Preparation method and application of dual-response fluorescent probe for detecting polarity and peroxynitrite

Abstract

The invention belongs to the technical field of fluorescent probes, and particularly relates to a preparation method and property research of a dual-response fluorescent probe for detecting polarity and peroxynitrite. The preparation method of the fluorescent probe is simple, the product yield is high, and the fluorescent probe is suitable for large-scale popularization and application. The present invention relates to a method for preparing a probe (Z)-4-(1-cyano-2-(6-(4-(diphenylamino) phenyl)-4-phenyl-4H-dithieno [3, 2-b: 2 ', 3'-d] pyrrol-2-yl) vinyl)-1-(4-(4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) benzyl) pyridine-1-onium bromide (DTBP). When excitation is carried out at 600 nanometers, the fluorescence intensity is gradually enhanced along with the reduction of the polarity of the solution; in a solvent of PBS and acetone in equal proportion, when excitation is carried out at 470 nanometers, the probe reacts with ONOO to generate strong fluorescence. Along with the increase of the ONOO concentration, the fluorescence intensity is gradually enhanced. The probe can specifically detect ONOO, and has a good linear relationship. The probe also has relatively strong fluorescence response to polarity, and the fluorescence emission intensity is gradually increased along with the reduction of the polarity.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, specifically relating to a method for preparing a polar and peroxynitrite dual-responsive fluorescent probe and its application. Background Technology

[0002] Cellular polarity is a core parameter regulating cellular physiological functions and maintaining microenvironmental homeostasis, deeply involved in key physiological processes such as membrane fusion, protein conformational changes, enzyme activity regulation, and peptide aggregation. Abnormal changes in cellular polarity are closely related to cellular dysfunction and disease development, not only associated with common diseases such as tumors, diabetes, and Alzheimer's disease, but also involving various pathological states such as cardiovascular disease, cirrhosis, and neurodegenerative diseases. Due to the complexity and transient nature of polarity changes, accurate monitoring of its dynamics is crucial for elucidating physiological mechanisms and disease pathology. Fluorescent probes, with their advantages of high sensitivity, non-invasiveness, real-time monitoring, and high spatiotemporal resolution, have become core tools for polarity detection, and related research has become a hot topic in the fields of chemistry, biology, and medicine.

[0003] peroxynitrite (ONOO) − As the most reactive and toxic member of the reactive oxygen species family, it is mainly generated through the rapid in-situ reaction of nitric oxide and superoxide anions, and is present as ONOO at physiological pH. − / ONOOH acid-base pair coexistence, because O2 − Its short lifespan, extremely short half-life, and nanoscale steady-state concentration within cells make its biological detection extremely challenging. − Excessive amounts of nitrated amino acids possess dual functions of physiological regulation and pathological damage. They can regulate cell signaling and participate in immune defense through the hydration of nitrated amino acid residues, but in excess, they can damage biomolecules through their own and secondary free radicals. They are closely related to various pathological processes such as cancer, neurodegenerative diseases, inflammation, and drug-induced toxicity. Therefore, developing probes with high selectivity, high sensitivity, rapid response, good biocompatibility, and suitability for live cell, tissue, and in vivo imaging is crucial for revealing the effects of nitrated amino acids on cell and tissue metabolism. − Understanding pathological mechanisms, discovering disease biomarkers, and guiding the early diagnosis and treatment of related diseases are of great significance.

[0004] Fluorescent probes, with their flexible synthetic design and diverse recognition mechanisms, combine high selectivity and high sensitivity, accurately avoiding interference from other active species to achieve specific recognition of ONOO⁻. Their rapid response ensures fast detection of target molecules. Near-infrared fluorescent probes further highlight their core advantages in biomedical imaging, possessing deep tissue penetration capabilities, low background fluorescence interference and low phototoxicity, and photobleaching properties. They enable high-resolution dynamic monitoring, and some probes exhibit subcellular targeting and multimodal imaging capabilities, further improving detection accuracy and applicability. Polarity and ONOO⁻, as important indicators of the intracellular microenvironment, are closely related to cellular homeostasis and disease progression. The non-invasiveness and good biocompatibility of fluorescent probes allow for in-situ tracking of polarity and ONOO⁻ dynamics under physiological conditions, providing a direct tool for disease mechanism research. In conclusion, near-infrared fluorescent probes with dual polarity and ONOO⁻ responses are highly necessary, enabling precise detection and dynamic monitoring of target molecules in vivo, providing strong support for early disease diagnosis and pathological mechanism analysis, and possessing broad prospects for medical applications. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing and studying the properties of a dual-response fluorescent probe for detecting polarity and peroxynitrite. DTBP has the advantages of simple synthetic route, good selectivity, high sensitivity, and large Stokes shift, and can detect ONOO under physiological conditions. − Effective detection of polarity.

[0006] The dual-response near-infrared fluorescent probe (Z)-4-(1-cyano-2-(6-(4-(diphenylamino)phenyl)-4-phenyl-4H-dithienro[3,2-b:2',3'-d]pyrrolo-2-yl)vinyl)-1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)benzyl)pyridine-1-onium bromide (DTBP) of this invention has the following structural formula:

[0007] The fluorescent probe synthesis process in this invention is as follows:

[0008] The preparation steps of the probe DTBP are as follows:

[0009] 3,3'-dibromo-2,2'-dithiophene, aniline, sodium tert-butoxide, 1,1'-binaphthyl-2,2'-bis(diphenylphosphine)dipalladium were sonicated to fully dissolve in toluene and refluxed for 12 hours. After the reaction was completed, the reaction solution was cooled to room temperature and evaporated to dryness to obtain the crude product. The crude product was separated and purified by column chromatography and dried to obtain 4-phenyl-4H-dithiophene[3,2-b:2',3'-d]pyrrole (A1, yield 73.2%).

