Mitochondrial membrane and mitochondrial DNA double-targeting fluorescent probe compounds, and preparation method and application thereof
Patent Information
- Application Number
- CN202610828813.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-06-09
- Publication Date
- 2026-10-27
Smart Images
Figure CN122978183A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of fluorescent compounds targeting mitochondrial DNA. More specifically, it relates to a class of fluorescent probe compounds that target both the mitochondrial membrane and mitochondrial DNA, their preparation methods, and applications. Background Technology
[0002] Mitochondria are the only organelles in eukaryotes that contain their own DNA (mitochondrial DNA, mtDNA). They have a double-membrane structure, and their primary function is to synthesize adenosine triphosphate (ATP) through oxidative phosphorylation, providing the chemical energy needed for cellular activities. Since the cellular environment is constantly changing and consuming energy, mitochondrial health directly determines the cell's ability to adapt to these dynamic changes. Therefore, maintaining mitochondrial homeostasis is crucial for cellular metabolism. mtDNA differs significantly from nuclear DNA in genetics and topology, and is indispensable for functions such as ATP production, redox balance, maintenance of cellular energy homeostasis, and regulation of cell death signals. Any factor affecting the structure, copy number, or integrity of mtDNA can interfere with transcription and replication, impairing mtDNA stability and consequently affecting fundamental physiological processes such as metabolism, differentiation, and cell division. Furthermore, the main site of reactive oxygen species (ROS) production in cells is the mitochondria, while mtDNA is distributed in the mitochondrial matrix, close to the electron transport chain (ETC). Therefore, mtDNA is highly susceptible to damage, and this vulnerability subsequently leads to mtDNA homeostasis imbalance, which is associated with various diseases such as neurodegenerative diseases, metabolic disorders, cardiovascular diseases, and cancer. In conclusion, real-time monitoring of mitochondrial and mtDNA status in living cells is crucial for understanding mitochondrial regulatory mechanisms under physiological and pathological conditions.
[0003] Small molecule fluorescent probes are low-invasive, enabling in-situ, real-time monitoring of the dynamic behavior of biomolecules without interfering with the normal physiological state of biological systems, providing a powerful tool for visualizing life processes. To date, various fluorescent probes for mtDNA imaging have been reported, with PicoGreen, SYBR Green, and SYBR Gold being the mainstream commercial probes. However, these dyes also exhibit significant non-specific staining for nuclear DNA, severely limiting their specificity and practicality in mtDNA detection. This makes accurate identification and quantification of mtDNA in living cells extremely difficult, thus hindering research using mtDNA as an indicator to assess cellular health. Furthermore, some novel mtDNA probes have been developed. Currently, there are high-resolution imaging probes targeting the dynamic behavior of mtDNA, such as N-arylpyridinium-anthocyanin derivative probes suitable for two-photon and super-resolution imaging of the cell nucleus and mtDNA, and probes that specifically respond to mitochondrial G-quadruplex DNA. Nevertheless, these probes can only achieve mtDNA imaging and still need to be used in conjunction with commercially available mitochondrial labeling dyes to achieve double staining of mitochondrial membrane and nuclear mtDNA, which increases the complexity of staining.
[0004] Therefore, there is an urgent need to develop highly specific and easy-to-use multifunctional fluorescent probes to achieve detailed observation of mitochondria and mtDNA. Summary of the Invention
[0005] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of existing small molecule fluorescent probes, such as significant non-specific staining of nuclear DNA, difficulty in simultaneously and specifically recognizing mitochondrial membrane and mtDNA in living cells, difficulty in achieving spatial resolution of two colors for two existing probes, the need for multiple complex operations in conjunction with commercially available dyes, and the tendency to affect the physiological state of cells. The primary objective is to provide a class of fluorescent probe compounds or their pharmaceutically acceptable salts.
[0006] A second objective of this invention is to provide a method for preparing the fluorescent probe compound.
[0007] A third object of the present invention is to provide the use of the fluorescent probe compound or a pharmaceutically acceptable salt thereof in the preparation of mtDNA-specific recognition and labeling reagents.
[0008] A fourth object of the present invention is to provide the use of the fluorescent probe compound or a pharmaceutically acceptable salt thereof in the preparation of diagnostic reagents for mitochondrial dysfunction.
[0009] The fifth objective of this invention is to provide a reagent.
[0010] The above-mentioned objective of this invention is achieved through the following technical solution: This invention comprises a class of fluorescent probe compounds or pharmaceutically acceptable salts thereof, said fluorescent probe compounds having the following structure:
[0011] In the above structure, R is a heteroatom, and the heteroatom is selected from S or O. 1 R 2 Each is independently selected from C 1~6 alkyl.
[0012] Furthermore, the R 1 R 2 Each is independently selected from C 1~3 Alkyl group. This structure has little effect on the fluorescence properties of the probe compound.
[0013] Furthermore, the R 1 R 2 Each is independently selected from methyl, ethyl, or n-propyl.
[0014] Preferably, the fluorescent probe compound has any of the following structures: .
[0015] This invention also protects a method for preparing the fluorescent probe compound or a pharmaceutically acceptable salt thereof, the preparation of the fluorescent probe compound comprising the following steps: S1. 6-hydroxy-2H-benzofuran-3-one, compound 1 and a basic reagent are mixed in a first organic solvent and reacted under heating conditions. After the reaction is complete, the intermediate compound 2 is obtained by post-treatment. S2. The intermediate compound 2 and compound 3 obtained in step S1 and the basic reagent are mixed in a second organic solvent and reacted under heating conditions. After the reaction is complete, the target compound is obtained through post-treatment. Wherein, the structure of compound 1 is as follows: The structure of the intermediate compound 2 is as follows: ; The structure of compound 3 is as follows: .
[0016] Furthermore, the preparation method includes at least one of the following conditions: (1) In step S1 or step S2, the temperature at which the reaction is completed is 70~120 ℃.
[0017] (2) The alkaline reagent is selected from at least one of sodium carbonate, potassium carbonate, sodium bicarbonate, sodium hydroxide, and potassium hydroxide; (3) The first organic solvent is selected from at least one of methanol, ethanol, and glacial acetic acid; (4) The second organic solvent is selected from at least one of acetonitrile, N,N-dimethylformamide, dichloromethane, tetrahydrofuran, and toluene.
[0018] Furthermore, in step S1 or step S2, the temperature at which the reaction is completed is 70~90 ℃, preferably 75~85 ℃.
[0019] Further, in step S1, the molar ratio of 6-hydroxy-2H-benzofuran-3-one to compound 1 is 1:(0.8~2), preferably 1:(1.2~1.6).
[0020] Further, in step S1, the molar ratio of the 6-hydroxy-2H-benzofuran-3-one to the basic reagent is 1:(0.1~0.3), preferably 1:(0.15~0.25).
[0021] Further, in step S1, the mixing ratio of the 6-hydroxy-2H-benzofuran-3-one and the first organic solvent is 1 mol: (1~3) mL, preferably 1 mol: (1.5~2.5) mL.
[0022] Further, in step S2, the molar ratio of intermediate compound 2 and compound 3 is 1:(0.8~2), preferably 1:(1.3~1.7).
[0023] Further, in step S2, the molar ratio of the intermediate compound 2 and the basic reagent is 1:(0.8~3), preferably 1:(1~2.6).
[0024] Further, in step S2, the mixing ratio of the intermediate compound 2 and the second organic solvent is 1 mol: (5~35) mL, preferably 1 mol: (7~30) mL.
[0025] Furthermore, in step S1, the reaction takes 3 to 8 hours to complete, preferably 5 to 7 hours.
