A Fluorescent Probe Based on Triphenylamine and Indole Derivatives, Its Preparation Method and Application

Abstract

The present invention belongs to the field of chemical detection, relates to a fluorescent probe, and particularly refers to a fluorescent probe based on triphenylamine and indole derivatives, and a preparation method and application thereof. It has the following structural formula: #imgabs0#. The fluorescent probe of the present invention can respond to sulfur dioxide and viscosity respectively under two different excitation wavelengths. The recognition processes of the two are independent and do not interfere with each other. It has good biocompatibility, can specifically localize in the sub-organelle mitochondria, and has the ability to image sulfur dioxide and viscosity in mitochondria. The probe BDI can realize the visualization imaging of intracellular SO2 and viscosity during mitochondrial dysfunction and apoptosis, and can be regarded as a rapid-response colorimetric analysis tool for detecting related analytes in real samples and organisms. It has high specificity and sensitivity and has broad application prospects in the field of bioimaging.

Description

Technical Field

[0001] The present invention belongs to the field of chemical detection, relates to a fluorescent probe, and particularly refers to a fluorescent probe based on triphenylamine and indole derivatives, and a preparation method and application thereof. Background Art

[0002] Sulfur dioxide (SO 2 ) is a toxic environmental pollutant widely present in the air, mainly generated by the combustion of fossil fuels. When exogenous sulfur dioxide is ingested into an organism through food or air, sulfur dioxide easily reacts with water to form sulfite and bisulfite. Endogenous sulfur dioxide is also produced in organisms through the oxidation of hydrogen sulfide (H 2 S) and the decomposition of sulfur-containing amino acids. Many research results have found that normal levels of sulfur dioxide have various important physiological functions such as dilating blood vessels, regulating cardiovascular function, and maintaining the intracellular redox balance. However, abnormal levels of sulfur dioxide and its derivatives can induce cardiovascular diseases, neurological diseases, and cancers. Viscosity is also an important biological indicator, participating in various physiological activities such as intracellular chemical signal and nutrient transport, metabolism, and apoptosis. An increase in mitochondrial matrix viscosity may lead to mutations in the mitochondrial reticular structure, hindering the transmission of signaling molecules. Changes in cell viscosity have also been proven to be closely related to many diseases, such as hypertension, diabetes, atherosclerosis, and cancer. Therefore, developing a method that can simultaneously detect intracellular sulfur dioxide and viscosity levels is of great significance for the prevention, diagnosis, and treatment of related diseases.

[0003] At present, most of the traditional detection methods for sulfur dioxide, such as spectrophotometry, titration, electrochemistry analysis, ion chromatography, etc., are complex in operation, poor in selectivity and damage the samples. Moreover, traditional viscosity detection methods, such as capillary viscometers, falling ball viscometers, rotational viscometers, cannot even be used to detect viscosities in the microscopic range. In contrast, organic small molecule fluorescent probes have the advantages of good selectivity, high sensitivity, rapid response, no damage to samples and the ability to achieve real-time monitoring. They are an effective means for detecting sulfur dioxide and viscosity in organisms. At present, many fluorescent probes for detecting sulfur dioxide or viscosity alone have been reported. Viscosity probes are mainly designed based on molecular rotors such as cyanine (Cy), boron dipyrromethene (BODIPY), cyanovinyl julolidine (CVJ) and diphenylamine thiophene aldehyde (DPT). The intramolecular rotation of these molecular rotors is restricted in a high-viscosity environment, resulting in fluorescence changes. At the same time, such molecular rotor probes can also undergo Michael addition with sulfur dioxide through the double bonds in the molecule, thus realizing the recognition of sulfur dioxide. However, there are still few probes that can detect both of them simultaneously. Patent CN 110437219 A discloses a fluorescent probe with dual functions of detecting viscosity and sulfur dioxide. The excitation wavelengths used in the detection of viscosity and the detection of SO 2 In the application, the excitation wavelength is 405 nm, and the detection wavelength ranges are 450 - 750 nm and 430 - 750 nm. The repetition rate of the wavelength ranges of the two is high, and there will be a certain mutual influence during simultaneous detection; Patent CN114149359 A discloses a two-photon fluorescent probe for dual detection of sulfur dioxide and viscosity. This fluorescent probe utilizes the twisted intramolecular charge transfer (TICT) mechanism between indolium salt and carbazole to achieve two-photon excitation response to viscosity. The carbon-carbon double bond connected to the indolium salt can undergo a nucleophilic addition reaction to achieve the response to sulfur dioxide. For the above two probes for detecting sulfur dioxide and viscosity, their emission wavelengths are in the visible light region and are easily interfered by the background fluorescence of the detection matrix. In order to prepare a visual, highly anti-interference fluorescent probe that can independently detect sulfur dioxide and viscosity simultaneously, our research group has conducted long-term exploration. Summary of the Invention

[0004] To solve the above technical problems, the present invention proposes a fluorescent probe based on triphenylamine and indole derivatives, its preparation method and application.

[0005] The technical solution of the present invention is realized as follows:

[0006] The fluorescent probe (BDI) based on triphenylamine and indole derivatives has the following structural formula:

[0007] .

[0008] The preparation method of the above fluorescent probe has a synthetic route as follows:

[0009] .

