A bifunctional fluorescent probe based on nopinel acridine capable of simultaneously detecting hypochlorous acid and viscosity, its preparation and application.

Abstract

This invention discloses a nopinel-based bifunctional fluorescent probe capable of simultaneously detecting hypochlorous acid and viscosity, along with its preparation method and applications. The probe is 2-(2-(5-(4-(3,3-dimethyl-1,2,3,4-tetrahydro-2,4-geomylidene-acridin-9-yl)phenyl)furan-2-yl)vinyl)-6-methoxy-3-methylbenzothiazolium iodide, abbreviated as TA-PFV-TI. This probe can specifically detect the viscosity and hypochlorous acid of a solution. Under 365 nm ultraviolet light irradiation, the probe's orange fluorescence gradually intensifies with increasing solution viscosity. The linear detection range for viscosity is 1.28 cP–1411.98 cP, with a response time within 1 second and a detection limit of 1.00 cP. Upon addition of hypochlorous acid to the probe solution, its fluorescence color changes from orange to bright blue. The linear detection range for hypochlorous acid is 0–200 μM, with a response time of 5 min and a detection limit of 2 nM. The applicable pH range is 5–9. This probe, as a bifunctional fluorescent probe capable of simultaneously detecting hypochlorous acid and viscosity, exhibits numerous advantages such as good selectivity, high sensitivity, and wide applicability, demonstrating promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, and relates to a nopinel acridine bifunctional fluorescent probe that can simultaneously detect hypochlorous acid and viscosity, as well as its preparation method and application. Background Technology

[0002] Hypochlorous acid is a weak acid that, during inflammation, is produced by neutrophils via myeloperoxidase (MPO) catalysis, reacting hydrogen peroxide (H₂O₂) with chloride ions (Cl₂). - The reaction produces hypochlorous acid. As an important member of the reactive oxygen species (ROS) family, hypochlorous acid plays a crucial role in the body's immune defense, effectively killing pathogenic microorganisms such as bacteria, viruses, and fungi, and is one of the host's first lines of defense against infection. Due to its strong oxidizing properties and broad-spectrum bactericidal capabilities, hypochlorous acid and its salts are also widely used in medical disinfection, drinking water treatment, food processing, and environmental sanitation. In recent years, slightly acidic hypochlorous acid aqueous solutions with a pH of 5.0-6.5 have been widely used in clinical settings such as wound care, oral disinfection, and ophthalmic irrigation due to their advantages of low irritation to human tissues and high safety.

[0003] However, despite its important physiological functions, hypochlorous acid's abnormal accumulation or uncontrolled release in the human body can cause serious damage to biomolecules. Hypochlorous acid can oxidize methionine, cysteine, and histidine residues in proteins, disrupting enzyme activity; attack the lipid bilayer, inducing lipid peroxidation; and undergo addition or breakage reactions with DNA, inducing gene mutations. Numerous studies have shown that hypochlorous acid-mediated oxidative stress is closely linked to various inflammation-related diseases, such as atherosclerosis, rheumatoid arthritis, neurodegenerative diseases (e.g., Alzheimer's disease), chronic lung disease, and cancer. Therefore, developing highly selective and sensitive detection methods, especially fluorescent probes capable of real-time, in-situ monitoring of hypochlorous acid levels in complex biological systems, is of great significance for a deeper understanding of its biological mechanisms of action, assessing the degree of oxidative damage, and early diagnosis and treatment of related diseases. Intracellular viscosity is an important physical parameter reflecting cellular metabolic state, mitochondrial function, and pathological changes (e.g., apoptosis, fibrosis), and is often coupled with oxidative stress processes. Therefore, constructing a bifunctional fluorescent probe that can simultaneously respond to changes in hypochlorous acid and viscosity can help to more comprehensively analyze the dynamics of the microenvironment in the process of inflammation or disease.

