A polar-viscosity sensitive small molecule fluorescent probe for lipid droplet visualization, a preparation method and application thereof

Abstract

The application provides a new type of small molecule fluorescent probe for polar-viscosity sensitive lipid droplet visualization, a preparation method and application thereof, the fluorescent probe is 7-(4-(dimethylamino)phenyl)-2,3-dihydrothieno[3,4-b][1,4]dioxane-5-formaldehyde; the application develops a new type of polar and viscosity sensitive fluorescent probe with a novel pi bridge structure, has good selectivity, strong anti-interference ability, and various interference solvents have little influence on the probe; the probe has a large stokes shift, low background interference, high sensitivity, and can be better applied to biological imaging; the application realizes visualization of lipid droplets.

Description

Technical Field

[0001] The present invention relates to a fluorescent probe for dual-color visualization of polarity-viscosity sensitive lipid droplets, as well as spectral testing and cell imaging; and belongs to the field of organic small molecule fluorescent probes. Background Art

[0002] In biological systems, polarity, viscosity, redox state, and internal environmental temperature are crucial for maintaining the normal physicochemical properties of biomolecules. As two extremely important microenvironment-related parameters, polarity and viscosity play a crucial role in various physiological processes within cells. Each organelle resides in an optimal microenvironment for its normal physiological activities, which results in different polarity and viscosity for each organelle. Because lipid droplets provide a more hydrophobic and viscous environment compared to the cytosol, fluorescent probes sensitive to polarity or viscosity can serve as potential tools for targeting lipid droplets for imaging or functional studies. Abnormal lipid droplet metabolism is often closely associated with cancer, diabetes, obesity, viral proliferation, and other diseases. Therefore, studying lipid droplets in cells is highly necessary.

[0003] Fluorescence imaging has the advantages of being non-invasive, highly sensitive, highly selective, allowing real-time observation, high-resolution imaging, and multi-channel imaging, making it a promising approach for broad application in life science research. Many fluorescent probes for imaging lipid droplets alone have been reported recently, but few probes for simultaneously imaging LDs based on their polarity and viscosity have been reported.

[0004] CN114213388B discloses the use of a fluorescent probe based on a thiophene compound for detecting polarity and viscosity values, but the fluorescent probe is not sensitive enough to polarity and viscosity and does not accurately locate lipid droplets in HeLa cells. Summary of the Invention

[0005] In response to the shortcomings of the existing technology, the present invention uses 3,4-ethylenedioxythiophene as a new π bridge to synthesize a molecular push-pull type (D-π-A) fluorescent probe that can visualize the polarity and viscosity of lipid droplets, achieving the following invention objectives:

[0006] Provides a lipid droplet visualization fluorescent probe with high sensitivity to polarity and viscosity, and precise positioning of lipid droplets in HeLa cells.

[0007] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0008] A polarity-viscosity sensitive small molecule fluorescent probe for lipid droplet visualization, the chemical structure of the fluorescent probe is as follows:

[0009]

[0010] The fluorescent probe is prepared by dissolving 2-bromo-3,4-ethylenedioxythiophene-5-carboxaldehyde, 4-(dimethylamino)phenylboronic acid pinacol ester, {[1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II)} and potassium acetate in a 1,4-dioxane solution, reacting the mixture at 78-82° C. for 11.5-12.5 hours in a N2 atmosphere, and filtering, extracting, drying and purifying the obtained crude product to obtain the probe 7-(4-(dimethylamino)phenyl)-2,3-dihydrothieno[3,4-b][1,4]dioxane-5-carboxaldehyde.

[0011] The method of filtration, extraction, drying and purification comprises filtering the crude product to remove potassium acetate, extracting with dichloromethane and water, drying the obtained organic phase, and purifying by chromatography.

[0012] The molar ratio of the 2-bromo-3,4-ethylenedioxythiophene-5-carboxaldehyde to 4-(dimethylamino)phenylboronic acid pinacol ester is 1:0.9-1.1; the molar ratio of the 2-bromo-3,4-ethylenedioxythiophene-5-carboxaldehyde to {[1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloride} is 10:0.9-1.1; the molar ratio of the 2-bromo-3,4-ethylenedioxythiophene-5-carboxaldehyde to potassium acetate is 1:1.2-1.3; and the mass volume ratio of the potassium acetate to 1,4-dioxane is 1 g:31-33 mL.

[0013] The preparation method of 2-bromo-3,4-ethylenedioxythiophene-5-carboxaldehyde comprises suspending 2-(3,4-ethylenedioxythiophene) carboxaldehyde in acetonitrile, cooling the mixture to -0.8 to 0.2°C, adding N-bromosuccinimide, and reacting the mixture at room temperature for 58 to 62 hours to obtain an orange transparent solution mixture, which is then extracted, dried, and purified.

