A fluorescent probe for detecting microenvironment polarity, a preparation method thereof, and a special detection kit

Abstract

The present invention proposes a lipid droplet-targeted fluorescent probe for detecting microenvironment polarity, a preparation method thereof, and a dedicated detection kit. The fluorescent probe is 5,5-difluoro-1,3,9-trimethyl-10-phenyl-7-((E)-3-((E)-1,3,3-trimethylindoline-2-ylidene)prop-1-en-1-yl)-5H-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaboronic acid. The present invention synthesizes the BFZ probe using fluoroboron dipyrimidine (BODIPY) as an electron acceptor (A) and Fisher's aldehyde as a donor (D), and the donor and the acceptor are connected by a π bridge. Based on the intramolecular charge transfer mechanism and the ICT mechanism, the probe exhibits a higher fluorescence intensity under low-polarity conditions and a lower fluorescence reflection intensity under high-polarity conditions, and has a significant red shift. The probe has good targeting ability for lipid droplets and high photostability. Common species such as reactive oxygen species, amino acids, and enzymes will not interfere with the probe's selectivity for polarity.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical analysis and biological analysis, and in particular to a fluorescent probe for detecting microenvironment polarity, a preparation method thereof, and a special detection kit. Background Art

[0002] The microenvironment is a crucial factor in maintaining the specific physiological functions of organelles and plays an indispensable role in the physiological processes of cells. Factors influencing the homeostasis of the intracellular microenvironment primarily include viscosity, temperature, polarity, hypoxia, and acid-base status. Polarity, reflecting a complex series of mechanisms, is a crucial parameter for establishing specific functional domains and maintaining normal cellular function. Most cellular activities, such as membrane permeability, directional migration, and activation of immune responses, are often intertwined with polarity. Because the intracellular microenvironment is complex and variable, studies have shown that changes in polarity in different regions are considered reliable indicators of different diseases.

[0003] Lipid droplets (LDs) are specialized organelles that store lipids. They consist of a neutral lipid core, a monolayer of phospholipids, and specialized surface proteins (such as perilipin). LDs are considered dynamic organelles, involved in lipid storage and metabolism, signal transduction, and apoptosis. Abnormal changes in LD polarity have been linked to a variety of diseases, including cancer, inflammatory disorders, and metabolic diseases. Therefore, early diagnosis and detection of diseases based on changes in LD polarity are of great significance.

[0004] Existing fluorescent probes for detecting polarity are not very sensitive. For example, the Chinese invention patent is titled "A polarity-resistant blue-shifted lipid droplet-targeted fluorescent probe" with application number 2023106328659. The probe disclosed in the patent has an excitation wavelength of 575 nm and an emission wavelength of 615 nm, which are not within the near-infrared imaging range. The probe stabilizes after 40 minutes of incubation with cells, which is a long time. Moreover, when the polarity changes, only the fluorescence intensity changes, and the emission wavelength remains unchanged. In solutions of different polarities, as the polarity increases, the fluorescence intensity decreases and the emission wavelength does not red-shift. The sensitivity to polarity is determined only by the fluorescence intensity. Summary of the Invention

[0005] In order to solve the above-mentioned defects, the purpose of the present invention is to provide a fluorescent probe for detecting microenvironment polarity, a preparation method thereof, and a dedicated detection kit.

[0006] The technical solution of the present invention is achieved as follows: a fluorescent probe for detecting polarity, the fluorescent probe is 5-difluoro-1,3,9-trimethyl-10-phenyl-7-((E)-3-((E)-1,3,3-trimethylindolin-2-ylidene)prop-1-en-1-yl)-5H-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaboronic acid, and its structural formula is shown in Formula I:

[0007]

[0008] The present invention provides a method for preparing a fluorescent probe for detecting polarity, comprising the following steps:

[0009] Step 1: Add 2,4-dimethylpyrrole, benzaldehyde represented by formula III and trifluoroacetic acid to a round-bottom flask containing tetrahydrofuran, protect with inert gas, stir at room temperature for 12 hours, add 2,3-dichloro-5,6-dicyanobenzoquinone, stir at room temperature for 4 hours for oxidation, finally add a catalyst, add boron trifluoride ether dropwise, and continue stirring at room temperature for 24 hours to obtain the compound represented by formula II.

[0010]

[0011] Step 2: Fischer's aldehyde, fluoroboranedipyrimidine of Formula II, piperidine, magnesium perchlorate, and glacial acetic acid are placed in a 30 mL round-bottom flask in sequence, and a condensation reaction is carried out in an organic solvent. The mixture is heated under reflux at 120° C. with stirring for 6 hours under the protection of an inert gas to obtain the compound of Formula I.

