CN111334067B - Self-flashing fluorescent dye for super-resolution fluorescence imaging and synthesis and application thereof - Google Patents

Self-flashing fluorescent dye for super-resolution fluorescence imaging and synthesis and application thereof Download PDF

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CN111334067B
CN111334067B CN201811550585.9A CN201811550585A CN111334067B CN 111334067 B CN111334067 B CN 111334067B CN 201811550585 A CN201811550585 A CN 201811550585A CN 111334067 B CN111334067 B CN 111334067B
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徐兆超
乔庆龙
刘晓刚
郑加柱
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Dalian Institute of Chemical Physics of CAS
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Abstract

The invention provides a self-flashing fluorescent dye for super-resolution fluorescence imaging and synthesis and application thereof, the dye has the characteristics of high quantum yield and high stability by introducing azetidine into 4, 5-site of silarhodamine to inhibit intramolecular rotation, and simultaneously, fluorescent and non-fluorescent forms of the probe can be mutually converted through intramolecular action under normal physiological conditions, so that the probe can be applied to living cell super-resolution imaging, the structural formula of the probe is shown as (1), and the dye can be combined with an antibody to carry out specific labeling imaging on tubulin cells. The dye can be widely applied to the fields of protein labeling, super-resolution fluorescence imaging and the like.

Description

Self-flashing fluorescent dye for super-resolution fluorescence imaging and synthesis and application thereof
Technical Field
The invention belongs to the technical field of fluorescent dyes, and particularly relates to a self-flashing fluorescent dye for super-resolution fluorescence imaging, and synthesis and application thereof.
Background
Optical microscopes have long been an important tool for biomedical research due to their advantages of being non-contact, non-invasive, etc. However, the resolution of the conventional optical microscope is larger than 200nm, and the conventional optical microscope cannot be used for clearly observing biological structures with the size within 200 nm. The super-resolution optical imaging is the most important breakthrough in the field of optical microscopic imaging in the century, breaks away from the limitation of optical diffraction limit, and provides an unprecedented tool for life science research. The current super-resolution technology mainly comprises a structured light illumination microscope (SIM), a stimulated emission depletion microscope (STED) and a random optical positioning reconstruction microscope (STORM). These techniques are largely restricted by dye molecules, where STED and Storm usually require intense laser irradiation and addition of thiol additives to induce the fluorophore to switch on and off, which can cause significant damage to the cell structure and affect the accurate observation of the structure. The fluorescent and non-fluorescent forms of the self-flashing fluorescent dye can be mutually converted through intramolecular action, so that the flashing characteristic is realized by virtue of the molecular spontaneous switch under the physiological environment, and the use of any chemical additive or light activation is avoided. Only low-power exciting light is needed, so that single molecule positioning can be carried out under mild experimental conditions, the minimum side effect on cells is achieved, the observation time can be prolonged, and living cells can be subjected to super-resolution imaging. However, at present, such self-blinking molecules are deficient, and a systematic study is lacking to determine whether the self-blinking performance meets the imaging requirements, so a set of molecular design model is urgently needed to be established to meet the super-resolution multi-color fluorescence imaging.
Disclosure of Invention
The invention provides a self-flashing fluorescent dye for super-resolution fluorescence imaging, and synthesis and application thereof.
The invention provides a self-flashing fluorescent dye for super-resolution fluorescence imaging, which is used for the self-flashing fluorescent dye for super-resolution fluorescence imaging, takes silicorhodamine as a fluorescent group and-NHS as a binding site, and has the following structure:
Figure BDA0001910533200000021
the self-flashing fluorescent dye for super-resolution fluorescence imaging has high light stability and high quantum yield, and the fluorescent quantum yield reaches 0.8 (in water).
The invention relates to a self-flashing fluorescent dye for super-resolution fluorescence imaging, which has a self-flashing function and can reduce the laser energy (40W/cm) of dSTORM application2)。
A synthetic method of self-flashing fluorescent dye for super-resolution fluorescence imaging is provided, and the synthetic route of the probe is as follows:
Figure BDA0001910533200000022
the specific synthesis steps are as follows:
(1) synthesis of intermediate 1:
3-methyl-4-bromobenzoic acid, N-bromosuccinimide (NBS), azoDiisobutyronitrile (AIBN) dissolved in carbon tetrachloride (CCl)4) Heating and refluxing for 40-80 h; after the reaction is finished, the solvent is removed by reduced pressure distillation, and the white target substance 1 is obtained by separation and purification through a 200-mesh 300-mesh silica gel column by using petroleum ether and ethyl acetate as developing agents in the volume ratio of 20: 1;
(2) synthesis of intermediate 2:
the intermediate 1 was dissolved in 10% sodium carbonate solution and stirred at 70 ℃ for 1-4 h. After the reaction is finished, adding dilute hydrochloric acid, filtering and drying after precipitation is separated out; separating and purifying by a 200-mesh 300-mesh silica gel column by using dichloromethane and methanol with the volume ratio of 20:1 as developing agents to obtain a white target 2;
(3) synthesis of intermediate 3:
dissolving the intermediate 2, tert-butyl alcohol, anhydrous magnesium sulfate and concentrated sulfuric acid in dichloromethane, and stirring at room temperature for 24-72 h. After the reaction is finished, the solvent is removed by reduced pressure distillation, and the colorless oily target substance 3 is obtained by separation and purification through a 200-mesh 300-mesh silica gel column by using petroleum ether and ethyl acetate as developing agents in the volume ratio of 100: 1;
(4) synthesis of intermediate 4:
