A fluorescent probe for detecting HSO3 - / ClO - and viscosity, and application thereof

Abstract

The application belongs to the technical field of fluorescent probes, and particularly relates to a fluorescent probe for detecting HSO3 ‑ / ClO ‑ and viscosity, a preparation method and property research of the fluorescent probe. The preparation method of the fluorescent probe in the application is simple, the product yield is high, and the method is suitable for scale promotion and use. The probe (E) -4- (2- (6- (4- (diphenylamino) phenyl) -2, 3- dihydro-1H- xanthene-4-yl) vinyl) -3- ethylbenzothiazole-3- onium iodide (TXET) is first used to detect HSO3 ‑ , and the fluorescence intensity (I ‑ ) gradually increases with the increase of the HSO3 500 concentration. When the HSO3 ‑ concentration is within 0-0.13 millimoles per liter, a good linear relationship is obtained. When ClO ‑ is detected under a low polarity condition, the fluorescence intensity (I ‑ ) gradually increases with the increase of the ClO 530 concentration. Secondly, when the ClO ‑ concentration is within 40-80 micromoles per liter, a good linear relationship is obtained. Finally, the fluorescence intensity (I 720 ) gradually increases with the increase of the viscosity, and a good linear relationship is obtained within 7.9-880 centipoises. The biological experiment results show that the probe TXET can be used to monitor the change levels of HSO3 ‑ / ClO ‑ and viscosity in cells and animals, and can be used as a sensitive tool for identifying liver damage.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of fluorescent probes, and particularly relates to a fluorescent probe for detecting HSO3 - / ClO - and viscosity, a preparation method thereof and application thereof. BACKGROUND

[0002] Hypochlorous acid (HOCl) is one of the biologically important reactive oxygen species (ROS). Due to its sub-acidity, HOCl usually undergoes spontaneous hydrolysis reaction in neutral solution at pH 7.0, thereby forming free ClO - ion. Biologically, ClO - ion is considered to be produced in neutrophils activated by hydrogen peroxide and chloride ion under the catalysis of heme enzyme myeloperoxidase (MPO). However, abnormal production of hypochlorite can cause tissue damage and diseases, such as liver injury, atherosclerosis, lung injury, rheumatoid, cardiovascular disease, neuronal degeneration, cancer, and it is crucial to detect the level of ClO - in these diseases. Sulfur dioxide (SO2) and ClO - are closely related to redox balance. SO2 is a reducing substance in cells, mainly produced by oxidation of sulfur-containing amino acids or H2S. In addition, as an environmental pollutant, SO2 mainly exists in the form of SO3 2- / HSO3 - . Abnormal level of SO2 metabolism in cells can lead to imbalance of redox homeostasis, and imbalance of redox homeostasis is closely related to inflammation, ferroptosis and the like, and it is crucial to detect the level of SO2 in these diseases.

[0003] Cell viscosity is a basic factor for some cell processes, such as diffusion of biologically active substances and transport of signals, and is closely related to cell viscosity. Abnormal change of cell viscosity can be related to certain diseases, such as ferroptosis, kidney injury, liver injury and the like can cause increase of cell viscosity, and monitoring of cell viscosity is also crucial.

[0004] Fluorescent probes have become an indispensable tool in clinical medicine and biomedical research, because they can provide dynamic information about the localization and quantity of the biological molecules of interest in living cells, tissues and even animals. So far, a number of fluorescent probes have been reported for detecting ClO - or SO2 in biological systems, respectively. But there is no probe that can monitor ClO - , SO2 in the living organisms at the same time. In summary, it is necessary to develop a fluorescent probe for detecting HSO3 - / ClO - and viscosity, for detection of HSO3 - / ClO - and viscosity. SUMMARY

[0005] The present application is directed to a fluorescent probe for detecting HSO3 - / ClO - and viscosity, a preparation method and property research thereof. (E)-4-(2-(6-(4-(diphenylamino)phenyl)-2,3-dihydro-1H-xanthene-4-yl)vinyl)-3-ethylbenzothiazol-3-ium iodide (TXET) has the characteristics of simple synthetic route, good selectivity, high sensitivity, large Stokes shift, etc., and can effectively detect HSO3 - / ClO - and viscosity under physiological conditions.

[0006] The fluorescent probe (E)-4-(2-(6-(4-(diphenylamino)phenyl)-2,3-dihydro-1H-xanthene-4-yl)vinyl)-3-ethylbenzothiazol-3-ium iodide (TXET) in the present application has the following molecular structure:

[0007]

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

[0009]

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

[0011] Anhydrous N,N-dimethylformamide (DMF) is added with chloroform and phosphorus tribromide, stirred at 0℃ for 1 hour, then cyclohexanone is added in chloroform to mix thoroughly, the mixed solution is added to the reaction system, vacuum extraction, then reaction at room temperature for 18 hours; after the reaction is completed, the pH is adjusted to neutral with saturated sodium bicarbonate (NaHCO3), then the organic phase is extracted with dichloromethane, the organic phase is collected and rotary evaporation is performed to remove the solvent, silica gel column purification (eluent dichloromethane: ethanol = 30:1), vacuum drying to obtain a light yellow oily product 2-chloro-1-cyclooctene-1-formaldehyde (A1, yield 50.4%).

[0012] 4-(diphenylamino)phenylboronic acid and 4-bromo-2-hydroxybenzaldehyde were dissolved in THF by ultrasonic, and then potassium carbonate aqueous solution and tetrabutylammonium bromide were added and dissolved by ultrasonic. After stirring at room temperature for 30 minutes, tetrakis(triphenylphosphine)palladium was added and dissolved by ultrasonic. The reaction system was vacuumized and heated to reflux for about 6 hours. After the reaction was completed, the reaction solution was cooled to room temperature, extracted with dichloromethane, and the organic phase was collected and rotary evaporated to remove the solvent. The product, 4'-(diphenylamino)-3-hydroxy-[1,1'-biphenyl]-4-carbaldehyde (A2, yield 87.8%), was obtained in the form of fluorescent green solid by silica gel column purification (eluent: petroleum ether:dichloromethane = 4:1).

[0013] A1 and A2 were dissolved in anhydrous DMF, vacuumized, and then stirred at room temperature for 20 hours. After the reaction was completed, the reaction solution was rotary evaporated to obtain the crude product. The product, 6-(4-(diphenylamino)phenyl)-2,3-dihydro-1H-xanthene-4-carbaldehyde (A3, yield 69.6%), was obtained in the form of yellow solid by column chromatography separation and purification (eluent: petroleum ether: ethyl acetate = 15:1) and vacuum drying.