[0014] The extraction, drying and purification method comprises adding ethyl acetate to the mixture, extracting it twice with a sodium carbonate aqueous solution, then extracting it twice with water, drying it, and then purifying it by chromatography.

[0015] The molar ratio of the 2-(3,4-vinyldioxythiophene)formaldehyde to N-bromosuccinimide is 1:1.05-1.15; the mass volume ratio of the 2-(3,4-vinyldioxythiophene)formaldehyde to acetonitrile is 1 g:24-26 mL.

[0016] The application of the fluorescent probe in the preparation of biological imaging reagents includes fluorescence spectrum detection and fluorescence cell imaging.

[0017] The synthetic route of the probe EDOT-LDs is as follows:

[0018]

[0019] Compared with the prior art, the present application has the following beneficial effects:

[0020] The present application develops a new type of polar and viscosity sensitive fluorescent probe with a π bridge structure, which has good selectivity, strong anti-interference ability and little influence of various interference solvents on the probe.

[0021] The probe has a large stokes shift, low background interference, high sensitivity to viscosity and can be better applied to biological imaging.

[0022] The present application realizes visualization of lipid droplets, and the probe can be used for monitoring changes of lipid droplets in a biological system based on the fact that it presents yellow in lipid droplets. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The UV absorption and fluorescence spectrum of the probe EDOT-LDs in different solvents are shown in the following figures. 1 HNMR spectrum.

[0024] Figure 2 The UV absorption and fluorescence spectrum of the probe EDOT-LDs in different solvents are shown in the following figures.

[0025] In the figures, (a) is the UV absorption spectrum, Figure 2 (b) is the fluorescence spectrum. Figure 2

[0026] Figure 3 The fluorescence spectrum and the fold line graph of the fluorescence intensity of the probe EDOT-LDs with the change of polarity are shown in the following figures.

[0027] In the figures, (a) is the fluorescence spectrum of the probe EDOT-LDs with the change of polarity, Figure 3 (b) is the fold line graph of the fluorescence intensity of the probe EDOT-LDs in different proportions of methanol / 1,4-dioxane mixed solvents.

[0028] Figure 3 (b) is the fold line graph of the fluorescence intensity of the probe EDOT-LDs in different proportions of methanol / 1,4-dioxane mixed solvents.

[0029] Figure 4 The fluorescence spectrum and the fold line graph of the fluorescence intensity of the probe EDOT-LDs with the change of viscosity are shown in the following figures.

[0030] In the figures, (a) is the fluorescence spectrum of the probe EDOT-LDs with the change of viscosity, Figure 4 (b) is the fold line graph of the fluorescence intensity of the probe in different proportions of methanol / glycerol mixed solvents.

[0031] Figure 4 (b) is the fold line graph of the fluorescence intensity of the probe in different proportions of methanol / glycerol mixed solvents.

[0032] Figure 5 The fluorescence spectrum and the column chart of the probe EDOT-LDs with the change of pH are shown in the following figures.​

[0033] in Figure 5 (a) shows the fluorescence spectrum of the probe EDOT-LDs as the pH changes;

[0034] Figure 5 (b) is a bar graph of the fluorescence intensity of the probe EDOT-LDs in solutions with different pH values;

[0035] Figure 6 Selectivity profile and histogram of probe EDOT-LDs;

[0036] in Figure 6 (a) is the selectivity map of the probe EDOT-LDs; Figure 6 (b) is the selectivity bar graph of the probe EDOT-LDs;

[0037] Figure 6 The interfering solvents corresponding to the curves from bottom to top in (a) are glutathione, Na2CO3, MgSO4, MgCl2, Na3HSO, valine, K3PO4, ZnCl2, blank, CaCl2, HCl, arginine, and H2O2;

[0038] Figure 6 In (b), 1. blank; 2. valine; 3. glutathione; 4. ZnCl2; 5. Na2CO3; 6. MgSO4; 7. MgCl2; 8. arginine; 9. K3PO4; 10. Na3HSO4; 11. HCl; 12. H2O2; 13. CaCl2;

[0039] Figure 7 Fluorescence imaging of lipid droplets in cells using the probe EDOT-LDs;

[0040] in Figure 7 A in the image is the BODIPY channel: λ ex =405nm,λ em =470–500nm; Figure 7 B in the figure is the EDOT-LDs channel image: λ ex =405nm,λ em =530–570 nm (yellow channel); Figure 7 C in Figure 7 A and Figure 7 The merged image of B in ; Figure 7 D in the figure is the bright field image; Figure 7 E in the figure is the correlation image of EDOT-LDs and BODIPY intensity; Figure 7 F in the figure is the confocal curve image of EDOT-LDs and BODIPY. DETAILED DESCRIPTION

[0041] The present invention will be further described below with reference to the embodiments and drawings, but the present invention is not limited to the following embodiments.

