Bifunctional fluorescent probe ER-Pro based on BODIPY mother nucleus as well as preparation method and application of bifunctional fluorescent probe ER-Pro

Abstract

The invention discloses a bifunctional fluorescent probe ER-Pro based on a BODIPY mother nucleus as well as a preparation method and application of the bifunctional fluorescent probe ER-Pro. The synthesized probe ER-Pro keeps high-sensitivity fluorescence response to the endoplasmic reticulum, the preparation method is simple and efficient, and through the optimized synthesis method, the BODIPY mother nucleus is conjugated to a key element targeted by the endoplasmic reticulum, and the targeting efficiency of the endoplasmic reticulum and GRP78 protein of the endoplasmic reticulum is improved. The fluorescent probe provided by the invention can be used for simultaneously observing the distribution state of endoplasmic reticulum protein GRP78 and the liquid-liquid phase separation process of the endoplasmic reticulum protein GRP78 in real time, provides effective evidence for visual detection of endoplasmic reticulum stress, and has great potential in biomedical research due to efficient targeting and excellent optical performance; the method is expected to become a powerful tool for studying the endoplasmic reticulum function and the disease related mechanism, and particularly has wide application prospects in the fields of cytobiology, pathology, drug screening and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent compounds and their preparation and application, and particularly relates to a bifunctional fluorescent probe ER-Pro based on a boron-dipyrromethene core, and its preparation method and application. Background Art

[0002] The endoplasmic reticulum (ER) is the main organelle for synthesizing and processing cellular proteins in mammals and plays an important role in maintaining cellular homeostasis. When ER function is abnormally stressed, the unfolded protein response (UPR) of the endoplasmic reticulum is triggered, and this mechanism is activated to clear unfolded or misfolded proteins in the cell to restore ER homeostasis. However, long-term and excessive ER stress responses may lead to serious diseases, such as cardiometabolic diseases including atherosclerosis, diabetes, and liver injury, as well as neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease. Glucose regulatory protein 78 (GRP78, also known as BiP) is a multifunctional protein and also a marker protein for endoplasmic reticulum stress. The expression level of GRP78 in cells can largely represent the endoplasmic reticulum stress level in cells, and upregulation of GRP78 expression can promote the correct folding of proteins. Based on the above characteristics, many fluorescence imaging tools are used to target the endoplasmic reticulum to achieve real-time observation of the endoplasmic reticulum, thereby facilitating a better understanding of the pathogenesis of diseases. Therefore, it is very necessary to conduct visual research on the distribution status of the endoplasmic reticulum and its marker proteins, which is of great significance for the monitoring and diagnosis of neurodegenerative diseases. This technology takes the endoplasmic reticulum as the research object, uses a fluorescent probe technology, and uses a bio-inspired chromophore as a fluorescence imaging tool to perform fluorescence imaging in living cells to achieve the purpose of visually identifying the endoplasmic reticulum and the endoplasmic reticulum protein GRP78, filling the gap in the rapid detection of the distribution status of the endoplasmic reticulum and its marker proteins by fluorescence methods and is expected to be used as an effective drug screening platform.

[0003] Fluorinated boron dipyrrole fluorescent chromophores (BODIPY) The main characteristics of these dyes include their strong and bright colors, high fluorescence quantum yields, large molar absorption coefficients, high sensitivity, good photostability, and fluorescence emission in a wide wavelength range, etc., which are very ideal fluorescent probe chromophores for biological imaging. At present, researchers have developed a variety of methods for chemically synthesizing BODIPY and its analogs, and their applications in the fields of fluorescence imaging, biosensing, optical devices, etc. have attracted more and more attention.

[0004] However, how to modify BODIPY and at which position the modification can obtain the target detection marker protein still requires further in-depth research. Moreover, since the yield of each step of the reaction directly affects the total yield of the final product, in multi-step synthesis, low-yield steps will significantly increase the material cost and synthesis complexity. At the same time, the final step of the synthesis requires an efficient purification method to ensure the purity and activity of the probe, and impurities or unreacted starting materials may interfere with the biological activity test of the probe. Summary of the Invention

[0005] Object of the Invention: Through rich functional modifications of the BODIPY fluorophore, the present invention obtains a new bifunctional fluorescent probe ER-Pro. The probe enables their absorption and emission to be even redshifted to the red / near-infrared (NIR) region. The conjugated endoplasmic reticulum recognition site can not only efficiently target the endoplasmic reticulum, but also target the distribution state and phase separation process of the GRP78 marker protein, realizing the visualization detection of endoplasmic reticulum stress in real time, and greatly improving the targeting efficiency.

