Small-molecule fluorescent probe compound as well as preparation method and application thereof

Abstract

The invention relates to the technical field of fluorescence imaging, and discloses a small-molecule fluorescence probe compound as well as a preparation method and application thereof. The small-molecule fluorescent probe compound provided by the invention can be combined with mitochondrial OPA1 protein to be positioned in mitochondria and generate green fluorescence, and is relatively weak in sensitivity to mitochondrial membrane potential; the compound has relatively low cytotoxicity and relatively strong photobleaching resistance, and can be applied to ultrahigh-resolution microscope imaging to realize visual monitoring of mitochondria; the preparation method of the micromolecular fluorescent probe compound is simple in steps and high in yield; comprising a fluorescent probe of the micromolecular fluorescent probe compound, can be applied to mitochondrial tracing in fixed cells or tissue slices and ultra-high resolution microscope imaging of SIM, STED and the like, and has a wide application space in biological function research of lysosomes and mitochondria.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescence imaging, and particularly relates to a small molecule fluorescent probe compound, a preparation method thereof, and an application thereof. Background Art

[0002] A small molecule fluorescent probe is a molecule that changes its fluorescence emission in response to a binding event, a chemical reaction, or a direct environmental change, and is widely used in drug discovery, cell imaging, environmental analysis, and various medical applications. It has become a powerful tool for uses such as cell biology research, new drug R & D, environmental pollutant detection, and cancer detection.

[0003] In the field of cell research, positively charged probes are usually used to study mitochondrial function and activity within cells. However, the targeting effect of positively charged probes on mitochondria is affected by various factors, such as mitochondrial membrane potential and intracellular ion concentration. In an unstable environment, the probe may drift or separate from mitochondria, losing its mitochondrial localization ability, resulting in signal distortion or attenuation, thereby affecting the accuracy and reliability of measurement results. Therefore, developing new mitochondrial probe targeting strategies to achieve accurate and reliable localization and monitoring of mitochondria is of great significance for biological research and drug development in the field of mitochondria. Summary of the Invention

[0004] The present invention aims to at least solve the above-mentioned technical problems existing in the prior art. For this reason, one of the purposes of the present invention is to provide a small molecule fluorescent probe compound;

[0005] The second purpose of the present invention is to provide a preparation method of this small molecule fluorescent probe compound.

[0006] The third purpose of the present invention is to provide a fluorescent probe.

[0007] The fourth purpose of the present invention is to provide an application of the small molecule fluorescent probe compound or the fluorescent probe.

[0008] The fifth purpose of the present invention is to provide a method for monitoring the mitochondrial fusion process.

[0009] In order to achieve the above purposes, the technical solutions adopted by the present invention are as follows:

[0010] The first aspect of the present invention provides a small molecule fluorescent probe compound, and its structural formula is shown in formula (Ⅰ):

[0011]

[0012] Wherein, R - is selected from a halogen ion, a p-toluenesulfonate group, a trifluoromethanesulfonate group, a tetrafluoroborate group, a hexafluorophosphate group, a bis(trifluoromethanesulfonyl)imide group;

[0013] R 1 selected from

[0014] In some embodiments of the present invention, the R - is selected from halide ions, p-toluenesulfonate or trifluoromethanesulfonate, tetrafluoroborate, hexafluorophosphate, bis(trifluoromethanesulfonyl)imide.

[0015] In some specific embodiments of the present invention, the R - is selected from iodide ions, p-toluenesulfonate or trifluoromethanesulfonate, tetrafluoroborate, hexafluorophosphate, bis(trifluoromethanesulfonyl)imide.

[0016] In some embodiments of the present invention, the small molecule fluorescent probe compound is represented by formula (A), formula (B) or formula (C):

[0017]

[0018] OPA1 protein is a connecting protein between the inner and outer mitochondrial membranes and plays a key role in maintaining mitochondrial morphology and function. According to the properties of the OPA1 protein pocket, through virtual screening, the present invention uses thiazolo[4,5-c]pyridinium salt as the core structure and hydrophobic fragments triphenylamine and its derivatives as the main modification groups. By adjusting the lipophilicity, molecular size and charge distribution of thiazolo[4,5-c]pyridinium salt and other properties, a small molecule fluorescent probe compound of formula (I) with enhanced binding ability to OPA1 protein and fluorescence properties is obtained. The docking results show that the small molecule fluorescent probe compound of formula (I) can interact with the cytoplasmic domain of OPA1 through salt bridges, π-π stacking and hydrophobic interactions, etc.