[0010] The preparation method is as follows:

[0011] (1) Dissolve commercially available 5-(4-(diphenylamino)phenyl)thiophene-2-carbaldehyde and 2,3,3-trimethylindole in ethanol according to a molar ratio of 1:1.5 - 4, add methanesulfonic acid (in a molar ratio of 1:4 - 10 with 5-(4-(diphenylamino)phenyl)thiophene-2-carbaldehyde), react under reflux at 90 °C for 9 - 15 h. After cooling, add triethylamine dropwise until the color changes from purple to yellow, remove ethanol by reduced pressure distillation, and obtain an orange solid as intermediate 1 by column chromatography separation; the eluent used for column chromatography separation is petroleum ether and dichloromethane, with a volume ratio of (1 - 5):1;

[0012] (2) Dissolve intermediate 1 and benzyl bromide in acetonitrile according to a molar ratio of 1:1.5 - 4, react under reflux at 90 °C for 9 - 15 h. After cooling, remove acetonitrile by reduced pressure distillation, and obtain a black product as the target product BDI by column chromatography separation; the eluent used for column chromatography separation is dichloromethane and methanol, with a volume ratio of (15 - 50):1.

[0013] The fluorescent probe prepared in this application can respond to sulfur dioxide and viscosity respectively under two different excitation wavelengths. The recognition processes of the two are independent and do not interfere with each other. This probe can specifically recognize sulfur dioxide, has high sensitivity and is not easily interfered by other reactive species. Therefore, it has the following uses:

[0014] The above fluorescent probe is used in the preparation of a fluorescent probe for dual recognition of sulfur dioxide and viscosity.

[0015] The above fluorescent probe is used in the specific recognition of sulfur dioxide in alcoholic beverages.

[0016] The above alcoholic beverage is any one of white wine, yellow rice wine and red wine.

[0017] The above fluorescent probe is used for the recognition of sulfur dioxide and viscosity for non-disease diagnosis and treatment purposes.

[0018] The above probe BDI has good cell compatibility, can achieve mitochondrial localization, and can realize the visualization imaging of intracellular SO 2 and viscosity during mitochondrial dysfunction and apoptosis, and has great application potential in the field of bioimaging.

[0019] The detection mechanism of the above probe is as follows: The fluorescent probe BDI of the present invention utilizes the strong nucleophilicity of sulfur dioxide to generate a new product BDI-SO through a Michael addition reaction with the double bond in the probe structure2 Specific selectivity for sulfur dioxide is achieved. The reaction route of the mechanism is as follows:

[0020] .

[0021] The fluorescent probe of this application can also rapidly and sensitively respond to changes in environmental viscosity and is not affected by environmental polarity. The probe has good biocompatibility, can specifically localize in the subcellular organelle mitochondria, and has the ability to image sulfur dioxide and viscosity in mitochondria. Probe BDI has good cell imaging results for the viscosity in the biological microenvironment and can image the increase in the concentration of endogenous SO 2 during viscosity-induced mitochondrial dysfunction.

[0022] The present invention has the following beneficial effects:

[0023] 1. The fluorescent probe of this application can respond to sulfur dioxide and viscosity respectively under two different excitation wavelengths. The recognition processes of the two are independent and do not interfere with each other. Under the excitation condition of 400 nm, probe BDI can rapidly and accurately recognize sulfur dioxide, with advantages such as short response time, good selectivity, low detection limit (54 nM), strong anti-interference ability, and a wide pH applicable range. At the same time, the probe can respond to viscosity under the excitation condition of 585 nm. As the solution viscosity increases, the fluorescence emission intensity of probe BDI at 730 nm also continuously increases, and the relationship between the two perfectly conforms to the Forster-Hoffmann equation, which proves that probe BDI has the ability to quantitatively detect viscosity. The probe can also rapidly and sensitively respond to changes in environmental viscosity and is not affected by environmental polarity. In addition, probe BDI can be used to detect sulfur dioxide in alcoholic beverages such as white wine, yellow rice wine, and red wine.

[0024] 2. Probe BDI of the present invention itself is dark blue in Tris-CH 3 CN solution. When sodium sulfite is added, the solution color quickly turns colorless, enabling naked-eye recognition of sulfur dioxide. The naked-eye color changes from dark blue to colorless before and after recognizing sulfur dioxide, and the solution color changes from colorless to pale blue fluorescence under 365 nm. The solution color change is obvious and easy to distinguish, achieving visual detection (attached Figure 15 ). It can also utilize the strong nucleophilicity of sulfur dioxide to generate a new product BDI-SO through a Michael addition reaction with the double bond in the probe structure 2 Specific selectivity for sulfur dioxide is achieved, and its recognition mechanism is confirmed by high-resolution mass spectrometry. The theoretical molecular weight of the new product BDI-SO 2 after recognizing sulfur dioxide is 667.2095 (negative ion peak), and the high-resolution detected value is 667.2106 (negative ion peak, attached Figure 18 ).

