141, it has the characteristics of highly sensitive quantitative or qualitative detection of environmental viscosity.

[0034] (7) The response principle of the probe to polarity changes: The D-π-A structure with excited-state intramolecular charge transfer (ESICT) characteristics makes the probe highly sensitive to polarity changes.

[0035] (8) Detection of polarity changes by the probe: In an aqueous solution of 1,4-dioxane, the solution polarity level (Δf) varies with the volume fraction of water (f). W It increases with the increase of polarity level. It increases with the increase of polarity level from 0.0205 (f) W =0%) increased to 0.3200(f W =100%), the fluorescence intensity at 714 nm decreased significantly by 543 times, and the maximum emission peak also redshifted to 786 nm. The fluorescence intensity (F) of MQA-DNP showed a good linear relationship with Δf in the range of 0.0205 to 0.3200 (R = 100%). 2 =0.9913).

[0036] (9) The probe utilizes the positive charge of the quinoline cation in the molecule to combine with the negative membrane potential in the mitochondria through electrostatic interaction, enabling the probe to specifically target the mitochondria; it is applied to the visualization imaging of H2S, viscosity and polarity in inflammation, non-alcoholic fatty liver and cancer cells / tissue sections / organs and clinical patient tissue samples.

[0037] (10) The detection method is simple and only requires a fluorescence spectrophotometer, a laser confocal microscope, and a multi-mode live imaging instrument. Attached Figure Description

[0038] Figure 1 For the NMR characterization of the probe MQA-DNP of this invention, 1 H-NMR spectrum.

[0039] Figure 2 For the NMR characterization of the probe MQA-DNP of this invention, 13 C-NMR spectrum.

[0040] Figure 3 The mass spectrometry characterization of the probe MQA-DNP of this invention is shown in the HR-MS spectrum.

[0041] Figure 4 This is the fluorescence emission spectrum of the probe MQA-DNP of this invention in a CH3CN / PBS mixed system as a function of H2S concentration.

[0042] Figure 5 The fluorescence intensity (I) of the probe MQA-DNP of this invention 634 nmLinear relationship between H2S concentration and H2S concentration.

[0043] Figure 6 This is a bar chart showing the selectivity of the probe MQA-DNP of this invention for H2S.

[0044] Figure 7 This is a high-resolution mass spectrum of H2S identified by the probe MQA-DNP of this invention.

[0045] Figure 8 This is the fluorescence emission spectrum of the probe MQA-DNP of the present invention in a water / glycerol mixture as the glycerol volume content changes.

[0046] Figure 9 This is a linear relationship graph showing the logF of the probe MQA-DNP of this invention as a function of logη in a water / glycerol mixture.

[0047] Figure 10 This is the fluorescence emission spectrum of the probe MQA-DNP of the present invention in a water / 1,4-dioxane mixed system, showing the change with water volume content.

[0048] Figure 11 This is a linear relationship between the fluorescence intensity of the probe MQA-DNP of this invention and the polarity parameter Δf in a water / 1,4-dioxane mixed system.

[0049] Figure 12 This is a fluorescence co-localization imaging image of live cells co-stained with the probe MQA-DNP of this invention and a commercially available mitochondrial-specific dye (MTG).

[0050] Figure 13 This is a fluorescence imaging image of the changes in H2S, viscosity, and polarity of the probe MQA-DNP of this invention in a lipopolysaccharide (LPS)-stimulated HeLa cell inflammation model.

[0051] Figure 14 This is a fluorescence imaging image of the probe MQA-DNP of the present invention in fatty liver tissue sections, showing H2S, viscosity, and polarity.

[0052] Figure 15 This is a fluorescence imaging image of H2S, viscosity, and polarity of the probe MQA-DNP of this invention in normal cells (HL 7702, RAW264.7) and cancer cells (A549, HeLa).