[0019] Due to the connection of the triphenylamine and its derivative structures, the whole molecule of the small molecule fluorescent probe compound of formula (I) forms a twisted intramolecular charge transfer (TICT) structure, and the triphenylamine and its derivative structures act as molecular rotors. When the small molecule fluorescent probe compound of formula (I) is not bound to OPA1 protein, the freely rotating rotor mainly causes the dissipation of excited state energy through non-radiative processes, resulting in weak fluorescence emission of the probe; when the small molecule fluorescent probe compound of formula (I) binds to OPA1 protein, the hydrophobic cavity of the protein will restrict the rotation of the molecule, thereby reducing non-radiative energy dissipation and resulting in strong fluorescence emission.

[0020] Studies have shown that the stromal domain of OPA1 can insert into the inner membrane to promote inner membrane fusion. After the small molecule fluorescent probe compound of formula (I) binds to the stromal domain of OPA1, under the protection of the protein and the inner membrane lipid components pair, it has the ability to resist photobleaching, can resist a certain laser intensity, and is applicable to super-resolution microscopy imaging such as stimulated emission depletion microscopy (STED).

[0021] Therefore, the small molecule fluorescent probe compound of formula (I) can be localized to mitochondria by targeting the mitochondrial inner membrane protein OPA1; then, by binding to the stromal domain of the OPA1 protein, the molecular rotor is restricted, emitting strong fluorescence; and by utilizing the protection of the protein and the inner membrane lipid components, it exhibits the property of resisting photobleaching, thus possessing the ability to visualize and monitor mitochondria based on microscopy imaging.

[0022] In some embodiments of the present invention, the excitation wavelength of the small molecule fluorescent probe compound in an alkaline environment with pH = 7 - 9 is 480 - 520 nm.

[0023] In some specific embodiments of the present invention, the excitation wavelength of the small molecule fluorescent probe compound in an alkaline environment with pH = 7 - 9 is 480 - 500 nm.

[0024] In some specific embodiments of the present invention, the small molecule fluorescent probe compound emits green fluorescence at the excitation wavelength.

[0025] The second aspect of the present invention provides a preparation method of the small molecule fluorescent probe compound described in the first aspect of the present invention, comprising the following steps:

[0026] S1. React compound (x) with acetic anhydride and triethylamine to obtain intermediate (a)

[0027] S2. Subject intermediate (a) to a cyclization reaction with phosphorus pentasulfide to obtain intermediate (b)

[0028] S3. Subject intermediate (b) to a condensation reaction with compound (z) to obtain intermediate (c);

[0029] S4. React intermediate (c) with a methylation reagent to obtain the small molecule fluorescent probe compound;

[0030] wherein, in step S3, the compound (z) is selected from 4 - diphenylaminobenzaldehyde, 4 - bis(p - methoxyanilino)benzaldehyde, or 5 - (diphenylamino)thiophene - 2 - carbaldehyde;

[0031] The structural formula of the obtained intermediate (c) is as shown in formula (c 1 ) and formula (c2 ) or formula (c 3 ) as shown below:

[0032] In some embodiments of the present invention, in step S1, the molar ratio of the compound (x), acetic anhydride (Ac 2 O) and triethylamine (Et 3 N) is 1:(1 - 2):(2 - 3).

[0033] In some specific embodiments of the present invention, in step S1, the molar ratio of the compound (x), acetic anhydride and triethylamine is 1:(1 - 1.5):(2 - 2.5).

[0034] In some embodiments of the present invention, in step S1, the yield of the intermediate (a) is 80% - 95%.

[0035] In some embodiments of the present invention, in step S2, the molar ratio of the intermediate (a) to phosphorus pentasulfide (P 2 S 5 ) is 1:(1 - 2).

[0036] In some specific embodiments of the present invention, in step S2, the molar ratio of the intermediate (a) to phosphorus pentasulfide (P 2 S 5 ) is 1:(1 - 1.5).

[0037] In some embodiments of the present invention, in step S2, the yield of the intermediate (b) is 30% - 50%.