[0010] Under a nitrogen atmosphere, phosphorus oxychloride was dissolved in N,N-dimethylformamide (DMF) cooled in an ice-water bath. After stirring for 1 hour, compound A1 was added, and the reaction was carried out at 70°C for 2 hours. After the reaction was completed, the mixture was cooled to room temperature, the pH was adjusted to neutral with sodium hydroxide solution, and then extracted with dichloromethane. The organic phases were combined, the solvent was removed by rotary evaporation to obtain the crude product, and finally purified by column chromatography. After drying in a vacuum drying oven, 4-phenyl-4H-dithieno[3,2-b:2',3'-d]pyrrole-2-carboxaldehyde (A2, yield 78.1%) was obtained.

[0011] N-bromosuccinimide and A2 were dissolved in tetrahydrofuran (THF) under ice-water bath conditions and reacted at room temperature in the dark for 12 hours. After the reaction was completed, the solvent was directly evaporated to obtain the product. The product was dried in a vacuum drying oven to give 6-bromo-4-phenyl-4H-dithieno[3,2-b:2',3'-d]pyrrole-2-carboxaldehyde (A3, yield 85.3%).

[0012] 4-(diphenylamino)phenylboronic acid and A3 were sonicated to fully dissolve them in tetrahydrofuran (THF). Potassium carbonate aqueous solution and tetra(triphenylphosphine)palladium were added, and the mixture was sonicated to dissolve them. The mixture was refluxed for 22 hours under nitrogen protection. After the reaction was complete, the reaction solution was cooled to room temperature and evaporated to dryness to obtain the crude product. The crude product was separated and purified by column chromatography, and after drying, 6-(4-(diphenylamino)phenyl)-4-phenyl-4H-dithieno[3,2-b:2',3'-d]pyrrole-2-carboxaldehyde (A4, yield 74.1%) was obtained.

[0013] 4-Pyridineacetonitrile and pinacol ester of 4-bromomethylphenylboronic acid were dissolved in acetonitrile and sonicated until completely dissolved. The reaction mixture was then heated under reflux for 12 hours. After the reaction was completed, the mixture was cooled to room temperature, and recrystallized in diethyl ether, resulting in a solid precipitate. The precipitate was collected by filtration and dried in a vacuum drying oven to give 4-(cyanomethyl)-1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)benzyl)pyridine-1-onium bromide (B1, yield 80.5%).

[0014] A4 and B1 were added to anhydrous ethanol and sonicated to dissolve completely. The mixture was then refluxed for 5 hours. At the end of the reaction, the reaction solution was cooled to room temperature and evaporated to dryness to obtain the crude product. The crude product was separated and purified by column chromatography and dried to obtain a black solid (Z)-4-(1-cyano-2-(6-(4-(diphenylamino)phenyl)-4-phenyl-4H-dithieno[3,2-b:2',3'-d]pyrrolo-2-yl)vinyl)-1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)benzyl)pyridine-1-onium bromide (DTBP, yield 24.6%).

[0015] The detection mechanism of the fluorescent probe of the present invention is as follows: The probe DTBP is a molecule with a donor (D)-π-acceptor (A) structure, typically exhibiting a significant solvochromatic effect; its photophysical properties vary with solvent polarity. Simultaneously, due to ONOO... − Possessing both strong oxidizing and nucleophilic properties, specific reactive sites in this probe structure can selectively respond to these properties. Based on these characteristics, we designed and synthesized a novel fluorescent probe that can simultaneously monitor changes in the polarity of the solution microenvironment and specifically detect peroxynitrite, demonstrating potential applications in chemical sensing and bioanalysis.

[0016] Figure 3 It is a probe DTBP (1×10 -5 The UV-Vis absorption spectra of the probe (mol / L) in different solvents. The UV absorption spectra of this probe show significant differences in different solvents.

[0017] Figure 4 It is a probe DTBP (1×10 -5 Fluorescence emission spectra of mol / L in different solvents at an excitation wavelength of 600 nm.

[0018] Figure 5 It is a probe DTBP (1×10 -5 UV-Vis absorption spectra of 1,4-dioxane and DMSO mixed solvents in different proportions (mol / L).

[0019] Figure 6 It is a probe DTBP (1×10 -5 Fluorescence emission spectra of 1,4-dioxane and DMSO mixed solvents in different proportions (mol / L).

[0020] Figure 7 It is a probe DTBP (1×10 -5Fluorescence intensity at 730 nm emission wavelength under excitation wavelength of 600 nm in mixed solvents of 1,4-dioxane and DMSO in different proportions (mol / L).

[0021] Figure 8 It is a probe DTBP (1×10 -5 The selectivity of metal ions for detection polarity was studied in PBS buffer solution (1–14) with a concentration of mol / L. From left to right: 1, blank; 2, Na+. + 3. K + 4. Mg 2+ 5. Ag + 6. Cu 2+ 7. Ni 2+ 8. Mn 2+ 9. Ca 2+ 10. Fe 2+ 11. Fe 3+ ;12, Co 2+ 13. Zn 2+ 14. Cr 3+ 15. 99% 1,4-dioxane + 1% DMSO. Metal ions had no effect on the fluorescence intensity of DTBP, indicating that the DTBP probe has good polarity selectivity.

