Construction of an indole derivative fluorescent probe for in vivo detection of viscosity and sulfur dioxide

Abstract

The application provides a kind of construction of indole derivative fluorescent probe for detecting viscosity and sulfur dioxide in vivo, and belongs to the field of biological detection.The fluorescent probe (denoted as BIC) of the application utilizes the strong nucleophilicity of sulfur dioxide, and realizes the specificity selection of sulfur dioxide by generating new product BIC-SO2 through Michael addition reaction with double bond in the structure of probe; in addition, benzene ring and indole in BIC structure can freely rotate in low viscosity environment, resulting in non-radiation consumption of excited state energy, showing very weak fluorescence; in high viscosity solution, the free rotation of probe is inhibited, resulting in the recovery and enhancement of probe fluorescence, showing very strong fluorescence. Therefore, the fluorescent probe of the application can simultaneously detect viscosity and sulfur dioxide in vivo.

Description

Technical Field

[0001] This invention relates to the field of biological detection, specifically to the construction of an indole derivative fluorescent probe for in vivo detection of viscosity and sulfur dioxide. Background Technology

[0002] Sulfur dioxide (SO2) is one of the four important endogenous gaseous signaling molecules. At physiological concentrations, SO2 has significant regulatory effects on the human body, including vasodilation, anti-inflammation, and blood pressure regulation. However, there is often a delicate balance between maintaining life activities and posing health risks. Excessive SO2 intake can cause significant harm to human health, impairing the normal function of the respiratory, cardiovascular, and nervous systems, and may lead to allergies, asthma, and even lung cancer.

[0003] In biological systems, viscosity is an important microenvironmental parameter within cells. It regulates core cellular physiological processes by influencing interactions between biomolecules, the transport of chemical signals, and the diffusion of bioactive metabolites within living cells, playing a crucial role, especially in the degeneration of subcellular organelles. Abnormal fluctuations in cellular viscosity levels are associated with a variety of diseases, such as malignant tumors, Alzheimer's disease, prions, and Parkinson's disease.

[0004] In recent years, fluorescent probes have been used for the detection of SO2 and viscosity due to their advantages such as high sensitivity, good selectivity, rapid response, non-invasiveness and real-time monitoring capabilities. However, the fluorescent probes currently developed can only achieve the detection of a single target. There are still very few probes that can simultaneously detect SO2 and viscosity, and the recognition wavelength ranges all show a high degree of overlap, which affects the effect of simultaneous detection. Summary of the Invention

[0005] This invention provides the construction of an indole derivative fluorescent probe for in vivo detection of viscosity and sulfur dioxide. The indole derivative fluorescent probe of this invention has strong anti-interference ability and can detect sulfur dioxide and viscosity at two different excitation wavelengths.

[0006] This invention provides an indole derivative fluorescent probe having the structural formula shown in Formula I: Formula I; X includes I or Br.

[0007] This invention also provides a method for preparing the indole derivative fluorescent probe described in the above technical solution, characterized by comprising the following steps: A condensation reaction was carried out by mixing 1,8-naphthoimide, iodoethane, a strong base, and a first polar organic solvent to give compound 2; Compound 2, Grignard reagent, and second polar organic solvent are mixed to carry out Grignard reaction. Then, the resulting Grignard reaction system is mixed with acid to remove the second polar organic solvent from the mixture. The mixture is then mixed with iodine salt solution or bromine salt solution to carry out salt formation reaction to obtain compound 3. The indole derivative fluorescent probe was obtained by mixing the compound 3, trans-3,5-dimethoxy-4-hydroxycinnamaldehyde, a catalyst and an alcohol solvent and carrying out a substitution reaction. Compound 2 has the structural formula shown in Formula II; Compound 3 has the structural formula shown in Formula III; Formula II; Formula III.

[0008] Preferably, the molar ratio of 1,8-naphthoimide to iodoethane is 3:10.

[0009] Preferably, the molar ratio of the 1,8-naphthoimide to the strong base is 3:10, and the strong base includes KOH.

[0010] Preferably, the condensation reaction is carried out at a temperature of 85-90°C for 10-12 hours.

[0011] Preferably, the molar ratio of compound 2 to Grignard reagent is 1:1.2~1.5, and the Grignard reagent includes methyl magnesium chloride.

[0012] Preferably, the reaction temperature is 55~60℃ and the time is 1~3h.

[0013] Preferably, the molar ratio of compound 3 to trans-3,5-dimethoxy-4-hydroxycinnamaldehyde is 1:1.5.

[0014] Preferably, the substitution reaction is carried out at a temperature of 80-85°C for 6-12 hours.

[0015] The present invention also provides the application of the indole derivative fluorescent probe described in the above technical solution or the indole derivative fluorescent probe prepared by the preparation method described in the above technical solution in the detection of sulfur dioxide and viscosity for non-diagnostic or therapeutic purposes.