adding cuprous iodide and tripotassium phosphate into a two-mouth bottle, and then respectively adding n-butyl alcohol, ethylene glycol, 1-bromo-3-iodobenzene and azetidine. Repeatedly vacuumizing and introducing nitrogen for three times; stirring at 100 deg.C for 18-48 h; cooling to room temperature after the reaction is finished, adding a saturated ammonium chloride solution, extracting with ethyl acetate, collecting the organic phase, drying with anhydrous sodium sulfate, distilling the organic phase under reduced pressure, and separating and purifying the reaction product by a 200-mesh 300-mesh silica gel column by using petroleum ether and ethyl acetate in a volume ratio of 2:1 as developing agents to obtain a colorless oily intermediate 4;
(5) synthesis of intermediate 5:
adding the intermediate 4 into a 25mL Schlenk bottle, repeatedly vacuumizing and introducing nitrogen for three times, adding tetrahydrofuran and cooling to-78 ℃; then adding n-butyllithium (2.4M), reacting for 15-20min, and then adding dichlorodimethylsilane; the reaction gradually returns to room temperature; after the reaction is finished, adding saturated ammonium chloride to quench the reaction, extracting with ethyl acetate, collecting the organic phase, drying with anhydrous sodium sulfate, distilling the organic phase under reduced pressure, and passing the reaction product through a silica gel column of 200-mesh and 300-mesh; petroleum ether and ethyl acetate in a volume ratio of 70:1 are used as developing agents for separation and purification to obtain an intermediate 5;
(6) synthesis of intermediate 6:
dissolving the intermediate 5 and N-bromosuccinimide (NBS) in N, N-Dimethylformamide (DMF), reacting at room temperature for 1-4h, removing the solvent by reduced pressure distillation after the reaction is finished, and separating and purifying by using 200-300 silica gel column and petroleum ether and ethyl acetate as developing agents in a volume ratio of 30:1 to obtain a white target substance 6;
(7) synthesis of intermediate 7:
adding the intermediate 6 into a 25mL Schlenk bottle, repeatedly vacuumizing and introducing nitrogen for three times, adding tetrahydrofuran and cooling to-18 ℃; then adding isobutyl lithium (1.6M), reacting for 20-30min, and then adding dimethylcarbamoyl chloride; the reaction gradually returns to room temperature; after the reaction is finished, adding saturated ammonium chloride to quench the reaction, extracting with ethyl acetate, collecting the organic phase, drying with anhydrous sodium sulfate, distilling the organic phase under reduced pressure, and passing the reaction product through a 200-mesh 300-mesh silica gel column; separating and purifying by using dichloromethane as a developing agent to obtain a yellow solid intermediate 7;
(8) synthesis of intermediate 8:
adding the intermediate 3 into a 25mL Schlenk bottle, repeatedly vacuumizing and introducing nitrogen for three times, adding anhydrous tetrahydrofuran by using a syringe, and cooling to-78 ℃; then adding isobutyl lithium, reacting for 30min, and then adding the intermediate 7; gradually returning to room temperature and stirring for 12-24 h; after the reaction is finished, adding saturated chloride, quenching the reaction by ammonium, extracting the reaction product by ethyl acetate, collecting an organic phase, drying the organic phase by anhydrous sodium sulfate, carrying out vacuum distillation on the organic phase, and passing the reaction product through a 200-mesh and 300-mesh silica gel column; dichloromethane and methanol in a volume ratio of 30:1 are used as developing agents for separation and purification to obtain a blue solid intermediate 8;
(9) synthesis of intermediate 9:
intermediate 8 was dissolved in trifluoroacetic acid (CF)3COOH), at room temperature for 2-4 days. After the reaction is finished, the solvent is removed by reduced pressure distillation, and the blue solid intermediate 9 is obtained by separation and purification through a 200-mesh 300-mesh silica gel column by taking dichloromethane and methanol with the volume ratio of 10:1 as developing agents;
(10) synthesis of intermediate 10:
dissolving the intermediate 9, N, N-disuccinimidyl carbonate, 4-Dimethylaminopyridine (DMAP) and triethylamine in DMF, and stirring at room temperature for 1-3 h; after the reaction is finished, the solvent is removed by reduced pressure distillation, and the white powder intermediate 10 is obtained by separation and purification through a 200-mesh 300-mesh silica gel column by using petroleum ether and ethyl acetate as developing agents in the volume ratio of 10: 1;
(11) synthesis of Probe BG-SiRho:
the intermediate 10, N-diisopropylethylamine and the intermediate 11 are dissolved in DMF and stirred for 12-16h at room temperature. After the reaction is finished, the solvent is removed by reduced pressure distillation, and the white powder probe BG-SiRho is obtained by separation and purification through a 200-mesh 300-mesh silica gel column by using dichloromethane and methanol with the volume ratio of 10:1 as eluent;
in the step (1):
the mass ratio of the 3-methyl-4-bromobenzoic acid to the N-bromosuccinimide is 1: 0.45-1.8;
the mass ratio of the 3-methyl-4-bromobenzoic acid to the azodiisobutyronitrile is 1: 0.0078-0.0312;
the mass-to-volume ratio of the 3-methyl-4-bromobenzoic acid to the carbon tetrachloride is 1:5-20 g/mL.
In the step (2):
the mass-to-volume ratio of the intermediate 1 to the 10% sodium carbonate solution is 1:4-16 g/mL.
In the step (3):
the mass ratio of the intermediate 2 to the tertiary butanol is 1: 0.7-2.8;
the mass ratio of the intermediate 2 to the anhydrous magnesium sulfate is 1: 0.9-3.6;
the mass-to-volume ratio of the intermediate 2 to the concentrated sulfuric acid is 1: 0.1-0.4;
the mass-to-volume ratio of the intermediate 2 to the dichloromethane is 1:13-52 g/mL.
In the step (4):
the mass ratio of the 1-bromo-3-iodobenzene to the cuprous iodide is 1: 0.2-2;
the mass ratio of the 1-bromo-3-iodobenzene to the tripotassium phosphate is 1: 4-10;
the mass-to-volume ratio of the 1-bromo-3-iodobenzene to the azacyclobutane is 1: 0.2-1;
the volume ratio of the 1-bromo-3-iodobenzene to the ethylene glycol is 1:1-1.5 (g: mL).
In the step (5):
the mass-to-volume ratio of the intermediate 4 to the n-butyllithium is 1:1-4 g/mL;
the mass-to-volume ratio of the intermediate 4 to the dichlorodimethylsilane is 1:0.13-0.5 g/mL;
the mass-to-volume ratio of the intermediate 4 to tetrahydrofuran is 1:10-20 g/mL.
In the step (6):
the mass ratio of the intermediate 5 to the N-bromosuccinimide is 1: 0.56-2.23;
the mass-to-volume ratio of the intermediate 5 to the N, N-dimethylformamide is 1:7.5-30 g/mL. In the step (7):
the mass-to-volume ratio of the intermediate 4 to the isobutyllithium is 1:1.36-5.4 (g: mL);
the mass-to-volume ratio of the intermediate 4 to the dimethylcarbamoyl chloride is 1:0.12-0.48 g/mL;
the mass-to-volume ratio of the intermediate 4 to tetrahydrofuran is 1:18-70 g/mL.
In the step (8):
the mass-to-volume ratio of the intermediate 3 to the isobutyl lithium is 1:0.85-3.4 g/mL;
the mass ratio of the intermediate 3 to the intermediate 7 is 1: 0.07-0.30;
the mass-to-volume ratio of the intermediate 3 to tetrahydrofuran is 1:18-72 g/mL.
