CN112724085A - Pyrrole isoquinoline aggregation-induced fluorescent molecular probe and preparation and application thereof - Google Patents

Pyrrole isoquinoline aggregation-induced fluorescent molecular probe and preparation and application thereof Download PDF

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CN112724085A
CN112724085A CN202110030361.0A CN202110030361A CN112724085A CN 112724085 A CN112724085 A CN 112724085A CN 202110030361 A CN202110030361 A CN 202110030361A CN 112724085 A CN112724085 A CN 112724085A
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邹宏斌
李进彪
张帅众
劳嘉欣
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Abstract

The invention provides a pyrrole isoquinoline aggregation-induced fluorescent molecular probe and preparation and application thereof. The synthetic method of the pyrrole isoquinoline derivative is simple, and the synthesized fluorescent probe has various emission wavelengths, large Stokes shift, good light stability and aggregation-induced fluorescence property. Meanwhile, the fluorescent probe molecules can be specifically combined with lipid drops in cells to realize the labeling and fluorescence imaging of the lipid drops in the cells. The structure of the pyrrole isoquinoline derivative is as follows:

Description

Pyrrole isoquinoline aggregation-induced fluorescent molecular probe and preparation and application thereof
Technical Field
The invention relates to the field of new material fluorescent probes, in particular to a pyrrole isoquinoline aggregation-induced fluorescent molecular probe, and preparation and application thereof, which are suitable for lipid drop labeling imaging in cells.
Background
Lipid Droplets (LDs) are organelles of cells rich in neutral lipids such as triglycerides and cholesterol, and participate in life processes such as fat storage and metabolism. Recent studies have shown that LDs are highly mobile organelles whose activities are closely related to lipid storage metabolism, signal transduction, and apoptosis. Dysregulation of LDs has been shown to be closely associated with a variety of diseases such as viral infection, inflammation, obesity, and cancer. Therefore, specific labeling of LDs in a complex cellular environment is of great importance for their intracellular localization and analysis.
Conventional LDs probes suffer from drawbacks such as background interference, small stokes shift, aggregation-induced quenching, single emission wavelength, etc., resulting in incomplete imaging of LDs and loss of information. 2001Tang et al found and reported molecules having Aggregation-Induced Emission (AIE) phenomenon, and solved the Aggregation-Induced quenching problem, thereby making fluorescent materials have wider application in various fields. Over 20 years of research, a plurality of organic fluorescent molecules with AIE properties are developed and successfully applied to the fields of organic photoelectric devices, biological imaging, photodynamic therapy and the like. Compared with the traditional fluorescent molecules, the AIE molecules have the advantages of good cell biocompatibility, high brightness, strong specificity, good light stability and the like in LDs imaging. Therefore, more and more organic AIE small molecules are being developed as LDs fluorescent probes.
Many challenges are faced in the current research of LDs fluorescent probes for developing small molecules with AIE properties, such as: 1. the structure of the mother nucleus is single, so that the excitation wavelength and the emission wavelength are single, and the method is not suitable for multiple labeling in a complex cell environment; 2. the synthetic procedures are cumbersome, many AIE molecules are engineered from tetraphenylethylene or tetraphenylsilole, requiring complex synthetic procedures. 3. The stokes shift is small and the excitation wavelength interferes with the signal at the emission wavelength.
Disclosure of Invention
Aiming at the defects or the improvement requirements of the prior art, the invention provides a pyrrole isoquinoline aggregation-induced fluorescent molecular probe, and the structure of the pyrrole isoquinoline derivative is as follows:
Figure BDA0002891873920000011
the fluorescent molecular probe has AIE properties, and different compounds generate corresponding fluorescent signals at different emission wavelengths under the excitation wavelength, wherein the excitation wavelength of the compound I-1 is 355nm, the emission wavelength is 400-500 nm, and blue fluorescence is represented; the excitation wavelength of the compound I-2 is 370nm, the emission wavelength is 500-600 nm, and green fluorescence is represented; the excitation wavelength of the compound I-3 is 365nm, the emission wavelength is 450-580 nm, and blue fluorescence is represented; the excitation wavelength of the compound I-4 is 368nm, the emission wavelength is 550-700 nm, and red fluorescence is represented; the excitation wavelength of the compound I-5 is 361nm, the emission wavelength is 500-600 nm, and green fluorescence is shown. The fluorescent molecular probe has the advantages of good photostability, large Stokes displacement, high fluorescence intensity and the like.
