Synthesis and application of a class of symmetric fluorine boron fluorescent dyes with a-d-a configuration

Abstract

The application relates to synthesis and application of a kind of symmetric fluorine boron fluorescent dye with A-D-A configuration, and belongs to the field of fluorescent dye.The molecular structure is as follows: wherein R=H, CH3, OCH3.At present, fluorine boron fluorescent dyes are all D-A structure, and A-D-A fluorine boron fluorescent dye has not been reported yet.Phenothiazine has strong electron supply ability, and is expected to be conjugated with two electron acceptors to obtain A-D-A fluorescent dye.In addition, compared with traditional fluorine boron dyes, the fluorine boron dye has long wavelength emission, large Stokes shift, high fluorescence quantum efficiency and solid fluorescence property.In addition, it can respond to HClO, and has good biological application prospect.

Description

Technical Field

[0001] This invention relates to a class of symmetrical fluoroboron fluorescent dyes based on the ADA configuration, including their synthesis, photophysical property studies, and applications in bioimaging, and belongs to the field of fluorescent dyes. Background Technology

[0002] In recent years, heteroatom-modified organic dyes, such as BODIPY, have attracted widespread attention due to their outstanding optical properties, including relatively narrow absorption and emission bands, high molar absorptivity, high fluorescence quantum yield, satisfactory photostability and chemical stability, and insensitivity to solvent polarity and pH. They have been widely used in biosensors, photodynamic therapy, bioimaging, laser dyes, and optoelectronic materials. However, due to the tight π-π packing between molecules, classic BODIPY suffers severe fluorescence quenching in the solid state. Furthermore, most BODIPYs exhibit very small Stokes shifts (typically <30 nm), which inevitably leads to self-quenching and measurement errors in fluorescence sensing. Therefore, developing novel red-emitting fluorine-boron fluorescent dyes with long-wavelength emission and large Stokes shifts is of great significance.

[0003] Phenothiazine (PTZ), first synthesized by Bernthsen in 1883, has been widely used in the manufacture of dyes, pigments, pharmaceuticals, dye-sensitized solar cells, and photocopying materials. Phenothiazine contains electron-rich nitrogen and sulfur heteroatoms, exhibiting a heterocyclic structure and strong electron-donating ability. The non-planar butterfly conformation of phenothiazine effectively prevents intermolecular π-π stacking, thus preventing strong solid-state fluorescence. Therefore, introducing the phenothiazine moiety into BODIPY analogs is a robust strategy for developing red fluorescent dyes with large Stokes shifts. Furthermore, fluoroboron dyes with a DA configuration using the phenothiazine group as an electron donor have been reported, but symmetrical fluoroboron fluorescent dyes with this ADA configuration have not yet been reported.

[0004] In this patent, we designed and synthesized a series of novel symmetrical fluoroboron fluorescent dyes with ADA configurations by introducing different substituents at the aryl end of the aniline group, using the phenothiazine group as the electron donor moiety. These dyes possess the advantages of long-wavelength emission and large Stokes shift, effectively avoiding self-absorption and background fluorescence interference. Furthermore, these dyes can specifically recognize HClO by oxidizing the electron-donating divalent sulfur atom on the phenothiazine group into an electron-withdrawing sulfoxide group, achieving ratiometric detection of HClO under single-wavelength excitation. This has been successfully applied to bioimaging in HeLa cells and zebrafish. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing research by developing a class of symmetrical fluorine-boron fluorescent dyes with an ADA configuration. This dye possesses advantages such as a large Stokes shift, long-wavelength emission, and high fluorescence quantum efficiency, and can specifically recognize HClO. Its structure is as follows:

[0006]

[0007] Where R = H, CH3, OCH3.

[0008] When R = H, the synthetic route of the representative compound of this invention is as follows:

[0009]

[0010] (a) Sodium tert-butoxide, bis(2-diphenylphosphine) ether, and palladium acetate were dissolved in toluene, and the mixture was refluxed under argon protection for 12 h. After the reaction was complete, the reaction solution was poured into water, stirred briefly, and then extracted with ethyl acetate. The organic layer was washed with saturated brine and water, and dried over anhydrous Na2SO4. After removing the solvent by rotary evaporation, the crude product was purified by column chromatography to obtain a brownish-red oily liquid compound 1.

[0011] (b) Compound 1, S8, and elemental iodine were added to a round-bottom flask. Under argon protection, the reactants were melted and reacted at 195°C for 30 min. After the reaction was complete, ethyl acetate was added to dissolve the resulting mixture, which was then washed with saturated brine and water, and dried over anhydrous Na2SO4. After removing the solvent by rotary evaporation, the crude product was purified by column chromatography to obtain a brownish-yellow oily liquid compound 2.