[0022] Figure 9 It is a probe DTBP (1×10 -5 The selectivity of anions for detection polarity was studied in PBS buffer solution (1–20) with a concentration of mol / L. From left to right: 1, blank; 2, F. − 3. Cl − ; 4. Br − 5. I − 6. HSO3 − 7. SO3 2− 8. SCN − 9. HS − 10. S 2− 11. S2O8 2− 12. SO4 2− 13. S2O5 2− 14. PO4 3− 15. H2PO4 − 16. CO3 2− 17. HCO3 − 18. NO2 − 19. NO3 − 20. Cr2O7 2−21. 99% 1,4-dioxane + 1% DMSO. The anions had no effect on the fluorescence intensity of DTBP, indicating that the DTBP probe has good polarity selectivity.

[0023] Figure 10 It is a probe DTBP (1×10 -5 The selectivity of small biomolecules for detection polarity was studied in PBS buffer solution (1-21) at mol / L. From left to right: 1. Blank; 2. Ascorbic acid; 3. Serine; 4. Leucine; 5. Aspartic acid; 6. Glutamic acid; 7. Methionine; 8. Alanine; 9. Tryptophan; 10. Valine; 11. Glycine; 12. Phenylalanine; 13. Arginine; 14. Lysine; 15. Threonine; 16. Glutamine; 17. Cysteine; 18. Homocysteine; 19. Proline; 20. Glutathione; 21. Isoleucine; 22. 99% 1,4-dioxane + 1% DMSO. None of the small biomolecules affected the fluorescence intensity of DTBP, indicating that the DTBP probe has good selectivity for polarity.

[0024] Figure 11 It is a probe DTBP (1×10 -5 The selectivity of reactive oxygen species (ROS) for detection polarity was studied in PBS buffer solutions (1–5) at concentrations of mol / L. From left to right: 1. Blank; 2. H₂O₂; 3. ClO₂. − 4. 1 O2; 5, •OH; 6, 99% 1,4-dioxane + 1% DMSO. Reactive oxygen species had no effect on the fluorescence intensity of DTBP, indicating that the DTBP probe has good polarity selectivity.

[0025] Figure 12 It is a probe DTBP (1×10 -5 In Acetone-PBS systems containing different amounts of acetone (mol / L) at an excitation wavelength of 470 nm, the same concentration of ONOO was added. − The fluorescence intensity changes before and after are shown in the graph. This probe, with an Acetone volume percentage of 50%, was treated with 0.18 mmol / L ONOO. − The fluorescence intensity changed significantly before and after.

[0026] Figure 13 It is a probe DTBP (1×10 -5 In a mixture of PBS and Acetone in equal proportions, different concentrations of ONOO were added. − The UV-Vis absorption spectrum of (0−0.18 mmol / L).

[0027] Figure 14 It is a probe DTBP (1×10 -5 In a mixture of PBS and Acetone in equal proportions, different concentrations of ONOO were added. − The fluorescence emission spectrum of the probe at (0−0.18 mmol / L) can be obtained. − As the concentration increases, the fluorescence intensity gradually increases.

[0028] Figure 15 Yes, it is a probe DTBP (1×10) -5 Add ONOO (mol / L) to an equal proportion of PBS and Acetone mixed solvent. − Concentration range: 0–0.18 mmol / L 650 Linear relationship graph.

[0029] Figure 16 It is a probe DTBP (1×10 -5 Different concentrations of ONOO were added to an equal ratio of PBS and Acetone mixed solvent (mol / L). − The graph shows the change in fluorescence intensity over time.

[0030] Figure 17 It is a probe DTBP (1×10 -5 Add ONOO (mol / L) to an equal proportion of PBS and Acetone mixed solvent. − Changes in fluorescence intensity with pH value before and after.

[0031] Figure 18 It is a probe DTBP (1×10 -5 In a mixed solvent of PBS and Acetone in equal proportions (mol / L), metal ions were used to detect ONOO. − Anti-interference study. From left to right: 1, blank; 2, Na. + 3. K + 4. Mg 2+ 5. Cu 2+ 6. Ni 2+ 7. Mn 2+ 8. Ca 2+ 9. Fe 2+ 10. Fe 3+ 11. Co 2+ 12. Zn 2+ 13. Cr 3+ Metal ions had no effect on the fluorescence intensity of DTBP, indicating that the DTBP probe was effective in detecting ONOO. − It exhibits good resistance to interference from metal ions during the process.

[0032] Figure 19 It is a probe DTBP (1×10 -5 In a mixed solvent of PBS and Acetone in equal proportions (mol / L), anion pairs were used to detect ONOO. − Anti-interference study. From left to right: 1, blank; 2, F − 3. Cl − ; 4. Br − 5. I − 6. HSO3 − 7. SO3 2− 8. SCN − 9. HS − 10. S2O8 2− 11. SO4 2− 12. S2O5 2− 13. PO4 3− 14. H2PO4 − 15. CO3 2− 16. HCO3 − 17. NO2 − 18. NO3 − 19. Cr2O7 2− Anions had no effect on the fluorescence intensity of DTBP, indicating that the DTBP probe was effective in detecting ONOO. − It exhibits good resistance to interference from anions during the process.