[0012]

[0013] Preferably, in step 1, the molar ratio of 2,4-dimethylpyrrole, benzaldehyde and catalyst is 1:1-3:1-3.

[0014] Preferably, in step 1, the molar ratio of 2,4-dimethylpyrrole, benzaldehyde and catalyst is 1:1.5:1.5 or 1:3:3.

[0015] Preferably, the catalyst is an organic base; the organic base is triethylamine.

[0016] Preferably, the organic solvent in step 2 is anhydrous toluene.

[0017] The present invention also provides a special detection kit comprising the compound represented by formula I in claim 1 and a solvent.

[0018] The concentration of the compound represented by formula I is 1 mM,

[0019] The solvent is ethanol or dimethyl sulfoxide.

[0020] Preferably, a buffer solution is also included.

[0021] The buffer solution is a phosphate buffer solution with a pH value of 6.0 to 8.0, wherein the phosphate is selected from at least one of Na2HPO4, NaH2PO4 and KH2PO4; and the concentration of the phosphate is 0.01 to 0.5M.

[0022] The application of the microenvironment polarity detection kit provided by the present invention in measuring microenvironment polarity, especially in detecting changes in the microenvironment polarity of a biological system, falls within the protection scope of the present invention.

[0023] The method for determining the polarity of a microenvironment comprises the following steps:

[0024] 1) Detecting changes in polarity in the sample to be tested:

[0025] a) Polarity-responsiveness testing was performed using 645 nm as the excitation wavelength and 680 nm as the emission wavelength. Organic solvents of varying polarity and mixed systems of 1,4-dioxane and water in varying ratios were prepared. 10 μL of the probe BFZ mother solution (1 mmol / L) was added to a 1.5 mL EP tube. 990 μL of the solvents of varying polarity were then added to obtain a final volume of 1 mL. Spectral curves were plotted with wavelength as the horizontal axis and fluorescence intensity as the vertical axis.

[0026] In the above detection method, the volume of the series of solvents of different polarities is 990 μL, and in the mixed system of 1,4-dioxane and water in different proportions, the volume ratios of 1,4-dioxane and water are 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% respectively.

[0027] The volumes of the series of mixed solutions of 1,4-dioxane and water in different ratios were all 1 mL.

[0028] The volume ratio of the reagent stock solution in the polarity detection kit is 1 mL:10 μL.

[0029] The polarity test kit provided by the present invention has the following characteristics:

[0030] 1) Near-infrared emission wavelength (712nm), strong tissue penetration.

[0031] 2) Sensitive to polarity and not affected by viscosity or other biologically related substances.

[0032] 3) Fluorescence intensity and emission wavelength change with the change of polarity in the microenvironment.

[0033] Beneficial effects of the present invention:

[0034] The present invention synthesizes a polarity-sensitive probe using boron dipyrrole (BODIPY) as an electron acceptor (A) and Fischer's aldehyde as a donor (D), with the donor and acceptor connected by a π bridge. Based on the intramolecular charge transfer mechanism, the probe exhibits higher fluorescence intensity under low-polarity conditions and lower fluorescence intensity under high-polarity conditions, accompanied by a significant red shift. The probe also has lipid droplet targeting and can be used in cell experiments to specifically detect changes in the polarity of intracellular lipid droplets. Whether under physiological conditions or under drug stimulation, the probe can detect changes in the polarity of the organism's microenvironment.

[0035] The D-π-A structure gives the probe an intramolecular charge transfer effect, which makes the probe exhibit near-infrared emission and polarity-sensitive properties. The probe emits strong fluorescence in low-polarity solvents, while the fluorescence is significantly weakened in high-polarity solvents due to dipole-dipole interactions.

[0036] The excitation wavelength of this probe is 645nm and the emission wavelength is 680nm. It can be used for near-infrared imaging and has better imaging penetration depth and visualization effect.

[0037] This probe achieved stable imaging within 30 minutes, with a shorter imaging time and subsequent cell incubation time of 30 minutes.