[0025] 3. The fluorescence probe based on triphenylamine and indole derivatives proposed by the present invention has an emission wavelength of 730 nm after responding to viscosity. Compared with the fluorescence probes in the above two invention patents, it has the advantages of strong tissue penetration, small light loss, and the ability to overcome background fluorescence interference. The fluorescence probe of the present invention has good biocompatibility, can specifically localize in the subcellular organelle mitochondria, and has the ability to image sulfur dioxide and viscosity in mitochondria. The probe BDI can achieve visual imaging of intracellular SO 2 and viscosity during mitochondrial dysfunction and apoptosis, and can be regarded as a rapid-response colorimetric analysis tool with high specificity and sensitivity, and has broad application prospects in the field of bioimaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0027] Figure 1 1H NMR spectrum of the fluorescence probe BDI of the present invention (the solvent is CDCl 3 ).

[0028] Figure 2 13C NMR spectrum of the fluorescence probe BDI of the present invention (the solvent is CDCl 3 ).

[0029] Figure 3 High-resolution mass spectrum of the fluorescence probe BDI of the present invention (positive ion mode, the solvent is CH 3 OH).

[0030] Figure 4 Fluorescence selectivity curve of the fluorescence probe BDI of the present invention for identifying sulfur dioxide, the excitation wavelength is 360 nm, and the emission wavelength is 434 nm.

[0031] Figure 5 Fluorescence anti-interference diagram of the fluorescence probe BDI of the present invention for identifying sulfur dioxide, the excitation wavelength is 360 nm, and the emission wavelength is 434 nm.

[0032] Figure 6 Fluorescence titration diagram of the fluorescence probe BDI of the present invention for identifying sulfur dioxide, the excitation wavelength is 360 nm, and the emission wavelength is 434 nm.

[0033] Figure 7This is the graph of the lowest detection limit for the recognition of sulfur dioxide by the fluorescent probe BDI of the present invention. The excitation wavelength is 360 nm and the emission wavelength is 434 nm.

[0034] Figure 8 This is the fluorescence kinetics graph of the fluorescent probe BDI of the present invention for the recognition of sulfur dioxide. The excitation wavelength is 360 nm and the emission wavelength is 434 nm.

[0035] Figure 9 This is the graph of the pH applicable range for the recognition of sulfur dioxide by the fluorescent probe BDI of the present invention. The excitation wavelength is 360 nm and the emission wavelength is 434 nm.

[0036] Figure 10 This is the fluorescence graph of the fluorescent probe BDI of the present invention in different polar solvents. The excitation wavelength is 585 nm and the emission wavelength is 730 nm.

[0037] Figure 11 This is the fluorescence graph of the fluorescent probe BDI of the present invention for different viscosity solutions. The excitation wavelength is 585 nm and the emission wavelength is 730 nm.

[0038] Figure 12 This is the linear relationship graph between the fluorescence intensity of the fluorescent probe BDI of the present invention and the solution viscosity. The excitation wavelength is 585 nm and the emission wavelength is 730 nm.

[0039] Figure 13 This is the fluorescence graph of the fluorescent probe BDI of the present invention in ethanol solution at different temperatures. The excitation wavelength is 585 nm and the emission wavelength is 730 nm.

[0040] Figure 14 This is the fluorescence graph of the fluorescent probe BDI of the present invention in low viscosity solution at different temperatures. The excitation wavelength is 585 nm and the emission wavelength is 730 nm.

[0041] Figure 15 This is the fluorescence graph of the fluorescent probe BDI of the present invention in high viscosity solution at different temperatures. The excitation wavelength is 585 nm and the emission wavelength is 730 nm.

[0042] Figure 16 This is the graph of the naked-eye color change of the solution before and after the recognition of sulfur dioxide by the fluorescent probe BDI of the present invention.

[0043] Figure 17 This is the graph of the color change of the solution under a 365 nm fluorescent lamp before and after the recognition of sulfur dioxide by the fluorescent probe BDI of the present invention.

[0044] Figure 18 This is the high-resolution mass spectrometry graph (negative ion mode, solvent is CH 3 OH) for the mechanism verification after the recognition of sulfur dioxide by the fluorescent probe BDI of the present invention.

[0045] Figure 19 This is the co-localization imaging diagram of the fluorescent probe BDI of the present invention with a commercial green mitochondrial dye in living biological cells.

[0046] Figure 20 This is the fluorescence imaging diagram of the fluorescent probe BDI of the present invention for exogenous and endogenous sulfur dioxide and viscosity changes in the biological microenvironment in living biological cells. Detailed implementation manners

[0047] Next, in combination with the embodiments of the present invention, the technical solutions of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0048] In the process of preparing the fluorescent probe BDI in the present invention, chemical reagents, solvents, active small molecules, etc. are all purchased from Aladdin Reagent Co., Ltd. and Adamas Reagent Co., Ltd. In the process of structural confirmation and analytical performance testing of the fluorescent probe BDI, a Bruke DTX-400 nuclear magnetic resonance spectrometer is used, the solvent is deuterated chloroform, and the nuclear magnetic resonance hydrogen spectrum and carbon spectrum are recorded with TMS as the internal standard. A Q-Exactive HR-MS mass spectrometer of Thermo Company in the United States is used to record high-resolution mass spectrometry data. A fluorescence spectrometer F-7000 of Hitachi Company in Japan is used to record the fluorescence spectrum. An NDJ-7 rotational viscometer is used to test the viscosity.

