CN116693563A - Synthesis and application of symmetrical fluorine boron fluorescent dye with A-D-A configuration - Google Patents

Synthesis and application of symmetrical fluorine boron fluorescent dye with A-D-A configuration Download PDF

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CN116693563A
CN116693563A CN202310567878.2A CN202310567878A CN116693563A CN 116693563 A CN116693563 A CN 116693563A CN 202310567878 A CN202310567878 A CN 202310567878A CN 116693563 A CN116693563 A CN 116693563A
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compound
reaction
fluorescent dye
sptz
dichloromethane
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宋相志
王宏
韩少辉
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Central South University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic System
    • C07F5/02—Boron compounds
    • C07F5/022—Boron compounds without C-boron linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/003—Thiazine dyes
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    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B21/00—Thiazine dyes
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    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
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    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
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    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1059—Heterocyclic compounds characterised by ligands containing three nitrogen atoms as heteroatoms
    • C09K2211/107—Heterocyclic compounds characterised by ligands containing three nitrogen atoms as heteroatoms with other heteroatoms
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    • Y02E10/50—Photovoltaic [PV] energy
    • Y02E10/542—Dye sensitized solar cells

Abstract

The invention relates to synthesis and application of symmetrical boron fluoride fluorescent dye with A-D-A configuration, belonging to the field of fluorescent dye. The molecular structure is as follows:wherein r=h, CH 3 ，OCH 3 . At present, the fluorine boron fluorescent dye has a D-A structure, and reports of A-D-A fluorine boron fluorescent dye are not yet seen. The phenothiazine has strong electron supply capacity and is hopeful to conjugate with two electron acceptors to obtain A-D-A fluorescenceA dye. In addition, such fluoroboric dyes have long wavelength emission, large Stokes shift, high fluorescence quantum efficiency, and solid fluorescence properties as compared to conventional fluoroboric dyes. And the method can respond to HClO and has good biological application prospect.

Description

Synthesis and application of symmetrical fluorine boron fluorescent dye with A-D-A configuration
Technical Field
The invention relates to a symmetrical boron fluoride fluorescent dye based on an A-D-A configuration, which comprises the application of the fluorescent dye in the aspects of synthesis, photophysical property research and biological imaging, and belongs to the field of fluorescent dyes.
Background
In recent years, heteroatom-modified organic dyes such as BODIPY have been widely focused on due to their outstanding optical properties such as relatively narrow absorption and emission bands, large molar absorption coefficient, high fluorescence quantum yield, satisfactory photostability and chemical stability, and insensitivity to polarity and pH of solvents, and have been widely used as biosensors, photodynamic therapy, bioimaging, laser dyes, photoelectric materials, and the like. However, classical BODIPY is severely fluorescence quenched in the solid state due to the close pi-pi packing between molecules. Furthermore, most BODIPYs exhibit very small stokes shifts (typically <30 nm), which inevitably leads to measurement errors in self-quenching and fluorescence sensing. Therefore, development of a novel red light-emitting fluoroboron-based fluorescent dye with long wavelength emission and large Stokes shift has important significance.
Phenothiazine (PTZ) was first synthesized by Bernthsen in 1883 and has been widely used in the manufacture of dyes, pigments, pharmaceuticals, dye sensitized solar cells and copy materials. Phenothiazine contains nitrogen and sulfur heteroatoms rich in electrons, has a heterocyclic structure and has strong electron supply capability. The non-planar butterfly molecular conformation of phenothiazines can effectively prevent pi-pi accumulation between molecules, resulting in intense solid fluorescence. Thus, the introduction of phenothiazine moieties in BODIPY analogs is a robust strategy to develop red fluorescent dyes with large Stokes shifts. Furthermore, D-A configuration of the fluoroboric dye using a phenothiazine group as an electron donor has been reported, but such A-D-A configuration of the symmetrical fluoroboric fluorescent dye has not been reported.
In the patent, a phenothiazine group is taken as an electron donor part of a fluorescent dye, different substituents are introduced at the tail end of an aniline aryl group, and a series of novel symmetrical boron fluoride fluorescent dyes with A-D-A configuration are designed and synthesized, and have the advantages of long wavelength emission and large Stokes displacement, so that self-absorption and background fluorescence interference are effectively avoided. In addition, the dye can specifically identify HClO, oxidize the divalent sulfur atom which gives electrons on the phenothiazine group into an electron-withdrawing sulfoxide group, realize the ratio detection of HClO under single wavelength excitation, and be successfully applied to HeLa cells and zebra fishes for biological imaging.