[0004] Fluorescent probes, with their high sensitivity, excellent spatiotemporal resolution, and non-invasive detection characteristics, have become important tools for bioimaging and environmental analysis. Despite significant progress in the research and application of fluorescent probes, developing multifunctional fluorescent probes remains a challenge. To address the shortcomings of existing fluorescent probes for detecting hypochlorous acid and viscosity, such as long response time, low sensitivity, narrow detection range, high cytotoxicity, and the ability to perform only single detection, this invention aims to develop a bifunctional fluorescent probe with fast response time, high sensitivity, wide detection range, applicability to cell imaging, and the ability to simultaneously detect hypochlorous acid and viscosity. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the technical problem this invention aims to solve is to provide a nopinel-acrylidine bifunctional fluorescent probe capable of simultaneously detecting hypochlorous acid and viscosity. This probe features rapid response, high sensitivity, and a wide pH adaptability range, enabling qualitative and quantitative detection of hypochlorous acid in water, plants, and aquatic animals, and can be applied to fluorescence imaging of hypochlorous acid and viscosity in cells. Another technical problem this invention aims to solve is to provide a method for preparing this nopinel-acrylidine bifunctional fluorescent probe capable of simultaneously detecting hypochlorous acid and viscosity. A further technical problem this invention aims to solve is to provide an application of this nopinel-acrylidine bifunctional fluorescent probe capable of simultaneously detecting hypochlorous acid and viscosity.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by this invention is: a nopineyl acridine bifunctional fluorescent probe capable of simultaneously detecting hypochlorous acid and viscosity, with the following structural formula:

[0007]

[0008] The molecular formula is C 37 H 33 IN2O2S, chemically named 2-(2-(5-(4-(3,3-dimethyl-1,2,3,4-tetrahydro-2,4-geomethylacridin-9-yl)phenyl)furan-2-yl)vinyl)-6-methoxy-3-methylbenzothiazolyl iodide, abbreviated as TA-PFV-TI.

[0009] The method for preparing a nopinel-based bifunctional fluorescent probe capable of simultaneously detecting hypochlorous acid and viscosity involves reacting 5-(4-(3,3-dimethyl-1,2,3,4-tetrahydro-2,4-bridged methylene acridine-9-yl)phenyl)furan-2-carboxaldehyde with 6-methoxy-2,3-dimethylbenzothiazolium iodide to obtain the compound TA-PFV-TI.

[0010] A method for preparing a nopinel acridine-based bifunctional fluorescent probe capable of simultaneously detecting hypochlorous acid and viscosity, specifically comprising the following steps:

[0011] (1) 0.25 mmol of 5-(4-(3,3-dimethyl-1,2,3,4-tetrahydro-2,4-bridged methylene acridine-9-yl)phenyl)furan-2-carboxaldehyde, 0.25-1.00 mmol of 6-methoxy-2,3-dimethylbenzothiazolium iodide, 10-50 mL of anhydrous ethanol, and 0.15-0.5 mmol of sodium ethoxide were added sequentially to a reaction flask. After reacting at 60-70 °C for 10-30 min, the mixture was heated to reflux and the reaction was continued for 10-20 h.

[0012] (2) After the reaction is completed, the ethanol is recovered by distillation. The distillation residue is dissolved in 30-50 mL of toluene, washed with water and saturated saline until neutral, dried with anhydrous sodium sulfate and then the toluene is evaporated to obtain crude TA-PFV-TI product.

[0013] (3) The crude product of TA-PFV-TI was recrystallized to obtain the fluorescent probe TA-PFV-TI, an orange powder.

[0014] Compound TA-PFV-TI specifically reacts with hypochlorous acid. Under 365 nm UV irradiation, the probe solution changes from orange to bright blue fluorescence upon the addition of hypochlorous acid. The linear detection range for hypochlorous acid is 0-200 μM, with a response time within 5 min and a detection limit of 2 nM. The applicable pH range is 5-9. Compound TA-PFV-TI also specifically identifies solution viscosity. Under 365 nm UV irradiation, the orange fluorescence of the probe significantly increases with increasing solution viscosity. The linear detection range for viscosity is 1.28 cP-1411.98 cP, with a response time within 1 s and a detection limit of 1.00 cP.

[0015] Beneficial Effects: Compared with existing technologies, this invention uses 5-(4-(3,3-dimethyl-1,2,3,4-tetrahydro-2,4-bridged methylene acridine-9-yl)phenyl)furan-2-carboxaldehyde as a raw material, reacting it with 6-methoxy-2,3-dimethylbenzothiazol-3-iodide to prepare the compound TA-PFV-TI. This compound can specifically recognize hypochlorous acid and viscosity, and can sensitively detect the content of hypochlorous acid and the viscosity of the solution. As a bifunctional fluorescent probe for detecting hypochlorous acid and viscosity, it has many advantages such as good selectivity, high sensitivity, and wide application range, and has good application prospects. Attached Figure Description

[0016] Figure 1 The fluorescence spectra of TA-PFV-TI reacting with hypochlorous acid of different concentrations are shown.

[0017] Figure 2 These are fluorescence spectra of TA-PFV-TI in glycerol systems of different viscosities;

[0018] Figure 3 This is a bar chart showing the fluorescence intensity ratio at 440 nm and 590 nm after TA-PFV-TI reacts with different analytes such as metal ions, anions, and common amino acids.