[0014] 2-methylbenzothiazole and ethyl iodide were added to ethanol and heated to reflux for about 12 hours. After the reaction was completed, the reaction solution was cooled to room temperature, a large amount of precipitate was separated and washed by filtration, and then dried in a vacuum drying box to obtain 3-ethyl-2-methylbenzothiazolium iodide (A4, yield 86.4%) in the form of off-white solid.

[0015] A3 and A4 were dissolved in anhydrous ethanol and ultrasonically dissolved. After being heated to reflux for 8 hours, the reaction solution was cooled to room temperature, a precipitate was generated, and then the precipitate was filtered and washed with ethanol. After drying, the product, (E)-4-(2-(6-(4-(diphenylamino)phenyl)-2,3-dihydro-1H-xanthen-4-yl)vinyl)-3-ethylbenzothiazolium iodide (TXET, yield 62.4%), was obtained in the form of deep blue solid.

[0016] The detection mechanism of the fluorescent probe of the present application is as follows:

[0017]

[0018] Due to the photoinduced electron transfer effect of the probe molecule itself, the fluorescent molecule TXET exhibits fluorescence quenching. When HSO3 - is detected, a Michael addition reaction occurs to eliminate the photoinduced electron transfer effect of the fluorescent molecule, and strong green fluorescence is emitted. When ClO -Response after the occurrence of carbon-carbon double bond cleavage, resulting in free fluorophore, so that the probe in low polarity solvents show strong yellow fluorescence, while in high viscosity red fluorescence quenching. Based on the above function, a new type of detection HSO3 - / ClO - And viscosity of fluorescent probe was designed and synthesized to detect HSO3 - / ClO - And viscosity in solvent.

[0019] Figure 3 Is the UV-Vis absorption spectrum of probe TXET (3x10 -6 mol / L) in different solvents. The UV absorption spectrum of the probe in different solvents has no obvious difference.

[0020] Figure 4 Is the fluorescence emission spectrum of probe TXET (3x10 -6 mol / L) in different solvents under the excitation wavelength of 620 nm. The probe has fluorescence in lower polarity solvents, and the fluorescence is quenched in higher polarity solvents

[0021] Figure 5 Is the fluorescence intensity change chart of probe TXET (3x10 -5 mol / L) before and after adding the same concentration of HSO3 - In DMF-PBS system with different content of DMF under the excitation wavelength of 400 nm. The fluorescence intensity of the probe changes obviously before and after adding 0.13 mmol / L of HSO3 - In the volume percentage of DMF is 50%.

[0022] Figure 6 Is the fluorescence intensity change chart of probe TXET (3x10 -5 mol / L) before and after adding different concentrations of HSO3 - In the mixed solvent of equal proportion of PBS and DMF. The fluorescence intensity of the probe changes not obviously with time after adding HSO3 - After 15 minutes.

[0023] Figure 7 Is the UV-Vis absorption spectrum of probe TXET (3x10 -5 mol / L) before and after adding different concentrations of HSO3 - In the mixed solvent of equal proportion of PBS and DMF. With the increase of HSO3 - Concentration, a new absorption peak appears at 400 nm and gradually increases, and the absorption peak at 600 nm decreases.

[0024] Figure 8 Is the UV-Vis absorption spectrum of probe TXET (3x10-5 Different concentrations of HSO3 were added to a mixed solvent of PBS and DMF in equal proportions at an excitation wavelength of 400 nm (mol / L). - The fluorescence emission spectrum of HSO3. Fluorescence intensity increases with HSO3. - The concentration increases and the effect is enhanced.

[0025] Figure 9 It is a probe TXET (3×10) -5 Add HSO3 (mol / L) to an equal proportion of PBS and DMF mixed solvent. - Concentration range: 0 mmol / L - 0.13 mmol / L 500 The linear relationship diagram shows a good linear relationship.

[0026] Figure 10 It is a probe TXET (3×10) -5 Add HSO3 (mol / L) to an equal proportion of PBS and DMF mixed solvent. - The fluorescence intensity changed with pH before and after detection. This probe can detect HSO3 under neutral and alkaline conditions. - The effect is good.

[0027] Figure 11 It is a probe TXET (3×10) -5 The study investigated the anti-interference properties of TXET (mol / L) against small biological molecules. From left to right, the substances are: Blank, Ser, Met, Gln, Trp, Thr, Arg, Gly, Asp, GSH, Val, Leu, AA, Lys, Ala, Pro, Phe, Ile, and Cys. None of the interfering substances affected the fluorescence intensity of TXET, indicating that the probe TXET exhibits good anti-interference properties against small biological molecules.

[0028] Figure 12 It is a probe TXET (3×10) -5 Study on the anti-interference effect of (mol / L) on metal ions. From left to right: Blank, K + Na + Ca 2+ Mg 2+ Zn 2+ Fe 2+ Fe 3+ Cu 2+ Ag + Sn 2+ Co 2+ Ni 2+ Mn 2+ Cd 3+ Cr 3+The interfering substances had no effect on the fluorescence intensity of TXET, indicating that the probe TXET has good anti-interference properties against small biological molecules.

[0029] Figure 13 It is a probe TXET (3×10) -5 Study on the anti-interference effect of (mol / L) on anions. From left to right: Blank, F - Cl - ,Br - I - SO4 2- S 2- HS - SCN - PO4 3- H2PO4 - P2O7 4- C2O4 2- CO3 2- HCO3 - Cr2O7 2- CrO4 2- NO3 - NO2 - BF4 - The interfering substances had no effect on the fluorescence intensity of TXET, indicating that the probe TXET has good anti-interference properties against small biological molecules.

[0030] Figure 14 It is a probe TXET (3×10) -5 In THF-PBS systems with different tetrahydrofuran (THF) contents, the same concentration of ClO was added at an excitation wavelength of 400 nm. - The fluorescence intensity changes before and after are shown in the graph. This probe, with a THF volume percentage of 80%, was added with 80 μmol / L ClO₂. - The fluorescence intensity changed significantly before and after.

[0031] Figure 15 It is a probe TXET (3×10) -5 Different concentrations of ClO were added to a THF-PBS mixed solvent containing 80% THF (mol / L). - The graph shows the change in fluorescence intensity over time before and after the addition of ClO. - After 3 minutes, the fluorescence intensity did not change significantly over time.

