Micromolecular fluorescent probe for specific imaging autophagy as well as preparation method and application of micromolecular fluorescent probe

Abstract

The invention discloses a micromolecular fluorescent probe for specific imaging autophagy and a preparation method and application thereof, the fluorescent probe comprises a fluorophore, a linking group and an autophagy targeting group, the fluorophore is an intelligent fluorescence reporter group, the linking group adopts cysteine as a linking unit, and the autophagy targeting group targets LC3 protein; the probe can be used for specifically imaging an autophagy process of a biological sample and screening an autophagy regulator. The micromolecular fluorescent probe has the advantages of being good in cell membrane permeability, simple and convenient in dyeing process and easy to be compatible with other fluorescence labeling technologies, can perform specific recognition and selective imaging on an autophagy related structure, improves the targeting property and imaging credibility of the probe in a complex cell environment, and can be used for detecting autophagy related structures. The method can be used for visual tracing and detection of autophagy flow in living cells under physiological and pathological conditions, and is beneficial for promoting a small-molecule fluorescent probe to analyze an autophagy regulation mechanism and biological functions in real time.

Description

Technical Field

[0001] This invention relates to a small molecule fluorescent probe, and more particularly to a small molecule fluorescent probe for specific imaging of autophagy, its preparation method, and its application. Background Technology

[0002] Autophagy is a highly conserved intracellular degradation pathway that plays a crucial role in maintaining cellular homeostasis and physiological function through the renewal and clearance of proteins, organelles, and invading pathogens via lysosomes. Abnormal regulation of autophagy is closely related to the development and progression of various diseases, including neurodegenerative diseases, tumors, metabolic disorders, and aging. Therefore, accurate, real-time, and dynamic monitoring of autophagy at the living cell level is a significant technical challenge in basic research and disease mechanism studies related to autophagy.

[0003] Currently, autophagy imaging studies widely employ reporter systems based on genetically encoded fluorescent proteins, such as the mRFP-GFP-LC3 dual-fluorescent labeling method, which can be used to trace autophagosome formation and its fusion with lysosomes. However, this type of method relies on exogenous gene transfection or stable expression, and suffers from drawbacks such as complex procedures, uneven expression levels, and potential interference with endogenous autophagy pathways. Furthermore, its applicability is limited in primary cells, tissue samples, or cell systems that are difficult to transfect, restricting its application in a wider range of systems.

[0004] To overcome the limitations imposed by gene manipulation, various small-molecule fluorescent probes have been developed in recent years for autophagy-related imaging. Small-molecule probes offer advantages such as no need for transfection, high cell membrane permeability, and ease of manipulation, thus expanding the application scenarios of autophagy imaging to some extent. However, most existing small-molecule autophagy fluorescent probes do not directly identify key molecular events in the autophagy process, but mainly rely on changes in the intracellular microenvironment (such as pH, polarity, or lysosomal acidification) to generate fluorescence signals. In the complex cellular environment, these probes often struggle to distinguish autophagy-related structures from other acidic organelles, exhibiting technical shortcomings such as insufficient targeting, low specificity, and inaccurate response to autophagic flux.

[0005] Therefore, while fully leveraging the inherent advantages of small-molecule fluorescent probes, such as good cell membrane permeability, simple staining procedures, and easy compatibility with other fluorescent labeling technologies, a key technical problem urgently needing to be solved in the field of small-molecule autophagy fluorescent probes is how to achieve specific recognition and selective imaging of autophagy-related structures, thereby improving the targeting and imaging reliability of probes in complex cellular environments. This is of great significance for promoting the real-time analysis of autophagy regulatory mechanisms and biological functions under physiological or pathological conditions using small-molecule fluorescent probes. Summary of the Invention

[0006] Purpose of the invention: The purpose of this invention is to provide a small molecule fluorescent probe for specific imaging autophagy, its preparation method, and its application.