In the step (9):
the mass-to-volume ratio of the intermediate 8 to the trifluoroacetic acid is 1:74-300 g/mL.
In the step (10):
the mass ratio of the intermediate 9 to the N, N-disuccinimidyl carbonate is 1: 0.8-3.2;
the mass ratio of the intermediate 9 to the 4-dimethylaminopyridine is 1: 0.1-0.3;
the mass-to-volume ratio of the intermediate 9 to triethylamine is 1:0.6-2.4 g/mL;
the mass-to-volume ratio of the intermediate 9 to DMF was 1:147-600 g/mL.
The invention provides a synthetic method of self-flashing fluorescent dye for super-resolution fluorescence imaging, which has the advantages of convenient operation, low cost and the like.
An application of self-flashing fluorescent dye for super-resolution fluorescence imaging in the fluorescence imaging field of cells, tissues and living bodies.
The invention has the following advantages:
the probe is capable of binding to antibodies and performing super-resolution imaging of proteins of cells.
The probe has high light stability and high quantum yield, and the fluorescence quantum yield reaches 0.8 (in water).
The probe has self-flashing function, and can reduce the laser energy (40W/cm) of dSTORM application2)。
The synthesis method of the probe has the advantages of convenience in operation, low cost and the like.
Drawings
Figure 1 nuclear magnetic hydrogen spectrum of intermediate 3 prepared in example 1.
Figure 2 nuclear magnetic hydrogen spectrum of intermediate 4 prepared in example 1.
Figure 3 nuclear magnetic hydrogen spectrum of intermediate 5 prepared in example 1.
Figure 4 nuclear magnetic hydrogen spectrum of intermediate 6 prepared in example 1.
Figure 5 nuclear magnetic hydrogen spectrum of intermediate 7 prepared in example 1.
Figure 6 nuclear magnetic hydrogen spectrum of intermediate 8 prepared in example 1.
Figure 7 nuclear magnetic hydrogen spectrum of intermediate 9 prepared in example 1.
FIG. 8 shows the hydrogen nuclear magnetic spectrum of the fluorescent dye NHS-SiRho prepared in example 1.
Fig. 9 fluorescence emission spectra of the dye NHS-SiRho prepared in example 1 at different pH (pH 2-12) with wavelength on the abscissa, fluorescence intensity on the ordinate, and fluorescent dye concentration of 5 μ M.
FIG. 10 super-resolution imaging of intracellular tubulin by the dye NHS-SiRho conjugated antibody prepared in example 1.
Detailed Description
Example 1
Synthesis of intermediate 1
Figure BDA0001910533200000081
3-methyl-4-bromobenzoic acid (5.03g, 23mmol), NBS (4.5g, 25mmol), AIBN (76.8mg, 0.47mmol) were dissolved in 50mL of carbon tetrachloride (CCl)4) And heating and refluxing for 72 h. After the reaction, the solvent was removed by distillation under reduced pressure, and the white target product (6g, 86% yield) was isolated and purified by silica gel column (200-300 mesh) using petroleum ether and ethyl acetate (20:1) as developing agents.
Synthesis of intermediate 2
Figure BDA0001910533200000082
1(6g, 20mmol) was dissolved in 50mL 10% sodium carbonate solution and stirred at 70 ℃ for 2 h. And after the reaction is finished, adding dilute hydrochloric acid, filtering and drying after precipitation is separated out. The white target 3g was isolated and purified by silica gel column (200-300 mesh) using dichloromethane and methanol (20:1) as developing solvent, yield 65%.
Synthesis of intermediate 3
Figure BDA0001910533200000091
2(2.3g, 10mmol), t-butanol (3.2g, 44mmol), anhydrous magnesium sulfate (4.18g), concentrated sulfuric acid (0.47mL) were dissolved in 60mL of dichloromethane and stirred at room temperature for 48 h. After the reaction, the solvent was distilled off under reduced pressure, and the product was separated and purified by means of a silica gel column (200-300 mesh) using petroleum ether and ethyl acetate (100:1) as developing agents to give 2.75g of a colorless oily target substance in a yield of 80%. The nuclear magnetic spectrum of the intermediate 3 prepared in example 1 is shown in figure 1, and specific hydrogen spectrum data are as follows:
1H NMR(400MHz,CDCl3)δ8.14(d,J＝2.1Hz,1H),7.71(dd,J＝8.3,2.1Hz,1H),7.55(d,J＝8.3Hz,1H),4.50(s,2H),1.59(s,9H),1.32(s,9H).
synthesis of intermediate 4
Figure BDA0001910533200000092
Mixing CuI (135mg,0.707mmol), K3PO4(4.5g, 21mmol) was added to a 50mL two-necked flask, and 10mL of n-butanol, 1mL of ethylene glycol, 1-bromo-3-iodobenzene (900mg, 7mmol), and azetidine (600. mu.L, 8mmol) were added, respectively. Repeatedly vacuumizing and introducing nitrogen for three times. Stirring was carried out at 100 ℃ for 24 h. After the reaction was completed, the reaction mixture was cooled to room temperature, 10mL of a saturated ammonium chloride solution was added, extraction was performed with ethyl acetate, the organic phase was collected and dried over anhydrous sodium sulfate, the organic phase was distilled under reduced pressure, and the reaction product was separated and purified by means of a silica gel column (200 mesh, 300 mesh) using petroleum ether and ethyl acetate (2:1) as developing agents to obtain 500mg of a colorless oily liquid with a yield of 50%. The nuclear magnetic spectrum of the intermediate 4 prepared in example 1 is shown in fig. 2, and specific hydrogen spectrum data are as follows:
1H NMR(400MHz,CDCl3)δ7.03(t,J＝8.0Hz,1H),6.81(dd,J＝7.9,0.7Hz,1H),6.54(t,J＝1.9Hz,1H),6.33(dd,J＝8.1,1.7Hz,1H),3.86(t,J＝7.3Hz,4H),2.37(m,J＝14.5,7.2Hz,2H).