[0038] In some embodiments of the present invention, in step S3, the molar ratio of the intermediate (b) to the compound (z) is 1:(0.5 - 1.5).

[0039] In some specific embodiments of the present invention, in step S3, the molar ratio of the intermediate (b) to the compound (z) is 1:(0.8 - 1.2).

[0040] In some embodiments of the present invention, in step S3, the yield of the intermediate (c) is 30% - 80%.

[0041] In some embodiments of the present invention, in step S4, the molar ratio of the intermediate (c) to the methylation reagent is 1:(2 - 3).

[0042] In some specific embodiments of the present invention, in step S4, the molar ratio of the intermediate (c) to the methylation reagent is 1:(2 - 2.5).

[0043] In some embodiments of the present invention, in step S4, the yield of the small molecule fluorescent probe compound is 80%-95%.

[0044] In some embodiments of the present invention, in step S1, the reaction temperature of the reaction is 10-40°C, and the reaction time is 5-20 h.

[0045] In some specific embodiments of the present invention, in step S1, the reaction temperature of the reaction is 16-37°C, and the reaction time is 6-18 h.

[0046] In some embodiments of the present invention, in step S1, a solvent is further included, and the solvent includes tetrahydrofuran (THF).

[0047] In some embodiments of the present invention, after the reaction in step S1, it further includes dissolving the product in water, extracting with ethyl acetate (EA), washing the combined organic layer with NaHCO 3 and NaCl, removing ethyl acetate under reduced pressure, and purifying by column chromatography.

[0048] In some embodiments of the present invention, in step S2, the reaction temperature of the reaction is 120-200°C, and the reaction time is 1-10 h.

[0049] In some specific embodiments of the present invention, in step S2, the reaction temperature of the reaction is 120-180°C, and the reaction time is 1-6 h.

[0050] In some embodiments of the present invention, in step S2, a solvent is further included, and the solvent includes toluene (PhMe).

[0051] In some embodiments of the present invention, after the reaction in step S2, it further includes drying under reduced pressure, post-treatment with NaHCO 3 and purifying by column chromatography.

[0052] In some embodiments of the present invention, in step S3, the reaction includes adding an alkali solution to make the pH of the reaction system 13-14.

[0053] In some specific embodiments of the present invention, in step S3, the concentration of the alkali solution is 0.2-1 mol / L.

[0054] In some specific embodiments of the present invention, in step S3, the alkali source of the alkali solution includes alkali metal hydroxides.

[0055] In some embodiments of the present invention, in step S4, the reaction temperature of the reaction is 60-100°C, and the reaction time is 5-20 h.

[0056] In some specific embodiments of the present invention, in step S4, the reaction temperature of the reaction is 76 - 86 °C, and the reaction time is 6 - 18 h.

[0057] The third aspect of the present invention provides a fluorescent probe, comprising the small molecule fluorescent probe compound described in the first aspect of the present invention or a pharmaceutically acceptable salt thereof.

[0058] In some embodiments of the present invention, the fluorescent probe targets and binds to the OPA1 protein on the inner mitochondrial membrane.

[0059] The fourth aspect of the present invention provides the use of the small molecule fluorescent probe compound described in the first aspect of the present invention, or the fluorescent probe described in the third aspect, in in vivo mitochondrial tracing and / or imaging.

[0060] In some embodiments of the present invention, the in vivo includes fixed cells or tissue sections.

[0061] In some specific embodiments of the present invention, the fixed cells include human osteosarcoma cells (U - 2OS), human cervical cancer cells (Hela).

[0062] In some specific embodiments of the present invention, the fixed tissue section includes an animal muscle tissue section.

[0063] In some embodiments of the present invention, the fluorescent probe further includes a pharmaceutically acceptable excipient.

[0064] In some specific embodiments of the present invention, the pharmaceutically acceptable excipient includes a buffer solution, an antifreeze, or a preservative.

[0065] In some embodiments of the present invention, the imaging includes structured illumination microscopy (SIM), stimulated emission depletion microscopy (STED).

[0066] The fifth aspect of the present invention provides a method for monitoring the mitochondrial fusion process, comprising the following steps:

[0067] Formulating the fluorescent probe described in the third aspect of the present invention into a solution for sample staining, and placing the washed sample under a microscope to monitor the dynamic changes and interactions of lysosomes and mitochondria; wherein, the sample includes fixed cells or tissue sections.