[0053] Figure 16 This is a fluorescence imaging image of H2S, viscosity, and polarity of the probe MQA-DNP of this invention in normal mouse tissue sections (heart, liver, spleen, lung, and kidney) and tumor tissue sections.

[0054] Figure 17This is a fluorescence imaging image of H2S, viscosity, and polarity of the probe MQA-DNP of this invention in normal mouse organs (heart, liver, spleen, lung, and kidney) and tumors.

[0055] Figure 18 This is a fluorescence imaging image of the probe MQA-DNP of the present invention in benign and malignant tissue sections from clinical cancer patients, showing H2S, viscosity, and polarity. Detailed Implementation

[0056] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0057] Example 1

[0058] Preparation and characterization of a fluorescent probe targeting mitochondria for the detection of hydrogen sulfide and viscosity / polarity:

[0059]

[0060] (1) 4-hydroxybenzaldehyde (300 mg, 2.45 mmol) was added to anhydrous N,N-dimethylformamide (5 mL), followed by potassium carbonate (680 mg, 4.92 mmol). The mixture was stirred at room temperature for 5 min. After adding 1-bromo-2,4-dinitrobenzene (668 mg, 2.70 mmol), the mixture was stirred under nitrogen for 24 h to form a yellow mixture. After filtration, the solvent was removed under reduced pressure, and the mixture was purified by column chromatography (hexane / ethyl acetate, 8:1, v / v). The yellow solid 4-(2,4-dinitrophenoxy)benzaldehyde, i.e., compound 1 (650 mg, 92%), was collected.

[0061] (2) Compound 1 (650 mg, 2.26 mmol) was dissolved in 10 mL of methanol and cooled at 0 °C for 5 minutes. Sodium borohydride (86 mg, 2.66 mmol) was added. The mixture was stirred at 0 °C under nitrogen for 4 h, and the reaction was quenched with deionized water. The yellow crude product was extracted with dichloromethane (50 mL × 3), and purified by column chromatography (n-hexane / ethyl acetate, 4:1, v / v) to obtain a yellow solid (4-(2,4-dinitrophenoxy)phenyl)methanol, i.e., compound 2 (590 mg, 90%).

[0062] (3) Compound 2 (150 mg, 0.56 mmol) was dissolved in dichloromethane, and PBr3 (159 μL, 1.68 mmol) was added dropwise at 0 °C. The mixture was stirred for 6 h, and deionized water was added to quench the reaction. The mixture was then extracted with ethyl acetate (50 mL × 3). After washing with saturated NaHCO3 solution, the product was dried over sodium sulfate, and the solvent was removed under reduced pressure. The crude solid product was purified by chromatography to obtain a yellow solid 1-(4-(bromomethyl)phenoxy)-2,4-dinitrobenzene, i.e., compound 3 (98 mg, 47%). 1 H NMR (600MHz, CDCl3) δ8.85 (d, 1H), 8.34 (dd, J1=2.4Hz, J2=9.6Hz, 1H), 7.52 (d, J=8.4Hz, 2H), 7.12 (d, J=8.4Hz, 2H), 7.08 (d, J=9.6Hz, 1H), 4.53 (s, 2H). 13 C NMR (151MHz, CDCl3) δ155.69,153.55,141.72,139.75,136.28,131.45,128.84,122.13,120.73,118.84,32.11.