[0006] The technical problem to be solved by the present invention is to provide a simple and efficient preparation method of a bifunctional fluorescent probe ER-Pro based on the BODIPY core.

[0007] The last technical problem to be solved by the present invention is to provide the application of the bifunctional fluorescent probe ER-Pro in targeting the endoplasmic reticulum and / or fluorescence imaging. The probe can realize the observation of the liquid-liquid phase separation phenomenon of GRP78 protein in vitro and in cells.

[0008] Technical Solution: To solve the above technical problems, the present invention provides a bifunctional fluorescent probe ER-Pro based on the BODIPY core, and its structural formula is as follows:

[0009]

[0010] The probe ER-Pro of the present invention is a green fluorescent probe, which can not only be used to target the endoplasmic reticulum and its marker protein GRP78, but also realize the visualization imaging of the liquid-liquid phase separation of GRP78 protein in vitro and in cells.

[0011] The present invention also includes the preparation method of the bifunctional fluorescent probe ER-Pro based on the BODIPY core. The method includes: modifying p-carboxybenzaldehyde on the BODIPY core, condensing it with p-toluenesulfonamide, stirring the reaction mixture overnight, collecting the organic part, and purifying to obtain the probe ER-Pro.

[0012] The ER-Pro of the present invention is synthesized by a two-step method of conjugating p-toluenesulfonamide with the BODIPY core.

[0013] Specifically, it includes the following steps: Add compound 3, N-(2-aminoethyl)-4-methylbenzenesulfonamide, HOBt·H2O, triethylamine, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride into DMF, stir the reaction mixture overnight, and obtain the probe molecule ER-Pro after purification; the structural formula of the compound 3 is:

[0014]

[0015] Among them, the preparation method of the compound 3 includes the following steps: Dissolve p-carboxybenzaldehyde in dichloromethane, add 2,4-dimethylpyrrole and the catalyst trifluoroacetic acid, stir the reaction mixture overnight at room temperature, after the aldehyde is completely consumed, add tetrachlorobenzoquinone to the reaction mixture, stir and then add triethylamine and boron trifluoride diethyl ether, stir the obtained mixture at room temperature again, collect the organic phase, and purify to obtain a purplish-red solid, which is the intermediate compound 3.

[0016] Among them, the mass ratio of p-carboxybenzaldehyde, 2,4-dimethylpyrrole, and tetrachlorobenzoquinone is (0.8 - 1.2):(0.9 - 1.7):(1.2 - 2.0), and the volume ratio of dichloromethane, triethylamine, and boron trifluoride diethyl ether is (2.6 - 3.4):(0.8 - 1.2):(0.8 - 1.2).

[0017] Preferably, the mass ratio of p-carboxybenzaldehyde, 2,4-dimethylpyrrole, and tetrachlorobenzoquinone is 1:1.3:1.6, and the volume ratio of dichloromethane, triethylamine, and boron trifluoride diethyl ether is 3:1:1.

[0018] Among them, the molar ratio of the compound 3, N-(2-aminoethyl)-4-methylbenzenesulfonamide, HOBt . H2O, triethylamine, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is (14.6 - 15.4):(11.96 - 12.04):(6.6 - 7.4):(9.56 - 10.44); the volume ratio of N,N-dimethylformamide to triethylamine is (27.1 - 27.9):1.

[0019] Preferably, the mass ratio of the compound 3, N-(2-aminoethyl)-4-methylbenzenesulfonamide, HOBt . H2O, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 15:12:7:10, and the volume ratio of N,N-dimethylformamide to triethylamine is 27.5:1.

[0020] The content of the present invention also includes the application of the bifunctional fluorescent probe ER-Pro in targeting the endoplasmic reticulum and / or fluorescence imaging.

[0021] Among them, the targeting endoplasmic reticulum includes the localization of the endoplasmic reticulum marker protein GRP78.

[0022] Among them, the fluorescence imaging is co-localization fluorescence imaging.

[0023] Among them, the application includes observing the liquid-liquid phase separation phenomenon of GRP78 protein in vitro or in cells.

[0024] Specifically, in the endoplasmic reticulum stress model induced by thapsigargin, it was detected that the GRP78 protein underwent liquid-liquid phase separation, and the phenomena of droplet fusion and fission occurred.

