CN117362268A - Lysosome targeted polar fluorescent probe and preparation method and application thereof - Google Patents

Abstract

The invention discloses a preparation method of a lysosome targeting polar fluorescent probe and application thereof in biological imaging, belonging to the technical field of chemical analysis and detection, wherein the polar fluorescent probe has the following structure:the lysosome targeted polar fluorescent probe has excellent polar sensing property and obvious aggregation-induced emission characteristic. With the increase of the polarity of the solvent, the fluorescence emission of the probe PHPT shows remarkable red shift and remarkable enhancement of fluorescence intensity, and a good linear relation is shown between the maximum emission wavelength of the probe PHPT and the polarization rate of the solvent within the range of 0.214-0.353. The probe also has good photostability. More importantly, the polarity of the probe PHPTThe detection properties are not disturbed by pH and various biological small molecules. The probe PHPT has low cytotoxicity and is suitable for biological imaging. PHPT has the capability of precisely targeting lysosomes, can be used for detecting polarity reduction of cells and polarity increase in autophagy process, and can be used for monitoring physiological activities related to polarity change in cells.

Description

Lysosome targeted polar fluorescent probe and preparation method and application thereof

Technical Field

The invention belongs to the technical field of analytical chemistry, and particularly relates to a preparation method of a lysosome targeting polar fluorescent probe and application of the lysosome targeting polar fluorescent probe in biological imaging.

Background

The change of microenvironment in a biological system has obvious influence on various biological processes, and has important significance on the detection of related states in the diagnosis and analysis of diseases, the determination of pathological mechanisms of diseases and the research and development of new drug targets. Polarity is an important parameter of the microenvironment in biological systems that can severely affect the rate and direction of chemical reactions. Polarity is critical to establishing and reflecting a number of complex physiological and pathological roles including activating functional proteins and immune responses, triggering signal transduction and membrane rearrangements, changes in polarity of protein binding, stimulating cell migration and cell proliferation, affecting protein interactions and enzyme stability and membrane compartment permeability. Abnormal changes in polarity have been shown to be associated with a variety of diseases such as inflammation, organ failure and cancer. Therefore, the polarity-sensitive dye can visualize the polarity distribution, change and adjustment in a biological system, can promote the research on biochemical reactions and deepen our understanding of important biological processes. In addition, polarity sensitive dyes are also used to distinguish critical subcellular microstructures.

Lysosomes play an important role in cellular processes including protein degradation, secretion, plasma membrane repair, and autophagy. The polarity of lysosomes affects the interaction activity between enzyme and substrate at the cellular level, and the level of lysosomes polarity in cancer cells is lower than that of normal cells, and the polarity changes during lysosomal autophagy. Therefore, a novel method capable of rapidly and sensitively monitoring the polarity change of lysosomes is developed, and has important significance for cell biology and related pathology research [ J.Yin, L.Huang, L.Wu, J.Li, T.D.James, W.Lin, small molecule based fluorescent chemosensors for imaging the microenvironment within specific cellular regions, chem.Soc.Rev.2021,51:12098.N.Jiang,J.Fan,F.Xu,X.Peng,H.Mu,J.Wang,X.Xiong,Ratiometric fluorescence imaging of cellular polarity:decrease in mitochondrial polarity in cancer cells,Angew.Chem.Int.Ed.2015,54:2510-2514.M.Li,J.Fan,H.Li,J.Du,S.Long,X.Peng,A ratiometric fluorescence probe for lysosomal polarity,Biomaterials 2018,164:98-105 ].

Disclosure of Invention

The invention aims at a preparation method of a lysosome targeting polar fluorescent probe and application of the lysosome targeting polar fluorescent probe in biological imaging.

The fluorescent probe has the following molecular structure:

the synthesis process of the fluorescent probe in the invention is as follows:

a1:5- (4- (diphenylamino) phenyl) thiophene-2-carbaldehyde;

b1:1- (4- (dimethylamino) -2-hydroxyphenyl) ethan-1-one;

PHPT: (E) -1- (4- (dimethylamino) -2-hydroxyphenyl) -3- (5- (4- (biphenylamino) phenyl) thiophen-2-yl) prop-2-en-1-one.