Disclosure of Invention
Aiming at the defects of the prior researches, the invention develops a symmetrical fluorine boron fluorescent dye with an A-D-A configuration. The dye has the advantages of large Stokes shift, long wavelength emission and high fluorescence quantum efficiency, and can specifically identify HClO. The structure is as follows:
wherein r=h, CH 3 ，OCH 3 。
When r=h, the synthetic route of representative compounds of the present invention is as follows:
(a) Dissolving m-aminoanisole and m-bromoanisole in toluene, adding sodium tert-butoxide, bis (2-diphenylphosphinophenyl) ether and palladium acetate, and reflux-reacting for 12h under the protection of argon. After the reaction, the reaction solution was poured into water, stirred for a while, and then extracted with ethyl acetate. The organic layer was washed with saturated brine and water, and dried Na 2 SO 4 And (5) drying. After the solvent is removed by rotary evaporation, the crude product is purified by column chromatography to obtain a brown red oily liquid compound 1;
(b) Compound 1, S 8 Elemental iodine was added to a round bottom flask. The reactants were melt reacted at 195 ℃ for 30min under argon protection. After the reaction was completed, ethyl acetate was added to dissolve the resulting mixture, which was washed with saturated brine and water, and anhydrous Na 2 SO 4 And (5) drying. After the solvent was removed by rotary evaporation, the crude product was purified by column chromatography to give compound 2 as a brown-yellow oil;
(c) Compound 2, ethyl iodide, sodium hydroxide and catalytic amounts of KI were dissolved in anhydrous DMSO and reacted at 70 ℃ for 6h. After the reaction was completed, the reaction mixture was poured into water, and extracted with DCM. The organic layer was washed with saturated brine and water, and dried Na 2 SO 4 And (5) drying. After the solvent was removed by rotary evaporation, the crude product was purified by column chromatography to give green oily liquid compound 3;
(d) Phosphorus oxychloride was slowly added to anhydrous DMF at 0 ℃. After the dropwise addition was completed, stirring was continued for 15min, and the solution became pink. Compound 3 was dissolved in an appropriate amount of anhydrous DMF, added to the reaction system, and reacted at 80 ℃ for 3 hours. After the reaction, the reaction solution was poured into ice water and saturated NaHCO 3 The solution was adjusted to neutral pH and extracted with DCM. The organic layer was washed with saturated brine and water, and dried Na 2 SO 4 And (5) drying. After the solvent is removed by rotary evaporation, the crude product is purified by column chromatography to obtain a yellow solid product compound 4;
(e) Compound 4 was dissolved with anhydrous DCM, and the reaction was continued for 1h at-78 ℃ under argon, after which the reaction was continued at room temperature for 10h. After the reaction was completed, the reaction solution was slowly poured into ice water, and then DCM was added for extraction. The organic layer was washed with saturated brine and water, and dried Na 2 SO 4 And (5) drying. After the solvent is removed by rotary evaporation, the crude product is purified by column chromatography to obtain a yellow solid product compound 5;
(f) Compound 5 was dissolved in absolute ethanol, aniline was added thereto, and the reaction was carried out at room temperature for 12 hours. After the reaction was completed, the product was filtered to give a dark red solid, which was washed with cold ethanol and dried to give crude product 6a, which was used directly in the next step. The crude product 6a was dissolved in 1, 2-dichloroethane, N-diisopropylethylamine and boron trifluoride diethyl etherate were added and reacted under reflux for 5 hours. After the reaction is finished, removing the solvent by rotary evaporation, and purifying the crude product by column chromatography to obtain a red solid product SPTZ-H;
the mass ratio of the substances of the intermediate aminoanisole, the m-bromoanisole, the sodium tert-butoxide, the bis (2-diphenylphosphinophenyl) ether and the palladium acetate in the step (a) is 1:1.1:1.5:0.1:0.05.
compound 1, S in said step (b) 8 The mass ratio of the iodine simple substance is 1:0.32:0.01.