The invention also aims to provide a preparation method of the pyrrole isoquinoline aggregation-induced fluorescent molecular probe, which is realized by the following steps:
a) reacting 4-chlorobenzoyl chloride with 1H-pyrrole in anhydrous tetrahydrofuran under the catalysis of methyl magnesium bromide to generate M1;
b) in N, N-dimethylformamide, the intermediate M1 reacts with bromoacetonitrile under the catalysis of sodium hydride to generate an intermediate M2;
c) in N, N-dimethylformamide, treating a reaction system with acetic acid, M-chloroperoxybenzoic acid and methyl iodide to obtain compounds I-1, I-2 and I-3 respectively by using an intermediate M2 under the catalysis of potassium tert-butoxide;
d) in 2, 4-dioxane, compounds I-2 and I-3 react with (4- (diphenylamino) phenyl) boric acid under the catalysis of tetrakis (triphenylphosphine) palladium and lithium tert-butoxide to respectively obtain compounds I-4 and I-5. The synthetic route is as follows:
Figure BDA0002891873920000021
Figure BDA0002891873920000022
in step a), the preferred conditions are: 1H-pyrrole (0.603g, 9.0mmol) was dissolved in 5 ml of anhydrous tetrahydrofuran under nitrogen protection and added dropwise to a methylmagnesium bromide n-hexane solution (3.0M, 4 ml) at 50 ℃ and after completion of the addition, the solution was refluxed for another 30 minutes. Then, the reaction solution was cooled to room temperature, and 4-chlorobenzoyl chloride dissolved in 10 ml of anhydrous tetrahydrofuran was added dropwise thereto and reacted at room temperature overnight. After the completion of the reaction of the starting materials was monitored by thin layer chromatography, the reaction solution was poured into 20 ml of saturated aqueous ammonium chloride solution and extracted with 30 ml of ethyl acetate, the aqueous phase was extracted twice with 30 ml of ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, the solvent was removed by distillation under reduced pressure, and the resulting crude product was purified by silica gel column chromatography to give a white intermediate M1.
In step b), the preferred conditions are: intermediate M1(0.820g, 4.0mmol) was dissolved in 5 mL of anhydrous N, N-dimethylformamide under nitrogen and NaH (0.230 g, 4.8mmol in 60% kerosene) was added at 0 ℃. After the reaction solution was stirred at 0 ℃ for 30 minutes, bromoacetonitrile (0.571g, 4.8mmol) dissolved in 5 ml of anhydrous N, N-dimethylformamide was added dropwise. The reaction was slowly warmed to room temperature and stirred for an additional 2 hours. The reaction was added dropwise to 100 ml of ice water, filtered, and the residue was recrystallized from ethanol to give intermediate M2.
In step c), the preferred conditions are: intermediate M2(49mg, 0.2mmol) was dissolved in 2 ml of anhydrous N, N-dimethylformamide under nitrogen protection, potassium tert-butoxide (45mg, 0.4mmol) was added at-5 ℃ and the mixture was stirred at-5 ℃ for 5 minutes, then acetic acid (60mg, 1mmol), M-chloroperoxybenzoic acid (172mg, 1mmol) and iodomethane (122mg, 1mmol) were added to the reaction mixture to quench the reaction and the reaction was stirred at room temperature for another 10 minutes. After the raw materials are completely reacted by monitoring through thin layer chromatography, pouring the reaction liquid into 10 ml of water and extracting with 10 ml of ethyl acetate, extracting the water phase twice with 10 ml of ethyl acetate, combining organic phases, drying by anhydrous sodium sulfate, removing the solvent by reduced pressure distillation, and purifying the obtained crude product by silica gel column chromatography to respectively obtain a white solid I-1, a green solid I-2 or a light yellow solid I-3.