[0016] The fluorescent probe of this invention (denoted as BIC) utilizes the strong nucleophilicity of sulfur dioxide to achieve specific selection of sulfur dioxide by undergoing a Michael addition reaction with the double bond in the probe structure to generate a new product, BIC-SO2. Furthermore, the benzene ring and indole in the BIC structure can rotate freely in low-viscosity environments, leading to non-radiative energy consumption of the excited state and exhibiting very weak fluorescence. In high-viscosity solutions, the free rotation of the probe is suppressed, resulting in the recovery and enhancement of the probe fluorescence, exhibiting very strong fluorescence. Therefore, the fluorescent probe of this invention can simultaneously detect in vivo viscosity and sulfur dioxide.

[0017] The fluorescent probe of this invention can respond to sulfur dioxide and viscosity separately under two different excitation wavelengths. The recognition processes for these two substances are independent and do not interfere with each other, enabling independent and simultaneous detection of viscosity and sulfur dioxide. Under excitation at 420 nm, the probe BIC can rapidly and accurately identify sulfur dioxide, exhibiting advantages such as short response time, high selectivity, low detection limit (39 nM), strong anti-interference ability, and wide pH applicability. Simultaneously, the probe can respond to viscosity under excitation at 600 nm. As the solution viscosity increases, the fluorescence emission intensity of the probe BIC at 720 nm continuously increases, demonstrating its ability to quantitatively detect viscosity. The fluorescence emission peaks are at 720 nm and 490 nm, with a significant emission difference of 230 nm between the two peaks. This is highly advantageous for imaging the viscosity and SO2 derivatives of biological systems in different emission channels. The probe can also respond rapidly and sensitively to changes in environmental viscosity, unaffected by environmental polarity.

[0018] Compared to previously published fluorescent probes, the fluorescent probe of this invention has the advantages of strong tissue penetration, low light loss, and the ability to overcome background fluorescence interference. The fluorescent probe of this invention also exhibits good biocompatibility, can specifically locate within the subcellular organelle mitochondria, and has the ability to image sulfur dioxide and viscosity in mitochondria. This allows for more precise applications in biological systems, providing a powerful tool for studying cell physiology and pathology, and possesses significant value for environmental monitoring and biomedical research. Attached Figure Description

[0019] Figure 1 The hydrogen NMR spectrum of the fluorescent probe BIC (solvent is CDCl3); Figure 2 The carbon NMR spectrum of the fluorescent probe BIC (solvent is CDCl3); Figure 3 High-resolution mass spectrum of the fluorescent probe BIC (positive ion mode, solvent CH3CN); Figure 4The fluorescence selectivity curve for the fluorescent probe BIC to recognize sulfur dioxide is shown. The excitation wavelength is 420 nm and the emission wavelength is 490 nm. Figure 5 Fluorescence anti-interference diagram for the fluorescent probe BIC to recognize sulfur dioxide, with excitation wavelength of 420 nm and emission wavelength of 490 nm; Figure 6 A fluorescence titration diagram for the BIC fluorescent probe to recognize sulfur dioxide was obtained, with an excitation wavelength of 420 nm and an emission wavelength of 490 nm. Figure 7 The limit of detection for the fluorescent probe BIC to recognize sulfur dioxide is plotted with an excitation wavelength of 420 nm and an emission wavelength of 490 nm. Figure 8 The fluorescence kinetics of the fluorescent probe BIC for recognizing sulfur dioxide is shown, with an excitation wavelength of 420 nm and an emission wavelength of 490 nm. Figure 9 The pH range for the fluorescent probe BIC to recognize sulfur dioxide is shown in the diagram, with an excitation wavelength of 420 nm and an emission wavelength of 490 nm. Figure 10 The fluorescence spectra of the fluorescent probe BIC in different polar solvents are shown. The excitation wavelength is 600 nm and the emission wavelength is 720 nm. Figure 11 The fluorescence spectrum of the fluorescent probe BIC in solutions of different viscosities is shown. The excitation wavelength is 600 nm and the emission wavelength is 720 nm. Figure 12 The graph shows the linear relationship between different viscosities of the fluorescent probe BIC solution and the strongest absorption, with an excitation wavelength of 600 nm and an emission wavelength of 720 nm. Figure 13 The survival rate of L-02 and HepG-2 cells after incubation with different concentrations of the fluorescent probe BIC; Figure 14 This is an image showing the co-localization of the fluorescent probe BIC with a commercially available green mitochondrial dye in living cells. Figure 15 This is a fluorescence imaging image of the fluorescent probe BIC in the detection of exogenous and endogenous sulfur dioxide in living cells. Figure 16 This is a fluorescence imaging image of the fluorescent probe BIC detecting viscosity changes in a biological microenvironment; Figure 17 This is a fluorescence imaging image of the fluorescent probe BIC in different animal models to detect changes in sulfur dioxide and viscosity. Detailed Implementation

[0020] This invention provides an indole derivative fluorescent probe having the structural formula shown in Formula I: Formula I; X includes I or Br.