[0012] (c) Compound 2, iodoethane, sodium hydroxide, and a catalytic amount of KI were dissolved in anhydrous DMSO and reacted at 70°C for 6 h. After the reaction was complete, the reaction mixture was poured into water and extracted with DCM. The organic layer was washed with saturated brine and water and dried over anhydrous Na2SO4. After removing the solvent by rotary evaporation, the crude product was purified by column chromatography to obtain a green oily liquid compound 3.

[0013] (d) Phosphorus oxychloride was slowly added to anhydrous DMF at 0°C. After the addition was complete, stirring was continued for 15 min, and the solution turned pink. Compound 3 was dissolved in an appropriate amount of anhydrous DMF and added to the reaction system, and the reaction was carried out at 80°C for 3 h. After the reaction was completed, the reaction solution was poured into ice water, the pH was adjusted to neutral with saturated NaHCO3 solution, and DCM was added for extraction. The organic layer was washed successively with saturated brine and water, and dried over anhydrous Na2SO4. After removing the solvent by rotary evaporation, the crude product was purified by column chromatography to obtain the yellow solid product compound 4.

[0014] (e) Compound 4 was dissolved in anhydrous DCM. The mixture was reacted with boron tribromide added at -78°C under argon protection for 1 h, then the reaction was continued at room temperature for 10 h. After the reaction was complete, the reaction solution was slowly poured into ice water, and then extracted with DCM. The organic layer was washed successively with saturated brine and water, and dried over anhydrous Na₂SO₄. After removing the solvent by rotary evaporation, the crude product was purified by column chromatography to obtain the yellow solid product, compound 5.

[0015] (f) Compound 5 was dissolved in anhydrous ethanol, and aniline was added. The mixture was reacted at room temperature for 12 h. After the reaction was complete, the product was filtered to obtain a dark red solid. The solid was washed with cold ethanol and dried to obtain crude product 6a, which was used directly in the next step. Crude product 6a was dissolved in 1,2-dichloroethane, and N,N-diisopropylethylamine and boron trifluoride diethyl ether were added. The mixture was refluxed for 5 h. After the reaction was complete, the solvent was removed by rotary evaporation, and the crude product was purified by column chromatography to obtain a red solid product SPTZ-H.

[0016] In step (a), the molar ratio of intermediate aminoanisole, m-bromoanisole, sodium tert-butoxide, bis(2-diphenylphosphophenyl) ether, and palladium acetate is 1:1.1:1.5:0.1:0.05.

[0017] In step (b), the molar ratio of compound 1, S8, and iodine is 1:0.32:0.01.

[0018] In step (c), the molar ratio of compound 2, iodoethane, and sodium hydroxide is 1:10:2.5.

[0019] In step (d), the molar ratio of compound 3 to POCl3 is 1:5.8.

[0020] In step (e), the molar ratio of compound 4 to BBr3 is 1:6.7.

[0021] In step (f), the molar ratio of compound 5, aniline, N,N-diisopropylethylamine, and boron trifluoride ether is 1:6.9:3.75:3.75.

[0022] In steps (a), (b), (c), (d), (e), and (f), the eluent used in the column chromatography is (V) 石油醚 / V 二氯甲烷 =1 / 1), (V 石油醚 / V 二氯甲烷 =1 / 2), (V 石油醚 / V 二氯甲烷 =4 / 1), (V 石油醚 / V 二氯甲烷 =1 / 1), (V 石油醚 / V二氯甲烷 =1 / 2), (V 石油醚 / V 二氯甲烷 =1 / 3).

[0023] The photophysical property testing method for the fluorescent dyes of this invention is as follows: Dyes SPTZ-H, SPTZ-CH3, and SPTZ-OCH3 are dissolved in toluene, tetrahydrofuran, dichloromethane, dioxane, methanol, acetonitrile, and dimethyl sulfoxide, respectively, to prepare a solution of 1.0 × 10⁻⁶. - 5 The absorption wavelength, emission wavelength, molar extinction coefficient, and fluorescence quantum efficiency of a solution of M were determined at room temperature. Specific implementation methods are detailed in the implementation examples.

[0024] The method for testing the response of the fluorescent dye to HClO according to the present invention is as follows: the dye SPTZ-OCH3 is dissolved in THF / PBS (3:7, v / v, 10mM, pH=7.40) and tested at room temperature. It can perform qualitative and quantitative detection of HClO; the specific implementation method is described in detail in the implementation examples.

