A simultaneous response to ClO - Tetraphenylethylene-aromatic ring hybrid fluorescent probes with varying viscosity, their preparation methods, and applications

Abstract

This invention belongs to the field of fluorescent probes and discloses a probe that can simultaneously respond to ClO ‑ Tetraphenylethylene-thiophene hybrid fluorescent probes with varying viscosity and their preparation methods and applications. The probes utilize a tetraphenylethylene-aromatic ring hybrid structure as the chromophore, with dinitrile isophorone and diaminomaleitrile as ClO₂ groups, respectively. ‑ Based on the recognition group, fluorescent probe molecules were designed and synthesized, with the following general structural formula: This type of fluorescent probe is effective against ClO₂. ‑ It exhibits rapid response, high specificity, and high sensitivity, with a strong responsiveness to viscosity, and can simultaneously detect ClO. ‑ Changes in viscosity levels and the environment provide new insights and potential detection tools for the early diagnosis of atherosclerosis.

Description

Technical Field

[0001] This invention relates to the field of fluorescent probes, specifically to a novel fluorescent probe based on a tetraphenylethylene-aromatic ring hybrid structural unit for detecting changes in HClO levels and viscosity, its preparation method, and its application. Background Technology

[0002] Hypochlorous acid / hypochlorite ion (HClO / ClO) - Hydrogen peroxide (H₂O₂) is an endogenously produced reactive oxygen species, catalyzed by myeloperoxidase (MPO) and the reaction of hydrogen peroxide (H₂O₂) with chloride ions (Cl₂). - Hypochlorous acid (HClO) is produced through a process where its strong oxidizing properties protect the body from microbial invasion. Abnormal hypochlorous acid levels can react with numerous molecules in the body, such as DNA, RNA, fatty acids, cholesterol, and proteins, thereby promoting the progression of a range of diseases, including cardiovascular disease, osteoarthritis, neurodegeneration, and cancer. Therefore, detecting changes in HClO levels in the body is crucial for further research into the complex relationship between HClO and disease processes.

[0003] Cell viscosity is an important indicator of the cellular microenvironment, closely related to organismal homeostasis, and plays a crucial role in signal transduction, regulation of intracellular diffusion, transport, and interactions of biomolecules. Abnormal cell viscosity can lead to serious diseases such as inflammation, fatty liver, Alzheimer's disease, and Parkinson's syndrome, most of which are accompanied by changes in reactive oxygen species (ROS), including HClO. Therefore, there is an urgent need for a method that can simultaneously detect ROS levels and changes in cell viscosity.

[0004] Atherosclerosis (AS) is considered a chronic inflammatory condition and a major contributing factor to many cardiovascular and cerebrovascular diseases. Current detection methods mostly only provide accurate diagnosis in the middle or late stages of the disease, often causing patients to miss the optimal treatment window. Foam cells (FCs) have been reported to form in large numbers in the early stages of AS and are a recognized biomarker for the disease. FC formation is accompanied by the production of large amounts of HClO and the accumulation of lipid droplets, leading to increased cell viscosity. Therefore, developing a convenient, sensitive, and rapid method for detecting HClO and cell viscosity is essential for the early diagnosis of AS. In recent decades, fluorescent probe technology has become increasingly sophisticated, with its unique advantages such as high sensitivity, specificity, in-situ detection, and high spatiotemporal resolution making it a powerful tool for analysis and detection. However, there are currently no reports on dual-responsive fluorescent probes for the early diagnosis of AS, specifically targeting both HClO and cell viscosity. Therefore, developing a novel dual-responsive fluorescent probe for AS represents a significant innovative challenge. Summary of the Invention

[0005] Based on the aforementioned research background, this invention designs and synthesizes a dual-response probe for HClO and cell viscosity based on tetraphenylethylene-aromatic ring hybridization, used to detect changes in reactive oxygen species levels and viscosity under different physiological states of cells. Therefore, the purpose of this invention is to provide a dual-response fluorescent probe for HClO and cell viscosity and its preparation method, and another objective is to provide applications of this probe in analytical and biological detection.

[0006] To achieve the objective of this invention, tetraphenylethylene and aromatic aldehydes are linked by carbon-carbon single bonds to form a viscosity-responsive molecular rotor. This is achieved by introducing dinitrile isophorone and diaminomaleonitrile to influence the viscosity of ClO₂. - Specific identification.

[0007] The general structural formula of the fluorescent probe of the present invention is as follows:

[0008]

[0009] The above ClO based on tetraphenylethylene-aromatic ring hybridization - The synthesis route for the dual-responsive fluorescent probe based on cell viscosity is as follows:

[0010]

[0011] Step I: Weigh malononitrile and isophorone, and carry out a condensation reaction at reflux temperature using anhydrous toluene as solvent and ammonium acetate and acetic acid as catalysts to obtain dinitrile isophorone 1;

[0012] Step II: Dissolve 4,4'-disubstituted benzophenone and 4-bromobenzophenone in anhydrous tetrahydrofuran, add Zn powder, place the reaction in a mixed solution of dry ice and acetone, slowly add titanium tetrachloride dropwise, and react at room temperature after the addition is complete. After the system stabilizes, carry out the McMurry coupling reaction at reflux temperature to obtain intermediate 2.

