Preparation method and application of a fluorescent probe capable of simultaneously detecting polarity and peroxynitrite

Abstract

The application belongs to the technical field of fluorescent probe, and particularly relates to a preparation method and property research of a fluorescent probe for simultaneously detecting polarity and peroxynitrite double response. The preparation method of the fluorescent probe is simple, the product yield is high, and the method is suitable for scale popularization and use. The probe (E)-1-(2-carboxyethyl)-4-(2-(5-(4-(diphenylamino)phenyl)thiophene-2-yl)vinyl)quinolin-1-ium iodide (PBQI) is gradually enhanced in fluorescence intensity when being excited at 520 nanometers along with the decrease of solution polarity; when being excited at 400 nanometers in a low-polarity environment, the probe reacts with peroxynitrite to generate strong fluorescence. The fluorescence intensity is gradually enhanced along with the increase of peroxynitrite concentration. The probe can detect peroxynitrite in a low-polarity environment, and has a good linear ratio relationship. The probe also has a strong fluorescence response to polarity, and the fluorescence emission intensity is gradually increased along with the decrease of polarity.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probes, and in particular relates to a preparation method and application of a fluorescent probe capable of simultaneously detecting polarity and peroxynitrite dual responses. Background Art

[0002] Polarity is a crucial parameter for establishing and maintaining cellular homeostasis within the cellular microenvironment. Immune responses, cell activation, and differentiation can all lead to changes in cell polarity. Abnormal polarity is closely associated with cellular disorders and even disease. Abnormal polarity changes can cause many diseases, such as tumors, diabetes, Alzheimer's disease, and cancer, and have therefore garnered widespread attention.

[0003] Peroxynitrite anion (ONOO - ) is a highly reactive oxygen-nitrogen species (RONS) produced by the in situ reaction of free superoxide radicals with nitric oxide through diffusion and control, and plays a vital role in cellular redox homeostasis. Excessive peroxynitrite can affect the normal function of cells. Studies have shown that peroxynitrite is involved in intracellular redox balance, inflammation and immune response, and signal transduction pathways, thereby inducing cell death or necrosis. Abnormal changes in peroxynitrite levels are closely related to a variety of diseases, such as tumors, diabetes, rheumatism, cardiovascular and neurodegenerative diseases. Therefore, it is important to design and synthesize ratiometric fluorescent probes for the detection of peroxynitrite.

[0004] Fluorescence imaging has attracted widespread attention due to its advantages, including simple synthesis and operation, high sensitivity, noninvasiveness, and low toxicity. However, the fluorescence of probes is easily quenched by photobleaching. However, fluorescent probes with aggregation-induced emission properties can overcome this limitation and are suitable for non-destructive detection of microenvironmental changes. Despite the wide variety of fluorescent probes, some have shortcomings such as short fluorescence emission wavelengths that fail to reach the near-infrared region, poor tissue penetration resulting in incomplete fluorescence signal collection, and background interference. A growing number of studies have shown that changes in the physiological microenvironment, such as decreased polarity, have been considered effective tumor markers. Furthermore, recent studies have also demonstrated that a significant increase in peroxynitrite levels is observed during tumor progression, indicating its potential as a tumor marker. Therefore, the development of near-infrared fluorescent probes that can simultaneously detect polarity and peroxynitrite is highly desirable. Summary of the Invention

[0005] The present invention provides a method for preparing a dual-responsive fluorescent probe capable of simultaneously detecting polarity and peroxynitrite, and its application. (E)-1-(2-carboxyethyl)-4-(2-(5-(4-(diphenylamino)phenyl)thiophen-2-yl)vinyl)quinolin-1-ium iodide (PBQI) has the advantages of a simple synthetic route, good selectivity, high sensitivity, and a large Stokes shift. It can effectively detect polarity under physiological conditions and effectively respond to ONOO in low-polarity environments. - Concentration changes.

[0006] The fluorescent probe (E)-1-(2-carboxyethyl)-4-(2-(5-(4-(diphenylamino)phenyl)thiophen-2-yl)vinyl)quinolin-1-ium iodide (PBQI) of the present invention has the following molecular structure:

[0007]

[0008] The synthesis process of the fluorescent probe in the present invention is as follows:

[0009]

[0010] The preparation steps of the probe PBQI are as follows:

[0011] 3-Iodo-1-propionic acid and 4-methylquinoline were dissolved in toluene, ultrasonically dissolved, vacuumed, and heated for 12 hours. After the reaction was complete, the mixture was cooled to room temperature to form a precipitate. Filtered, washed with toluene, and dried in a vacuum oven to yield an off-white solid, 1-(2-carboxyethyl)-4-methylquinolin-1-ium iodide (A1, 89% yield).