[0026] Furthermore, in step S2, the reaction takes 5 to 10 hours to complete, preferably 7 to 9 hours.
[0027] Further, in step S1, the post-processing includes cooling, vacuum concentration, and chromatography, specifically including the following steps: After the reaction is complete, the mixture is cooled to room temperature and concentrated under vacuum to obtain a crude product. Then, the crude product is purified by chromatography using a column (methanol (MeOH / ) and dichloromethane (DCM) as eluents, with a volume ratio of 1:80, i.e., MeOH / DCM=1:80) to obtain intermediate compound 2.
[0028] Further, in step S2, the post-processing includes cooling, vacuum concentration, and chromatography, specifically including the following steps: After the reaction is complete, the reaction solution is cooled to room temperature and concentrated under vacuum to obtain a crude product. Then, the crude product is purified by chromatography (using MeOH and DCM as eluents, with a volume ratio of 1:20, i.e., MeOH / DCM=1:20) to obtain the target compound.
[0029] The present invention also protects the use of the fluorescent probe compound or a pharmaceutically acceptable salt thereof in the preparation of reagents for the simultaneous and specific recognition and labeling of mitochondrial membrane and mitochondrial DNA.
[0030] Furthermore, the reagent is used for imaging the mitochondrial membrane and mitochondrial DNA of cells.
[0031] Furthermore, the cells include living cells.
[0032] Furthermore, the imaging includes real-time tracking imaging of mitochondrial membranes and mtDNA in living cells.
[0033] This invention also protects the use of the fluorescent probe compound or a pharmaceutically acceptable salt thereof in the preparation of diagnostic reagents for mitochondrial dysfunction.
[0034] The present invention also protects a reagent comprising one or more of the fluorescent probe compound or a pharmaceutically acceptable salt thereof.
[0035] Compared with the prior art, the present invention has the following beneficial effects: This invention constructs a series of novel bifunctional fluorescent probes for mitochondria and mtDNA by modifying the fluorescent backbone of benzofuranone derivatives, with Mt3P as the representative molecule. Compared with traditional mitochondrial and mtDNA probes, Mt3P exhibits dual-color fluorescence properties in both lipid and DNA environments, enabling it to specifically and sensitively locate mitochondrial membranes and mtDNA simultaneously in living cells. Furthermore, this invention demonstrates the ultra-high resolution tracing capabilities of Mt3P for mitochondrial membranes and mtDNA, as well as its compatibility with various application scenarios, through imaging using laser scanning confocal microscopy (LSCM) and structured illumination super-resolution microscopy (SIM). It is expected to provide a reliable visualization tool for monitoring cellular mitochondrial status and find wide application in the diagnosis and drug screening of mitochondrial-related diseases. Attached Figure Description
[0036] Figure 1 The fluorescence spectra of Mf3P(A), Mt2P(B), Mt3P(C) and Mt6P(D) are shown. The spectra were measured in PBS solution (pH=7.4) with n=3.
[0037] Figure 2 The graph shows the linear test data of Mf3P(A), Mt3P(B) and Mt6P(C). All tests were conducted in PBS solution (pH=7.4), n=3.
[0038] Figure 3 Statistical graph of selective test data for Mf3P(A), Mt3P(B), and Mt6P(C); Ion: CO3 2- (1) SO4 2- (2) Br - (3) Cl - (4) CH3COO - (5) K + (6) Na + (7) Ca + (8) Fe 2+ (9) Mg 2+ (10) Zn 2+ (11) Cu 2+ (12) NH 4+ (13); Amino acids: Lys (14), Pro (15), Met (16), Gln (17), Thr (18), Ala (19), Phe (20), Ile (21), Gly (22), Leu (23), Glu (24), Arg (25), His (26), Tyr (27), Asp (28), Cys (29), GSH (30); All tests were conducted in PBS solution (pH=7.4), n=3.
[0039] Figure 4 Figure A shows the kinetic test data of AMf3P, Mt3P, and Mt6P; Figure B shows the stability test data of Mf3P, Mt3P, and Mt6P; all tests were conducted in PBS solution (pH=7.4), n=3.
[0040] Figure 5 Statistical graphs showing the effects of SDS and DNA environment on the fluorescence of Mf3P(A), Mt3P(B), and Mt6P(C); all tests were performed in PBS solution (pH=7.4), n=3.
[0041] Figure 6 Figure A shows the imaging of live HeLa cells using Mf3P, Mt3P, and Mt6P (5 μM); Figures B and C are confocal images (B) and fluorescence intensity analysis (C) of live HeLa cells after DNase (20 μg / mL) digestion for 1 h followed by incubation with Mt3P (5 μM) for 1 h. (63x oil, λ) ex =488 nm, λ em=510-560 nm; λ ex =561 nm, λ em =600-650 nm, scale bar in the figure = 10 μm, n = 3).
[0042] Figure 7 Statistical graph of Mt3P cytotoxicity data (3 replicates per group, i.e., n=3).
[0043] Figure 8 Confocal imaging (A) and linear colocalization analysis (B) of HeLa live cells incubated with MT3P (5 μM) and PicoGreen for 1 h are presented as statistical graphs (63x oil, λ). ex =488 nm, λ em =510-560 nm; λ ex =561 nm, λ em =600-650 nm, scale bar in the figure = 10, 2 μm).
[0044] Figure 9 Figure A shows the colocalization of Mt3P with organelle markers, 63x oil, scale bar = 10 μm; Figures B and E show the statistical data of the colocalization analysis of MT3P organelle markers. Figure B shows the colocalization analysis of Mt3P using MitoTracker (MitoTracker: λ). ex =640 nm, λ em Figure C shows the statistical data of Mt3P (641-700 nm); Figure C shows the co-localization analysis of Mt3P and ER-Blue (ER-Blue: λ ex =405 nm; λ em Figure D shows the statistical data of Mt3P (406-480 nm); Figure D is the co-localization analysis of Mt3P and Lipi-Blue (Lipi-Blue: λ ex =405 nm; λ em =406-480 nm) data statistics; Figure E shows the co-localization analysis of Mt3P and Lyso-Blue (Lyso-Blue: λ ex =405 nm; λ em Statistical chart of data (406-480 nm).
[0045] Figure 10Figure A shows a schematic diagram of different drug-induced cell death programs; Figure B shows confocal images (100x oil) of HeLa live cells treated with FCCP (5 μM) for 0 min, 15 min, and 30 min, followed by incubation with Mt3P (5 μM) for 1 h; Figures C and E show confocal images (63x oil) of HeLa live cells treated with Erastin (10 μM) (C) and Oligomycin A (10 μM) (D) for 0 h, 60 min, and 120 min, followed by incubation with Mt3P (5 μM) for 1 h, and a statistical graph (λ) of fluorescence intensity analysis (E). ex =488 nm, λ em =510-560 nm; λ ex =561 nm, λ em =600-650 nm, scale bar in the figure = 10 or 2 μm, n = 3).
[0046] Figure 11 Super-resolution microscopy image of HeLa live cells incubated with Mt3P (5 μM) for 1 h under structured illumination (100x oil, λ). ex =488 nm, λ em =505-545 nm; λ ex =561 nm, λ em =575-643 nm; scale bar in the figure = 1 or 10 μm).
