Vinyl quinoline fluorescent compound and application thereof

Abstract

The invention discloses a vinyl quinoline fluorescent compound and an application of the vinyl quinoline fluorescent compound. The fluorescent compound is (E)-1-(2-ethoxyl)-2-pyrene-1-vinyl quinoline-1-iodised salt. The fluorescent compound does not need to be washed, can avoid influence of washing on viscosity and damage to cells when being used for observing mitochondrial viscosity, and is wider in applicability.

Description

Technical Field

[0001] The invention relates to a vinylquinoline fluorescent compound and application thereof, belonging to the field of fluorescent compounds. Background Art

[0002] As a key parameter characterizing the internal friction of fluids, viscosity holds significant value in both chemical industry production and life science research. It not only influences the efficiency of mass and heat transfer in chemical production processes but also reflects the dynamic changes in the intracellular microenvironment. Viscosity is closely related to physiological processes such as biomacromolecule interactions, metabolite transport, and cellular signaling. Therefore, developing precise viscosity measurement methods is of great significance to both chemical engineering and life science research.

[0003] As the energy metabolism center of eukaryotic cells, the functional integrity of mitochondria is crucial for maintaining cellular homeostasis. Mitochondrial viscosity is an important indicator for assessing their functional status, sensitively reflecting mitochondrial respiratory chain activity, membrane fluidity, and matrix microenvironment stability. Studies have shown that abnormal mitochondrial viscosity often indicates functional impairment, and such changes are closely associated with the development and progression of various pathological processes.

[0004] Traditional viscosity detection techniques (such as rotational viscometry) usually require milliliter-level sample volumes and it is difficult to achieve in situ detection of viscosity at the subcellular level. In contrast, fluorescent probe technology based on molecular rotation has become an important tool for solution viscosity detection due to its advantages such as high sensitivity, rapid response and spatial resolution. However, existing viscosity-responsive mitochondrial fluorescent probes generally have problems such as limited response amplitude and insufficient mitochondrial targeting, making it difficult to accurately monitor dynamic changes in mitochondrial viscosity. In addition, the washing steps in the conventional staining process can easily cause disturbances in the cell microenvironment, affecting the reliability of the test results. Therefore, the development of wash-free fluorescent probes with mitochondrial targeting function has important scientific significance and application value for achieving accurate monitoring of mitochondrial viscosity in living cells. Summary of the Invention

[0005] Purpose of the invention: The first purpose of the present invention is to provide a vinylquinoline fluorescent compound; the second purpose of the present invention is to provide an application of the above-mentioned vinylquinoline fluorescent compound in the preparation of a reagent for detecting changes in liquid sample viscosity or mitochondrial viscosity.

[0006] Technical solution: The vinylquinoline fluorescent compound of the present invention has the structural formula:

[0007]

[0008] The synthetic route of the compound is:

[0009]

[0010] The vinylquinoline fluorescent compound of the present invention is used in preparing a reagent for detecting viscosity changes of liquid samples.

[0011] The vinylquinoline fluorescent compound of the present invention is used in preparing a reagent for detecting changes in mitochondrial viscosity.

[0012] The reagent for detecting changes in viscosity of a liquid sample or mitochondria of the present invention comprises the detection probe of the vinylquinoline fluorescent compound.

[0013] Furthermore, the vinylquinoline fluorescent compound does not require washing during detection.

[0014] Furthermore, the concentration of the detection probe is 1-15 μM.

[0015] Furthermore, the solvent of the reagent includes one or more combinations of DMF, DMSO, EtOH, H2O, MeCN or 1,4-dioxane.

[0016] The reagent of the present invention is used for imaging mitochondria of living cells cultured in vitro for purposes other than disease diagnosis or treatment.

[0017] Furthermore, the light excitation of the reagent during use is 400-600 nm, and the emission spectrum ranges from 500-700 nm.