[0012] 4-Bromotriphenylamine and 5-formyl-2-thiopheneboronic acid were ultrasonically dissolved in a tetrahydrofuran (THF) solution. Aqueous potassium carbonate solution and tetrakis(triphenylphosphine)palladium were added and ultrasonically dissolved. The mixture was evacuated, filled with nitrogen protection, and heated to reflux for about 18 hours. After completion of the reaction, the reaction solution was cooled to room temperature and extracted with dichloromethane and water. The organic phase was dried over anhydrous sodium sulfate (Na2SO4) and spin-dried to obtain a crude product. The crude product was separated and purified by column chromatography to obtain a yellow solid product, 5-(4-(diphenylamino)phenyl)thiophene-2-carboxaldehyde (A2, 32.5% yield).

[0013] A1 and A2 were added to a round-bottom flask filled with anhydrous ethanol and sonicated to dissolve. Two drops of piperidine solution were added, and the mixture was heated to reflux for 12 hours. Upon completion of the reaction, the reaction mixture was cooled to room temperature. A large amount of precipitate formed, which was filtered, washed with cold ethanol, and dried in a vacuum oven to yield (E)-1-(2-carboxyethyl)-4-(2-(5-(4-(diphenylamino)phenyl)thiophen-2-yl)vinyl)quinolin-1-ium iodide (PBQI, 40.8% yield) as a dark purple solid.

[0014] The detection mechanism of the fluorescent probe of the present invention is as follows:

[0015]

[0016] Molecules with a donor (D)-π-acceptor (A) structure, the PBQI probe, typically exhibit a significant solvatochromic effect, with its photophysical properties varying with solvent polarity. Furthermore, the strong oxidizing and nucleophilic nature of peroxynitrite facilitates probe response. Based on these capabilities, a new class of fluorescent probes for polarity and peroxynitrite detection was designed and synthesized to reflect polarity changes and detect peroxynitrite in different solutions.

[0017] Figure 3 The probe PBQI (1×10 -5 mol / L) in different solvents. The absorption spectra of the probe in different solvents show obvious differences.

[0018] Figure 4 The probe PBQI (1×10 -5 mol / L) in different solvents at an excitation wavelength of 360 nm.

[0019] Figure 5 The probe PBQI (1×10 -5 mol / L) in different solvents at an excitation wavelength of 520 nm.

[0020] Figure 6 The probe PBQI (1×10 -5 mol / L) in mixed solvents of 1,4-dioxane and PBS at different ratios.

[0021] Figure 7 The probe PBQI (1×10 -5 mol / L) in a mixed solvent of 1,4-dioxane and PBS at an excitation wavelength of 520 nm.

[0022] Figure 8 The probe PBQI (1×10 -5mol / L) in 80% 1,4-dioxane and 20% PBS system, with an excitation wavelength of 520 nm and an emission wavelength of 700 nm.

[0023] Figure 9 The probe PBQI (1×10 -5 mol / L) under different pH values ​​and polarity conditions, the fluorescence intensity spectrum of the probe at 700 nm emission wavelength. It can be seen that the fluorescence intensity of the probe gradually increases with the increase of pH value and the decrease of polarity.

[0024] Figure 10 The probe PBQI (1×10 -5 mol / L) in a solvent of 80% 1,4-dioxane and 20% PBS, a 0-90 μM peroxynitrite solution was added, and the UV-visible absorption spectrum of the probe PBQI was obtained.

[0025] Figure 11 The probe PBQI (1×10 -5 Fluorescence emission spectra of the probe at an excitation wavelength of 400 nm after adding a 0-90 μM peroxynitrite solution to an 80% 1,4-dioxane and 20% PBS solution (100 mol / L). It can be seen that the fluorescence intensity of the probe gradually increases with increasing peroxynitrite concentration.

[0026] Figure 12 The probe PBQI (1×10 -5 Fluorescence emission spectra of the probe at an excitation wavelength of 520 nm after adding a 0-90 μM peroxynitrite solution to an 80% 1,4-dioxane and 20% PBS solvent (Figure 2). It can be seen that the fluorescence intensity of the probe gradually decreases with increasing peroxynitrite concentration.

[0027] Figure 13 The probe PBQI (1×10 -5 mol / L) at an excitation wavelength of 400 nm and an emission wavelength of 520 nm, the linear graph of the fluorescence intensity change after the probe responds to peroxynitrite.

[0028] Figure 14 The probe PBQI (1×10 -5 mol / L) after adding different concentrations of peroxynitrite solution, the fluorescence intensity change at 520 nm emission wavelength within 0-60 minutes.

[0029] Figure 15 The probe PBQI (1×10 -5mol / L) in a system of 80% 1,4-dioxane and 20% PBS, and the effect of using PBS with different pH values ​​on the detection of peroxynitrite.

[0030] Figure 16 The probe PBQI (1×10 -5 mol / L) at different pH and polarity on the detection of peroxynitrite.