[0063] (4) 4-(dimethylamine)cinnamaldehyde (876.2 mg, 5 mmol), 4-methylquinoline (715.9 mg, 5 mmol), and anhydrous N,N-dimethylformamide (8 mL) were mixed, and p-toluenesulfonic acid was added dropwise. The mixture was heated and stirred under reflux for 3 h. After cooling, the mixture was washed with dichloromethane (100 mL), deionized water (100 mL × 2), and saturated sodium chloride solution (100 mL), and then dried with anhydrous sodium sulfate. Column chromatography (dichloromethane / methanol, 200:1 → 100:1, v / v) yielded a yellowish-brown solid N,N-dimethyl-4-((1E,3E)-4-(quinoline-4-yl)but-1,3-dien-1-yl)aniline, i.e., compound MQA (745 mg, 50%). 1H NMR (600MHz, CDCl3) δ8.83 (d, J=4.8Hz, 1H), 8.18 (d, J=8.4Hz, 1H), 8.10 (d, J=8.4Hz, 1H), 7 .70(td, J1=1.2Hz, J2=8.4Hz, 1H), 7.55(td, J1=1.2Hz, J2=8.4Hz, 1H), 7.53(d, J=4.8Hz, 1H ), 7.39 (d, J=8.4Hz, 2H), 7.25 (dd, J1=9.6Hz, J2=15.6Hz, 1H), 7.22 (d, J=15.6Hz, 1H), 6.93 (dd, J1=9.6Hz, J2=15.6Hz, 1H), 6.77 (d, J=15.6Hz, 1H), 6.70 (d, J=8.4Hz, 2H), 3.00 (s, 6H). 13 C NMR (151MHz, CDCl3) δ150.54,150.04,148.84,143.08,136.65,136.31,130.06,129.13, 128.08,126.20,126.14,125.02,124.38,123.37,123.29,116.02,112.28,40.32.HR-MS m / z:calcd.for C 21 H 21 N2,300.1626; measured,301.1700.

[0064] (5) Add MQA (450 mg, 1.5 mmol) and compound 3 (525 mg, 1.5 mmol) to N,N-dimethylformamide (3 mL), stir at room temperature for 24 h, and then add 50 mL of water. Filter to obtain a solid, which is purified by column chromatography (dichloromethane / methanol, 50:1→20:1, v / v) to obtain a dark brown solid 4-(1E,3E)-4-(4-(dimethylamino)phenyl)but-1,3-dien-1-yl)-1-(4-(2,4-dinitrophenoxy)benzyl)quinoline-1-ammonium bromide, i.e., MQA-DNP (110 mg, 40%).

[0065] like Figure 1 As shown, 1H NMR (600MHz, CDCl3) δ10.12(d,J=7.2Hz,1H),8.77(d,J=3.0Hz,1H),8.47(d,J=8.4Hz,1 H),8.35(d,J=8.4Hz,1H),8.28(dd,J1=3.0Hz,J2=9.0Hz,1H),8.17(d,J=7.2Hz,1H),7.9 9(t,J=7.2Hz,1H),7.83-7.78(m,2H),7.61(d,J=9.0Hz,2H),7.44(d,J=9.0Hz,2H),7.31 (d,J=14.4Hz,1H),7.12-7.02(m,5H),6.66(d,J=9.0Hz,2H),6.56(s,2H),3.06(s,6H). Such as Figure 2 As shown, 13 C NMR (151MHz, CDCl3) δ 155.41, 153.86, 153.43, 151.88, 147.42, 147.02, 145.97, 141.57, 138.32, 135.01, 132.10, 130.23, 129.08, 128.77, 126.43, 125.88, 123.73, 123.34, 122.01, 121.11, 119.36, 119.17, 118.57, 114.99, 112.06, 58.56, 40.17. (e.g.) Figure 3 As shown, HR-MS:calcd.for C 34 H 29 N4O5 + ,573.2132; found 573.2133.

[0066] Example 2

[0067] The fluorescent probe MQA-DNP from Example 1 was diluted to a final concentration of 10 μmol / L with a mixed solvent of acetonitrile and water (1 / 1, v / v, pH 7.4). The excitation wavelength was fixed at 400 nm, and the fluorescence emission spectrum of MQA-DNP as a function of H2S concentration was recorded. Figure 4 As the H2S concentration increased from 0 μmol / L to 2000 μmol / L, the fluorescence intensity at 634 nm gradually increased, and the linear detection range for H2S was 0-1000 μmol / L (R0). 2 =0.9909)( Figure 5 The detection limit was 0.295 μmol / L, indicating that the probe can achieve a highly sensitive response to H2S.