[0021] This invention also provides a method for preparing the indole derivative fluorescent probe described in the above technical solution, comprising the following steps: A condensation reaction was carried out by mixing 1,8-naphthoimide, iodoethane, a strong base, and a first polar organic solvent to give compound 2; Compound 2, Grignard reagent, and second polar organic solvent are mixed to carry out Grignard reaction. Then, the resulting Grignard reaction system is mixed with acid to remove the second polar organic solvent from the mixture. The mixture is then mixed with iodine salt solution or bromine salt solution to carry out salt formation reaction to obtain compound 3. The indole derivative fluorescent probe was obtained by mixing the compound 3, trans-3,5-dimethoxy-4-hydroxycinnamaldehyde, a catalyst and an alcohol solvent and carrying out a substitution reaction. Compound 2 has the structural formula shown in Formula II; Compound 3 has the structural formula shown in Formula III; Formula II; Formula III.

[0022] In this invention, 1,8-naphthoimide, iodoethane, a strong base, and a first polar organic solvent are mixed and subjected to a condensation reaction to obtain compound 2.

[0023] In this invention, the mixing preferably includes a first mixing of 1,8-naphthoimide, a strong base and a first polar organic solvent, followed by a second mixing of the resulting mixture with iodoethane.

[0024] In this invention, the molar ratio of the 1,8-naphthoimide to iodoethane or bromoethane is preferably 3:10.

[0025] In this invention, the molar ratio of the 1,8-naphthoimide to the strong base is preferably 3:10, and the strong base preferably includes KOH.

[0026] In this invention, the preferred ratio of 1,8-naphthoimide to the first polar organic solvent is 50 mg: 1 mL; the first polar organic solvent preferably includes DMF.

[0027] In this invention, the condensation reaction is preferably carried out at a temperature of 85-90°C and for a time of 10-12 hours.

[0028] After the condensation reaction, the present invention preferably cools the system obtained by the condensation reaction to room temperature, mixes it with ice water, and filters it to obtain compound 2.

[0029] After obtaining compound 2, the present invention will mix compound 2, Grignard reagent, and second polar organic solvent to carry out Grignard reaction, then mix the obtained Grignard reaction system with acid to remove the second polar organic solvent from the obtained mixture, and then mix it with iodine salt solution or bromine salt solution to carry out salt formation reaction to obtain compound 3.

[0030] In this invention, the molar ratio of compound 2 to Grignard reagent is preferably 1:1.2 to 1.5, and in specific embodiments of this invention it can be 1:1.3 or 1:1.4; the Grignard reagent preferably includes methyl magnesium chloride; the Grignard reagent is preferably used in the form of a Grignard reagent solution; the concentration of the Grignard reagent solution is preferably 3 mol / L, and the solvent preferably includes THF.

[0031] In this invention, the preferred ratio of compound 2 to acid is 2.5 mmol: 20 mL, and the acid preferably includes hydrochloric acid with a concentration of 2 M.

[0032] The preferred temperature for the reaction is 55-60°C, and the preferred time is 1-3 hours.

[0033] In this invention, the temperature at which the obtained reaction system is mixed with acid is preferably room temperature.

[0034] In this invention, the preferred ratio of compound 2 to iodine salt solution or bromine salt solution is 2.5 mmol: 10 mL, and the iodine salt solution preferably includes saturated potassium iodide solution.

[0035] In this invention, the preferred temperature for the salt formation reaction is 60°C, and the preferred time is 30 min.

[0036] After the salt-forming reaction, the present invention preferably further includes: performing solid-liquid separation on the system obtained from the salt-forming reaction, and then drying the obtained solid to obtain compound 3.

[0037] After obtaining compound 3, the present invention mixes compound 3, trans-3,5-dimethoxy-4-hydroxycinnamaldehyde, catalyst and alcohol solvent to carry out a substitution reaction to obtain the indole derivative fluorescent probe.

[0038] In this invention, the molar ratio of compound 3 to trans-3,5-dimethoxy-4-hydroxycinnamaldehyde is preferably 1:1.5.

[0039] In this invention, the catalyst preferably comprises piperidine.

[0040] In this invention, the preferred ratio of compound 3 to alcohol solvent is 1 mmol: 20 mL; the alcohol solvent preferably includes ethanol.

[0041] The present invention does not impose any special limitation on the amount of the catalyst used; it can be adjusted according to the actual situation.

[0042] In this invention, the temperature of the substitution reaction is preferably 80~85℃, and the time is preferably 6~12h. In specific embodiments of this invention, it can be 8h, 9h, 10h or 11h.

[0043] After the substitution reaction, the present invention preferably performs rotary evaporation on the obtained substitution reaction system to obtain a residue; the residue is dissolved in dichloromethane and then mixed with brine to obtain an organic phase; the organic phase is mixed with a desiccant and dried, vacuum distilled and silica gel chromatography is performed.

[0044] The present invention also provides the application of the indole derivative fluorescent probe described in the above technical solution or the indole derivative fluorescent probe prepared by the preparation method described in the above technical solution in the detection of sulfur dioxide and viscosity for non-diagnostic or therapeutic purposes.

[0045] In this invention, the sulfur dioxide is preferably sulfur dioxide present in vivo. Since sulfur dioxide in vivo exists as bisulfite, when detecting sulfur dioxide in vivo, what is actually being detected is bisulfite.