[0049] Example 1

[0050] The preparation method of the fluorescent probe for identifying sulfur dioxide and viscosity in this example is as follows:

[0051] (1) Preparation of Intermediate 1 [(E)-4-(5-(2-(3,3-dimethyl-3H-indol-2-yl)vinyl)thiophen-2-yl)-N,N-diphenylaniline]

[0052] In a 50 mL round-bottom flask, commercially available 5-(4-(diphenylamino)phenyl)thiophene-2-carbaldehyde (355.5 mg, 1 mmol) and 2,3,3-trimethylindole (240.0 mg, 1.5 mmol) are dissolved in 10 mL of ethanol, and methanesulfonic acid (9.6 mg, 0.10 mmol) is added dropwise. The reaction is refluxed at 90 °C for 9 hours. After cooling to room temperature, triethylamine is added dropwise to the flask until the solution color changes from purple to yellow. Ethanol is removed by reduced pressure distillation, and column chromatography separation (the eluent is petroleum ether / dichloromethane = 1 / 1) is carried out to obtain an orange solid, which is Intermediate 1 (225.0 mg, with a yield of 45%).

[0053] (2) Preparation of Probe BDI

[0054] In a 50 mL round-bottom flask, intermediate 1 (496.7 mg, 1 mmol) and benzyl bromide (257.0 mg, 1.5 mmol) were dissolved in 10 mL of acetonitrile and refluxed at 90 °C for 9 hours. After cooling to room temperature, acetonitrile was removed by distillation under reduced pressure, and column chromatography separation (eluent: dichloromethane / methanol = 15 / 1) gave a black product, which was probe BDI (150.0 mg, yield 22.5%).

[0055] Nuclear magnetic resonance measurement: 1 H NMR (CDCl 3 , 400 MHz) δ 1.93(s, 6H), 6.03(s, 2H), 7.00(d, J = 4 Hz, 2H), 7.02(d, J = 4 Hz, 2H), 7.12(q, J = 8 Hz, 2H), 7.15(d, J = 4 Hz,4H), 7.20(d, J = 4 Hz, 1H), 7.23(d, J = 4 Hz, 2H), 7.29(q, J = 2 Hz, 1H), 7.33(q, J = 4 Hz, 4H), 7.37(q, J = 4 Hz, 2H), 7.45(q, J = 4 Hz, 1H), 7.48(d, J = 4 Hz, 2H),7.51(d, J = 4 Hz, 1H), 7.52(d, J = 4 Hz, 1H), 8.52(q, J = 4 Hz, 1H), 8.74(d, J = 4Hz, 1H); 13 C NMR (CDCl 3, 100 MHz) δ 27.38, 46.29, 52.00, 108.19, 114.21, 121.49, 122.70, 124.46, 125.22, 125.58, 126.66, 127.71, 128.71, 128.78, 129.45, 129.52, 129.60, 133.07, 138.61, 141.37, 142.82, 146.50, 147.80, 150.01, 158.08, 180.75. The 1H NMR spectrum is as shown in Figure 1 shown, and the 13C NMR spectrum is as shown in Figure 2 shown.

[0056] High-resolution mass spectrometry determination: HR-ESI-MS calcd for C 41 H 34 BrN 2 S + : 587.2515, found 587.2507. The high-resolution mass spectrum is as shown in Figure 3 shown.

[0057] Example 2

[0058] The preparation method of the fluorescent probe for identifying sulfur dioxide and viscosity in this example is as follows:

[0059] (1) Preparation of Intermediate 1 [(E)-4-(5-(2-(3,3-dimethyl-3H-indol-2-yl)vinyl)thiophen-2-yl)-N,N-diphenylaniline]

[0060] In a 50 mL round-bottom flask, commercially available 5-(4-(diphenylamino)phenyl)thiophene-2-carbaldehyde (355.5 mg, 1 mmol) and 2,3,3-trimethylindole (480.0 mg, 3 mmol) were dissolved in 10 mL of ethanol, and methanesulfonic acid (19.2 mg, 0.20 mmol) was added dropwise. The mixture was refluxed at 90 °C for 12 hours. After cooling to room temperature, triethylamine was added dropwise to the flask until the solution color changed from purple to yellow. Ethanol was removed by distillation under reduced pressure, and column chromatography separation (eluent: petroleum ether / dichloromethane = 2 / 1) was carried out to obtain an orange solid, which is Intermediate 1 (270.0 mg, yield 54%).

[0061] (2) Preparation of Probe BDI

[0062] In a 50 mL round-bottom flask, intermediate 1 (496.7 mg, 1 mmol) and benzyl bromide (513.0 mg, 3 mmol) were dissolved in 10 mL of acetonitrile and refluxed at 90 °C for 12 hours. After cooling to room temperature, the acetonitrile was removed by distillation under reduced pressure, and the product was separated by column chromatography (eluent: dichloromethane / methanol = 30 / 1) to obtain the black product, probe BDI (180.0 mg, yield 27%).