the mass ratio of the compound 2, the ethyl iodide and the sodium hydroxide in the step (c) is 1:10:2.5.
compound 3, POCl in step (d) 3 The mass ratio of the substances is 1:5.8.
compound 4, BBr in said step (e) 3 The mass ratio of the substances is 1:6.7.
the mass ratio of the compound 5, the aniline, the N, N-diisopropylethylamine and the boron trifluoride diethyl etherate in the step (f) is 1:6.9:3.75:3.75.
the eluent used in the column chromatography in the steps (a), (b), (c), (d), (e) and (f) is (V) Petroleum ether /V Dichloromethane (dichloromethane) ＝1/1)、(V Petroleum ether /V Dichloromethane (dichloromethane) ＝1/2)、(V Petroleum ether /V Dichloromethane (dichloromethane) ＝4/1)、(V Petroleum ether /V Dichloromethane (dichloromethane) ＝1/1)、(V Petroleum ether /V Dichloromethane (dichloromethane) ＝1/2)、(V Petroleum ether /V Dichloromethane (dichloromethane) ＝1/3)。
The method for testing the photophysical properties of the fluorescent dye comprises the following steps: the dye SPTZ-H, SPTZ-CH 3 ，SPTZ-OCH 3 Respectively dissolving in toluene, tetrahydrofuran, dichloromethane, dioxane, methanol, acetonitrile, and dimethyl sulfoxide to obtain 1.0X10 - 5 M, and the absorption wavelength, the emission wavelength, the molar extinction coefficient and the fluorescence quantum efficiency are carried out at room temperature. The specific implementation method is described in detail in the implementation example.
The method for testing the response of the fluorescent dye to HClO is as follows; the dye SPTZ-OCH 3 The test was performed at room temperature by dissolving in THF/PBS (3:7, v/v,10mM, pH=7.40). The method can carry out qualitative and quantitative detection on HClO, and the specific implementation method is described in detail in an implementation example.
The invention aims to synthesize a series of symmetrical fluorine boron fluorescent dyes with A-D-A configuration by taking a phenothiazine group as a donor part of the fluorescent dye and introducing different substituents. Compared with traditional boron fluoride dyes, the boron fluoride dyes have long wavelength emission (> 620 nm), large Stokes shift (> 135 nm), good fluorescence quantum yield (> 0.11) and solid fluorescence properties.
Fluorescent dye of the inventionThe response mechanism of the material to HClO is as follows: SPTZ-OCH in the presence of HClO 3 The sulfur (S) atoms in the moiety are oxidized to sulfoxide groups (s=o), the intermolecular ICT effect is diminished and the emission wavelength is blue shifted.
The fluorescent dye SPTZ-OCH of the invention 3 Has good selectivity and responsiveness to HClO and good biocompatibility.
Drawings
FIG. 1 shows the nuclear magnetic resonance hydrogen spectrum of the fluorescent dye SPTZ-H of the invention in deuterated DMSO, with chemical shifts on the abscissa and intensities on the ordinate.
FIG. 2 shows nuclear magnetic resonance carbon spectrum of the fluorescent dye SPTZ-H of the present invention in deuterated DMSO, chemical shift on the abscissa and intensity on the ordinate.
FIG. 3 shows the fluorescent dye SPTZ-CH of the present invention 3 Nuclear magnetic resonance hydrogen spectrum in deuterated DMSO, chemical shift on the abscissa, and intensity on the ordinate.
FIG. 4 shows the fluorescent dye SPTZ-CH of the present invention 3 Nuclear magnetic resonance carbon spectrum in deuterated DMSO, chemical shift on the abscissa, and intensity on the ordinate.
FIG. 5 shows the fluorescent dye SPTZ-OCH of the present invention 3 Nuclear magnetic resonance hydrogen spectrum in deuterated DMSO, chemical shift on the abscissa, and intensity on the ordinate.
FIG. 6 shows the fluorescent dye SPTZ-OCH of the present invention 3 Nuclear magnetic resonance carbon spectrum in deuterated DMSO, chemical shift on the abscissa, and intensity on the ordinate.
FIG. 7 shows normalized absorption spectra of the fluorescent dye SPTZ-H of the present invention in different solvents (toluene, tetrahydrofuran, methylene chloride, dioxane, methanol, acetonitrile, dimethyl sulfoxide), with wavelength on the abscissa and relative intensity on the ordinate.