In step d), the preferred conditions are: compound I-2(58mg, 0.2mmol, 1.0 equiv.) or compound I-3(58mg, 0.2mmol, 1.0 equiv.), 4- (dianilino) phenyl) boronic acid (87mg, 0.3mmo), tetrakis (triphenylphosphine) palladium (12mg, 0.01mmol) and 0.4 ml of a lithium tert-butoxide solution (2M) were stirred in 2 ml of 2, 4-dioxane for 36 hours under reflux. After the reaction of the raw materials is completely monitored by thin layer chromatography, the reaction solution is poured into 10 ml of water and extracted by 10 ml of ethyl acetate, the water phase is extracted twice by 10 ml of ethyl acetate, organic phases are combined, dried by anhydrous sodium sulfate, the solvent is removed by reduced pressure distillation, and the obtained crude product is purified by silica gel column chromatography to respectively obtain red solid I-4 or green solid I-5.
The invention further aims to provide application of the pyrrole isoquinoline aggregation-induced fluorescent molecular probe in cell lipid drop fluorescent labeling and imaging.
The fluorescent molecular probe is directly or dissolved in solvents such as dimethyl sulfoxide (DMSO), 1, 3-propylene glycol, physiological saline and the like to form a solution containing the fluorescent molecular probe, the solution is added into a culture medium containing cells, the fluorescent molecular probe is taken by the cells and gathered at lipid drops, and fluorescence is emitted under the excitation of ultraviolet light, so that the labeling of the lipid drops in the cells is completed, and the fluorescence imaging of the lipid drops of the cells is realized.
In some embodiments, the working concentration of the fluorescent probe in the culture medium is 0.1 to 1mol/L, preferably 1 to 100. mu. mol/L.
In some embodiments, the ultraviolet light has a wavelength of 200nm to 500nm, preferably 350nm to 450 nm.
The pyrrole isoquinoline aggregation-induced fluorescent molecular probe provided by the invention has the advantages of good photostability, large Stokes shift, high fluorescence intensity and the like. The synthetic method of the pyrrole isoquinoline derivative is simple, and the synthesized fluorescent probe has various emission wavelengths, large Stokes shift, good light stability and aggregation-induced fluorescence property. Meanwhile, the fluorescent probe molecules can be specifically combined with lipid drops in cells to realize the labeling and fluorescence imaging of the lipid drops.
Drawings
FIG. 1 is a graph showing fluorescence spectra of aggregation-induced fluorescent probe I-1 in different ratios of dimethylsulfoxide/water solution.
FIG. 2 is a graph showing fluorescence spectra of aggregation-induced fluorescent probe I-2 in different ratios of dimethylsulfoxide/water solution.
FIG. 3 is a graph showing fluorescence spectra of aggregation-induced fluorescent probe I-3 in different ratios of dimethylsulfoxide/water solution.
FIG. 4 is a graph showing fluorescence spectra of aggregation-induced fluorescent probe I-4 in different ratios of dimethylsulfoxide/water solution.
FIG. 5 is a graph showing fluorescence spectra of aggregation-induced fluorescent probe I-5 in different ratios of dimethylsulfoxide/water solution.
FIG. 6 is a graph of fluorescence images of labeled lipid droplets in cells from different concentrations of aggregation-inducing fluorescent probe I-4.
FIG. 7 is a photograph of fluorescence images of labeled lipid droplets in cells from aggregation-induced fluorescent probes I-1, I-2, I-3 and I-5.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention.
Example 1: preparation of the compound 7-chloropyrrolo [1,2-b ] isoquinolin-10 (5H) -one (I-1).
Figure BDA0002891873920000041
Step a): 1H-pyrrole (0.603g, 9.0mmol) was dissolved in 5 ml of anhydrous tetrahydrofuran under nitrogen protection and added dropwise to a methylmagnesium bromide n-hexane solution (3.0M, 4 ml) at 50 ℃ and after completion of the addition, the solution was refluxed for another 30 minutes. Then, the reaction solution was cooled to room temperature, and 4-chlorobenzoyl chloride dissolved in 10 ml of anhydrous tetrahydrofuran was added dropwise thereto and reacted at room temperature overnight. After the completion of the reaction of the starting materials was monitored by thin layer chromatography, the reaction solution was poured into 20 ml of saturated aqueous ammonium chloride solution and extracted with 30 ml of ethyl acetate, the aqueous phase was extracted twice with 30 ml of ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, the solvent was removed by distillation under reduced pressure, and the resulting crude product was purified by silica gel column chromatography to give a white intermediate M1.