[0046] In a specific embodiment of the present invention, the application is in the preparation of a detection device that can identify both sulfur dioxide and viscosity.

[0047] In a specific embodiment of the present invention, the application can be the preparation of a detection device for dual recognition of exogenous SO2 and viscosity in live cell imaging, wherein the live cells are preferably one or more of HepG-2, A549 and 4T1 cells.

[0048] In a specific embodiment of the present invention, the application can be the preparation of a device co-localized with mitochondria, wherein the mitochondria are preferably derived from HepG-2 cells.

[0049] In a specific embodiment of the present invention, the application can be in the preparation of a device for detecting the viscosity of SO2 derivatives and bleomycin-induced pulmonary fibrosis models in vivo in an acute liver injury model.

[0050] The following detailed description, in conjunction with embodiments, illustrates the construction of the indole derivative fluorescent probes for in vivo detection of viscosity and sulfur dioxide provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0051] Example 1 Synthesis of Intermediate 2: Compound 1 (500 mg, 3 mmol) and potassium hydroxide (560 mg, 10 mmol) were added to a round-bottom flask containing 10 mL of anhydrous DMF. The mixture was stirred thoroughly at 30 °C for 10 min. Then, iodoethane (750 μL, 10 mmol) was added to the reaction mixture. The temperature was then raised to 90 °C and the reaction was allowed to proceed for 10 h. After cooling to room temperature, the solution was poured into ice water, filtered, and the product was collected and dried. The purity of the product was sufficient to proceed directly to the next reaction.

[0052] Synthesis of Intermediate 3: Intermediate 2 (500 mg, 2.5 mmol) was dissolved in anhydrous THF (20 mL) with methyl magnesium chloride solution (3.0 M inTHF, 1 mL, 3 mmol). The reaction was carried out under a nitrogen atmosphere and heated to 60 °C for 1 h. After the reaction was complete, the mixture was cooled to room temperature, and 2 M hydrochloric acid (20 mL) was added. The THF was removed by rotary evaporation. A saturated potassium iodide solution (10 mL) was then added to the residue and mixed thoroughly. After standing for 30 min, the precipitate was filtered to obtain the product. After vacuum drying, intermediate 3 yielded an orange-red solid without further purification, with a yield of 76%.

[0053] Synthesis of probe BIC: Intermediate 3 (323 mg, 1 mmol) and trans-3,5-dimethoxy-4-hydroxycinnamaldehyde (312 mg, 1.5 mmol) were dissolved in ethanol (20 mL), and 3 drops of piperidine were added as a catalyst. The mixture was refluxed at 80 °C for 6 h. After the reaction was complete, the solvent was removed by rotary evaporation, and the residue was dissolved in dichloromethane (30 mL). The residue was washed with brine, and the resulting organic phase was dried over anhydrous sodium sulfate. Then, dichloromethane was removed by vacuum distillation. The crude product was purified by silica gel chromatography (CH2Cl2 / MeOH = 100 / 1, v / v) to give a dark blue probe BIC in 80% yield.

[0054] Figure 1 The image shows the hydrogen NMR spectrum of the fluorescent probe BIC (solvent: CDCl3).

[0055] Figure 2 The carbon NMR spectrum of the fluorescent probe BIC (solvent is CDCl3); Figure 3 This is a high-resolution mass spectrum of the fluorescent probe BIC (positive ion mode, solvent CH3CN).

[0056] 1 H NMR (500 MHz, CDCl3) δ 7.98 (d, J = 7.3 Hz, 1H), 7.78 (d, J= 8.1 Hz,1H), 7.73 - 7.70 (m, 1H), 7.63 (t, J = 7.7 Hz, 1H), 7.42 (t, J = 7.7 Hz, 1H), 7.30 (d, J = 8.2 Hz, 1H), 7.01 - 6.99 (m, 2H), 6.71 - 6.64 (mz, 3H), 6.23 (d, J =12.6 Hz, 1H), 5.32 (s, 1H), 4.00 (q, J = 7.2 Hz, 2H), 3.88 (s, 6H), 1.40 (t, J =7.1 Hz, 3H). 13 C NMR (100 MHz, CDCl3) δ 173.74, 152.35, 144.84, 142.47, 138.15,131.58, 130.94, 129.07, 128.75, 126.87, 126.76, 126.63, 125.08, 122.34,117.24, 105.87, 101.87, 55.62, 29.72, 13.26. HRMS-ESI: m / z calcd for C 25 H 24 NO3I [M - I] + 386.1751, found 386.1745. Preparation of 1 mM probe solution: Accurately weigh the probe (BIC) prepared in Example 1, dissolve the BIC in dimethyl sulfoxide (DMSO) solution to prepare a 1 mM solution for the test solutions used in the following experiments.

[0057] Fluorescence selectivity experiment For a fluorescent probe to achieve single recognition of the target species, it is necessary to test the fluorescence selectivity of the probe BIC for different active small molecules. The test solution for the BIC fluorescence selectivity experiment was PBS-CH3CN (PBS concentration was 10 mM, pH=7.40, and the volume ratio of PBS to CH3CN was 9 / 1), and the concentration of BIC in the test solution was 10 μM.