FIG. 8 shows normalized emission spectra of the fluorescent dye SPTZ-H of the present invention in different solvents (toluene, tetrahydrofuran, methylene chloride, dioxane, methanol, acetonitrile, dimethyl sulfoxide), with wavelength on the abscissa and relative intensity on the ordinate.
FIG. 9 shows the fluorescent dye SPTZ-CH of the present invention 3 In different solvents (toluene, tetrahydrofuran, dichloromethane, dioxane, methanol, acetonitrile)Dimethyl sulfoxide), the abscissa is the wavelength, and the ordinate is the relative intensity.
FIG. 10 shows the fluorescent dye SPTZ-CH of the present invention 3 Normalized emission spectra in different solvents (toluene, tetrahydrofuran, dichloromethane, dioxane, methanol, acetonitrile, dimethyl sulfoxide), with wavelength on the abscissa and relative intensity on the ordinate.
FIG. 11 shows the fluorescent dye SPTZ-OCH of the present invention 3 Normalized absorption spectra in different solvents (toluene, tetrahydrofuran, dichloromethane, dioxane, methanol, acetonitrile, dimethyl sulfoxide), with the abscissa being wavelength and the ordinate being relative intensity.
FIG. 12 shows the fluorescent dye SPTZ-OCH of the present invention 3 Normalized emission spectra in different solvents (toluene, tetrahydrofuran, dichloromethane, dioxane, methanol, acetonitrile, dimethyl sulfoxide), with wavelength on the abscissa and relative intensity on the ordinate.
FIG. 13 shows the 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, dimethyl sulfoxide).
FIG. 14 is a normalized emission spectrum of the fluorescent dye of the present invention in a solid state, with the emission wavelength on the abscissa and the fluorescence intensity on the ordinate.
FIG. 15 shows the fluorescent dye SPTZ-OCH of the present invention 3 (10. Mu.M) fluorescence emission spectra before and after response with 1200. Mu.M HClO in PBS buffer (0.01M, pH=7.4, 30% tetrahydrofuran). The abscissa is wavelength and the ordinate is fluorescence intensity.
FIG. 16 shows the fluorescent dye SPTZ-OCH of the present invention 3 (10. Mu.M) in PBS buffer (0.01M, pH=7.4, 30% tetrahydrofuran) with the relevant substances (Ca 2+ 、K + 、Mg 2+ 、NH 4 + 、S 2- 、CO 3 2- 、ONOO - 、SO 3 2- 、TBHP、F - 、NO 2 - 、Hcy、Cys、GSH、NO·、ROO·、H 2 O 2 、HO·、HClO) responds to the change in fluorescence spectrum of the dye solution, with the abscissa being wavelength and the ordinate being fluorescence intensity.
FIG. 17 shows the SPTZ-OCH fluorescent dye with different concentrations 3 MTT assay conditions incubated in HeLa cells for 24 hours, with concentrations on the abscissa and cell viability on the ordinate.
FIG. 18 shows the fluorescent dye SPTZ-OCH according to the invention 3 HeLa cell imaging experiments of (C).
FIG. 19 shows the fluorescent dye SPTZ-OCH of the present invention 3 Zebra fish imaging experiments.
FIG. 20 shows the fluorescent dye SPTZ-OCH of the present invention 3 Is a response process mechanism diagram of (1).
Examples of the embodiments
Example 1: synthesis of Compound 1
Meta-aminoanisole (4.92 g,40 mmol) and m-bromoanisole (8.23 g,44 mmol) were dissolved in 80mL of anhydrous toluene, sodium t-butoxide (5.77 g,60 mmol) was added, bis (2-diphenylphosphinophenyl) ether (2.15 g,4 mmol) and palladium acetate (449 mg,0.05 mmol) were added under argon atmosphere, and the mixture was heated under reflux for 12h. After the reaction was completed, the reaction mixture was poured into 100mL of water, stirred for a while, then extracted 3 times with EA (100 mL. Times.3), washed with saturated brine and water, and dried Na 2 SO 4 And (5) drying. After removal of the solvent by rotary evaporation, the crude product was purified by column chromatography (PE: dcm=1:1) to give a reddish brown oily liquid (5.52 g, 60.3%). H NMR (400 MHz, CDCl) 3 )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,CDCl 3 )δ160.73,144.32,130.13,110.70,106.55,103.87,55.24.HRMS(ESI)m/z[C 14 H 15 NO 2 +H] + Theoretical value: 230.1176; actual measurement value: 230.1097.