[0063] Nuclear magnetic resonance measurement: 1 H NMR (CDCl 3 , 400 MHz) δ 1.93(s, 6H), 6.03(s, 2H), 7.00(d, J = 4 Hz, 2H), 7.02(d, J = 4 Hz, 2H), 7.12(q, J = 8 Hz, 2H), 7.15(d, J = 4 Hz,4H), 7.20(d, J = 4 Hz, 1H), 7.23(d, J = 4 Hz, 2H), 7.29(q, J = 2 Hz, 1H), 7.33(q, J = 4 Hz, 4H), 7.37(q, J = 4 Hz, 2H), 7.45(q, J = 4 Hz, 1H), 7.48(d, J = 4 Hz, 2H),7.51(d, J = 4 Hz, 1H), 7.52(d, J = 4 Hz, 1H), 8.52(q, J = 4 Hz, 1H), 8.74(d, J = 4Hz, 1H); 13 C NMR (CDCl 3 , 100 MHz) δ 27.38, 46.29, 52.00, 108.19, 114.21,121.49, 122.70, 124.46, 125.22, 125.58, 126.66,127.71, 128.71, 128.78,129.45, 129.52, 129.60, 133.07, 138.61, 141.37, 142.82, 146.50, 147.80,150.01, 158.08, 180.75. The hydrogen nuclear magnetic resonance spectrum is as followsFigure 1 As shown, the nuclear magnetic resonance carbon spectrum is as Figure 2 shown.

[0064] High-resolution mass spectrometry determination: HR-ESI-MS calcd for C 41 H 34 BrN 2 S + : 587.2515, found 587.2507. The high-resolution mass spectrum is as Figure 3 shown.

[0065] Example 3

[0066] The preparation method of the fluorescent probe for identifying sulfur dioxide and viscosity in this example is as follows:

[0067] (1) Preparation of Intermediate 1 [(E)-4-(5-(2-(3,3-dimethyl-3H-indol-2-yl)vinyl)thiophen-2-yl)-N,N-diphenylaniline]

[0068] In a 50 mL round-bottom flask, commercially available 5-(4-(diphenylamino)phenyl)thiophene-2-carbaldehyde (355.5 mg, 1 mmol) and 2,3,3-trimethylindole (640.0 mg, 4 mmol) were dissolved in 10 mL of ethanol, and methanesulfonic acid (24 mg, 0.25 mmol) was added dropwise. The mixture was refluxed at 90 °C for 16 hours. After cooling to room temperature, triethylamine was added dropwise to the flask until the solution color changed from purple to yellow. Ethanol was removed by distillation under reduced pressure, and column chromatography separation (eluent: petroleum ether / dichloromethane = 5 / 1) was carried out to obtain an orange solid, which is Intermediate 1 (345.0 mg, yield 69%).

[0069] (2) Preparation of Probe BDI

[0070] In a 50 mL round-bottom flask, Intermediate 1 (496.7 mg, 1 mmol) and benzyl bromide (684.0 mg, 4 mmol) were dissolved in 10 mL of acetonitrile, and the mixture was refluxed at 90 °C for 15 hours. After cooling to room temperature, acetonitrile was removed by distillation under reduced pressure, and column chromatography separation (eluent: dichloromethane / methanol = 50 / 1) was carried out to obtain a black product, which is Probe BDI (233.0 mg, yield 35%).

[0071] Nuclear magnetic resonance determination: 1 H NMR (CDCl 3 , 400 MHz) δ 1.93(s, 6H), 6.03(s, 2H), 7.00(d, J = 4 Hz, 2H), 7.02(d,J = 4 Hz, 2H), 7.12 (q, J = 8 Hz, 2H), 7.15 (d, J = 4 Hz, 4H), 7.20 (d, J = 4 Hz, 1H), 7.23 (d, J = 4 Hz, 2H), 7.29 (q, J = 2 Hz, 1H), 7.33 (q, J = 4 Hz, 4H), 7.37 (q, J = 4 Hz, 2H), 7.45 (q, J = 4 Hz, 1H), 7.48 (d, J = 4 Hz, 2H), 7.51 (d, J = 4 Hz, 1H), 7.52 (d, J = 4 Hz, 1H), 8.52 (q, J = 4 Hz, 1H), 8.74 (d, J = 4 Hz, 1H); 13 C NMR (CDCl 3 , 100 MHz) δ 27.38, 46.29, 52.00, 108.19, 114.21, 121.49, 122.70, 124.46, 125.22, 125.58, 126.66, 127.71, 128.71, 128.78, 129.45, 129.52, 129.60, 133.07, 138.61, 141.37, 142.82, 146.50, 147.80, 150.01, 158.08, 180.75. The 1H NMR spectrum is as shown in Figure 1 the figure, and the 13C NMR spectrum is as shown in Figure 2 the figure.

[0072] High-resolution mass spectrometry determination: HR-ESI-MS calcd for C 41 H 34 BrN 2 S + : 587.2515, found 587.2507. The high-resolution mass spectrum is as shown in Figure 3 the figure.

[0073] Example of implementation effect

[0074] Preparation of 1 mM probe solution: Weigh accurately the probe (BDI) prepared in Example 1. Dissolve BDI in dimethyl sulfoxide (DMSO) solution to prepare a 1 mM solution for standby.