[0068] In some embodiments of the present invention, the concentration of the fluorescent probe solution is 0.1 - 5 μmol / L.

[0069] In some specific embodiments of the present invention, the concentration of the fluorescent probe solution is 1 - 2 μmol / L.

[0070] In some embodiments of the present invention, the dyeing time is 1 - 3 h.

[0071] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0072] 1) The small molecule fluorescent probe compound provided by the present invention can bind to mitochondrial OPA1 protein and localize in mitochondria, producing green fluorescence, and being less sensitive to mitochondrial membrane potential; this compound has low cytotoxicity and strong anti-photobleaching effect, and can be applied to super-resolution microscopy imaging to realize the visualization monitoring of mitochondria;

[0073] 2) The preparation method of the small molecule fluorescent probe compound provided by the present invention has simple steps and high yield, and is suitable for industrial application;

[0074] 3) The fluorescent probe provided by the present invention, including the small molecule fluorescent probe compound, can be applied to the mitochondrial tracing in fixed cells or tissue sections, as well as super-resolution microscopy imaging such as SIM and STED of living mitochondria, and has broad application prospects in the research of lysosome and mitochondrial biological functions. Description of the Drawings

[0075] Figure 1 It is the high performance liquid chromatography diagram of the small molecule fluorescent probe compound of formula (A) in Example 1;

[0076] Figure 2 It is the confocal imaging diagram after co-staining living cells with the small molecule fluorescent probe compound of formula (A) and a commercially available mitochondrial probe in Test Example 1;

[0077] Figure 3 It is the fluorescence spectrum diagram before and after the small molecule fluorescent probe compound of formula (A) binds to OPA1 protein in Test Example 2;

[0078] Figure 4 It is the STED (a) and SIM (b) imaging diagrams of the small molecule fluorescent probe compound of formula (A) in Test Example 3;

[0079] Figure 5 It is the confocal imaging diagram of mitochondrial staining in fixed mouse muscle tissue sections in Test Example 4;

[0080] Figure 6 It is the confocal imaging diagram of the co-localization experiment of mitochondrial staining in fixed mouse muscle tissue sections in Test Example 4. Detailed Embodiments

[0081] The content of the present invention will be further described in detail through specific embodiments below. Unless otherwise specified, the raw materials, reagents or devices used in the embodiments can be obtained from conventional commercial channels or can be obtained by existing technical methods. Unless otherwise specified, the test or testing methods are conventional methods in the art.

[0082] Example 1

[0083] In this example, a small molecule fluorescent probe compound was prepared, and the steps and synthetic route are shown as follows:

[0084] S11. Dissolve compound (x), acetic anhydride (1.5 equiv) and triethylamine (2.0 equiv) in dry tetrahydrofuran. After reacting at 25 °C for 12 h, pour the resulting mixture into water, extract it repeatedly with an appropriate amount of ethyl acetate, and then wash the combined organic layer with NaHCO 3 and NaCl. Remove ethyl acetate under reduced pressure. The crude product was purified by silica gel column chromatography to obtain intermediate (a) as a white solid with a yield of 90%;

[0085]

[0086] S21. Dissolve intermediate (a) in dry toluene, add phosphorus pentasulfide (1.5 equiv), react at 150 °C for 3 h, dry under reduced pressure, and perform post-treatment to obtain a crude product. Purify it by column chromatography to obtain intermediate (b) as a white solid with a yield of 43%; 3 After treatment, a crude product was obtained and purified by column chromatography to obtain intermediate (b) as a white solid with a yield of 43%;

[0087]

[0088] S31. Under the alkaline condition of 0.5 mol / L NaOH, carry out a condensation reaction between intermediate (b) and 4-diphenylaminobenzaldehyde (1.1 equiv), stir and react at room temperature for 12 h, and filter to obtain compound of formula (c 1 ) with a yield of 78%;

[0089]

[0090] S41. React intermediate (c 1 ) with methyl iodide (2.0 equiv) at 80 °C for 12 h to obtain the small molecule fluorescent probe compound of formula (A) with a yield of 94%.