Step b): under the protection of nitrogenIntermediate M1(0.820g, 4.0mmol) was dissolved in 5 ml of anhydrous N, N-dimethylformamide and NaH (60% in kerosene, 0.230g, 4.8mmol) was added at 0 ℃. After the reaction solution was stirred at 0 ℃ for 30 minutes, bromoacetonitrile (0.571g, 4.8mmol) dissolved in 5 ml of anhydrous N, N-dimethylformamide was added dropwise. The reaction was slowly warmed to room temperature and stirred for an additional 2 hours. The reaction solution was added dropwise to 100 ml of ice water, filtered, and the residue was recrystallized from ethanol to give intermediate M2(0.898g, 92% yield);1H NMR(500MHz,CDCl3):δ7.76(2H,dt,J＝8.5,2.0Hz),7.45(2H,dt,J＝8.5,2.0Hz),7.12(1H,dd,J＝2.5,1.5Hz),6.83(1H,dd,J＝4.0,1.5Hz),6.32(1H,dd,J＝4.0,2.5Hz),5.42(2H,s)；13C NMR(125MHz,CDCl3):δ184.96,138.47,137.07,130.59,129.55,128.63,123.85,114.93,110.45,36.99；HRMS Calcd.for C13H9ClN2O+H+:245.0482,found:245.0489。
step c): intermediate M2(49mg, 0.2mmol) was dissolved in 2 ml of anhydrous N, N-dimethylformamide under nitrogen, potassium tert-butoxide (45mg, 0.4mmol) was added at-5 deg.C, and after stirring the mixture at-5 deg.C for 5 minutes, acetic acid (60mg, 1mmol) was added to the reaction solution to quench the reaction and the reaction was stirred at room temperature for a further 10 minutes. After completion of the reaction of the starting materials was monitored by thin layer chromatography, the reaction solution was poured into 10 ml of water and extracted with 10 ml of ethyl acetate, the aqueous phase was extracted twice with 10 ml of ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure, and the resulting crude product was purified by silica gel column chromatography to obtain white solid I-1(37mg, 85%) respectively.1H NMR(500MHz,CDCl3):δ8.26(1H,d,J＝8.5Hz),7.46(1H,dd,J＝8.0,2.0Hz),7.35(1H,d,J＝2.0Hz),7.21(1H,dd,J＝4.0,1.5Hz),7.09(1H,m),6.46(1H,dd,J＝4.0,2.5Hz),5.38(2H,s)；13C NMR(125MHz,CDCl3):δ173.69,138.86,137.08,129.41,129.17,128.88,128.55,125.80,125.68,114.14,112.02,46.62；HRMS(ESI)m/z calcd for C12H8ClNO[M+H]+218.0373,found 218.0379。
Example 2: preparation of quinoline [10, b-iso-7, 5] chloro-1, 5-pyrrolidone (I-2)
Figure BDA0002891873920000051
The procedure of example 1 was followed, using m-chloroperoxybenzoic acid (172mg, 1mmol) instead of acetic acid, to give I-2 as a green solid (31mg, 68%).1H NMR(500MHz,CDCl3):δ8.29(1H,d,J＝8.5Hz),8.23(1H,d,J＝2.0Hz),7.78(1H,dd,J＝3.0,1.5Hz),7.73(1H,dd,J＝8.0,2.0Hz),7.35(1H,dd,J＝3.5,1.5Hz),6.54(1H,dd,J＝4.0,3.0Hz)；13C NMR(125MHz,CDCl3):δ172.14,157.62,141.96,135.42,133.78,130.99,130.71,127.70,127.26,123.90,121.58,115.31；HRMS(ESI)m/z calcd for C12H6ClNO2[M+H]+232.0165,found 232.0170。
Example 3: preparation of 7-chloro-5, 5-dimethylpyrrolo [1,2-b ] isoquinolin-10 (5H) -one (I-3)