[0075] Fluorescence selectivity experiment

[0076] The fluorescent probe should be able to achieve single recognition of the species to be detected. Therefore, it is necessary to test the fluorescence selectivity of probe BDI for different reactive small molecules. The test solution for the fluorescence selectivity experiment of probe BDI is Tris-CH 3 CN (10 mM, pH = 7.40, v / v = 7 / 3). As shown in the appendix Figure 4 When excited at 360 nm, the individual probe hardly shows fluorescence emission at 434 nm. When Na 2 SO 3 (10 eq.) is added, the fluorescence intensity at 434 nm increases significantly. When other reactive species (10 eq.): GSH, Cys, Hcy, GSSG, Ala, Arg, Asp, Glu, Gly, Leu, Lys, His, Ile, Met, Phe, Pro, Ser, Thr, Trp, Val, KCl, NaI, MgSO 4 、KNO 3 、NaNO 2 、Na 2 CO 3 、NaHCO 3 、NaOAc, ONOO - 、Na 2 S are added, only the solution with Na 2 S shows a slight increase in the fluorescence emission intensity (F434) at 434 nm, but the increase amplitude is much smaller than that of Na 2 SO 3 ; The fluorescence emission intensity (F434) at 434 nm of the solutions with other reactive species is the same as that of the individual probe at 434 nm (F434), without obvious enhancement. The above selectivity experiment results show that probe BDI has good selectivity for sulfur dioxide when excited at 360 nm.

[0077] Fluorescence interference experiment

[0078] To investigate the anti-interference ability of probe BDI in response to sulfur dioxide in a complex environment, other reactive small molecules were tested by fluorescence emission spectroscopy. The test solution for the fluorescence interference experiment of probe BDI is Tris-CH 3 CN (10 mM, pH = 7.40, v / v = 7 / 3). As shown in the appendix Figure 5As shown, under 360 nm excitation, 10 equivalents of GSH, Cys, Hcy, GSSG, Ala, Arg, Asp, Glu, Gly, Leu, Lys, His, Ile, Met, Phe, Pro, Ser, Thr, Trp, Val, KCl, NaI, MgSO 4 、KNO 3 、NaNO 2 、Na 2 CO 3 、NaHCO 3 、NaOAc, ONOO - 、Na 2 S were separately added to the individual probe solution, and then 10 equivalents of Na 2 SO 3 were added. The fluorescence emission intensity at 434 nm of the solution was detected respectively. As can be seen from the attached figure, only the fluorescence emission intensity at 434 nm of the probe solution added with Na 2 S increased slightly. This is mainly because in the aqueous solution system, S 2- is prone to undergo a disproportionation reaction with SO 3 2- . The physiological content of H 2 S in the organism is generally about 20 - 160 μM, while the content of SO 2 2 is generally as high as 1 - 3 M. Therefore, H 2 S will not interfere with the biological application of the probe BDI for recognizing SO 2 2 . In addition, the fluorescence emission intensity at 434 nm (F434) of the solution added with different reactive small molecules as interfering species is basically the same as that of the solution added with Na 2 SO 3 3 alone at 434 nm. This indicates that the probe BDI has strong anti-interference ability to other reactive small molecules when detecting sulfur dioxide.

[0079] Minimum detection limit experiment

[0080] Fluorescent probes are required to have high detection sensitivity. Therefore, the minimum detection limit is also an important indicator to measure the properties of the probe. The fluorescence emission spectrum was used to test the minimum detection limit of the probe BDI for sulfur dioxide. The test solution for the minimum detection limit experiment of the probe BDI was Tris-CH 3 CN (10 mM, pH = 7.40, v / v = 7 / 3). Under 360 nm excitation, the concentration of the probe BDI was fixed at 10 μM, and the concentration of Na 2 SO 3 in the solution was adjusted (from 0 μM to 20 μM), and the fluorescence emission intensity of the solution containing different concentrations of Na 2 SO3 The fluorescence emission intensity (F434) of the probe solution at 434 nm, as shown in the appendix Figure 6 、 7 Shown. The fluorescence intensity of the solution at 434 nm is in the range of 0 - 20 μM of Na 2 SO 3 Concentration shows a good linear relationship with the Na 2 SO 3 Concentration (R 2 = 0.999). According to the IUPAC rules, the lowest detection limit of the probe BDI for sulfur dioxide is calculated by the formula (3σ / k) to be 54 nM. The experimental results of the lowest detection limit indicate that the probe BDI has high sensitivity to sulfur dioxide and can achieve quantitative detection of extremely trace concentrations of sulfur dioxide.

[0081] Fluorescence kinetics experiment

[0082] Response time is also an important indicator to evaluate the quality of fluorescence probes. The fluorescence kinetics of the probe BDI towards sulfur dioxide was tested using fluorescence emission spectra. The test solution for the lowest detection limit experiment of the probe BDI was Tris-CH 3 CN (10 mM, pH = 7.40, v / v = 7 / 3). Under 360 nm excitation, with the concentration of the probe BDI fixed at 10 μM, the change in the fluorescence emission intensity (F434) at 434 nm of the solution without and with Na 2 SO 3 (100 μM) over time was measured. As shown in the appendix Figure 8 Shown, the fluorescence emission intensity at 434 nm of the single probe remains basically unchanged over time, while the fluorescence emission intensity at 434 nm of the solution with the addition of Na 2 SO 3 (100 μM) increases rapidly over time and reaches a plateau after 4 minutes. The results of the fluorescence kinetics experiment indicate that the probe BDI responds rapidly to sulfur dioxide and can achieve rapid detection.