[0007] Technical solution: The small molecule fluorescent probe for specific imaging autophagy includes a fluorescent group, a linker group, and an autophagy targeting group. The autophagy targeting group selectively targets the autophagy marker LC3 protein, and the linker group uses cysteine ​​as the linker unit.

[0008] Using cysteine ​​as a linker offers multiple advantages in probe construction, including a well-defined linking mechanism, high synthetic flexibility, and compatibility with various functional modules. This linking strategy spatially separates the target group from the fluorophore, thereby minimizing potential interference from the fluorophore to the autophagy directing group. To achieve a dynamic fluorescence reporter signal, a smart fluorophore is employed, possessing the following characteristics: it is activated to emit light when its internal rotation is restricted or in a hydrophobic environment, and it exhibits a significant spectral redshift response based on acidification.

[0009] The autophagy targeting group is preferably guanine as the autophagy targeting core. S-guanosine monophosphate is most preferred. A cysteine-based linker is introduced at the C8 site of S-guanosine monophosphate to couple a smart fluorescent reporter group. After membrane binding, the reporter group is activated by enhanced hydrophobic environment and restricted intramolecular rotation, thus exhibiting different fluorescence in response to pH changes during the conversion from autophagosome to autolysosome. This enables highly sensitive visualization of autophagic flux and real-time monitoring of the autophagy process.

[0010] The fluorescent probe preferably has the structural formula shown in formula (I):

[0011]

[0012] Equation (Ⅰ).

[0013] The fluorescent probe was named ATP3. It can change its fluorescence in response to changes in pH, showing yellow fluorescence under neutral conditions and red fluorescence under acidic conditions, thus distinguishing autophagosomes from autolysosomes.

[0014] The present invention also provides a method for preparing the fluorescent probe, the preparation route of which is as follows:

[0015] .

[0016] The present invention also provides the application of the fluorescent probe in the autophagy process of specific imaging biological samples.

[0017] The biological sample is preferably a live cell or a live animal.

[0018] The live cells are preferably under physiological or pathological conditions. Physiological live cell detection preferably uses basic autophagy imaging of adherent cells, suspension cells, and primary cells, while pathological live cell detection preferably uses autophagy imaging of cells subjected to oxygen-glucose deprivation, starvation induction, drug treatment, and gene defects.

[0019] The preferred method for detecting live animals is live two-photon imaging.

[0020] The present invention also provides the application of the fluorescent probe in screening autophagy regulators.

[0021] Beneficial Effects: Compared with existing technologies, the present invention has the following significant advantages: The small-molecule fluorescent probe for specific imaging autophagy provided by the present invention has: 1. Good cell membrane permeability, enabling detection of autophagy in living cells; 2. Strong fluorescence signal, high signal-to-noise ratio, and good sensitivity; 3. Autophagy specificity and high spatiotemporal resolution, enabling real-time and dynamic monitoring of the autophagy process in living cells; 4. Good pH responsiveness, enabling real-time tracing of the entire autophagy process through fluorescent labeling of autophagosomes and autolysosomes. The fluorescent probe enhances targeting and imaging reliability in complex cellular environments, and can be used for the visualization, tracing, and detection of autophagy flow in living cells under physiological and pathological conditions, which is beneficial for promoting the real-time analysis of autophagy regulatory mechanisms and biological functions using small-molecule fluorescent probes. Attached Figure Description

[0022] Figure 1 The synthetic route for the fluorescent group N3-F5;

[0023] Figure 2 The synthetic route for the autophagy fluorescent probe ATP3;

[0024] Figure 3 Characterization of the environmentally responsive fluorescence properties of the autophagy fluorescent probe ATP3;

[0025] Figure 4 The responsiveness of the autophagy fluorescent probe ATP3 to autophagy regulators;

[0026] Figure 5 The labeling efficiency and signal-to-noise ratio of the autophagy fluorescent probe ATP3;

[0027] Figure 6 Application of the autophagy fluorescent probe ATP3 in autophagy gene knockout cell lines;

[0028] Figure 7 The effect of oxygen-glucose deprivation on autophagic flux in brain vascular endothelial cells was detected using the autophagy fluorescent probe ATP3. Detailed Implementation

[0029] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0030] Unless otherwise specified, all raw materials used are commercially available products well known to those skilled in the art or prepared using methods well known to those skilled in the art.