[0047] Figure 12 Figure A shows a super-resolution microscopy image (100x oil, λ) of HeLa live cells after treatment with Doce (10 μM, 24 h), D-Gal (100 mM, 24 h), and H2O2 (0.2 Mm, 12 h) followed by incubation with MT3P (5 μM) for 1 h under structured light illumination. ex =488 nm, λ em =505-545 nm; λ ex =561 nm, λ em =575-643 nm; scale bar in the figure = 10 or 2 μm); BC plot is a statistical chart of the changes in p-STING / STING induced by different drugs (n=3). Detailed Implementation
[0048] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.
[0049] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.
[0050] Figure 1 A represents Figure 1 Figure A in the text, Figure 1 B indicates Figure 1 The naming meanings of the other figures follow the same pattern as Figure B in the diagram.
[0051] Example 1 Organic Synthesis of Probe Compounds 2.1.1 Synthesis of Mf3P, Mt2P, Mt3P, and Mt6P The synthetic routes of probe compounds (hereinafter referred to as probes) Mf3P, Mt2P, Mt3P and Mt6P are shown below. The synthetic routes mainly include the following steps: (1) 5-dimethylaminothiophene-2-carboxaldehyde is introduced into the active methylene group of 6-hydroxy-2H-benzofuran-3-one by Knoevenagel condensation reaction to obtain compound Mt; (2) under alkaline conditions, triphenylphosphine groups of different chain lengths are introduced into the hydroxyl end of compound Mt by nucleophilic substitution reaction to obtain compounds Mf3P, Mt2P, Mt3P and Mt6P.
[0052]
[0053] The synthesis of the above compounds specifically includes the following steps: Compound Mf: 150.13 mg (1 mol) of 6-hydroxy-2H-benzofuran-3-one, 202.73 mg (1.5 mol) of 5-(dimethylamino)furan-2-carboxaldehyde, and 21.28 mg (0.2 mol) of sodium carbonate were weighed and added sequentially to a 35 mL pressure-resistant tube. 2 mL of anhydrous methanol was added to fully dissolve and mix the mixture. The mixture was then heated to 80 °C using a magnetic stirrer and reacted for 6 h. After the reaction was stopped, the mixture was cooled to room temperature and concentrated under vacuum to obtain the crude product. The crude product was then purified by chromatography (using MeOH and DCM as eluents in a volume ratio of 1:80, i.e., MeOH / DCM = 1:80) to obtain 208.92 mg of a purple solid product, which was compound Mf, with a yield of 77%. NMR and mass spectrometry characterization: 1 H NMR (400MHz, DMSO-d6) δ 10.84 (s, 1H), 7.52 (d, J = 8.4 Hz, 1H), 7.19 (d, J = 3.8 Hz,1H), 6.72 – 6.65 (m, 2H), 6.60 (s, 1H), 5.52 (d, J = 3.8 Hz, 1H), 2.99 (s, 6H). 13C NMR (101 MHz, DMSO-d6) δ 179.59, 166.30, 165.27, 162.80, 141.60,139.50, 125.31, 125.20, 114.93, 112.75, 101.06, 98.76, 87.99, 38.33.HRMS(ESI) m / z: [M+H] + Calcd for C 13 H 15 NO4H + 272.0917; Found 272.0907. Compound Mf3P: 71.83 mg (0.25 mol) of compound Mf, 162.80 mg (0.4 mol) of (4-bromopropyl)triphenylphosphine bromide, and 68.83 mg (0.65 mol) of sodium carbonate were weighed and added sequentially to a 35 mL pressure-resistant tube. 2 mL of anhydrous acetonitrile was added to fully dissolve and mix the mixture. The mixture was then heated to 80 °C using a magnetic stirrer and reacted for 8 h. After the reaction was stopped, the reaction solution was cooled to room temperature and concentrated under vacuum to obtain the crude product. The crude product was then purified by column chromatography (using MeOH and DCM as eluents in a volume ratio of 1:20, i.e., MeOH / DCM = 1:20). 93.73 mg of a purplish-black colloidal product, namely compound Mf3P, was obtained, with a yield of 62%. NMR and mass spectrometry characterization: 1 H NMR (400 MHz, DMSO-d6)δ 7.95 – 7.71 (m, 15H), 7.59 (d, J = 8.5 Hz, 1H), 7.47 (d, J = 4.3 Hz, 1H), 7.13(s, 1H), 7.01 (d, J = 2.1 Hz, 1H), 6.72 (dd, J = 8.5, 2.0 Hz, 1H), 6.11 (d, J =4.3 Hz, 1H), 4.22 (t, J = 6.2 Hz, 2H), 3.08 (s, 6H), 1.97 (p, J = 6.7 Hz, 2H), 1.73 (q, J = 7.8 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 179.28, 166.77, 166.10,165.47, 141.95, 138.85, 135.41, 134.11, 134.01, 130.80, 130.67, 124.99,119.34, 118.66, 118.49, 116.25, 113.14, 110.07, 104.19, 97.57, 67.48, 29.37,19.84, 18.80.HRMS (ESI) m / z: [M] Calcd for C 37 H 35 NO3PSNa + 574.2155; Found 574.2138. Compound Mt: 150.13 mg (1 mmol) of 6-hydroxy-2H-benzofuran-3-one, 232.83 mg (1.5 mmol) of 5-dimethylaminothiophene-2-carboxaldehyde, and 21.28 mg (0.2 mmol) of sodium carbonate were weighed and added sequentially to a 35 mL pressure-resistant tube. 2 mL of anhydrous methanol was added to fully dissolve and mix the mixture. The mixture was then heated to 80 °C using a magnetic stirrer and reacted for 6 h. After the reaction was stopped, the reaction solution was cooled to room temperature and concentrated under vacuum to obtain the crude product. The crude product was then purified by column chromatography (using MeOH and DCM as eluents in a volume ratio of 1:80, i.e., MeOH / DCM = 1:80) to obtain 137.92 mg of a purple solid product, which was compound Mt, with a yield of 48%. NMR and mass spectrometry characterization: 1 H NMR (400MHz, DMSO-d6) δ 10.90 (s, 1H), 7.53 (d, J = 8.4 Hz, 1H), 7.42 (d, J = 4.3 Hz,1H), 7.05 (s, 1H), 6.74 – 6.66 (m, 2H), 6.06 (d, J = 4.3 Hz, 1H), 3.05 (s, 6H). 13 C NMR (101 MHz, DMSO-d6) δ 179.47, 166.53, 166.14, 165.37, 142.02,138.39, 125.54, 118.65, 115.14, 112.96, 109.59, 104.05, 98.69, 42.33. HRMSESI m / z calcd for C15 H 13 NO3S[M+H] + 288.0689, found 288.0689. Compound Mt3P: 20 mg (0.07 mmol) of compound Mt, 40.12 mg (0.1 mmol) of (3-bromopropyl)triphenylphosphine bromide, and 9.62 mg (0.07 mmol) of sodium carbonate were weighed and added sequentially to a 35 mL pressure-resistant tube. 2 mL of anhydrous acetonitrile was added to fully dissolve and mix the mixture. The mixture was then heated to 80 °C using a magnetic stirrer and reacted for 8 h. After the reaction was stopped, the reaction solution was cooled to room temperature and extracted with ethyl acetate and purified water. The organic layer was dried over MgSO4 and concentrated under vacuum to obtain the crude product. The crude product was then purified by column chromatography (using MeOH and DCM as eluents in a volume ratio of 1:20, i.e., MeOH / DCM = 1:20) to obtain 7 mg of purple solid product, which was