[0025] Reaction mechanism: Compound 1 and Compound 2 of the present invention introduce p-carboxybenzoic acid at the 8th position of the BODIPY core through steps such as dehydration and cyclization to obtain Compound 3. By activating the carboxyl group and amidation reaction, an endoplasmic reticulum potent localization group - p-toluenesulfonamide group is introduced onto Compound 3 to form the final ER-Pro probe.

[0026] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0027] 1. The compound probe ER-Pro of the present invention has excellent fluorescence properties. It can not only be used to target the endoplasmic reticulum and its marker protein GRP78, but also can realize the visualization imaging of the liquid-liquid phase separation of GRP78 protein in vitro and in cells. The probe ER-Pro synthesized by the present invention maintains a high-sensitivity fluorescence response to the endoplasmic reticulum, has low cytotoxicity, a high fluorescence quantum yield, a large molar absorption coefficient, a high signal-to-noise ratio, good biocompatibility, stable fluorescence properties, strong anti-background interference ability, and highly consistent localization to the endoplasmic reticulum compared with the commercial ER-Tracker Red. These excellent properties make it show extremely important application value in the fields of live cell imaging, fluorescence sensors, and biological fluorescence labeling, biosensors, drug research and development, etc.

[0028] 2. The synthesis method disclosed by the present invention is simple and efficient, which is beneficial to subsequent research and synthesis work; through the optimized synthesis method, the key elements of the BODIPY parent nucleus conjugated to the endoplasmic reticulum targeting are improved, the targeting efficiency of the endoplasmic reticulum and its GRP78 protein is increased, and a multifunctional fluorescence sensor ER-Pro is designed and synthesized.

[0029] 3. The fluorescent probe provided by the present invention expands the application scope of BODIPY-based probes, provides a simple fluorescent tool for the technical platform of endoplasmic reticulum localization, and simultaneously observes the distribution state of the endoplasmic reticulum protein GRP78 and its liquid-liquid phase separation process in real time, providing effective evidence for the visual detection of endoplasmic reticulum stress. Its high targeting efficiency and excellent optical properties make it have great potential in biomedical research and are expected to become a powerful tool for studying the functions of the endoplasmic reticulum and its related mechanisms with diseases, especially in the fields of cell biology, pathology, and drug screening, with broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Synthesis route diagram of the endoplasmic reticulum-targeted probe ER-Pro based on the BODIPY fluorophore.

[0031] Figure 2 Co-localization fluorescence imaging diagram of the probe ER-Pro and the commercial endoplasmic reticulum probe ER-Tracker Red.

[0032] Figure 3 Co-localization fluorescence imaging diagram of the probe ER-Pro and the endoplasmic reticulum marker protein GRP78.

[0033] Figure 4 Diagram of the liquid-liquid phase separation of the GRP78 protein induced by endoplasmic reticulum stress in HEK293T cells detected by the probe ER-Pro.

[0034] Figure 5 Diagram of the liquid-liquid phase separation of the GRP78 protein induced by TG in vitro detected by the probe ER-Pro.

[0035] Figure 6 Structure diagram and co-localization fluorescence imaging diagram of the probe ER-Pro of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0036] The present invention will be further described in detail below.

[0037] Example 1 Preparation of the probe molecule ER-Pro

[0038] 1. Synthesis of intermediates

[0039] Dissolve 1 g of 4-carboxybenzaldehyde in 30 mL of dichloromethane, then add 1.3 g of 2,4-dimethylpyrrole and 20 μL of the catalyst trifluoroacetic acid to a round-bottom flask, and stir the reaction mixture overnight at room temperature. After the aldehyde is completely consumed, monitored by thin-layer chromatography, add 1.6 g of tetrachlorobenzoquinone to the reaction mixture and stir for 5 hours. Add 10 mL of triethylamine, then add 10 mL of boron trifluoride diethyl etherate, and stir the resulting mixture at room temperature for another 8 hours. Terminate the reaction with water and extract with dichloromethane. Collect the organic phase, dry it over anhydrous Na2SO4, and evaporate the organic solvent under reduced pressure. The crude product is further purified by silica gel column chromatography (petroleum ether:dichloromethane:methanol = 3:3:1) to obtain a purple-red solid, which is the intermediate compound 3. The characterization data of this intermediate compound 3 are as follows:

[0040] 1 H NMR (400 MHz, Chloroform-d) δ 7.11 (d, J = 8.4 Hz, 2H), 6.94 (d, J = 8.4 Hz, 2H), 5.98 (s, 2H), 2.55 (s, 6H), 1.44 (s, 6H).