[0025] The purpose of this invention is to synthesize a series of symmetrical fluoroboron fluorescent dyes with ADA configuration by introducing different substituents using a phenothiazine group as the donor moiety of the fluorescent dye. Compared with traditional fluoroboron dyes, these fluoroboron dyes exhibit long-wavelength emission (>620 nm), large Stokes shift (>135 nm), good fluorescence quantum yield (>0.11), and solid-state fluorescence properties.

[0026] The response mechanism of the fluorescent dye of the present invention to HClO is as follows: In the presence of HClO, the sulfur (S) atom in the SPTZ-OCH3 moiety is oxidized to a sulfoxide group (S=O), the intermolecular ICT interaction is weakened, and the emission wavelength is blue shifted.

[0027] The fluorescent dye SPTZ-OCH3 described in this invention exhibits good selectivity and responsiveness to HClO, and good biocompatibility. Attached Figure Description

[0028] Figure 1 The image shows the 1H NMR spectrum of the fluorescent dye SPTZ-H in deuterated DMSO, with the x-axis representing chemical shift and the y-axis representing intensity.

[0029] Figure 2 The image shows the carbon NMR spectrum of the fluorescent dye SPTZ-H in deuterated DMSO, with the x-axis representing chemical shift and the y-axis representing intensity.

[0030] Figure 3 The image shows the 1H NMR spectrum of the fluorescent dye SPTZ-CH3 in deuterated DMSO, with the x-axis representing chemical shift and the y-axis representing intensity.

[0031] Figure 4 The image shows the carbon NMR spectrum of the fluorescent dye SPTZ-CH3 in deuterated DMSO, with the x-axis representing chemical shift and the y-axis representing intensity.

[0032] Figure 5 The image shows the 1H NMR spectrum of the fluorescent dye SPTZ-OCH3 in deuterated DMSO, with the x-axis representing chemical shift and the y-axis representing intensity.

[0033] Figure 6 The image shows the carbon NMR spectrum of the fluorescent dye SPTZ-OCH3 in deuterated DMSO, with the x-axis representing chemical shift and the y-axis representing intensity.

[0034] Figure 7 The normalized absorption spectra of the fluorescent dye SPTZ-H of the present invention in different solvents (toluene, tetrahydrofuran, dichloromethane, dioxane, methanol, acetonitrile, and dimethyl sulfoxide) are shown. The horizontal axis represents wavelength, and the vertical axis represents relative intensity.

[0035] Figure 8 The normalized emission spectra of the fluorescent dye SPTZ-H of the present invention in different solvents (toluene, tetrahydrofuran, dichloromethane, dioxane, methanol, acetonitrile, dimethyl sulfoxide) are shown. The horizontal axis represents wavelength and the vertical axis represents relative intensity.

[0036] Figure 9 The normalized absorption spectra of the fluorescent dye SPTZ-CH3 of the present invention in different solvents (toluene, tetrahydrofuran, dichloromethane, dioxane, methanol, acetonitrile, and dimethyl sulfoxide) are shown. The horizontal axis represents wavelength, and the vertical axis represents relative intensity.

[0037] Figure 10 The normalized emission spectra of the fluorescent dye SPTZ-CH3 of the present invention in different solvents (toluene, tetrahydrofuran, dichloromethane, dioxane, methanol, acetonitrile, dimethyl sulfoxide) are shown. The horizontal axis represents wavelength, and the vertical axis represents relative intensity.

[0038] Figure 11 The normalized absorption spectra of the fluorescent dye SPTZ-OCH3 of the present invention in different solvents (toluene, tetrahydrofuran, dichloromethane, dioxane, methanol, acetonitrile, dimethyl sulfoxide) are shown. The horizontal axis represents wavelength, and the vertical axis represents relative intensity.

[0039] Figure 12 The normalized emission spectra of the fluorescent dye SPTZ-OCH3 of the present invention in different solvents (toluene, tetrahydrofuran, dichloromethane, dioxane, methanol, acetonitrile, dimethyl sulfoxide) are shown. The horizontal axis represents wavelength and the vertical axis represents relative intensity.

[0040] Figure 13The absorption wavelength, emission wavelength, Stokes shift, fluorescence quantum efficiency, and molar extinction coefficient of the fluorescent dye SPTZ of the present invention in different solvents (toluene, tetrahydrofuran, dichloromethane, dioxane, methanol, acetonitrile, and dimethyl sulfoxide).