[0058] Figure 4 The fluorescence selectivity curve for sulfur dioxide recognition by the fluorescent probe BIC is shown. The excitation wavelength is 420 nm and the emission wavelength is 490 nm. Under 420 nm excitation, the probe alone showed almost no fluorescence emission at 490 nm. However, the fluorescence intensity at 490 nm was significantly enhanced upon the addition of Na₂HSO₃ (10 eq.). Furthermore, the test solution contained other active species (10 eq.): Br - Cl - ,ClO - ClO4 - F - H2PO4 - HPO3 2- HSO4 - I - OAc - PO4 3- PPi, SO3 2- SO4 2- S2O5 2- S2O3 2- S2O4 2- S 2- Ag + Cd 3+ Al 3+ Ca 2+ Co 3+ Cr 3+ Cu 2+ Fe 3+ Hg 2+ K + Mg 2+ Mn 2+ Na + Ni 2+ Pb 2+ Zn 2+ ,Cys,GSH,Hcy,H2O2,NO·,ROO - After excitation with Arg, Gly, and Glu, no significant enhancement was observed. The above selectivity experimental results show that the probe BIC exhibits good selectivity for sulfur dioxide under 420 nm excitation.

[0059] Fluorescence interference experiment To investigate the anti-interference ability of the probe BIC in response to sulfur dioxide in complex environments, other active small molecules were tested using fluorescence emission spectroscopy. The test solution for the BIC fluorescence interference experiment was PBS-CH3CN (PBS concentration was 10 mM, pH=7.40, volume ratio of PBS to CH3CN was 9 / 1), and the concentration of BIC in the test solution was 10 μM.

[0060] Under 420 nm excitation, 10 equivalents of Br were added to the individual probe solutions. - Cl -,ClO - ClO4 - F - H2PO4 - HPO3 2- HSO4 - I - OAc - PO4 3- PPi, SO3 2- SO4 2- S2O5 2- S2O3 2- S2O4 2- S 2- Ag + Cd 3+ Al 3+ Ca 2+ Co 3+ Cr 3+ Cu 2+ Fe 3+ Hg 2+ K + Mg 2+ Mn 2+ Na + Ni 2+ Pb 2+ Zn 2+ ,Cys,GSH,Hcy,H2O2,NO·,ROO - Arg, Gly, and Glu were used to detect the fluorescence emission intensity of the solution at 490 nm.

[0061] Figure 5 Fluorescence immunity of the fluorescent probe BIC for recognizing sulfur dioxide.

[0062] Depend on Figure 5 It can be seen that the fluorescence emission intensity (F490) at 490 nm of the solution with different active small molecules as interfering species is basically the same as that of the solution with Na2HSO3 added alone. This indicates that the probe BIC has a strong anti-interference ability against other active small molecules when detecting sulfur dioxide.

[0063] Limit of detection test Fluorescence emission spectroscopy was used to determine the limit of detection (LOD) of the probe BIC for sulfur dioxide. The test solution for the LOD experiment was PBS-CH3CN (PBS concentration was 10 mM, pH=7.40, and the volume ratio of PBS to CH3CN was 9 / 1).

[0064] Under 420 nm excitation, the probe BIC concentration was fixed at 10 μM. The Na₂HSO₃ concentration in the solution was adjusted (from 0 μM to 20 μM), and the fluorescence emission intensity (F490) at 490 nm was measured for probe solutions containing different concentrations of Na₂HSO₃. Figures 6-7 As shown.

[0065] Figure 6 The fluorescence titration pattern for the BIC fluorescent probe to recognize sulfur dioxide was obtained with an excitation wavelength of 420 nm and an emission wavelength of 490 nm.

[0066] Figure 7 The detection limit diagram for the fluorescent probe BIC to recognize sulfur dioxide is shown, with an excitation wavelength of 420 nm and an emission wavelength of 490 nm.

[0067] Depend on Figures 6-7 It was found that the fluorescence intensity at 490 nm in the solution showed a good linear relationship with the concentration of Na2HSO3 in the range of 0–20 μM (R² = 0.999). According to the IUPAC rules, the detection limit of probe BIC for sulfur dioxide was calculated to be 39 nM using the limit of detection formula (3σ / k). The results of the limit of detection experiment indicate that probe BIC has high sensitivity to sulfur dioxide and can achieve quantitative detection of trace concentrations of sulfur dioxide.

[0068] Table 1. Response time, detection limit, and detection targets for different fluorescent probes.

[0069] Fluorescence kinetics experiment Fluorescence emission spectroscopy was used to test the fluorescence kinetics of the probe BIC for sulfur dioxide. The limit of detection (LOD) assay solution for the probe BIC was PBS-CH3CN (PBS concentration 10 mM, pH 7.40, volume ratio of PBS to CH3CN 9 / 1). Under 420 nm excitation, with a fixed probe BIC concentration of 10 μM, the change in fluorescence emission intensity (F490) at 490 nm over time was measured for solutions containing and without Na2HSO3 (100 μM). Figure 8 As shown.