example 2: synthesis of Compound 2
Compound 1 (1 g,4.4 mmol), S 8 (350mg，1.4mmol)，I 2 (10 mg,0.04 mmol) was added to a round bottom flask. The reactants were melt reacted at 195 ℃ for 30min under argon protection. After the reaction was completed, the resulting mixture was dissolved in 100mL of EA, washed with saturated saline and waterAnhydrous Na 2 SO 4 And (5) drying. After removal of the solvent by rotary evaporation, the crude product was purified by column chromatography (PE: dcm=1:2) to give a brown oil (320 mg, 28.3%). 1 H NMR(400MHz,DMSO-d 6 )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). 13 C NMR(100MHz,DMSO-d 6 )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 NO 2 S] + Theoretical value: 259.0667; actual measurement value: 259.0649.
example 3: synthesis of Compound 3
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 7mL anhydrous DMSO. The resulting mixture was stirred at 70℃under argon for 6h. After the reaction was completed, the reaction mixture was poured into 50mL of water, and then extracted 3 times (3X 100 mL) with DCM, washed with saturated brine and water, and anhydrous Na 2 SO 4 And (5) drying. After removal of 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-d 6 )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,2H),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 C NMR(100MHz,DMSO-d 6 )δ159.97,155.32,146.39,145.31,127.92,127.85,113.89,112.00,109.20,107.47,105.41,103.08,56.38,55.74,41.87,13.27.HRMS(ESI)m/z[C 16 H 17 NO 2 S] + Theoretical value: 287.0980; actual measurement value: 287.0872.
example 4: synthesis of Compound 4
Phosphorus oxychloride (0.3 mL,3.2 mmol) was added slowly to anhydrous DMF (0.35 mL,4.5 mmol) at 0deg.C. After the addition was completed, the resulting mixture was stirred at this temperature for 15 minutes, and the solution turned pink. Compound 3 (160 mg,0.55 mmol) was then dissolvedIn a proper amount of anhydrous DMF, adding into the reaction system. The mixture was heated to 80℃and reacted for 3h. After the reaction was completed, the reaction mixture was poured into 50mL of ice water, and saturated NaHCO was used 3 The solution was adjusted to neutral pH and then extracted 3 times with DCM (3X 50 mL). The organic layer was washed with saturated brine and water, and dried Na 2 SO 4 And (5) drying. After removal of the solvent by rotary evaporation, the crude product was purified by column chromatography (PE: dcm=1:1) to give the product as a yellow solid (60 mg, 32.1%). 1 H NMR(400MHz,DMSO-d 6 )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 NO 4 S+Na] + Theoretical value: 366.0770; actual measurement value: 366.0625.
example 5: synthesis of Compound 5
Compound 4 (400 mg,1.2 mmol) was dissolved in 10mL anhydrous DCM, 2mol/L boron tribromide (4 mL,8 mmol) was added to the mixture by syringe at-78deg.C under argon, and after 1h the reaction was continued at room temperature for 10h. After the reaction was completed, the reaction solution was slowly poured into 100mL of ice water, and then extracted 2 times (2×80 mL) with DCM. The organic layer was washed with saturated brine and water, and dried Na 2 SO 4 And (5) drying. After removal of the solvent by rotary evaporation, the crude product was purified by column chromatography (PE: dcm=1:2) to give the product as a yellow solid (302 mg, 82.3%). 1 H NMR(400MHz,DMSO-d 6 )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). 13 C NMR(100MHz,DMSO-d 6 )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 NO 4 S] + Theoretical value: 315.0565; actual measurement value: 315.0802.