[0041] Figure 14 The normalized emission spectrum of the fluorescent dye of the present invention in the solid state is shown, with the horizontal axis representing the emission wavelength and the vertical axis representing the fluorescence intensity.

[0042] Figure 15 The fluorescence emission spectra of the fluorescent dye SPTZ-OCH3 (10 μM) of this invention before and after the response to PBS buffer (0.01 M, pH = 7.4, 30% tetrahydrofuran) and 1200 μM HClO are shown. The horizontal axis represents wavelength, and the vertical axis represents fluorescence intensity.

[0043] Figure 16 The fluorescent dye SPTZ-OCH3 (10 μM) of the present invention was reacted with related substances (Ca) in PBS buffer (0.01 M, pH = 7.4, 30% tetrahydrofuran). 2+ K + Mg 2+ NH4 + S 2- CO3 2- ONOO - SO3 2- TBHP, F - NO2 - The changes in the fluorescence spectrum of the dye solution after responding to Hcy, Cys, GSH, NO·, ROO·, H2O2, HO·, and HClO are shown on the x-axis as wavelength and the y-axis as fluorescence intensity.

[0044] Figure 17 The MTT assay results of different concentrations of the fluorescent dye SPTZ-OCH3 in HeLa cells after 24 hours of incubation are shown. The horizontal axis represents concentration, and the vertical axis represents cell viability.

[0045] Figure 18 This is an imaging experiment of HeLa cells using the fluorescent dye SPTZ-OCH3 of this invention.

[0046] Figure 19 This is a zebrafish imaging experiment using the fluorescent dye SPTZ-OCH3 of this invention.

[0047] Figure 20 This is a schematic diagram of the response mechanism of the fluorescent dye SPTZ-OCH3 of the present invention.

[0048] Specific implementation examples

[0049] Example 1: Synthesis of Compound 1

[0050] m-Aminoanisole (4.92 g, 40 mmol) and m-bromoanisole (8.23 g, 44 mmol) were dissolved in 80 mL of anhydrous toluene. Sodium tert-butoxide (5.77 g, 60 mmol) was added, followed by the addition of bis(2-diphenylphosphine) ether (2.15 g, 4 mmol) and palladium acetate (449 mg, 0.05 mmol) under argon protection. The mixture was heated to reflux for 12 h. After the reaction was complete, the reaction solution was poured into 100 mL of water, stirred briefly, and then extracted three times with EA (100 mL × 3). The extract was washed with saturated brine and water, and dried over anhydrous Na₂SO₄. After removing the solvent by rotary evaporation, the crude product was purified by column chromatography (PE:DCM = 1:1) to obtain a brownish-red oily liquid (5.52 g, 60.3%). H NMR (400MHz, CDCl3) δ7.15 (t, J = 8.0Hz, 1H), 6.71–6.59 (m, 2H), 6.52–6.43 (m, 1H), 3.75 (s, 3H). 13 C NMR(100MHz, CDCl3)δ160.73,144.32,130.13,110.70,106.55,103.87,55.24.HRMS(ESI)m / z[C 14 H 15 NO2+H] + Theoretical value: 230.1176; Measured value: 230.1097.

[0051] Example 2: Synthesis of Compound 2

[0052] Compound 1 (1 g, 4.4 mmol), S8 (350 mg, 1.4 mmol), and I2 (10 mg, 0.04 mmol) were added to a round-bottom flask. Under argon protection, the reactants were melted and reacted at 195 °C for 30 min. After the reaction was complete, the mixture was dissolved in 100 mL of EA, washed with saturated brine and water, and dried over anhydrous Na2SO4. After removing the solvent by rotary evaporation, the crude product was purified by column chromatography (PE:DCM = 1:2) to obtain a brownish-yellow oily liquid (320 mg, 28.3%). 1 H NMR (400MHz, DMSO-d6) δ8.42(s,1H),6.90(t,J=8.1Hz,1H),6.76(d,J=8.4Hz,1H),6.42(d ,J=8.2Hz,1H),6.32(dd,J=8.5,2.6Hz,1H),6.28–6.21(m,2H),3.73(s,3H),3.66(s,3H). 13C NMR(100MHz,DMSO-d6)δ159.61,155.13,143.10,142.32,127.71,127.33,108 .05,107.38,107.35,105.21,104.39,100.04,56.22,55.45.HRMS(ESI)m / z[C 14 H 13 NO2S] + Theoretical value: 259.0667; Measured value: 259.0649.