[0038] In solutions of different polarities, the fluorescence intensity of this probe decreases and the emission wavelength also red-shifts as the polarity increases. The sensitivity to polarity can be determined based on the changes in both emission wavelength and fluorescence intensity, which is more sensitive to polarity than other probes. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0040] Figure 1 The chemical reaction equation for preparing the compound represented by formula I of the present invention;

[0041] Figure 2 The fluorescence spectra of the probe in water and 1,4-dioxane at different volume ratios are shown;

[0042] Figure 3 is the fluorescence emission spectrum of the probe in the presence of various interfering substances;

[0043] Figure 4 For the cytotoxicity test of this probe;

[0044] Figure 5 This probe detects different cell polarity changes;

[0045] Figure 6 To locate lipid droplets in cells for this probe;

[0046] Figure 7 To localize lipid droplets in zebrafish for this probe;

[0047] Figure 8 This probe is used to detect changes in cellular lipid droplet polarity under drug stimulation (oleic acid);

[0048] Figure 9 is the hydrogen spectrum of formula I (400 MHz, CDCl3, 298 K);

[0049] Figure 10 This is the carbon spectrum of formula I (100 MHz, CDCl3, 298K). DETAILED DESCRIPTION

[0050] The present invention is described below by means of specific examples, but the present invention is not limited thereto.

[0051] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and biological materials described are commercially available unless otherwise specified.

[0052] Example 1. Preparation of 5,5-difluoro-1,3,9-trimethyl-10-phenyl-7-((E)-3-((E)-1,3,3-trimethylindolin-2-ylidene)prop-1-en-1-yl)-5H-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaboronic acid shown in Formula I.

[0053] according to Figure 1The preparation was carried out according to the chemical reaction flow chart shown in FIG. 1 , and the operation steps were as follows: 2,4-dimethylpyrrole (374 μL, 3.6 mmol), benzaldehyde represented by formula III (190 μL, 1.8 mmol) and 90 μL of trifluoroacetic acid were added to a round-bottom flask containing tetrahydrofuran (7.5 ml), protected by inert gas, and stirred at room temperature for 12 hours. 2,3-dichloro-5,6-dicyanobenzoquinone (405 mg, 1.6 mmol) was added and stirred at room temperature for 4 hours for oxidation. Finally, 4.5 ml of triethylamine was added to create an alkaline environment, and boron trifluoride etherate (4.5 ml) was added dropwise and continued to stir at room temperature for 24 hours. After the reaction was completed, the sediment produced during the reaction was filtered off by suction. The obtained liquid was extracted three times with ethyl acetate and water, the organic layer was retained, and the volatiles were removed under reduced pressure on a rotary evaporator. The crude product was extracted, separated and purified by silica gel column chromatography, eluted with ethyl acetate: petroleum ether (v:v, 120:1) to obtain the compound represented by Formula II.

[0054] Fischer's aldehyde, fluoroboranedipyrimidine represented by Formula II, piperidine, magnesium perchlorate, and glacial acetic acid were placed in a 30 mL round-bottom flask in sequence. Anhydrous toluene was used as the solvent. The mixture was heated under reflux at 120° C. under the protection of an inert gas and stirred for 6 hours. After the reaction was complete, ethyl acetate was used for extraction. The organic layer was placed in a round-bottom flask and the solvent was removed under reduced pressure on a rotary evaporator. The crude product was separated and purified by column chromatography and separated with petroleum ether / ethyl acetate (v:v, 5:1) to obtain the compound represented by Formula I.

[0055] The structural characterization data of the product are as follows:

[0056] 1 H NMR (400MHz, CDCl3, 298K), δ7.58: (t, J=13.5Hz, 1H), 7.46 (d, J=5.2Hz, 3H), 7.30 (d, J=6.8Hz, 2H), 7.22–7.16 (m, 2H), 6.93-6.86 (m, 2H), 6.70(d,J=7.6Hz,1H),6.50(s,1H),5.92(s,1H),5.68(d,J=12.4Hz,1H),3.20(s,3H),2.56(s,3H),1.61(s,6H),1.43(s,3H),1.36(s,3H).

[0057] 13C NMR (100MHz, CDCl3, 298K), δ161.19,155.99,150.15,144.60,143.04,139.15,138.38,136.55,135.98,135.78,130.62,128.87,128.74,128 .58,127.94,121.65,120.59,119.51,117.88,114.04,106.79,98.43, 46.21,31.62,29.30,28.72,26.93,22.68,14.76,14.42,14.15,14.07.

[0058] Combined with the above Figure 8 , Figure 9 It can be seen that the product structure is correct, which is 5,5-difluoro-1,3,9-trimethyl-10-phenyl-7-((E)-3-((E)-1,3,3-trimethylindolin-2-ylidene)prop-1-en-1-yl)-5H-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaboronic acid as shown in Formula I. The carbon spectrum of Formula I is shown in FIG. Figure 10 shown.