The preparation steps of the probe PHPT are as follows:

4-bromotriphenylamine and 5-aldehyde-2-thiopheneboronic acid are placed in a round bottom flask, and anhydrous tetrahydrofuran is added for ultrasonic dissolution. After which K is added ₂ CO ₃ Aqueous solution and Pd (PPh) ₃ ) ₄ The mixed solution was stirred well and then heated to reflux under argon for 18 hours. The mixture was cooled to room temperature, extracted with dichloromethane and water, the organic phase was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to conduct column chromatography purification (eluent V _{Petroleum ether} :V _{Acetic acid ethyl ester} =20:1), a yellow product A1 is obtained.

Weighing K ₂ CO ₃ And 4 '-amino-2' -hydroxyacetophenone are placed in a round bottom flask, 5mL of absolute ethyl alcohol is added for ultrasonic dissolution, and methyl iodide is added for uniform mixing. The mixed solution was heated to 55℃and refluxed for 36 hours. After the reaction is finishedThe mixed solution was cooled to room temperature, the solvent was removed under reduced pressure, and column chromatography purification was performed by adding silica gel (V _{Petroleum ether} :V _{Acetic acid ethyl ester} =1:1), and dried in vacuo for 24 hours to give the product as a white solid.

Compounds A1 and B1 were placed in a round bottom flask, dissolved by sonication with methanol, and then heated to reflux with 60% NaOH solution for 8 hours. After cooling the mixed solution to room temperature, ice water was added, and the solution was neutralized with dilute hydrochloric acid. Orange precipitate is separated out, and a solid is obtained through suction filtration. Purifying by column chromatography (eluent is V) _{Petroleum ether} :V _{Acetic acid ethyl ester} =20:1), and the product was dried in vacuo to give PHPT as an orange-red product.

The fluorescence probe detection mechanism of the invention is as follows:

under low polarity conditions (low water content), the probe PHPT emits weak and short fluorescence due to weak interactions with the solvent in which the molecules are dispersed. In highly polar solvents (higher water content), greater charge separation may occur due to interactions with the solvent, excited state energy dissipation, while in poor solvents, molecules aggregate, producing strong and long fluorescence; in this process, a large Stokes shift is generated, which may be useful for eliminating potential interference from bioluminescence.

FIG. 3 is an ultraviolet-visible absorption spectrum of (A) probe PHPT in different solvents. (B) fluorescence emission spectra of probe PHPT in different solvents. (C) Normalized fluorescence spectra of probe PHPT in different solvents (lambda _ex Slit width=455 nm: 10nm,5nm, probe test concentration of 1X 10 ^-5 mol/L). The change in absorption spectrum of the probe PHPT is small under different polarities, which indicates that the change in dipole moment of the ground state probe along with the polarity is small. The fluorescence emission spectrum was tested with 455nm as the excitation wavelength, and it was found that with increasing polarity, the emission wavelength of probe PHPT was significantly red shifted with concomitant increase in fluorescence intensity.

FIG. 4 shows that the prepared probe PHPT was found in different casesPhotophysical properties in solvent, including maximum absorbance lambda _abs,max Maximum emission lambda _em,max Fluorescence quantum yield phi. The above results indicate that from low polarity 1, 4-dioxane to high polarity DMSO, the fluorescence corresponding to the maximum emission wavelength was enhanced by nearly 27-fold. The difference in photophysical properties of probe PHPT in different solvents was studied with reference to fluorescein in 0.1M NaOH (Φ=79%) and the results showed that the fluorescence quantum yield of probe in DMSO (dimethyl sulfoxide) (Φ=1.4%) was much greater than that in 1, 4-dioxane (Φ=0.36%) and these phenomena indicated that the dipole moment of probe was significantly changed in the excited state.

FIG. 5 shows the particle size distribution of the probe PHPT in 1, 4-dioxane. (B) particle size distribution of Probe PHPT in DMSO. The particle size of the probe in DMSO is significantly larger than that in 1, 4-dioxane.