compound Mt3P, with a yield of 17%. NMR and mass spectrometry characterization: 1 H NMR (400 MHz, DMSO-d6) δ 7.94 – 7.75 (m, 16H), 7.61 (d, J = 8.5 Hz, 1H), 7.48 (d, J = 4.3 Hz, 1H), 7.14 (s, 1H), 7.05 (d, J = 2.0 Hz,1H), 6.85 – 6.80 (m, 1H), 6.12 (d, J = 4.3 Hz, 1H), 4.28 (t, J = 5.8 Hz, 2H), 3.76 (s, 2H), 3.08 (s, 6H), 2.04 (s, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 179.27,166.83, 165.94, 165.04, 141.93, 138.92, 135.49, 134.18, 134.07, 130.82,130.70, 119.21, 118.66, 118.35, 116.56, 110.17, 104.24, 97.92, 67.98, 42.37,22.38, 18.34. HRMS ESI m / z calcd for C 36 H 33 NO3PS + [M+H] +591.1992, found 591.1927. Compound Mt6P: 34 mg (0.12 mmol) of compound Mt, 50.45 mg (0.12 mmol) of (6-bromohexyl)triphenylphosphine bromide, and 16.35 mg (0.12 mmol) of potassium carbonate were weighed and added sequentially to a 35 mL pressure-resistant tube. 2 mL of anhydrous acetonitrile was added to fully dissolve and mix the mixture. The mixture was then heated to 80 °C using a magnetic stirrer and reacted for 8 h. After the reaction was stopped, the reaction solution was cooled to room temperature and extracted with ethyl acetate and purified water. The organic layer was dried over MgSO4 and concentrated under vacuum to obtain the crude product. The crude product was then purified by column chromatography (using MeOH and DCM as eluents in a volume ratio of 1:20, i.e., MeOH / DCM = 1:20) to obtain 64 mg of a purple solid product, which was compound Mt6P, with a yield of 85%. NMR and mass spectrometry characterization: 1 H NMR (400 MHz, DMSO-d6) δ 7.83 – 7.75 (m, 15H), 7.57 (d, J = 8.5 Hz, 1H), 7.46 (d, J = 4.3 Hz, 1H), 7.11 (s, 1H), 6.99 (d, J = 2.1 Hz, 1H), 6.75 (dd, J = 8.6, 2.1 Hz, 1H), 6.10 (d, J = 4.3 Hz, 1H), 4.09 (t, J = 6.4Hz, 2H), 3.57 (q, J = 7.7 Hz, 2H), 3.07 (s, 6H), 1.69 (t, J = 7.3 Hz, 2H), 1.55(d, J = 7.2 Hz, 4H), 1.47 – 1.41 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 179.31,166.75, 165.71, 141.97, 138.77, 135.39, 134.10, 134.00, 130.78, 130.65,125.00, 119.45, 118.60, 116.13, 113.05, 110.00, 97.44, 68.90, 65.39, 42.36,30.02, 29.86, 28.50, 25.11. HRMS ESI m / z calcd for C 39 H 39 NO3PS + [M] + 632.2388, found 632.2354. Compound Mt2Br: 30 mg (0.11 mmol) of compound Mt, 1 mL (12.53 mmol) of 1,2-dibromoethane, and 14.43 mg (0.11 mmol) of potassium carbonate were weighed and added sequentially to a 35 mL pressure-resistant tube. 2 mL of anhydrous acetonitrile was added to fully dissolve and mix the mixture. The mixture was then heated to 80 °C using a magnetic stirrer and reacted for 8 h. After the reaction was stopped, the mixture was cooled to room temperature and extracted with ethyl acetate and purified water. The organic layer was dried over MgSO4 and concentrated under vacuum to obtain the crude product. The crude product was then purified by column chromatography (using EA and PE as eluents in a volume ratio of 1:2, i.e., EA / PE = 1:2) to obtain 12.2 mg of a purple solid product, which was compound Mt2Br, with a yield of 30%. NMR and mass spectrometry characterization: 1 H NMR (400 MHz, CDCl3) δ 7.69 (d, J = 8.5 Hz, 1H), 7.48 (d, J = 4.4 Hz, 1H),7.10 (s, 1H), 6.78 (d, J = 2.2 Hz, 1H), 6.74 (dd, J = 8.6, 2.1 Hz, 1H), 5.92(dd, J = 4.4, 2.0 Hz, 2H), 4.38 (t, J = 6.3 Hz, 2H), 3.68 (t, J = 6.2 Hz, 2H), 3.09 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 180.17, 166.19, 164.31, 142.84,136.96, 125.30, 119.91, 117.24, 111.84, 110.20, 103.49, 103.16, 97.31, 68.24,42.28, 42.08. HRMS ESI m / z calcd for C 17 H 16 BrNO3S[M+H] + 394.0107, found 394.0103. Compound Mt2P: 26 mg (0.11 mmol) of Mt2Br, 17.3 mg (0.66 mmol) of triphenylphosphine, and 9.11 mg (0.66 mmol) of potassium carbonate were weighed and added sequentially to a 35 mL pressure-resistant tube. 2 mL of toluene was added to fully dissolve and mix the mixture. The mixture was then heated to 110 °C using a magnetic stirrer and reacted for 12 h. After the reaction was stopped, the reaction solution was cooled to room temperature and extracted with ethyl acetate and purified water. The organic layer was dried over MgSO4 and concentrated under vacuum to obtain the crude product. The crude product was then purified by column chromatography (using MeOH and DCM as eluents in a volume ratio of 1:20, i.e., MeOH / DCM = 1:20) to obtain 21.5 mg of a purple solid product, which was compound Mt2P, with a yield of 56%. NMR and mass spectrometry characterization: 1 H NMR (400 MHz, DMSO-d6) δ 7.80 – 7.37 (m, 15H), 7.14 (s, 1H), 6.87 (d, J = 2.1 Hz, 1H), 6.85 (d, J = 2.1 Hz, 1H), 6.12 (d, J = 1.3 Hz, 1H), 6.11 (d, J =1.2 Hz, 1H), 4.55 (s, 1H), 3.08 (s, 6H), 2.71 – 2.63 (m, 2H), 2.36 – 2.29 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 180.00, 168.02, 165.22, 141.98, 133.68,132.66, 132.54, 132.51, 132.00, 131.91, 129.30, 129.18, 125.85, 118.69,113.19, 104.43, 103.99, 42.20, 40.44, 29.48. HRMS ESI m / z calcd forC 35 H 31 NO3PS + [M+K] + 615.1394, found 615.1380. Example 2: Measurement of photophysical properties Probe stock solution preparation: Weigh an appropriate amount of probe into a 1.5 mL centrifuge tube, prepare a 20 mM stock solution with DMSO, and store it in a refrigerator at 4 ℃. When needed, dilute with phosphate-buffered saline (PBS) (pH=7.4) to the target concentration.
[0054] DNA preparation: Weigh an appropriate amount of calf thymus DNA and dissolve it in phosphate-buffered saline (PBS) (pH=7.4). Prepare fresh before use.
[0055] Unless otherwise stated, the final test volume of the 96-well plate involved in this embodiment is 200 μL, and each group is set with 3 replicates.
[0056] 2.1 Fluorescence Spectroscopy Test In a 96-well plate, probe solution to a final concentration of 20 μM was added, followed by DNA solution to a final concentration of 200 μg / mL. After incubation at room temperature in the dark for 30 min, fluorescence spectroscopy was performed on the sample wells using a microplate reader at room temperature. The wavelength corresponding to the maximum absorption peak in the absorption spectrum was used as the excitation wavelength to plot the fluorescence spectrum curve. The blank control group did not contain DNA solution and was prepared using phosphate-buffered saline (PBS) (pH=7.4) to prepare the test volume.