[0068] Example 3

[0069] The concentration of the fluorescent probe MQA-DNP from Example 1 was maintained at 10 μmol / L, and the fluorescence intensity of the probe in the presence of common ions and bioactive small molecules was investigated, as well as its selectivity for H2S. Figure 6 As shown, the addition of the following substances (1 mmol / L) to the CH3CN / PBS (1 / 1, v / v, 7.4) system had almost no interference with the fluorescence intensity of MQA-DNP. Figure 6 The substances in the sample are, in order: (1) blank, (2) H2S, (3) GSH, (4) Cys, (5) Hcy, (6) HSO3 - (7) SO3 2- (8)S2O3 2- (9)H2O2, (10)ClO - (11)NO2 - (12)K + (13)Ca 2+ (14)Na + (15)Mg 2+ (16)Cu 2+ (17)Zn 2 + (18)Fe 2+ (19)Fe 3+ (20)H2PO4 - (21)HPO4 2- (22)CO3 2- (23)HCO3 - (24)Br - (25)Cl - , (26)Trp, (27)Ala, (28)Arg, (29)Tyr, (30)Thr, (31)His, (32)Ile.

[0070] Example 4

[0071] The fluorescent probe MQA-DNP from Example 1, the solution after the reaction of MQA-DNP with H2S, and the compound MQA were subjected to mass spectrometry analysis to verify the interaction mechanism of the probe MQA-DNP with H2S. Figure 7As shown, the peak at m / z = 573.2133 is the mass spectrum peak of the probe MQA-DNP (theoretical value: 573.2132), and the peak at m / z = 301.1699 can be attributed to the mass spectrum peak of the new fluorophore released after the probe MQA-DNP reacts with H2S. It is almost identical to the mass spectrum peak of the pure compound MQA (m / z = 301.1700) (theoretical value: 300.1626). This indicates that after the probe MQA-DNP reacts with H2S, the 2,4-dinitrophenyl ether linked to quinoline is removed, thereby releasing a new fluorophore, which is the compound MQA.

[0072] Example 5

[0073] The fluorescent probe MQA-DNP from Example 1 was diluted to a final concentration of 10 μmol / L using a mixed solvent of water and glycerol. The excitation wavelength was fixed at 570 nm, and the fluorescence emission spectrum of the probe was recorded as a function of glycerol volume content (or viscosity coefficient η). Figure 8 The fluorescence intensity value (log F) of the probe at 740 nm was plotted, and a linear correlation curve was generated between the fluorescence intensity value (log F) of the probe and the viscosity coefficient (log η) in the water and glycerol mixture. Figure 9 As the glycerol volume ratio increased from 0% (0.89 cP) to 99% (945 cP), the fluorescence intensity at 740 nm gradually increased, reaching its maximum value when the glycerol volume ratio was 99%, indicating that the fluorescence intensity of the probe significantly increased with the increase of environmental viscosity.

[0074] Example 6

[0075] The fluorescent probe MQA-DNP from Example 1 was diluted with a mixed solvent of water and 1,4-dioxane to a final concentration of 10 μmol / L. The excitation wavelength was fixed at 570 nm, and the fluorescence emission spectrum of the probe as a function of water volume content was recorded. Figure 10 ), and plotted the linear correlation curve of the maximum fluorescence intensity of the probe in the water and 1,4-dioxane mixed system as a function of the polarity parameter (Δf). Figure 11 As the volume ratio of 1,4-dioxane increased from 0% (0.3200) to 100% (0.0205), the fluorescence intensity gradually increased, reaching its maximum value when the volume ratio of 1,4-dioxane was 100%, indicating that the fluorescence intensity of the probe significantly increased with decreasing environmental polarity.