[0031] Figure 17 The probe PBQI (1×10 -5 mol / L) in a 99% 1,4-dioxane and 1% PBS system, investigating the interference resistance of small biomolecules to detection polarity. From left to right: 1, blank; 2, alanine; 3, phenylalanine; 4, isoleucine; 5, arginine; 6, threonine; 7, tryptophan; 8, valine; 9, glycine; 10, aspartic acid; 11, glutamine; 12, leucine; 13, cysteine; 14, lysine; 15, methionine; 16, proline; 17, ascorbic acid; 18, glutathione; 19, 99% 1,4-dioxane + 1% PBS. None of the interfering substances affected the fluorescence intensity of PBQI, demonstrating that the probe PBQI exhibits excellent interference resistance to small biomolecules.

[0032] Figure 18 The probe PBQI (1×10 -5 mol / L) in a system of 99% 1,4-dioxane and 1% PBS, the anti-interference of metal ions on the detection of polarity. From left to right are 1, blank; 2, Fe 2+ ; 3. Cr 3+ ; 4. Zn 2+ 5. Cd 2+ ; 6. Mn 2+ ; 7. Na + 8. Ag + ; 9, Ca 2+ ; 10, Fe 3+ ; 11, Mg 2+ ; 12, Ni 2+ ;13.Co 2+ ; 14, Sn 2+ ; 15, Cu 2+ ; 16, K + 17. 99% 1,4-dioxane + 1% PBS. None of the interfering substances affected the fluorescence intensity of PBQI, indicating that the probe PBQI has good anti-interference properties against metal ions.

[0033] Figure 19 The probe PBQI (1×10 -5mol / L) in a system of 99% 1,4-dioxane and 1% PBS, the anti-interference of anions on the detection of polarity. From left to right are 1, blank; 2, S 2- ; 3. HS - ; 4. SO4 2- 5. HSO3 - ; 6. S2O5 2- ; 7. SCN - ;8, Cl - ; 9, Br - ; 10, I - ; 11. CrO4 2- ;12, Cr2O4 2- ;13, Cr2O7 2- ; 14, BF4 - 15. CO3 2- ; 16, HCO3 - ; 17, NO2 - ; 18, NO3 - 19. H2PO4 - ; 20, PO4 3- 21. 99% 1,4-dioxane + 1% PBS. None of the interfering substances affected the fluorescence intensity of PBQI, indicating that the probe PBQI has good anti-interference properties for anions.

[0034] Figure 20 The probe PBQI (1×10 -5 mol / L) in an 80% 1,4-dioxane and 20% PBS system, investigating the interference resistance of anions to the detection of peroxynitrite. From left to right: 1, blank; 2, threonine; 3, leucine; 4, tryptophan; 5, glycine; 6, alanine; 7, aspartic acid; 8, glutathione; 9, cysteine; 10, methionine; 11, phenylalanine; 12, valine; 13, glutamine; 14, lysine; 15, arginine; 16, proline; 17, ascorbic acid. None of the interfering compounds affected the fluorescence intensity of the PBQI probe, demonstrating its excellent interference resistance to small biological molecules in the detection of peroxynitrite.

[0035] Figure 21 The probe PBQI (1×10 -5 mol / L) in the system of 80% 1,4-dioxane and 20% PBS, the anti-interference of metal ions on the detection of peroxynitrite. From left to right are 1, blank; 2, Sn 2+ ; 3. K + ; 4. Cu 2+ 5. Ag + ; 6. Ca 2+; 7. Fe 3+ ;8, Mg 2+ ; 9. Ni 2+ ; 10, Na + ; 11, Zn 2+ ; 12, Cr 3+ ; 13. Fe 2+ The interferences had no effect on the fluorescence intensity of PBQI, indicating that the probe PBQI has good anti-interference ability against metal ions in the detection of peroxynitrite.

[0036] Figure 22 The probe PBQI (1×10 -5 mol / L) in the system of 80% 1,4-dioxane and 20% PBS, the anti-interference of anions on the detection of peroxynitrite. From left to right are 1, blank; 2, PO4 3- 3. H2PO4 - 4. P2O7 4- 5. BF4 - ; 6. HS - ; 7. S2O5 2- ;8, S 2- ;9HSO3 - ; 10, SO4 2- ; 11. CrO4 2- ;12, Cr2O7 2- ;13, C2O4 2- ; 14, F - ; 15, Cl - ; 16, Br - ; 17, I - 18. CO3 2- ; 19, HCO3 - ; 20, NO2 - ; 21, NO3 - The interferences had no effect on the fluorescence intensity of PBQI, indicating that the probe PBQI has good anti-interference ability against anions in the detection of peroxynitrite.