[0053] Example 3: Synthesis of Compound 3

[0054] Compound 2 (80 mg, 0.3 mmol), iodoethane (513 mg, 3 mmol), sodium hydroxide (100 mg, 2.5 mmol), and a catalytic amount of KI were dissolved in 7 mL of anhydrous DMSO. The resulting mixture was stirred at 70 °C under argon protection for 6 h. After the reaction was complete, the reaction mixture was poured into 50 mL of water, and then extracted three times (3 × 100 mL) with DCM. The mixture was washed with saturated brine and water, and dried over anhydrous Na₂SO₄. After removing the solvent by rotary evaporation, the crude product was purified by column chromatography (PE:DCM = 4:1) to give a green oily liquid (78 mg, 87.6%). 1 H NMR (400MHz, DMSO-d6) δ7.12(td,J=8.2,3.3Hz,1H),6.99(dd,J=8.2,3.3Hz,1H),6.63(ddd,J=12.1,8.2,3.2Hz,2 H),6.55–6.47(m,2H),3.89–3.83(m,2H),3.79(d,J=3.3Hz,3H),3.74(d,J=3.3Hz,3H),1.28(t,J=6.8,3.3Hz,3H). 13 CNMR(100MHz,DMSO-d6)δ159.97,155.32,146.39,145.31,127.92,127.85,113.89,1 12.00,109.20,107.47,105.41,103.08,56.38,55.74,41.87,13.27.HRMS(ESI)m / z[C 16 H 17 NO2S] + Theoretical value: 287.0980; Measured value: 287.0872.

[0055] Example 4: Synthesis of Compound 4

[0056] At 0°C, phosphorus oxychloride (0.3 mL, 3.2 mmol) was slowly added to anhydrous DMF (0.35 mL, 4.5 mmol). After the addition was complete, the mixture was stirred at this temperature for 15 min, and the solution turned pink. Then, compound 3 (160 mg, 0.55 mmol) was dissolved in an appropriate amount of anhydrous DMF and added to the reaction system. The mixture was heated to 80°C and reacted for 3 h. After the reaction was complete, the reaction solution was poured into 50 mL of ice water, the pH was adjusted to neutral with saturated NaHCO3 solution, and then extracted three times with DCM (3 × 50 mL). The organic layer was washed successively with saturated brine and water, and dried over anhydrous Na2SO4. After removing the solvent by rotary evaporation, the crude product was purified by column chromatography (PE:DCM = 1:1) to obtain a yellow solid product (60 mg, 32.1%). 1 H NMR (400MHz, DMSO-d6) δ10.13(s,2H),7.38(s,2H),6.77(s,2H),4.22(q,J=6.9Hz,2H),3.97(s,6H),1.40(t,J=6.9Hz,3H).HRMS(ESI)m / z[C 18 H 17 NO4S+Na] + Theoretical value: 366.0770; Measured value: 366.0625.

[0057] Example 5: Synthesis of Compound 5

[0058] Compound 4 (400 mg, 1.2 mmol) was dissolved in 10 mL of anhydrous DCM. Under argon protection at -78 °C, 2 mol / L boron tribromide (4 mL, 8 mmol) was added to the mixture using a syringe. After reacting for 1 h, the mixture was moved to room temperature and reacted for another 10 h. After the reaction was complete, the reaction solution was slowly poured into 100 mL of ice water, and then extracted twice with DCM (2 × 80 mL). The organic layer was washed successively with saturated brine and water, and dried over anhydrous Na₂SO₄. After removing the solvent by rotary evaporation, the crude product was purified by column chromatography (PE:DCM = 1:2) to obtain a yellow solid product (302 mg, 82.3%). 1 H NMR (400MHz, DMSO-d6) δ10.91(s,2H),9.99(s,2H),7.35(s,2H),6.65(s,2H),3.95(q,J=6.9Hz,2H),1.38(t,J=6.9Hz,3H). 13C NMR(100MHz,DMSO-d6)δ189.61,161.97,149.20,127.32,118.22,112.49,104.38,43.34,12.51.HRMS(ESI)m / z[C 16 H 13 NO4S] + Theoretical value: 315.0565; Measured value: 315.0802.