[0032] Figure 16 It is a probe TXET (3×10) -5 Different concentrations of ClO were added to a THF-PBS mixed solvent containing 80% THF (mol / L). -The ultraviolet-visible absorption spectrum of ClO. - As the concentration of [agent] increases, the absorption peak at 400 nm gradually decreases, and the absorption peak at 600 nm also gradually decreases.

[0033] Figure 17 It is a probe TXET (3×10) -5 Different concentrations of ClO were added to a THF-PBS mixed solvent containing 80% THF at an excitation wavelength of 400 nm (mol / L). - The fluorescence emission spectrum of [the material]. Fluorescence intensity I [is shown]. 530 With ClO - The concentration increases and the effect is enhanced.

[0034] Figure 18 It is a probe TXET (3×10) -5 (mol / L) ClO was added to a THF-PBS mixed solvent containing 80% THF. - Concentration range: 40 μmol / L - 80 μmol / L 530 The linear relationship diagram shows a good linear relationship.

[0035] Figure 19 It is a probe TXET (3×10) -5 Add ClO (mol / L) to a THF-PBS mixed solvent containing 80% THF. - The fluorescence intensity changes with pH value before and after detection. The probe detects ClO under neutral and alkaline conditions. - The effect is good.

[0036] Figure 20 It is a probe TXET (3×10) -5 The study investigated the anti-interference properties of TXET (mol / L) against small biological molecules. From left to right, the substances are: Blank, Ser, Met, Gln, Trp, Thr, Arg, Gly, Asp, GSH, Val, Leu, AA, Lys, Ala, Pro, Phe, Ile, and Cys. None of the interfering substances affected the fluorescence intensity of TXET, indicating that the probe TXET exhibits good anti-interference properties against small biological molecules.

[0037] Figure 21 It is a probe TXET (3×10) -5 Study on the anti-interference effect of (mol / L) on metal ions. From left to right: Blank, K + Na + Ca 2+ Mg 2+ Zn 2+ Fe 2+ Fe 3+ Cu2+ Ag + Sn 2+ Co 2+ Ni 2+ Mn 2+ Cd 3+ Cr 3+ The interfering substances had no effect on the fluorescence intensity of TXET, indicating that the probe TXET has good anti-interference properties against small biological molecules.

[0038] Figure 22 It is a probe TXET (3×10) -5 Study on the anti-interference effect of (mol / L) on anions. From left to right: Blank, F - Cl - ,Br - I - SO4 2- S 2- HS - SCN - PO4 3- H2PO4 - P2O7 4- C2O4 2- CO3 2- HCO3 - Cr2O7 2- CrO4 2- NO3 - NO2 - BF4 - The interfering substances had no effect on the fluorescence intensity of TXET, indicating that the probe TXET has good anti-interference properties against small biological molecules.

[0039] Figure 23 It is a probe TXET (3×10) -5 The UV-Vis absorption spectra of PBS and glycerol mixed solvents in different proportions (mol / L) were obtained. The absorption peak at 620 nm gradually increased with increasing glycerol volume.

[0040] Figure 24 It is a probe TXET (3×10) -5 Fluorescence emission spectra of PBS and glycerol mixtures in different proportions at an excitation wavelength of 620 nm (mol / L). Fluorescence at 720 nm gradually increases with increasing glycerol volume.

[0041] Figure 25 It is a probe TXET (3×10) -5 The logarithmic value of fluorescence intensity (logI) in the viscosity range of 7.9-880 centipoise (mol / L). 720The graph shows the linear relationship between the viscosity (logη) and the system viscosity. A good linear relationship is observed.

[0042] Figure 26 It is a probe TXET (3×10) -5 The fluorescence intensity of the probe (mol / L) in mixed solvents of PBS and glycerol at different ratios varies with pH. The probe is unaffected by pH when detecting viscosity.

[0043] Figure 27 It is a probe TXET (3×10) -5 The study investigated the anti-interference properties of the probe TXET against small biological molecules (mol / L). From left to right, the probes are: Blank, Ser, Met, Gln, Trp, Thr, Arg, Gly, Asp, Val, Leu, AA, Lys, Ala, Pro, Phe, Ile, and Cys. None of the interfering substances affected the fluorescence intensity of TXET, indicating that the probe TXET exhibits good anti-interference properties against small biological molecules.

[0044] Figure 28 It is a probe TXET (3×10) -5 Study on the anti-interference effect of (mol / L) on metal ions. From left to right: Blank, K + Na + Ca 2+ Mg 2+ Zn 2+ Fe 2+ Fe 3+ Cu 2+ Ag + Sn 2+ Co 2+ Ni 2+ Mn 2+ Cd 3+ Cr 3+ The interfering substances had no effect on the fluorescence intensity of TXET, indicating that the probe TXET has good anti-interference properties against small biological molecules.

[0045] Figure 29 It is a probe TXET (3×10) -5 Study on the anti-interference effect of (mol / L) on anions. From left to right: Blank, F - Cl - ,Br - I - SO4 2- S 2- HS - SCN - PO4 3- H2PO4 - P2O74- , C2O4 2- , CO3 2- , HCO3 - , Cr2O7 2- , CrO4 2- , NO3 - , NO2 - , BF4 - . All of the interferents have no effect on the fluorescence intensity of TXET, indicating that the probe TXET has good anti-interference ability to biological small molecules.

[0046] Figure 30 Sequentially showed the photophysical properties of the prepared probe TXET (3 x 10 -6 mol / L) in different solvents, including the maximum absorption peak wavelength λ abs,max , the maximum emission wavelength λ em,max , the Stokes shift and the fluorescence quantum yield Φ (with Rhodamine B as the reference).

[0047] Figure 31 (A) is the fluorescence imaging of human normal liver cells (L02) and different degrees of damaged liver cells after staining with probe TXET (1 x 10 -5 mol / L). (B) is the relative fluorescence intensity diagram in figure (A). Green channel: λ ex / λ em = 405 / (470-500) nanometers; yellow channel: λ ex / λ em = 405 / (550-600) nanometers; red channel: λ ex / λ em = 620 / (700-750) nanometers. At the cellular level, the probe TXET has good monitoring ability for the damage degree of L02 cells.