[0031] Example 1

[0032] This embodiment provides a small molecule fluorescent probe ATP3 for specific imaging of autophagy, comprising a fluorescent group, a linker group, and an autophagy-targeting group. The autophagy-targeting group selectively targets the autophagy marker LC3 protein, and the linker group uses cysteine ​​as the linker unit. The autophagy-targeting group uses guanine as the autophagy-targeting core. The fluorescent probe has the structural formula shown in formula (I):

[0033]

[0034] Equation (Ⅰ).

[0035] Based on the design concept of targeted autophagy, this embodiment selects guanine as the autophagy targeting core and introduces a cysteine-based linker at its C8 site to achieve site-specific coupling. Figure 3 A). This connection strategy spatially separates the target group from the fluorophore, thereby minimizing the potential interference of the fluorophore with the autophagy-directing function of the guanine group. To achieve a dynamic fluorescence reporter signal, this embodiment introduces a smart fluorophore. This fluorophore has the following characteristics: it is activated to emit light when its molecular spin is restricted or in a hydrophobic environment, and it can produce a significant spectral redshift response based on the acidification environment (…). Figure 3 A).

[0036] Example 2

[0037] This embodiment provides a method for preparing the fluorescent probe ATP3 described in Example 1, the preparation route as follows: Figure 1-2 As shown. Details are as follows.

[0038] 1. Chemical synthesis method of fluorescent group N3-F5.

[0039] Specific steps are as follows Figure 1 As shown: F5 (17 mg, 0.056 mmol), 6-azidohexanoic acid (10.5 mg, 0.067 mmol), HOBt (11 mg, 0.084 mmol), and EDCI (16 mg, 0.084 mmol) were dissolved in anhydrous dichloromethane (20 ml), followed by the addition of DIPEA (20 μL, 0.112 mmol). The reaction mixture was stirred at room temperature for 2 hours. After dichloromethane was removed by evaporation, a yellow oil was obtained, which was purified by rapid column chromatography (PE:EA = 1:2, v / v) to give a yellow foamy solid N3-F5 (15.5 mg, yield 62.5%).

[0040] N3-F5 characterization data: 1 H NMR (500 MHz, DMSO) δ 8.11 (d, J = 9 Hz, 2H), 8.08(d, J = 8.6 Hz, 1H), 7.71 (d, J = 8.7 Hz, 1H), 7.65 (d, J =8.5 Hz, 1H), 7.06(d, J = 9 Hz, 2H), 7.00 (dd, J = 9.0, 2.6 Hz, 1H), 6.57 (d, J = 2.5 Hz, 1H), 3.62 (s, 4H), 3.40 (t, J = 6.5 Hz, 4H), 3.28 (t, J = 6.5 Hz, 2H), 3.23 (t, J= 6.5 Hz, 2H), 2.44 (t, J = 7.5 Hz, 2H), 2.02 (m, 4H), 1.54 (m, 4H), 1.35 (m, 2H), 1.26 (m, 2H).

[0041] 2. Chemical synthesis method of small molecule fluorescent probe ATP3.

[0042] Specific steps are as follows Figure 2 As shown.

[0043] Step 1: Synthesize compound 1.

[0044]

[0045] In a dry 100 mL round-bottom flask, di-tert-butyl dicarbonate (34 mL, 147 mmol) and 4-dimethylaminopyridine (177 mg, 0.45 mmol) were added to a THF suspension of 2-amino-6-chloroguanine (5 g, 29 mmol) in 350 mL, and the reaction was carried out at room temperature. As the reaction proceeded, the starting materials gradually dissolved, and the reaction solution gradually became clear. After 30 minutes, the reaction solution became completely clear. The reaction was monitored by TLC until it was complete. The solvent was removed by vacuum distillation, and the crude product was separated and purified by silica gel column chromatography (PE:EtOAc=5:1) to obtain an orange oil. 20 mL of ethyl acetate was added to dissolve the oil, and then 300 mL of petroleum ether was added and sonicated thoroughly, precipitating a large amount of white solid. The solid was filtered to obtain compound 1 (9 g, yield 66%).