[0041] 13 C NMR (101 MHz, Chloroform-d) δ 142.60, 140.06, 138.69, 132.91, 129.36, 118.12, 113.26, 46.86, 14.65, 8.70.

[0042] 2. Synthesis of the target compound probe ER-Pro

[0043] Add 150 mg of intermediate compound 3, 115 mg of N-(2-aminoethyl)-4-methylbenzenesulfonamide (Leyan reagent https: / / www.leyan.com / , product number 1021362-5g), 68 mg of 1-hydroxybenzotriazole monohydrate (HOBt . H2O) (aladdin, product number H106176-25g), 182 μL of triethylamine (TEA) and 96 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) (CAS registration number: 25952-53-8) to 5 mL of N,N-dimethylformamide (DMF). Mix and stir overnight, then quench with water, extract with dichloromethane, collect the organic part, dry it over anhydrous Na2SO4, and evaporate the organic solvent under reduced pressure. Further purify the compound by silica gel column chromatography (petroleum ether:ethyl acetate = 1:1) to obtain the probe orange-red solid ER-Pro.

[0044] The synthetic route diagram is as Figure 1 shown.

[0045] 3. Analysis and Characterization of Probe ER-Pro:

[0046] 1H Nuclear Magnetic Resonance 1 H NMR(400MHz,Chloroform-d)δ8.09(s,1H),7.97(d,J=7.8Hz,2H),7.76(d,J=7.9Hz,3H),7.51(d,J=8.5Hz,1H),7.44(s,1H),7.34(d,J=8.0Hz,2H),7.29(d,J=7.8Hz,2H),5.97(s,1H),5.80(s,1H),4.21(s,1H),3.65–3.57(m,2H),3.22(s,2H),2.55(s,3H),2.40(s,3H),1.32(s,6H).

[0047] 13C Nuclear Magnetic Resonance 13 C NMR(101MHz,CDCl3)δ167.44(s),155.95(s),143.88(s),142.95(s),138.61(s),136.48(s),134.27(s),129.92(s),128.51(s),128.03(s),127.06(s),121.49(s),42.73(s),40.41(s),38.73(s),34.88(s),34.54(s),31.44(s),30.28(d,J=16.6Hz),29.71(s),28.94(s),23.75(s),23.00(s),21.55(s),14.61(s),14.08(s),10.98(s),1.04(s).

[0048] High Resolution Mass Spectrometry HRMS: Calculated:[M+H] + =564.22,Obsd,565.22

[0049] The UV absorption spectrum of ER-Pro was recorded at 25 °C using a UV spectrophotometer (Agilent, Cary5000). The results are shown in Table 1, and the maximum absorption wavelength λ abs ; its excitation and emission spectra were recorded using a fluorescence and phosphorescence spectrophotometer (Hitachi, F-7100) to obtain the maximum excitation wavelength λ ex , the maximum emission wavelength λ em ; the Stokes shift Stoke’s shift is Δλ = λ em –λ abs; ε is the molar extinction coefficient; Φ is the fluorescence quantum yield, which is the quantum yield determined relative to rhodamine 6G (Φ = 0.91 in ethanol); Brightness = Φ·ε.

[0050] Table 1 Spectral properties of ER-Pro

[0051]

[0052] Example 2 Co-localization fluorescence imaging of probe ER-Pro and commercial endoplasmic reticulum probe ER-Tracker Red

[0053] In this experiment, cell imaging of probe ER-Pro was performed. The cells used in the experiment were human embryonic kidney cells HEK293T. ER-Pro with a final concentration of 2 μM was co-incubated with 2 μM commercial endoplasmic reticulum dye ER-Tracker Red (Beyotime Biotechnology, catalog number C1041S-1) in 1 mL of HEK293T cells at a density of 3×10 6 cells / mL for 30 min, and the excess probe was rinsed off. Before imaging, the nucleus dye Hoechst 33342 with a final concentration of 2 μM (Beyotime Biotechnology, catalog number C1028) was used for 10 min, and the excess probe and dye were rinsed off. Fluorescence imaging was performed using a Zeiss LSM980 laser confocal microscope. The experimental results showed that probe ER-Pro maintained bright green fluorescence in the endoplasmic reticulum region, ER-Tracker Red maintained red fluorescence in the endoplasmic reticulum region, ER-Pro and ER-Tracker Red significantly overlapped in the cells, and the co-localization coefficient was as high as 90%, proving that this probe could effectively target the endoplasmic reticulum and the reticular structure of the endoplasmic reticulum could be successfully observed. These experimental results fully demonstrated the excellent performance of ER-Pro in endoplasmic reticulum targeting.