[0033] Figure 20 It is a probe DTBP (1×10 -5 In a mixed solvent of PBS and Acetone in equal proportions (mol / L), the biomolecules were used to detect ONOO. − The interference resistance study was conducted. From left to right: 1. Blank; 2. Ascorbic acid; 3. Serine; 4. Leucine; 5. Aspartic acid; 6. Glutamic acid; 7. Methionine; 8. Alanine; 9. Tryptophan; 10. Valine; 11. Glycine; 12. Phenylalanine; 13. Arginine; 14. Lysine; 15. Threonine; 16. Glutamine; 17. Cysteine; 18. Homocysteine; 19. Proline; 20. Glutathione. None of the small biomolecules affected the fluorescence intensity of DTBP, indicating that the probe DTBP is effective in detecting ONOO. − It exhibits good resistance to interference from small biological molecules during the process.

[0034] Figure 21 It is a probe DTBP (1×10 -5 In a mixed solvent of PBS and Acetone in equal proportions (mol / L), reactive oxygen species were used to detect ONOO. −Anti-interference study. From left to right: 1. Blank; 2. H2O2; 3. ClO − 4. 1 O2; 5, •OH; 6, NO. None of these reactive oxygen species affected the fluorescence intensity of DTBP, indicating that the DTBP probe effectively detects ONOO. − It exhibits good resistance to reactive oxygen species during the process.

[0035] Figure 22 The photophysical properties of the prepared probe DTBP in different solvents are presented sequentially, including the maximum absorption peak λ. abs,max Maximum emission λ em,max Stokes shift and fluorescence quantum yield Φ (with Cy5 as a reference).

[0036] In summary, using a simple organic synthesis method, we obtained the synthesis of ONOO. − A polar-responsive near-infrared fluorescent probe, (Z)-4-(1-cyano-2-(6-(4-(diphenylamino)phenyl)-4-phenyl-4H-dithiopheno[3,2-b:2',3'-d]pyrrolo-2-yl)vinyl)-1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)benzyl)pyridin-1-onium bromide (DTBP), was developed. This probe exhibited significant lyochromic effects in different solvents, a phenomenon directly observable to the naked eye, confirming its different optical properties in solvents of varying polarities. Furthermore, the fluorescence intensity of the probe gradually increased with increasing 1,4-dioxane solvent volume ratio. This is attributed to the aggregation-induced phenomenon caused by intramolecular torsion restriction due to probe aggregation in the solvent. ONOO was added to the DTBP probe in an equal volume mixture of PBS and acetone. − Subsequently, both the UV-Vis absorption spectrum and the fluorescence emission spectrum exhibited significant and regular changes, indicating ONOO. − The detection provides a high-quality analytical tool. The successful synthesis of the DTBP probe fills the research gap in the field of fluorescent probes that can simultaneously achieve the quantitative detection of polarity and peroxynitrite. Furthermore, the aggregation-induced emission properties of the probe endow it with excellent optical properties, while also possessing a large Stokes shift, showing promising application prospects in the field of biomedical detection. Attached Figure Description

[0037] Figure 1 This describes the preparation and design route for the probe (Z)-4-(1-cyano-2-(6-(4-(diphenylamino)phenyl)-4-phenyl-4H-dithieno[3,2-b:2',3'-d]pyrrolo-2-yl)vinyl)-1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)benzyl)pyridine-1-onium bromide (DTBP).

[0038] Figure 2 It is a probe DTBP to detect ONOO − and the mechanism of polar response

[0039] Figure 3 It is a probe DTBP (1×10 -5 UV-Vis absorption spectra of (mol / L) in different solvents.

[0040] Figure 4 It is a probe DTBP (1×10 -5 Fluorescence emission spectra of mol / L in different solvents at an excitation wavelength of 600 nm.

[0041] Figure 5 It is a probe DTBP (1×10 -5 UV-Vis absorption spectra of 1,4-dioxane and DMSO mixed solvents in different proportions (mol / L).

[0042] Figure 6 It is a probe DTBP (1×10 -5 Fluorescence emission spectra of 1,4-dioxane and DMSO mixed solvents in different proportions (mol / L).

[0043] Figure 7 It is a probe DTBP (1×10 -5 Fluorescence intensity at 730 nm emission wavelength under excitation wavelength of 600 nm in mixed solvents of 1,4-dioxane and DMSO in different proportions (mol / L).

[0044] Figure 8 It is a probe DTBP (1×10 -5 The selectivity of metal ions for detection polarity was studied in PBS buffer solution (1–14) with a concentration of mol / L. From left to right: 1, blank; 2, Na+. + 3. K + 4. Mg 2+ 5. Ag + 6. Cu 2+ 7. Ni 2+ 8. Mn 2+ 9. Ca 2+ 10. Fe 2+ 11. Fe 3+ ;12, Co 2+ 13. Zn 2+ 14. Cr 3+ ;15. 99% 1,4-dioxane+1% DMSO.

[0045] Figure 9 It is a probe DTBP (1×10 -5 The selectivity of anions for detection polarity was studied in PBS buffer solution (1–20) with a concentration of mol / L. From left to right: 1, blank; 2, F. − 3. Cl − ; 4. Br − 5. I − 6. HSO3 − 7. SO3 2− 8. SCN − 9. HS − 10. S 2− 11. S2O8 2− 12. SO4 2− 13. S2O5 2− 14. PO4 3− 15. H2PO4 − 16. CO3 2− 17. HCO3 − 18. NO2 − 19. NO3 − 20. Cr2O7 2− ;21. 99% 1,4-dioxane+1% DMSO.