[0046] Characterization data of compound 1: 1H NMR (500 MHz, DMSO) δ 9.01 (s, 1H), 1.61 (s, 9H), 1.42 (s, 18H).

[0047] Step 2: Synthesize compound 2.

[0048]

[0049] In a dry 500 mL round-bottom flask, a saturated sodium bicarbonate solution (120 mL) was added to a MeOH solution (150 mL) containing 8 g (17 mmol). The reaction was heated to 50 °C. The reaction was monitored by TLC until complete. Most of the methanol was removed by vacuum distillation, followed by extraction with ethyl acetate (100 mL), washing with water (100 mL), and then with saturated brine (30 mL). The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. The mixture was then purified by silica gel column chromatography (PE:EtOAc = 1:1) to give a white solid, compound 2 (3.3 g, yield 52.6%).

[0050] Characterization data of compound 2: 1 H NMR (500 MHz, DMSO) δ 14.14 (s, 1H), 8.77 (s, 1H), 1.40 (s, 18H).

[0051] Step 3: Synthesize compound 3.

[0052]

[0053] In a dry 100 mL round-bottom flask, p-fluorobenzyl bromide (0.96 mL, 8 mmol) was added to a DMF (30 mL) solution of compound 2 (2.45 g, 6.6 mmol) and potassium carbonate (2.75 g, 19.9 mmol). The reaction was allowed to proceed at room temperature. The reaction was monitored by TLC until complete. The reaction was quenched with water, extracted with ethyl acetate (30 mL), washed with water (50 mL × 3), washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was removed by vacuum distillation. The filtrate was then purified by silica gel column chromatography (PE: EtOAc = 5:1–3:1). Compound 3 (1.63 g, 54%) was obtained as a white solid.

[0054] Characterization data of compound 3: 1H NMR (500 MHz, DMSO) δ 8.94 (s, 1H), 7.41 – 7.37 (m, 2H), 7.20 – 7.15 (m, 2H), 5.53 (s, 2H), 1.31 (s, 18H).

[0055] Step 4: Synthesize compound 4.

[0056]

[0057] In a dry 50 mL single-necked flask, compound 3 (800 mg, 1.7 mmol) was added to 18 mL of 80% formic acid solution and reacted at 80 °C. The reaction was monitored by TLC until complete, then heating was stopped and the reaction was allowed to return to room temperature. The solvent was removed by vacuum distillation. This yielded a white compound 4 (410 mg, 93.2%).

[0058] Characterization data of compound 4: 1 H NMR (500 MHz, DMSO) δ 11.11 (s, 1H), 8.19 (s, 1H), 7.35 – 7.31 (m, 2H), 7.18 (m, 2H), 6.90 (s, 2H), 5.21 (s, 2H).

[0059] Step 5: Synthesize compound 5.

[0060]

[0061] In a dry 50 mL single-necked flask, compound 4 (810 mg, 3.1 mmol) was added to 8 mL of glacial acetic acid. Liquid bromine (200 μL, 3.7 mmol) was added with stirring in an ice bath, and the reaction was allowed to proceed at room temperature. The reaction was monitored by TLC until complete. 20 mL of ice water was added to the reaction mixture, precipitating a pale yellow solid. The solid was filtered, and the filter cake was washed twice with saturated sodium thiosulfate solution. The filter cake was dried to give a white solid, compound 5 (860 mg, 82%). The crude product was confirmed by LC-MS and directly added to the next reaction step.

[0062] Characterization data of compound 5: ESI-MS (m / z): [M+H]+=338.

[0063] Step 6: Synthesize compound 6.