[0059] Example 2: Fluorescence spectra of the compound represented by Formula I in the reaction of water and 1,4-dioxane in different volume ratios.

[0060] Weigh 4.5 mg of reagent 1 and prepare 7.5 mL of dimethyl sulfoxide solution as the mother solution (1 mM)

[0061] 10 μL of the probe BFZ stock solution (1 mmol / L) was added to a 1.5 mL EP tube. 990 μL of solvents of varying polarity were then added to obtain a final volume of 1 mL of the mixed solution. After incubation at 37°C for 30 minutes, the UV-visible absorption and fluorescence emission spectra were measured. Fluorescence emission spectra were measured at 645 nm with excitation and emission slit widths of 5 nm and a voltage of 700 V.

[0062] Figure 2 Fluorescence spectra of the probe reacting with water and 1,4-dioxane at different volume ratios.

[0063] Figure 2 The results show that the reagent 1 of the present invention has the following characteristics:

[0064] 1) The probe is light blue in solution and has low fluorescence, but as the volume ratio of 1,4-dioxane increases, the fluorescence intensity increases and the emission wavelength blue-shifts;

[0065] 2) The fluorescence intensity of the probe in 1,4-dioxane is much higher than that in water.

[0066] Example 3: Reaction of the compound represented by Formula I with other species

[0067] Add various substances into the buffer solution: (1) Pb 2+ (100μM); (2)Fe 3+ (100μM); (3) Cr 3+ (100 μM); (4) Mg 2+ (10μM); (5)Ca 2+ (100 μM); (6) Zn 2+ (100μM); (7)K + (10μM); (8)Na + (50μM); (9) Tyr (50U / mL); (10) NAC (100μM); (11) Hcy (100μM); (12) GSH (100μM); (13) Cys (10μM); (1 4)HSA(50μg / mL);(15)BSA(20μg / mL);(16)ALP(10U / mL);(17)VC(100μM);(18)Glu(10μM);(19)ONOO - (10 μM); (20)ClO - (42μM); (21)O 2- (40 μM); (22) 1 O2 (86μM); (23)·OH (100μM); (24) water; (25) Simvastatin (40μM); (26) Atorvaststin (40μM); (27) Rosuvastatin (40μM); (28)

[0068] 1,4-Dioxane. After reacting at 37°C for 30 minutes, the fluorescence emission spectrum was measured. The fluorescence emission spectrum was determined using an excitation wavelength of 645 nm, a slit width of 5 nm for both excitation and emission, and a voltage of 700 V.

[0069] To 1 mL of the solution containing the above-mentioned various substances, 10 μL of the stock solution of Reagent 1 (1 mM) was added.

[0070] Figure 3 The fluorescence emission spectra obtained when reagent 1 (10 μM) was mixed with various other substances.

[0071] The experimental results showed that only 1,4-dioxane could induce reagent 1 to produce an obvious light signal response, proving that the reagent was highly selective for polarity, while the presence of other species did not interfere with the determination of polarity.

[0072] Example 4: Cell Detection

[0073] 1) Cell culture:

[0074] L02, MCF-7, and PANC02-H7 cells were cultured in DMEM (1% penicillin-streptomycin, 10% fetal bovine serum, and 89% DMEM) in a 37°C incubator with 5% CO2. Cells were passaged every two days. A549, CNE, HTR-8, and HeLa cells were cultured in RPMI-1640 (1% penicillin-streptomycin, 10% fetal bovine serum, and 89% RPMI-1640) in a 37°C incubator with 5% CO2. Cells were passaged every two days.

[0075] 2) Toxicity assay

[0076] Cells were counted at 8×10 3 The cells were seeded into a 96-well plate at a density of 100 μL / well. After the cells were completely attached to the wall, 100 μL of BFZ solution of different concentrations (0, 3, 5, 7, 10, 15, 20 and 25 μM) was added to each well. Six replicates were set for each concentration of the probe for the experiment. The cells were then placed in a constant temperature incubator at 37°C and 5% carbon dioxide for 12 hours. The supernatant was then discarded, and 100 μL of 0.5 mg / mL MTT solution was added to each well and cultured in an incubator for 4 hours. Finally, 100 μL of dimethyl sulfoxide was added to each well to dissolve the formazan at the bottom. The absorbance value of each well was measured at a wavelength of 490 nm using an enzyme reader, as shown in FIG. Figure 4 .

