CN111334068A - Self-flashing super-resolution fluorescent dye based on SNAP-tag technology and synthesis and application thereof - Google Patents

Self-flashing super-resolution fluorescent dye based on SNAP-tag technology and synthesis and application thereof Download PDF

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CN111334068A
CN111334068A CN201811550693.6A CN201811550693A CN111334068A CN 111334068 A CN111334068 A CN 111334068A CN 201811550693 A CN201811550693 A CN 201811550693A CN 111334068 A CN111334068 A CN 111334068A
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CN111334068B (en
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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 super-resolution fluorescent dye based on SNAP-tag technology, and synthesis and application thereof, wherein the fluorescent dye has a structure (1) shown in the specification, and has the characteristics of high light stability, high quantum yield and the like by introducing azetidine to inhibit intramolecular rotation of silarhodamine. And due to the thermodynamic equilibrium of the intramolecular spiral of the dye molecule, the fluorescent dye can continuously flash, and is further applied to super-resolution fluorescence imaging. In addition, the probe can specifically mark a plurality of target proteins fused with SNAP-tag labels in living cells, so that the target proteins in the living cells can be traced. The SNAP-tag self-flashing super-resolution fluorescent dye disclosed by the invention has wide application in the fields of protein marking, protein-protein interaction, super-resolution fluorescence imaging and the like.