Figure BDA0002891873920000052
The preparation process according to example 1 was repeated, using methyl iodide (122mg, 1mmol) in place of acetic acid, to give I-3 as a pale yellow solid (43mg, 88%).1H NMR(500MHz,CDCl3):δ8.30(1H,d,J＝8.5Hz),7.49(1H,d,J＝2.0Hz),7.46(1H,dd,J＝8.5,2.0Hz),7.27(2H,m),6.49(1H,dd,J＝4.0,3.0Hz),1.87(6H,s)；13C NMR(125MHz,CDCl3):δ172.94,147.71,139.28,128.81,128.71,128.24,127.88,125.38,123.60,114.62,112.27,58.12,33.30；HRMS(ESI)m/z calcd for C14H12ClNO[M+H]+246.0686,found 246.0683。
Example 4: preparation of 7- (4- (diphenylamino) phenyl) pyrrolo [1,2-b ] isoquinoline-5, 10-dione (I-4)
Figure BDA0002891873920000061
Step d): compound I-2(58mg, 0.2mmol), (4- (diphenylamino) phenyl) boronic acid (87mg,0.3mmol), tetrakis (triphenylphosphine) palladium (12mg, 0.01mmol) and 0.4 ml of lithium tert-butoxide solution (2M) in 2 ml of 2, 4-dioxane were stirred under reflux for 36 h. After completion of the reaction of the starting materials was monitored by thin layer chromatography, the reaction solution was poured into 10 ml of water and extracted with 10 ml of ethyl acetate, the aqueous phase was extracted twice with 10 ml of ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure, and the resulting crude product was purified by silica gel column chromatography to obtain red solid I-4(67mg, 76%), respectively.1H NMR(500MHz,CDCl3):δ8.53(1H,d,J＝2.0Hz),8.29(1H,d,J＝8.0Hz),7.97(1H,dd,J＝8.0,2.0Hz),7.78(1H,dd,J＝3.5,1.5Hz),7.59(2H,dt,J＝9.0,2.0Hz),7.30(5H,m),7.16(6H,m),7.08(2H,dt,J＝9.0,2.0Hz),6.51(1H,dd,J＝3.5,3.0Hz)；13C NMR(125MHz,CDCl3):δ173.33,158.58,148.95,147.18,146.09,131.96,131.34,131.12,129.97,129.49,128.02,127.94,126.97,125.08,123.75,123.45,122.86,120.68,115.00；HRMS(ESI)m/z calcd for C30H20N2O2[M+H]+441.1603,found 441.1606。
Example 5: preparation of 7- (4- (dianilino) phenyl) -5, 5-dimethylpyrrolo [1,2-b ] isoquinolin-10 (5H) -one (I-5)
Figure BDA0002891873920000062
Following the preparation method of example 4, compound I-3(58mg, 0.2mmol) was used instead of compound I-2 to give I-5(76mg, 84%) as a green solid.1H NMR(500MHz,CDCl3):δ8.40(1H,d,J＝8.0Hz),7.68(1H,dd,J＝8.0,1.5Hz),7.66(1H,d,J＝1.0Hz),7.53(2H,dt,J＝9.0,2.0Hz),7.30(4H,s),7.26(1H,m),7.25(1H,dd,J＝3.5,1.5Hz),7.16(6H,m),7.08(2H,dt,J＝7.0,1.0Hz),6.50(1H,dd,J＝3.5,2.5Hz),1.93(6H,s)；13C NMR(125MHz,CDCl3):δ173.88,148.31,147.38,146.70,145.17,133.30,129.42,129.14,128.07,127.69,127.67,126.04,124.81,123.45,123.36,123.31,123.15,114.07,111.95,58.37,33.56.HRMS(ESI)m/z calcd for C32H26N2O[M+H]+455.2123,found 455.2126。
Example 6: and (3) characterization of fluorescence properties of each compound.
Table 1: maximum excitation wavelength, maximum emission wavelength, Stokes' shift, quantum yield, fluorescence lifetime of compounds I-1, I-2, I-3, I-4 and I-5.