[0013] Step III: Dissolve intermediate 2 in tetrahydrofuran, add a catalytic amount of Pd(dppf)Cl2 and initiate the catalyst with potassium carbonate solution, heat and stir until the catalyst is activated, then add 5-aldehyde-2-thiopheneboronic acid, and carry out the Suzuki-Miyaura coupling reaction at reflux temperature to obtain intermediate 3.

[0014] Step IV: Dissolve intermediates 1 and 3 obtained above in anhydrous ethanol, add piperidine dropwise, and carry out Knoevenagel condensation reaction at reflux temperature. After purification by column chromatography, the target fluorescent probe 4 is obtained.

[0015] Step V: Diaminomaleitrile and intermediate 3 were dissolved in anhydrous ethanol, glacial acetic acid was added, and Knoevenagel condensation reaction was carried out at reflux temperature. After purification by column chromatography, the target fluorescent probe 5 was obtained.

[0016] After comparing the absorption, emission wavelengths, and fluorescence quantum yields of the above probe molecules, two probe molecules, 3d and 6d, with the best optical properties and cell viscosity response were selected, and their structural formulas are shown below:

[0017]

[0018] Detection of ClO in solution using 3d and 6d probe molecules of the present invention - The level includes the following steps:

[0019] (1) Prepare 3d and 6d stock solutions (40 μM) of fluorescent probes using dimethyl sulfoxide; prepare a series of NaClO solutions (30-600 μM) with different concentration gradients using deionized water.

[0020] (2) The prepared fluorescent probe stock solution was mixed with a series of NaClO solutions of varying concentrations to achieve a final concentration of 25 μM. Fluorescence emission was measured using a fluorescence spectrophotometer. When excess NaClO solution was added, the fluorescence emission peak of probe molecule 3d at 642 nm gradually decreased, and a new emission peak appeared at 506 nm; the fluorescence emission peak of probe molecule 6d at 651 nm gradually decreased, and a new emission peak appeared at 491 nm. Simultaneously, the solution color gradually changed from red to yellow, thus detecting the ClO content in the solution. - The detection limits of the fluorescent probe at 3d and 6d were calculated to be 15.3 μM and 8.16 μM, respectively.

[0021] The method for detecting solution viscosity using 3d and 6d probe molecules of the present invention includes the following steps:

[0022] (1) Prepare stock solutions (20 mM) of fluorescent probes at 3d and 6d and after their reaction with NaClO using dimethyl sulfoxide; prepare a series of mixed solutions with different viscosity gradients using dimethyl sulfoxide and glycerol.

[0023] (2) The prepared stock solutions were diluted with dimethyl sulfoxide-glycerol mixture to a final probe molecule concentration of 25 μM, and their fluorescence emission was measured using a fluorescence spectrophotometer. The results showed that the 3d probe molecule was more sensitive to viscosity; as the glycerol content gradually increased, the fluorescence emission of the 3d probe molecule at 642 nm gradually increased; the fluorescence emission of the solution after the probe molecule reacted with NaClO gradually increased. Calculations showed that the fluorescence intensity of the probe molecule increased by 22.9 times and 21.5 times before and after the reaction with NaClO when the viscosity was between 2.2 cp and 510.3 cp, respectively, thus the viscosity of the solution was determined.

[0024] For intracellular ClO - Viscosity detection imaging

[0025] (1) A549 cells and RAW 264.7 cells were selected for experiments. Different concentrations of fluorescent probes 3d and 6d were incubated with the cells, and the imaging in the red channel was observed using a fluorescence microscope after a period of time. Comparison of the fluorescence and bright-field imaging results in the red channel revealed that probe molecule 3d crystallized and precipitated between cells, while probe molecule 6d had good cell membrane permeability and could pass through the cell membrane for staining and imaging. Therefore, probe molecule 6d was selected for subsequent cell detection and imaging experiments.

[0026] (2) Detection of intracellular viscosity changes: Cells were divided into a blank group and an experimental group with abnormal cell viscosity caused by monensin treatment. The probe 6d was co-incubated with the blank group and the experimental group, respectively, and the imaging results in the red channel were observed after a period of time. It was observed that the red fluorescence in the experimental group increased significantly and increased with the increase of monensin treatment concentration, indicating that the probe 6d can detect and image abnormal changes in intracellular viscosity.

[0027] (3) Detection of endogenous and exogenous ClO in imaging cells - ① Cells were incubated with different concentrations of NaClO. After a period of time, excess NaClO was washed off, and the cells were incubated for another 6 days. After a period of incubation, the imaging within the green channel was observed using a confocal microscope. The results showed that the fluorescence intensity within the green channel increased with increasing NaClO concentration. ② Cells were divided into a control group and an experimental group. In the experimental group, lipopolysaccharide (LPS) induced an inflammatory response. N-acetylcysteine ​​(NAC) was added to one group of experimental cells to scavenge reactive oxygen species (ROS). Cells were then incubated for 6 days, and the imaging within the green channel was observed using a confocal microscope. The results showed that the green fluorescence intensity in LPS-induced inflammatory cells significantly increased, and the fluorescence intensity significantly decreased after removal with NAC. These results indicate that the 6-day probe molecule can specifically detect endogenous and exogenous HClO in imaging cells.

