A compound with lysosome and mitochondria dual-color imaging function and its preparation method and application

Abstract

The present invention belongs to the field of fluorescence imaging technology, and specifically relates to a compound with lysosome and mitochondria dual-color imaging function, and a preparation method and application thereof. The compound provided by the present invention has different excitation wavelengths under different acid-base environments, has fluorescence of different colors, and can be used to prepare fluorescent probes for simultaneous imaging of different organelles. The fluorescent probe provided by the present invention can simultaneously dual-color image lysosomes and mitochondria in living cells or in vivo, showing green fluorescence in lysosomes and red fluorescence in mitochondria, and can monitor lysosomes and mitochondria in real time in living biological samples. At the same time, the fluorescent probe can avoid the problems of mutual interference, groping for staining conditions, etc. when using commercial probes to study lysosomes and mitochondria. In addition, the preparation process of the fluorescent probe is simple, the cost is low, and the structure is stable and easy to store, and it has a broad application space in the study of lysosome and mitochondrial biological functions.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescence imaging, and in particular relates to a compound with lysosome and mitochondria dual-color imaging function, and a preparation method and application thereof. Background Art

[0002] In eukaryotic cells, organelles are spatially compartmentalized to allow mutually incompatible biochemical processes to proceed smoothly. Studies have shown that organelles within cells are not independent individuals. They cooperate and coordinate with each other in performing corresponding physiological functions to form a complex cellular network. Lysosomes and mitochondria are two types of highly transported subcellular organelles. Healthy mitochondria and lysosomes are essential for maintaining cell homeostasis and exercising normal physiological functions of cells. Functional mitochondrial movement depends on many dynamic processes, including fusion, fission, migration, and direct contact with other organelles. As highly dynamic organelles widely present in most cells, lysosomes and mitochondria have been shown to interact in related biological events. For example, lysosomes are acidic organelles with a single membrane-enclosed cystic structure (pH 4.5-5.5) that can decompose and recycle a variety of wastes, including proteins, lipids, and cell debris. In order to maintain a healthy mitochondrial network in cells, damaged mitochondria need to be cleared in a timely manner through lysosome-mediated autophagy (also known as mitophagy) to maintain the quality and quantity of mitochondrial clusters in cells. Mitochondrial autophagy is a special form of autophagy. Cells rely on this unique way to remove damaged mitochondria and ensure the normal energy supply of mitochondria. In addition, studies have shown that specialized membrane contact sites are formed between mitochondria and lysosomes in cells to mediate the inter-organelle transfer of metabolites, providing unique spatial areas for executing and regulating their functions. Therefore, monitoring and in-depth study of the dynamic interactions between lysosomes and mitochondria in living cells and in vivo systems is crucial to understanding their roles in related biological events and metabolic diseases.

[0003] However, there are few reports on methods to study the interaction between mitochondria and lysosomes. Small molecule fluorescent probes have the characteristics of high resolution, strong sensitivity, good specificity, and can detect living cell samples in situ. With the help of fluorescence microscopy, the dynamic interaction between mitochondria and lysosomes can be monitored. At present, there are still relatively few reports on the dual-color imaging monitoring of lysosome-mitochondria interaction. Most studies use two different organic small molecules to achieve dual-color imaging of mitochondria and lysosomes, such as LysoTracker and MitoTracker, which have been used to detect mitophagy. Tian's group rationally designed two fluorescent probes (G-Mito and R-Lyso) based on the principle of Förster resonance energy transfer (FRET) to study the interaction between mitochondria and lysosomes. In addition, Diao's group constructed a propionic acid-reactive semi-cyanine small molecule fluorescent probe coupa, which can achieve dual-color distinction between mitochondria and lysosomes by changing the conjugated structure of the semi-cyanine in response to reactive sulfur (RSS) in the mitochondrial microenvironment. Except for Coupa, the above methods for studying the interaction between mitochondria and lysosomes inevitably use two different fluorescent probes. Although fluorescence excitation imaging of mitochondria and lysosomes using two different probes also shows good performance in studying mitochondrial-lysosomal interactions, these methods require two fluorescent dyes to simultaneously detect mitochondria and lysosomes, which brings operational troubles and increases the risk of mutual interference between probes. Coupa, as the only single-molecule reactive fluorescent probe, may interfere with the normal physiological function of mitochondria due to its reaction characteristics with active sulfur in mitochondria in vivo, causing potential toxicity, and its biocompatibility needs to be studied.