[0048] Figure 32 (A) is the evaluation of the ability of probe TXET (1 x 10 -5 mol / L) to diagnose liver damage in vivo and in vitro. (B) is the relative fluorescence intensity diagram in figure (A). Green channel: λ ex / λ em = 405 / (470-500) nanometers; yellow channel: λ ex / λ em = 405 / (550-600) nanometers; red channel: λ ex / λ em = 620 / (700-750) nanometers; **** P < 0.0001. The probe TXET can track the process of liver damage and treatment in vivo and in vitro.

[0049] Figure 33 (A) is the fluorescence imaging of probe TXET (1 x 10 -5 mol / L) monitoring the process of liver injury at tissue level. (B) is the relative fluorescence intensity map in (A). Green channel: λ ex / λ em =405 / (470-500) nm; Yellow channel: λ ex / λ em =405 / (550-600) nm; Red channel: λ ex / λ em =620 / (700-750) nm; ****P<0.0001 Probe TXET can successfully monitor the process of liver injury at tissue level.

[0050] In summary, by using simple organic synthesis method, we obtained a fluorescence probe (E)-4-(2-(6-(4-(diphenylamino)phenyl)-2,3-dihydro-1H-xanthene-4-yl)vinyl)-3-ethylbenzothiazol-3-ium iodide (TXET) for detecting HSO3 - / ClO - and viscosity. The solution color changed obviously (from dark blue to colorless) before and after adding HSO3 - . This indicates that the probe exhibits different optical properties after adding HSO3 - . And with the increase of HSO3 - concentration, the fluorescence intensity of the probe at 400 nm wavelength gradually increased, which is due to the Michael addition reaction between HSO3 - and the probe molecule after adding HSO3 - , which makes the photoinduced electron transfer effect of the fluorescent molecule disappear, and emits strong green fluorescence. Secondly, the solution color changed obviously (from dark blue to light yellow) before and after adding ClO - under low polarity conditions, and showed light yellow light under 365 nm hand lamp. This indicates that the probe exhibits different optical properties after adding ClO - . And with the increase of ClO - concentration, the fluorescence intensity of the probe at 530 nm wavelength gradually increased. The solvent color changed obviously (from dark blue to colorless) before and after adding ClO - under high viscosity conditions. This indicates that the probe exhibits different optical properties after adding ClO - . And with the increase of ClO - concentration, the fluorescence intensity of the probe at 720 nm wavelength gradually decreased. This is due to the probe molecule to ClO -The response of the probe makes the photo-induced electron transfer effect of the light-emitting molecule disappear, and strong yellow fluorescence is emitted, while the fluorescence at 720 nm gradually decreases. Finally, with the increase of viscosity, the fluorescence intensity of the probe at 720 nm gradually increases. At different concentrations of HSO3 - / ClO - and different viscosities, there are obvious regular changes in the fluorescence emission spectrum, which provides a good tool for detecting HSO3 - / ClO - and viscosity. The preparation of this fluorescent probe fills the gap in the field of fluorescent probes that can simultaneously quantitatively detect HSO3 - / ClO - and viscosity. At the same time, in the process of biological experiments, it can be found that with the occurrence and development of liver injury, the green, yellow and red fluorescence signals of the probe TXET all increase significantly. Notably, in the process of treating liver injury in mice, the three fluorescence signals decrease significantly. This indicates that the probe TXET can be used as a sensitive tool for identifying liver injury. BRIEF DESCRIPTION OF DRAWINGS

[0051] Figure 1 Preparation and design route of the probe (E)-4-(2-(6-(4-(diphenylamino)phenyl)-2,3-dihydro-1H-xanthene-4-yl)vinyl)-3-ethylbenzothiazol-3-ium iodide (TXET).

[0052] Figure 2 Mechanism of the probe TXET detecting HSO3 - , ClO - and responding to viscosity.

[0053] Figure 3 is the ultraviolet-visible absorption spectrum of the probe TXET (3×10 -6 mol / L) in different solvents.

[0054] Figure 4 is the fluorescence emission spectrum of the probe TXET (3×10 -6 mol / L) in different solvents under an excitation wavelength of 620 nm.

[0055] Figure 5 is the fluorescence intensity change chart of the probe TXET (3×10 -5 mol / L) in the DMF-PBS system containing different amounts of DMF under an excitation wavelength of 400 nm before and after adding 0.13 mmol / L HSO3 - .

[0056] Figure 6 is the fluorescence intensity change chart of the probe TXET (3×10 -5Different concentrations of HSO3 were added to an equal ratio of PBS and DMF mixed solvents (mol / L). - The graph shows the change in fluorescence intensity over time.

[0057] Figure 7 It is a probe TXET (3×10) -5 Different concentrations of HSO3 were added to an equal ratio of PBS and DMF mixed solvents (mol / L). - The ultraviolet-visible absorption spectrum.

[0058] Figure 8 It is a probe TXET (3×10) -5 Different concentrations of HSO3 were added to a mixed solvent of PBS and DMF in equal proportions at an excitation wavelength of 400 nm (mol / L). - The fluorescence emission spectrum of [the substance].

[0059] Figure 9 It is a probe TXET (3×10) -5 Add HSO3 (mol / L) to an equal proportion of PBS and DMF mixed solvent. - Concentration range: 0 mmol / L - 0.13 mmol / L 500 Linear relationship graph.

[0060] Figure 10 It is a probe TXET (3×10) -5 Add HSO3 (mol / L) to an equal proportion of PBS and DMF mixed solvent. - Changes in fluorescence intensity with pH value before and after.

[0061] Figure 11 It is a probe TXET (3×10) -5 Studies on the anti-interference properties of mol / L on small biological molecules. From left to right: Blank, Ser, Met, Gln, Trp, Thr, Arg, Gly, Asp, GSH, Val, Leu, AA, Lys, Ala, Pro, Phe, Ile, Cys.

[0062] Figure 12 It is a probe TXET (3×10) -5 Study on the anti-interference effect of (mol / L) on metal ions. From left to right: Blank, K + Na + Ca 2+ Mg 2+ Zn 2+ Fe 2+ Fe 3+ Cu 2+ Ag + Sn2+ Co 2+ Ni 2+ Mn 2+ Cd 3+ Cr 3+ .