example 6: synthesis of SPTZ-H
Compound 5 (50 mg,0.16 mmol) was dissolved in 15mL of anhydrous EtOH, aniline (100. Mu.L, 1.1 mmol) was added and reacted at room temperature for 12h. After the reaction was completed, the product was filtered to give a dark red solid, which was washed with cold EtOH and then dried to give crude product 6a, which was used directly in the next step. Crude product is producedThe material 6a was dissolved in 8mL C 2 H 4 Cl 2 DIEA (100. Mu.L, 0.6 mmol) was added and the mixture was reacted at 80℃under reflux for 10min. After adding BF 3 ·OEt 2 (76. Mu.L, 0.6 mmol) and the reaction mixture was continued to reflux for 5h. After the reaction is finished, removing C by rotary evaporation 2 H 4 Cl 2 After this time, the crude product was purified by column chromatography (PE: dcm=1:3) to give the product as a red solid (33 mg, 68.8%). 1 H NMR(400MHz,CDCl 3 )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-d 6 )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 B 2 F 4 N 3 O 2 S+Na] + Theoretical value: 584.1369; actual measurement value: 584.1141.
SPTZ-CH 3 ，SPTZ-OCH 3 the synthesis steps are the same as above.
SPTZ-CH 3 : red solid (42 mg) in 48% yield. 1 H NMR(400MHz,DMSO-d 6 )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 C NMR(100MHz,DMSO-d 6 )δ167.45,162.85,160.13,150.89,140.24,138.84,132.05,130.35,129.13,123.57,113.28,112.55,105.47,67.89,21.06,14.35.HRMS(ESI)m/z[C 30 H 25 B 2 F 4 N 3 O 2 S+Na] + Theoretical value: 588.1816; actual measurement value: 588.3853.
SPTZ-OCH 3 : red solid (38 mg), 43% yield. 1 H NMR(400MHz,DMSO-d 6 )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-d 6 )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-d 6 )δ163.19,161.88,159.92,151.25,136.78,129.99,124.94,122.86,115.17,113.25,105.15,55.86,30.31,12.22.HRMS(ESI)m/z[C 30 H 25 B 2 F 4 N 3 O 4 S+Na] + Theoretical value 644.1580; actual measurement value: 644.1489.
example 7: photophysical property test of dye
Weighing SPTZ dye of certain mass, respectively dissolving with solvents of different polarities to obtain 1.0X10 -5 M, and then testing the uv-vis absorption spectrum and fluorescence spectrum of the dye. The molar extinction coefficient of the dye is calculated from the measured absorbance and can be found from the following formula:
wherein A represents absorbance at the maximum absorption wavelength, b represents the thickness of the cuvette, and c represents the concentration of the dye. In addition, a proper amount of SPTZ dye is taken to be made into a film on a glass slide, and the solid fluorescence device is used for measuring the emission spectrum of the dye in a solid state.
Selecting fluorescein (phi) f =0.93, solvent: aqueous 0.1M NaOH) was used as a reference dye to determine the fluorescence quantum yield of the above fluorochromes in the selected solvents. The fluorescence quantum yield can be calculated by the following formula:
wherein phi is f X and s representing fluorescence quantum yield and subscripts representing the analyte and the reference dye, respectively, A x And A s Respectively representing the corresponding absorbance of the substance to be detected and the reference dye under the specific absorption wavelength, F x And F s Respectively represent the integral area of fluorescence spectrum of specific excitation wavelength for the substance to be detected and the reference dye, n x And n s Respectively representing the refractive index of the solvent used for the substance to be measured and the reference dye.
Example 8: dye SPTZ-OCH 3 Detection response study
The dye was dissolved in the test solution system THF/PBS (3:7, v/v,10mm, ph=7.40) to make 1.0×10 - 3 The fluorescence emission spectrum of the solution with mol/L is tested by adding HClO solution. Before addition of HClO, an emission peak was shown at 643 nm; after addition of HClO, a rapid increase in fluorescence intensity at 484nm and a decrease in fluorescence intensity at 643nm were observed. The detection time is short, and the qualitative and quantitative detection of HClO can be realized.
Example 9: dye SPTZ-OCH 3 Selectivity experiments of (2)
The dye was dissolved in the test solution system THF/PBS (3:7, v/v,10mm, ph=7.40) to make 1.0×10 - 3 mol/L solution, 1200. Mu.M of metal cation (Ca 2+ 、K + 、Mg 2+ 、Na + 、NH 4 + ) Anions (S) 2- 、CO 3 2- 、SO 3 2- 、SO 4 2- 、F - 、NO 2 - ) Biological thiols (Hcy, cys, GSH), reactive oxidizing species (NO, ROO, H 2 O 2 、HO·、ONOO - TBHP) did not cause a change in fluorescence, whereas the addition of HClO produced a good linear relationship in the ratio fluorescence signal. The fluorescent dye has better detection performance, and the applicability of the dye is proved.