[0037] Figure 23 The probe PBQI (1×10 -5 mol / L) in the system of 80% 1,4-dioxane and 20% PBS, the anti-interference of other active oxygen species on the detection of peroxynitrite. From left to right are H2O2, ClO - 、NO、 1 O2, ·OH. The interferences had no effect on the fluorescence intensity of PBQI, indicating that the probe PBQI has good anti-interference ability against other reactive oxygen species in the detection of peroxynitrite.

[0038] Figure 24 The prepared probe PBQI (1×10 -5 mol / L) in different solvents, including the maximum absorption peak wavelength λ abs,max , maximum emission peak wavelength λ em,max , Stokes shift and fluorescence quantum yield Φ (with rhodamine B as reference).

[0039] In summary, we obtained ONOO using a simple organic synthesis method. - The polarity-dual-responsive fluorescent probe (E)-1-(2-carboxyethyl)-4-(2-(5-(4-(diphenylamino)phenyl)thiophen-2-yl)vinyl)quinolin-1-ium iodide (PBQI) was developed. This probe exhibits significant solvatochromic changes in different solvents, which can be clearly observed with the naked eye. This indicates that the probe exhibits different optical properties in solvents of different polarities. Secondly, with the increase of 1,4-dioxane solvent, the fluorescence intensity of the probe gradually increases. This is due to the aggregation-induced phenomenon caused by the restricted intramolecular torsion caused by the aggregation of the probe in the solvent, which is also verified by the Tyndall effect. When the probe PBQI is added to a peroxynitrite solution in a low-polarity environment, the UV-visible absorption spectrum and fluorescence emission spectrum of the probe show obvious regular changes. This provides a good tool for the detection of peroxynitrite. The preparation of the probe PBQI fills the gap in the field of fluorescent probes that can simultaneously quantitatively detect polarity and peroxynitrite. The aggregation-inducing properties of the probe enable it to exhibit excellent optical properties and a large Stokes shift, which has good prospects for medical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a preparation and design route for the probe (E)-1-(2-carboxyethyl)-4-(2-(5-(4-(diphenylamino)phenyl)thiophen-2-yl)vinyl)quinolin-1-ium iodide (PBQI).

[0041] Figure 2 The probe PBQI detects ONOO - and the mechanism of response to polarity.

[0042] Figure 3 The probe PBQI (1×10 -5 mol / L) in different solvents.

[0043] Figure 4 The probe PBQI (1×10 -5 mol / L) in different solvents at an excitation wavelength of 360 nm.

[0044] Figure 5The probe PBQI (1×10 -5 mol / L) in different solvents at an excitation wavelength of 520 nm.

[0045] Figure 6 The probe PBQI (1×10 -5 mol / L) in mixed solvents of 1,4-dioxane and PBS at different ratios.

[0046] Figure 7 The probe PBQI (1×10 -5 mol / L) in a mixed solvent of 1,4-dioxane and PBS at an excitation wavelength of 520 nm.

[0047] Figure 8 The probe PBQI (1×10 -5 mol / L) in 80% 1,4-dioxane and 20% PBS system, with an excitation wavelength of 520 nm and an emission wavelength of 700 nm.

[0048] Figure 9 The probe PBQI (1×10 -5 mol / L) under different pH values ​​and polarity conditions, the fluorescence intensity spectrum of the probe at an emission wavelength of 700 nm.

[0049] Figure 10 The probe PBQI (1×10 -5 mol / L) in a solvent of 80% 1,4-dioxane and 20% PBS, a 0-90 μM peroxynitrite solution was added, and the UV-visible absorption spectrum of the probe PBQI was obtained.

[0050] Figure 11 The probe PBQI (1×10 -5 mol / L) in a solvent of 80% 1,4-dioxane and 20% PBS, with the addition of 0-90 μM peroxynitrite solution, and the fluorescence emission spectrum at an excitation wavelength of 400 nm.

[0051] Figure 12 The probe PBQI (1×10 -5 mol / L) in a solvent of 80% 1,4-dioxane and 20% PBS, with the addition of 0-90 μM peroxynitrite solution, and the fluorescence emission spectrum at an excitation wavelength of 520 nm.

[0052] Figure 13 The probe PBQI (1×10 -5mol / L) at an excitation wavelength of 400 nm and an emission wavelength of 520 nm, the linear relationship between the fluorescence intensity change of the probe and peroxynitrite.

[0053] Figure 14 The probe PBQI (1×10 -5 mol / L) after adding different concentrations of peroxynitrite solution, the fluorescence intensity change at 520 nm emission wavelength within 0-60 minutes.

[0054] Figure 15 The probe PBQI (1×10 -5 mol / L) in a system of 80% 1,4-dioxane and 20% PBS, and the effect of using PBS with different pH values ​​on the detection of peroxynitrite.