[0057] 2.2 Responsiveness In a 96-well plate, probe solution with a final concentration of 20 μM was added, followed by DNA solutions with final concentrations of 400 μg / mL, 300 μg / mL, 200 μg / mL, 120 μg / mL, 80 μg / mL, 40 μg / mL, and 0 μg / mL, respectively. After incubation at room temperature in the dark for 30 min, fluorescence spectra of the sample wells were measured using a microplate reader at an excitation wavelength of 500 nm at room temperature. Fluorescence spectral curves for each concentration were plotted, and a linear equation was plotted for the fluorescence intensity corresponding to the maximum emission wavelength. The limit of detection (LOD) was calculated using the following formula: LOD = (3.3 × σ) / k, where σ is the standard deviation of the blank value and k is the slope of the standard curve.
[0058] 2.2 Dynamics In a 96-well plate, probe solution with a final concentration of 20 μM and DNA solution with a final concentration of 200 μg / mL were rapidly added. Then, the plate was immediately subjected to kinetic tests at room temperature using a microplate reader with an excitation wavelength of 500 nm and an emission wavelength of 590 nm for a total duration of 30 min and an interval of 30 s. The kinetic curves were plotted.
[0059] 2.4 Stability Preparation of PBS solutions (pH=3-11): Adjust the pH of phosphate buffered PBS (pH=7.4) solutions to 3, 4, 5, 6, 7, 8, 9, 10, or 11 using 2 M HCl and 2 M NaOH solutions.
[0060] Probe working solution preparation: Dilute each probe with PBS solution of the corresponding pH to a final concentration of 20 μM, and use immediately after preparation.
[0061] DNA solution preparation: Prepare DNA solution to a final concentration of 200 μg / mL using PBS solution at the corresponding pH, and use immediately after preparation.
[0062] Add probe solution to a final concentration of 20 μM in a 96-well plate, then add DNA solution to a final concentration of 200 μg / mL. After incubating in the dark at room temperature for 30 min, use a microplate reader at room temperature with an excitation wavelength of 500 nm and an emission wavelength of 590 nm to measure the final fluorescence intensity of the sample wells and plot a fluorescence intensity line graph.
[0063] 2.5 Selectivity Preparation of biological macromolecule solutions: Bovine serum albumin (BSA), pepsin, trypsin, thrombin, acetylcholinesterase (AChE), RNA, and DNA were selected and prepared into solutions with phosphate-buffered saline (PBS) (pH=7.4) to a final concentration of 200 μg / mL. The solutions were prepared fresh each time they were used.
[0064] Preparation of amino acid and ion stock solutions: Lysine (Lys), proline (Pro), methionine (Met), glutamine (Gln), threonine (Thr), alanine (Ala), phenylalanine (Phe), isoleucine (Ile), glycine (Gly), leucine (Leu), glutamic acid (Glu), arginine (Arg), histidine (His), tyrosine (Tyr), aspartic acid (Asp), cysteine (Cys), and glutathione (GSH) were selected sequentially and a 10 mM biomolecule solution was prepared using phosphate-buffered saline (PBS) (pH=7.4). Sodium chloride, potassium carbonate, sodium sulfate, copper bromide, sodium acetate, calcium chloride, zinc chloride, ammonium chloride, and ferrous sulfate heptahydrate were selected sequentially and a 10 mM anion and cation (CO, SO, Br) solution was prepared using phosphate-buffered saline (PBS) (pH=7.4). - Cl - CH3COO - K + Na + Ca + Fe 2+ Mg 2+ Zn 2+ Cu 2+ NH 4+ The mother liquor was prepared using phosphate-buffered saline (PBS) (pH=7.4) to a final concentration of 1 mM.
[0065] In a 96-well plate, a probe solution with a final concentration of 20 μM was added, followed by a biomacromolecule solution with a final concentration of 200 μg / mL and an amino acid and ion solution with a final concentration of 1 mM. After incubation at room temperature in the dark for 30 min, the final fluorescence intensity of the sample wells was measured using a microplate reader at room temperature with an excitation wavelength of 500 nm and an emission wavelength of 590 nm. A normalized fluorescence intensity bar chart was then plotted.
[0066] 2.6 Effects of SDS and DNA environment on probe fluorescence SDS and DNA solution preparation: Weigh appropriate amounts of sodium dodecyl sulfate (SDS) and DNA, and prepare a 0.2% SDS solution and a 200 μg / mL DNA solution using phosphate-buffered saline (PBS) (pH=7.4).
[0067] In a 96-well plate, probe solution with a final concentration of 20 μM was added, followed by 0.2% SDS solution and 200 μg / mL DNA solution. After incubation at room temperature in the dark for 30 min, fluorescence spectra were measured in the sample wells using a microplate reader at an excitation wavelength of 500 nm at room temperature, and fluorescence spectral curves were plotted. The blank control group did not contain DNA solution and was prepared using phosphate-buffered saline (PBS) (pH=7.4) to prepare the test volume.
[0068] 2.7 Experimental Results The results are as follows Figure 1 As shown, the free probe (20 μM) produced only weak fluorescence at 590 nm, while its fluorescence signal at 590 nm was significantly enhanced after binding to DNA, with Mf3P, Mt3P, and Mt6P exhibiting higher fluorescence enhancement folds. This result indicates that the probe can "turn on" fluorescence by inducing a conformational change upon binding to DNA, thereby inhibiting twisted intramolecular charge transfer (TICT).
[0069] like Figure 2 As shown, the fluorescence intensity of Mf3P, Mt3P, and Mt6P at 590 nm showed a good linear relationship with the DNA concentration in the range of 0 to 400 μg / mL. Based on this, their limits of detection (LOD) for DNA were calculated to be 1.24 μg / mL, 3.38 μg / mL, and 0.62 μg / mL, respectively. The low LOD indicates that they have high sensitivity.
[0070] In addition to their high sensitivity, Mf3P and Mt3P also exhibit excellent selectivity for nucleic acids in complex biological environments, which is a key prerequisite for the detection of intracellular mtDNA. Figure 3 As shown, this invention investigated the effects of six common biomacromolecules (bovine serum albumin, pepsin, trypsin, thrombin, acetylcholinesterase, and RNA) as potential interfering agents (20 μM) on the fluorescence response of the probe (20 μM). Except for nucleic acids, these substances did not cause significant fluorescence changes in Mf3P and Mt3P. Furthermore, 12 common ions (1-13 being CO, SO, Br₂, etc.) were also investigated. - Cl - CH3COO - K + Na + Ca + Fe 2+ Mg 2+ Zn 2+ Cu 2+ NH 4+The coexistence of Mf3P and 17 amino acids (14-30 being Lys, Pro, Met, Gln, Thr, Ala, Phe, Ile, Gly, Leu, Glu, Arg, His, Tyr, Asp, Cys, and GSH, respectively) did not produce significant interference. These results indicate that Mf3P and Mt3P possess good selectivity and can achieve specific recognition of target nucleic acids in complex systems containing various biological components.
[0071] The stability of the complex intracellular pH environment and the binding kinetics of fluorescence response are also important factors affecting practical applications. For example... Figure 4 As shown in Figure B, the fluorescence signal intensities of Mf3P, Mt3P, and Mt6P remained almost constant within the pH range of 4–11. This result indicates that Mf3P, Mt3P, and Mt6P are adaptable to the acid-base environments of different organelles in the cell, such as lysosomes to mitochondria. Furthermore, as… Figure 4 As shown in Figure A, the three probes Mf3P, Mt3P, and Mt6P can immediately trigger fluorescence "on" after binding to DNA, and the signal does not decay significantly within 30 minutes, demonstrating good binding stability and response speed.