[0064]

[0065] In a dry 100 mL double-necked flask, compound 5 (530 mg, 1.55 mmol), N-acetylcysteine ​​(1 g, 6.2 mmol), and potassium carbonate (2.1 g, 15.5 mmol) were added to completely displace the nitrogen atmosphere. Then, 60 mL of dry DMF was added to the reaction flask. The nitrogen atmosphere was completely displaced again, and the reaction was heated to 90 °C. After the reaction was monitored by LC-MS until complete, the mixture was cooled to room temperature, and the potassium carbonate was filtered off. The filtrate was distilled under reduced pressure to remove the DMF, yielding a pale yellow oil. 10 mL of methanol was added to dissolve the oil, followed by 10 mL of dichloromethane and 15 mL of ethyl acetate. The mixture was sonicated thoroughly until a white solid precipitated. The solid was filtered to give compound 6 (500 mg, yield 76.8%) as a white solid.

[0066] Characterization data of compound 6: 1 H NMR (500 MHz, DMSO) δ 7.71 (s, 1H), 7.26 (t, J =8.4 Hz, 2H), 7.16 (t, J = 8.4 Hz, 2H), 5.09 (s, 2H), 4.26 (s, 1H), 3.17 (s,1H), 1.86 (s, 3H).

[0067] 13 C NMR (125 MHz, DMSO) δ 173.54 (s), 169.27 (s), 162.89 (s), 160.96 (s), 129.82 (s), 129.75 (s), 129.66 (s), 129.59 (s), 116.00 (s), 115.83 (s), 54.38 (d, J = 4.0 Hz), 43.96 (s), 36.23 (s), 23.31 (s).

[0068] ESI-HRMS: m / z =[M+H]+calculated 421.1094, found 421.1094.

[0069] Step 7: Synthesize compound 7.

[0070]

[0071] In a dry 25 mL round-bottom flask, compound 6 (50 mg, 0.12 mmol), propyneamine (45 μL, 0.7 mmol), TCFH (50 mg, 0.18 mmol), and NMI (33 μL, 0.3 mmol) were added to DMF (5 mL), and the mixture was reacted at room temperature for 24 hours. The solvent was removed by vacuum distillation to obtain a brown oily substance, which was separated using a FLASH-C18 column (H2O: MeOH). The collected pale yellow-brown liquid was further purified by vacuum distillation to obtain a light brown oily substance. The crude product, confirmed by LC-MS, was directly added to step 8 of the reaction.

[0072] Characterization data of compound 7: ESI-MS (m / z): [M+H]+=458.

[0073] Step 8: Synthesize ATP3.

[0074]

[0075] Compound 7 (75 mg, 11 μmol) and 5.5 mg (11 μmol) of N3-F5 were dissolved in a mixture of methanol (2 ml), tetrahydrofuran (0.5 ml), and water (0.5 ml). Copper sulfate pentahydrate (0.25 mg, 1 μmol) and sodium ascorbate (0.6 mg, 3 μmol) were then added, and the reaction mixture was stirred at room temperature for 12 hours. The reaction solution was concentrated under reduced pressure and purified using a rapid purification system (water / methanol gradient 30% to 100%, run time 30 min, flow rate 1 mL / min, detection at 254 nm) to obtain a yellow, foamy solid ATP3 (2.3 mg, yield 11.9%).

[0076] ATP3 characterization data: 1H NMR (500 MHz, DMSO) δ 8.51 (d, J = 7.5 Hz, 2H), 8.51(s, 2H), 8.10 (d, J = 8.6 Hz, 2H), 8.07 (d, J = 8.6 Hz, 1H), 7.89 (s, 1H),7.69 (d, J = 9.2 Hz, 1H), 7.63 (d, J = 8.5 Hz, 1H), 7.19 (d, J = 5.4 Hz, 2H), 7.13 (t, J = 8.7 Hz, 2H), 7.05 (d, J = 8.3 Hz, 2H), 7.03 – 7.01 (m, 1H), 7.01(s, 1H), 6.83(s, 1H), 5.34 – 5.30(m, 1H), 5.06(s, 2H), 4.52 – 4.46 (m, 2H), 4.22 (t, J = 6.5 Hz, 2H), 3.62 – 3.56 (m, 4H), 3.22 – 3.19 (m, 4H), 3.16 (t,J =5 Hz, 4H), 2.34 – 2.30 (t, J =7 Hz, 2H), 2.04 – 2.00 (m, 4H), 1.98 – 1.96(m, 2H), 1.86(t, 3H), 1.53 – 1.51 (m, 2H), 1.36 – 1.33(m, 2H).