TABLE 1
Figure BDA0002891873920000071
Experimental results show that the compounds I-1, I-2, I-3, I-4 and I-5 prepared in the invention can emit fluorescence with different wavelengths under the excitation of ultraviolet light with different wavelengths in a solid state. The emission wavelength range of the compound is from 446nm to 610nm, the compound has luminescence in the whole wavelength range, the Stokes shift of each compound is different from 91nm to 249nm, and the Stokes shift is larger. Wherein the absolute quantum yield of the compounds I-4 and I-5 reaches 32.35 percent and 27.47 percent, and each compound has the fluorescence lifetime of nanosecond level. Fluorescence property characterization experiments show that the compounds have good fluorescence property and can be used as fluorescent probes for fluorescence imaging.
Example 7: detection of the AIE properties of each fluorescent probe.
(1) Taking the fluorescent probe I-1 prepared in example 1, dissolving the fluorescent probe in dimethyl sulfoxide to prepare 1mmol/L stock solution, taking 40 microliters of the stock solution out, adding the stock solution into a 5 ml centrifuge tube, configuring 4 ml of dimethyl sulfoxide/water solution with different water contents and different water concentrations of 10 mu mol/L, wherein the water ratios are 0%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 95%, respectively, and testing the fluorescence properties of the solutions, and the fluorescence spectrum is shown in figure 1. When the proportion of water in the solution is 0%, the maximum emission wavelength is 446nm, and as the proportion of water in the solution gradually increases, the fluorescent probe I-1 starts to aggregate in the solution, and the fluorescence peak intensity of the fluorescent probe I-1 also gradually increases, indicating that the fluorescent probe shows aggregation-induced fluorescence. When the water proportion reached 70%, a slight decrease in the intensity of the fluorescence peak appeared.
(2) The I-2 fluorescent probe prepared in example 2 was dissolved in dimethyl sulfoxide to prepare a 1mmol/L stock solution, 40. mu.L of the stock solution was taken out and added into a 5 mL centrifuge tube to prepare 4 mL of dimethyl sulfoxide/water solution with different water contents of 10. mu. mol/L, wherein the water ratios are 0%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 95%, respectively, and the fluorescence properties of the solutions were tested, and the fluorescence spectra are shown in FIG. 2. When the proportion of water in the solution is 0%, the maximum emission wavelength is 519nm, and as the proportion of water in the solution gradually increases, the fluorescent probe I-2 starts to aggregate in the solution, and the fluorescence peak intensity also gradually increases, which indicates that the fluorescent probe shows aggregation-induced fluorescence properties.
(3) The I-3 fluorescent probe prepared in example 3 was dissolved in dimethyl sulfoxide to prepare a 1mmol/L stock solution, 40. mu.L of the stock solution was taken out and added into a 5 mL centrifuge tube to prepare 4 mL of dimethyl sulfoxide/water solution with different water contents of 10. mu. mol/L, wherein the water ratios are 0%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 95%, respectively, and the fluorescence properties of the solutions were tested, and the fluorescence spectra are shown in FIG. 3. When the proportion of water in the solution is 0%, the maximum emission wavelength is 496nm, and as the proportion of water in the solution gradually increases, the fluorescent probe I-3 starts to aggregate in the solution, and the fluorescence peak intensity also gradually increases, indicating that the fluorescent probe shows aggregation-induced fluorescence properties. When the water proportion reached 80%, a slight decrease in the intensity of the fluorescence peak appeared.
(4) The I-4 fluorescent probe prepared in example 4 was dissolved in dimethyl sulfoxide to prepare a 1mmol/L stock solution, 40. mu.L of the stock solution was taken out and added into a 5 mL centrifuge tube to prepare 4 mL of dimethyl sulfoxide/water solution with different water contents of 10. mu. mol/L, wherein the water ratios are 0%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 95%, respectively, and the fluorescence properties of the solutions were tested, and the fluorescence spectra are shown in FIG. 4. When the proportion of water in the solution was 0%, the maximum emission wavelength was 476nm, and as the proportion of water in the solution gradually increased, the fluorescent probe I-4 began to aggregate in the solution, and its fluorescence peak intensity appeared to slightly decrease. When the proportion of water in the solution reaches 50%, the maximum emission wavelength red shifts to 530nm, and as the proportion of water in the solution further gradually increases, the aggregation of the fluorescent probe I-4 in the solution is accelerated, and the fluorescence peak intensity of the fluorescent probe I-4 is obviously increased, which indicates that the fluorescent probe shows aggregation-induced fluorescence.