[0063] Figure 13 is the anti-interference of probe TXET (3 x 10 -5 mol / L) to anions. From left to right, respectively, Blank, F - Cl - Br - I - SO4 2- S 2- HS - SCN - PO4 3- H2PO4 - P2O7 4- C2O4 2- CO3 2- HCO3 - Cr2O7 2- CrO4 2- NO3 - NO2 - BF4 - .

[0064] Figure 14 is the fluorescence intensity change chart of probe TXET (3 x 10 -5 mol / L) before and after adding 80 μmol / L ClO - in THF-PBS system with different content of THF at the excitation wavelength of 400 nm.

[0065] Figure 15 is the fluorescence intensity change chart of probe TXET (3 x 10 -5 mol / L) before and after adding different concentrations of ClO - in THF-PBS mixed solvent containing 80% THF.

[0066] Figure 16 is the UV-Vis absorption spectrum of probe TXET (3 x 10 -5 mol / L) before and after adding different concentrations of ClO - in THF-PBS mixed solvent containing 80% THF.

[0067] Figure 17 is the UV-Vis absorption spectrum of probe TXET (3 x 10 -5Different concentrations of ClO were added to a THF-PBS mixed solvent containing 80% THF at an excitation wavelength of 400 nm (mol / L). - The fluorescence emission spectrum of [the substance].

[0068] Figure 18 It is a probe TXET (3×10) -5 (mol / L) ClO was added to a THF-PBS mixed solvent containing 80% THF. - Concentration range: 40 μmol / L - 80 μmol / L 530 Linear relationship graph.

[0069] Figure 19 It is a probe TXET (3×10) -5 Add ClO (mol / L) to a THF-PBS mixed solvent containing 80% THF. - Changes in fluorescence intensity with pH value before and after.

[0070] Figure 20 It is a probe TXET (3×10) -5 Studies on the anti-interference properties of mol / L on small biological molecules. From left to right: Blank, Ser, Met, Gln, Trp, Thr, Arg, Gly, Asp, GSH, Val, Leu, AA, Lys, Ala, Pro, Phe, Ile, Cys.

[0071] Figure 21 It is a probe TXET (3×10) -5 Study on the anti-interference effect of (mol / L) on metal ions. From left to right: Blank, K + Na + Ca 2+ Mg 2+ Zn 2+ Fe 2+ Fe 3+ Cu 2+ Ag + Sn 2+ Co 2+ Ni 2+ Mn 2+ Cd 3+ Cr 3+ .

[0072] Figure 22 It is a probe TXET (3×10) -5 Study on the anti-interference effect of (mol / L) on anions. From left to right: Blank, F - Cl - ,Br - I- , SO4 2- , S 2- , HS - , SCN - , PO4 3- , H2PO4 - , P2O7 4- , C2O4 2- , CO3 2- , HCO3 - , Cr2O7 2- , CrO4 2- , NO3 - , NO2 - , BF4 - .

[0073] Figure 23 is the UV-Vis absorption spectra of probe TXET (3 × 10 -5 mol / L) in different proportion of PBS and glycerol mixed solvents.

[0074] Figure 24 is the fluorescence emission spectra of probe TXET (3 × 10 -5 mol / L) in different proportion of PBS and glycerol mixed solvents at an excitation wavelength of 620 nm.

[0075] Figure 25 is the linear relationship between the fluorescence intensity (logI -5 ) and the system viscosity (logη) of probe TXET (3 × 10 720 mol / L) in the viscosity range of 7.9-880 centipoise.

[0076] Figure 26 is the change of fluorescence intensity of probe TXET (3 × 10 -5 mol / L) with pH value in different proportion of PBS and glycerol mixed solvents.

[0077] Figure 27 is the anti-interference study of probe TXET (3 × 10 -5 mol / L) to biological small molecules. From left to right, respectively, Blank, Ser, Met, Gln, Trp, Thr, Arg, Gly, Asp, Val, Leu, AA, Lys, Ala, Pro, Phe, Ile, Cys.

[0078] Figure 28 is the anti-interference study of probe TXET (3 × 10 -5 mol / L) to metal ions. From left to right, respectively, Blank, K + , Na +, Ca 2+ , Mg 2+ , Zn 2+ , Fe 2+ , Fe 3+ , Cu 2+ , Ag + , Sn 2+ , Co 2+ , Ni 2+ , Mn 2+ , Cd 3+ , Cr 3+ .

[0079] Figure 29 are the anti-interference studies of the probe TXET (3 x 10 -5 mol / L) to anions. From left to right, they are Blank, F - , Cl - , Br - , I - , SO4 2- , S 2- , HS - , SCN - , PO4 3- , H2PO4 - , P2O7 4- , C2O4 2- , CO3 2- , HCO3 - , Cr2O7 2- , CrO4 2- , NO3 - , NO2 - , BF4 - .

[0080] Figure 30 Sequentially show the photophysical properties of the prepared probe TXET (3 x 10 -6 mol / L) in different solvents, including the maximum absorption peak wavelength λ abs,max , the maximum emission peak wavelength λ em,max , the Stokes shift and the fluorescence quantum yield Φ (with Rhodamine B as the reference).

[0081] Figure 31 (A) is the fluorescence imaging of human normal liver cells (L02) and liver cells with different degrees of damage after staining with the probe TXET (1 x 10 -5 mol / L). (B) is the relative fluorescence intensity diagram in (A). Green channel: λ ex / λ em = 405 / (470-500) nanometers; Yellow channel: λ ex / λ em= 405 / (550-600) nm; red channel: λ ex / λ em = 620 / (700-750) nm.

[0082] Figure 32 (A) is the ability evaluation of probe TXET (1 x 10 -5 mol / L) in vivo and in vitro diagnosis of liver injury. (B) is the relative fluorescence intensity chart in (A). Green channel: λ ex / λ em = 405 / (470-500) nm; yellow channel: λ ex / λ em = 405 / (550-600) nm; red channel: λ ex / λ em = 620 / (700-750) nm; ****P<0.0001.

[0083] Figure 33 (A) is the fluorescence imaging of probe TXET (1 x 10 -5 mol / L) in monitoring the process of liver injury at the tissue level. (B) is the relative fluorescence intensity chart in (A). Green channel: λ ex / λ em = 405 / (470-500) nm; yellow channel: λ ex / λ em = 405 / (550-600) nm; red channel: λ ex / λ em = 620 / (700-750) nm; ****P<0.0001.