[0055] Figure 16 The probe PBQI (1×10 -5 mol / L) under different pH and polarity conditions, the fluorescence intensity change spectrum of the probe PBQI in response to peroxynitrite.

[0056] Figure 17 The probe PBQI (1×10 -5 mol / L) in a 99% 1,4-dioxane and 1% PBS system to investigate the interference effects of small biomolecules on detection polarity. From left to right: 1. Blank; 2. Alanine; 3. Phenylalanine; 4. Isoleucine; 5. Arginine; 6. Threonine; 7. Tryptophan; 8. Valine; 9. Glycine; 10. Aspartic acid; 11. Glutamine; 12. Leucine; 13. Cysteine; 14. Lysine; 15. Methionine; 16. Proline; 17. Ascorbic acid; 18. Glutathione; 19. 99% 1,4-dioxane + 1% PBS.

[0057] Figure 18 The probe PBQI (1×10 -5 mol / L) in a system of 99% 1,4-dioxane and 1% PBS, the anti-interference of metal ions on the detection of polarity. From left to right are 1, blank; 2, Fe 2+ ; 3. Cr 3+ ; 4. Zn 2+ 5. Cd 2+ ; 6. Mn 2+ ; 7. Na + 8. Ag + ; 9, Ca 2+ ; 10, Fe 3+ ; 11, Mg 2+ ; 12, Ni 2+ ;13.Co2+ ; 14, Sn 2+ ; 15, Cu 2 + ; 16, K + ; 17. 99% 1,4-dioxane+1% PBS.

[0058] Figure 19 The probe PBQI (1×10 -5 mol / L) in a system of 99% 1,4-dioxane and 1% PBS, the anti-interference of anions on the detection of polarity. From left to right are 1, blank; 2, S 2- ; 3. HS - ; 4. SO4 2- 5. HSO3 - ; 6. S2O5 2- ; 7. SCN - ;8, Cl - ; 9, Br - ; 10, I - ; 11. CrO4 2- ;12, Cr2O4 2- ;13, Cr2O7 2- ; 14, BF4 - 15. CO3 2- ; 16, HCO3 - ; 17, NO2 - ; 18, NO3 - 19. H2PO4 - ; 20, PO4 3- ;21. 99% 1,4-dioxane+1%PBS.

[0059] Figure 20 The probe PBQI (1×10 -5 mol / L) in an 80% 1,4-dioxane and 20% PBS system to investigate the interference of anions with the detection of peroxynitrite. From left to right: 1. Blank; 2. Threonine; 3. Leucine; 4. Tryptophan; 5. Glycine; 6. Alanine; 7. Aspartic acid; 8. Glutathione; 9. Cysteine; 10. Methionine; 11. Phenylalanine; 12. Valine; 13. Glutamine; 14. Lysine; 15. Arginine; 16. Proline; 17. Ascorbic acid.

[0060] Figure 21 The probe PBQI (1×10 -5 mol / L) in the system of 80% 1,4-dioxane and 20% PBS, the anti-interference of metal ions on the detection of peroxynitrite. From left to right are 1, blank; 2, Sn2+ ; 3. K + ; 4. Cu 2+ 5. Ag + ; 6. Ca 2+ ; 7. Fe 3+ ;8, Mg 2+ ; 9. Ni 2+ ; 10, Na + ; 11, Zn 2+ ; 12, Cr 3+ ; 13. Fe 2+ The interferences had no effect on the fluorescence intensity of PBQI, indicating that the probe PBQI has good anti-interference ability against metal ions in the detection of peroxynitrite.

[0061] Figure 22 The probe PBQI (1×10 -5 mol / L) in the system of 80% 1,4-dioxane and 20% PBS, the anti-interference of anions on the detection of peroxynitrite. From left to right are 1, blank; 2, PO4 3- 3. H2PO4 - 4. P2O7 4- 5. BF4 - ; 6. HS - ; 7. S2O5 2- ;8, S 2- ;9HSO3 - ; 10, SO4 2- ; 11. CrO4 2- ;12, Cr2O7 2- ;13, C2O4 2- ; 14, F - ; 15, Cl - ; 16, Br - ; 17, I - 18. CO3 2- ; 19, HCO3 - ; 20, NO2 - ; 21, NO3 - .

[0062] Figure 23 The probe PBQI (1×10 -5 mol / L) in the system of 80% 1,4-dioxane and 20% PBS, the anti-interference of other active oxygen species on the detection of peroxynitrite. From left to right are H2O2, ClO - 、NO、 1 O2, ·OH.

[0063] Figure 24The prepared probe PBQI (1×10 -5 mol / L) in different solvents, including the maximum absorption peak wavelength λ abs,max , maximum emission peak wavelength λ em,max , Stokes shift and fluorescence quantum yield Φ (with rhodamine B as reference).