[0019] Figure 4 These are the fluorescence spectra of TA-PFV-TI in different solvents. Detailed Implementation

[0020] The present invention will be further described below with reference to specific embodiments.

[0021] Example 1

[0022] Preparation of TA-PFV-TI:

[0023] The reaction formula for preparing TA-PFV-TI is as follows:

[0024]

[0025] 98.4 mg of 5-(4-(3,3-dimethyl-1,2,3,4-tetrahydro-2,4-bridged methylene acridine-9-yl)phenyl)furan-2-carboxaldehyde, 48.5 mg of 6-methoxy-2,3-dimethylbenzothiazolium iodide, 15 mL of anhydrous ethanol, and 10.2 mg of sodium ethoxide were added sequentially to a reaction flask. The mixture was stirred at 60 °C for 10 min, and then the temperature was slowly increased to reflux for 10 h. After the reaction was completed, the ethanol was recovered by distillation. The distillation residue was dissolved in 40 mL of toluene, washed with water and saturated brine until neutral, dried with anhydrous sodium sulfate, and the toluene was evaporated off. The crude TA-PFV-TI product was recrystallized from the dichloromethane-petroleum ether mixture to obtain compound TA-PFV-TI, an orange powder. 1 H NMR (600MHz, DMSO) δ: 6.38 (s, 1H), 6.36 (s, 1H), 6.30 (d, J = 5.5Hz, 1H), 6.28 (s, 1H), 6. 24 (d, J=2.6Hz, 1H), 6.18 (s, 1H), 5.94 (s, 1H), 5.93-5.89 (m, 2H), 5.81 (d, J=2.2Hz, 1H ), 5.74 (s, 1H), 5.70 (s, 3H), 5.60 (s, 1H), 2.55 (s, 3H), 2.14 (s, 3H), 1.38 (d, J=2.1Hz, 2H), 1.32 (s, 1H), 0.95 (s, 1H), 0.53 (tt, J=5.9, 3.0Hz, 2H), 0.35 (s, 3H), 1.08 (s, 3H); 13C NMR (150MHz, DMSO) δ168.72, 166.55, 159.82, 158.01, 151.09, 136.78, 132.63, 130.24, 130.07, 129.04, 126.93, 126.82, 125.90, 125.42, 123.58 ,118.96,118.15,111.38,110.58,107.15,56.80,56.67,49.07,40.54, 39.33, 36.88, 36.76, 31.04, 30.35, 26.10, 21.83, 17.37.HRMS (m / z): [M] + Calculated for C 37 H 33 N2O2S + :569.23, found:569.2263.

[0026] Example 2

[0027] The compound TA-PFV-TI was prepared into 1×10 -5 Hypochlorous acid was dissolved in PBS buffer solution (pH = 7.4, 1‰ Tween 80) to prepare solutions with concentrations of 0, 20, 40, 60, 80, 100, 120, 140, 160, 180, and 200 μmol / L. The fluorescence emission spectra of the probe TA-PFV-TI in the presence of different concentrations of hypochlorous acid were measured using a fluorescence spectrophotometer by fluorescence titration. The results are shown below. Figure 1 As shown in the figure, the results indicate that as the concentration of hypochlorous acid in the solution gradually increases from 0 mol / L to 200 μmol / L, the fluorescence emission intensity of this compound at 440 nm gradually increases, while the fluorescence intensity at 590 nm gradually decreases. This suggests that this compound can be used as a ratiometric fluorescent probe for the sensitive detection of hypochlorous acid. The linear detection range for hypochlorous acid is 0-200 μM, the response time is within 5 min, the detection limit is 2 nM, and the applicable pH range is 5-9.

[0028] Example 3

[0029] TA-PFV-TI is made into 1×10 -30%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 99.9% glycerol solutions were prepared by dissolving different amounts of glycerol in PBS buffer solution (pH = 7.4). 100 μL of TA-PFV-TI PBS buffer solution was added to 10 mL of glycerol solution. The fluorescence emission spectra of TA-PFV-TI in glycerol solutions of different concentrations were measured using a fluorescence spectrophotometer. The results are shown below. Figure 2 As shown, the fluorescence emission intensity of the probe at 590 nm gradually increased as the glycerol concentration in the solution gradually increased from 0 to 99.9%. This indicates that the compound can be used as a fluorescent probe for sensitive detection of solution viscosity, with a linear detection range of 1.28 cP-1411.98 cP, a response time within 1 s, and a detection limit of 1.00 cP.