[0077] Example 3

[0078] This embodiment is a characterization of the environmentally responsive fluorescence properties of the probe ATP3 described in Example 1.

[0079] The ATP3 probe was systematically characterized by solution-phase spectrometry to elucidate its photophysical properties and environmental response behavior. First, in phosphate buffer, ATP3 exhibited only negligible fluorescence emission, indicating extremely low background fluorescence in an aqueous environment. Subsequently, to assess the activation effect of the hydrophobic interface on fluorescence, ATP3 was measured in PBS containing 10 mg / mL cetyltrimethylammonium bromide. A significant increase in fluorescence intensity was observed compared to pure PBS, and acidification of the solution caused a significant red shift in the emission spectrum. Figure 3 B).

[0080] Furthermore, by altering the composition of the water / acetonitrile mixed solvent to adjust the polarity, it was found that the fluorescence intensity of ATP3 significantly increased with increasing acetonitrile ratio (decreasing polarity); under these conditions, lowering the pH of the aqueous phase of the mixed solvent also resulted in a redshift in the emission spectrum. Figure 3 C).

[0081] In addition, to investigate the effect of restricted intramolecular motion, ATP3 was dissolved in a high-viscosity glycerol / water mixture for measurement. It showed strong fluorescence enhancement in 90% glycerol medium, and acidification of the medium also caused a red shift in the spectrum. Figure 3 D).

[0082] The above results demonstrate that the ATP3 probe possesses the following key characteristics: 1. Extremely low background fluorescence in aqueous environments; 2. Its fluorescence emission can be significantly activated by hydrophobic environments, low-polarity solvents, or high-viscosity media; 3. In the activated state, its emission spectrum exhibits a specific redshift response to environmental acidification. These photophysical properties provide a foundation for the subsequent specific detection and imaging of autophagy-related microenvironmental changes in living cells using the ATP3 probe.

[0083] Example 4

[0084] This embodiment describes the detection of the response of ATP3 to drug-induced autophagy using live-cell imaging.

[0085] To evaluate the performance of the ATP3 probe in monitoring autophagic flux, autophagy was pharmacologically inhibited using wortmannin (Wort) or bafilomycin A1 (BafA1) under rapamycin (Rapa)-induced autophagy conditions. Wortmannin, as a PI3K inhibitor, was used to inhibit autophagosome formation; bafilomycin A1, as a V-ATPase inhibitor, was used to block the fusion of autophagosomes and lysosomes.

[0086] The experiment was divided into the following treatment groups: 1) untreated control group; 2) rapamycin alone treatment group (10 μM, 4 h); 3) rapamycin (10 μM, 4 h) combined with womanpemicin (1 μM, 6 h) treatment group; 4) rapamycin (10 μM, 4 h) combined with bafloxacin A1 (100 nM, 2 h) treatment group. After treatment and washing, cells in each group were stained with ATP3 probe (2.5 μM) at 37°C for 25 minutes.

[0087] After staining, cells were imaged using a confocal microscope in a dual-channel configuration: a green fluorescence channel (excitation wavelength 493 nm, emission wavelength 517 nm) and a red fluorescence channel (excitation wavelength 590 nm, emission wavelength 618 nm). Based on the characteristic changes in fluorescence signal of the ATP3 probe in an acidic environment, the following criteria were established: yellow dot-like structures (i.e., co-localization of green and red fluorescence) in the dual-channel superimposed image were identified as autophagosomes; dot-like structures showing only red fluorescence (green fluorescence quenched) were identified as autolysosomes. Quantitative analysis of autophagosomes and autolysosomes in cells was performed using Imaris software to assess the state of autophagic flux. Figure 4 B,C).