[0018] Beneficial Effects: Compared with existing technologies, the present invention has the following significant advantages: Disclosed is a fluorescent compound, vinylquinoline fluorescent compound (E)-1-(2-hydroxyethyl)-2-pyrene-1-vinylquinoline-1-iodine salt, and its use in preparing a reagent for detecting changes in liquid samples or mitochondrial viscosity. This fluorescent compound does not require washing, thus avoiding the effects of washing on viscosity and damage to cells when used to observe mitochondrial viscosity, and has a wider applicability. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Absorption and fluorescence spectra of QP (10 μM) in different solvents.

[0020] Figure 2 Absorption and fluorescence spectra of QP (10 μM) in Gly-H2O solutions with different ratios.

[0021] Figure 3 is the fluorescence intensity of QP (10 μM) at 615 nm in different ions. + ,3.Na + ,4.Zn 2+ ,5.Ca 2+,6.F - ,7.Cl - ,8.Br - ,9.HCO3 - ,10.NO3 - ,11.NO2 - ,12.HPO4 2- ,13.CO3 2- ,14.SO3 2- ,15.PO4 3- , 16.10μM Cys, 17.10μM glucose, 18.10μM H2O2. Anion and cation concentrations are: 100μM.

[0022] Figure 4 This is a confocal fluorescence image of HeLa cells co-stained with QP (2 μM, 15 min) and the commercial mitochondrial deep red fluorescent probe MTDR (0.2 μM, 15 min).

[0023] Figure 5 Confocal fluorescence images (A) and fluorescence intensity (B) of normal and nystatin-treated HeLa cells stained with QP (2 μM, 15 min). DETAILED DESCRIPTION

[0024] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0025] The absorption spectrum was measured using a Hitachi U-2910 spectrophotometer, the fluorescence spectrum was measured using a Hitachi F-2700 spectrophotometer, and the cell imaging instrument was a Lecia confocal microscope.

[0026] In the following examples, the materials and reagents used were obtained from commercial sources unless otherwise specified.

[0027] Example 1 Synthesis of fluorescent compounds

[0028] (E)-1-(2-Hydroxyethyl)-2-pyrene-1-vinylquinoline-1-iodine salt (i.e., compound QP)

[0029] The reaction route is as follows:

[0030]

[0031] 1) Synthesis of quinoline iodide salt (Compound 2)

[0032] Dissolve compound 1 (2-methylquinoline, 10 mmol, 1.43 g) and iodoethanol (1.76 mL, 10 mmol) in 25 mL of anhydrous ethanol in a flask and stir at room temperature for 1 hour. Reflux the mixture at 100°C for 8 hours, cool, filter, and wash three times with anhydrous EtOH. Dry to obtain a brown solid, compound 2.

[0033] 2) Synthesis of compound QP

[0034] Compound 2 (0.315 g, 1 mmol) and compound 3 (0.23 g, 1 mmol) were dissolved in 25 mL of methanol and stirred in a flask for 1 hour. Two drops of piperidine were added. After stirring, the mixture was refluxed at 90°C for 10 hours. After cooling to room temperature, it was washed with petroleum ether. Column chromatography using a CH2Cl2 / CH3OH mixture (12:1 to 7:1, v / v) as the eluent yielded a yellow solid, Compound QP.

[0035] 1 H NMR (400MHz, DMSO-d6) δ (ppm): 9.28 (d, J = 16.0Hz, 1H), 9.18 (d, J = 8.0Hz, 1H), 9.04 (q, J = 10.6Hz, 2H), 8.87 (d, J = 8.0Hz, 1H), 8. 64(d,J=8.0Hz,1H),8.30-8.49(m,8H),8.19(q,J=8.0Hz,2H),7.99(t,J=8.0Hz,1H),5.53-5.42(m,3H),4.14(d,J=4.0Hz,2H). 13 C NMR (100MHz, DMSO-d6), δ (ppm): 156.84, 144.70, 143.09, 139.21, 135.27, 133.46, 131.26, 130.66, 130.39, 129.76, 129.45 ,129.12,128.74,127.86,127.30,127.06,126.80,125.92,125.70,124.48,124.05,123.54,122.34,119.97,60.05,53.35.