[0076] Example 7

[0077] To observe whether the probe MQA-DNP could target and accumulate in the mitochondria of live cells, a co-localization experiment was conducted using the probe and the commercially available mitochondrial-specific dye MitoTracker Green (MTG). Adherent HeLa cells were incubated with MTG (final concentration 0.8 μmol / L) at pH 7.4 in a 37°C, 5% CO2 incubator for 30 min. Afterward, the cells were washed three times with PBS buffer (pH 7.4) to remove excess dye. Then, the probe MQA-DNP (final concentration 5 μmol / L) was added, and co-incubation continued for another 30 min. The co-localization of the two was observed under a laser confocal microscope. The excitation wavelength for MQA-DNP was fixed at 561 nm, and red fluorescence imaging was selected, collecting the red channel range of 700-754 nm. The excitation wavelength for MTG was fixed at 488 nm, and the red channel range of 490-530 nm was collected. Figure 12 The fluorescent probe MQA-DNP exhibits a typical red rod-shaped mitochondrial morphology and overlaps well with MTG, producing yellow overlapping fluorescence. Furthermore, their Pearson colocalization coefficient (PC) is 0.92. This indicates that the fluorescent probe MQA-DNP and MTG have significant colocalization imaging capabilities, enabling them to target and localize within mitochondria.

[0078] Example 8

[0079] Adherent HeLa cells were incubated with the fluorescent probe MQA-DNP (final concentration 5 μmol / L) from Example 1 at pH 7.4 in an incubator at 37°C and 5% CO2 for 30 min. The probe was then imaged using a laser confocal microscope in two channels, where the H2S channel (E...) was used to visualize the fluorescence. x =405nm, E m =580-680nm) is used to observe the probe's detection of H2S; viscosity / polarity channel (E x =561nm, E m =700-754nm) is used to observe the probe's detection of viscosity and polarity. For example Figure 13 As shown, the probe itself emits weak fluorescence in both channels. Then, lipopolysaccharide (LPS) is added to stimulate cells for 180 min to induce inflammation. It can be observed that the fluorescence emission in both channels of the cell is enhanced, indicating that LPS can induce a significant increase in intracellular H2S and viscosity levels and a decrease in polarity levels. Moreover, the probe MQA-DNP can simultaneously detect intracellular H2S, viscosity and polarity in the inflammatory model cells.

[0080] Example 9

[0081] The fluorescent probe MQA-DNP (final concentration 20 μmol / L) from Example 1 was incubated into sections of normal liver tissue and fatty liver tissue, respectively. Fluorescence imaging of the probe was observed under a laser confocal microscope. The probe showed significant fluorescence enhancement in fatty liver sections, while fluorescence in normal liver tissue sections was negligible. Figure 14 This indicates that the early H2S and viscosity levels in fatty liver tissue are significantly higher than those in normal liver tissue, while the polarity level is lower. Furthermore, the probe MQA-DNP enables efficient visual identification of fatty liver tissue.

[0082] Example 10

[0083] The fluorescent probe MQA-DNP from Example 1 was incubated in normal cells (HL 7702, RAW264.7) and cancer cells (A549, HeLa), respectively. Figure 15 ), mouse normal tissue sections (heart, liver, spleen, lung, kidney) and tumor tissue sections ( Figure 16 ), in normal mouse organs (heart, liver, spleen, lung, kidney) and tumors ( Figure 17 The fluorescence imaging of the probe was observed under a laser confocal microscope and a small animal in vivo imaging system. The probe showed significant fluorescence enhancement in cancer cells / tissue sections / organs, while the fluorescence in normal cells / tissue sections / organs was negligible. This indicates that the H2S and viscosity levels in tumor tissues are significantly higher than those in normal tissues, while the polarity level is lower than that in normal tissues. Moreover, the probe MQA-DNP can achieve highly sensitive visual identification of tumors.