[0077] The results showed that when the probe was used for cytotoxicity testing, the cell survival rate was higher than 90% in both normal cells and cancer cells when the probe concentration was 10 μM. When the probe concentration was increased to 25 μM, the cell activity still remained above 80%, indicating that the probe has good biocompatibility and low toxicity.

[0078] 3) Detecting changes in different cell polarity

[0079] Human breast cancer cells MCF-7 cells, human cervical cancer cells HeLa cells, human chorionic trophoblast cells HTR-8 cells, and human liver cancer cells L02 cells were divided into four groups and pretreated: (1) Before imaging, MCF-7 cells were digested with trypsin, collected, and counted. 5The cells were seeded at a density of 100 cells / well in a 20 mm confocal culture dish and incubated in a constant temperature incubator with set temperature and carbon dioxide concentration for 24 h. The cells were then incubated in a culture medium containing 1 μmol / L probe BFZ for 30 min. (2) Before imaging, HeLa cells were digested with trypsin, collected, and counted. The number of cells was 1.0 × 10 5 The cells were seeded at a density of 100 cells / well in a 20 mm confocal culture dish and incubated in a constant temperature incubator with set temperature and carbon dioxide concentration for 24 h. The cells were then incubated in a culture medium containing 1 μmol / L probe BFZ for 30 min. (3) Before imaging, HTR-8 cells were digested with trypsin, collected, and counted. The number of cells was 1.0 × 10 5 The cells were seeded at a density of 100 cells / well in a 20 mm confocal culture dish and incubated in a constant temperature incubator with set temperature and carbon dioxide concentration for 24 h. The cells were then incubated in a culture medium containing 1 μmol / L probe BFZ for 30 min. (4) Before imaging, L02 cells were digested with trypsin, collected, and counted. The number of cells was 1.0 × 10 5 The cells were seeded at a density of 1 μg / well in a 20 mm confocal culture dish and incubated in a constant temperature incubator with set temperature and carbon dioxide concentration for 24 h. The cells were then incubated in a culture medium containing 1 μmol / L probe BFZ for 30 min in the incubator.

[0080] After the cells were incubated with the probe, the culture medium was discarded and 1 mL of PBS was added to image the cells under a confocal laser microscope. The microscope was set to an excitation wavelength of 633 nm and an emission wavelength of 650-750 nm. Figure 5 .

[0081] The results showed that the fluorescence intensity of different cells detected by the kit was different according to the polarity of cancer cells and normal cells; Figure 5 The results showed that the polarity of cancer cells was lower than that of normal cells.

[0082] 4) Locating lipid droplets in cells

[0083] The cells were analyzed by using a PCR product containing probe BFZ (1 μmol / L) and commercial tracers for mitochondria (Rhodamine 123 1 μM), lysosomes (Lyso Tracker Green DND-26 500 nM), and endoplasmic reticulum (ER-Tracker TMGreen 200nM)), commercial lipid droplet tracer (BODIPY 493 / 503 1μM), and commercial nucleus tracer (Hoechst 33342 1μM) were co-incubated in A549 cells for 30 minutes. After the incubation time, the culture medium was discarded and the dishes were washed three times with PBS. Before imaging, 1mL of 1640 culture medium was added to each dish for imaging. The light source wavelengths collected by the above commercial tracers were: Rhodamine 123 (excitation wavelength of 496nm, emission wavelength collection range of 510-610nm), Lyso TrackerGreen DND-26 (excitation wavelength of 496nm, emission wavelength collection range of 510-610nm), ER-Tracker TM Green (excitation wavelength is 496nm, emission wavelength is collected in the range of 510-610nm), BODIPY 493 / 503 (excitation wavelength is 496nm, emission wavelength is collected in the range of 500-600nm), Hoechst 33342 (excitation wavelength is 405nm, emission wavelength is collected in the range of 420-490nm), such as Figure 6 .

[0084] The results showed that the colocalization coefficient of lipid droplets detected by the kit was higher than 0.8, and it increased to 0.90 after oleic acid stimulation. Figure 6 It can be seen that the kit designed by the present invention can be localized in lipid droplets in cells.

[0085] 5) Localizing lipid droplets in zebrafish

[0086] Zebrafish were fed in E3 embryo culture medium and divided into two groups. One group of zebrafish was fed normally, and the other group of zebrafish was co-incubated with probes BFZ (1 μmol / L) and BODIPY 493 / 503 (2 μmol / L) for 2 hours. After the incubation was completed, the culture medium was discarded, and the fish were washed with PBS three times. Finally, an appropriate amount of PBS was added and the fish were imaged under a confocal microscope. Figure 7 .