Description

Self-flashing super-resolution fluorescent dye based on SNAP-tag technology and synthesis and application thereof
Technical Field
The invention belongs to the technical field of fluorescent dyes, and particularly relates to a self-flashing super-resolution fluorescent dye based on an SNAP-tag technology, and synthesis and application thereof.
Background
The protein labeling technology combines genetic coding characteristics of proteins and excellent photophysical characteristics of small molecules, and has wide application in researching functions of the proteins and interaction between the proteins. The fluorescence microscopic imaging technology is used as a main tool for in-situ and nondestructive tracking of dynamic life activities, and the resolution cannot break through the limit of diffraction limit for a long time. In recent years, the resolution of fluorescence microscopic imaging is improved to 20nm by the super-resolution imaging technology, and new opportunities are brought to related researches on proteins. Researchers can extract information from finer structures such as protein-protein interactions, protein distribution, etc.
Currently, when a single-molecule positioning microscope is used for super-resolution image construction of a fluorescence-labeled biomolecule, strong laser irradiation and addition of additives such as thiol are generally required to induce the fluorescence group to be switched on and off. However, strong laser irradiation can cause serious cell damage, and the added thiol can also have great influence on cell structures, and the conditions limit the potential application of the super-resolution technology. The self-flashing fluorescent dye has no color and fluorescence in a ground state due to thermodynamic equilibrium of a spiro ring in a molecule, and can continuously flash through an intramolecular switch. Therefore, under near-physiological conditions, fluorophores capable of spontaneous blinking without the need for high power laser irradiation or any additives are of paramount importance for living cell super-resolution imaging.
Disclosure of Invention
The invention provides a self-flashing super-resolution fluorescent dye based on an SNAP-tag technology, and synthesis and application thereof, and relates to a high-brightness SNAP-tag self-flashing fluorescent dye which can specifically identify SNAP-tag protein and carry out super-resolution imaging.
A high brightness self-blinking fluorescent dye with SNAP-tag, the fluorescent dye having the following structure:
Figure BDA0001910561010000021
the invention provides a high-brightness SNAP-tag-used self-flashing fluorescent dye, which has high light stability and high quantum yield, and the fluorescence quantum yield reaches 0.8 (in water).
The invention provides a high-brightness self-flashing fluorescent dye for SNAP-tag protein, which has a self-flashing function and can reduce the laser energy (40W/cm) of application of dSTORM2)。
A synthesis method of self-flashing fluorescent dye with SNAP-tag with high brightness comprises the following steps:
Figure BDA0001910561010000022
the specific synthesis steps are as follows:
(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 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.
In the step (11):
the mass-to-volume ratio of the intermediate 10 to the N, N-diisopropylethylamine is 1: 1-2;
the mass ratio of the intermediate 10 to the intermediate 11 is 1: 0.33-1.2;
the mass-to-volume ratio of the intermediate 10 to DMF was 1:250-500 g/mL.
The synthesis method of the high-brightness self-flashing fluorescent dye for the SNAP-tag protein has the advantages of convenience in operation, low cost and the like.
The application of a high-brightness self-flashing super-resolution fluorescent dye for marking SNAP-tag protein in the field of fluorescence imaging of cells, tissues and living bodies.
The dyes of the present invention have the following characteristics:
the dye has the advantages of low cost of synthetic raw materials, simple method and the like.
The dye can specifically recognize SNAP-tag protein and perform super-resolution imaging.
The dye has high light stability and high quantum yield, and the fluorescence quantum yield reaches 0.8 (in water).
The dye has self-flashing function, and can reduce the laser energy (40W/cm) of dSTORM application2)。
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.
Figure 8 nuclear magnetic hydrogen spectrum of intermediate 10 prepared in example 1.
FIG. 9 NMR spectrum of BG-SiRho dye prepared in example 1.
FIG. 10 is a graph showing UV absorption spectra of BG-SiRho dye prepared in example 1 at various pH values (pH 2-12), wavelength on the abscissa, absorbance on the ordinate, and fluorescent dye concentration of 5. mu.M.
FIG. 11 shows fluorescence emission spectra of BG-SiRho dye prepared in example 1 at different pH values (pH 2-12), with wavelength on the abscissa, fluorescence intensity on the ordinate, and fluorescent dye concentration of 5 μ M.
FIG. 12 super-resolution imaging of SNAP-tag protein transfected into nuclei by the dye BG-SiRho prepared in example 1.
FIG. 13 super-resolution imaging of intracellular mitochondria after transfection of SNAP-tag protein by the dye BG-SiRho prepared in example 1.
Detailed Description
Example 1
Synthesis of intermediate 1
Figure BDA0001910561010000091
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 BDA0001910561010000092
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 BDA0001910561010000093
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 BDA0001910561010000101
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. Cooling to room temperature after the reaction is finished, adding 10mL of saturated ammonium chloride solution, extracting with ethyl acetate, collecting the organic phase, drying with anhydrous sodium sulfate, distilling the organic phase under reduced pressure, passing the reaction product through a silica gel column (200 meshes and 300 meshes) with petroleum ether andethyl acetate (2:1) was isolated and purified as a developing solvent to obtain 500mg of a colorless oily liquid in 50% yield. 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 BDA0001910561010000102
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 BDA0001910561010000111
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 BDA0001910561010000121
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 BDA0001910561010000122
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 BDA0001910561010000131
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 intermediate 10
Figure BDA0001910561010000141
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 white powdery target 10m g was obtained by separation and purification through a silica gel column (200-300 mesh) using petroleum ether and ethyl acetate (10:1) as developing agents, with a yield of 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).
synthesis of dye BG-SiRho
Figure BDA0001910561010000142
10(10mg, 0.018mmol), N-diisopropylethylamine (20. mu.L), 11(6mg, 0.02mmol) were dissolved in 5mL of DMF and stirred at room temperature for 12 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 methylene chloride and methanol (10:1) as developing agents to give 10mg of a target white powder in a yield of 80%. The nuclear magnetic spectrum of the dye BG-SiRho prepared in example 1 is shown in FIG. 9, and the specific hydrogen spectrum data are as follows:
1H NMR(400MHz,MeOD)δ7.83(s,2H),7.66(d,J＝8.0Hz,1H),7.49(d,J＝8.1Hz,2H),7.36(d,J＝8.0Hz,2H),7.00(d,J＝8.7Hz,2H),6.88(d,J＝7.9Hz,1H),6.68(d,J＝2.6Hz,2H),6.40(dd,J＝8.7,2.6Hz,2H),5.53(s,2H),5.39(s,2H),4.57(s,3H),3.85(t,J＝7.2Hz,8H),2.39–2.29(m,4H),0.56(s,3H),0.45(s,3H).
through detection, the structure of the dye is shown as the formula BG-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 2
Synthesis of intermediate 1
Figure BDA0001910561010000151
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 BDA0001910561010000161
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 BDA0001910561010000162
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 BDA0001910561010000163
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 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 250mg of a colorless oily liquid with a yield of 50%.
Synthesis of intermediate 5
Figure BDA0001910561010000171
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 BDA0001910561010000172
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 BDA0001910561010000181
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 BDA0001910561010000182
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 BDA0001910561010000191
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 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 62%.
Synthesis of intermediate 10
Figure BDA0001910561010000192
9(17mg, 0.034mmol), 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 and a yield of 52%.
Synthesis of dye BG-SiRho
Figure BDA0001910561010000201
10(10mg, 0.018mmol), N-diisopropylethylamine (10. mu.L), 11(3mg, 0.01mmol) were dissolved in 5mL of DMF and stirred at room temperature for 12 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 methylene chloride and methanol (10:1) as developing agents to give the desired product as a white powder in an amount of 5mg in a yield of 80%.
Through detection, the structure of the dye is shown as the formula BG-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 BDA0001910561010000202
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 BDA0001910561010000211
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 BDA0001910561010000212
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 BDA0001910561010000213
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 BDA0001910561010000221
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 BDA0001910561010000222
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 BDA0001910561010000231
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 BDA0001910561010000232
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 BDA0001910561010000241
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 with a silica gel column (200-300 mesh) using methylene chloride and methanol (10:1) as developing agents, in a yield of 60％。
Synthesis of intermediate 10
Figure BDA0001910561010000242
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%.
Synthesis of dye BG-SiRho
Figure BDA0001910561010000251
10(5mg, 0.009mmol), N-diisopropylethylamine (20. mu.L), 11(6mg, 0.02mmol) were dissolved in 5mL of DMF and stirred at room temperature for 16 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 methylene chloride and methanol (10:1) as developing agents to give the desired product as a white powder in an amount of 5mg in a yield of 80%.
The structure of the compound is shown as BG-SiRho through detection.
BG-SiRho is dissolved in DMSO solution to prepare 2mM mother solution, and test solutions with different concentrations are prepared according to requirements to detect ultraviolet absorption, fluorescence spectrum change and intracellular fluorescence imaging.
BG-SiRho uv absorption spectra at different pH (pH 2-8). Dissolving 400 mu L of BG-SiRho mother liquor in 80mL of water, adjusting the pH value by using sodium hydroxide solution and hydrochloric acid solution, taking out 4mL of test solution after the band is stable, and carrying out ultraviolet absorption spectrum test.
Uv absorption at a final BG-SiRho concentration of 2 μ M at various phs (pH 2-8) as shown in figure 10, the spectrum is greatly altered, with lower pH absorbing more strongly at 650nm, indicating that the probe remains mostly dark under normal physiological conditions (pH 7.0-7.4).
Fluorescence emission spectroscopy of BG-SiRho at various pH (pH 2-8). Dissolving 400 mu L of BG-SiRho mother liquor in 80mL of water, adjusting the pH value by using sodium hydroxide solution and hydrochloric acid solution, taking out 4mL of test solution after the band is stable, and carrying out fluorescence spectrum test.
The fluorescence of BG-SiRho at a final concentration of 2 μ M at different pH values is shown in FIG. 11, which shows that the fluorescence spectrum is greatly changed, the fluorescence is weak in neutral and alkaline environments, and the fluorescence is enhanced in acidic conditions, indicating that most of the probes remain in a non-fluorescent dark state under physiological conditions.
Example 4
The expression of SNAP-tag protein in Hela cell nucleus is induced by plasmids. And then adding BG-SiRho to incubate for 20 minutes, and performing a living cell super-resolution fluorescence imaging experiment.
A fluorescence imaging graph is shown in figure 12 after the Hela cells are incubated by cell culture solution with the probe BG-SiRho final concentration of 1 mu M for 10 minutes, the BG-SiRho fluorescent dye has a good marking effect on SNAP-tag protein of cell nuclei, and has a good super-resolution imaging effect, and strong laser quenching fluorescent molecules are not needed in the imaging process.
Example 5
The SNAP-tag protein is expressed in mitochondria of Hela cells through plasmid induction. And then adding BG-SiRho to incubate for 20 minutes, and performing a living cell super-resolution fluorescence imaging experiment.
After the Hela cells are incubated by the cell culture solution with the probe BG-SiRho final concentration of 1 mu M for 10 minutes, fluorescence imaging is shown in figure 8, and the BG-SiRho fluorescent dye in figure 13 has a good marking effect on SNAP-tag protein of mitochondria and a good super-resolution imaging effect.