[0072] To achieve simultaneous labeling of mitochondria and mtDNA, this invention investigated the fluorescence emission behavior of the probe in simulated membrane (SDS) and DNA environments. Figure 5 As shown, the emission spectra of Mf3P, Mt3P, and Mt6P (20 μM each) in the SDS environment show a blue shift, while those in the DNA environment show a red shift. This indicates that these probes exhibit a blue shift in fluorescence emission in the membrane lipid environment of mitochondria, emitting green light, and a red shift in fluorescence emission in the mtDNA environment, emitting red light. This is beneficial for achieving dual-color signals during cell imaging.
[0073] Example 3 Cell Imaging 3.1 Live-cell imaging With 1×10 per hole 5 HeLa cells were seeded at a density of 1 / mL on 35 mm confocal culture dishes and incubated for 12–24 h. The culture medium was removed, and 1 mL of 5 μM probe solution was added and incubated for 1 h. After incubation, the probe solution was removed, and the cells were washed once with sterile phosphate-buffered saline (PBS) (pH 7.4), and 1 mL of complete culture medium was added. Immediate imaging was performed using a laser scanning confocal microscope (63x oil). The blank control group received no fluorescent probe, only 1 mL of complete culture medium; all other procedures were identical to the experimental group. Images were processed using Zeiss ZEN microscopy software and analyzed using ImageJ.
[0074] 3.2 Cytotoxicity With 8×10 per hole 4HeLa cells were seeded in 96-well plates at a cell density of 0-100 μM / mL and incubated for 12-24 h. The culture medium was removed, and 200 μL of Mt3P probe solution (9 concentrations ranging from 0-100 μM) was added for incubation for 48 h. After incubation, the probe solution was removed, and the cells were washed once with sterile phosphate-buffered saline (PBS) (pH=7.4), and then incubated for 2 h with 200 μL of 100 μM resazurin solution. After incubation, absorbance (OD) was measured using a microplate reader, and cytotoxicity was calculated. Three replicates were used for each group. The calculation formula is as follows: Cell viability (%) = (OD0.05 / mL) / (OD0.05 / mL) probe -OD blackground ) / (OD ctrl -OD blackground ) × 100%.
[0075] 3.3 Imaging of Mt3P enzyme treatment With 1×10 per hole 5 HeLa cells were seeded at a density of 1 / mL on 35 mm confocal culture dishes and incubated for 12–24 h. The culture medium was removed, and 1 mL of 20 μg / mL DNase solution was added and incubated for 1 h. After incubation, the DNase solution was removed, and the cells were washed once with sterile phosphate-buffered saline (PBS) (pH=7.4). Then, 1 mL of 5 μM Mt3P probe solution was added and incubated for 1 h. After incubation, the probe solution was removed, and the cells were washed once with sterile phosphate-buffered saline (PBS) (pH=7.4). 1 mL of complete culture medium was added, and imaging was immediately performed using a laser scanning confocal microscope (63x oil). The blank control group did not receive DNase solution, but only 1 mL of DMEM was added; all other procedures were identical to the experimental group. Images were processed using Zeiss ZEN microscopy software and analyzed using ImageJ.
[0076] 3.4 Co-staining experiments of Mt3P with commercially available probes With 1×10 per hole 5HeLa cells were seeded at a density of 1 / mL on 35 mm confocal culture dishes and incubated for 12–24 h. The culture medium was removed, and 1 mL of 5 μM Mt3P probe solution was added and incubated for 1 h. After incubation, the probe solution was removed, and the cells were washed once with sterile phosphate-buffered saline (PBS) (pH=7.4). PicoGreen was added and incubated for 1 h. MitoTracker™ Deep Red FM, LipiTracker Blue, ERTracker Blue, and LysoTracker Blue working solutions were added and incubated for 30 min. After incubation, the probe solution was removed, and the cells were washed once with sterile phosphate-buffered saline (PBS) (pH=7.4). 1 mL of complete culture medium was added, and imaging was immediately performed using a laser scanning confocal microscope (63x oil). Images were processed using Zeiss ZEN microscopy software and analyzed using ImageJ.
[0077] 3.5 Mt3P tracing for live-cell imaging under different cell death programs With 1×10 per hole 5 HeLa cells were seeded at a density of 1 / mL on 35 mm confocal culture dishes and incubated for 12–24 h. After incubation, the culture medium was removed, and 1 mL of 5 μM 4-trifluoromethoxyphenylhydrazone (FCCP) solution was added, followed by incubation for 0 min, 15 min, and 30 min. After incubation, the FCCP solution was removed, and the cells were washed once with sterile phosphate-buffered saline (PBS) (pH=7.4), and 1 mL of 5 μM Mt3P probe solution was added, followed by incubation for 1 h. After incubation, the probe solution was removed, and the cells were washed once with sterile phosphate-buffered saline (PBS) (pH=7.4), and 1 mL of complete culture medium was added. Immediate imaging was performed using a laser scanning confocal microscope (100x oil). The blank control group did not receive FCCP solution, but only 1 mL of complete culture medium was added; all other procedures were identical to the experimental group. Images were processed using Zeiss ZEN microscopy software and analyzed using ImageJ.
[0078] With 1×10 per hole 5HeLa cells were seeded at a density of 1 / mL on 35 mm confocal culture dishes and incubated for 12–24 h. The culture medium was removed, and 10 μM Oligomycin A and Erastin solutions were added for incubation at 0 h, 1 h, and 2 h, respectively. After incubation, the Oligomycin A / Erastin solution was removed, and the cells were washed once with sterile phosphate-buffered saline (PBS) (pH=7.4), and 1 mL of 5 μM Mt3P probe solution was added for incubation for 1 h. After incubation, the probe solution was removed, and the cells were washed once with sterile phosphate-buffered saline (PBS) (pH=7.4), and 1 mL of complete culture medium was added. Immediately after incubation, images were captured using a laser scanning confocal microscope (63×). The blank control group did not receive Oligomycin A / Erastin solution, but only 1 mL of complete culture medium was added; all other procedures were identical to the experimental group. Images were processed using Zeiss ZEN microscopy software and analyzed using ImageJ.
[0079] 3.6 Live-cell imaging of mtDNA leakage induced by Mt3P tracer drugs Following the cell seeding procedure described above, use 1 × 10⁶ cells per well. 5 HeLa cells were seeded at a density of 1 / mL on 35 mm confocal culture dishes and incubated for 12–24 h. After incubation, the culture medium was removed, and 1 mL of 100 mM D-galactose (D-Gal) solution and 10 μM docetaxel (Doce) solution were added and incubated for 24 h, followed by incubation with 0.2 mM hydrogen peroxide (H2O2) solution for 12 h. After incubation, the D-galactose / docetaxel / hydrogen peroxide solution was removed, and the cells were washed once with sterile phosphate-buffered saline (PBS) (pH=7.4). 1 mL of 5 μM Mt3P probe solution was added and incubated for 1 h. After incubation, the probe solution was removed, and the cells were washed once with sterile phosphate-buffered saline (PBS) (pH=7.4). 1 mL of complete culture medium was added, and imaging was immediately performed using a structured light illumination microscope (100 x). The blank control group underwent no treatment, only 1 mL of complete culture medium was added, and the remaining procedures were exactly the same as the experimental group. Images were processed using ImageJ.