[0046] Figure 10 It is a probe DTBP (1×10 -5 Selectivity of biomolecules for detection polarity in PBS buffer solution (1–21) (mol / L). From left to right: 1. Blank; 2. Ascorbic acid; 3. Serine; 4. Leucine; 5. Aspartic acid; 6. Glutamic acid; 7. Methionine; 8. Alanine; 9. Tryptophan; 10. Valine; 11. Glycine; 12. Phenylalanine; 13. Arginine; 14. Lysine; 15. Threonine; 16. Glutamine; 17. Cysteine; 18. Homocysteine; 19. Proline; 20. Glutathione; 21. Isoleucine; 22. 99% 1,4-dioxane + 1% DMSO.

[0047] Figure 11 It is a probe DTBP (1×10 -5 The selectivity of reactive oxygen species (ROS) for detection polarity was studied in PBS buffer solutions (1–5) at concentrations of mol / L. From left to right: 1. Blank; 2. H₂O₂; 3. ClO₂. − 4. 1 O2; 5, •OH; 6, 99% 1,4-dioxane + 1% DMSO.

[0048] Figure 12It is a probe DTBP (1×10 -5 In Acetone-PBS systems containing different amounts of acetone (mol / L) at an excitation wavelength of 470 nm, the same concentration of ONOO was added. − The graph shows the changes in fluorescence intensity before and after.

[0049] Figure 13 It is a probe DTBP (1×10 -5 In a mixture of PBS and Acetone in equal proportions, different concentrations of ONOO were added. − The UV-Vis absorption spectrum of (0−0.18 mmol / L).

[0050] Figure 14 It is a probe DTBP (1×10 -5 In a mixture of PBS and Acetone in equal proportions, different concentrations of ONOO were added. − Fluorescence emission spectrum of (0−0.18 mmol / L).

[0051] Figure 15 Yes, it is a probe DTBP (1×10) -5 Add ONOO (mol / L) to an equal proportion of PBS and Acetone mixed solvent. − Concentration range: 0–0.18 mmol / L 650 Linear relationship graph.

[0052] Figure 16 It is a probe DTBP (1×10 -5 Different concentrations of ONOO were added to an equal ratio of PBS and Acetone mixed solvent (mol / L). − The graph shows the change in fluorescence intensity over time.

[0053] Figure 17 It is a probe DTBP (1×10 -5 Add ONOO (mol / L) to an equal proportion of PBS and Acetone mixed solvent. − Changes in fluorescence intensity with pH value before and after.

[0054] Figure 18 It is a probe DTBP (1×10 -5 In a mixed solvent of PBS and Acetone in equal proportions (mol / L), metal ions were used to detect ONOO. − Anti-interference study. From left to right: 1, blank; 2, Na. + 3. K + 4. Mg 2+ 5. Cu2+ 6. Ni 2+ 7. Mn 2+ 8. Ca 2+ 9. Fe 2+ 10. Fe 3+ 11. Co 2+ 12. Zn 2+ 13. Cr 3+ .

[0055] Figure 19 It is a probe DTBP (1×10 -5 In a mixed solvent of PBS and Acetone in equal proportions (mol / L), anion pairs were used to detect ONOO. − Anti-interference study. From left to right: 1, blank; 2, F − 3. Cl − ; 4. Br − 5. I − 6. HSO3 − 7. SO3 2− 8. SCN − 9. HS − 10. S2O8 2− 11. SO4 2− 12. S2O5 2− 13. PO4 3− 14. H2PO4 − 15. CO3 2− 16. HCO3 − 17. NO2 − 18. NO3 − 19. Cr2O7 2− .

[0056] Figure 20 It is a probe DTBP (1×10 -5 In a mixed solvent of PBS and Acetone in equal proportions (mol / L), the biomolecules were used to detect ONOO. − Anti-interference study. From left to right: 1. Blank; 2. Ascorbic acid; 3. Serine; 4. Leucine; 5. Aspartic acid; 6. Glutamic acid; 7. Methionine; 8. Alanine; 9. Tryptophan; 10. Valine; 11. Glycine; 12. Phenylalanine; 13. Arginine; 14. Lysine; 15. Threonine; 16. Glutamine; 17. Cysteine; 18. Homocysteine; 19. Proline; 20. Glutathione.

[0057] Figure 21 It is a probe DTBP (1×10 -5In a mixed solvent of PBS and Acetone in equal proportions (mol / L), reactive oxygen species were used to detect ONOO. − Anti-interference study. From left to right: 1. Blank; 2. H2O2; 3. ClO − 4. 1 O2; 5、•OH; 6、NO.

[0058] Figure 22 The photophysical properties of the prepared probe DTBP in different solvents are presented sequentially, including the maximum absorption peak λ. abs,max Maximum emission λ em,max Stokes shift and fluorescence quantum yield Φ (with Cy5 as a reference). Specific implementation examples

[0059] Example 1: Synthesis of compound A1 3,3'-dibromo-2,2'-dithiophene, aniline, sodium tert-butoxide, 1,1'-binaphthyl-2,2'-bis(diphenylphosphine)dipalladium were sonicated to fully dissolve in toluene and refluxed for 12 hours. After the reaction was completed, the reaction solution was cooled to room temperature and evaporated to dryness to obtain the crude product. The crude product was separated and purified by column chromatography and dried to obtain 4-phenyl-4H-dithiophene[3,2-b:2',3'-d]pyrrole (A1, yield 73.2%).