[0059] Example 6: Synthesis of compound SPTZ-H

[0060] Compound 5 (50 mg, 0.16 mmol) was dissolved in 15 mL of anhydrous EtOH, and aniline (100 μL, 1.1 mmol) was added. The mixture was reacted at room temperature for 12 h. After the reaction was complete, the product was filtered to obtain a dark red solid. The solid was washed with cold EtOH and then dried to obtain crude product 6a, which was used directly in the next step. Crude product 6a was dissolved in 8 mL of C2H4Cl2, and DIEA (100 μL, 0.6 mmol) was added. The mixture was refluxed at 80 °C for 10 min. Then BF3·OEt2 (76 μL, 0.6 mmol) was added, and the reaction mixture was refluxed for another 5 h. After the reaction was complete, C2H4Cl2 was removed by rotary evaporation, and the crude product was purified by column chromatography (PE:DCM = 1:3) to obtain a red solid product (33 mg, 68.8%). 1 H NMR(400MHz, CDCl3)δ8.26(s,2H),7.53–7.50(m,4H),7.49(s,2H),7.47(dd,J=4.1,1.9Hz ,2H),7.17(s,2H),6.70(s,2H),5.31(s,2H),4.06(q,J=7.1Hz,2H),1.54(t,J=7.0Hz,3H). 13 C NMR(100MHz,DMSO-d6)δ164.10,160.27,151.02,142.66,130.43,129.94,129.13,121.25,113.36,112.55,105.50,56.12,12.70.HRMS(ESI)m / z[C 28 H 21 B2F4N3O2S+Na] + Theoretical value: 584.1369; Measured value: 584.1141.

[0061] The synthesis steps for SPTZ-CH3 and SPTZ-OCH3 are the same as above.

[0062] SPTZ-CH3: Red solid (42 mg), yield 48%. 1 H NMR (400MHz, DMSO-d6) δ8.91(t,J=3.2Hz,2H),7.69(tdd,J=9.5,6.7,3.8Hz,2H),7.56(s,2H),7.50(d,J =8.1Hz,4H),7.34(s,2H),6.84(s,2H),4.14(dd,J=5.7,3.8Hz,2H),2.37(s,6H),1.39(d,J=6.8Hz,3H). 13 CNMR(100MHz,DMSO-d6)δ167.45,162.85,160.13,150.89,140.24,138.84,132.05,13 0.35,129.13,123.57,113.28,112.55,105.47,67.89,21.06,14.35.HRMS(ESI)m / z[C 30 H 25 B2F4N3O2S+Na] + Theoretical value: 588.1816; Measured value: 588.3853.

[0063] SPTZ-OCH3: Red solid (38 mg), yield 43%. 1 H NMR(400MHz, DMSO-d6)δ8.88(s,2H),7.55(t,J=4.5Hz,6H),7.13–7.06(m,4H),6.83( d,J=2.5Hz,2H),4.14(q,J=7.0Hz,2H),3.82(d,J=1.7Hz,6H),1.38(t,J=6.8Hz,3H). 13 C NMR (100MHz, DMSO-d6) δ159.92,129.99,124.94,122.86,115.17,113.25,105.15,55.86,31.62,30.31. 13 C NMR(100MHz,DMSO-d6)δ163.19,161.88,159.92,151.25,136.78,129.99,12 4.94,122.86,115.17,113.25,105.15,55.86,30.31,12.22.HRMS(ESI)m / z[C 30 H 25 B2F4N3O4S+Na] + Theoretical value: 644.1580; Measured value: 644.1489.

[0064] Example 7: Photophysical property testing of dyes

[0065] A certain mass of SPTZ dye was weighed and dissolved in solvents of different polarities to prepare a solution of 1.0 × 10⁻⁶. -5 A solution of M was prepared, and then the UV-Vis absorption and fluorescence spectra of the dye were measured. The molar extinction coefficient of the dye was calculated based on the measured absorbance, and can be obtained from the following formula:

[0066]

[0067] Where A represents the absorbance at the maximum absorption wavelength, b represents the thickness of the cuvette, and c represents the dye concentration. Additionally, an appropriate amount of SPTZ dye was prepared as a thin film on a glass slide, and the emission spectrum of the dye in its solid state was measured using a solid-state fluorescence apparatus.

[0068] Select fluorescein (Φ) f =0.93, solvent: 0.1M NaOH aqueous solution) was used as the reference dye to determine the fluorescence quantum yield of the above fluorescent dye in the selected solvent. The fluorescence quantum yield can be calculated by the following formula:

[0069]

[0070] Where, Φ f The fluorescence quantum yield is expressed as x, s, and s, where x and s represent the analyte and reference dye, respectively. x and A s F represents the absorbance of the analyte and the reference dye at a specific absorption wavelength, respectively. x and F s n represents the integrated area of ​​the fluorescence spectrum of the analyte and the reference dye at a specific excitation wavelength, respectively. x and n s These represent the refractive indices of the solvents used for the analyte and the reference dye, respectively.