synthesis of intermediate 5
Figure BDA0001910533200000101
4(1.055g, 5mmol) was added to a 25mL Schlenk flask, vacuum was repeatedly applied three times with nitrogen, and cooled to-78 ℃. Then, 2mL of n-butyllithium (2.4M) was added, reacted for 15min, and dichlorodimethylsilane (252. mu.L, 2.1mmol) was added. The reaction was gradually returned to room temperature. After the reaction, saturated ammonium chloride is added to quench the reaction, the reaction solution is extracted by ethyl acetate, an organic phase is collected and dried by anhydrous sodium sulfate, the organic phase is subjected to reduced pressure distillation, and a reaction product is separated and purified by a silica gel column (200-300 meshes) by using petroleum ether and ethyl acetate (70:1) as developing agents to obtain 2g of colorless oily liquid with the yield of 80 percent. The nuclear magnetic spectrum of the intermediate 5 prepared in example 1 is shown in fig. 3, and specific hydrogen spectrum data are as follows:
1H NMR(400MHz,CDCl3)δ7.19(t,J＝7.6Hz,2H),6.89(d,J＝7.2Hz,2H),6.60(d,J＝2.1Hz,2H),6.45(dd,J＝8.0,1.7Hz,2H),3.85(t,J＝7.2Hz,8H),2.39–2.27(m,4H),0.49(s,6H).
synthesis of intermediate 6
Figure BDA0001910533200000102
5(0.322g, 1mmol) and NBS (0.36g, 2mmol) were dissolved in 5mL of N, N-Dimethylformamide (DMF) and reacted at room temperature for 2 h. After the reaction, the solvent was removed by distillation under reduced pressure, and the white target compound (0.5 g) was isolated and purified by silica gel column (200-300 mesh) using petroleum ether and ethyl acetate (30:1) as developing agents, with a yield of 90%. The nuclear magnetic spectrum of the intermediate 6 prepared in example 1 is shown in fig. 4, and specific hydrogen spectrum data are as follows:
1H NMR(400MHz,CDCl3)δ7.30(d,J＝8.5Hz,2H),6.51(d,J＝2.9Hz,2H),6.31(dd,J＝8.5,3.0Hz,2H),3.81(t,J＝7.2Hz,8H),2.38–2.26(m,4H),0.71(s,6H).
synthesis of intermediate 7
Figure BDA0001910533200000111
6(0.42g, 0.9mmol) was added to a 25mL Schlenk flask, vacuum was repeatedly applied three times with nitrogen, and cooled to-18 ℃. Then 1.14mL of isobutyllithium (1.6M) was added and reacted for 30min, followed by addition of dimethylcarbamoyl chloride (92. mu.L, 1 mmol). The reaction was gradually returned to room temperature. After the reaction, saturated ammonium chloride is added to quench the reaction, the reaction solution is extracted by ethyl acetate, an organic phase is collected and dried by anhydrous sodium sulfate, the organic phase is subjected to reduced pressure distillation, and a reaction product is separated and purified by a silica gel column (200-300 meshes) by taking dichloromethane as a developing agent to obtain 0.3g of yellow solid with the yield of 60%. The nuclear magnetic spectrum of the intermediate 7 prepared in example 1 is shown in fig. 5, and specific hydrogen spectrum data are as follows:
1H NMR(400MHz,CDCl3)δ8.36(d,J＝8.7Hz,2H),6.51(dd,J＝8.7,2.5Hz,2H),6.48(d,J＝2.5Hz,2H),4.02(t,J＝7.3Hz,8H),2.47–2.35(m,4H),0.43(s,6H).
synthesis of intermediate 8
Figure BDA0001910533200000121
3(0.275g, 0.8mmol) was added to a 25mL Schlenk flask, vacuum was repeatedly applied three times with nitrogen, 10mL of anhydrous tetrahydrofuran was added via syringe, and the mixture was cooled to-78 ℃. Then, 0.47mL of isobutyllithium (1.6M) was added and reacted for 30min, followed by addition of 7(40mg, 0.114 mmol). Gradually return to room temperature and stir for 12 h. After the reaction was completed, the reaction was quenched by adding saturated ammonium chloride, extracted with ethyl acetate, the organic phase was collected and dried over anhydrous sodium sulfate, the organic phase was distilled under reduced pressure, and the reaction product was separated and purified by a silica gel column (200-300 mesh) using methylene chloride and methanol (30:1) as developing agents to obtain 34mg of a blue solid with a yield of 50%. The nuclear magnetic spectrum of the intermediate 8 prepared in example 1 is shown in fig. 6, and specific hydrogen spectrum data are as follows:
1H NMR(400MHz,MeOD)δ8.06(d,J＝1.4Hz,1H),7.94(dd,J＝7.9,1.6Hz,1H),7.14(d,J＝7.9Hz,1H),6.87(d,J＝2.6Hz,2H),6.85(d,J＝9.4Hz,2H),6.25(dd,J＝9.4,2.5Hz,2H),4.28(s,8H),4.09(s,2H),2.54–2.38(m,4H),1.55(s,9H),0.83(s,9H),0.46(d,J＝5.0Hz,6H).
synthesis of intermediate 9
Figure BDA0001910533200000122
8(34mg, 0.057mmol) was dissolved in 5mL trifluoroacetic acid (CF)3COOH), stirred at room temperature for 2 days. After the reaction, the solvent was distilled off under reduced pressure, and the blue target 17mg was isolated and purified by means of a silica gel column (200-300 mesh) using methylene chloride and methanol (10:1) as developing agents in a yield of 60%. The nuclear magnetic spectrum of the intermediate 9 prepared in example 1 is shown in fig. 7, and specific hydrogen spectrum data are as follows:
1H NMR(400MHz,DMSO-d6)δ7.92(s,1H),7.75(d,J＝7.9Hz,1H),7.01(d,J＝8.6Hz,2H),6.78(d,J＝8.0Hz,1H),6.34(dd,J＝8.7,2.5Hz,2H),5.41(s,2H),3.79(t,J＝7.3Hz,8H),2.28(dt,J＝14.5,7.1Hz,4H),0.56(s,3H),0.44(s,3H).
synthesis of NHS-SiRho
Figure BDA0001910533200000131
9(17mg, 0.034mmol), N-disuccinimidyl carbonate (27mg, 0.1mmol), DMAP (5.1mg), triethylamine (20. mu.L) were dissolved in 5mL of DMF and stirred at room temperature for 1 h. After the reaction, the solvent was removed by distillation under reduced pressure, and the product was separated and purified by a silica gel column (200-300 mesh) using petroleum ether and ethyl acetate (10:1) as developing agents to give the desired white powder in a yield of 10mg and 53%. The nuclear magnetic spectrum of the intermediate 10 prepared in example 1 is shown in fig. 8, and specific hydrogen spectrum data are as follows:
1H NMR(400MHz,CDCl3)δ8.10(s,1H),8.03(d,J＝8.1Hz,1H),7.15(d,J＝8.1Hz,1H),6.88(d,J＝8.6Hz,2H),6.66(d,J＝2.6Hz,2H),6.32(dd,J＝8.6,2.6Hz,2H),5.29(s,2H),3.89(t,J＝7.2Hz,8H),2.91(s,4H),2.41–2.29(m,4H),0.59(s,3H),0.51(s,3H).
the structure of the compound is shown as NHS-SiRho
Dissolving the fluorescent dye in a DMSO solution to prepare 2mM mother liquor, and preparing test solutions with different concentrations according to requirements to detect the fluorescence spectrum change and intracellular fluorescence imaging.