[0004] Therefore, the current research goal in this field is to invent a single-molecule fluorescent probe that can simultaneously target lysosomes and mitochondria and emit fluorescence of different colors to achieve simultaneous distinction and dual-color imaging of lysosomes and mitochondria. However, the design of a single-molecule fluorescent probe for dual-color imaging of lysosomes and mitochondria using the dual luminescence mechanism of monomers and aggregations and its application have not been reported. Summary of the invention

[0005] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the first aspect of the present invention aims to provide a compound having a dual-color imaging function of lysosomes and mitochondria.

[0006] The purpose of the second aspect of the present invention is to provide a method for preparing the compound described in the first aspect of the present invention.

[0007] The purpose of the third aspect of the present invention is to provide the use of the compound described in the first aspect of the present invention.

[0008] The fourth aspect of the present invention aims to provide a fluorescent probe.

[0009] The fifth aspect of the present invention aims to provide a method for detecting dynamic changes of lysosomes and mitochondria.

[0010] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

[0011] The first aspect of the present invention provides a compound, the structural formula of the compound is shown as formula (IV).

[0012] Formula (IV).

[0013] The R - At least one selected from the group consisting of iodide ion, bromide ion, chloride ion, p-toluenesulfonate ion, and trifluoromethanesulfonate ion.

[0014] Preferably, the R - It is iodide ion.

[0015] Preferably, the compound has an excitation wavelength of 483-492 nm in an acidic environment; more preferably, it is 488 nm.

[0016] Preferably, the compound has an excitation wavelength of 556-566 nm in a weakly alkaline environment; more preferably, it is 561 nm.

[0017] Preferably, the pH of the acidic environment is 4.5-5.5.

[0018] Preferably, the pH of the weakly alkaline environment is 7-9.

[0019] In the first aspect of the present invention, the chemical name of the probe is (E)-2-(3,4-dimethoxystyryl)-3,7-dimethylbenzo[1,2-d:4,3-d']bis(thiazole)-3-ium, abbreviated as MISO.

[0020] The compound MISO provided by the present invention is obtained by a Knoevenagel condensation reaction using an onium salt of N-methylbenzisodithiazole as an electron acceptor and veratraldehyde (3, 4-dimethoxybenzaldehyde) as an electron donor. Since MISO contains N cations, the molecule has a certain positive charge, so that MISO has the ability to target mitochondria. In addition, in the parent nucleus of MISObenzisodithiazole, there is also an unmethylated N atom, which makes the molecule have a certain alkalinity and selectively distributes in a weakly acidic environment such as lysosomes.

[0021] The compound MISO provided by the present invention can be used as a fluorescent probe. After entering the cell, it can simultaneously target lysosomes and mitochondria and produce fluorescence of different colors. Under normal physiological conditions, MISO is distributed in the acidic lysosomes (pH 4.5-5.5) in the form of monomers and produces green fluorescence ( λ ex = 481nm, λ em ≈ 560 nm), but in weakly alkaline conditions such as mitochondria, MISO will aggregate, causing the fluorescence to red-shift and produce red fluorescence ( λ ex = 561nm, λ em ≈ 620nm). Due to its good fluorescence properties, low cytotoxicity, and strong resistance to photobleaching, this probe can be used to study the interaction between mitochondria and lysosomes in living cells and the mutual changes between the two types of organelles during copper death.

[0022] The second aspect of the present invention provides a method for preparing the compound described in the first aspect of the present invention, comprising the following steps:

[0023] S1: The compound of formula (I) reacts with Ac2O, Et3N and Lawesson's reagent to obtain a compound of formula (II);

[0024] S2: The compound of formula (II) undergoes a cyclization reaction under the catalysis of K3Fe(CN)6 and NaOH to obtain a compound of formula (III);

[0025] S3: The compound of formula (III) reacts with a methylating agent under heating conditions to obtain a methylated intermediate compound of formula (III), which then undergoes a condensation reaction with 3, 4-dimethoxybenzaldehyde under heating conditions to obtain a final product;

[0026] The structures of the compound of formula (I), the compound of formula (II) and the compound of formula (III) are shown below:

[0027] .