[0064] Specific implementation examples

[0065] Example 1: Synthesis of Compound A1

[0066] 3-Iodo-1-propionic acid was added to a round-bottom flask containing 4-methylquinoline and toluene. The mixture was ultrasonically dissolved and then heated under reflux for 12 hours under a nitrogen atmosphere. After the reaction was complete, the mixture was cooled to room temperature. A precipitate formed, which was filtered, washed with toluene, and dried in a vacuum oven to obtain 1-(2-carboxyethyl)-4-methylquinolin-1-ium iodide A1 as an off-white solid (89%).

[0067] Example 2: Synthesis of Compound A2

[0068] Weigh 4-bromotriphenylamine and 5-formyl-2-thiophene boronic acid, ultrasonically make it fully dissolve in tetrahydrofuran solution, add potassium carbonate aqueous solution and tetrakis(triphenylphosphine)palladium in sequence, and ultrasonically dissolve, evacuate, make it be in nitrogen atmosphere, reflux for about 18 hours. After reaction finishes, reaction solution is cooled to room temperature, extracted with dichloromethane and water, collect organic phase and use anhydrous sodium sulfate (Na2SO4) drying, be spin-dried for and obtain crude product. Separate and purify by column chromatography, obtain yellow solid product 5-(4-(diphenylamino)phenyl)thiophene-2-carboxaldehyde A2 (32.5%) after drying.

[0069] Example 3: Synthesis of probe PBQI

[0070] A1 and A2 were added to a round-bottom flask filled with anhydrous ethanol and sonicated to dissolve. Two drops of piperidine solution were added, and the mixture was heated under reflux for 12 hours. Upon completion of the reaction, the reaction mixture was cooled to room temperature. A large amount of precipitate formed, which was filtered, washed with cold ethanol, and dried in a vacuum oven to yield (E)-1-(2-carboxyethyl)-4-(2-(5-(4-(diphenylamino)phenyl)thiophen-2-yl)vinyl)quinolin-1-ium iodide PBQI as a dark purple solid (40.8%).

[0071] Example 4: Detection of ONOO by Probe PBQI - and application of solution polarity