[0070] Figure 8 The fluorescence kinetics of the fluorescent probe BIC for recognizing sulfur dioxide is shown. The excitation wavelength is 420 nm and the emission wavelength is 490 nm.

[0071] Depend on Figure 8It was found that the fluorescence emission intensity at 490 nm of the probe alone remained essentially constant over time, while the fluorescence emission intensity at 490 nm of the solution containing Na2HSO3 (100 μM) increased rapidly over time, reaching a plateau after 30 seconds. The fluorescence kinetics results indicate that the probe BIC responds rapidly to sulfur dioxide, enabling rapid detection.

[0072] The effect of pH on the detection ability of the probe for sulfur dioxide Fluorescence emission spectroscopy was used to investigate the recognition ability of the probe BIC for sulfur dioxide in different pH environments. The pH test solution for the probe BIC was PBS-CH3CN with a pH range of 1 to 14 (PBS concentration of 10 mM, pH=7.40, volume ratio of PBS to CH3CN of 9 / 1), and the concentrations of probe and Na2HSO3 were 10 μM and 100 μM, respectively.

[0073] Figure 9 The pH range for the fluorescent probe BIC to recognize sulfur dioxide is shown in the diagram. The excitation wavelength is 420 nm and the emission wavelength is 490 nm.

[0074] Depend on Figure 9 It was found that within the pH range of 4–9, the fluorescence emission intensity at 490 nm of the probe alone did not change significantly; however, when Na₂HSO₃ was present, the fluorescence emission intensity at 490 nm of the test solution was significantly enhanced within the pH range of 7–8. The pH test results indicate that the probe BIC can recognize sulfur dioxide under physiological conditions.

[0075] Interference experiment of solutions with different polarities To investigate the effect of different polarities on the viscosity recognition of the probe BIC, fluorescence emission spectroscopy was used to test solutions of different polarities. Thirteen different solutions, including glycerol, methanol, ethanol, acetonitrile, DCM, DMSO, DMF, THF, 1,4-dioxane, toluene, ethyl acetate, chloroform, and water, were selected, and the probe BIC was added at a concentration of 10 μM. The fluorescence emission at 720 nm was then detected.

[0076] Figure 10 The fluorescence spectra of the fluorescent probe BIC in different polar solvents are shown. The excitation wavelength is 600 nm and the emission wavelength is 720 nm.

[0077] like Figure 10 It was found that only the glycerol solution produced strong fluorescence emission at 720 nm, while other solutions of different polarities did not show significant fluorescence changes. The polarity interference experiment results indicate that the probe BIC is only sensitive to viscosity and is not affected by solution polarity.

[0078] Fluorescence experiments with different viscosities Since the probe BIC molecule exhibits the highest fluorescence intensity in glycerol, its viscosity response was investigated. The test solution for the probe BIC viscosity response experiment (with a BIC concentration of 10 μM) was a water-glycerol system, and the glycerol content of the solution ranged from 0% to 99%. The viscosity was measured using a rotational viscometer.

[0079] Figure 11 The fluorescence spectrum of the fluorescent probe BIC in solutions of different viscosities is shown. The excitation wavelength is 600 nm and the emission wavelength is 720 nm.

[0080] Figure 12 The graph shows the linear relationship between different viscosities of the fluorescent probe BIC and the strongest absorption, with an excitation wavelength of 600 nm and an emission wavelength of 720 nm.

[0081] Depend on Figures 11-12 It can be seen that as the glycerol content of the system increases from 0% to 99%, the fluorescence intensity at 720 nm (F720) of the solution significantly increases. According to the Forster-Hoffmann equation (log(I)=C+x logη), there is a good linear relationship between logη and F720 (R2=0.994). The probe viscosity response test results show that the probe BIC can achieve quantitative detection of viscosity.

[0082] Cytotoxicity assay of probe BIC L-02 and HepG-2 cells were spaced at 5 × 10⁶ cells per well. 3 Cells were cultured at a density of 1000 cells / well in 96-well plates and incubated overnight at 37°C with 5% CO2 to allow complete cell adhesion. Gradual concentrations of BIC (0, 10, 20, 30, 40, 50 μM) were added to the cells, followed by incubation at 37°C with 5% CO2 for 24 h. Then, 20 μL of MTT (formazan) solution (5 mg / mL PBS solution) was added to each well, and after incubation for 4 h, the culture medium was aspirated. 150 μL of DMSO was added to each well to dissolve the formazan. The wells were then placed in a microplate reader, and the absorbance at 570 nm was measured by shaking at 37°C for 5 min. Cell viability was then calculated.

[0083] Figure 13 The survival rate of L-02 and HepG-2 cells after incubation with different concentrations of the fluorescent probe BIC was determined.

[0084] Depend on Figure 13 It can be seen that the probe BIC did not exhibit significant cytotoxicity at the tested concentration.