FIG. 6 (A) ultraviolet visible absorption spectra of probe PHPT in different proportions of water and 1, 4-dioxane. (B) Fluorescence emission spectra of probe PHPT in different proportions of water and 1, 4-dioxane. (C) luminescence color graph of probe PHPT. (D) Maximum emission wavelength (lambda) of probe PHPT _em,max ) Relation (lambda) to the solvent polarizability (Deltaf) _ex Slit width=450 nm: 10nm,5nm, probe test concentration of 1X 10 ^-5 mol/L). As the water proportion increases, the probe PHPT absorbance spectrum changes little. As the water content in the mixed solvent increases from 0% to 30% (polarity increase), the maximum emission wavelength of the probe PHPT red-shifts from 569nm to 643nm, and the fluorescence signal corresponding to the maximum emission wavelength increases. Under 365nm hand-held ultraviolet lamp irradiation, the probe emits light to be orange in pure 1, 4-dioxane and light red in a mixed solvent of 1, 4-dioxane and 30% water, which is consistent with the result of the color coordinate representation of the light emission. In a highly polar environment, a significant red shift in the maximum emission wavelength may be due to an increased degree of charge separation of the probe PHPT, and excited state energy dissipation. Furthermore, as the water content increases, the solvent polarizability is in the range of 0.214 to 0.353 (2% H ₂ O–20％H ₂ O), the maximum emission wavelength of the probe PHPT has a good linear relationship with the polarizability (Δf) of the solvent (FIG. 2.6D, R) ² = 0.9899). With the increase of the water content, the detection is performedThe fluorescence of needle PHPT at 640nm was enhanced by about 48 times. This is in contrast to the typical ICT effect phenomenon, probably due to limited intramolecular torsion caused by aggregation of the probe in the solvent as the water content of the poor solvent increases.

FIG. 7 shows the particle size distribution of the probe PHPT in 1, 4-dioxane. (B) Particle size distribution of probe PHPT in a mixed solvent of 1, 4-dioxane and 30% water. Insert: the tyndall effect of the probe in different solvents under the irradiation of a green laser pen. The average particle size of the probe PHPT in the mixed solvent of 30% water is significantly larger than that in the pure 1, 4-dioxane. Meanwhile, under the irradiation of a laser pen, a remarkable Tyndall effect of the probe PHPT in a mixed solvent of 30% of water can be observed. This further indicates the formation of aggregates, indicating that the probe PHPT has excellent aggregation-induced emission properties.

FIG. 8 is an (A) ultraviolet visible absorption spectrum of probe PHPT in solvents of different pH values; (B) Fluorescence emission spectrum (lambda) _ex Slit width=450 nm: 10nm,5nm, probe test concentration of 1X 10 ^-5 mol/L, the test system is 1, 4-dioxane+30% PBS buffer solutions with different pH values). In a mixed solvent of 1, 4-dioxane and 30% water, the absorption spectrum of the probe PHPT hardly changes under different pH conditions, and the fluorescence spectrum changes little under acidic (pH=4) or alkaline (pH=10) conditions. The detection property of the probe PHPT is hardly interfered by pH, and the probe can detect the polarity under the physiological pH condition.

FIG. 9 shows the change in fluorescence intensity of probe PHPT at 640nm in pure 1, 4-dioxane and a mixed solvent of 1, 4-dioxane and 30% water over 1 hour (lambda _ex Slit width=450 nm: 10nm,5nm, probe test concentration of 1X 10 ^-5 mol/L). In two different solvents, the fluorescence intensity of probe PHPT at 640nm within 1 hour is little changed under the continuous irradiation of 450nm excitation light. The probe PHPT has good light stability in both low-polarity environment and high-polarity environment, and fluorescence cannot be attenuated with time.

FIG. 10 shows the interference resistance of probe PHPT. 1-22 are respectively Blank, ca ²⁺ 、Cu ²⁺ 、Fe ²⁺ 、K ⁺ 、Ag ⁺ 、Al ³⁺ 、Zn ²⁺ 、ClO ^- 、H ₂ O ₂ 、Glycerol、F ^- 、NO ₃ ^- 、HCO ₃ ^- 、S ^2- 、HSO ₃ ^- 、I ^- 、SO ₄ ^2- 、Br ^- 、Cys、GSH、Hcy(λ _ex Slit width=450 nm: 10nm,5nm, probe test concentration of 1X 10 ^-5 mol/L, test system is PBS buffer solution at ph=7.4). In the presence of various biological small molecules, the fluorescence of the probe is almost the same as that in PBS buffer solution, which indicates that the fluorescence of the probe PHPT is not interfered by the biological small molecules.