[0080] 3.7 Experimental Results To verify the imaging capability of the probe in live cells, the HeLa cancer cell line was selected as the model system. Fluorescence imaging experiments were performed on HeLa cells using Mf3P, Mt3P, and Mt6P (5 μM). Figure 6 As shown in Figure A, both Mf3P and Mt3P can achieve fluorescence imaging of intracellular mitochondrial-like organelles, and the green and red light channels are not completely superimposed. This indicates that both Mf3P and Mt3P can observe different sub-organelle structures in mitochondria (such as mitochondrial membrane and mtDNA) through dual channels.
[0081] The inventors selected the Mt3P probe as a representative probe for subsequent research. Live cells were treated with 20 μg / mL DNase for 1 hour to partially degrade mtDNA (refer to Yafu Wang et al., mtDNA-Specific Ultrasensitive Near-Infrared Fluorescent Probe Enables the Differentiation of Healthy and Apoptotic Cells. DOI: 10.1021 / acs.analchem.1c05582). Figure 6 As shown in B and 6C, the red fluorescence intensity used to indicate mtDNA in the DNase-treated group was significantly lower than that in the untreated group; however, the green fluorescence intensity showed no significant change compared to the untreated group. The Red / Green ratio in the DNase-treated group was reduced by approximately 27% compared to the untreated group. This result indicates that Mt3P can indeed achieve mtDNA fluorescence imaging through the red fluorescence channel, which is consistent with the in vitro spectral results. However, due to the limited permeability of DNase in living cells, it cannot completely degrade mtDNA.
[0082] In biological research, the potential cytotoxicity of any fluorescent probe must be taken into account, as cytotoxicity is a critical prerequisite for ensuring experimental reliability and biosafety. Therefore, this invention uses the resazure reduction method to determine the cytotoxicity of Mt3P in the HeLa cell line. Figure 7 As shown, the working concentration of Mt3P in the cell imaging experiments was 5 μM. Cytotoxicity results indicated that the cell viability remained above 80% after 48 h of incubation at this concentration. Since the probe incubation time in the cell imaging experiments was only 1 h, far shorter than the toxicity test duration, the cytotoxicity of the probe at 5 μM for 1 h can be considered negligible. This result demonstrates that Mt3P exhibits low cytotoxicity and good biosafety at conventional imaging concentrations, making it suitable for subsequent live-cell fluorescence imaging and biological function studies.
[0083] like Figure 8 As shown, Mt3P exhibits superior mitochondrial targeting compared to the commercially available DNA probe PicoGreen. Furthermore, the fluorescence signals of Mt3P and PicoGreen at mtDNA show strong spatial overlap, indicating precise localization of mtDNA by Mt3P. However, PicoGreen stains nuclear DNA significantly, while Mt3P stains almost no nuclear DNA. These results collectively demonstrate that Mt3P possesses excellent targeting selectivity for mtDNA and exhibits low nuclear DNA staining interference.
[0084] To further evaluate the compatibility of Mt3P with various application scenarios, this invention conducted co-staining experiments using a set of commercially available organelle-targeting dyes. Figure 9 This includes the mitochondrial probe MitoTracker™ Deep Red FM (MitoTracker), the lipid droplet probe LipiTracker-Blue (Lipi-Blue), the endoplasmic reticulum probe ERTracker Blue (ER-Blue), and the lysosomal probe LysoTracker Blue (Lyso-Blue). For example... Figure 9 As shown, the probe's ability to label mitochondria in cells was validated by co-incubation with the commercially available mitochondrial membrane dye MitoTracker™ Deep Red FM, with a Pearson correlation coefficient of 0.940 obtained from co-localization analysis. When co-incubated with other organelle probes, they all exhibited bright fluorescence in their respective channels with negligible spectral crosstalk, supporting the direct integration of Mt3P into multi-organelle imaging workflows.
[0085] FCCP is a widely used chemical uncoupling agent in cell biology and mitochondrial research. Its main function is to disrupt the mitochondrial membrane potential, thereby inhibiting ATP synthesis. It is often used to study mitochondrial function, cellular energy metabolism, and autophagy. This invention pretreated HeLa cells with 5 μM FCCP for 15 min and 30 min, respectively, to investigate the imaging ability of Mt3P on mitochondrial morphological changes and mtDNA dynamics during autophagy. The flowchart is shown below. Figure 10 As shown in Figure A. The measurement results are as follows. Figure 10 As shown in Figure B, although no significant changes were observed in the overall fluorescence intensity of the FCCP-treated group in the green fluorescence channel (mitochondrial membrane) and the red fluorescence channel (mtDNA), the imaging results revealed a clear dynamic evolution of mitochondrial morphology. After 15 min of treatment, the mitochondrial network began to break down, its morphology changing from long tubular to short rod-shaped, with a few loop structures appearing. After 30 min of FCCP treatment, the mitochondria further fragmented, with some structures swelling and becoming spherical. Notably, even under membrane potential collapse, the signals of Mt3P in the mitochondrial membrane region and mtDNA remained clearly discernible, and the spatial distribution characteristics remained stable. This indicates that Mt3P has good binding stability after targeting and binding to the mitochondrial membrane and mtDNA, and no obvious signal diffusion or release phenomenon was observed even under the stress condition of membrane potential loss.
[0086] To compare the differences in mitochondrial morphology and mtDNA dynamics under apoptosis and ferroptosis states, HeLa cells were treated with Erastin (10 μM) and Oligomycin A (10 μM) for 60 min and 120 min, respectively, to induce different degrees of ferroptosis and apoptosis. Figure 10 As shown in Figure C, with prolonged Erastin incubation time, the mitochondrial morphology (green light channel) in the treated group changed significantly, while the fluorescence signal intensity of mtDNA (red light channel) was significantly weakened compared to the control group. After 120 minutes of incubation, the mitochondria exhibited characteristics such as reduced volume, denser structure, and partial incomplete outer membrane, consistent with ferroptosis-related ultrastructural changes in mitochondria. Notably, the weakening of the mtDNA signal may originate from leakage from the mitochondrial matrix into the cytoplasm, leading to a local decrease in fluorescence intensity, rather than DNA degradation itself. Figure 10 As shown in Figure D, with prolonged Oligomycin A incubation time, the mitochondrial membrane structure in the treated group exhibited significant fragmentation, manifested as tubular networks breaking into short rod-shaped or granular structures. However, the fluorescence signal intensity of mtDNA did not show a significant change compared to the control group. This phenomenon may stem from the inhibition of ATP synthase by Oligomycin A, which blocks the return of protons to the matrix, leading to the accumulation of protons continuously pumped out by the electron transport chain in the intermembrane space, thereby causing mitochondrial membrane potential hyperpolarization. Previous studies have shown that a moderately elevated membrane potential can inhibit the mitochondrial permeability transition pore (mPTP) to maintain the integrity of the mitochondrial outer membrane, thereby preventing mtDNA release. Therefore, under these conditions, Oligomycin A mainly induces morphological remodeling due to mitochondrial kinetic imbalance, without triggering mtDNA leakage. Figure 10 E summarized the dynamic changes in mitochondrial membrane and mtDNA fluorescence intensity after treatment with Erastin and Oligomycin A, reflecting the differences in mitochondrial membrane structural integrity and mtDNA localization under different stress conditions. These results collectively indicate that mitochondria play a core regulatory role in all three cell death processes—autophagy, apoptosis, and ferroptosis—and the dynamic changes in their morphology and mtDNA localization are key to understanding cell fate determination. The Mt3P probe can simultaneously and clearly visualize the dynamic changes in mitochondrial membrane structure and mtDNA under autophagy, ferroptosis, and apoptosis-induced conditions, and the signal is unaffected by changes in mitochondrial membrane potential (MMP). Compared with traditional mitochondrial probes, Mt3P maintains a high signal-to-noise ratio and targeting specificity even under MMP depolarization or hyperpolarization, significantly expanding the applicability of dynamic monitoring of mitochondria and mtDNA in live cells.