[0085] Colocalization imaging experiment of probe BIC and subcellular organelle mitochondria The probe BIC was applied to human liver cancer cells (HepG-2), and co-localization imaging was performed using commercially available green mitochondrial dye. The specific steps are as follows: 1) Spread HepG-2 cells at a rate of 1 × 10⁶ cells per dish 5 Cells were cultured at a density of 10 μM in 35 mm confocal culture dishes containing 1.2 mL of complete culture medium and incubated overnight at 37 °C and 5% CO2 to allow complete cell adhesion. Cells were then incubated for 30 min in medium containing 10 μM commercial green mitochondrial dye (purchased from Beyotime) (89% high glucose DMEM (purchased from Seville) + 10% serum (purchased from Sijiqing) + 1% penicillin antibody (purchased from Hyclone)). Cells were then washed once with PBS. Next, cells were incubated for 30 min in medium containing 10 μM probe BIC (89% high glucose DMEM + 10% serum + 1% penicillin antibody), and washed once with PBS. Finally, cells were co-incubated with DAPI nuclear dye for 15 min, and after washing with PBS, fresh PBS was added to each culture dish.

[0086] 2) Observe the cells under an inverted fluorescence microscope.

[0087] Figure 14 This is an image showing the co-localization of the fluorescent probe BIC with a commercially available green mitochondrial dye in living cells.

[0088] from Figure 14 As can be seen, the white light channel shows good cell morphology, while the green and red light channels both emit strong fluorescence and overlap well. The Pearson coefficient is 0.84, indicating that the probe BIC can specifically locate in the subcellular organelle mitochondria and has the ability to image sulfur dioxide and viscosity in mitochondria.

[0089] Imaging experiment of BIC probe for detecting exogenous and endogenous SO2 in living cells Studies have shown that GSH and Na2S2O3 can generate HSO3 intracellularly. - Therefore, it was used as the detection group for endogenous imaging. Experiments were conducted using the probe BIC for both exogenous and endogenous SO2 imaging, and the results are as follows... Figure 15 As shown ( Figure 15 (The image shows a fluorescence image of the fluorescent probe BIC detecting exogenous and endogenous sulfur dioxide in living cells.) The specific experimental steps are as follows: HepG-2 cells were stored at 1 × 10⁶ cells per dish. 5 Four groups of cells were cultured at a density of 1.2 mL of complete culture medium in 35 mm confocal culture dishes and incubated overnight at 37°C and 5% CO2 to allow the cells to adhere completely. 1) Control group: Add culture medium containing 10 μM probe BIC and incubate with cells for 30 min. Under a microscope, strong red fluorescence was observed and no fluorescence was observed in the green channel.

[0090] 2) HSO3 - Experimental group: Cells were first co-incubated with medium containing 10 μM probe BIC (89% high glucose DMEM + 10% serum + 1% penicillin antibody) for 30 min, then washed once with PBS, and then medium containing 100 μM Na2HSO3 was added and cultured for another 30 min. The culture medium was aspirated, and the cells were washed once with PBS. Strong green fluorescence and very weak red fluorescence were observed under a microscope, indicating that probe BIC can perform cell imaging of exogenous SO2.

[0091] 3) GSH experimental group: Cells were first co-incubated with medium containing 10 μM probe BIC (89% high glucose DMEM + 10% serum + 1% penicillin antibody) for 30 min, then washed once with PBS, and then 500 μM GSH (which reduces or regulates HSO3 through direct chemical reactions, enzymatic metabolism, and maintaining redox balance) was added. - The cells were cultured in a medium containing 89% high glucose DMEM, 10% serum, and 1% penicillin and antibiotics for 30 minutes. The culture medium was then removed, and the cells were washed once with PBS. Red fluorescence was observed under a microscope, with no green fluorescence.

[0092] 4) GSH+Na2S2O3 experimental group: Cells were first co-incubated with medium containing 10 μM probe BIC (89% high glucose DMEM + 10% serum + 1% penicillin antibody) for 30 min, then washed once with PBS. Next, medium containing 500 μM probe GSH (89% high glucose DMEM + 10% serum + 1% penicillin antibody) was added, and the cells were cultured for another 30 min. Then, medium containing 250 μM Na2S2O3 (89% high glucose DMEM + 10% serum + 1% penicillin antibody) was added, and the cells were co-incubated for another 30 min. The culture medium was aspirated, and the cells were washed once with PBS. Strong green fluorescence was observed under a microscope, with no obvious red fluorescence. This indicates that probe BIC can detect endogenously produced SO2 within cells.

[0093] Imaging experiment of BIC probe for detecting viscosity in living cells For cell adhesion imaging studies, this invention constructs three adhesion models: 1) A549 lung cell fibrosis model: Overactivation of intracellular TGF-β signaling can lead to severe cell fibrosis and increased intracellular viscosity; 2) Non-alcoholic hepatocyte model: High-lipid culture medium can induce lipotoxicity in cells, which further triggers TGF-β activation due to cellular inflammation, resulting in increased intracellular viscosity; 3) Cellular inflammation model: LPS and Nys induce the activation of the TLR4 / NF-κB pathway in 4T1 cells, leading to the accumulation of protein aggregates and organelle debris, which induces cellular inflammation and increases intracellular viscosity.

[0094] Figure 16 This is a fluorescence imaging image of the fluorescent probe BIC detecting viscosity changes in a biological microenvironment.