FIG. 11 is a graph showing the cell viability statistics of (A). (incubation time: 12 hours, probe PHPT concentration: 0. Mu.M, 2.5. Mu.M, 5. Mu.M, 10. Mu.M, 20. Mu.M, 40. Mu.M) (B) A549 cells were incubated with probe PHPT (10. Mu.M) followed by time-dependent fluorescence imaging (red channel: lambda.) _ex ＝488nm，λ _em Green channel = 590-680 nm: lambda (lambda) _ex ＝488nm，λ _em =500-550 nm, scale: 25 μm). After 12 hours of incubation with different concentrations of PHPT (0. Mu.M, 2.5. Mu.M, 5. Mu.M, 10. Mu.M, 20. Mu.M, 40. Mu.M), the A549 cell viability was above 85%. The result shows that the probe PHPT has small cytotoxicity and is suitable for biological imaging. A549 cells were co-incubated with the probe PHPT followed by time series. The fluorescence images recorded in the red channel and the green channel show that the fluorescence intensity of the probe PHPT in the A549 cells hardly changes within 30min, which indicates that the probe PHPT has good light stability in the cells.

FIG. 12 shows images of the green, red, and blue channels of (A) probe PHPT co-stained with commercial lysosomal dyes. (B) Fluorescence profile of the probe in a specific region (red channel: lambda) _ex ＝488nm，λ _em Green channel = 590-680 nm: lambda (lambda) _ex ＝488nm，λ _em =500-550 nm, blue channel: lambda (lambda) _ex ＝408nm，λ _em =450-490 nm, scale: 10 μm). The images of the probes in the red channel and the green channel are well overlapped with the images of the commercial lysosome dye in the blue channel, and the co-localization system is adoptedThe numbers are 0.87 and 0.89 respectively, which indicates that the probe PHPT has good lysosome targeting ability.

FIG. 13 shows real-time confocal imaging of (A) DMSO (3. Mu.L) induced A549 cells after staining with PHPT (10. Mu.M). (B) Statistics of fluorescence intensity semi-quantitative analysis at different times within 30 min. To verify the feasibility of probe PHPT to monitor intracellular polarity changes, the probe PHPT stained a549 cells were treated with DMSO (3 μl), and fluorescence images of the cells in the red and green channels were recorded every 10 min. After DMSO is added, the fluorescence signal of the red channel is obviously weakened, and the fluorescence of the green channel is gradually strengthened along with the time extension. This is the same phenomenon as when the polarity of the probe in solution decreases, indicating that the addition of DMSO causes a decrease in cell polarity and that the probe PHPT can be used to monitor cell polarity changes.

FIG. 14 shows real-time confocal imaging of lipopolysaccharide (50. Mu.g/mL) induced A549 cells after staining with PHPT (10. Mu.M) over 90min (A). (B) Statistics of fluorescence intensity semi-quantitative analysis at different times within 90 min. We further investigated whether the probe PHPT was suitable for monitoring the polarity change caused by inflammation. After staining A549 cells with probe PHPT, further treating the cells with lipopolysaccharide (50 μg/mL), photographing at intervals of 30min to record images of green channel and red channel, and after treating with LPS, the fluorescence of the green channel only changes slightly and the fluorescence of the red channel gradually weakens along with the extension of time. The LPS-induced decrease in inflammatory cell polarity was shown, and the probe PHPT could be used to monitor polarity changes in inflammatory cell models.

FIG. 15 shows real-time confocal imaging of probe PHPT (10. Mu.M) stained A549 cells under (A) starvation. (B) Statistics of fluorescence intensity semi-quantitative analysis at different times within 2 hours. To further investigate whether probes could be used to monitor autophagy processes, we used starvation induction to build an autophagy model. A549 cells were stained with the probe PHPT and placed in PBS-only dishes, and the cells were observed for fluorescence change 2 hours later. The red channel fluorescence was initially weaker, and then the fluorescence increased significantly over time. The fluorescence intensity of the green channel varies inversely, and shows a tendency that fluorescence decreases significantly with the time of starvation. Indicating that the polarity of lysosomes gradually increases during starvation-induced autophagy. The results of the cell experiments prove that the probe PHPT has the capability of accurately positioning lysosomes, can be used for monitoring the cell polarity change under the physiological conditions of inflammation and autophagy in real time, and provides a new strategy for monitoring the cell polarity change under various physiological conditions in real time without loss.

Drawings

FIG. 1A preparation route of probe PHPT.