[0028] Preferably, in step S1, the compound of formula (I) is first reacted with Ac2O and Et3N at 16-37°C for 6-18h, and then reacted with Lawesson's reagent at 100-140°C for 1-3h.

[0029] Preferably, in the step S3, the heating condition is 60-90°C.

[0030] In some embodiments of the present invention, the methylating agent is methyl iodide.

[0031] In some embodiments of the present invention, the preparation method comprises: firstly dissolving the compound of formula (I) p-phenylenediamine, Ac2O and Et3N (2.4 equiv, 22.3 mmol, 3.1 mL) at room temperature and stirring for 12 h, and then filtering and collecting the precipitate. Then the precipitate (1.7 g, 8.8 mmol) is dissolved in dry THF, Lawesson's reagent is added, and stirred at 120°C for 2 h to obtain compound (II); compound (II) undergoes cyclization reaction under K3Fe(CN)6, NaOH catalysis to obtain compound (III); compound (III) and iodomethane are heated to obtain a methylated intermediate compound of formula (III), and then condensed with 3, 4-dimethoxybenzaldehyde under heating to obtain the final product MISO. The preparation reaction process is shown in the following formula:

[0032] .

[0033] Reagents and conditions: (a): i) Ac2O, Et3N; ii) Lawesson, THF, 100~140℃, 80~90%; (b) K3Fe(CN)6, NaOH, 20~30%; (c) : i) MeI, MeCN, 70~90℃; ii) veratraldehyde,EtOH, 60~90℃, 30~40%.

[0034] The third aspect of the present invention provides use of the compound described in the first aspect of the present invention in preparing a product, wherein the product includes a fluorescent probe.

[0035] Preferably, the fluorescent probe targets lysosomes and mitochondria;

[0036] Preferably, the fluorescent probe is used to monitor the dynamic changes of lysosomes and mitochondria and the interaction between the two in situ and in real time.

[0037] In some embodiments of the present invention, the fluorescent probe can simultaneously image lysosomes and mitochondria in two colors in living cells or in vivo. Wherein, the cells are cultured human cervical cancer cells (HeLa) or human osteosarcoma cells (U2OS). The single-molecule fluorescent probe simultaneously targets lysosomes and mitochondria. The excitation wavelength in the lysosome is 488 nm, which can make the lysosome show green fluorescence; the excitation wavelength in the mitochondria is 561 nm, which can make the mitochondria show red fluorescence.

[0038] Further preferably, the fluorescent probe can detect the number, size, and morphology of lysosomes and mitochondria in situ and visualize the dynamic changes of fusion and fission of lysosomes and mitochondria in real time. More preferably, the fluorescent probe can be used for in situ, real-time visualization monitoring of lysosome and mitochondrial interaction.

[0039] The fourth aspect of the present invention provides a fluorescent probe, comprising the compound described in the first aspect of the present invention.

[0040] Preferably, the fluorescent probe includes auxiliary materials required for storage and transportation, and the auxiliary materials include but are not limited to buffer, antifreeze, and preservative.

[0041] A fifth aspect of the present invention provides a method for detecting dynamic changes of lysosomes and mitochondria.

[0042] The fluorescent probe described in the fourth aspect of the present invention is prepared into a solution, used to treat cells, and then detected under a microscope after washing.

[0043] In some embodiments of the present invention, the steps of simultaneously dual-color imaging of lysosomes and mitochondria of living cells by the fluorescent probe are as follows: the fluorescent probe is configured into a fluorescent probe solution with a cell culture medium, which is added to a laser confocal culture dish containing living cells for treatment, and after the treatment, the living cells are washed with a culture medium, and then the dynamic changes and interactions of lysosomes and mitochondria in the living cells are monitored in real time under a fluorescence microscope; more preferably, the concentration of the fluorescent probe used is 5~15 µM, and the staining time is 1~3 h.