Fluorescence emission spectroscopy of NHS-SiRho at different pH (pH 2-8). Dissolving 400 mu of LNHS-Sirho mother liquor in 80mL of water, adjusting the pH value by using a sodium hydroxide solution and a hydrochloric acid solution, taking out 4mL of test solution after the band is stable, and carrying out fluorescence spectrum test.
The fluorescence of NHS-SiRho with a final concentration of 2 μ M at different pH values is shown in FIG. 9, the fluorescence spectrum of NHS-SiRho at different pH values is greatly changed, the fluorescence is weak in neutral and alkaline environments, and the fluorescence is enhanced under acidic conditions, which indicates that most of the probes are kept dark under physiological conditions.
Example 2
Synthesis of intermediate 1
Figure BDA0001910533200000141
3-methyl-4-bromobenzoic acid (5g, 24mmol), NBS (2.25g, 12.5mmol), AIBN (38mg, 0.24mmol) were dissolved in 30mL of carbon tetrachloride (CCl)4) And heating and refluxing for 40 h. After the reaction, the solvent was removed by distillation under reduced pressure, and the white target product (3g, yield 85%) was isolated and purified by silica gel column (200-300 mesh) using petroleum ether and ethyl acetate (20:1) as developing agents.
Synthesis of intermediate 2
Figure BDA0001910533200000142
1(6g, 20mmol) was dissolved in 50mL 10% sodium carbonate solution and stirred at 70 ℃ for 2 h. And after the reaction is finished, adding dilute hydrochloric acid, filtering and drying after precipitation is separated out. The white target 1.5g was isolated and purified by silica gel column (200-300 mesh) using dichloromethane and methanol (20:1) as developing solvent, yield 64%.
Synthesis of intermediate 3
Figure BDA0001910533200000151
2(2.3g, 10mmol), t-butanol (1.6g, 22mmol), anhydrous magnesium sulfate (2g), concentrated sulfuric acid (0.25mL) were dissolved in 60mL of dichloromethane and stirred at room temperature for 24 h. After the reaction, the solvent was distilled off under reduced pressure, and the product was separated and purified by means of a silica gel column (200-300 mesh) using petroleum ether and ethyl acetate (100:1) as developing agents to give 1.4g of a colorless oily target in a yield of 81%.
Synthesis of intermediate 4
Figure BDA0001910533200000152
Mixing CuI (70mg,035mmol), K3PO4(2.3g, 10mmol) was added to a 50mL two-necked flask, and 5mL of n-butanol, 0.5mL of ethylene glycol, 1-bromo-3-iodobenzene (900mg, 7mmol), and azetidine (300. mu.L, 4mmol) were added, respectively. Repeatedly vacuumizing and introducing nitrogen for three times. Stirring was carried out at 100 ℃ for 18 h. After the reaction was complete, the mixture was cooled to room temperature and 10mL of the solution was addedSaturated ammonium chloride solution, using ethyl acetate to extract, collecting organic phase, drying with anhydrous sodium sulfate, decompressing and distilling the organic phase, separating and purifying the reaction product by silica gel column (200-300 mesh) by using petroleum ether and ethyl acetate (2:1) as developing agents to obtain 250mg of colorless oily liquid with 50 percent of yield.
Synthesis of intermediate 5
Figure BDA0001910533200000161
4(1g, 5mmol) was added to a 25mL Schlenk flask and the flask was repeatedly evacuated and purged with nitrogen three times and cooled to-78 ℃. Then, 1mL of n-butyllithium (2.4M) was added, reacted for 15min, and dichlorodimethylsilane (130. mu.L, 1.05mmol) was added. The reaction was gradually returned to room temperature. After the reaction, saturated ammonium chloride is added to quench the reaction, the reaction solution is extracted by ethyl acetate, an organic phase is collected and dried by anhydrous sodium sulfate, the organic phase is subjected to reduced pressure distillation, and a reaction product is separated and purified by a silica gel column (200-300 meshes) by using petroleum ether and ethyl acetate (70:1) as developing agents to obtain 1g of colorless oily liquid with the yield of 81 percent.
Synthesis of intermediate 6
Figure BDA0001910533200000162
5(0.3g, 1mmol) and NBS (0.18g, 1mmol) were dissolved in 5mL of N, N-Dimethylformamide (DMF) and reacted at room temperature for 1 h. After the reaction, the solvent was removed by distillation under reduced pressure, and the product was separated and purified by means of a silica gel column (200-mesh 300-mesh) using petroleum ether and ethyl acetate (30:1) as developing agents to give 0.6g of a white target in 93% yield.
Synthesis of intermediate 7
Figure BDA0001910533200000163
6(0.42g, 0.9mmol) was added to a 25mL Schlenk flask, vacuum was repeatedly applied three times with nitrogen, and cooled to-18 ℃. Then 0.7mL of isobutyllithium (1.6M) was added and reacted for 30min, followed by addition of dimethylcarbamoyl chloride (46. mu.L, 0.5 mmol). The reaction was gradually returned to room temperature. After the reaction is finished, adding saturated ammonium chloride to quench the reaction, extracting with ethyl acetate, collecting an organic phase, drying with anhydrous sodium sulfate, distilling the organic phase under reduced pressure, and separating and purifying a reaction product by a silica gel column (200-300 meshes) by taking dichloromethane as a developing agent to obtain 0.15g of yellow solid with the yield of 60%.
Synthesis of intermediate 8
Figure BDA0001910533200000171
3(0.28g, 0.8mmol) was added to a 25mL Schlenk flask, vacuum was repeatedly applied three times with nitrogen, 10mL of anhydrous tetrahydrofuran was added via syringe, and the mixture was cooled to-78 ℃. Then, 0.24mL of isobutyllithium (1.6M) was added and reacted for 30min, followed by addition of 7(20mg, 0.6 mmol). Gradually return to room temperature and stir for 12 h. After the reaction was completed, the reaction was quenched by adding saturated ammonium chloride, extracted with ethyl acetate, the organic phase was collected and dried over anhydrous sodium sulfate, the organic phase was distilled under reduced pressure, and the reaction product was separated and purified by a silica gel column (200-300 mesh) using methylene chloride and methanol (30:1) as developing agents to obtain 18mg of a blue solid in a yield of 58%.