[0084] Example 11

[0085] The fluorescent probe MQA-DNP (final concentration 20 μmol / L) from Example 1 was incubated into human benign (breast and thyroid) and malignant (breast and thyroid) tissue sections, respectively. Fluorescence imaging of the probe was performed under a laser confocal microscope. Significant fluorescence enhancement was observed in the malignant cancer tissue, while the fluorescence signal in the benign tissue sections was negligible. Figure 18 The results indicate that the levels of H2S and viscosity in malignant cancer tissues are significantly higher than those in benign tissues, while the polarity level is lower. Furthermore, the probe MQA-DNP has excellent visual identification capabilities for cancer.

[0086] The embodiments described above are merely specific examples of the present invention, and while the descriptions are detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A fluorescent probe MQA-DNP targeting mitochondria for detecting hydrogen sulfide and viscosity / polarity, characterized in that, The structural formula is: 。 2. The method for preparing the fluorescent probe MQA-DNP as described in claim 1, characterized in that, Includes the following steps: Step 1: Add 4-hydroxybenzaldehyde and potassium carbonate to anhydrous N,N-dimethylformamide, stir at room temperature, add 1-bromo-2,4-dinitrobenzene, continue stirring under nitrogen, filter, remove solvent under reduced pressure, purify by silica gel column, and collect the yellow solid 4-(2,4-dinitrophenoxy)benzaldehyde, i.e., compound 1. Step 2: Dissolve compound 1 in methanol, cool to room temperature, add sodium borohydride, stir, then quench the reaction, extract the yellow crude product with dichloromethane, concentrate by rotary evaporation, and purify by silica gel column chromatography to obtain the yellow solid (4-(2,4-dinitrophenoxy)phenyl)methanol, i.e., compound 2. Step 3: Dissolve compound 2 in dichloromethane, add phosphorus tribromide dropwise, stir, then quench the reaction, extract with ethyl acetate, wash with saturated sodium bicarbonate solution, dry on sodium sulfate, remove solvent under reduced pressure, and purify by silica gel column chromatography to obtain a yellow solid 1-(4-(bromomethyl)phenoxy)-2,4-dinitrobenzene, i.e., compound 3. Step 4: Add 4-(dimethylamine)cinnamaldehyde and 4-methylquinoline to anhydrous N,N-dimethylformamide and p-toluenesulfonic acid solution, stir and reflux, cool and concentrate, then dissolve in dichloromethane, wash with deionized water and saturated sodium chloride solution, then dry with anhydrous sodium sulfate, and after solvent evaporation, obtain yellowish-brown solid N,N-dimethyl-4-((1E,3E)-4-(quinoline-4-yl)but-1,3-dien-1-yl)aniline, i.e., compound MQA, by silica gel column chromatography; Step 5: Add MQA and compound 3 to N,N-dimethylformamide, stir at room temperature, filter, and recrystallize in a dichloromethane / methanol mixed solvent to obtain a dark brown solid 4-(1E,3E)-4-(4-(dimethylamino)phenyl)but-1,3-dien-1-yl)-1-(4-(2,4-dinitrophenoxy)benzyl)quinoline-1-ammonium bromide, i.e., MQA-DNP.

3. The method for preparing the fluorescent probe MQA-DNP as described in claim 2, characterized in that, In step 1, the molar ratio of 4-hydroxybenzaldehyde to potassium carbonate is 1:

2.

4. The method for preparing the fluorescent probe MQA-DNP as described in claim 2, characterized in that, In step 3, the molar ratio of compound 2 to phosphorus tribromide is 1:

3.

5. The method for preparing the fluorescent probe MQA-DNP as described in claim 2, characterized in that, In step 5, the molar ratio of MQA to compound 3 is 1:

1.

6. The method for preparing the fluorescent probe MQA-DNP as described in claim 2, characterized in that, In step 5, the volume ratio of the dichloromethane / methanol mixed solvent is in the range of 50:1-20:

1.

7. Application of the fluorescent probe MQA-DNP as described in claim 1 for non-therapeutic and non-diagnostic purposes, targeting mitochondria to simultaneously detect hydrogen sulfide, viscosity, and polarity.

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