FIG. 2 the polar response mechanism of probe PHPT.

FIG. 3 (A) ultraviolet visible absorption spectra of probe PHPT in different solvents. (B) fluorescence emission spectra of probe PHPT in different solvents. (C) Normalized fluorescence spectra of probe PHPT in different solvents. (lambda) _ex Slit width=455 nm: 10nm,5nm, probe test concentration of 1X 10 ^-5 mol/L)

FIG. 4 photophysical properties of probe PHPT in different solvents.

FIG. 5 (A) shows the particle size distribution of probe PHPT in 1, 4-dioxane. (B) particle size distribution of Probe PHPT in DMSO.

FIG. 6 (A) ultraviolet visible absorption spectra of probe PHPT in different proportions of water and 1, 4-dioxane. (B) Fluorescence emission spectra of probe PHPT in different proportions of water and 1, 4-dioxane. (C) luminescence color graph of probe PHPT. (D) Maximum emission wavelength (lambda) of probe PHPT _em Max) and solvent polarizability (Δf). (lambda) _ex Slit width=450 nm: 10nm,5nm, probe test concentration of 1X 10 ^-5 mol/L)

FIG. 7 (A) shows the particle size distribution of probe PHPT in 1, 4-dioxane. (B) Particle size distribution of probe PHPT in a mixed solvent of 1, 4-dioxane and 30% water. Insert: and (3) images of the tyndall effect of the probe in different solvents under the irradiation of a green laser pen.

FIG. 8 ultraviolet visible absorption spectra of probe PHPT in solvents of different pH values; (B) fluorescence emission spectrum. (lambda) _ex Slit width=450 nm: 10nm,5nm, probe test concentration of 1X 10 ^-5 mol/L, the test system is 1, 4-dioxane +30% PBS buffer solution with different pH values

FIG. 9 Probe PHPT at pure 1The fluorescence intensity at 640nm in a mixed solvent of 4-dioxane and 1, 4-dioxane with 30% water was changed within 1 hour. (lambda) _ex Slit width=450 nm: 10nm,5nm, probe test concentration of 1X 10 ^-5 mol/L)

FIG. 10 interference resistance study of probe PHPT. 1-22 are respectively blank, ca ²⁺ 、Cu ²⁺ 、Fe ²⁺ 、K ⁺ 、Ag ⁺ 、Al ³⁺ 、Zn ^{2 +} 、ClO ^- 、H ₂ O ₂ Glycerol (Glycerol), F ^- 、NO ₃ ^- 、HCO ₃ ^- 、S ^2- 、HSO ₃ ^- 、I ^- 、SO ₄ ^2- 、Br ^- 、Cys、GSH、Hcy(λ _ex Slit width=450 nm: 10nm,5nm, probe test concentration of 1X 10 ^-5 mol/L, test system is PBS buffer solution at ph=7.4).

FIG. 11 (A) is a statistical plot of cell viability. (incubation time: 12 hours; probe PHPT concentration: 0. Mu.M, 2.5. Mu.M, 5. Mu.M, 10. Mu.M, 20. Mu.M, 40. Mu.M) (B) fluorescence imaging of A549 cells with probe PHPT (10. Mu.M) over time. (Red channel: lambda) _ex ＝488nm，λ _em Green channel = 590-680 nm: lambda (lambda) _ex ＝488nm，λ _em =500-550 nm, scale: 25 μm).

FIG. 12 (A) shows images of the green, red, and blue channels of the co-stained probe PHPT with commercial lysosomal dyes. (B) fluorescence profile of the probe in a specific region. (Red channel: lambda) _ex ＝488nm，λ _em Green channel = 590-680 nm: lambda (lambda) _ex ＝488nm，λ _em =500-550 nm, blue channel: lambda (lambda) _ex ＝408nm，λ _em =450-490 nm, scale: 10 μm).

FIG. 13 (A) real-time confocal imaging of DMSO (3. Mu.L) induced A549 cells after staining with PHPT (10. Mu.M). (B) Statistics of fluorescence intensity semi-quantitative analysis at different times within 30 min.

FIG. 14 (A) real-time confocal imaging of lipopolysaccharide (50. Mu.g/mL) induced A549 cells after staining with PHPT (10. Mu.M) within 90 min. (B) Statistics of fluorescence intensity semi-quantitative analysis at different times within 90 min.