[0028] (4) Detection and imaging of foam cells. Cells were co-incubated with oxidized low-density lipoprotein to induce cell differentiation into foam cells (FCs). Nile River staining was used to verify the successful induction of FCs. The probe molecule 6d was co-incubated with FCs, and the imaging was observed using confocal microscopy after a period of time. The results showed that compared with normal cells, the fluorescence intensity in the green and red channels of FC cells was significantly increased. This result confirms that 6d can effectively distinguish normal cells from foam cells through synchronous changes in viscosity and HClO, providing a new approach and a potential detection tool for the early diagnosis of atherosclerosis.

[0029] The innovation of this invention lies in the fact that the 3d and 6d compounds of this invention use a tetraphenylethylene-thiophene hybrid structure as the chromophore and molecular rotor, and the methoxy and hydroxyl groups as auxochromes together with dinitrile isoflavone to form a strong push-pull electron system, which greatly enhances the fluorescence emission wavelength. The dinitrile group site serves as the recognition site for HClO, enabling the probe to simultaneously detect changes in the levels of HClO and cell viscosity. This provides a new approach and a promising detection tool for exploring complex cellular events in life activities and for the early diagnosis of AS.

[0030] This invention has the following advantages: the probe molecule undergoes fluorescence quenching in a low-viscosity environment, and exhibits gradually enhanced near-infrared fluorescence emission as the environmental viscosity increases; it reacts with ClO... - After interaction, the emission wavelength undergoes a significant blue shift, emitting yellow-green fluorescence. The fluorescence intensity increases significantly with increasing environmental viscosity. This type of fluorescent probe is effective against ClO₂. - It exhibits rapid response, strong anti-interference ability, high specificity, and high sensitivity, with a strong response to viscosity, and can simultaneously detect ClO. - The probe molecule 6d exhibits good cell membrane permeability, specifically responding to changes in intracellular viscosity and exogenous HClO levels without interference between these changes. 6d can detect changes in endogenous HClO levels in imaging inflammatory cells. Furthermore, 6d can effectively distinguish between normal cells and foam cells by observing simultaneous changes in viscosity and HClO, providing a new approach and a promising detection tool for the early diagnosis of atherosclerosis. In addition, the probe synthesis method is simple, the reaction is green and safe, the raw materials are inexpensive and readily available, and it is easy to promote. Attached Figure Description

[0031] Figure 1 These are the UV spectra of the fluorescent probe of this invention at 3d and 6d.

[0032] Figure 2 The fluorescence spectra of the fluorescent probe of this invention at 3d and 6d are shown.

[0033] Figure 3 The figures show the fluorescence intensity curves of the fluorescent probes of the present invention at 3d and 6d versus NaClO concentration, where (a) is the fluorescence intensity curve of probe 3d at 382nm for different NaClO concentrations; (b) is the linear relationship between the fluorescence intensity of probe 3d and NaClO concentration; (c) is the fluorescence intensity curve of probe 6d at 331nm for different NaClO concentrations; and (d) is the linear relationship between the fluorescence intensity of probe 6d and NaClO concentration.

[0034] Figure 4The image shows the viscosity response of the fluorescent probe 3d before and after recognizing HClO, where (a) is the fluorescence intensity curve of probe 3d at different viscosities; (b) is the linear relationship between the fluorescence intensity and viscosity of probe 3d; (c) is the fluorescence intensity curve of probe 3d after reacting with NaClO at different viscosities; and (d) is the linear relationship between the fluorescence intensity and viscosity of probe 3d after reacting with NaClO.

[0035] Figure 5 The image shows the viscosity response of the fluorescent probe 6d before and after recognizing HClO. (a) shows the fluorescence intensity curve of probe 6d at different viscosities; (b) shows the linear relationship between the fluorescence intensity and viscosity of probe 6d; (c) shows the fluorescence intensity curve of probe 6d after reacting with NaClO at different viscosities; and (d) shows the linear relationship between the fluorescence intensity and viscosity of probe 6d after reacting with NaClO.

[0036] Figure 6 Anti-interference experiments of the fluorescent probe of this invention at 3d and 6d;

[0037] Figure 7 pH stability experiments of the fluorescent probe of this invention at 3d and 6d;

[0038] Figure 8 To investigate the cell penetration ability of the fluorescent probe of this invention at 3d;

[0039] Figure 9 To investigate the cell penetration ability of the fluorescent probe of this invention at 6 days;

[0040] Figure 10 The image shows the cell viscosity detection imaging of the fluorescent probe of the present invention after 6 days. (A) shows the fluorescence imaging in the red channel of cells treated with different concentrations of monensin and incubated with the probe for 6 days; (B) and (C) show the fluorescence intensity quantification in the red channel of A549 and RAW 264.7 cells, respectively.

[0041] Figure 11 The image shows the detection of endogenous HClO in cells by the fluorescent probe of the present invention after 6 days. (A) shows the fluorescence imaging in the green channel of cells treated with different concentrations of NaClO and incubated with the probe for 6 days. (B) and (C) show the fluorescence intensity quantification in the green channel of A549 cells and RAW264.7 cells, respectively.