[0072] Polarity detection in solution: Figure 3 The probe PBQI (1×10 5mol / L) in different solvents. The UV-visible absorption spectra of the probe in different solvents have obvious differences. Figure 4 The probe PBQI (1×10 -5 mol / L) in different solvents at an excitation wavelength of 360 nm. Figure 5 The probe PBQI (1×10 -5 mol / L) in different solvents at an excitation wavelength of 520 nm. Figure 6 The probe PBQI (1×10 -5 mol / L) in mixed solvents of 1,4-dioxane and PBS at different ratios. Figure 7 The probe PBQI (1×10 - 5 Figure 2 shows the fluorescence emission spectrum of a 1,4-dioxane and PBS mixture at an excitation wavelength of 520 nm. The fluorescence intensity gradually increases with the increase of 1,4-dioxane content. Figure 8 The probe PBQI (1×10 -5 mol / L) in an 80% 1,4-dioxane and 20% PBS system, with an excitation wavelength of 520 nm and an emission wavelength of 700 nm. The fluorescence intensity gradually increases with the increase of 1,4-dioxane content. Figure 9 The probe PBQI (1×10 -5 mol / L) at a wavelength of 700 nm. The fluorescence intensity of the probe increases with increasing solvent polarity. The fluorescence intensity gradually increases with increasing pH and decreases at a pH of 9. Figure 10 The probe PBQI (1×10 -5 mol / L) in a solvent of 80% 1,4-dioxane and 20% PBS, a 0-90 μM peroxynitrite solution was added, and the UV-visible absorption spectrum of the probe PBQI was obtained. Figure 11 The probe PBQI (1×10 - 5 Figure 2 shows the fluorescence emission spectrum of a 0-90 μM peroxynitrite solution added to an 80% 1,4-dioxane and 20% PBS solution at an excitation wavelength of 400 nm. It can be seen that the fluorescence intensity gradually increases with increasing peroxynitrite concentration. Figure 12 The probe PBQI (1×10 -5Fluorescence emission spectra of 0-90 μM peroxynitrite solution added to 80% 1,4-dioxane and 20% PBS (100 mol / L) at an excitation wavelength of 520 nm. It can be seen that the fluorescence intensity gradually decreases with increasing peroxynitrite concentration. Figure 13 The probe PBQI (1×10 -5 mol / L) at an excitation wavelength of 400 nm and an emission wavelength of 520 nm, the linear relationship between the fluorescence intensity change of the probe PBQI and peroxynitrite can be seen from the figure. Figure 14 The probe PBQI (1×10 -5 Figure 2 shows the fluorescence intensity changes at 520 nm from 0 to 60 minutes after the addition of peroxynitrite solutions of different concentrations (10 mol / L). As can be seen from the figure, the probe responds to peroxynitrite quickly, and the fluorescence intensity gradually stabilizes after 20 minutes. Figure 15 The probe PBQI (1×10 -5 mol / L) in a system of 80% 1,4-dioxane and 20% PBS, and the effect of using PBS of different pH on the detection of peroxynitrite. As can be seen from the figure, the probe PBQI is suitable for monitoring the changes of peroxynitrite at physiological pH. Figure 16 The probe PBQI (1×10 -5 mol / L) under different pH and polarity conditions, the fluorescence intensity of the probe PBQI in response to peroxynitrite increases. The figure shows that as the pH increases and the polarity decreases, the fluorescence intensity at 520 nm gradually increases after the probe reacts with peroxynitrite. Figure 17 The probe PBQI (1×10 - 5 mol / L) in a 99% 1,4-dioxane and 1% PBS system, studying the anti-interference properties of small biomolecules in detecting polarity. From left to right: 1, blank; 2, alanine; 3, phenylalanine; 4, isoleucine; 5, arginine; 6, threonine; 7, tryptophan; 8, valine; 9, glycine; 10, aspartic acid; 11, glutamine; 12, leucine; 13, cysteine; 14, lysine; 15, methionine; 16, proline; 17, ascorbic acid; 18, glutathione; 19, 99% 1,4-dioxane + 1% PBS. As can be seen from the figure, none of the interfering substances affected the fluorescence intensity of the PBQI probe, demonstrating that the PBQI probe exhibits excellent anti-interference properties for small biomolecules. Figure 18 The probe PBQI (1×10 -5mol / L) in a system of 99% 1,4-dioxane and 1% PBS, the anti-interference of metal ions on the detection of polarity. From left to right are 1, blank; 2, Fe 2+ ; 3. Cr 3+ ; 4. Zn 2+ 5. Cd 2+ ; 6. Mn 2+ ; 7. Na + 8. Ag + ; 9, Ca 2+ ; 10, Fe 3+ ; 11, Mg 2+ ; 12, Ni 2+ ;13.Co 2+ ; 14, Sn 2+ ; 15, Cu 2+ ; 16, K + 17. 99% 1,4-dioxane + 1% PBS. This indicates that the interfering substances have no effect on the fluorescence intensity of the probe PBQI, indicating that the probe PBQI has good anti-interference properties against metal ions. Figure 19 The probe PBQI (1×10 -5 mol / L) in a system of 99% 1,4-dioxane and 1% PBS, the anti-interference of anions on the detection of polarity. From left to right are 1, blank; 2, S 2- ; 3. HS - ; 4. SO4 2- 5. HSO3 - ; 6. S2O5 2- ; 7. SCN - ;8, Cl - ; 9, Br - ; 10, I - ; 11. CrO4 2- ;12, Cr2O4 2- ;13, Cr2O7 2- ; 14, BF4 - 15. CO3 2- ; 16, HCO3 - ; 17, NO2 - ; 18, NO3 - 19. H2PO4 - ; 20, PO4 3- 21. 99% 1,4-dioxane + 1% PBS. This indicates that the interfering substances have no effect on the fluorescence intensity of the probe PBQI, indicating that the probe PBQI has good anti-interference properties against anions. Figure 20 The probe PBQI (1×10 -5mol / L) in an 80% 1,4-dioxane and 20% PBS system, demonstrating the anti-interference ability of small biomolecules in the detection of peroxynitrite. From left to right: 1, blank; 2, threonine; 3, leucine; 4, tryptophan; 5, glycine; 6, alanine; 7, aspartic acid; 8, glutathione; 9, cysteine; 10, methionine; 11, phenylalanine; 12, valine; 13, glutamine; 14, lysine; 15, arginine; 16, proline; 17, ascorbic acid. None of the interfering compounds affected the fluorescence intensity of the PBQI probe, demonstrating its excellent anti-interference ability against small biomolecules in the detection of peroxynitrite. Figure 21 The probe PBQI (1×10 -5 mol / L) in the system of 80% 1,4-dioxane and 20% PBS, the anti-interference of metal ions on the detection of peroxynitrite. From left to right are 1, blank; 2, Sn 2+ ; 3. K + ; 4. Cu 2+ 5. Ag + ; 6. Ca 2+ ; 7. Fe 3+ ;8, Mg 2+ ; 9. Ni 2+ ; 10, Na + ; 11, Zn 2+ ; 12, Cr 3+ ; 13. Fe 2+ The interferences had no effect on the fluorescence intensity of PBQI, indicating that the probe PBQI has good anti-interference ability against metal ions in the detection of peroxynitrite. Figure 22 The probe PBQI (1×10 -5 mol / L) in the system of 80% 1,4-dioxane and 20% PBS, the anti-interference of anions on the detection of peroxynitrite. From left to right are 1, blank; 2, PO4 3- 3. H2PO4 - 4. P2O7 4- 5. BF4 - ; 6. HS - ; 7. S2O5 2- ;8, S 2- ; 9. HSO3 - ; 10, SO4 2- ; 11. CrO4 2- ;12, Cr2O7 2- ;13, C2O4 2- ; 14, F - ; 15, Cl - ; 16, Br - ; 17, I- 18. CO3 2- ; 19, HCO3 - ; 20, NO2 - ; 21, NO3 - The interferences had no effect on the fluorescence intensity of PBQI, indicating that the probe PBQI has good anti-interference ability against anions in the detection of peroxynitrite. Figure 23 The probe PBQI (1×10 -5 mol / L) in the system of 80% 1,4-dioxane and 20% PBS, the anti-interference of other active oxygen species on the detection of peroxynitrite. From left to right are H2O2, ClO - 、NO、 1 O2, ·OH. The interferences had no effect on the fluorescence intensity of PBQI, indicating that the probe PBQI has good anti-interference ability against other reactive oxygen species in the detection of peroxynitrite. Figure 24 The prepared probe PBQI (1×10 -5 mol / L) in different solvents, including the maximum absorption peak wavelength λ abs,max , maximum emission peak wavelength λ em,max , Stokes shift and fluorescence quantum yield Φ (with rhodamine B as reference).