FIG. 15 (A) real-time confocal imaging of probe PHPT (10. Mu.M) stained A549 cells in starved state. (B) Statistics of fluorescence intensity semi-quantitative analysis at different times within 2 hours.

Examples of the embodiments

Example 1: synthesis of Compound A1

4-Bromotrianiline (2.4 mmol,778 mg) and 5-formyl-2-thiopheneboronic acid (2 mmol,311 mg) were placed in a 50mL round bottom flask and dissolved by sonication with the addition of 8mL anhydrous tetrahydrofuran. After which 2mol/L K are added ₂ CO ₃ Aqueous solution (3.2 mL) and Pd (PPh) ₃ ) ₄ (116 mg,0.1 mmol) and the mixture was stirred well and then heated under reflux under argon for 18 hours. The mixture was cooled to room temperature and extracted with dichloromethane and water, and the organic phase was taken up in anhydrous Na ₂ SO ₄ Drying, removing solvent under reduced pressure, and purifying by column chromatography (eluent is V) _{Petroleum ether} :V _{Acetic acid ethyl ester} =20:1) to give yellow product A1 (249 mg, yield 35.0%). A1 structural characterization data are as follows: ¹ H NMR(600MHz,DMSO-d ₆ )δ9.86(s,1H),8.00(d,1H),7.69(d,2H),7.60(d,1H),7.36(t,4H),7.13(t,2H),7.09(d,4H),6.96(d,2H). ¹³ C NMR(151MHz,DMSO-d ₆ )δ183.6,152.9,148.5,146.4,140.9,139.4,129.7,127.4,125.5,124.9,124.1,123.9,121.7.

example 2: synthesis of Compound B1

Weighing K ₂ CO ₃ (5 mmol,691.1 mg) and 4 '-amino-2' -hydroxyacetophenone (5 mmol,755.8 mg) were placed in a round bottom flask, and after ultrasonic dissolution with 5mL absolute ethanol, 12.5mL methyl iodide (CH) was added ₃ I) Mixing well. The mixed solution was heated to 55℃and refluxed for 36 hours. After the reaction was completed, the mixed solution was cooled to room temperature, the solvent was removed under reduced pressure, and column chromatography purification was performed by adding silica gel (V _{Petroleum ether} :V _{Acetic acid ethyl ester} After 24 hours of vacuum drying, the product was obtained as a white solid (418.1 mg, 46.7% yield). B1 structural characterization data are as follows: ¹ H NMR(600MHz,DMSO-d ₆ )δ12.90(s,1H),7.65(d,1H),6.30(dd,1H),6.02(d,1H),3.01(s,6H),2.46(s,3H). ¹³ C NMR(151MHz,DMSO-d ₆ )δ201.4,164.5,156.3,133.3,109.9,104.6,97.3,26.0.

example 3: synthesis of probe PPBI

Compounds A1 (0.44 mmol,155 mg) and B1 (0.44 mmol,78.2 mg) were placed in a round-bottomed flask, and after adding 8mL of methanol for ultrasonic dissolution, 1mL of 60% NaOH solution was added thereto, and heated under reflux for 8 hours. After cooling the mixed solution to room temperature, ice water was added, and the solution was neutralized with dilute hydrochloric acid. Orange precipitate is separated out, and a solid is obtained through suction filtration. Purifying by column chromatography (eluent is V) _{Petroleum ether} :V _{Acetic acid ethyl ester} After vacuum drying, the product gave PHPT (68 mg, 30.0% yield) as orange-red product. The probe PPBI structure characterization data are as follows: ¹ H NMR(600MHz,DMSO-d ₆ )δ13.83(s,1H),7.96(d,1H),7.87(d,1H),7.63(t,4H),7.50(dd,3H),7.35(t,5H),7.16–7.06(m,8H),6.99(d,2H),3.05(s,8H). ¹³ C NMR(151MHz,CDCl ₃ )δ189.4,166.2,155.8,148.2,147.4,147.2,139.0,135.4,133.3,131.1,129.4,127.2,126.7,124.9,123.5,123.6,123.0,118.8,110.6,104.0,98.1,40.0.HR-MS m/z:calcd for C ₃₃ H ₂₈ N ₂ O ₂ S[M+H] ⁺ ,517.1905；found,517.1952.

example 4: application of probe PHPT in biological imaging

Drug and starvation induced cell polarity change: in the DMSO-induced cell polarity change experiment, cells were stained with probe PHPT (10 μM) for 1 hour, washed three times with PBS, and DMSO was added to the petri dish and fluorescence images (0 min, 5min, 10min, 15min, 20min, 30 min) were recorded every 5 min. The parameters at the time of cell image acquisition were set to red channel: the excitation light source is 488nm, and the collection range is 590-680nm; green channel: the excitation light source is 488nm and the collection range is 500-550nm.