[0044] The beneficial effects of the present invention are:

[0045] The compound provided by the present invention has different excitation wavelengths and different colors of fluorescence under different acid-base environments, and can be used to prepare fluorescent probes for simultaneous imaging of different organelles. The fluorescent probe provided by the present invention can simultaneously dual-color image lysosomes and mitochondria in living cells or in vivo, showing green fluorescence in lysosomes and red fluorescence in mitochondria. The fluorescent probe provided by the present invention can monitor lysosomes and mitochondria in real time in living biological samples. At the same time, the fluorescent probe provided by the present invention can avoid the problems of mutual interference when using commercial mitochondrial probe MitoTracker and lysosomal probe LysoTracker to study lysosomes and mitochondria, and spend a lot of time exploring staining conditions. In addition, the preparation process of the fluorescent probe is simple, low-cost, and the structure is stable and easy to store, and it has a broad application space in the study of lysosomal and mitochondrial biological functions. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0047] Figure 1 is the fluorescent probe MISO in the embodiment of the present invention. 1 H-NMR spectrum.

[0048] Figure 2 is the fluorescent probe MISO in the embodiment of the present invention. 13 C-NMR spectrum.

[0049] Figure 3 This is the HRMS (ESI) image of the fluorescent probe MISO in an embodiment of the present invention.

[0050] Figure 4 This is the HPLC chart of the fluorescent probe MISO in the embodiment of the present invention.

[0051] Figure 5 Characterization of the dual-color luminescence characteristics of the fluorescent probe MISO in the embodiment of the present invention; (a) Normalized UV absorption spectra of the fluorescent probe MISO (5 μM) in methanol, EA and water; (b) Normalized fluorescence emission spectra of the fluorescent probe MISO (2 μM) in methanol, EA and water; (c) Normalized fluorescence emission spectra of 50 μM MISO in aqueous solution (pH = 8.0, λ ex = 488 nm) within 30 minutes; (d) dynamic light scattering (DLS) analysis of the aggregation state of the fluorescent probe MISO in water; (e) red fluorescence emission of the fluorescent probe MISO in the solid state; (f) fluorescence emission spectra of the fluorescent probe MISO in glycerol solvents with different concentrations.

[0052] Figure 6 MISO dual-color imaging of lysosomes and mitochondria; (a) Live cell imaging results of fluorescent probe MISO; (b) Co-localization experimental results of fluorescent probe MISO with commercially available lysosome probe LysoTracker deepred and commercially available mitochondrial probe MitoTrackerdeepred.

[0053] Figure 7 The fluorescent probe MISO monitors the interaction between lysosomes and mitochondria in living cells in situ and in real time.

[0054] Figure 8 Experimental results of using the fluorescent probe MISO to monitor the mitophagy process in living cells, including: (a) Schematic diagram of MISO monitoring the interaction between mitochondria and lysosomes during mitophagy. (b) Confocal microscopy was used to observe the interaction between mitochondria and lysosomes in cells induced by starvation after culturing in serum-free medium for 0 hours, 4 hours, and 6 hours. Scale bar: 10 μm. DETAILED DESCRIPTION

[0055] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0056] In the following examples, the materials, reagents, etc. used were obtained from commercial sources unless otherwise specified.

[0057] Example 1 Preparation and characterization of a compound with dual-color imaging function of lysosomes and mitochondria

[0058] The compound preparation reaction process provided in this embodiment is shown in the following formula:

[0059]

[0060] Reagents and conditions: (a): i) Ac2O, Et3N; ii) Lawesson, THF, 100~140℃, 80~90%; (b) K3Fe(CN)6, NaOH, 20~30%; (c) : i) MeI, MeCN, 70~90℃; ii) veratraldehyde,EtOH, 60~90℃, 30~40%.

[0061] ① Compound Ⅰ (1 g, 9.3 mmol), Ac2O (2.2 equiv, 20.5 mmol, 1.9 mL) and Et3N (2.4 equiv, 22.3 mmol, 3.1 mL) were dissolved in dry THF (6.0 mL). After the mixture was stirred at room temperature for 12 h, the precipitate was collected by filtration and dried under reduced pressure to obtain 1.7 g of the intermediate. The intermediate was then dissolved in dry THF again, Lawesson's reagent (1.2 equiv, 10.6 mmol, 4.3 g) was added to the flask and stirred at 120°C for 2 h. After cooling to room temperature, the mixture was poured into water and repeatedly extracted with EA (3 × 40 mL). The composite organic layer was further washed with NaHCO3 and NaCl. EA was removed under reduced pressure, and the crude product was purified by silica gel column chromatography with PE / EA (4:1) as eluent to give a yellow solid II (1.8 g, yield 85.5%). 1 H NMR (400 MHz, DMSO- d 6) δ 11.57 (s, 2H), 7.79 (s, 4H), 2.58 (s, 6H).