[0088] Confocal imaging results showed that, compared with the untreated control group, rapamycin treatment alone significantly enhanced the signal intensity of the green and red fluorescence channels and induced the formation of numerous yellow dot-like structures, indicating increased autophagosome generation. A small number of dot-like structures were also observed in the untreated control group, demonstrating that the ATP3 probe can detect basal-level autophagy. In the woumacil-based combined treatment group, both the fluorescence intensity of the dual channels and the number of dot-like structures were significantly reduced, consistent with the expectation of inhibited autophagosome formation. In the bafloxacin A1 combined treatment group, compared with rapamycin treatment alone, an increase in the accumulation of yellow dot-like structures (autophagosomes) was observed, while the number of red-only dot-like structures (autolysosomes) decreased, clearly reflecting the stagnation of autophagic flux caused by the blockage of the autophagosome-lysosome fusion process. Figure 4 A).

[0089] This embodiment demonstrates that the ATP3 probe can sensitively distinguish and visualize autophagosomes and autolysosomes, and is suitable for monitoring the dynamic changes of autophagic flux, including basal autophagy, induced autophagy, and pharmacological perturbations.

[0090] Example 5

[0091] This embodiment detects the labeling efficiency and signal-to-noise ratio of the autophagy imaging probe ATP3.

[0092] After Example 4 demonstrated that the probe ATP3 described in Example 1 could be used for autophagy flux visualization, this example compares its performance with that of the conventional mRFP-GFP-LC3 detection method.

[0093] First, the uniformity of staining was evaluated. The mRFP-GFP-LC3 assay relies on gene transfection, often leading to uneven expression among cells and affecting reproducibility. In contrast, ATP3, as a chemical probe, requires no transfection step, thus achieving uniform and stable staining across the cell population. To verify this, we used adenovirus (AV) expression of mRFP-GFP-LC3 and ATP3 fluorescent probe staining to detect basal autophagy levels in HeLa cells. Thirty-six hours after adenovirus infection, confocal imaging showed that only about 12% of cells exhibited obvious fluorescent spots, while using ATP3 for live cell staining required only a short 25-minute incubation to label almost all cells and produce bright fluorescent spots. Figure 5 (A, B). This result demonstrates that ATP3 staining is uniform, easy to perform, and suitable for rapid detection of autophagy in live cells.

[0094] The imaging contrast was then evaluated. Random region sampling analysis of fluorescence images obtained from AV-mRFP-GFP-LC3 infection and ATP3 staining showed that the background fluorescence intensity of the ATP3-stained group was approximately 30% lower than that of the mRFP-GFP-LC3 group. Furthermore, the ATP3 fluorescent probe exhibited a higher signal-to-noise ratio in both the green and red channels, approximately 20-fold higher than that of the AV-mRFP-GFP-LC3 group. Figure 5 (C-5E). These results fully demonstrate that ATP3 has superior imaging contrast and can more clearly image autophagy levels; moreover, ATP3 effectively suppresses background signals due to its fluorescence quenching design under acidic conditions.

[0095] This embodiment confirms that ATP3 is superior to the traditional mRFP-GFP-LC3 detection method in monitoring autophagic flux, with the main advantages including: (1) lower background and higher imaging contrast; and (2) consistent performance across cell populations. These characteristics make ATP3 a reliable and widely applicable probe for autophagy research, applicable to a variety of experimental conditions.

[0096] Example 6

[0097] This embodiment uses ATP3 to detect autophagy flux under autophagy gene knockout conditions.

[0098] To verify the specificity of the ATP3 probe in detecting autophagy, this embodiment used CRISPR-Cas9 gene editing technology to construct stable HEK293 cell lines with ATG5, ATG7, FIP200, and LC3B gene knockouts. After incubating the above gene knockout cell lines and wild-type control cells with the ATP3 probe (2.5 μM) at 37°C for 25 minutes, dual-channel confocal imaging analysis was performed.