(5) The I-5 fluorescent probe prepared in example 5 was dissolved in dimethyl sulfoxide to prepare a 1mmol/L stock solution, 40. mu.L of the stock solution was taken out and added into a 5 mL centrifuge tube to prepare 4 mL of dimethyl sulfoxide/water solution with different water contents of 10. mu. mol/L, wherein the water ratios are 0%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 95%, respectively, and the fluorescence properties of the solutions were tested, and the fluorescence spectra are shown in FIG. 5. When the proportion of water in the solution is 0%, the maximum emission wavelength is 520nm, and as the proportion of water in the solution gradually increases, the fluorescent probe I-5 starts to gather in the solution, and the fluorescence peak intensity slightly decreases. When the proportion of water in the solution reaches 50%, the maximum emission wavelength red shifts to 610nm, and as the proportion of water in the solution further gradually increases, the aggregation of the fluorescent probe I-5 in the solution is accelerated, and the fluorescence peak intensity of the fluorescent probe I-5 is obviously increased, which indicates that the fluorescent probe shows aggregation-induced fluorescence.
Example 8: lipid droplets in cells were detected by imaging.
(1)4T1 cells at 5X 10 per well4The density of cells was seeded on 24-well plates and allowed to adhere overnight. After 4 hours of pretreatment with 220. mu.g/mL oleic acid at 37 ℃, cells were treated with fluorescent probe I-4 at final concentrations of 1,5 and 10. mu.M. The cells were further incubated at 37 ℃ for 30 minutes and then washed twice with PBS. Then, nile red was added at a final concentration of 5 μ M, incubated at 37 ℃ for 30 minutes, washed twice with PBS, and imaged by confocal fluorescence microscopy. Fluorescence imaging results are shown in FIG. 6, and the imaging results of the fluorescent probe I-4 (excitation 405nm, emission 500-550 nm) at three concentrations of 1,5 and 10 μ M are highly coincident with the imaging results of the positive control Nile red (excitation 550nm, emission 580-640 nm), which indicates that the fluorescent probe I-4 can specifically label lipid droplets in cells at a concentration of 1-10 μ M and realize fluorescence imaging.
(2)4T1 cells at 5X 10 per well4The density of cells was seeded on 24-well plates and allowed to adhere overnight. At 37 ℃ with 2After 4 hours of pretreatment with 20. mu.g/mL oleic acid, cells were treated with fluorescent probes I-1, I-2, I-3 and I-5 at a final concentration of 5. mu.M. Then, nile red was added at a final concentration of 5 μ M, incubated at 37 ℃ for 30 minutes, washed twice with PBS, and imaged by confocal fluorescence microscopy. The fluorescence imaging results are shown in FIG. 7, and the imaging results of the fluorescent probes I-1 (excitation 405nm, emission 420-480 nm), I-2 (excitation 405nm, emission 490-550 nm), I-3 (excitation 405nm, emission 470-530 nm) and I-5 (excitation 405nm, emission 580-640 nm) are highly coincident with the imaging result of the positive control Nile red (excitation 550nm, emission 580-640 nm), which indicates that the fluorescent probes I-1, I-2, I-3 and I-5 can specifically label lipid droplets in cells at a concentration of 5 μ M and realize fluorescence imaging.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.