Embodiment case 10: dye SPTZ-OCH 3 Is of the order of (2)
HeLa cells were cultured in DMEM medium (10% fetal bovine serum and 1% penicillin-streptomycin) and incubated at 37℃in a cell incubator containing 5% carbon dioxide for 24 hours. After the cells grow to a proper form, inoculating the cells into a 96-well plate, and adding dyes SPTZ-OCH with different concentration gradients 3 Incubation was continued for 24h. Then, 0.5mg/mL of thiazole blue was added for 4 hours, the culture broth was aspirated and 100. Mu.L of DMSO was added. The absorbance at 490nm was recorded with a microplate reader and cell viability was calculated from absorbance ratio.
Embodiment case 11: dye SPTZ-OCH 3 Is a biological imaging experiment of (2)
HeLa cell imaging experiments: heLa cells were divided into experimental and control groups. Control group: after the cells grew to the proper morphology, the culture was removed, rinsed three times with PBS, and 10. Mu.M of probe SPTZ-OCH was added to HeLa cells 3 Incubating for 30min, and performing fluorescence imaging experiments; experimental group: after the cells grew to the proper morphology, the culture was removed, rinsed three times with PBS, and 10. Mu.M of probe SPTZ-OCH was added to HeLa cells 3 Incubation was continued for 30min, and further incubation was continued for 10min with 500.0. Mu.M HClO added to HeLa cells, and fluorescence imaging experiments were performed. Blue collection channel: lambda (lambda) em =450-500 nm, excitation wavelength: 405nm; red collection channel: lambda (lambda) em =600-650 nm, excitation wavelength: 488nm.
Zebra fish fluorescence imaging experiments: zebra fish embryos are incubated in embryo culture for 2 days, while the temperature is maintained at around 28 ℃. After the embryo comes out of the membrane, a fluorescence imaging experiment is performed. Control group: add 10.0. Mu.M probe SPTZ-OCH to confocal dish 3 Incubating the fish with zebra fish for 30min, rinsing the fish with PBS for 3 times, and performing a fluorescence imaging experiment; control group: add 10.0. Mu.M probe SPTZ-OCH to confocal dish 3 Incubation with zebra fish for 30min, rinsing with PBS 3 times, adding 500.0 μM HClO into confocal dish, and incubating for 10min. Blue collection channel: lambda (lambda) em =450-500 nm, excitation wavelength: 405nm; red collection channel: lambda (lambda) em =600-650 nm, excitation wavelength: 488nm.

Claims (9)

1. The synthesis and application of symmetrical fluorine boron fluorescent dye with A-D-A configuration comprises the following steps:
wherein r=h, CH 3 ，OCH 3 。
2. The process for preparing a SPTZ dye as claimed in claim 1:
(a) Dissolving m-aminoanisole and m-bromoanisole in toluene, adding sodium tert-butoxide, bis (2-diphenylphosphinophenyl) ether and palladium acetate, and reflux-reacting for 12h under the protection of argon. After the reaction, the reaction solution was poured into water, stirred for a while, and then extracted with ethyl acetate. The organic layer was washed with saturated brine and water, and dried Na 2 SO 4 And (5) drying. After the solvent is removed by rotary evaporation, the crude product is purified by column chromatography to obtain a brown red oily liquid compound 1;
(b) Compound 1, S 8 Elemental iodine was added to a round bottom flask. The reactants were melt reacted at 195 ℃ for 30min under argon protection. After the reaction was completed, ethyl acetate was added to dissolve the resulting mixture, which was washed with saturated brine and water, and anhydrous Na 2 SO 4 And (5) drying. After the solvent was removed by rotary evaporation, the crude product was purified by column chromatography to give compound 2 as a brown-yellow oil;
(c) Compound 2, ethyl iodide, sodium hydroxide and catalytic amounts of KI were dissolved in anhydrous DMSO and reacted at 70 ℃ for 6h. After the reaction was completed, the reaction mixture was poured into water, and extracted with DCM. The organic layer was washed with saturated brine and water, and dried Na 2 SO 4 And (5) drying. After the solvent was removed by rotary evaporation, the crude product was purified by column chromatography to give green oily liquid compound 3;