[0071] Example 8: Study on the detection response of dye SPTZ-OCH3

[0072] The dye was dissolved in the test solution system THF / PBS (3:7, v / v, 10 mM, pH = 7.40) to prepare a solution of 1.0 × 10⁻⁶ mM. - 3 A mol / L solution was tested by adding HClO solution to measure the change in fluorescence emission spectrum with concentration. Before adding HClO, an emission peak was observed at 643 nm; after adding HClO, a rapid increase in fluorescence intensity at 484 nm was observed, while the fluorescence intensity at 643 nm decreased. The detection time is short, enabling qualitative and quantitative detection of HClO.

[0073] Example 9: Selectivity Experiment of Dye SPTZ-OCH3

[0074] The dye was dissolved in the test solution system THF / PBS (3:7, v / v, 10 mM, pH = 7.40) to prepare a solution of 1.0 × 10⁻⁶ mM. - 3 A solution of mol / L, to which 1200 μM of metal cation (Ca) is added. 2+ K + Mg 2+ Na + NH4 + ), anion (S 2- CO3 2- SO3 2- SO4 2- F - NO2 - Biothiols (Hcy, Cys, GSH), reactive oxides (NO·, ROO·, H2O2, HO·, ONOO) - Neither TBHP nor HClO caused any change in fluorescence, but the addition of HClO produced a ratio fluorescence signal with a good linear relationship. This demonstrates that the fluorescent dye of the present invention has good detection performance, confirming its applicability.

[0075] Implementation Case 10: Biotoxicity Experiment of Dye SPTZ-OCH3

[0076] HeLa cells were cultured in DMEM medium (10% fetal bovine serum and 1% penicillin-streptomycin) and incubated at 37°C in a cell culture incubator containing 5% carbon dioxide for 24 h. Once the cells reached the appropriate morphology, they were seeded into 96-well plates, and different concentration gradients of the dye SPTZ-OCH3 were added, followed by incubation for another 24 h. Then, 0.5 mg / mL thiazolyl blue was added and incubated for 4 h. The culture medium was then aspirated and 100 μL of DMSO was added. The absorbance at 490 nm was recorded using a microplate reader, and cell viability was calculated using the absorbance ratio.

[0077] Implementation Case 11: Bioimaging Experiment with Dye SPTZ-OCH3

[0078] HeLa cell imaging experiment: HeLa cells were divided into experimental and control groups. Control group: After cells reached a suitable morphology, the culture medium was removed, and the cells were washed three times with PBS. 10 μM of the probe SPTZ-OCH3 was added to the HeLa cells, and they were incubated together for 30 min before fluorescence imaging. Experimental group: After cells reached a suitable morphology, the culture medium was removed, and the cells were washed three times with PBS. 10 μM of the probe SPTZ-OCH3 was added to the HeLa cells, and they were incubated together for 30 min. 500.0 μM HClO was then added to the HeLa cells, and incubation continued for another 10 min before fluorescence imaging. Blue collection channel: λ em =450-500nm, excitation wavelength: 405nm; red collection channel: λ em =600-650nm, excitation wavelength: 488nm.

[0079] Zebrafish fluorescence imaging experiment: Zebrafish embryos were incubated in embryo culture medium for 2 days, with the temperature maintained at approximately 28℃. After hatching, fluorescence imaging was performed. Control group: 10.0 μM probe SPTZ-OCH3 was added to a confocal culture dish and zebrafish were incubated together for 30 min, rinsed three times with PBS, and then fluorescence imaging was performed. Control group: 10.0 μM probe SPTZ-OCH3 was added to a confocal culture dish and zebrafish were incubated together for 30 min, rinsed three times with PBS, and then 500.0 μM HClO was added to the confocal culture dish for another 10 min, followed by fluorescence imaging. Blue collection channel: λ em =450-500nm, excitation wavelength: 405nm; red collection channel: λ em =600-650nm, excitation wavelength: 488nm.

Claims

1. A class of symmetrical fluorine-boron fluorescent dyes, SPTZ-R, with the ADA configuration, has the following structure: Where R = H, CH3, OCH3.