[0087] The above findings indicate that Mt3P is a bifunctional fluorescent probe capable of simultaneously targeting both the mitochondrial membrane and mtDNA. While its targeting ability depends on mitochondrial membrane potential, it can achieve high-fidelity synchronous imaging of mitochondrial membrane structure and mtDNA dynamics under various pathological stress conditions, including FCCP-induced depolarization and Oligomycin A-induced hyperpolarization. Notably, mtDNA, as a widely recognized endogenous risk-associated molecular pattern (DAMP) in recent years, can be recognized by the cytoplasmic DNA sensor cGAS once leaked into the cytoplasm. This activates the cGAS–STING innate immune pathway, triggering the production of type I interferon and pro-inflammatory factors. This pathway plays a crucial role in pathological processes such as aging, neurodegenerative diseases, autoimmune diseases, ischemia-reperfusion injury, and the tumor microenvironment. To systematically evaluate the tracing ability of Mt3P in different mtDNA leakage models, this invention pretreated HeLa cells with D-galactose (D-Gal), H2O2, and docetaxel (Doce) to simulate typical pathological scenarios such as replicative senescence, oxidative stress, and chemotherapy-induced mitochondrial damage. Structured illumination super-resolution microscopy (SIM) breaks through the optical diffraction limit, improving the resolution to approximately 100 nm. It combines high-speed imaging, low phototoxicity, good live-cell compatibility, and multicolor imaging capabilities, making it suitable for high spatiotemporal resolution observation of subcellular organelle dynamic processes. Figure 11 Therefore, this invention employs structured light illumination ultra-high resolution microscopy (SIM) combined with Mt3P staining for imaging. Figure 11 The results showed that both mitochondrial membrane structure and mtDNA localization exhibited clear, high-contrast signals, and their spatial distributions were clearly distinguishable, fully demonstrating Mt3P's precise labeling ability for mitochondrial dual components under ultra-high resolution conditions. Furthermore, after treatment with D-Gal, Doce, and H2O2, the mitochondrial membrane morphology in the D-Gal and Doce treatment groups showed significant fragmentation, changing from typical long tubular structures to short rod-shaped or even spherical shapes. Simultaneously, the fluorescence signal intensity of mtDNA was significantly weakened compared to the control group, suggesting mtDNA leakage. In addition, circular mitochondrial structures and punctate autophagosome-like structures formed by mtDNA aggregation were observed in the ultra-high resolution SIM images. Figure 12A) suggests that mitochondrial damage under stress conditions may trigger a selective autophagy clearance mechanism. H2O2 treatment did not cause significant changes in mitochondrial membrane structure or weakening of mtDNA signaling under the current conditions, possibly because the cellular antioxidant system effectively counteracted oxidative damage or the stimulation intensity did not reach the mPTP activation threshold. Subsequently, this invention used Western blotting to detect the ratio of p-STING to STING expression to verify whether mtDNA leakage occurred after Doce and D-Gal treatment of HeLa live cells, thereby activating the cGAS-STING pathway. The results showed that the phosphorylation level of STING (p-STING) was significantly upregulated in both the D-Gal and Doce treatment groups, further supporting that mitochondrial stress-induced mtDNA cytoplasmic leakage is a key upstream event for cGAS–STING pathway activation. Figure 12 BC).
[0088] This invention successfully developed an "on-off" type dual-targeting fluorescent probe, Mt3P, for mitochondrial membrane and mtDNA, which can achieve specific and highly sensitive simultaneous localization of mitochondrial membrane and mtDNA. Due to activation by ICT and inhibition by TICT, Mt3P exhibits different emission spectra in lipid and DNA environments, enabling simultaneous regional imaging of mitochondrial membrane structure and mtDNA. In vitro experimental results show that the fluorescence intensity of Mt3P at 590 nm has a good linear relationship with mtDNA concentration (R² = 0.999), with a detection limit as low as 3.38 μg / mL, and possesses the ability to rapidly detect DNA, making it suitable for applications in complex biological systems. Molecular docking, density functional theory calculations, and kinetic simulations confirm that Mt3P strongly binds to mtDNA in the DNA environment via the minor groove, exhibiting HOMO (Homogeneous Occurrence) properties. The reduction in the LUMO band gap results in a red shift of the emission peak, while in a nonpolar environment, a blue shift occurs due to the positive solvation colorimetric effect. Mt3P exhibits excellent biocompatibility, with negligible damage to mtDNA. Furthermore, cell imaging results show that Mt3P can be efficiently enriched in the mitochondria of healthy cells, enabling the tracking of mitochondrial state and dynamic changes in mtDNA under different environmental conditions. Combined with structured light illumination super-resolution microscopy (SIM), it further achieves high spatiotemporal dynamic imaging at approximately 100 nm resolution, clearly distinguishing the fine spatial relationship between mitochondrial membrane structure and mtDNA, providing a reliable visualization tool for monitoring cellular mitochondrial state.
[0089] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.
Claims
1. A class of fluorescent probe compounds or pharmaceutically acceptable salts thereof, characterized in that, The fluorescent probe compound has the following structure: In the above structure, R is a heteroatom, and the heteroatom is selected from S or O. 1 R 2 Each is independently selected from C 1~6 alkyl.
2. The fluorescent probe compound according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The R 1 R 2 Each is independently selected from C 1~3 alkyl.
3. The fluorescent probe compound according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The R 1 R 2 Each is independently selected from methyl, ethyl, or n-propyl.
4. The fluorescent probe compound according to claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The fluorescent probe compound has any of the following structures: 。 5. A method for preparing the fluorescent probe compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, characterized in that, The preparation of the fluorescent probe compound includes the following steps: S1. 6-hydroxy-2H-benzofuran-3-one, compound 1 and a basic reagent are mixed in a first organic solvent and reacted under heating conditions. After the reaction is complete, the intermediate compound 2 is obtained by post-treatment. S2. The intermediate compound 2 and compound 3 obtained in step S1 and the basic reagent are mixed in a second organic solvent and reacted under heating conditions. After the reaction is complete, the target compound is obtained through post-treatment. Wherein, the structure of compound 1 is as follows: The structure of the intermediate compound 2 is as follows: ; The structure of compound 3 is as follows: .
6. The preparation method according to claim 1, characterized in that, The preparation method includes at least one of the following conditions: (1) In step S1 or step S2, the temperature at which the reaction is completed is 70~120 ℃. (2) The alkaline reagent is selected from at least one of sodium carbonate, potassium carbonate, sodium bicarbonate, sodium hydroxide, and potassium hydroxide; (3) The first organic solvent is selected from at least one of methanol, ethanol, and glacial acetic acid; (4) The second organic solvent is selected from at least one of acetonitrile, N,N-dimethylformamide, dichloromethane, tetrahydrofuran, and toluene.
7. Use of the fluorescent probe compound according to any one of claims 1 to 4 in the preparation of a reagent for simultaneously and specifically recognizing and labeling mitochondrial membrane and mitochondrial DNA.
8. The application according to claim 7, characterized in that, The reagent is used for imaging the mitochondrial membrane and mitochondrial DNA of cells.
9. The use of the fluorescent probe compound of any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof in the preparation of diagnostic reagents for mitochondrial dysfunction.
10. A reagent, characterized in that, The reagent comprises one or more of the fluorescent probe compounds of any one of claims 1 to 4 or their pharmaceutically acceptable salts.