[0042] Figure 12 The image shows the detection of exogenous HClO in cells using the fluorescent probe of this invention for 6 days. (A) shows the fluorescence imaging in the blue and green channels of cells in the blank group, inflammation group, and NAC clearance group after incubation with the probe for 6 days; (B) and (C) show the quantification of fluorescence intensity in the green channel of RAW 264.7 cells and A549 cells, respectively.

[0043] Figure 13 The above describes the imaging of foam cells using the fluorescent probe 6d of this invention, where (A) is the fluorescence imaging of cell differentiation induced by different concentrations of ox-LDL; (B) is the quantification of fluorescence intensity in the red channel in (A); (C) is the fluorescence imaging of normal cells and FC cells in the three color channels after incubation with the probe 6d; and (D) is the quantification of fluorescence intensity in the green and red channels of normal cells and FC cells. Detailed Implementation

[0044] The invention is further illustrated by the following specific examples, but it should be noted that the scope of the invention is not limited to these embodiments in any way. Unless otherwise specified, all percentages mentioned below are mass percentages.

[0045] Example 1: Preparation of dinitrile isophorone (intermediate 1)

[0046] Isophorone (2.17 mL, 1 eq) was placed in a 50 mL round-bottom flask, and 30 mL of anhydrous toluene was added. Malononitrile (2.87 g, 3 eq) was weighed and added to the flask. Glacial acetic acid (125 μL, 0.15 eq) was added dropwise with stirring, followed by ammonium acetate (265 mg, 0.3 eq). The mixture was refluxed in an oil bath at 80 °C for 8 h. The reaction was monitored by TCL. After the reaction was complete, the mixture was cooled, the solvent was evaporated under reduced pressure, and the product was purified by column chromatography to obtain 2.21 g of a white solid, with a yield of 81.9%.

[0047] Preparation of intermediates 3a and 6a in Example 2

[0048] Weigh 4,4'-disubstituted benzophenone (1 eq) and 4-bromobenzophenone (1.2 eq) into a 100 mL round-bottom flask, add 50 mL of anhydrous tetrahydrofuran, and weigh zinc powder (5 eq) into the flask. Pre-cool to -78 °C in a dry ice-acetone mixture, and slowly add titanium tetrachloride (4 eq) dropwise over an ice bath, completing the addition within 10 min. Stir at room temperature for 30 min, and after the system stabilizes, reflux at 80 °C for 3 h. Monitor the reaction by TLC. After the reaction is complete, cool to room temperature, evaporate part of the tetrahydrofuran under reduced pressure, transfer the reaction system to a beaker in an ice bath, add 200 mL of deionized water, adjust the pH to pH = 7 with NaHCO3, extract three times with dichloromethane, combine the organic phases, wash three times with saturated NaCl solution, add anhydrous MgSO4 to the organic phase for drying, filter, concentrate the organic phase, and purify by column chromatography to obtain 3a and 6a, with yields of 40.3% and 30.4%, respectively.

[0049] Intermediate 3a, pale yellow solid, yield: 40.3%, melting point: 139.1-146.5℃. 1HNMR (400MHz, CDCl3) δ7.23–7.19(m,2H),7.11–7.08(m,2H),7.09(t,J=1.8Hz,1H),7.00(d,J=2.2Hz,1H),7.00–6 .97(m,1H),6.95–6.90(m,4H),6.90–6.85(m,2H),6.68–6.64(m,2H),6.64–6.60(m,2H),3.75(s,3H),3.72(s,3H). 13 CNMR(101MHz, CDCl3)δ158.30,158.21,143.81,143.32,140.80,137.93,136.04,135.95,133.06(*2),132.57(*2),132.54(* 2),131.36(*2),130.87(*2),127.83(*2),126.32,119.98,113.23(*2),113.05(*2),55.14,55.10.HR-MS(ESI):Calculated for C 28 H 24 BrO2[M+H] + :471.0960,found 471.0946.

[0050] Intermediate 6a, pale white solid, yield: 30.4%, melting point: 191.2-197.4℃. 1 HNMR (400MHz, DMSO) δ9.43(s,1H),9.39(s,1H),7.30(d,J=8.5Hz,2H),7.13–7.09(m,2H),7.07(dd,J=6.7,3.2H z,1H),6.97(d,J=6.9Hz,2H),6.89(d,J=8.5Hz,2H),6.79(dd,J=8.5,6.0Hz,4H),6.56(dd,J=16.9,8.6Hz,4H). 13 C NMR(101MHz,DMSO)δ156.63,156.54,144.20,144.00,141.90,136.86,134.37,134.30,133.42(*2),132.62(*2),132 .56(*2),131.31(*2),131.14(*2),129.90,128.32(*2),119.57,115.25(*2),115.08(*2).HR-MS(ESI):Calculated for C 26 H 20BrO2[M+H] + :443.0647,found 443.0601.

[0051] Example 3 Preparation of intermediates 3b and 6b

[0052] Weigh intermediate 3a / 6a (1 eq) and Pd(dppf)Cl2 (0.1 eq) separately into a 100 mL two-necked flask, add 30 mL of tetrahydrofuran, and weigh K2CO3 (6 eq) and dissolve it in 10 mL of deionized water.