[0095] The specific experimental steps are as follows: A549, HepG-2, and 4T1 cells were respectively 1×10⁶ per dish 5 Cells were cultured at a density of 1,000 cells in 35 mm confocal culture dishes containing 1.2 mL of complete culture medium and incubated overnight at 37°C and 5% CO2 to allow the cells to adhere completely.

[0096] 1) A549 lung cell fibrosis model: TGF-β was co-incubated with cells for 30 min, followed by washing with PBS once, and then medium containing 10 μM probe BIC was added for co-incubation with cells. Under a microscope, it was observed that the red fluorescence gradually increased over time. Figure 16 (a) Figure 16 In this context, DAPI stands for nuclear dye, merge is a fluorescence overlay image, and control refers to the absence of the probe BIC.

[0097] 2) Non-alcoholic hepatocyte model: Cells were cultured in high-fat medium for 48 h, washed once with PBS, and then co-incubated with HepG-2 cells in high-fat medium containing 10 μM probe BIC. A gradual increase in red fluorescence was observed over time. Figure 16 (b)

[0098] 3) Cellular inflammation model: LPS and Nys were co-incubated with cells in different dishes for 30 min, followed by washing with PBS once, and then culture medium containing 10 μM probe BIC was added for co-incubation with 4T1 cells. Compared with the control group, the experimental group showed strong red fluorescence ( Figure 16 c)).

[0099] Imaging experiment of detecting sulfur dioxide and viscosity in living organisms using probe BIC. To detect changes in sulfur dioxide and viscosity in vivo, CCl4-induced acute liver injury and bleomycin-induced pulmonary fibrosis models were constructed, respectively.

[0100] Figure 17 This is a fluorescence imaging image of the fluorescent probe BIC in different animal models to detect changes in sulfur dioxide and viscosity.

[0101] The specific steps are as follows: 1) The CCl4-induced acute liver injury model can induce HSO3 in liver tissue within a short period of time. - The content increased. After safe euthanasia via intraperitoneal administration, the liver was removed, sectioned, and imaged under a fluorescence microscope. The damaged group showed strong green fluorescence and relatively dim red fluorescence. Figure 17 (a) This indicates that the BIC probe can detect changes in SO2 in vivo.

[0102] 2) Bleomycin-induced pulmonary fibrosis model leads to increased cell tissue viscosity. After safe euthanasia via intraperitoneal administration, lung tissue was extracted, sectioned, and imaged under a fluorescence microscope after HE staining. It was found that the higher the degree of pulmonary fibrosis in mice over time, the stronger the red fluorescence. Figure 17 (b) This indicates that the BIC probe can detect annual changes in vivo.

[0103] The above experimental results show that the probe of this invention can simultaneously detect changes in sulfur dioxide and viscosity levels in the body, providing a new approach for the prevention and diagnosis of related diseases.

[0104] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An indole derivative fluorescent probe, characterized in that, It has the structural formula shown in Equation I: Equation I; X includes I or Br.

2. The method for preparing the indole derivative fluorescent probe according to claim 1, characterized in that, Includes the following steps: A condensation reaction was carried out by mixing 1,8-naphthoimide, iodoethane, a strong base, and a first polar organic solvent to give compound 2; Compound 2, Grignard reagent, and second polar organic solvent are mixed to carry out Grignard reaction. Then, the resulting Grignard reaction system is mixed with acid to remove the second polar organic solvent from the mixture. The mixture is then mixed with iodine salt solution or bromine salt solution to carry out salt formation reaction to obtain compound 3. The indole derivative fluorescent probe was obtained by mixing the compound 3, trans-3,5-dimethoxy-4-hydroxycinnamaldehyde, a catalyst and an alcohol solvent and carrying out a substitution reaction. Compound 2 has the structural formula shown in Formula II; Compound 3 has the structural formula shown in Formula III; Formula II; Formula III.

3. The preparation method according to claim 2, characterized in that, The molar ratio of 1,8-naphthoimide to iodoethane is 3:

10.

4. The preparation method according to claim 2 or 3, characterized in that, The molar ratio of the 1,8-naphthoimide to the strong base is 3:10, and the strong base includes KOH.

5. The preparation method according to claim 2, characterized in that, The condensation reaction is carried out at a temperature of 85-90°C for 10-12 hours.

6. The preparation method according to claim 2, characterized in that, The molar ratio of compound 2 to Grignard reagent is 1:1.2~1.5, and the Grignard reagent includes methyl magnesium chloride.

7. The preparation method according to claim 2, characterized in that, The reaction temperature is 55~60℃ and the time is 1~3h.

8. The preparation method according to claim 2, characterized in that, The molar ratio of compound 3 to trans-3,5-dimethoxy-4-hydroxycinnamaldehyde is 1:1.

5.

9. The preparation method according to claim 2, characterized in that, The substitution reaction is carried out at a temperature of 80-85°C for 6-12 hours.

10. The use of the indole derivative fluorescent probe of claim 1 or the indole derivative fluorescent probe prepared by any one of claims 2 to 9 in the detection of sulfur dioxide and viscosity for non-diagnostic or therapeutic purposes.

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