[0084] DETAILED DESCRIPTION

[0085] Example 1: Synthesis of compound A1

[0086] Into anhydrous N,N-dimethylformamide (6 mL) was added chloroform (25 mL) and phosphorus tribromide (4.5 mL), stirred at 0°C for 1 hour, then added cyclohexanone (2 mL) in chloroform and mixed well, the mixture was added to the reaction system, vacuumed, and then reacted at room temperature for 18 hours; after the reaction was completed, the pH was adjusted to neutral with saturated NaHCO3, and then the organic phase was extracted with dichloromethane, the organic phase was collected and rotary evaporated to remove the solvent, and purified by silica gel column (eluent: dichloromethane: ethanol = 30: 1), and then vacuum dried to obtain 1.83 g of light yellow oily product 2-chloro-1-cyclooctene-1-carboxaldehyde (A1, yield 50.4%).

[0087] Example 2: Synthesis of compound A2

[0088] Dissolve 4-(diphenylamino)benzene boronic acid (2.4 mmol, 693.94 mg) and 4-bromo-2-hydroxybenzaldehyde (2 mmol, 402.11 mg) in tetrahydrofuran (THF) by ultrasonic, add potassium carbonate aqueous solution (2 mol / L) and tetrabutylammonium bromide (1 mmol, 322 mg) and dissolve by ultrasonic, stir at room temperature for 30 minutes, then add tetrakis(triphenylphosphine)palladium (0.002 mmol, 2.4 mg) and dissolve by ultrasonic, vacuumize, heat to reflux for about 6 hours; after the reaction is completed, cool to room temperature, extract with dichloromethane (DCM), collect the organic phase and rotary evaporate the solvent, purify by silica gel column (eluent is petroleum ether: dichloromethane = 4:1) to obtain 641.70 mg of fluorescent green solid product 4'-(diphenylamino)-3-hydroxy-[1,1'-biphenyl]-4-carbaldehyde (A2, yield is 87.8%).

[0089] Example 3: Synthesis of compound A3

[0090] Dissolve A1 (2 mmol, 375.96 mg) and A2 (2 mmol, 730.28 mg), cesium carbonate (1.2 mmol, 166 mg) in anhydrous DMF, vacuumize, then react at room temperature for 20 hours; after the reaction is completed, rotary evaporate the reaction liquid to obtain the crude product, separate and purify by column chromatography, the eluent is petroleum ether: ethyl acetate = 15:1, vacuum dry to obtain 633.63 mg of yellow solid product 6-(4-(diphenylamine)phenyl)-2,3-dihydro-1H-xanthene-4-carbaldehyde (A3, yield is 69.6%).

[0091] Example 4: Synthesis of compound A4

[0092] Add 2-methylbenzothiazole (10 mmol, 1.49 g) and iodoethane (10 mmol, 1.56 g) in ethanol, heat to reflux for about 12 hours; after the reaction is completed, cool the reaction solution to room temperature, a large amount of precipitate is precipitated and vacuum filter, wash with ethanol for 3 times, put into a vacuum drying box to dry, obtain 2.64 g of off-white solid 3-ethyl-2-methylbenzothiazolium iodide (A4, yield is 86.4%).

[0093] Example 5: Synthesis of probe TXET

[0094] A3 (0.5 mmol, 227.60 mg) and A4 (0.5 mmol, 152.49 mg) were dissolved in anhydrous ethanol, sonicated until fully dissolved, and heated under reflux for 8 hours. At the end of the reaction, the original reaction solution was cooled to room temperature, and a precipitate was formed. The precipitate was filtered, washed with ethanol, and dried to give 238.40 mg of the dark blue product (E)-4-(2-(6-(4-(diphenylamino)phenyl)-2,3-dihydro-1H-oxanthracene-4-yl)vinyl)-3-ethylbenzothiazol-3-onium iodide (TXET, yield 64.2%).

[0095] Example 6: Detection of HSO3 in solvent using probe TXET - and ClO - And the application of viscosity.

[0096] HSO3 in solution - Detection: Figure 5 It is a probe TXET (3×10) -5 0.13 mmol / L HSO3 was added to DMF-PBS systems with different DMF contents at an excitation wavelength of 400 nm. - The graph shows the changes in fluorescence intensity before and after. When N,N-dimethylformamide is 50% by volume, 0.13 mmol / L HSO3 is added. - There was a significant difference in fluorescence intensity before and after. Figure 6 It is a probe TXET (3×10) -5 Different concentrations of HSO3 were added to an equal ratio of PBS and DMF mixed solvents (mol / L). - The graph shows the change in fluorescence intensity over time before and after the addition of HSO3. - In the last 15 minutes, the fluorescence intensity did not change significantly. Figure 7 It is a probe TXET (3×10) -5 Different concentrations of HSO3 were added to an equal ratio of PBS and DMF mixed solvents (mol / L). - The ultraviolet-visible absorption spectrum of the probe was observed when different concentrations of HSO3 were added. - The ultraviolet absorption spectra show significant differences. With HSO3 - With increasing concentration, the absorption peak at 400 nm gradually increases, while the absorption peak at 600 nm gradually decreases. Figure 8 It is a probe TXET (3×10) -5 Different concentrations of HSO3 were added to a mixed solvent of PBS and DMF in equal proportions at an excitation wavelength of 400 nm (mol / L). - The fluorescence emission spectrum of HSO3. - With increasing concentration, fluorescence intensity (I) 500 The strength of this factor is also gradually increasing.Figure 9 It is a probe TXET (3×10) -5 Add HSO3 (mol / L) to an equal proportion of PBS and DMF mixed solvent. - Concentration range: 0 mmol / L - 0.13 mmol / L 500 The linear relationship graph shows that, as can be seen from the graph, the addition of HSO3... - The concentration range of 0 mmol / L to 0.13 mmol / L showed a good linear relationship. Figure 10 It is a probe TXET (3×10) -5 Add HSO3 (mol / L) to an equal proportion of PBS and DMF mixed solvent. - The fluorescence intensity changed with pH before and after. The probe detected HSO3 under neutral and alkaline conditions. - The effect is quite good. Figure 11 It is a probe TXET (3×10) -5 The study investigated the anti-interference properties of the probe TXET against small biological molecules (mol / L). From left to right, the probes are: Blank, Ser, Met, Gln, Trp, Thr, Arg, Gly, Asp, Val, Leu, AA, Lys, Ala, Pro, Phe, Ile, and Cys. None of the interfering substances affected the fluorescence intensity of TXET, indicating that the probe TXET exhibits good anti-interference properties against small biological molecules. Figure 12 It is a probe TXET (3×10) -5 Study on the anti-interference effect of (mol / L) on metal ions. From left to right: Blank, K + Na + Ca 2+ Mg 2+ Zn 2+ Fe 2+ Fe 3+ Cu 2+ Ag + Sn 2+ Co 2+ Ni 2+ Mn 2+ Cd 3+ Cr 3+ The interfering substances had no effect on the fluorescence intensity of TXET, indicating that the probe TXET has good anti-interference properties against small biological molecules. Figure 13 It is a probe TXET (5×10 -5 Study on the anti-interference effect of (mol / L) on anions. From left to right: Blank, F - Cl - ,Br - I - SO4 2- S2- , HS - , SCN - , PO4 3- , H2PO4 - , P2O7 4- , C2O4 2- , CO3 2- , HCO3 - , Cr2O7 2- , CrO4 2- , NO3 - , NO2 - , BF4 - . All of the interferents have no effect on the fluorescence intensity of TXET, which indicates that the probe TXET has good anti-interference ability to biological small molecules.