[0053] Add to a flask, stir at 60°C for 30 min under N2 protection, weigh 1.5 eq of 5-aldehyde-2-thiophene boric acid, dissolve in 10 mL of tetrahydrofuran, and slowly add dropwise to the reaction system, controlling the addition to be completed within 5 min. Heat to 80°C and reflux overnight, cool to room temperature, evaporate tetrahydrofuran under reduced pressure, extract three times with dichloromethane, combine the organic phases, wash three times with saturated NaCl solution, add anhydrous MgSO4 to the organic phase to dry, filter, concentrate the organic phase, and purify by column chromatography to obtain 3b and 6b.

[0054] Fluorophore 3b, yellow solid, yield: 77.9%, melting point: 138.6-146.7℃. 1 H NMR(400MHz, CDCl3)δ9.85(s,1H),7.69(d,J=4.0Hz,1H),7.45–7.40(m,2H),7.33(d,J=4.0Hz,1H),7.15–7.09(m,3H),7.09–7.05(m,2 H),7.03(dd,J=7.6,1.9Hz,2H),7.00–6.95(m,2H),6.95–6.91(m,2H),6.70–6.65(m,2H),6.65–6.61(m,2H),3.74(s,3H),3.74(s,3H). 13 C NMR(101MHz, CDCl3)δ182.72,158.40,158.27,154.38,145.98,143.87,142.00,141.24(*2),138.19,137.46,136.0,132.64(*2),132.61(*2), 132.19(*2),131.42(*2),130.49,127.88(*2),126.38,125.66(*2),123.75,113.25(*2),113.05(*2),55.14,55.11.HR-MS(ESI):Calculated for C 33 H 27 O3S[M+H]+ :503.1681, found 503.1669.

[0055] Fluorophore 6b, yellow solid, yield: 63.2%, melting point: 216.3-222.5℃. 1 H NMR (400MHz, DMSO) δ9.88(s,1H),9.40(s,1H),9.37(s,1H),8.00(d,J=4.0Hz,1H),7.67(d,J=4.0Hz,1H),7.58(d,J=8.4Hz,2H),7.13(dt, J=13.8,6.9Hz,3H),7.01(d,J=8.4Hz,2H),6.99–6.93(m,2H),6.81(d,J=8.5Hz,2H),6.76(d,J=8.5Hz,2H),6.53(dd,J=16.3,8.6Hz,4H). 13 C NMR(101MHz,DMSO)δ184.38,156.68,156.52,152.96,146.22,144.30,142.10,142.08,139.75,137.27,134.40,134.38,132.63(*2),132 .58(*2),132.22(*2),131.36(*2),130.29,128.37(*2),126.66,126.01(*2),125.44,115.26(*2),115.05(*2).HR-MS(ESI):Calculated for C 31 H 23 O3S[M+H] + :475.1368,found 475.1339.

[0056] Example 4: Preparation of fluorescent probes at 3d and 6d

[0057] Intermediates 3b / 6b (1 eq) and dinitrile isophorone (1.2 eq) were weighed separately and placed in 50 mL round-bottom flasks. 35 mL of anhydrous ethanol was added, and two drops of piperidine were added dropwise with stirring. The mixture was refluxed overnight in an oil bath at 80 °C. After cooling to room temperature, the solvent was evaporated under reduced pressure, and the residues were purified by column chromatography to obtain 3d and 6d.

[0058] Probe molecule 3d, dark reddish-brown solid, yield: 92.9%, melting point: 171.2-178.4℃. 1HNMR (400MHz, CDCl3) δ7.36 (d, J = 8.4Hz, 2H), 7.21 (d, J = 3.9Hz, 1H), 7.15 (d, J = 5. 7Hz,1H),7.13–7.11(m,3H),7.09(d,J=7.3Hz,1H),7.06–7.02(m,4H),7.00–6.97 (m,2H),6.93(d,J=8.7Hz,2H),6.79(s,1H),6.74(d,J=15.8Hz,1H),6.65(dd,J=1 4.1,8.8Hz,4H),3.75(s,3H),3.74(s,3H),2.58(s,2H),2.42(s,2H),1.07(s,6H). 13 C NMR (101MHz, CDCl3) δ168.82,158.33,158.21,153.53,147.23,144.88,143.99,140.92(*2),1 40.42,138.39,136.16,132.65(*2),132.62(*2),132.09(*2),131.44(*2),131.21,131.02,1 29.98,128.06,127.75(*2),126.32,125.05(*2),123.96,123.02,113.74,113.22(*2),113.0 4(*2),112.94,77.93,55.14,55.11,42.95,39.16,32.01,28.04(*2).HR-MS(ESI):Calculated for C 45 H 39 N₂O₂S[M+H] + :671.2732,found 671.2713.