[0060] Example 2: Synthesis of compound A2 Under a nitrogen atmosphere, phosphorus oxychloride was dissolved in N,N-dimethylformamide (DMF) cooled in an ice-water bath. After stirring for 1 hour, compound A1 was added, and the reaction was carried out at 70°C for 2 hours. After the reaction was completed, the mixture was cooled to room temperature, the pH was adjusted to neutral with sodium hydroxide solution, and then extracted with dichloromethane. The organic phases were combined, the solvent was removed by rotary evaporation to obtain the crude product, and finally purified by column chromatography. After drying in a vacuum drying oven, 4-phenyl-4H-dithieno[3,2-b:2',3'-d]pyrrole-2-carboxaldehyde (A2, yield 78.1%) was obtained.

[0061] Example 3: Synthesis of compound A3 N-bromosuccinimide and A2 were dissolved in tetrahydrofuran (THF) under ice-water bath conditions and reacted at room temperature in the dark for 12 hours. After the reaction was completed, the solvent was directly evaporated to obtain the product. The product was dried in a vacuum drying oven to give 6-bromo-4-phenyl-4H-dithieno[3,2-b:2',3'-d]pyrrole-2-carboxaldehyde (A3, yield 85.3%).

[0062] Example 4: Synthesis of compound A4 4-(diphenylamino)phenylboronic acid and A3 were sonicated to fully dissolve them in tetrahydrofuran (THF). Potassium carbonate aqueous solution and tetra(triphenylphosphine)palladium were added, and the mixture was sonicated to dissolve them. The mixture was refluxed for 22 hours under nitrogen protection. After the reaction was complete, the reaction solution was cooled to room temperature and evaporated to dryness to obtain the crude product. The crude product was separated and purified by column chromatography, and after drying, 6-(4-(diphenylamino)phenyl)-4-phenyl-4H-dithieno[3,2-b:2',3'-d]pyrrole-2-carboxaldehyde (A4, yield 74.1%) was obtained.

[0063] Example 5: Synthesis of Compound B1 4-Pyridineacetonitrile and pinacol ester of 4-bromomethylphenylboronic acid were dissolved in acetonitrile and sonicated until completely dissolved. The reaction mixture was then heated under reflux for 12 hours. After the reaction was completed, the mixture was cooled to room temperature, and recrystallized in diethyl ether, resulting in a solid precipitate. The precipitate was collected by filtration and dried in a vacuum drying oven to give 4-(cyanomethyl)-1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)benzyl)pyridine-1-onium bromide (B1, yield 80.5%).

[0064] Example 6: Synthesis of compound DTBP A4 and B1 were added to anhydrous ethanol and sonicated to dissolve completely. The mixture was then refluxed for 5 hours. At the end of the reaction, the reaction solution was cooled to room temperature and evaporated to dryness to obtain the crude product. The crude product was separated and purified by column chromatography and dried to obtain a black solid (Z)-4-(1-cyano-2-(6-(4-(diphenylamino)phenyl)-4-phenyl-4H-dithieno[3,2-b:2',3'-d]pyrrolo-2-yl)vinyl)-1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)benzyl)pyridine-1-onium bromide (DTBP, yield 24.6%).

[0065] Example 7: Detection of ONOO by DTBP probe − Applications of solution polarity