[0062] ② Potassium ferrocyanide (5.0 equiv, 40.9 mmol, 13.5 g) was dissolved in H2O (50.0 mL), and a 10% NaOH (40 mL) solution containing compound II (1.8 g, 8.2 mmol) was slowly dripped into the potassium ferrocyanide solution. After 2 h, the brown precipitate was filtered off, and the crude product was purified by PE / EA (8:1) silica gel column chromatography to obtain a white solid III (0.47 g, yield 26.7%). 1 H NMR (500 MHz, DMSO- d 6) δ 8.57 (s, 2H), 2.83 (s, 6H).

[0063] ③ Compound III (0.47 g, 2.1 mmol) and MeI (3 equiv, 6.3 mmol, 0.4 mL) were dissolved in 2.0 mL of dry MeCN and stirred at 80°C for 8 h. After the mixture was cooled to room temperature, the precipitated product was collected by filtration, veratraldehyde (1 equiv, 2.1mmol, 0.35 g) was added, and refluxed in a flask containing ethanol for 12 h. After cooling to room temperature, the obtained precipitate was dissolved in dry ethanol for recrystallization, collected by filtration, and dried under reduced pressure to obtain the final product MISO (0.39 g, yield 35.5%). 1 H NMR (400 MHz, DMSO- d 6) δ 8.38 (d, J = 9.2 Hz, 1H), 8.29 (d, J = 9.2Hz, 1H), 8.21 (d, J = 15.6 Hz, 1H), 7.93 (d, J = 15.6 Hz, 1H), 7.67 (d, J = 2.0 Hz,1H), 7.60 (dd, J = 8.4, 2.0 Hz, 1H), 7.14 (d, J = 8.4 Hz, 1H), 4.44 (s, 3H), 3.90 (s, 3H), 3.88 (s, 3H), 2.95 (s, 3H). 13 C NMR (126 MHz, DMSO- d6) δ 170.71,169.41, 153.17, 152.57, 149.50, 149.22, 139.74, 128.52, 126.88, 126.23,123.21, 120.73, 114.91, 111.72, 111.03, 111.00, 56.00, 55.88, 37.24, 20.00;mass spectrum, m / z 383.08796 (M - I) + (C 20 H 19 N2O2S2 + requires 383.08825); HPLCpurify: 98.44%.

[0064] Figure 1 is the H NMR spectrum of MISO, Figure 2 is the NMR carbon spectrum of MISO, Figure 3 This is the high-resolution mass spectrum of MISO. Figure 4 Table 1 shows the results of the high performance liquid chromatography of MISO.

[0065] Table 1

[0066]

[0067] Example 2 Characterization of the dual-color luminescence characteristics of the fluorescent probe MISO

[0068] UV absorption and fluorescence spectra of the fluorescent probe MISO in different solvents ( Figure 5 The spectrum shows that the UV absorption of MISO in different solvents is significantly different. The maximum absorption in water is about 420 nm, while the maximum absorption in EA is about 470 nm, and methanol is in the middle. In addition, MISO emits strong fluorescence in ethyl acetate, while its fluorescence in other solvents is weak. This may be because the MISO structure belongs to the D-π-A structure, has intramolecular charge transfer properties, and is more sensitive to changes in the polarity of the surrounding environment.

[0069] However, when a highly viscous medium such as glycerol is added to water, its fluorescence spectrum changes significantly (the absorption peak at 480 nm gradually weakens over time, and a new emission peak appears at 600 nm, Figure 5 Dynamic light scattering analysis of the solution ( Figure 5 Middle (d) shows that the molecular aggregates form aggregates under alkaline conditions, but not under acidic conditions. In addition, MISO exhibits orange-red fluorescence in its solid state ( Figure 5e). In order to further study the aggregation-induced emission effect of MISO, this example studies the effect of viscosity (high viscosity will limit the intramolecular motion) on the fluorescence spectrum of MISO. The fluorescence intensity of MISO increases with the increase of the glycerol ratio. When glycerol is not added (glycerol concentration is 0%), the probe hardly emits fluorescence, indicating that the orange-red fluorescence emission of MISO is caused by the restriction of intramolecular motion ( Figure 5 These results suggest that MISO may exhibit shorter-wavelength fluorescence emission (monomer luminescence) in the weakly acidic environment of lysosomes and longer-wavelength emission (aggregate luminescence) in the weakly alkaline environment of mitochondria, respectively, demonstrating the potential of MISO in dual-color imaging of these two organelles.