[0059] Probe molecule 6d, red solid, yield: 81.4%, melting point: 211.6-218.3℃. 1HNMR (400MHz, DMSO) δ9.38 (s, 1H), 9.34 (s, 1H), 7.52–7.50 (d, J = 3.1Hz, 1H), 7. 50–7.02(d,J=8.4,1H),7.48–7.45(m,2H),7.42–7.40(m,1H),7.16–7.13(m,2H ),7.12–7.07(m,2H),6.99–6.95(m,4H),6.87–6.85(m,1H),6.79(s,2H),6.73( s,2H),6.55(s,2H),6.51–6.48(m,2H),2.60(s,2H),2.51(s,2H),1.01(s,6H). 13 CNMR(101MHz,DMSO)δ170.38,156.61,156.46,155.95,146.18,145.00,144.38,141.79 ,141.13,137.41,134.48(*2),132.62(*4),132.57(*2),132.15,131.37(*2),131.25,1 31.02,128.93,128.35(*2),126.63,125.48,125.21(*2),122.84,115.22(*2),115.04 (*2),114.53,113.64,76.14,42.69,38.57,32.11,27.89(*2).HR-MS(ESI):Calculated for C 43 H 33 N2O2S[MH] - :641.2268,found 641.2269.

[0060] Example 5: Determination of probe molecular spectra

[0061] The prepared probe stock solution was mixed with an equal volume of deionized water to achieve a final probe molecule concentration of 20 μM. 4 mL of the fluorescent probe solution (DMSO:H₂O = 1:1, pH = 7.4) was added to a bi-directionally transparent quartz cuvette, and its UV absorption spectrum was measured using a UV-Vis spectrophotometer. The prepared probe stock solution was then mixed with an equal volume of NaClO solution (800 μM) to achieve a final probe molecule concentration of 25 μM. 4 mL of the probe solution mixture was added to a bi-directionally transparent quartz cuvette, and its UV absorption spectrum was measured using a UV-Vis spectrophotometer. The measurement results are as follows: Figure 1 As shown. According to Figure 1 The maximum UV absorption wavelength of the fluorescent probe was observed to be 486 nm on day 3, which is similar to that of ClO. -After the reaction, the emission wavelength blue-shifted to 382 nm; the maximum UV absorption wavelength of the fluorescent probe at 6 days was 491 nm, which is similar to that of ClO. - The emission wavelength is blue-shifted to 311 nm after the reaction.

[0062] Example 6: Determination of fluorescence emission spectra of probe molecules

[0063] The prepared probe stock solution was mixed with an equal volume of NaClO solution (800 μM) to achieve a final probe molecule concentration of 25 μM. 4 mL of the mixed probe solution was added to a four-sided transparent quartz cuvette, and the fluorescence emission of the probe solution was measured using a fluorescence spectrophotometer. The fluorescence emission spectra are shown below. Figure 2 As shown. According to Figure 2 Fluorescent probe 3d was observed to have a fluorescence emission peak at 642 nm. After adding excess NaClO, the fluorescence emission of the probe molecule at 642 nm disappeared, and a new emission peak appeared at 506 nm. Fluorescent probe 6d had a fluorescence emission peak at 651 nm. After adding excess NaClO, the fluorescence emission of the probe molecule at 651 nm disappeared, and a new emission peak appeared at 516 nm.

[0064] The detection limit is an important indicator for evaluating the sensitivity of a fluorescent probe. To obtain the 3-day and 6-day detection limits of the fluorescent probe for ClO₂... - To determine the detection limit, the prepared fluorescent probe stock solution was mixed with equal volumes of NaClO solutions at a series of concentration gradients (50-800 μM) to achieve a final probe molecule concentration of 20 μM. The fluorescence intensity of the samples at different concentration gradients within the yellow region was measured using a fluorescence spectrophotometer. The NaClO concentration gradients were plotted on the x-axis, and the fluorescence emission intensity on the y-axis. Origin was used to fit the plots, and the results are shown below. Figure 3 As shown. According to Figure 3 It was observed that probe molecules 3d and 6d both reacted with ClO - It exhibits a good linear relationship, and the linear equation for 3d is: I 382 =0.213c ClO- -2.43, the squared correlation coefficient is R 2 =0.984; The linear equation for 6d is: I 331 =0.695c ClO- +17.2, the squared correlation coefficient is R 2 =0.989. The effects of probes 3d and 6d on ClO were calculated using the formula. - The limit of detection (LOD) is calculated using the formula LOD = 3s / k, where s is the standard deviation of 10 consecutive probe measurements and k is the slope of the linear equation. This yields a detection limit of 15.3 μM for probe 3 days and 8.16 μM for probe 6 days.

[0065] Example 7: Determination of the viscosity response curve of the probe molecule

[0066] Viscosity response curves are an important indicator for evaluating the performance of viscosity-responsive probes. To determine the viscosity response curves of probe molecules, a series of probe solutions with viscosity gradients were prepared by mixing the prepared probe stock solution and the probe stock solution after reaction with NaClO with glycerol. Fluorescence curves of samples with different viscosity gradients were measured using a fluorescence spectrophotometer. The logarithms of different viscosity coefficients were plotted on the x-axis, and the logarithms of fluorescence emission intensity were plotted on the y-axis. Origin was used to fit the graphs, and 3-day and 6-day viscosity response line graphs were generated. The results are shown below. Figure 4 , Figure 5 As shown. According to Figure 4 , Figure 5 The probe molecule 3d was observed to exhibit a good linear relationship with viscosity, and the linear equation is: lgI 486 =0.555lgη+0.289; the squared correlation coefficient is R 2 =0.989. The sensitivity of probe molecules 3d and 6d to viscosity increased significantly (k value increased) after reacting with NaClO, which is beneficial for detecting the synchronous changes of the two.