[0091]

[0092] The small molecule fluorescent probe compound of formula (A) in Example 1 was characterized by nuclear magnetic resonance 1 1H NMR and 13 13C NMR, and the results were as follows:

[0093] 1 1H NMR (500 MHz, DMSO-d 6 ) δ 9.72 (s, 1H), 8.79 (q, J = 6.6 Hz, 2H), 7.88 (d, J = 16.0 Hz, 1H), 7.74 (d, J = 8.5 Hz, 2H), 7.62 (d, J = 16.0 Hz, 1H), 7.40 (t, J = 7.7 Hz, 4H), 7.18 (t, J = 7.4 Hz, 2H), 7.16–7.12 (m, 4H), 6.93 (d, J = 8.4 Hz, 2H), 4.44 (s, 3H);

[0094] 13 13C NMR (126 MHz, DMSO-d 6 ) δ 174.61, 151.00, 150.15, 149.68, 146.13, 142.19, 139.20, 137.90, 130.00, 129.88, 127.19, 125.51, 124.62, 120.71, 120.38, 117.47, 47.80.

[0095] Figure 1 The HPLC chromatogram of the small molecule fluorescent probe compound of formula (A) in Example 1 is shown below, and Table 1 shows the results of the HPLC chromatogram of the small molecule fluorescent probe compound of formula (A) in Example 1:

[0096] Table 1 Results of the HPLC chromatogram of the small molecule fluorescent probe compound of formula (A) in Example 1

[0097] Time (min) Peak area Symmetry factor Peak area % 20.768 16479.2 0.81 99.70

[0098] Example 2

[0099] In this example, a small molecule fluorescent probe compound was prepared, and the steps and synthetic route are shown as follows:

[0100] S11. Dissolve compound (x), acetic anhydride (1.5 equiv) and triethylamine (2.0 equiv) in dry tetrahydrofuran, react at 25 °C for 12 h, then pour the resulting mixture into water, extract repeatedly with an appropriate amount of ethyl acetate, and wash the combined organic layer with NaHCO 3 and NaCl, remove ethyl acetate under reduced pressure, and purify the crude product by silica gel column chromatography to obtain intermediate (a) as a white solid with a yield of 90%;

[0101]

[0102] S21. Dissolve intermediate (a) in dry toluene, add phosphorus pentasulfide (1.5 equiv), react at 150 °C for 3 h, dry under reduced pressure, and use NaHCO 3 for post-treatment to obtain the crude product, which is purified by column chromatography to obtain white solid intermediate (b) with a yield of 43%;

[0103]

[0104] S31. Under the alkaline condition of 0.5 mol / L NaOH, carry out a condensation reaction between intermediate (b) and 4 - bis(p - methoxyanilino)benzaldehyde (1.1 equiv), stir and react at room temperature for 12 h, and filter to obtain the compound of formula (c 2 ) with a yield of 69%;

[0105]

[0106] S41. React intermediate (c 2 ) with methyl iodide (2.0 equiv) at 80 °C for 12 h to obtain the small - molecule fluorescent probe compound of formula (B) with a yield of 87%.

[0107]

[0108] Use nuclear magnetic resonance to characterize the small - molecule fluorescent probe compound of formula (B) in Example 2 1 for \(^1H\) NMR and 13 \(^{13}C\) NMR. The results are as follows:

[0109] 1 \(^1H\) NMR (400 MHz, DMSO - d 6 ) δ 9.70 (s, 1H), 8.87–8.74 (m, 2H), 7.86 (d, J = 16.0 Hz, 1H), 7.73–7.66 (m, 2H), 7.57 (d, J = 16.0 Hz, 1H), 7.25–7.15 (m, 4H), 7.09–7.00 (m, 4H), 6.90–6.80 (m, 2H), 4.43 (s, 3H), 2.31 (s, 6H);

[0110] 13 \(^{13}C\) NMR (101 MHz, DMSO - d 6 ) δ 174.67, 151.04, 150.13, 150.07, 143.52, 142.35, 139.05, 137.84, 134.03, 130.35, 129.95, 126.23, 125.74, 120.64, 119.07, 116.85, 47.77, 20.48.。

[0111] Example 3

[0112] In this example, a small molecule fluorescent probe compound was prepared. The steps and synthetic route are shown as follows:

[0113] S11. Dissolve compound (x), acetic anhydride (1.5 equiv) and triethylamine (2.0 equiv) in dry tetrahydrofuran. After reacting at 25 °C for 12 h, pour the resulting mixture into water, extract it repeatedly with an appropriate amount of ethyl acetate, and then wash the combined organic layer with NaHCO 3 and NaCl. Remove ethyl acetate under reduced pressure. The crude product was purified by silica gel column chromatography to obtain intermediate (a) as a white solid with a yield of 90%;

[0114]

[0115] S21. Dissolve intermediate (a) in dry toluene, add phosphorus pentasulfide (1.5 equiv), react at 150 °C for 3 h, dry under reduced pressure, and perform post-treatment to obtain a crude product, which was purified by column chromatography to obtain intermediate (b) as a white solid with a yield of 43%; 3 After treatment, a crude product was obtained and purified by column chromatography to obtain intermediate (b) as a white solid with a yield of 43%;

[0116]

[0117] S31. Under the alkaline condition of 0.5 mol / L NaOH, carry out a condensation reaction between intermediate (b) and 5-(diphenylamino)thiophene-2-carbaldehyde (1.1 equiv), stir and react at room temperature for 12 h, and filter to obtain compound of formula (c 3 ) with a yield of 38%;

[0118]

[0119] S41. React intermediate (c 3 ) with methyl iodide (2.0 equiv) at 80 °C for 12 h to obtain the small molecule fluorescent probe compound of formula (C) with a yield of 81%.

[0120]

[0121] The small molecule fluorescent probe compound of formula (C) in Example 3 was characterized by 1 1H NMR and 13 13C NMR. The results were as follows:

[0122] 1 1H NMR (400 MHz, DMSO-d 6)δ9.62(s,1H),8.76–8.68(m,2H),8.04(d,J=15.5Hz,1H),7.50–7.40(m,5H),7.30–7.21(m,6H),7.04(d,J=15.5Hz,1H),6.46(d,J=4.1Hz,1H),4.41(s,3H);

[0123] 13 C NMR(101MHz,DMSO-d 6 )δ173.89,157.40,151.14,150.26,146.14,138.55,137.49,135.51,134.63,129.94,128.73,125.50,124.59,120.51,114.96,114.17,47.74.。

[0124] Test Example 1

[0125] Human osteosarcoma cells U-2OS were placed in a culture medium (DMEM culture solution and 10 v / v% fetal bovine serum) and cultured in an incubator at 37 °C, 5% CO 2 and 20% O 2 for 48 h. The small molecule fluorescent probe compound of formula (A) prepared in Example 1 was diluted with U-2OS cell culture medium to a final concentration of 0.5 μmol / L, and a co-localization experiment was performed together with a commercially available mitochondrial probe Mito-Tracker Deep Red FM (0.1 μmol / L). After the prepared probe was added to the culture dish and cultured for another 2 h, the sample was washed 3 times with the culture medium, and laser confocal microscopy imaging was performed.

[0126] Figure 2 Figure is the confocal imaging diagram after co-staining live cells with the small molecule fluorescent probe compound of formula (A) and the commercially available mitochondrial probe in Test Example 1. Among them, Figure 2 (a) is the signal of the small molecule fluorescent probe compound of formula (A), Figure 2 (b) is the signal of the commercially available mitochondrial probe Mito-Tracker Deep Red FM, Figure 2 (c) is the merged image of the two. Among them, the excitation wavelength of the small molecule fluorescent probe compound of formula (A) is 488 nm, producing green fluorescence, and the excitation wavelength of the commercially available mitochondrial probe is 640 nm, producing red fluorescence. It can be Figure 2 seen that the mitochondrial localization of the small molecule fluorescent probe compound of formula (A) is similar to that of the commercially available mitochondrial probe. The co-localization rate was calculated by Pearson's constant, and the co-localization rate reached 0.89, indicating that the small molecule fluorescent probe compound of formula (A) prepared in Example 1 can indicate mitochondria in live cells.