(d) Phosphorus oxychloride was slowly added to anhydrous DMF at 0 ℃. After the dropwise addition was completed, stirring was continued for 15min, and the solution became pink. Compound 3 was dissolved in an appropriate amount of anhydrous DMF, added to the reaction system, and reacted at 80 ℃ for 3 hours. After the reaction, the reaction solution was poured into ice water and saturated NaHCO 3 The solution was adjusted to neutral pH and extracted with DCM. The organic layer was washed with saturated brine and water, and dried Na 2 SO 4 And (5) drying. After the solvent is removed by rotary evaporation, the crude product is purified by column chromatography to obtain a yellow solid product compound 4;
(e) Compound 4 was dissolved with anhydrous DCM, and the reaction was continued for 1h at-78 ℃ under argon, after which the reaction was continued at room temperature for 10h. After the reaction was completed, the reaction solution was slowly poured into ice water, and then DCM was added for extraction. The organic layer was washed with saturated brine and water, and dried Na 2 SO 4 And (5) drying. After the solvent is removed by rotary evaporation, the crude product is purified by column chromatography to obtain a yellow solid product compound 5;
(f) Compound 5 was dissolved in absolute ethanol, aniline was added thereto, and the reaction was carried out at room temperature for 12 hours. After the reaction was completed, the product was filtered to give a dark red solid, which was washed with cold ethanol and dried to give crude products 6a-c, which were used directly in the next step. The crude product 6 was dissolved in 1, 2-dichloroethane, N-diisopropylethylamine and boron trifluoride diethyl etherate were added and reacted under reflux for 5 hours. After the reaction is finished, the solvent is removed by rotary evaporation, and the crude product is purified by column chromatography to obtain a red solid product SPTZ-R.
3. The method for preparing the fluorescent dye SPTZ according to claim 2, wherein: the mass ratio of the substances of the intermediate aminoanisole, the m-bromoanisole, the sodium tert-butoxide, the bis (2-diphenylphosphinophenyl) ether and the palladium acetate in the step (a) is 1:1.1:1.5:0.1:0.05.
4. the method for preparing the fluorescent dye SPTZ according to claim 2, wherein: compound 1, S in said step (b) 8 The mass ratio of the iodine simple substance is 1:0.32:0.01.
5. the method for preparing the fluorescent dye SPTZ according to claim 2, wherein: the mass ratio of the compound 2, the ethyl iodide and the sodium hydroxide in the step (c) is 1:10:2.5.
6. the method for preparing the fluorescent dye SPTZ according to claim 2, wherein: compound 3, POCl in step (d) 3 The mass ratio of the substances is 1:5.8.
7. the method for preparing the fluorescent dye SPTZ according to claim 2, wherein: compound 4, BBr in said step (e) 3 The mass ratio of the substances is 1:6.7.
8. the method for preparing the fluorescent dye SPTZ according to claim 2, wherein: the mass ratio of the compound 5, the aniline, the N, N-diisopropylethylamine and the boron trifluoride diethyl etherate in the step (f) is 1:6.9:3.75:3.75.
9. the method for preparing the fluorescent dye SPTZ according to claim 2, wherein: the eluent used in the column chromatography in the steps (a), (b), (c), (d), (e) and (f) is (V) Petroleum ether /V Dichloromethane (dichloromethane) ＝1/1)、(V Petroleum ether /V Dichloromethane (dichloromethane) ＝1/2)、(V Petroleum ether /V Dichloromethane (dichloromethane) ＝4/1)、(V Petroleum ether /V Dichloromethane (dichloromethane) ＝1/1)、(V Petroleum ether /V Dichloromethane (dichloromethane) ＝1/2)、(V Petroleum ether /V Dichloromethane (dichloromethane) ＝1/3)。
CN202310567878.2A 2023-05-19 2023-05-19 Synthesis and application of symmetrical fluorine boron fluorescent dye with A-D-A configuration Pending CN116693563A (en)

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