[0042] Compound 1 is 2-(3,4-vinyldioxythiophene)carboxaldehyde;

[0043] Compound 2 is 2-bromo-3,4-ethylenedioxythiophene-5-carbaldehyde.

[0044] Example 1 Synthesis of probe compound EDOT-LDs

[0045]

[0046] (1) Preparation of compound 2

[0047] Compound 1 (2.0 g, 11.8 mol) was suspended in dry acetonitrile (50 mL), cooled to 0°C, and N-bromosuccinimide (2.3 g, 12.9 mol) was added. The mixture was reacted at room temperature for 60 h to obtain an orange transparent solution. The mixture was transferred to a separatory funnel along with 150 mL of ethyl acetate and extracted twice with 10% aqueous Na2CO3 (100 mL each) and then with water (100 mL each). Anhydrous sodium sulfate (200 g) was then added and stirred to dryness for 1 h. The anhydrous sodium sulfate solid was removed by filtration using a Buchner funnel. The mixture was then purified by column chromatography using petroleum ether and ethyl acetate (50:1 by volume) as the eluent to obtain compound 2 in a 62% yield.

[0048] (2) Preparation of compound 3

[0049] 2-Bromo-3,4-ethylenedioxythiophene-5-carbaldehyde (2 mmol, 0.4958 g), 4-(dimethylamino)phenylboronic acid pinacol ester (2 mmol, 0.5943 g), {[1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II)} (0.2 mmol, 0.1463 g) and potassium acetate (2.5 mmol, 0.2453 g) were dissolved in 1,4-dioxane (8 mL) solution and reacted at 80°C under N2 atmosphere for 12 h. The crude product was filtered to remove potassium acetate and extracted with dichloromethane (400 mL) and water (200 mL). The organic phase after extraction was freed from moisture with anhydrous sodium sulfate (150 g) and purified by column chromatography using petroleum ether and ethyl acetate (volume ratio of 12:1) as eluents to obtain the probe EDOT-LDs, i.e., 7-(4-(dimethylamino)phenyl)-2,3-dihydrothieno[3,4-b][1,4]dioxane-5-carbaldehyde, with a yield of 53%.

[0050] Example 2 Determination of Absorption and Fluorescence Spectra of Probe EDOT-LDs in Different Solvents

[0051] The probe EDOT-LDs prepared in Example 1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 mmol / L stock solution. 20 μL of the stock solution was taken each time and added to 2 mL of different solvents to perform fluorescence intensity and UV absorption tests. Figure 2 As shown in (b) and Table 1, as the polarity increases, the maximum emission wavelength increases from 481 nm (blue light range) to 590 nm (yellow light range), red-shifting by 109 nm.

[0052] Table 1 Maximum UV absorption and maximum emission wavelength of EDOT-LDs in various organic solvents

[0053]

[0054] Example 3 Determination of the Fluorescence Spectrum of Probe EDOT-LDs as It Changes with Polarity

[0055] The probe EDOT-LDs prepared in Example 1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 mmol / L stock solution. 20 μL of the stock solution was taken each time and added to 2 mL of a methanol / 1,4-dioxane mixed solvent of different proportions. The fluorescence properties were measured at the excitation wavelength λ ex =405nm, voltage U=440V, fluorescence spectrum is as follows Figure 3 As shown in Figure 3, as the volume percentage of methanol in the mixed solvent increases from 0% to 100% (the polarity of the solution gradually increases), the fluorescence intensity in the spectrum gradually decreases, accompanied by a red shift in the maximum emission.

[0056] Figure 3 In (a), the curves from top to bottom correspond to the volume percentages of methanol in the methanol / 1,4-dioxane mixed solvent, respectively: 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%.

[0057] Example 4 Determination of Fluorescence Spectra of Probe EDOT-LDs as Viscosity Changes

[0058] The probe EDOT-LDs prepared in Example 1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 mmol / L stock solution. 20 μL of the stock solution was taken each time and added to 2 mL of a methanol / glycerol mixture of different volume ratios to measure its fluorescence properties. ex =405nm, voltage U=530V, fluorescence spectrum is as follows Figure 4As shown in (a), as the volume percentage of glycerol in the mixed solvent increases from 0% to 100%, the viscosity gradually increases, the fluorescence intensity in the spectrum gradually increases, and is accompanied by a red shift in the maximum emission.