[0083] Effect of pH on the recognition ability of the probe towards sulfur dioxide

[0084] pH is also an important criterion to measure the versatility of the probe. The recognition ability of the probe BDI towards sulfur dioxide in different pH environments was investigated using fluorescence emission spectra. The test solution for the pH experiment of the probe BDI was Tris-CH 3 CN (10 mM, v / v = 7 / 3) with pH = 4, 5, 6, 7.4, 8, 9, 10. The concentrations of the probe and Na 2 SO 3 Were 10 μM and 100 μM respectively. As shown in the appendixFigure 9 As shown, in the range of pH = 4 - 9, the fluorescence emission intensity of the single probe at 434 nm did not change significantly; while in the presence of Na 2 SO 3 , in the range of pH = 6 - 9, the fluorescence emission intensity of the test solution at 434 nm increased significantly. The test results of the acidity and alkalinity (pH) indicate that the probe BDI can recognize sulfur dioxide under physiological conditions.

[0085] Interference experiment of solutions with different polarities

[0086] To investigate the influence of different polarities on the viscosity recognition of the probe BDI, fluorescence emission spectra were used to test solutions with different polarities. Eleven different solutions such as glycerol, methanol, ethanol, acetonitrile, DMSO, DMF, THF, PBS, Heppes, Tris and water were selected, 10 μM of the probe BDI was added, and the fluorescence emission at 730 nm was detected. As attached Figure 10 shown, only the solution in the glycerol system produced strong fluorescence emission at 730 nm, while the solutions in other systems with different polarities did not show obvious fluorescence changes. The results of the polarity interference experiment indicate that the probe BDI is only sensitive to viscosity and is not interfered by the solution polarity.

[0087] Fluorescence experiment of different viscosities

[0088] Since the probe BDI molecule has a typical viscosity-responsive rotor (triphenylamine group), its viscosity response was investigated. The test solution for the viscosity response experiment of the probe BDI was the ethanol-glycerol system, and the glycerol content in the solution was 0% - 99%. A rotational viscometer was used to measure the viscosity. As attached Figure 11 、 12 shown, as the glycerol content in the system increased from 0% to 99%, the fluorescence intensity of the solution at 730 nm increased significantly. According to the Forster-Hoffmann equation (log (I) = C + x log η), a good linear relationship was presented between log η and log (I) 730 with (R 2 = 0.998). The test results of the probe viscosity response indicate that the probe BDI can achieve quantitative detection of viscosity.

[0089] Temperature influence experiment

[0090] Since the temperature can affect the liquid viscosity and thus the fluorescence intensity, temperature is also a factor that needs to be investigated. Three systems of pure ethanol, ethanol-glycerol (v / v = 5 / 5), and ethanol-glycerol (v / v = 2 / 8) were selected, with the probe concentration fixed at 10 μM, and fluorescence emission tests were carried out at different temperatures. As attached Figure 13 、 14As shown in Fig. 15, as the temperature decreases from 30 °C to -10 °C, the fluorescence emission intensity of the probe at 730 nm in the pure ethanol system increases by about 3.8 times, the fluorescence emission intensity of the probe at 730 nm in the ethanol-glycerol (v / v = 5 / 5) system increases by about 2.6 times, and the fluorescence emission intensity of the probe at 730 nm in the ethanol-glycerol (v / v = 5 / 5) system increases by about 5.4 times. The temperature influence experiment results show that the probe BDI can also respond to the viscosity changes caused by temperature influence.

[0091] Localization imaging experiment of probe BDI in subcellular organelle mitochondria

[0092] Apply the probe BDI of the present invention to human cervical cancer cells (HeLa), and add a commercial green mitochondrial dye for localization contrast imaging (attached Figure 19 ), and the specific steps are as follows:

[0093] 1) A certain amount of living cells are incubated with a medium containing 10 μM commercial green mitochondrial dye for 30 min. Wash with PBS, and then incubate with a medium containing 10 μM probe BDI for 30 min;

[0094] 2) The above cells are washed with PBS, and then incubated with a medium containing 100 μM Na 2 SO 3 for 30 min. After washing with PBS, add PBS buffer. Observe the white light channel, green light channel and blue light channel under a microscope. It can be seen from the attachment Figure 19 that the cell morphology in the white light channel is good, and both the green light channel and the blue light channel emit strong fluorescence and overlap well. The Pearson coefficient is 0.9780, indicating that the probe BDI can specifically localize in the subcellular organelle mitochondria and has the ability to image sulfur dioxide and viscosity in mitochondria.

[0095] Probe BDI for exogenous and endogenous SO 2 and viscosity imaging experiments

[0096] Nystatin is called a membrane permeability enhancer, which can induce mitochondrial dysfunction and increase the viscosity of the cell microenvironment. Since a large amount of reactive oxygen species are generated in cells during mitochondrial dysfunction, and SO 2 as a cellular antioxidant has the function of scavenging excessive ROS to maintain the cellular redox balance, and ultimately leads to an increase in the SO 2 level with the increase in viscosity. Here, the probe BDI was applied to exogenous and endogenous SO 2 and viscosity imaging experiments (attached Figure 20 ), and the specific experimental steps are as follows:

[0097] 1) Blank control group: A certain amount of live cells were added to a medium containing 10 μM probe BDI and incubated in an incubator at 37 °C and 5% CO 2 for 30 min. After washing with PBS, PBS buffer was added. Under the microscope, imaging in the white light channel, blue light channel, and red light channel was observed. Among them, the cell morphology was good in the white light channel, and there was no fluorescence in both the blue light channel and the red light channel (see Figure 20 , a1, b1, c1).