[0066] Figure 3 It is a probe DTBP (1×10 -5 The UV-Vis absorption spectra of the probe (mol / L) in different solvents. The UV absorption spectra of this probe show significant differences in different solvents. Figure 4 It is a probe DTBP (1×10 -5 Fluorescence emission spectra of mol / L in different solvents at an excitation wavelength of 600 nm. Figure 5 It is a probe DTBP (1×10 -5 UV-Vis absorption spectra of 1,4-dioxane and DMSO mixed solvents in different proportions (mol / L). Figure 6It is a probe DTBP (1×10 -5 Fluorescence emission spectra of 1,4-dioxane and DMSO mixed solvents in different proportions (mol / L). Figure 7 It is a probe DTBP (1×10 -5 Fluorescence intensity at 730 nm emission wavelength under excitation wavelength of 600 nm in mixed solvents of 1,4-dioxane and DMSO in different proportions (mol / L). Figure 8 It is a probe DTBP (1×10 -5 The selectivity of metal ions for detection polarity was studied in PBS buffer solution (1–14) with a concentration of mol / L. From left to right: 1, blank; 2, Na+. + 3. K + 4. Mg 2+ 5. Ag + 6. Cu 2+ 7. Ni 2 + 8. Mn 2+ 9. Ca 2+ 10. Fe 2+ 11. Fe 3+ ;12, Co 2+ 13. Zn 2+ 14. Cr 3+ 15. 99% 1,4-dioxane + 1% DMSO. Metal ions had no effect on the fluorescence intensity of DTBP, indicating that the DTBP probe has good polarity selectivity. Figure 9 It is a probe DTBP (1×10 -5 The selectivity of anions for detection polarity was studied in PBS buffer solution (1–20) with a concentration of mol / L. From left to right: 1, blank; 2, F. − 3. Cl − ; 4. Br − 5. I − 6. HSO3 − 7. SO3 2− 8. SCN − 9. HS − 10. S 2− 11. S2O8 2− 12. SO4 2− 13. S2O5 2− 14. PO4 3− 15. H2PO4 − 16. CO3 2− 17. HCO3 − 18. NO2 − 19. NO3− 20. Cr2O7 2− 21. 99% 1,4-dioxane + 1% DMSO. The anions had no effect on the fluorescence intensity of DTBP, indicating that the DTBP probe has good polarity selectivity. Figure 10 It is a probe DTBP (1×10 -5 The selectivity of small biomolecules for detection polarity was studied in PBS buffer solution (1-21) at mol / L. From left to right: 1. Blank; 2. Ascorbic acid; 3. Serine; 4. Leucine; 5. Aspartic acid; 6. Glutamic acid; 7. Methionine; 8. Alanine; 9. Tryptophan; 10. Valine; 11. Glycine phenyl; 12. Phenylalanine; 13. Arginine; 14. Lysine; 15. Threonine; 16. Glutamine; 17. Cysteine; 18. Homocysteine; 19. Proline; 20. Glutathione; 21. Isoleucine; 22. 99% 1,4-dioxane + 1% DMSO. None of the small biomolecules affected the fluorescence intensity of DTBP, indicating that the DTBP probe has good selectivity for polarity. Figure 11 It is a probe DTBP (1×10 -5 The selectivity of reactive oxygen species (ROS) for detection polarity was studied in PBS buffer solutions (1–5) at concentrations of mol / L. From left to right: 1. Blank; 2. H₂O₂; 3. ClO₂. − 4. 1 O2; 5, •OH; 6, 99% 1,4-dioxane + 1% DMSO. Reactive oxygen species had no effect on the fluorescence intensity of DTBP, indicating that the DTBP probe has good polarity selectivity. Figure 12 It is a probe DTBP (1×10 -5 In Acetone-PBS systems containing different amounts of acetone (mol / L) at an excitation wavelength of 470 nm, the same concentration of ONOO was added. − The fluorescence intensity changes before and after are shown in the graph. This probe, with an Acetone volume percentage of 50%, was treated with 0.18 mmol / L ONOO. − The fluorescence intensity changed significantly before and after. Figure 13 It is a probe DTBP (1×10 -5 In a mixture of PBS and Acetone in equal proportions, different concentrations of ONOO were added. − The UV-Vis absorption spectrum of (0−0.18 mmol / L). Figure 14 It is a probe DTBP (1×10 -5 In a mixture of PBS and Acetone in equal proportions, different concentrations of ONOO were added. −The fluorescence emission spectrum of the probe at (0−0.18mmol / L) can be obtained. − As the concentration increases, the fluorescence intensity gradually increases. Figure 15 Yes, it is a probe DTBP (1×10) -5 Add ONOO (mol / L) to an equal proportion of PBS and Acetone mixed solvent. − Concentration range: 0–0.18 mmol / L 650 Linear relationship graph. Figure 16 It is a probe DTBP (1×10 -5 Different concentrations of ONOO were added to an equal ratio of PBS and Acetone mixed solvent (mol / L). − The graph shows the change in fluorescence intensity over time. Figure 17 It is a probe DTBP (1×10 -5 Add ONOO (mol / L) to an equal proportion of PBS and Acetone mixed solvent. − Changes in fluorescence intensity with pH value before and after. Figure 18 It is a probe DTBP (1×10 -5 In a mixed solvent of PBS and Acetone in equal proportions (mol / L), metal ions were used to detect ONOO. − Anti-interference study. From left to right: 1, blank; 2, Na. + 3. K + 4. Mg 2+ 5. Cu 2+ 6. Ni 2+ 7. Mn 2+ 8. Ca 2+ 9. Fe 2+ 10. Fe 3+ 11. Co 2+ 12. Zn 2+ 13. Cr 3+ Metal ions had no effect on the fluorescence intensity of DTBP, indicating that the DTBP probe was effective in detecting ONOO. − It exhibits good resistance to interference from metal ions during the process. Figure 19 It is a probe DTBP (1×10 -5 In a mixed solvent of PBS and Acetone in equal proportions (mol / L), anion pairs were used to detect ONOO. − Anti-interference study. From left to right: 1, blank; 2, F − 3. Cl − ; 4. Br − 5. I − 6. HSO3 − 7. SO32− 8. SCN − 9. HS − 10. S2O8 2− 11. SO4 2− 12. S2O5 2− 13. PO4 3− 14. H2PO4 − 15. CO3 2− 16. HCO3 − 17. NO2 − 18. NO3 − 19. Cr2O7 2− Anions had no effect on the fluorescence intensity of DTBP, indicating that the DTBP probe was effective in detecting ONOO. − It exhibits good resistance to interference from anions during the process. Figure 20 It is a probe DTBP (1×10 -5 In a mixed solvent of PBS and Acetone in equal proportions (mol / L), the biomolecules were used to detect ONOO. − The interference resistance study was conducted. From left to right: 1. Blank; 2. Ascorbic acid; 3. Serine; 4. Leucine; 5. Aspartic acid; 6. Glutamic acid; 7. Methionine; 8. Alanine; 9. Tryptophan; 10. Valine; 11. Glycine phenyl; 12. Phenylalanine; 13. Arginine; 14. Lysine; 15. Threonine; 16. Glutamine; 17. Cysteine; 18. Homocysteine; 19. Proline; 20. Glutathione. None of the small biological molecules affected the fluorescence intensity of DTBP, indicating that the probe DTBP is effective in detecting ONOO. − It exhibits good resistance to interference from small biological molecules during the process. Figure 21 It is a probe DTBP (1×10 -5 In a mixed solvent of PBS and Acetone in equal proportions (mol / L), reactive oxygen species were used to detect ONOO. − Anti-interference study. From left to right: 1. Blank; 2. H2O2; 3. ClO − 4. 1 O2; 5, •OH; 6, NO. None of these reactive oxygen species affected the fluorescence intensity of DTBP, indicating that the DTBP probe effectively detects ONOO. − It exhibits good resistance to reactive oxygen species during the process. Figure 22 The photophysical properties of the prepared probe DTBP in different solvents are presented sequentially, including the maximum absorption peak λ. abs,max Maximum emission λ em,max Stokes shift and fluorescence quantum yield Φ (with Cy5 as a reference).