[0059] Figure 4 In (a), the curves from bottom to top correspond to the volume percentages of glycerol in the methanol / glycerol mixed solvent, respectively: 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100%.

[0060] Example 5 Determination of Spectral Data of Probe EDOT-LDs as a Function of pH

[0061] The probe EDOT-LDs prepared in Example 1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 mmol / L stock solution. 20 μL of the stock solution was taken each time, 1 mL of 1,4-dioxane was added, and then 1 mL of PBS buffer solution of different pH values ​​was added. The fluorescence properties were measured, and the excitation wavelength λ ex =405nm, voltage U=530V, fluorescence spectrum is as follows Figure 5 As shown by Figure 5 It can be seen that after adding the probe EDOT-LDs, the influence of different pH on the probe is also very small.

[0062] Example 6 Probe EDOT-LDs Selectivity Profile Determination

[0063] The probe EDOT-LDs prepared in Example 1 was dissolved in dimethyl sulfoxide (DMSO) to make a 1 mmol / L stock solution. All 10 mmol / L interfering solutions were prepared with deionized water. 20 μL of each stock solution was added to 2 mL of DMSO:PBS (1:1 volume ratio), and 20 μL of each interfering solution was added. The excitation wavelength λ ex =405nm, emission wavelength is 571nm, and the fluorescence spectrum is as follows Figure 6 As shown. Figure 6 It can be seen that after adding the probe EDOT-LDs, the influence of various interfering solvents on the probe is also very small.

[0064] Example 7: Cellular Imaging of Lipid Droplets in Cells Using EDOT-LDs

[0065] The probe EDOT-LDs was applied to dual-channel imaging of lipid droplets and lysosomes in HeLa cells ( Figure 7 The specific operation steps are as follows: EDOT-LDs (final concentration of 10 μM) and commercial dye BODIPY (final concentration of 500 nM) were added to the HeLa cell culture medium (initial cell density of 1×10 4 / ml) and co-cultured for 30 minutes, and then the imaging of lipid droplets in the cells was detected, which proved that EDOT-LDs could enter the cells and target lipid droplets.

Claims

1. A polarity-viscosity sensitive small molecule fluorescent probe for lipid droplet visualization, characterized by: The fluorescent probe is 7-(4-(dimethylamino)phenyl)-2,3-dihydrothieno[3,4-b][1,4]dioxane-5-carbaldehyde, and its chemical structure is as follows: 。 2. A method for preparing a polarity-viscosity sensitive small molecule fluorescent probe for lipid droplet visualization, characterized by: The fluorescent probe is prepared by dissolving 2-bromo-3,4-ethylenedioxythiophene-5-carboxaldehyde, 4-(dimethylamino)phenylboronic acid pinacol ester, {[1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II)} and potassium acetate in a 1,4-dioxane solution, reacting the mixture at 78-82° C. for 11.5-12.5 hours in a N2 atmosphere, and filtering, extracting, drying and purifying the obtained crude product to obtain the probe 7-(4-(dimethylamino)phenyl)-2,3-dihydrothieno[3,4-b][1,4]dioxane-5-carboxaldehyde.

3. The preparation method according to claim 2, wherein: The molar ratio of the 2-bromo-3,4-ethylenedioxythiophene-5-carboxaldehyde to 4-(dimethylamino)phenylboronic acid pinacol ester is 1:0.9-1.1; the molar ratio of the 2-bromo-3,4-ethylenedioxythiophene-5-carboxaldehyde to {[1,1'-bis(diphenylphosphino)ferrocene]palladium(II) dichloride} is 10:0.9-1.1; the molar ratio of the 2-bromo-3,4-ethylenedioxythiophene-5-carboxaldehyde to potassium acetate is 1:1.2-1.3; and the mass volume ratio of the potassium acetate to 1,4-dioxane is 1 g:31-33 mL.

4. The preparation method according to claim 2, wherein: The preparation method of 2-bromo-3,4-ethylenedioxythiophene-5-carboxaldehyde comprises suspending 2-(3,4-ethylenedioxythiophene) carboxaldehyde in acetonitrile, cooling the mixture to -0.8 to 0.2°C, adding N-bromosuccinimide, and reacting the mixture at room temperature for 58 to 62 hours to obtain an orange transparent solution mixture, which is then extracted, dried, and purified.

5. The preparation method according to claim 4, characterized in that: The molar ratio of the 2-(3,4-vinyldioxythiophene)formaldehyde to N-bromosuccinimide is 1:1.05-1.15; the mass volume ratio of the 2-(3,4-vinyldioxythiophene)formaldehyde to acetonitrile is 1 g:24-26 mL.

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