[0054] Example 3 Co-localization fluorescence imaging of probe ER-Pro and endoplasmic reticulum marker protein GRP78

[0055] In this experiment, a commercial Human HSPA5 gene ORF cDNA cloning and expression plasmid (which contains the gene encoding the expression of GRP78 protein, Sino Biological, catalog number HG12063-ACR) was used, and Harvest TM 293F expression system (Shanghai OPM Biotech Co., Ltd., catalog number AC601500) was used to express GRP78 protein. Expi293F cells were transferred to a confocal culture dish for cultivation, 2 mL of 293F Hi-exp medium was added, and after mixing evenly, it was placed in a carbon dioxide incubator at 37 °C to adhere to the wall. When cultured to 3×10 6cells / mL, transfect the Human HSPA5 gene ORF cDNA cloning and expression plasmid (1 μg plasmid is transfected per 1 mL of cells), and add 4 μL of OPM-TR01 transfection reagent; the plasmid and the transfection reagent are respectively diluted with 293F Hi-exp medium at 5% of the transfection system volume). Slowly drip the diluted transfection reagent into the diluted plasmid, mix well, and incubate statically for 15 min; slowly add the incubated plasmid-transfection reagent complex to the Expi293F cells to be transfected, gently shake the flask while adding, and then culture in a carbon dioxide incubator at 37 °C for 24 hours. Pour out the transfection solution and wash twice, centrifuge to collect the cells, and place them in an ultrasonic cell disruptor for cell disruption; collect the supernatant of the disrupted solution, filter through a 0.22 μm filter membrane, and use an AKTA protein purification system (Cytiva, AKTAPure 25M1) for protein purification to obtain the purified protein GRP78.

[0056] Incubate the DMSO solution of ER-Pro with a final concentration of 60 μM and the Tris-NaCl solution (pH 8.0) of the purified GRP78 protein with a final concentration of 6 μM at room temperature for 24 h, and add thapsigargin (TG) with a final concentration of 20 μM as an inducer to induce protein aggregation. Place 5 μL of the protein-containing solution on a 24 x 60 mm glass slide, and image the aggregated protein. See the experimental results in Figure 3 , Figure 3 . The results show that the probe ER-Pro maintains bright green fluorescence and has obvious overlap in the GRP78 protein aggregation region, proving that this probe can effectively target the endoplasmic reticulum marker protein GRP78.

[0057] Example 4 Detection of liquid-liquid phase separation of GRP78 protein induced by endoplasmic reticulum stress in U-2OS cells by probe ER-Pro

[0058] In this invention, fluorescence recovery after photobleaching (FRAP) experiments were carried out intracellularly using the probe ER-Pro and long-term dynamic imaging was performed using a CD7 high-throughput live cell imaging system (Zeiss, Celldiscoverer 7). The cells used in the experiment were human osteosarcoma U-2OS cells (Wuhan Puresee Life Science Co., Ltd., product number CL-0236). The DMSO solution of ER-Pro with a final concentration of 2 μM and 1 mL with a density of 3×10 6cells / mL cells were co-incubated for 30 min and then treated with TG at a final concentration of 20 μM for 10 h to induce droplet formation. FRAP experiments were imaged using an LSM980 laser confocal microscope, and probe-labeled droplets were bleached at 488 nm using 90% laser intensity in selected areas during the experiment. The recovery rate was recorded within the specified time, and the fluorescence intensity was normalized to the intensity before photobleaching.

[0059] 1mL density is 3×10 6 U-2OS cells / mL were incubated with DMSO solution of ER-Pro at a final concentration of 2 μM for 30 min and treated with TG solution at a final concentration of 20 μM for 2 h. Long-term dynamic imaging was performed using the CD7 high-throughput live cell imaging system.

[0060] like Figure 4 The results of the FRAP experiment showed that the fluorescence intensity dropped to the minimum at about 11 seconds, and then gradually recovered to near the initial level over time. At the same time, the fusion and fission of intracellular droplets were successfully observed through the CD7 high-throughput live cell imaging system, proving that this probe can effectively observe the liquid-liquid phase separation phenomenon of intracellular GRP78 protein.