[0097] ClO - - detection: ClO - - detection: Figure 14 is the fluorescence intensity change chart of probe TXET (3 x 10 -5 mol / L) in THF-PBS system containing 80% THF at 400 nm excitation wavelength before and after adding 80 μmol / L ClO - - There is a significant difference in fluorescence intensity before and after adding 80 μmol / L ClO - - in THF-PBS system containing 80% THF. Figure 15 is the fluorescence intensity change chart of probe TXET (3 x 10 -5 mol / L) in THF-PBS system containing 80% THF before and after adding different concentrations of ClO - - The fluorescence intensity does not change significantly with time 3 minutes after adding ClO - - in THF-PBS system containing 80% THF. Figure 16 is the UV-Vis absorption spectrum of probe TXET (3 x 10 -5 mol / L) in THF-PBS system containing 80% THF before and after adding different concentrations of ClO - - There is a significant difference in UV-Vis absorption spectrum of the probe before and after adding different concentrations of ClO - - The absorption peak at 400 nm gradually decreases with the increase of ClO - - concentration, and the absorption peak at 600 nm also gradually decreases. Figure 17 is the fluorescence emission spectrum of probe TXET (3 x 10 -5 mol / L) in THF-PBS system containing 80% THF before and after adding different concentrations of ClO - - The fluorescence emission spectrum of the probe changes significantly with the increase of ClO -The fluorescence intensity (I 530 ) is gradually enhanced. Figure 18 is the probe TXET (3 x 10 -5 mol / L) in THF-PBS mixed solvent containing 80% THF added with ClO - The linear relationship graph of I 530 in the concentration range of 40-80 μmol / L. It can be seen from the graph that the fluorescence intensity of TXET is linearly related to the concentration of ClO - in the concentration range of 40-80 μmol / L. Figure 19 is the probe TXET (3 x 10 -5 mol / L) in THF-PBS mixed solvent containing 80% THF added with ClO - The change of fluorescence intensity before and after adding ClO - with pH value. The probe has a good effect on detecting ClO Figure 20 is the probe TXET (3 x 10 -5 mol / L) on biological small molecules. From left to right, they are Blank, Ser, Met, Gln, Trp, Thr, Arg, Gly, Asp, Val, Leu, AA, Lys, Ala, Pro, Phe, Ile, Cys. The interference substances have no effect on the fluorescence intensity of TXET, which shows that the probe TXET has good anti-interference property on biological small molecules. Figure 21 is the probe TXET (3 x 10 -5 mol / L) on metal ions. From left to right, they are Blank, K + , Na + , Ca 2 + , Mg 2+ , Zn 2+ , Fe 2+ , Fe 3+ , Cu 2+ , Ag + , Sn 2+ , Co 2+ , Ni 2+ , Mn 2+ , Cd 3+ , Cr 3+ . The interference substances have no effect on the fluorescence intensity of TXET, which shows that the probe TXET has good anti-interference property on biological small molecules. Figure 22 is the probe TXET (3 x 10 -5 mol / L) on anions. From left to right, they are Blank, F - , Cl - , Br- I - SO4 2- S 2- HS - SCN - PO4 3- H2PO4 - P2O7 4- C2O4 2- CO3 2- HCO3 - Cr2O7 2- CrO4 2- NO3 - NO2 - BF4 - The interfering substances had no effect on the fluorescence intensity of TXET, indicating that the probe TXET has good anti-interference properties against small biological molecules.

[0098] Viscosity testing in solvents: Figure 23 It is a probe TXET (3×10) -5 The UV-Vis absorption spectra of the probe in mixed solvents of PBS and glycerol at different ratios (mol / L) were analyzed. The UV absorption spectra of the probe showed significant differences at different viscosities. Figure 24 It is a probe TXET (3×10) -5 Fluorescence emission spectra of PBS and glycerol mixed solvents in different proportions at an excitation wavelength of 620 nm (mol / L). The fluorescence intensity gradually increases with increasing solvent viscosity. Figure 25 It is a probe TXET (3×10) -5 The logarithmic value of fluorescence intensity (logI) in the viscosity range of 7.9-880 centipoise (mol / L). 720 The graph shows the linear relationship between the viscosity of the system and the logarithm of the viscosity (logη). As can be seen from the graph, a good linear relationship is observed. Figure 26 It is a probe TXET (3×10) -5 The fluorescence intensity of the probe (mol / L) in mixed solvents of PBS and glycerol at different ratios varies with pH. The figure shows that the effect of pH on the probe is almost negligible. Figure 27 It is a probe TXET (3×10) -5 The study investigated the anti-interference properties of TXET (mol / L) against small biological molecules. From left to right, the substances are: Blank, Ser, Met, Gln, Trp, Thr, Arg, Gly, Asp, GSH, Val, Leu, AA, Lys, Ala, Pro, Phe, Ile, and Cys. None of the interfering substances affected the fluorescence intensity of TXET, indicating that the probe TXET exhibits good anti-interference properties against small biological molecules. Figure 28 It is a probe TXET (3×10) -5Anti-interference of TXET (3 x 10 -5 mol / L) to metal ions. From left to right are Blank, K + , Na + , Ca 2+ , Mg 2+ , Zn 2+ , Fe 2+ , Fe 3+ , Cu 2+ , Ag + , Sn 2+ , Co 2+ , Ni 2+ , Mn 2+ , Cd 3+ , Cr 3+ . Interfering substances have no effect on the fluorescence intensity of TXET, indicating that the probe TXET has good anti-interference to biological small molecules. Figure 29 Anti-interference of TXET (3 x 10 -5 mol / L) to anions. From left to right are Blank, F - , Cl - , Br - , I - , SO4 2- , S 2- , HS - , SCN - , PO4 3- , H2PO4 - , P2O7 4- , C2O4 2- , CO3 2- , HCO3 - , Cr2O7 2- , CrO4 2- , NO3 - , NO2 - , BF4 - . Interfering substances have no effect on the fluorescence intensity of TXET, indicating that the probe TXET has good anti-interference to biological small molecules. Figure 30 The prepared probe TXET (3 x 10 -6 mol / L) in different solvents successively shows the photophysical properties, including the maximum absorption peak wavelength λ abs,max , the maximum emission wavelength λ em,max , the Stokes shift and the fluorescence quantum yield Φ (with rhodamine B as reference).