[0098] 2) Exogenous SO 2 Experimental group: A certain amount of live cells were added to a medium containing 10 μM probe BDI and incubated in an incubator at 37 °C and 5% CO 2 for 30 min. After washing with PBS, a medium containing 100 μM probe Na 2 SO 3 was added and the cells were cultured for another 30 min. After washing with PBS, PBS buffer was added. Under the microscope, imaging in the white light channel, blue light channel, and red light channel was observed. Among them, the cell morphology was good in the white light channel, the blue light channel showed strong blue fluorescence, and there was no fluorescence in the red light channel (see Figure 20 , a2, b2, c2), indicating that probe BDI can perform cell imaging on exogenous SO 2 .

[0099] 3) Viscosity and endogenous SO 2 Experimental group: A certain amount of live cells were added to a medium containing 10 μM nystatin and incubated in an incubator at 37 °C and 5% CO 2 for 30 min. After washing with PBS, a medium containing 10 μM probe BDI was added and the cells were cultured for another 30 min. After washing with PBS, PBS buffer was added. Under the microscope, imaging in the white light channel, blue light channel, and red light channel was observed. Among them, the cell morphology was good in the white light channel, the blue light channel showed strong blue fluorescence, and the red light channel showed strong red fluorescence (see Figure 20 , a3, b3, c3), indicating that probe BDI has good cell imaging results for the viscosity in the biological microenvironment and can image the increase in the concentration of endogenous SO 2 induced during viscosity-induced mitochondrial dysfunction. The above experimental results show that probe BDI can achieve visual imaging of intracellular SO 2 and viscosity during mitochondrial dysfunction and apoptosis. Probe BDI can be regarded as a rapid-response colorimetric analysis tool for detecting relevant analytes in real samples and organisms, with high specificity and sensitivity.

[0100] Application research of probe BDI in the detection of SO 2 in alcoholic beverages

[0101] Adjust the pH of three liquor samples to 7.40, add sodium sulfite at different concentrations (0, 1, 2, 3 μM) to them, and then add the probe BDI to detect the change in fluorescence intensity at 434 nm of the solution. The results are shown in Table 1:

[0102] Table 1 Detection results of probe BDI in liquor

[0103]

[0104] As can be seen from Table 1, the recovery rates of the three liquor samples for SO 2 are all between 95% and 106%, which proves that the probe BDI can be used for the detection of SO 2 in liquor samples.

[0105] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A fluorescent probe based on triphenylamine and indole derivatives, which has the following structural formula: 。 2. A preparation method of the fluorescent probe based on triphenylamine and indole derivatives according to claim 1, characterized in that, the steps are as follows: (1) Dissolve 5-(4-(diphenylamino)phenyl)thiophene-2-carbaldehyde and 2,3,3-trimethylindole in ethanol, add methanesulfonic acid, carry out a reflux reaction, after cooling, add triethylamine dropwise until the color changes from purple to yellow, distill off ethanol under reduced pressure, and obtain an orange solid, namely intermediate 1, by the first column chromatography separation; the molar ratio of methanesulfonic acid to 5-(4-(diphenylamino)phenyl)thiophene-2-carbaldehyde is 1:4 - 10; (2) Dissolve intermediate 1 and benzyl bromide in acetonitrile, carry out a reflux reaction, after cooling, distill off acetonitrile under reduced pressure, and obtain a black product, namely the target product BDI, by the second column chromatography separation.

3. The preparation method of the fluorescent probe based on triphenylamine and indole derivatives according to claim 2, characterized in that: in the step (1), the molar ratio of 5-(4-(diphenylamino)phenyl)thiophene-2-carbaldehyde to 2,3,3-trimethylindole is 1:1.5 - 4.

4. The preparation method of the fluorescent probe based on triphenylamine and indole derivatives according to claim 2 or 3, characterized in that: the eluent used in the first column chromatography is petroleum ether and dichloromethane with a volume ratio of (1 - 5):

1.

5. The preparation method of the fluorescent probe based on triphenylamine and indole derivatives according to claim 4, characterized in that: in the step (2), the molar ratio of intermediate 1 to benzyl bromide is 1:1.5 - 4.

6. The preparation method of the fluorescent probe based on triphenylamine and indole derivatives according to claim 5, characterized in that: the eluent used in the second column chromatography is dichloromethane and methanol with a volume ratio of (15 - 50):

1.

7. The preparation method of the fluorescent probe based on triphenylamine and indole derivatives according to any one of claims 2, 3, 5 or 6, characterized in that: the temperature of the reflux reaction is 90 °C and the time is 9 - 15 h.

8. Application of the fluorescent probe according to claim 1 in the preparation of a fluorescent probe for dual recognition of sulfur dioxide and viscosity.

9. Application of the fluorescent probe according to claim 1 in the specific recognition of sulfur dioxide in alcoholic beverages.

10. Application of the fluorescent probe according to claim 1 in the recognition of sulfur dioxide and viscosity for non-disease diagnosis and treatment purposes.

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