[0127] Test Example 2

[0128] 1) Dissolve the recombinant human OPA1 protein fragment (Ser607-Lys960) in a buffer solution (PBS: pH 7.4, 0.02% NLS, 1 mmol / L EDTA, 4% Trehalose, 1% Mannitol) to obtain a protein stock solution with a concentration of 1 μg / μL;

[0129] 2) Dissolve the small molecule fluorescent probe compound of formula (A) prepared in Example 1 with dimethyl sulfoxide, prepare a stock solution of 1 mmol / L, and then dilute it to 2 μmol / L with PBS buffer (pH 7.4) to obtain a small molecule fluorescent probe compound solution of formula (A) for testing;

[0130] 3) Dropwise add the protein stock solution to the small molecule fluorescent probe compound solution of formula (A) so that the protein content in the test solution is 10 ng / μL, and record the fluorescence emission spectrum of the mixture.

[0131] Figure 3 is the fluorescence spectrum of the small molecule fluorescent probe compound of formula (A) before and after binding to OPA1 protein in Test Example 2. From Figure 3 it can be seen that at the same concentration, when the small molecule fluorescent probe compound of formula (A) is not bound to OPA1 protein, there is only weak fluorescence at different excitation wavelengths, and the intensity is close to 0. However, after the small molecule fluorescent probe compound of formula (A) binds to OPA1 protein, strong fluorescence can be emitted, indicating that the small molecule fluorescent probe compound of formula (A) prepared in Example 1 has great potential in recognizing mitochondrial OPA1 protein.

[0132] Test Example 3

[0133] Incubate adherent human osteosarcoma cells U-2OS with the small molecule fluorescent probe compound of formula (A) prepared in Example 1 (final concentration of 2 μmol / L) in an incubator at 37 °C, 5% CO 2 and 20% O 2 for 1 h, and then perform imaging under structured illumination microscopy (SIM) and stimulated emission depletion microscopy (STED) respectively (λ ex = 488 nm).

[0134] Figure 4 is the STED (a) and SIM (b) imaging diagrams of the small molecule fluorescent probe compound of formula (A) in Test Example 3. From Figure 4 it can be seen that the small molecule fluorescent probe compound of formula (A) prepared in Example 1 can be imaged under different super-resolution microscopes, and the imaging effects are all consistent with the protein localization of OPA1.

[0135] Test Example 4

[0136] The small molecule fluorescent probe compound of formula (A) prepared in Example 1 was formulated into a solution and used to stain mouse fixed muscle tissue sections together with the commercially available mitochondrial probe Mito-Tracker Deep Red FM. After washing the samples, analysis was carried out under a 60-fold confocal microscope.

[0137] Figure 5 It is a confocal imaging diagram of mitochondrial staining of mouse fixed muscle tissue sections in Test Example 4, where Figure 5 (a) is the DAPI signal, with an excitation wavelength of 640 nm, Figure 5 (b) is the signal of the small molecule fluorescent probe compound of formula (A), with an excitation wavelength of 488 nm, Figure 5 (c) is the signal of the commercially available mitochondrial probe Mito-Tracker Deep Red FM, with an excitation wavelength of 640 nm, Figure 5 (d) is the merged image of the three. It can be seen from Figure 5 that in the cross-section of mouse fixed muscle tissue stained with the small molecule fluorescent probe compound of formula (A), obvious punctate signals were observed, while in the cross-section of mouse fixed muscle tissue stained with the commercially available mitochondrial probe Mito-Tracker Deep Red FM, almost no mitochondria were observed.

[0138] To determine whether the punctate signals observed in the fixed tissue sections stained with the small molecule fluorescent probe compound of formula (A) belong to the mitochondria in mouse muscle tissue cells, the outer mitochondrial membrane protein TOMM20 was labeled using immunofluorescence technology to characterize the mitochondria of tissue cells, and to study whether the small molecule fluorescent probe compound of formula (A) co-localizes with the antibody-labeled TOMM20.

[0139] Figure 6 It is a confocal imaging diagram of the mitochondrial staining co-localization experiment of mouse fixed muscle tissue sections in Test Example 4, where Figure 6 (a) is the DAPI signal, with an excitation wavelength of 640 nm, Figure 6 (b) is the signal of the small molecule fluorescent probe compound of formula (A), with an excitation wavelength of 488 nm, Figure 6 (c) is the TOMM20 signal, with an excitation wavelength of 640 nm, Figure 6 (d) is the merged image of the three. It can be seen from Figure 6 that in the mouse fixed muscle tissue sections, the punctate signals stained with the small molecule fluorescent probe compound of formula (A) coincide with the position of the antibody-labeled TOMM20, indicating that the small molecule fluorescent probe compound of formula (A) has ultra-high selectivity for mitochondria in fixed tissue sections, can stain mitochondria in living tissues well, and has the potential for application in visual monitoring of mitochondrial morphology in tissue sections.