[0030] Example 4

[0031] TA-PFV-TI is formulated into 1×10 -5 A 100 μM solution was prepared by dissolving various analytes, including metal ions, anions, and common amino acids, in PBS buffer (pH = 7.4, 1‰ Tween 80). Fluorescence emission spectra of the TA-PFV-TI in the presence of these analytes were measured using a fluorescence spectrophotometer via fluorescence titration. The results are shown below. Figure 3 As shown. After the compound TA-PFV-TI reacts with hypochlorous acid, the fluorescence intensity ratio at 440 nm and 590 nm is significantly increased, while the addition of Fe... 3+ Cu 2+ Mg 2+ Ca 2+ Zn 2+ Ni 2+ Metal ions, F - I - ,Br - HS - NO 2- Comparative observations were conducted with different analytes, including anions and amino acids such as Tyr, Gln, and Cys, and it was found that these analytes had no significant effect on the fluorescence spectrum of TA-PFV-TI. This indicates that the compound can be used as a ratiometric fluorescent probe for the selective detection of hypochlorous acid.

[0032] Example 5

[0033] TA-PFV-TI is made into 1×10 -3Prepare a 100 μL PBS buffer solution (pH = 7.4) along with glycerol and other organic solvents. Add 100 μL of TA-PFV-TI in PBS buffer solution to 10 mL of the solvent. Measure the fluorescence emission spectra of TA-PFV-TI in different solvents using a fluorescence spectrophotometer, as shown below. Figure 4 As shown, this compound exhibits a distinct fluorescence emission peak at 590 nm in glycerol solution, while no significant change occurs in other solvents such as water, dimethyl sulfoxide, and tetrahydrofuran. This indicates that the compound can also be used as an open-type fluorescent probe for detecting solution viscosity.

Claims

1. A nopinel-based bifunctional fluorescent probe capable of simultaneously detecting hypochlorous acid and viscosity, its preparation method, and its application, characterized in that... The fluorescent probe is 2-(2-(5-(4-(3,3-dimethyl-1,2,3,4-tetrahydro-2,4-geomylidene-9-yl)phenyl)furan-2-yl)vinyl)-6-methoxy-3-methylbenzothiazolium iodide, abbreviated as TA-PFV-TI, and its structural formula is:

2. The method for preparing a nopinel-based bifunctional fluorescent probe capable of simultaneously detecting hypochlorous acid and viscosity, as described in claim 1, is characterized in that... Using 5-(4-(3,3-dimethyl-1,2,3,4-tetrahydro-2,4-bridged methylene acridine-9-yl)phenyl)furan-2-carboxaldehyde as a starting material, it was reacted with 6-methoxy-2,3-dimethylbenzothiazolyl iodide to obtain the compound TA-PFV-TI.

3. The method for preparing a nopinel-based bifunctional fluorescent probe capable of simultaneously detecting hypochlorous acid and viscosity according to claim 2, characterized in that, Specific preparation methods include: (1) 0.25 mmol of 5-(4-(3,3-dimethyl-1,2,3,4-tetrahydro-2,4-bridged methylene acridine-9-yl)phenyl)furan-2-carboxaldehyde, 0.25-1.00 mmol of 6-methoxy-2,3-dimethylbenzothiazolium iodide, 10-50 mL of anhydrous ethanol, and 0.15-0.5 mmol of sodium ethoxide were added sequentially to a reaction flask. After reacting at 60-70 °C for 10-30 min, the mixture was heated to reflux and the reaction was continued for 10-20 h. (2) After the reaction is completed, the ethanol is recovered by distillation. The distillation residue is dissolved in 30-50 mL of toluene, washed with water and saturated saline until neutral, dried with anhydrous sodium sulfate and then the toluene is evaporated to obtain crude TA-PFV-TI product. (3) The crude product of TA-PFV-TI was recrystallized to obtain the fluorescent probe TA-PFV-TI, an orange powder.

4. The application of the norpinel acridine bifunctional fluorescent probe as described in claim 1 in the detection of viscosity and hypochlorous acid.

5. The application according to claim 4, characterized in that, The fluorescent probe TA-PFV-TI reacts specifically with hypochlorous acid. When hypochlorous acid is added to the probe solution under 365nm UV light, its fluorescence color changes from orange to bright blue. The linear detection range for hypochlorous acid is 0-200μM, the response time is within 5min, the detection limit is 2nM, and the applicable pH range is 5-9.

6. The application according to claim 4, characterized in that, The probe TA-PFV-TI can also specifically identify solution viscosity. Under 365nm UV light irradiation, the orange fluorescence intensity of the probe increases significantly with increasing solution viscosity. The linear detection range for viscosity is 1.28cP-1411.98cP, the response time is within 1s, and the detection limit is 1.00cP.

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