Synthesis of intermediate 9
Figure BDA0001910533200000172
8(34mg, 0.06mmol) was dissolved in 5mL trifluoroacetic acid (CF)3COOH), stirred at room temperature for 2 days. After the reaction, the solvent was distilled off under reduced pressure, and the blue target product was isolated and purified by means of a silica gel column (200-300 mesh) using methylene chloride and methanol (10:1) as developing agents to give 9mg of a blue color, yield 33%.
Synthesis of NHS-SiRho
Figure BDA0001910533200000181
9(17mg, 0.035mmol), N-disuccinimidyl carbonate (14mg, 0.05mmol), DMAP (2.5mg), triethylamine (10. mu.L) were dissolved in 5mL of DMF and stirred at room temperature for 1 h. After the reaction, the solvent was removed by distillation under reduced pressure, and the product was separated and purified by a silica gel column (200-300 mesh) using petroleum ether and ethyl acetate (10:1) as developing agents to give the desired white powder in an amount of 5mg with a yield of 25%.
Through detection, the structure of the dye is shown as the formula NHS-SiRho, the fluorescence emission wavelength of the dye in water is about 660nm, the absorption wavelength of the dye is about 650nm, and the dye can realize fluorescence self-switching.
Example 3
Synthesis of intermediate 1
Figure BDA0001910533200000182
3-methyl-4-bromobenzoic acid (2.5g, 12mmol), NBS (4.5g, 25mmol), AIBN (76.8mg, 0.47mmol) were dissolved in 50mL of carbon tetrachloride (CCl)4) And heating and refluxing for 80 h. After the reaction, the solvent was removed by distillation under reduced pressure, and the white target product 5g was isolated and purified by silica gel column (200-300 mesh) using petroleum ether and ethyl acetate (20:1) as developing agents, with a yield of 80%.
Synthesis of intermediate 2
Figure BDA0001910533200000191
1(3g, 10mmol) was dissolved in 50mL of 10% sodium carbonate solution and stirred at 70 ℃ for 2 h. And after the reaction is finished, adding dilute hydrochloric acid, filtering and drying after precipitation is separated out. The white target 2g was isolated and purified by silica gel column (200-300 mesh) using dichloromethane and methanol (20:1) as developing solvent, yield 70%.
Synthesis of intermediate 3
Figure BDA0001910533200000192
2(1.2g, 5mmol), t-butanol (3.2g, 44mmol), anhydrous magnesium sulfate (4.18g), concentrated sulfuric acid (0.47mL) were dissolved in 60mL of dichloromethane and stirred at room temperature for 72 h. After the reaction, the solvent was removed by distillation under reduced pressure, and the product was separated and purified by means of a silica gel column (200-300 mesh) using petroleum ether and ethyl acetate (100:1) as developing agents to give 1g of a colorless oily target in a yield of 70%.
Synthesis of intermediate 4
Figure BDA0001910533200000193
Mixing CuI (135mg,0.707mmol), K3PO4(4.5g, 21mmol) was added to a 50mL two-necked flask, and 10mL of n-butanol, 1mL of ethylene glycol, 1-bromo-3-iodobenzene (450mg, 3.5mmol), and azetidine (600. mu.L, 8mmol) were added, respectively. Repeatedly vacuumizing and introducing nitrogen for three times. Stirring was carried out at 100 ℃ for 48 h. After the reaction was completed, the reaction mixture was cooled to room temperature, 10mL of a saturated ammonium chloride solution was added, extraction was performed with ethyl acetate, the organic phase was collected and dried over anhydrous sodium sulfate, the organic phase was distilled under reduced pressure, and the reaction product was separated and purified by means of a silica gel column (200 mesh and 300 mesh) using petroleum ether and ethyl acetate (2:1) as developing agents to obtain 300mg of a colorless oily liquid with a yield of 60%.
Synthesis of intermediate 5
Figure BDA0001910533200000201
4(0.5g, 2.5mmol) was added to a 25mL Schlenk flask, vacuum was repeatedly applied three times with nitrogen, and cooled to-78 ℃. Then, 2mL of n-butyllithium (2.4M) was added, reacted for 15min, and dichlorodimethylsilane (252. mu.L, 2.1mmol) was added. The reaction was gradually returned to room temperature. After the reaction, adding saturated ammonium chloride to quench the reaction, extracting with ethyl acetate, collecting the organic phase, drying with anhydrous sodium sulfate, distilling the organic phase under reduced pressure, and separating and purifying the reaction product by a silica gel column (200-300 mesh) by using petroleum ether and ethyl acetate (70:1) as developing agents to obtain 1g of colorless oily liquid with the yield of 80%.
Synthesis of intermediate 6
Figure BDA0001910533200000202
5(0.16g, 0.5mmol) and NBS (0.36g, 2mmol) were dissolved in 5mL of N, N-Dimethylformamide (DMF) and reacted at room temperature for 4 h. After the reaction, the solvent was removed by distillation under reduced pressure, and the white target compound (0.5 g) was isolated and purified by silica gel column (200-300 mesh) using petroleum ether and ethyl acetate (30:1) as developing agents, with a yield of 90%.
Synthesis of intermediate 7
Figure BDA0001910533200000211
6(0.21g, 0.45mmol) was added to a 25mL Schlenk flask, vacuum was repeatedly applied three times with nitrogen, and cooled to-18 ℃. Then 1.14mL of isobutyllithium (1.6M) was added and reacted for 30min, followed by addition of dimethylcarbamoyl chloride (92. mu.L, 1 mmol). The reaction was gradually returned to room temperature. After the reaction is finished, adding saturated ammonium chloride to quench the reaction, extracting with ethyl acetate, collecting an organic phase, drying with anhydrous sodium sulfate, distilling the organic phase under reduced pressure, and separating and purifying a reaction product by a silica gel column (200-300 meshes) by taking dichloromethane as a developing agent to obtain 0.15g of yellow solid with the yield of 60%.