[0061] Example 5 Detection of GRP78 protein production induced by TG in vitro by probe ER-Pro Liquid-Liquid Phase Separation

[0062] The present invention uses the probe ER-Pro to carry out fluorescence recovery after photobleaching (FRAP) experiments in vitro. A DMSO solution with a final concentration of 60 μM ER-Pro was mixed with 6 μM Harvest TM The GRP78 protein expressed by the 293F expression system was incubated with a Tris-NaCl solution (pH 8.0) for 24 h and then treated with 20 μM TG for 6 h to induce droplet formation. The experiment was performed using the built-in FRAP module of a Nikon AX confocal microscope equipped with a 100X oil immersion objective. The probe-labeled protein was bleached at 488 nm for 20 s using a laser intensity of 70%. The recovery rate within the specified time was recorded, and the fluorescence intensity was normalized to the intensity before photobleaching. The experimental results are shown in Figure 2. Figure 5 The results showed that the fluorescence intensity of the droplets decreased after strong photobleaching, but over time, the fluorescence intensity of the bleached area gradually recovered to near the initial level. The fusion and fission of the droplets were observed in vitro, proving that this probe can effectively observe the liquid-liquid phase separation phenomenon of GRP78 protein in vitro.

Claims

1. A bifunctional fluorescent probe ER-Pro based on a fluoroborane dipyrrole core, characterized in that: Its structural formula is as follows:

2. The method for preparing the bifunctional fluorescent probe ER-Pro based on fluoroborane dipyrrole core according to claim 1, characterized in that: The method comprises: modifying p-carboxybenzaldehyde on the BODIPY mother core, and condensing it with p-toluenesulfonamide, stirring the reaction mixture overnight, collecting the organic part, and purifying to obtain the probe ER-Pro.

3. The method for preparing the bifunctional fluorescent probe ER-Pro based on fluoroborane dipyrrole core according to claim 2, characterized in that: The specific steps include: Compound 3, N-(2-aminoethyl)-4-methylbenzenesulfonamide, HOBt·H2O, triethylamine and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were added to DMF, and the reaction mixture was stirred overnight. After purification, the probe molecule ER-Pro was obtained; the structural formula of the compound 3 is:

4. The method for preparing the bifunctional fluorescent probe ER-Pro based on fluoroborane dipyrrole core according to claim 3, characterized in that: The preparation method of the compound 3 comprises the following steps: dissolving p-carboxybenzaldehyde in dichloromethane, adding 2,4-dimethylpyrrole and a catalyst trifluoroacetic acid, stirring the reaction mixture overnight at room temperature, adding tetrachlorobenzoquinone to the reaction mixture after the aldehyde is completely consumed, adding triethylamine and boron trifluoride ether after stirring, stirring the resulting mixture again at room temperature, collecting the organic phase, and purifying to obtain a purple-red solid, which is the intermediate compound 3.

5. The method for preparing the bifunctional fluorescent probe ER-Pro based on fluoroborane dipyrrole core according to claim 4, characterized in that: The mass ratio of p-carboxybenzaldehyde, 2,4-dimethylpyrrole and tetrachlorobenzoquinone is (0.8-1.2):(0.9-1.7):(1.2-2.0), and the volume ratio of dichloromethane, triethylamine and boron trifluoride ether is (2.6-3.4):(0.8-1.2):(0.8-1.2).

6. The method for preparing the bifunctional fluorescent probe ER-Pro based on fluoroborane dipyrrole core according to claim 3, characterized in that: The compound 3, N-(2-aminoethyl)-4-methylbenzenesulfonamide, HOBt . The molar ratio of H2O, triethylamine and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is (14.6-15.4):(11.96-12.04):(6.6-7.4):(9.56-10.44); the volume ratio of N,N-dimethylformamide to triethylamine is (27.1-27.9):

1.

7. Use of the bifunctional fluorescent probe ER-Pro according to claim 1 in endoplasmic reticulum targeting and / or fluorescence imaging.

8. The use according to claim 7, characterized in that: The targeting of the endoplasmic reticulum includes targeting the localization of the endoplasmic reticulum marker protein GRP78.

9. The use according to claim 7, characterized in that: The fluorescence imaging is co-localization fluorescence imaging.

10. The use according to claim 7, characterized in that: The application includes observing the liquid-liquid phase separation phenomenon of GRP78 protein in vitro or in cells.

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