[0087] The results showed that when zebrafish were co-incubated with BODIPY493 / 503 and BFZ at room temperature, bright green (BODIPY493 / 503 fluorescence emission) and red (BFZ fluorescence emission) signals were observed in the corresponding channels, respectively. Furthermore, the high overlap between the two channels further demonstrated the promising potential of the probe BFZ for tracking zebrafish LDs. These results demonstrate that the probe can localize lipid droplets in cells and zebrafish.

[0088] 6) Detect changes in cell lipid droplet polarity under drug stimulation (oleic acid)

[0089] A549 cells and MCF-7 cells were digested with trypsin, and the cells were collected and counted at 1.0×10 5 The cells were seeded at a density of 100 μg / well in a 20 mm confocal culture dish. The two cell types were seeded in two culture dishes and treated as follows: (1) A549 cells: The control group was cultured normally with culture medium; the experimental group was incubated with 100 μM oleic acid for 30 min. Then, the cells were co-incubated with the probes BFZ (1 μmol / L) and BODIPY493 / 503 (2 μmol / L) for 30 min. After the incubation time, the culture medium was discarded and the dish was washed three times with PBS. Before confocal imaging, 1 mL of 1640 culture medium was added. The confocal excitation wavelength was 633 nm and the emission wavelength range was 650-750 nm. (2) MCF-7 cells: The control group was cultured normally with culture medium; the experimental group was incubated with 100 μM oleic acid for 30 min. Then, the probes BFZ (1 μmol / L) and BODIPY493 / 503 (2 μmol / L) were co-incubated for 30 min. After the incubation time was completed, the culture medium was discarded and the dish was washed 3 times with PBS. 1 mL of DMEM culture medium was added before confocal imaging. The confocal excitation wavelength was 633 nm and the emission wavelength range was 650-750 nm. Figure 8 .

[0090] The results showed that the kit can detect the changes in lipid droplet polarity after oleic acid treatment; Figure 8 It can be seen that the lipid droplet content in cells increased and the fluorescence intensity was enhanced after oleic acid treatment.

[0091] Finally, it should be noted that the above examples illustrate only the case where Reagent 1 was used as the fluorescent reagent, the concentrations of 1 and phosphate buffer in the reaction system were 10 μM and 10 mM, respectively, and the reaction time was 30 min. Results for other fluorescent reagent concentrations and reaction times are not listed here, but they are not intended to limit the present invention. Any person skilled in the art will readily be able to make various modifications and variations without departing from the spirit and scope of the present invention.

Claims

1. A method for preparing a fluorescent probe for detecting polarity, characterized in that: The following steps are involved: Step 1: Add 2,4-dimethylpyrrole, benzaldehyde represented by formula III and trifluoroacetic acid to a round-bottom flask containing tetrahydrofuran, protect with inert gas, stir at room temperature for 12 hours, add 2,3-dichloro-5,6-dicyanobenzoquinone, stir at room temperature for 4 hours for oxidation, finally add a catalyst, add boron trifluoride ether dropwise, and continue stirring at room temperature for 24 hours to obtain the compound represented by formula II. Step 2: Fischer's aldehyde, fluoroboranedipyrimidine of Formula II, piperidine, magnesium perchlorate, and glacial acetic acid are placed in a 30 mL round-bottom flask in sequence, and a condensation reaction is carried out in an organic solvent. The mixture is heated under reflux at 120° C. with stirring for 6 hours under the protection of an inert gas to obtain the compound of Formula I.

2. The method for preparing a fluorescent probe for detecting polarity according to claim 1, wherein: In step 1, the molar ratio of 2,4-dimethylpyrrole, benzaldehyde and catalyst is 1:1-3:1-3.

3. The method for preparing a fluorescent probe for detecting polarity according to claim 2, wherein: In step 1, the molar ratio of 2,4-dimethylpyrrole, benzaldehyde and catalyst is 1:1.5:1.5 or 1:3:

3.

4. The method for preparing a fluorescent probe for detecting polarity according to claim 1, wherein: The catalyst is an organic base; the organic base is triethylamine.

5. The method for preparing a fluorescent probe for detecting polarity according to claim 1, wherein: The organic solvent in step 2 is anhydrous toluene.

6. Use of the compound of formula I prepared in claim 1 in detecting changes in microenvironment polarity.

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