Synthesis of intermediate 8
Figure BDA0001910533200000212
3(0.14g, 0.4mmol) was added to a 25mL Schlenk flask, vacuum was repeatedly applied three times with nitrogen, 10mL of anhydrous tetrahydrofuran was added via syringe, and the mixture was cooled to-78 ℃. Then, 0.47mL of isobutyllithium (1.6M) was added and reacted for 30min, followed by addition of 7(40mg, 0.114 mmol). Gradually return to room temperature and stir for 12 h. After the reaction was completed, the reaction was quenched by adding saturated ammonium chloride, extracted with ethyl acetate, the organic phase was collected and dried over anhydrous sodium sulfate, the organic phase was distilled under reduced pressure, and the reaction product was separated and purified by a silica gel column (200-300 mesh) using methylene chloride and methanol (30:1) as developing agents to obtain 34mg of a blue solid with a yield of 50%.
Synthesis of intermediate 9
Figure BDA0001910533200000221
8(17mg, 0.03mmol) was dissolved in 5mL trifluoroacetic acid (CF)3COOH), stirred at room temperature for 4 days. After the reaction, the solvent was distilled off under reduced pressure, and the blue target 9mg was obtained by separation and purification through a silica gel column (200-300 mesh) using methylene chloride and methanol (10:1) as developing agents in a yield of 60%.
Synthesis of NHS-SiRho
Figure BDA0001910533200000222
9(9mg, 0.017mmol), N-disuccinimidyl carbonate (27mg, 0.1mmol), DMAP (5.1mg), triethylamine (20. mu.L) were dissolved in 5mL of DMF and stirred at room temperature for 3 hours. After the reaction, the solvent was removed by distillation under reduced pressure, and the product was separated and purified by a silica gel column (200-300 mesh) using petroleum ether and ethyl acetate (10:1) as developing agents to give the desired white powder in a yield of 5mg and 50%.
Through detection, the structure of the dye is shown as the formula NHS-SiRho, the fluorescence emission wavelength of the dye in water is about 660nm, the absorption wavelength of the dye is about 650nm, and the dye can realize fluorescence self-switching.
Example 4
NHS-SiRho in the antibody connecting solution and antibody connection, then to the cell tubulin super resolution imaging experiment.
The imaging of the probe NHS-SiRho on tubulin is shown in fig. 10, and has an obvious self-blinking effect in the imaging process, the probe does not need to be quenched by strong laser energy, and an imaging picture shows that the probe obviously marks tubulin cells and has a good imaging effect.

Claims (13)

1. A self-flashing fluorescent dye for super-resolution fluorescence imaging is characterized in that the self-flashing fluorescent dye has the following structure:
Figure FDA0003181870730000011
2. a method of synthesizing a self-scintillating fluorescent dye for super-resolution fluorescence imaging according to claim 1, comprising the steps of:
(1) synthesis of intermediate 1:
dissolving 3-methyl-4-bromobenzoic acid, N-bromosuccinimide (NBS), Azobisisobutyronitrile (AIBN) in carbon tetrachloride (CCl)4) Heating and refluxing for 40-80 h; after the reaction is finished, the solvent is removed by reduced pressure distillation, and the white intermediate 1 is obtained by separation and purification through a 200-mesh 300-mesh silica gel column by using petroleum ether and ethyl acetate as developing agents in the volume ratio of 20: 1;
(2) synthesis of intermediate 2:
dissolving the intermediate 1 in 10% sodium carbonate solution, and stirring at 70 deg.C for 1-4 h; after the reaction is finished, adding dilute hydrochloric acid, filtering and drying after precipitation is separated out; separating and purifying by a 200-mesh 300-mesh silica gel column by using dichloromethane and methanol with the volume ratio of 20:1 as developing agents to obtain a white intermediate 2;
(3) synthesis of intermediate 3:
dissolving the intermediate 2, tert-butyl alcohol, anhydrous magnesium sulfate and concentrated sulfuric acid in dichloromethane, and stirring at room temperature for 24-72 h; after the reaction is finished, the solvent is removed by reduced pressure distillation, and the colorless oily intermediate 3 is obtained by separation and purification through a 200-mesh 300-mesh silica gel column by using petroleum ether and ethyl acetate as developing agents in the volume ratio of 100: 1;
(4) synthesis of intermediate 4:
adding cuprous iodide and tripotassium phosphate into a two-mouth bottle, and respectively adding n-butyl alcohol, ethylene glycol, 1-bromo-3-iodobenzene and azetidine; repeatedly vacuumizing and introducing nitrogen for three times; stirring at 100 deg.C for 18-48 h; cooling to room temperature after the reaction is finished, adding a saturated ammonium chloride solution, extracting with ethyl acetate, collecting the organic phase, drying with anhydrous sodium sulfate, distilling the organic phase under reduced pressure, and separating and purifying the reaction product by a 200-mesh 300-mesh silica gel column by using petroleum ether and ethyl acetate in a volume ratio of 2:1 as developing agents to obtain a colorless oily intermediate 4;
(5) synthesis of intermediate 5
Adding the intermediate 4 into a 25mL Schlenk bottle, repeatedly vacuumizing and introducing nitrogen for three times, adding tetrahydrofuran and cooling to-78 ℃; then adding 2.4M n-butyllithium, reacting for 15-20min, and then adding dichlorodimethylsilane; the reaction gradually returns to room temperature; after the reaction is finished, adding saturated ammonium chloride to quench the reaction, extracting with ethyl acetate, collecting the organic phase, drying with anhydrous sodium sulfate, distilling the organic phase under reduced pressure, and passing the reaction product through a silica gel column of 200-mesh and 300-mesh; petroleum ether and ethyl acetate in a volume ratio of 70:1 are used as developing agents for separation and purification to obtain an intermediate 5;
(6) synthesis of intermediate 6:
dissolving the intermediate 5 and N-bromosuccinimide in N, N-dimethylformamide, reacting at room temperature for 1-4h, removing the solvent by reduced pressure distillation after the reaction is finished, and separating and purifying by using petroleum ether and ethyl acetate as developing agents in a volume ratio of 30:1 through a 200-300 silica gel column to obtain a white intermediate 6;
(7) synthesis of intermediate 7:
adding the intermediate 6 into a 25mL Schlenk bottle, repeatedly vacuumizing and introducing nitrogen for three times, adding tetrahydrofuran and cooling to-18 ℃; then adding 1.6M of isobutyl lithium, reacting for 20-30min, and then adding dimethylcarbamoyl chloride; the reaction gradually returns to room temperature; after the reaction is finished, adding saturated ammonium chloride to quench the reaction, extracting with ethyl acetate, collecting the organic phase, drying with anhydrous sodium sulfate, distilling the organic phase under reduced pressure, and passing the reaction product through a 200-mesh 300-mesh silica gel column; separating and purifying by using dichloromethane as a developing agent to obtain a yellow solid intermediate 7;
(8) synthesis of intermediate 8:
adding the intermediate 3 into a 25mL Schlenk bottle, repeatedly vacuumizing and introducing nitrogen for three times, adding anhydrous tetrahydrofuran by using a syringe, and cooling to-78 ℃; then adding isobutyl lithium, reacting for 30min, and then adding the intermediate 7; gradually returning to room temperature and stirring for 12-24 h; after the reaction is finished, adding saturated chloride, quenching the reaction by ammonium, extracting the reaction product by ethyl acetate, collecting an organic phase, drying the organic phase by anhydrous sodium sulfate, carrying out vacuum distillation on the organic phase, and passing the reaction product through a 200-mesh and 300-mesh silica gel column; dichloromethane and methanol in a volume ratio of 30:1 are used as developing agents for separation and purification to obtain a blue solid intermediate 8;
(9) synthesis of intermediate 9:
intermediate 8 was dissolved in trifluoroacetic acid (CF)3COOH), stirring for 2-4 days at room temperature; after the reaction is finished, the solvent is removed by reduced pressure distillation, and the blue solid intermediate 9 is obtained by separation and purification through a 200-mesh 300-mesh silica gel column by taking dichloromethane and methanol with the volume ratio of 10:1 as developing agents;
(10) synthesis of NHS-SiRho:
dissolving the intermediate 9, N, N-disuccinimidyl carbonate, 4-Dimethylaminopyridine (DMAP) and triethylamine in (DMF), and stirring at room temperature for 1-3 h; after the reaction is finished, the solvent is removed by reduced pressure distillation, and white powder NHS-SiRho is obtained by separation and purification through a 200-mesh 300-mesh silica gel column by using petroleum ether and ethyl acetate as developing agents in the volume ratio of 10: 1;
the synthesis route is as follows:
Figure FDA0003181870730000031
Figure FDA0003181870730000041
3. the method for synthesizing self-flashing fluorescent dye for super-resolution fluorescence imaging according to claim 2, wherein in the step (1):
the mass ratio of the 3-methyl-4-bromobenzoic acid to the N-bromosuccinimide is 1: 0.45-1.8;
the mass ratio of the 3-methyl-4-bromobenzoic acid to the azodiisobutyronitrile is 1: 0.0078-0.0312;
the mass-to-volume ratio of the 3-methyl-4-bromobenzoic acid to the carbon tetrachloride is 1:5-20 g/mL.
4. The method for synthesizing self-flashing fluorescent dye for super-resolution fluorescence imaging according to claim 2, wherein in the step (2):
the mass-to-volume ratio of the intermediate 1 to the 10% sodium carbonate solution is 1:4-16 g/mL.
5. The method for synthesizing self-flashing fluorescent dye for super-resolution fluorescence imaging as claimed in claim 2, wherein in the step (3):
the mass ratio of the intermediate 2 to the tertiary butanol is 1: 0.7-2.8;
the mass ratio of the intermediate 2 to the anhydrous magnesium sulfate is 1: 0.9-3.6;
the mass-to-volume ratio of the intermediate 2 to the concentrated sulfuric acid is 1: 0.1-0.4;
the mass-to-volume ratio of the intermediate 2 to the dichloromethane is 1:13-52 g/mL.
6. The method for synthesizing self-flashing fluorescent dye for super-resolution fluorescence imaging according to claim 2, wherein in the step (4):
the mass ratio of the 1-bromo-3-iodobenzene to the cuprous iodide is 1: 0.2-2;
the mass ratio of the 1-bromo-3-iodobenzene to the tripotassium phosphate is 1: 4-10;
the mass-to-volume ratio of the 1-bromo-3-iodobenzene to the azacyclobutane is 1: 0.2-1;
the volume ratio of the 1-bromo-3-iodobenzene to the ethylene glycol is 1:1-1.5 g/mL.
7. The method for synthesizing self-scintillating fluorescent dye for super-resolution fluorescence imaging according to claim 2, characterized in that in the step (5):
the mass-to-volume ratio of the intermediate 4 to the n-butyllithium is 1:1-4 g/mL;
the mass-to-volume ratio of the intermediate 4 to the dichlorodimethylsilane is 1:0.13-0.5 g/mL;
the mass-to-volume ratio of the intermediate 4 to tetrahydrofuran is 1:10-20 g/mL.
8. The method for synthesizing self-flashing fluorescent dye for super-resolution fluorescence imaging as claimed in claim 2, wherein in the step (6):
the mass ratio of the intermediate 5 to the N-bromosuccinimide is 1: 0.56-2.23;
the mass-to-volume ratio of the intermediate 5 to the N, N-dimethylformamide is 1:7.5-30 g/mL.
9. The method for synthesizing self-scintillating fluorescent dye for super-resolution fluorescence imaging according to claim 2, characterized in that in the step (7):
the mass-to-volume ratio of the intermediate 6 to the isobutyl lithium is 1:1.36-5.4 g/mL;
the mass-to-volume ratio of the intermediate 6 to the dimethylcarbamoyl chloride is 1:0.12-0.48 g/mL;
the mass-to-volume ratio of the intermediate 6 to tetrahydrofuran is 1:18-70 g/mL.
10. The method for synthesizing self-scintillating fluorescent dye for super-resolution fluorescence imaging according to claim 2, characterized in that in the step (8):
the mass-to-volume ratio of the intermediate 3 to the isobutyl lithium is 1:0.85-3.4 g/mL;
the mass ratio of the intermediate 3 to the intermediate 7 is 1: 0.07-0.30;
the mass-to-volume ratio of the intermediate 3 to tetrahydrofuran is 1:18-72 g/mL.
11. The method for synthesizing self-scintillating fluorescent dye for super-resolution fluorescence imaging according to claim 2, characterized in that in the step (9):
the mass-to-volume ratio of the intermediate 8 to the trifluoroacetic acid is 1:74-300 g/mL.
12. The method for synthesizing self-scintillating fluorescent dye for super-resolution fluorescence imaging according to claim 2, characterized in that in the step (10):
the mass ratio of the intermediate 9 to the N, N-disuccinimidyl carbonate is 1: 0.8-3.2;
the mass ratio of the intermediate 9 to the 4-dimethylaminopyridine is 1: 0.1-0.3;
the mass-to-volume ratio of the intermediate 9 to triethylamine is 1:0.6-2.4 g/mL;
the mass-to-volume ratio of the intermediate 9 to DMF was 1:147-600 g/mL.
13. Use of the self-blinking fluorescent dye for super-resolution fluorescence imaging according to claim 1 in the field of fluorescence imaging in cells, tissues and living bodies.
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