[0099] The results showed that in ATG5, ATG7, FIP200, or LC3B knockout cells, the number of fluorescent dot structures presented by the ATP3 probe was significantly reduced compared to wild-type control cells. Figure 6 A). This phenomenon indicates that autophagic flux is effectively inhibited under gene knockout conditions, further demonstrating the high correlation between the ATP3 probe signal and the autophagy process. Quantitative analysis of autophagosomes and autolysosomes in cells using Imaris software, comparing the number of punctate structures in each group of cells, clearly distinguishes the autophagy activity levels between wild-type and gene knockout cell lines. Figure 6 B,C).

[0100] This embodiment demonstrates that the ATP3 probe can stably and specifically reflect changes in autophagy flux caused by the deletion of core autophagy-related genes under genetic perturbation conditions, and is suitable for research and functional evaluation of autophagy mechanisms based on genetic manipulation.

[0101] Example 7

[0102] In this embodiment, ATP3 was used to monitor changes in autophagic flux induced by oxygen-glucose deprivation.

[0103] To verify the ability of the ATP3 probe to monitor the dynamic changes in autophagy under simulated ischemia-reperfusion injury pathophysiological conditions, this embodiment applies it to an oxygen-glucose deprivation model of human brain microvascular endothelial cells.

[0104] Human brain microvascular endothelial cells were divided into a control group and an experimental group. The experimental group underwent oxygen-glucose deprivation treatment for 3 hours, 6 hours, and 12 hours, respectively. After treatment, live cells were stained with an ATP3 probe (2.5 μM) (37℃, 25 min), and dual-channel time-series imaging was performed under a confocal microscope using both green and red fluorescence channels. Figure 7 A).

[0105] Imaging results showed that with prolonged oxygen-glucose deprivation treatment, the number of red fluorescent dot structures representing autophagosomes gradually increased, while the number of yellow fluorescent dot structures (i.e., red-green fluorescence co-localization) representing autophagosomes decreased accordingly. This dynamic change in the composition of these dot structures indicates a sustained enhancement of autophagic flux. Figure 7 (B, C). This result confirms the existence of functional autophagic flux under oxygen-glucose deprivation conditions.

[0106] This embodiment demonstrates that the ATP3 probe can achieve real-time and sensitive visualization of the dynamics of autophagy flux in living cells under oxygen-glucose deprivation stress, and is suitable for monitoring changes in autophagy activity in disease-related pathological models.

Claims

1. A small molecule fluorescent probe for specific imaging of autophagy, characterized in that, It includes a fluorescent group, a linker group, and an autophagy-targeting group, wherein the autophagy-targeting group selectively targets the autophagy marker LC3 protein, and the linker group uses cysteine ​​as the linker unit.

2. The fluorescent probe according to claim 1, characterized in that, The autophagy targeting group uses guanine as the autophagy targeting core.

3. The fluorescent probe according to claim 2, characterized in that, It has the structural formula shown in equation (Ⅰ): Equation (Ⅰ).

4. A method for preparing the fluorescent probe according to claim 3, characterized in that, The preparation route is as follows: 。 5. The application of the fluorescent probe of claim 1 in the autophagy process of specific imaging biological samples.

6. The application according to claim 5, characterized in that, The biological sample is a living cell or a living animal.

7. The application according to claim 6, characterized in that, The living cells are under physiological or pathological conditions.

8. The application according to claim 7, characterized in that, Physiological live cell detection includes basic autophagy imaging of adherent cells, suspension cells, and primary cells, while pathological live cell detection includes autophagy imaging in cells subjected to oxygen and glucose deprivation, starvation induction, drug treatment, and genetically defective cells.

9. The application according to claim 7, characterized in that, The method for detecting live animals is live two-photon imaging.

10. The use of the fluorescent probe of claim 1 in screening autophagy regulators.

Images