[0067] In summary, using a simple organic synthesis method, we obtained the synthesis of ONOO. − A polar-responsive near-infrared fluorescent probe, (Z)-4-(1-cyano-2-(6-(4-(diphenylamino)phenyl)-4-phenyl-4H-dithiopheno[3,2-b:2',3'-d]pyrrolo-2-yl)vinyl)-1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)benzyl)pyridin-1-onium bromide (DTBP), was developed. This probe exhibited significant lyochromic effects in different solvents, a phenomenon directly observable to the naked eye, confirming its different optical properties in solvents of varying polarities. Furthermore, the fluorescence intensity of the probe gradually increased with increasing 1,4-dioxane solvent volume ratio. This is attributed to the aggregation-induced phenomenon caused by intramolecular torsion restriction due to probe aggregation in the solvent. ONOO was added to the DTBP probe in an equal volume mixture of PBS and acetone. − Subsequently, both the UV-Vis absorption spectrum and the fluorescence emission spectrum exhibited significant and regular changes, indicating ONOO. − The detection provides a high-quality analytical tool. The successful synthesis of the DTBP probe fills the research gap in the field of fluorescent probes that can simultaneously achieve the quantitative detection of polarity and peroxynitrite. Furthermore, the aggregation-induced emission properties of the probe endow it with excellent optical properties, while also possessing a large Stokes shift, showing promising application prospects in the field of biomedical detection.

Claims

1. The structural formula of a dual-response fluorescent probe for detecting polarity and peroxynitrite (Z)-4-(1-cyano-2-(6-(4-(diphenylamino)phenyl)-4-phenyl-4H-dithienro[3,2-b:2',3'-d]pyrrolo-2-yl)vinyl)-1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)benzyl)pyridine-1-onium bromide DTBP is as follows: 。 2. The preparation method of the dual-responsive fluorescent probe for detecting polarity and peroxynitrite ions according to claim 1 is as follows: 3,3'-dibromo-2,2'-bisthiophene, aniline, sodium tert-butoxide, 1,1'-binaphthyl-2,2'-bisdiphenylphosphine, and tris(dibenzylideneacetone)dipalladium were sonicated to fully dissolve in toluene and refluxed for 12 hours. After the reaction was completed, the reaction solution was cooled to room temperature and evaporated to dryness to obtain the crude product. The crude product was separated and purified by column chromatography and dried to obtain 4-phenyl-4H-bisthiopheno[3,2-b:2',3'-d]pyrrole A1. Under a nitrogen atmosphere, phosphorus oxychloride was dissolved in N,N-dimethylformamide DMF cooled in an ice-water bath. After stirring for 1 hour, compound A1 was added, and the mixture was reacted at 70°C for 2 hours. After the reaction was completed, the mixture was cooled to room temperature, the pH was adjusted to neutral with sodium hydroxide solution, and then extracted with dichloromethane. The organic phases were combined, the solvent was removed by rotary evaporation to obtain the crude product, and finally separated and purified by column chromatography. After drying in a vacuum drying oven, 4-phenyl-4H-dithiopheno[3,2-b:2',3'-d]pyrrole-2-carboxaldehyde A2 was obtained. Under ice-water bath conditions, N-bromosuccinimide and A2 were dissolved in tetrahydrofuran (THF) and reacted at room temperature in the dark for 12 hours. After the reaction was completed, the solvent was directly evaporated to obtain the product. The product was then dried in a vacuum drying oven to obtain 6-bromo-4-phenyl-4H-dithieno[3,2-b:2',3'-d]pyrrole-2-carboxaldehyde A3. 4-(diphenylamino)phenylboronic acid and A3 were sonicated to fully dissolve them in tetrahydrofuran (THF). Potassium carbonate aqueous solution and tetra(triphenylphosphine)palladium were added and sonicated to dissolve them. After nitrogen protection, the mixture was refluxed for 22 hours. After the reaction was completed, the reaction solution was cooled to room temperature and evaporated to dryness to obtain the crude product. The crude product was separated and purified by column chromatography and dried to obtain 6-(4-(diphenylamino)phenyl)-4-phenyl-4H-dithiopheno[3,2-b:2',3'-d]pyrrole-2-carboxaldehyde A4. 4-Pyridineacetonitrile and 4-bromomethylphenylboronic acid pinacol ester were dissolved in acetonitrile and sonicated until completely dissolved. The reaction mixture was then heated under reflux for 12 hours. After the reaction was completed, the mixture was cooled to room temperature and recrystallized with diethyl ether to precipitate a solid precipitate. The precipitate was collected by filtration and dried in a vacuum drying oven to obtain 4-(cyanomethyl)-1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)benzyl)pyridine-1-onium bromide B1. Add A4 and B1 to anhydrous ethanol, sonicate to dissolve completely, and reflux for 5 hours; at the end of the reaction, cool the reaction solution to room temperature and evaporate to dryness to obtain crude product, which is separated and purified by column chromatography, and dried to obtain black solid (Z)-4-(1-cyano-2-(6-(4-(diphenylamino)phenyl)-4-phenyl-4H-dithieno[3,2-b:2',3'-d]pyrrolo-2-yl)vinyl)-1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)benzyl)pyridine-1-onium bromide (DTBP); The synthesis path is as follows: 。

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