[0036] Example 2 Photophysical properties test experiment

[0037] Test solutions containing 10 μM QP in different solvents were prepared using DMF, DMSO, EtOH, Gly, H2O, MeCN, and 1,4-dioxane. The absorption spectra of the above solutions were measured using a UV-visible spectrophotometer, and the fluorescence emission spectra were measured using a fluorescence spectrometer. The results are shown in Figure 1 .

[0038] It can be seen from the figure that the fluorescent compound QP has an absorption peak at around 460nm, and the absorption peak range is 400-600nm (see Figure 1 A). It has a fluorescence peak in the range of 500-700nm (see Figure 1 B), but not obvious in H2O and 1,4-dioxane. The above results show that the fluorescent compound can be excited by 400-600nm light and its emission spectrum ranges from 500-700nm.

[0039] Example 3 Viscosity Test Experiment of Fluorescent Compound QP

[0040] (1) Take the organic fluorescent compound QP prepared in Example 1 and prepare a probe stock solution with a concentration of 1 mM using DMSO;

[0041] (2) Glycerol and water were mixed in different proportions (0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% glycerol by volume) to prepare mixed solutions with different viscosity values;

[0042] (3) adding the probe stock solution prepared in step (1) to the mixed solutions of different viscosity values ​​prepared in step (2) to prepare test solutions, so that the final concentration of the fluorescent probe QP in each solution is 10 μM;

[0043] (4) The above solution was tested for absorption and fluorescence emission spectra using a UV-visible spectrophotometer and a fluorescence spectrometer to obtain corresponding curves. The absorption wavelength was 400-600 nm, and the emission spectrum wavelength was 550-750 nm.

[0044] The absorption and fluorescence spectra of QP (10 μM) in Gly-H2O solutions with different ratios are shown in Figure 2 ,from Figure 2 As can be seen from A, with the increase of Gly concentration, the absorbance value changes little and has no regularity. Figure 2 As can be seen in B, as the Gly ratio increases, the fluorescence intensity increases, which indicates that the probe has a significant response to viscosity.

[0045] Example 4 Experiment on the specific response of fluorescent compound QP to viscosity

[0046] (1) Take QP and prepare a probe stock solution with a concentration of 1 mM using DMSO;

[0047] (2) Prepare PBS solutions of different ions and other analytes (including K + 、Na + 、Zn 2+ , Ca 2+ 、F -、Cl - Br - 、HCO3 - 、NO3 - 、NO2 - 、HPO4 2- 、CO3 2- 、SO3 2- PO4 3- , 10 μM Cys, 10 μM glucose, and 10 μM H2O2), where the anion and cation concentrations were 100 μM;

[0048] (3) adding the probe stock solution prepared in step (1) to the solution prepared in step (2) to prepare a test solution, so that the final concentration of the fluorescent probe QP in each solution is 10 μM;

[0049] (4) The fluorescence emission spectrum of the above solution is tested using an ultraviolet-visible spectrophotometer and a fluorescence spectrometer to obtain the corresponding curve.

[0050] Measure the fluorescence intensity at 615 nm (fluorescence peak position) and obtain Figure 3 The bar chart shown.

[0051] As can be seen from the figure, the fluorescence intensity does not change significantly among different ions and other analytes, which indicates that the probe can respond specifically to viscosity, further proving that the probe can detect solution viscosity in complex environments.

[0052] Example 5 Experiment of Probe Staining Cells

[0053] Human cervical cancer cells (HeLa) were obtained from Thermo Fisher Scientific.

[0054] HeLa cells were adherently cultured in high-glucose culture medium (ThermoFisher) containing 10% fetal bovine serum in a 37°C, 5% CO2 saturated humidity incubator. The culture medium was replaced every 2-3 days and subculture was performed.