[0067] Example 8: Probe Selectivity and Interference Resistance Experiment

[0068] Due to the complex composition of the internal environment, certain amino acids and metal ions may interfere with probe detection. To verify the selectivity and anti-interference ability of probes at 3d and 6d, 50 μM probe stock solutions were prepared, and the following analytes (2 mM) that may cause interference were added respectively: (1) blank, (2) Cys, (3) Hcy, (4) GSH, (5) Phe, (6) Glu, (7) Alu, (8) Arg, (9) Lys, (10) Pro, (11) Try, (12) NaCl, (13) MgCl2, (14) ZnCl2, (15) FeSO4, (16) CuSO4, (17) NaBr, (18) KI, (19) Na2S2O3, (20) KO2 (500 μM), (21) H2O2, (22) 1 O2(10mM NCS+2mM H2O2),(23)·OH(10mM Fe 2+ +2mM H2O2), (24) NaClO, (25) glycerol (90%), to make the final probe concentration 25μM. Fluorescence emission of the samples at different excitation wavelengths was measured using a fluorescence spectrophotometer, and the results are as follows: Figure 6 As shown. According to Figure 6 The probe molecules 3d and 6d were observed to respond only to NaClO and glycerol, with no significant response to other reactive oxygen species, indicating that the probe molecules are resistant to ClO.- It exhibits extremely high specificity in terms of viscosity.

[0069] Example 9: pH Sensitivity Experiment of the Probe

[0070] Solutions with pH values ​​of 1-14 were prepared by diluting concentrated HCl and NaOH with deionized water. The probe stock solution was then diluted with solutions of different pH values ​​to achieve a final probe concentration of 25 μM. Fluorescence intensity was recorded at different time points using a fluorescence spectrophotometer. The results were plotted using Origin software, as shown below. Figure 7 As shown in the figure, the 3d and 6d probe molecules exhibit stable fluorescence properties within the normal physiological pH range (7-9), demonstrating their ability to perform cell imaging.

[0071] Example 10: Cellular fluorescence imaging experiment using probes

[0072] The probe can specifically respond to ClO in solution at 3d and 6d. - And viscosity changes, to identify ClO - Subsequently, the near-infrared fluorescence emission shifted from blue to yellow; with increasing environmental viscosity, the fluorescence emission intensity significantly increased. This study aimed to investigate the detection of intracellular ClO₂ by the probe at 3d and 6d. - The ability of probe molecules to detect changes in viscosity was investigated using A549 and RAW 264.7 cells as test cells.

[0073] (1) First, the cell penetration ability of the probe molecules was investigated. Different concentrations of fluorescent probes were incubated with cells for 4 hours after 3 days, and the imaging in the red channel was observed using a fluorescence microscope. Figure 8 As shown, comparing the fluorescence and bright-field imaging results in the red channel, it was found that probe molecule 3d crystallized and precipitated between cells, indicating that probe molecule 3d is difficult to enter cells and cannot be used for intracellular detection imaging.

[0074] The cell penetration ability of probe 6d was investigated. Cells were incubated with different concentrations of the fluorescent probe 6d for 4 hours, followed by DIPA staining for 30 minutes. Imaging within the blue and red channels was observed using a confocal microscope. Figure 9 As shown, a clear cell outline was observed in the red channel, and the fluorescence intensity increased with increasing probe concentration, indicating that 6d has good cell membrane permeability and can smoothly enter the cell for imaging. Meanwhile, under normal conditions, the red fluorescence is weak, indicating low background fluorescence, which is beneficial for detecting changes in intracellular viscosity.

[0075] (2) Secondly, changes in intracellular viscosity were detected and imaged: Cells were divided into a blank group and an experimental group with abnormal cell viscosity caused by monensin treatment. The probe 6d (20μM) was co-incubated with the blank group and the experimental groups treated with different concentrations of monensin for 2 hours, and the imaging results in the red channel were observed after a period of time. For example... Figure 10 As shown, a significant increase in red fluorescence was observed in the experimental group, which was enhanced with increasing monensin treatment concentration, indicating that the probe molecule can detect and image abnormal changes in intracellular viscosity over 6 days.

[0076] (3) Subsequently, the endogenous and exogenous ClO in the imaging cells were detected. - ① After incubating cells with different concentrations of NaClO for 4 hours, excess NaClO was washed away, and the cells were incubated with 6d (20 μM) for another 2 hours. The imaging within the green channel was then observed using a confocal microscope. For example... Figure 11 As shown, the fluorescence intensity in the green channel increases with increasing NaClO concentration during incubation; ② Cells were divided into a blank group and an experimental group. Inflammatory responses were induced in the experimental group cells using 2 μg / mL LPS. 0.2 mMMN-acetylcysteine ​​(NAC) was added to one experimental group cell to scavenge reactive oxygen species (ROS). Cells were then incubated for 2 hours for 6 days, and the imaging in the green channel was observed using a confocal microscope. Figure 12 As shown, the intensity of green fluorescence in LPS-induced inflammatory cells increased significantly, and the fluorescence intensity decreased significantly after removal with NAC. These results indicate that the 6d probe molecule can specifically detect endogenous and exogenous HClO in imaging cells.