[0140] In addition, the present invention provides a small molecule fluorescent probe compound, which targets mitochondria by binding to mitochondrial localization proteins, rather than targeting mitochondria by using the electrostatic attraction between the positive and negative charges of the inner mitochondrial membrane as in existing mitochondrial probes. Therefore, it has a lower sensitivity to mitochondrial membrane potential (MMP), overcoming the defect that the ability of existing mitochondrial probes to target mitochondria is easily affected by changes in mitochondrial membrane potential and cannot directly stain mitochondria without MMP in fixed cells and tissues.

Claims

1. A small molecule fluorescent probe compound, characterized in that: Its structural formula is shown in formula (I): Among them, R - Selected from halide, p-toluenesulfonate, tetrafluoroborate, hexafluorophosphate, bis(trifluoromethanesulfonyl)imide or trifluoromethanesulfonate; R1 is selected from 2. The small molecule fluorescent probe compound according to claim 1, characterized in that: The small molecule fluorescent probe compound is shown in formula (A), formula (B) or formula (C):

3. The method for preparing the small molecule fluorescent probe compound according to claim 1 or 2, characterized in that: The following steps are involved: S1. Compound (x) Reaction with acetic anhydride and triethylamine to obtain intermediate (a) S2, allowing intermediate (a) to undergo a cyclization reaction with phosphorus pentasulfide to obtain intermediate (b) S3, allowing the intermediate (b) to undergo a condensation reaction with the compound (z) to obtain the intermediate (c); S4, reacting the intermediate (c) with a methylating agent to obtain the small molecule fluorescent probe compound; Wherein, in step S3, the compound (z) is selected from 4-diphenylaminobenzaldehyde, 4-di-p-methoxyanilinobenzaldehyde or 5-(diphenylamino)thiophene-2-carboxaldehyde; The structural formula of the intermediate (c) obtained is shown in formula (c1), formula (c2) or formula (c3):

4. The preparation method according to claim 3, characterized in that: In step S1, the molar ratio of the compound (x), acetic anhydride and triethylamine is 1:(1-2):(2-3); And / or, in step S2, the molar ratio of the intermediate (a) to phosphorus pentasulfide is 1:(1-2); and / or, in step S3, the molar ratio of the intermediate (b) to the compound (z) is 1:(0.5-1.5); And / or, in step S4, the molar ratio of the intermediate (c) to the methylating agent is 1:(2-3).

5. The preparation method according to claim 3, characterized in that: In step S1, the reaction temperature is 10-40°C and the reaction time is 5-20h; And / or, in step S2, the reaction temperature of the reaction is 120-200° C., and the reaction time is 1-10 h; And / or, in step S3, the reaction includes adding alkali solution to make the pH of the reaction system 13-14; And / or, in step S4, the reaction temperature of the reaction is 60-100° C., and the reaction time is 5-20 h.

6. A fluorescent probe, characterized in that It comprises the small molecule fluorescent probe compound or a pharmaceutically acceptable salt thereof as described in claim 1 or 2.

7. The fluorescent probe according to claim 6, characterized in that The fluorescent probe targets and binds to the mitochondrial inner membrane OPA1 protein.

8. Use of the small molecule fluorescent probe compound according to claim 1 or 2, or the fluorescent probe according to claim 6 or 7 in tracing and / or imaging of living mitochondria.

9. A method for monitoring mitochondrial fusion process, characterized in that: The following steps are involved: The fluorescent probe described in claim 6 or 7 is prepared into a solution for sample staining, and the cleaned sample is placed under a microscope to monitor the dynamic changes and interactions between lysosomes and mitochondria; wherein the sample includes fixed cells or tissue sections.

10. The monitoring method according to claim 9, characterized in that: The concentration of the fluorescent probe solution is 0.1-5 μmol / L; And / or, the dyeing time is 1-3h.

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