[0070] The above results prove that the fluorescent probe MISO has dual-color luminescence characteristics of monomer luminescence and aggregated luminescence.

[0071] Example 3 Results of live cell fluorescence imaging using the fluorescent probe MISO

[0072] Human osteosarcoma U2OS cells were placed in culture medium (DMEM culture medium and 10v / v% embryonic bovine serum) and cultured in an incubator at 37°C, 5% CO2 and 20% O2 for 48 h. The fluorescent probe MISO (diluted to a final concentration of 10 μM with U2OS cell culture medium) was added to the culture dish and cultured for 2 h. The sample was washed 3 times with culture medium and imaged with a laser confocal microscope. The results are shown in Figure 6 a. The channel with excitation wavelength of 488 (MISO, λ ex =488 nm) to obtain green dot-shaped fluorescence signals; the channel with an excitation wavelength of 561 (MISO, λ ex = 561 nm) to obtain red rod-shaped fluorescence signals. These results show that the fluorescent probe MISO is capable of dual-color imaging and imaging of two different organelles.

[0073] In order to observe whether the two different organelles of the fluorescent probe MISO dual-color imaging are lysosomes and mitochondria, this example conducted a co-localization experiment of the probe MISO (10 μM) with the commercially available lysosome probe LysoTracker DeepRed (0.1 μM) and the commercially available mitochondrial probe MitoTracker DeepRed (0.1 μM). The results are shown in Figure 6 Middle b. Figure 6 In middle b, the fluorescent probe MISO shows a green dot-shaped fluorescent signal with a classic lysosomal morphology (MISO, λ ex= 488 nm) and the commercially available lysosomal probe LysoTracker DeepRed ( λ ex = 488 nm) can be well overlapped, and the fluorescent probe MISO ( λ ex = 488 nm) with LysoTracker DeepRed ( λ ex = 488 nm) had a colocalization coefficient (R) as high as 0.88; on the other hand, the fluorescent probe MISO showed a red rod-shaped fluorescence signal in the classic mitochondrial morphology (MISO, λ ex = 561 nm) and the commercially available mitochondrial probe MitoTracker DeepRed (MitoTracker, λ ex = 647 nm) can also overlap well, and the fluorescent probe MISO ( λ ex = 561 nm) and MitoTracker DeepRed (MitoTracker, λ ex = 647nm) had a colocalization coefficient (R) as high as 0.90, indicating that the fluorescent probe MISO can simultaneously image lysosomes and mitochondria in two colors, emitting green fluorescence in lysosomes and red fluorescence in mitochondria.

[0074] Example 4 Real-time monitoring of dynamic changes of lysosomes and mitochondria in living cells using fluorescent probe MISO

[0075] Adherent human osteosarcoma U2OS cells were incubated with the fluorescent probe MISO (final concentration of 10 μM) in an incubator at 37 °C, 5% CO2 and 20% O2 for 2 h, and the dynamic changes of lysosomes and mitochondria were monitored in situ under a laser confocal microscope. λ ex = 488 nm) to observe lysosomes; the red channel ( λ ex = 561 nm) to observe mitochondria ( Figure 7 ). In this example, the dynamic changes of lysosomes and mitochondria over time can be observed, including fusion, fusion and fission, and fission. Figure 7Indicated by the white arrows in the middle). This example observes four different interactions between lysosomes and mitochondria. MISO was used to monitor the dynamic interactions between lysosomes and mitochondria. Consistent with the dynamic properties of lysosomes and mitochondria, this example uses MISO to observe the contact between these two types of subcellular organelles. This example observes some short-term dynamic physical interactions between mitochondria and lysosomes. As shown in regions one and two, some mitochondria and lysosomes make short contact and then quickly leave. Region three shows the process in which some mitochondria and lysosomes first contact and then slowly leave. Region four records the process in which some lysosomes and mitochondria slowly approach and then aggregate.