[0055] When the cells grow to the logarithmic phase, culture them in sections:

[0056] ① Soak the coverslip in anhydrous ethanol for 30 minutes, dry it with an alcohol burner, and place it in a disposable 35mm Petri dish for later use;

[0057] ② Wash the cells in the 100 mL cell flask three times with PBS, digest them with 1 mL of 0.25% trypsin (Gibco) for 5 minutes, carefully pour out the trypsin, add fresh culture medium, pipette evenly and count the cells. The cell density is controlled by the amount of culture medium added to make the final cell concentration of 1×10 cells per mL. 5Then, inoculate the culture dish containing the cover glass and culture it in a 5% CO2 incubator to allow the cells to grow closely to the culture dish. After the HeLa cells have grown and covered the cover glass, they are used for cell experiments.

[0058] A 1 mM probe stock solution was prepared in DMSO. Active HeLa cells were incubated in a culture medium containing 2 μM QP (high-glucose culture medium with 10% fetal bovine serum, ThermoFisher) (incubation conditions: 37°C, 5% CO2) for 15 minutes. A 0.2 μM MTDR solution was then added and the cells were stained for 15 minutes. The cells were then observed using a laser confocal microscope. The staining locations, fluorescence distribution and brightness changes, and colocalization information were recorded. The results are shown in the table. Figure 4 .

[0059] in, Figure 4 Confocal microscopy images of viable HeLa cells stained with the probes QP (2μM, 15 minutes) and MTDR (0.2μM, 30 minutes). QP was excited at 488nm, with fluorescence collected between 500-630nm. MTDR was excited at 638nm, with fluorescence collected between 640-740nm. As can be seen, the two probes exhibit significant overlap, with a colocalization coefficient of 85%, indicating that the probes stain mitochondria.

[0060] Example 6: Observation of Cell Viscosity Changes Using Probe QP

[0061] The patch culture steps are the same as those in Example 5. After patch culture, a probe mother solution with a concentration of 1 mM is prepared with DMSO. The cultured active HeLa cells are incubated in a culture medium containing 2 μM QP (high-glucose culture medium containing 10% fetal bovine serum, ThermoFisher) (incubation conditions: 37°C, 5% CO2) for 15 minutes, 0.5 mM nystatin (to enhance cell mitochondrial viscosity) solution is added, treated for 45 minutes, and observed with a laser confocal microscope. The control group was only stained without any treatment. The stained area, fluorescence distribution and brightness changes in the cells were recorded. The results are shown in the table. Figure 5 .

[0062] in, Figure 5 A is a confocal microscopy image of normal HeLa cells and HeLa cells treated with 0.5 mM nystatin to enhance mitochondrial viscosity stained with probe QP (2 μM, 15 min). Figure 5 B is a fluorescence intensity control. The excitation wavelength of QP in the green light channel is 488 nm, and the fluorescence collection wavelength is 520-630 nm. Over time, the fluorescence intensity of the QP probe increases, confirming that this probe can monitor changes in intracellular viscosity.

[0063] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes may be made to it in form and detail without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. A vinylquinoline fluorescent compound, characterized in that The structural formula of the vinylquinoline fluorescent compound is:

2. The vinylquinoline fluorescent compound according to claim 1, wherein The synthetic route of the compound is:

3. Use of the vinylquinoline fluorescent compound according to claim 1 in preparing a reagent for detecting changes in viscosity of a liquid sample.

4. Use of the vinylquinoline fluorescent compound according to claim 1 in the preparation of a reagent for detecting changes in mitochondrial viscosity.

5. A reagent for detecting changes in liquid sample or mitochondrial viscosity, characterized in that: The detection probe in the reagent is the vinylquinoline fluorescent compound according to claim 1.

6. The reagent according to claim 5, characterized in that The vinylquinoline fluorescent compound does not require washing during detection.

7. The reagent according to claim 5, characterized in that The concentration of the detection probe is 1-15 μM.

8. The reagent according to claim 5, characterized in that The solvent of the reagent includes one or more combinations of DMF, DMSO, EtOH, H2O, MeCN or 1,4-dioxane.

9. Use of the reagent according to any one of claims 5 to 8 for imaging mitochondria in living cells cultured in vitro for purposes other than disease diagnosis or treatment.

10. The use according to claim 9, characterized in that The light excitation of the reagent during use is 400-600 nm, and the emission spectrum ranges from 500-700 nm.

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