Claims (10)

1. A pyrrole isoquinoline aggregation induced fluorescent molecular probe is characterized in that the structure of the pyrrole isoquinoline derivative is as follows:
Figure FDA0002891873910000011
2. the method for preparing the pyrrole isoquinoline aggregation-induced fluorescent molecular probe according to claim 1, which is characterized by comprising the following steps:
a) reacting 4-chlorobenzoyl chloride with 1H-pyrrole in anhydrous tetrahydrofuran under the catalysis of methyl magnesium bromide to generate M1;
b) in N, N-dimethylformamide, the intermediate M1 reacts with bromoacetonitrile under the catalysis of sodium hydride to generate an intermediate M2;
c) in N, N-dimethylformamide, treating a reaction system with acetic acid, M-chloroperoxybenzoic acid and methyl iodide to obtain compounds I-1, I-2 and I-3 respectively by using an intermediate M2 under the catalysis of potassium tert-butoxide;
d) in 2, 4-dioxane, compounds I-2 and I-3 react with (4- (diphenylamino) phenyl) boric acid under the catalysis of tetrakis (triphenylphosphine) palladium and lithium tert-butoxide to respectively obtain compounds I-4 and I-5;
the synthetic route is as follows:
Figure FDA0002891873910000012
3. the preparation method according to claim 2, wherein in step a), 1H-pyrrole is dissolved in anhydrous tetrahydrofuran under the protection of nitrogen and dropwise added into methyl magnesium bromide n-hexane solution at 50 ℃, after the dropwise addition, the solution is refluxed for 30 minutes, then 4-chlorobenzoyl chloride dissolved in anhydrous tetrahydrofuran is dropwise added after the reaction liquid is cooled to room temperature and reacted overnight at room temperature, after the reaction of the raw materials is completely monitored by thin layer chromatography, the reaction liquid is poured into saturated ammonium chloride aqueous solution and extracted with ethyl acetate, the aqueous phase is extracted twice with ethyl acetate, the organic phases are combined, dried by anhydrous sodium sulfate, the solvent is removed by reduced pressure distillation, and the obtained crude product is purified by silica gel column chromatography to obtain a white intermediate M1.
4. The preparation method according to claim 2, wherein in step b), intermediate M1 is dissolved in anhydrous N, N-dimethylformamide under the protection of nitrogen, NaH is added at 0 ℃, the reaction solution is stirred and reacted for 30 minutes at 0 ℃, bromoacetonitrile dissolved in anhydrous N, N-dimethylformamide is added dropwise, the reaction solution is slowly raised to room temperature and stirred for 2 hours again, the reaction solution is added dropwise into ice water, the mixture is filtered, and the residue is recrystallized from ethanol to obtain intermediate M2.
5. The preparation method according to claim 2, wherein in step c), intermediate M2 is dissolved in anhydrous N, N-dimethylformamide under the protection of nitrogen, potassium tert-butoxide is added at-5 ℃, the mixture is stirred at-5 ℃ for 5 minutes, then acetic acid, M-chloroperoxybenzoic acid and methyl iodide are respectively added into the reaction solution to quench the reaction and stir at room temperature for 10 minutes, after the reaction of the raw materials is monitored by thin layer chromatography, the reaction solution is poured into water and extracted with ethyl acetate, the aqueous phase is extracted twice with ethyl acetate, the organic phases are combined, anhydrous sodium sulfate is dried, the solvent is removed by reduced pressure distillation, and the obtained crude product is purified by silica gel column chromatography to obtain white solid I-1, green solid I-2 and light yellow solid I-3 respectively.
6. The preparation method according to claim 2, wherein in the step d), the compound I-2 or the compound I-3, (4- (diphenylamino) phenyl) boronic acid, tetrakis (triphenylphosphine) palladium and lithium tert-butoxide solution are stirred and refluxed in 2, 4-dioxane for 36 hours, after the completion of the reaction of the raw materials is monitored by thin layer chromatography, the reaction solution is poured into water and extracted with ethyl acetate, the aqueous phase is extracted twice with ethyl acetate, the organic phases are combined, anhydrous sodium sulfate is dried, the solvent is removed by reduced pressure distillation, and the obtained crude product is purified by silica gel column chromatography to obtain a red solid I-4 or a green solid I-5 respectively.
7. The use of the pyrroloisoquinoline aggregation-induced fluorescent molecular probe of claim 1 in fluorescent labeling and imaging of cellular lipid droplets.
8. The use of claim 7, wherein the fluorescent molecular probe is taken up by the cells and accumulated at the lipid drop, and fluoresces under the excitation of ultraviolet light, thereby completing the labeling and imaging of the lipid drop in the cell.
9. The use according to claim 7, wherein the working concentration of the fluorescent probe is between 0.1 μmol/L and 1 mol/L.
10. Use according to claim 7, wherein the ultraviolet light has a wavelength of 200nm to 500 nm.
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