Claims (14)

1. A self-flashing super-resolution fluorescent dye based on SNAP-tag technology is characterized in that the self-flashing fluorescent dye has the following structure:
Figure FDA0001910561000000011
2. the method for synthesizing SNAP-tag self-blinking super-resolution fluorescent dye with high brightness according to claim 1, comprising the following steps:
(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 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 200-300-mesh silica gel column by using dichloromethane and methanol as eluent in the volume ratio of 10: 1.
3. The method for synthesizing a high-brightness SNAP-tag self-flashing super-resolution fluorescent dye 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 a high-brightness SNAP-tag self-flashing super-resolution fluorescent dye 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 a high-brightness SNAP-tag self-flashing super-resolution fluorescent dye according to 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 a high-brightness SNAP-tag self-flashing super-resolution fluorescent dye 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 a high-brightness SNAP-tag self-flashing super-resolution fluorescent dye according to claim 2, wherein 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 a high-brightness SNAP-tag self-flashing super-resolution fluorescent dye according to 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 a high-brightness SNAP-tag self-blinking super-resolution fluorescent dye according to claim 2, wherein 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.
10. The method for synthesizing a high-brightness SNAP-tag self-blinking super-resolution fluorescent dye according to claim 2, wherein 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 a high-brightness SNAP-tag self-blinking super-resolution fluorescent dye according to claim 2, wherein 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 a high-brightness SNAP-tag self-blinking super-resolution fluorescent dye according to claim 2, wherein 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. The method for synthesizing a high-brightness SNAP-tag self-blinking super-resolution fluorescent dye according to claim 2, wherein in the step (11):
the mass-to-volume ratio of the intermediate 10 to the N, N-diisopropylethylamine is 1: 1-2;
the mass ratio of the intermediate 10 to the intermediate 11 is 1: 0.33-1.2;
the mass-to-volume ratio of the intermediate 10 to DMF was 1:250-500 g/mL.
14. Use of a high brightness self-blinking super-resolution fluorochrome as defined in claim 1 for SNAP-tag protein labeling in the field of fluorescence imaging of cells, tissues and living bodies.
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