In Lipopolysaccharide (LPS) -induced cell polarity change experiments, cells were stained with probe PHPT (10 μM) for 1 hour, washed three times with PBS, A549 cells were incubated with LPS (50 μg/mL), fluorescence images were recorded every 30min, and fluorescence image changes (0 min, 30min, 60min, 90 min) were recorded over 90 min. The parameters at the time of cell image acquisition were set to red channel: the excitation light source is 488nm, and the collection range is 590-680nm; green channel: setting an excitation light source 488nm and setting a collection range of 500-550nm.

In starvation induced cell polarity change experiments, cells were stained with probe PHPT (10 μM) for 1 hour, washed three times with PBS phosphate buffer, and the dishes were tested without culture medium and with a small amount of PBS phosphate buffer. Fluorescence images after 1 hour and 2 hours of starvation of cells were recorded and fluorescence intensities were collected. The parameters at the time of cell image acquisition were set to red channel: the excitation light source is 488nm, and the collection range is 590-680nm; green channel: setting an excitation light source 488nm and setting a collection range of 500-550nm.

The probe PHPT can be used to monitor cell polarity decrease caused by dimethyl sulfoxide and lipopolysaccharide, and can be further used to monitor polarity increase during autophagy. The probe PHPT with intramolecular charge transfer and aggregation induction effects provides an effective tool for researching physiological activities related to the polarity change in cells, and is expected to provide a new way for diagnosing lysosomes and diseases related to the polarity change in cells.

Claims (2)

1. A lysosome targeted polar fluorescent probe PHPT, having the structural formula:

2. the preparation method of the lysosome targeting polar fluorescent probe PHPT according to claim 1 comprises the following steps:

placing 4-bromotriphenylamine and 5-aldehyde-2-thiopheneboronic acid into a round bottom flask, and adding anhydrous tetrahydrofuran for ultrasonic dissolution; then adding 2mol/L potassium carbonate aqueous solution and Pd (PPh) ₃ ) ₄ Stirring the mixed solution uniformly, and then heating and refluxing for 18 hours under the protection of argon; the mixture is cooled to room temperature, extracted with dichloromethane and water, the organic phase is dried with anhydrous sodium sulfate, the solvent is removed under reduced pressure for column chromatography purification, and a yellow product A1 is obtained;

weighing K ₂ CO ₃ Placing 4 '-amino-2' -hydroxyacetophenone into a round bottom flask, adding absolute ethanol, dissolving by ultrasonic, and adding methyl iodide (CH) ₃ I) Uniformly mixing; heating and refluxing the mixed solution for 36 hours; after the reaction was completed, the mixed solution was cooled to room temperature, the solvent was removed under reduced pressure, and column chromatography purification was performed by adding silica gel (V _{Petroleum ether} :V _{Acetic acid ethyl ester} =1:1), dried in vacuo for 24 hours to give the product as a white solid;

placing the compounds A1 and B1 into a round-bottom flask, adding methanol for ultrasonic dissolution, then adding NaOH solution, and heating and refluxing for 8 hours; cooling the mixed solution to room temperature, adding ice water, and regulating the solution to be neutral by using dilute hydrochloric acid; orange precipitate is separated out, and a solid is obtained through suction filtration; purifying by column chromatography (eluent is V) _{Petroleum ether} :V _{Acetic acid ethyl ester} =20:1), the product was dried in vacuo to give an orange-red product;

the synthetic route is as follows:

a1:5- (4- (diphenylamino) phenyl) thiophene-2-carbaldehyde

B1:1- (4- (dimethylamino) -2-hydroxyphenyl) ethan-1-one

PHPT: (E) -1- (4- (dimethylamino) -2-hydroxyphenyl) -3- (5- (4- (biphenylamino) phenyl) thiophen-2-yl) prop-2-en-1-one.