[0099] In biological experiments, Figure 31 Anti-interference of TXET (1 x 10 -5Fluorescence imaging of human normal hepatocytes (L02) and hepatocytes with different degrees of injury after staining with probe TXET (1 x 10 - / ClO - The fluorescence intensity of the probe TXET (1 x 10 Figure 32 -4) increased with the increase of the concentration of HSO3 -5 The fluorescence intensity of the probe TXET (1 x 10 Figure 33 -4) decreased after the treatment of liver injury. -5 The fluorescence imaging of the probe TXET (1 x 10 Figure 32 -4) on the tissue level to monitor the process of liver injury can obtain consistent results with -4) can be used as a sensitive tool to identify liver injury.

[0100] In summary, by using a simple organic synthesis method, we obtained a fluorescent probe (E)-4-(2-(6-(4-(diphenylamino)phenyl)-2,3-dihydro-1H-xanthene-4-yl)vinyl)-3-ethylbenzothiazolium iodide (TXET) to detect HSO3 - / ClO - and viscosity. The solution color (from blue to colorless) changed significantly before and after the addition of HSO3 - This indicates that the probe exhibits different optical properties after the addition of HSO3 - The fluorescence intensity of the probe at 500 nm wavelength gradually increased with the increase of the concentration of HSO3 - This is because after the addition of HSO3 - , HSO3 - undergoes a Michael addition reaction with the probe molecule, making the photoinduced electron transfer effect of the fluorescent molecule disappear, and emitting strong green fluorescence. Secondly, the solution color (from blue to light yellow) changed significantly before and after the addition of ClO - This indicates that the probe exhibits different optical properties after the addition of ClO - The fluorescence intensity of the probe at 530 nm wavelength gradually increased with the increase of the concentration of ClO - The solution color (from blue to light colorless) changed significantly before and after the addition of ClO - This indicates that the probe exhibits different optical properties after the addition of ClO - The fluorescence intensity of the probe at 530 nm wavelength gradually increased with the increase of the concentration of ClO -With the increase of concentration, the fluorescence intensity of the probe at 720 nm wavelength gradually decreased, which was due to the response of the probe molecules to CIO - -4 that made the photoinduced electron transfer effect of the luminescent molecules disappear, and strong yellow fluorescence was emitted, and the red light gradually decreased. Finally, with the increase of viscosity, the fluorescence intensity of the probe at 720 nm wavelength gradually increased. Under different concentrations of HSO3 - / ClO - and different viscosities, there were obvious regular changes in the fluorescence emission spectrum, which provided a good tool for detecting the changes of HSO3 - / ClO - and viscosity. The preparation of this fluorescent probe filled the blank in the field of fluorescent probes that could simultaneously quantitatively detect HSO3 - / ClO - and viscosity. At the same time, in the process of biological experiments, it was found that with the occurrence and development of liver injury, the green, yellow and red fluorescence signals of the probe TXET all increased significantly. It is worth noting that in the process of treating liver injury in mice, the three fluorescence signals decreased significantly, which indicated that the probe TXET could be used as a sensitive tool for identifying liver injury.

Claims

1. A fluorescent probe for detecting HSO3 - / ClO - and viscosity, having the structural formula:

2. The method of claim 1, wherein the method further comprises: determining a concentration of HSO3 - / ClO - and viscosity simultaneously using the fluorescent probe. comprising the steps of: adding chloroform and phosphorus tribromide in anhydrous N,N-dimethylformamide (DMF), stirring at 0°C for 1 hour, then adding cyclohexanone in chloroform and mixing thoroughly, adding the mixture into the reaction system, vacuumizing, and reacting at room temperature for 18 hours; after the reaction is completed, adjusting the pH to neutral with saturated NaHCO3, then extracting the organic phase with dichloromethane, collecting the organic phase and rotary-evaporating the solvent, and purifying with a silica gel column to obtain the product A1 in light yellow oil; ultrasonically dissolving 4-(diphenylamino)phenylboronic acid and 4-bromo-2-hydroxybenzaldehyde in a tetrahydrofuran (THF) solution, adding an aqueous potassium carbonate solution and tetrabutylammonium bromide and ultrasonically dissolving, stirring at room temperature for 30 minutes, then adding tetrakis(triphenylphosphine)palladium into the reaction system and ultrasonically dissolving, vacuumizing, heating to reflux for 6 hours; after the reaction is completed, cooling to room temperature, extracting with dichloromethane (DCM), collecting the organic phase and rotary-evaporating the solvent, and purifying with a silica gel column to obtain the product A2 in fluorescent green solid; dissolving A1 and A2 and cesium carbonate in anhydrous DMF, vacuumizing, and then reacting at room temperature for 20 hours; after the reaction is completed, rotary-evaporating the reaction solution to obtain the crude product, separating and purifying by column chromatography, and drying to obtain the product A3 in yellow solid; adding 2-methylbenzothiazole and iodoethane into ethanol, heating to reflux for 12 hours; after the reaction is completed, cooling the reaction solution to room temperature, filtering and washing, and drying in a vacuum drying box to obtain A4 in off-white solid; dissolving A3 and A4 in anhydrous ethanol, ultrasonically dissolving, heating to reflux for 8 hours; after the reaction is completed, cooling the reaction solution to room temperature, filtering and washing with ethanol, and drying to obtain the product TXET in dark blue; The synthesis route is as follows:

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