2. The method for preparing the SPTZ-R dye according to claim 1: (a) Sodium tert-butoxide, bis(2-diphenylphosphine) ether and palladium acetate were added to dissolve m-aminoanisole and m-bromoanisole in toluene. The mixture was refluxed under argon protection for 12 h. After the reaction was completed, the reaction solution was poured into water and stirred for a while. Then, ethyl acetate was added for extraction. The organic layer was washed with saturated brine and water, dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The crude product was purified by column chromatography to obtain brownish-red oily liquid compound 1. (b) Compound 1, S8, and elemental iodine were added to a round-bottom flask. Under argon protection, the reactants were melted and reacted at 195 °C for 30 min. After the reaction was completed, ethyl acetate was added to dissolve the resulting mixture. The mixture was washed with saturated brine and water, dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The crude product was then purified by column chromatography to obtain a brownish-yellow oily liquid compound 2. (c) Compound 2, iodoethane, sodium hydroxide, and a catalytic amount of KI were dissolved in anhydrous DMSO and reacted at 70 °C for 14 h. After the reaction was completed, the reaction mixture was poured into water, extracted with DCM, and the organic layer was washed with saturated brine and water, dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The crude product was then purified by column chromatography to obtain a green oily liquid compound 3. (d) At 0 °C, phosphorus oxychloride was slowly added to anhydrous DMF. After the addition was complete, stirring was continued for 15 min until the solution turned pink. Compound 3 was dissolved in an appropriate amount of anhydrous DMF and added to the reaction system. The reaction was carried out at 80 °C for 3 h. After the reaction was completed, the reaction solution was poured into ice water, and the pH was adjusted to neutral with saturated NaHCO3 solution. DCM was added for extraction. The organic layer was washed successively with saturated brine and water, dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The crude product was purified by column chromatography to obtain the yellow solid product compound 4. (e) Compound 4 was dissolved in anhydrous DCM. The mixture was reacted with boron tribromide added at -78 °C under argon protection for 1 h. The reaction was then allowed to continue at room temperature for 10 h. After the reaction was complete, the reaction solution was slowly poured into ice water, and then extracted with DCM. The organic layer was washed successively with saturated brine and water, dried over anhydrous Na2SO4, and the solvent was removed by rotary evaporation. The crude product was then purified by column chromatography to obtain a yellow solid compound 5. (f) Compound 5 was dissolved in anhydrous ethanol, and an aniline derivative was added. The mixture was reacted at room temperature for 12 h. After the reaction was completed, the product was filtered to obtain a dark red solid product. The product was washed with cold ethanol and dried to obtain crude product 6a-c, which was directly used in the next step. Crude product 6 was dissolved in 1,2-dichloroethane, and N,N-diisopropylethylamine and boron trifluoride diethyl ether were added. The mixture was refluxed for 5 h. After the reaction was completed, the solvent was removed by rotary evaporation. The crude product was purified by column chromatography to obtain the red solid product SPTZ-R.

3. The method for preparing the fluorescent dye SPTZ-R according to claim 2, characterized in that... In step (a), the molar ratio of intermediate aminoanisole, m-bromoanisole, sodium tert-butoxide, bis(2-diphenylphosphine) ether, and palladium acetate is 1:1.1:1.5:0.1:0.

05.

4. The method for preparing the fluorescent dye SPTZ according to claim 2, characterized in that: In step (b), the molar ratio of compound 1,S8 to elemental iodine is 1:0.32:0.

01.

5. The method for preparing the fluorescent dye SPTZ according to claim 2, characterized in that: In step (c), the molar ratio of compound 2, iodoethane, and sodium hydroxide is 1:10:2.

5.

6. The method for preparing the fluorescent dye SPTZ according to claim 2, characterized in that: In step (d), the molar ratio of compound 3 to POCl3 is 1:5.

8.

7. The method for preparing the fluorescent dye SPTZ according to claim 2, characterized in that: In step (e), the molar ratio of compound 4 to BBr3 is 1:6.

7.

8. The method for preparing the fluorescent dye SPTZ according to claim 2, characterized in that: In step (f), the molar ratio of compound 5, aniline, N,N-diisopropylethylamine, and boron trifluoride ether is 1:6.9:3.75:3.

75.

9. The method for preparing the fluorescent dye SPTZ according to claim 2, characterized in that: In steps (a), (b), (c), (d), (e), and (f), the eluent used in column chromatography is V, respectively. 石油醚 / V 二氯甲烷 = 1 / 1、V 石油醚 / V 二氯甲烷 = 1 / 2, V 石油醚 / V 二氯甲烷 = 4 / 1、 V 石油醚 / V 二氯甲烷 = 1 / 1、 V 石油醚 / V 二氯甲烷 = 1 / 2、 V 石油醚 / V 二氯甲烷 = 1 / 3.

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