[0077] (4) Finally, the foam cells were examined. Cells were co-incubated with different concentrations of oxidized low-density lipoprotein (ox-LDL) for 24 hours to induce cell differentiation into foam cells (FCs). The success of FC induction was verified using Nile River stained lipid droplets. Figure 13 As shown in Figures A and B, when the ox-LDL concentration was 20 μg / mL, a large number of lipid droplets accumulated within the cells, proving that the cells had been induced to differentiate into FC at this concentration. Cells were divided into a control group and an experimental group. Cells in the experimental group were incubated with 20 μg / mL ox-LDL for 24 h to induce FC differentiation, and then co-incubated with probe molecules for 6 days and 2 h respectively. Imaging was observed using a confocal microscope. Figure 13 As shown in C and D, compared with normal cells, the fluorescence intensity in the green and red channels of FC cells increased significantly. This result confirms that normal cells and foam cells can be effectively distinguished by synchronous changes in viscosity and HClO at 6d, providing a new idea and a potential detection tool for the early diagnosis of atherosclerosis.

Claims

1. A tetraphenyl ethene-arene hybrid fluorescent probe which is simultaneously responsive to CIO - and viscosity, characterized by, selected from the following compounds:

2. The simultaneous response to CIO - and viscosity of tetraphenyl ethylene-arene ring hybrid fluorescent probe, characterized in that, Use of probe molecules for ClO - Detection of level and viscosity changes for non-diagnostic treatment.

3. The simultaneous response to CIO - and viscosity of tetraphenyl ethylene-arene ring hybrid fluorescent probe, characterized in that, The solution has a ClO - The horizontal change detection method comprises the following steps: (1) the fluorescent probe 3d, 6d or NaClO stock solution was prepared with dimethyl sulfoxide and PBS buffer solution respectively; (2) The prepared 3d or 6d stock solution is mixed with different concentrations of NaClO stock solution, and the wavelength is detected by using a fluorescence spectrophotometer. After the probe molecule 3d reacts with NaClO, the fluorescence emission peak at 642 nm gradually weakens, and a new emission peak gradually appears at 506 nm. After the probe molecule 6d reacts with NaClO, the fluorescence emission peak at 651 nm gradually weakens, and a new emission peak gradually appears at 491 nm. Accordingly, the specific detection of ClO - horizontal variation is realized.

4. The simultaneous response to CIO - and viscosity of tetraphenyl ethylene-arene hybrid fluorescent probe, characterized in that, The solution viscosity change detection method comprises the following steps: (1) dimethyl sulfoxide was used to prepare the stock solution of fluorescent probe 3d or 6d and the stock solution after reaction of the two with NaClO respectively; a series of mixed solvents A with viscosity gradient were prepared with dimethyl sulfoxide and glycerol; (2) the prepared fluorescent probe 3d or 6d stock solution was evenly divided and added into the mixed solvents A with different viscosities and mixed, and wavelength detection was performed by using a fluorescence spectrophotometer; the probe molecule 3d is at 642 nm, and its fluorescence emission gradually increases with the increase of the solution viscosity; the probe molecule 3d stock solution after reaction with NaClO was evenly divided and added into the mixed solvents A with different viscosities and mixed, and wavelength detection was performed by using a fluorescence spectrophotometer; the probe molecule 3d is at 506 nm, and its fluorescence emission gradually increases with the increase of the solution viscosity; the probe molecule 6d is at 651 nm, and its fluorescence emission gradually increases with the increase of the solution viscosity; the probe molecule 6d stock solution after reaction with NaClO was evenly divided and added into the mixed solvents A with different viscosities and mixed, and wavelength detection was performed by using a fluorescence spectrophotometer; the probe molecule 6d is at 516 nm, and its fluorescence emission gradually increases with the increase of the solution viscosity.

5. The simultaneous response to CIO - and viscosity of tetraphenyl ethylene-arene hybrid fluorescent probe, characterized in that, The fluorescent probe 6d is used for detecting intracellular ClO - And specific imaging of viscosity for non-diagnostic treatment, the specific method is as follows: 6d is incubated with cells, the abnormal change of intracellular viscosity is detected and imaged through the change of fluorescence intensity in the red channel; 6d is incubated with cells and different concentrations of NaClO, and the exogenous HClO detection in the imaged cells is realized through the change of fluorescence intensity in the green channel.

6. The simultaneous response to CIO - and viscosity of tetraphenyl ethylene-arene ring hybrid fluorescent probe, characterized in that, The fluorescent probe 6d is used for detecting imaging foam cells, and is used for non-diagnostic treatment, and the specific method is as follows: the fluorescent probe 6d is incubated with foam cells, and the synchronous change of the fluorescence intensity of the green channel and the red channel in the cells is used to distinguish normal cells and foam cells.

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