[0073] In summary, using a simple organic synthesis method, we have developed a dual-responsive fluorescent probe, PBQI, for polarity and peroxynitrite. This probe exhibits distinct solvatochromism in different solvents, which is clearly visible to the naked eye, demonstrating that the probe exhibits different optical properties in solvents of varying polarity. Furthermore, with increasing amounts of 1,4-dioxane, the fluorescence intensity at 700 nm gradually increases, accompanied by a slight blue shift. This is attributed to aggregation-induced binding due to restricted intramolecular torsion caused by probe aggregation in the solvent, a phenomenon also confirmed by the Tyndall effect. When peroxynitrite solution is added to a low-polarity environment, the probe's UV-visible absorption and fluorescence emission spectra exhibit distinct regular changes, providing a promising tool for peroxynitrite detection. The preparation of the probe PBQI fills a gap in the field of fluorescent probes capable of simultaneously and quantitatively detecting polarity and peroxynitrite. Furthermore, the probe's aggregation-induced properties result in excellent optical properties, including a large Stokes shift, and hold great promise for medical applications.

Claims

1. A fluorescent probe (E)-1-(2-carboxyethyl)-4-(2-(5-(4-(diphenylamino)phenyl)thiophen-2-yl)vinyl)quinolin-1-ium iodide for simultaneous detection of polarity and peroxynitrite, the structural formula of which is:

2. A method for preparing a fluorescent probe (E)-1-(2-carboxyethyl)-4-(2-(5-(4-(diphenylamino)phenyl)thiophen-2-yl)vinyl)quinolin-1-ium iodide for simultaneous detection of polarity and peroxynitrite according to claim 1, comprising the following steps: (1) Preparation of 1-(2-carboxyethyl)-4-methylquinolin-1-ium iodide A1: 3-iodo-1-propionic acid and 4-methylquinoline were dissolved in toluene, ultrasonically dissolved, and then vacuumed and heated for 12 h. After the reaction was completed, the mixture was cooled to room temperature. A precipitate was formed, which was filtered and washed with toluene. The precipitate was dried in a vacuum oven to obtain an off-white solid A1. (2) Preparation of 5-(4-(diphenylamino)phenyl)thiophene-2-carboxaldehyde A2: 4-bromotriphenylamine and 5-formyl-2-thiopheneboronic acid were ultrasonically dissolved in tetrahydrofuran solution, and potassium carbonate aqueous solution and tetrakis(triphenylphosphine)palladium were added and ultrasonically dissolved and vacuumed, and the mixture was refluxed for 18 hours. After the reaction, the reaction solution was cooled to room temperature and extracted with dichloromethane and water. The organic phase was dried over anhydrous sodium sulfate and dried to obtain a crude product, which was separated and purified by column chromatography and dried to obtain a yellow solid product A2. (3) Preparation of target fluorescent probe: A1 was added to anhydrous ethanol and heated under reflux until dissolved. A2 was then added to the reaction system to completely dissolve it and then heated under reflux for 12 hours. At the end of the reaction, the reaction solution was cooled to room temperature. A large amount of precipitate was precipitated and filtered and washed. The precipitate was then dried in a vacuum drying oven to obtain a dark purple solid (E)-1-(2-carboxyethyl)-4-(2-(5-(4-(diphenylamino)phenyl)thiophen-2-yl)vinyl)quinolin-1-ium iodide PBQI. The synthesis path is as follows:

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