[0076] Example 5 Real-time monitoring of mitochondrial autophagy in living cells using fluorescent probe MISO:

[0077] Adherent human osteosarcoma U2OS cells were incubated with the fluorescent probe MISO (final concentration of 10 μM) in an incubator at 37 °C, 5% CO2 and 20% O2 for 2 h, and the dynamic changes of lysosomes and mitochondria were monitored in situ under a laser confocal microscope. λ ex = 488 nm) to observe lysosomes ( Figure 7 ); Red channel ( λ ex = 561 nm) to observe mitochondria ( Figure 8 ). Adherent human osteosarcoma U2OS cells were serum starved for 0 h, 4 h, and 6 h, and then live U2OS cells were stained with 10 μM MISO and then imaged under a confocal microscope. Using MISO, we can directly observe the various stages of mitochondrial autophagy ( Figure 8 In cells without nutrient deprivation, MISO-stained mitochondria were striped, and the green dot-like fluorescence signals (lysosomes) in the cytoplasm did not overlap with the red filamentous fluorescence signals (mitochondria). When the cells were deprived of serum for 4 h, the MISO-stained mitochondria showed obvious shrinkage and the dot-like signals increased, but at this time the mitochondria and lysosomes were still not co-localized. After 6 h of starvation treatment, most of the MISO-stained mitochondria turned into a small sphere. And these mitochondria with red fluorescence signals and lysosomes with green fluorescence signals almost overlapped. This process is consistent with the current literature records ( Figure 8 (as shown in a).

[0078] The compound provided by the present invention has different excitation wavelengths and different colors of fluorescence under different acid-base environments, and can be used to prepare fluorescent probes for simultaneous imaging of different organelles. The fluorescent probe provided by the present invention can simultaneously dual-color image lysosomes and mitochondria in living cells or in vivo, showing green fluorescence in lysosomes and red fluorescence in mitochondria. The fluorescent probe provided by the present invention can monitor lysosomes and mitochondria in real time in living biological samples. At the same time, the fluorescent probe provided by the present invention can avoid the problems of mutual interference when using commercial mitochondrial probe MitoTracker and lysosomal probe LysoTracker to study lysosomes and mitochondria, and spend a lot of time exploring staining conditions. In addition, the preparation process of the fluorescent probe is simple, low-cost, and the structure is stable and easy to store, and it has a broad application space in the study of lysosomal and mitochondrial biological functions.

Claims

1. A compound, characterized in that: The structural formula of the compound is shown in formula (IV): Formula (IV); The R - At least one selected from the group consisting of halogen ions, p-toluenesulfonate ions, and trifluoromethanesulfonate ions.

2. The compound according to claim 1, characterized in that: The halogen ion is an iodide ion.

3. The compound according to claim 2, characterized in that: The compound has an excitation wavelength of 483-492 nm in an acidic environment; The compound has an excitation wavelength of 556-566 nm in a weakly alkaline environment.

4. A method for preparing the compound according to any one of claims 1 to 3, comprising the following steps: S1: The compound of formula (I) reacts with Ac2O, Et3N and Lawesson's reagent to obtain a compound of formula (II); S2: The compound of formula (II) undergoes a cyclization reaction under base catalysis to obtain a compound of formula (III); S3: The compound of formula (III) reacts with a methylating agent under heating conditions to obtain a methylated intermediate, which then undergoes a condensation reaction with 3, 4-dimethoxybenzaldehyde under heating conditions to obtain a final product; The structures of the compound of formula (I), the compound of formula (II) and the compound of formula (III) are shown below: 。 5. The preparation method according to claim 4, characterized in that: In the step S1, the compound of formula (I) is first reacted with Ac2O and Et3N at 16-37°C for 6-18h, and then reacted with Lawesson's reagent at 100-140°C for 1-3h.

6. The preparation method according to claim 5, characterized in that: In the step S3, the heating condition is 60-90°C.

7. Use of the compound according to any one of claims 1 to 3 in the preparation of fluorescent products.

8. The use according to claim 7, characterized in that: The fluorescent product is targeted to lysosomes and mitochondria.

9. A fluorescent probe, characterized in that: The invention comprises the compound according to any one of claims 1 to 3.

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