Use of a 1,6-diynic compound in the preparation of a mitochondrial fluorescent probe

Abstract

The application discloses application of a 1,6-diynyl compound as a mitochondrial fluorescent probe. The application discovers a new use of the 1,6-diynyl compound, and opens up a new application field. The benzisoindole dimer compound has different excitation wavelengths and emission wavelengths, and can meet the needs of different cell mitochondrial fluorescent probes. The benzisoindole dimer compound can be well positioned as a probe molecule. The benzisoindole dimer compound has good adaptability as a probe molecule in most microenvironments.

Description

Technical Field

[0001] This application relates to the application of a 1,6-diyne compound in the preparation of mitochondrial fluorescent probes, and belongs to the field of biochemistry. Background Technology

[0002] Small molecule fluorescent probes are widely used in live-cell imaging, from the specific identification of small signal molecules to human health, and can be used to explore the "black box" of life sciences. Due to their simple structure, high selectivity, high sensitivity, non-invasiveness, and suitability for real-time analysis of in vivo systems, small molecule fluorescent probes are becoming increasingly important in fluorescence microscopy, newly developed fluorescence imaging modalities, and live-cell imaging based on super-resolution technology. Among them, fluorescein, coumarins, rhodamine, anthocyanins, BODIPYs, and their derivatives have greatly helped in exploring the complex roles of small molecules, important cations, anions, metal ions, and biomacromolecules in biological research. While these molecules are powerful, they also have some drawbacks, such as photobleaching, phototoxicity, and high background. Given the inherent limitations of existing probes and the further demands of future biological research, the design and synthesis of a diverse range of fluorescent probes is essential.

[0003] Mitochondria, often referred to as the cell's "energy factories," are among the most important organelles in eukaryotic cells. Their functions are diverse yet interconnected, including the production of adenosine triphosphate (ATP), balancing reactive oxygen species (ROS), regulating cellular metabolic processes, autophagy, and apoptosis. Mitochondrial dysfunction is associated with many diseases, such as keratitis, diabetes, and neurodegenerative diseases (Parkinson's disease and Alzheimer's disease). Due to the crucial role of mitochondria, live-cell imaging using fluorescent probes targeting mitochondria has been widely used to study their structure and function. While significant progress has been made in this field, limitations remain, particularly in probe design strategies. Cationic dyes, with their negative membrane potential (-180 mV), are commonly used to target mitochondria. However, cationic dyes can alter membrane potential, affecting mitochondrial and cellular homeostasis. Therefore, non-invasive, sensitive fluorescent probes remain urgently needed. Summary of the Invention

[0004] According to one aspect of this application, the use of 1,6-diyne compounds in the preparation of mitochondrial fluorescent probes is provided.

[0005] Application of a 1,6-diyne compound in the preparation of mitochondrial fluorescent probes, wherein the 1,6-diyne compound has the structural formula shown in Formula I:

[0006]

[0007] Wherein, X is selected from nitrogen atom, phosphorus atom, arsenic atom, tellurium atom, and boron atom;

[0008] R' is selected from C1–C 30 Alkyl, substituted C1–C 30 Alkyl, C6–C 30 Aryl, substituted C6–C 30 Aryl, C3–C 30 heteroaryl, substituted C3–C 30 Heteroaryl, amino, substituted amino, and optionally heteroatom groups selected from the following: CO, O, S, SO, SO2, NR are inserted at any position. a -N = , = N-;

[0009] Among them, R 1a R 4a Independently selected from hydrogen, deuterium, C1–C 30 Alkyl, substituted C1–C 30 Alkyl, C6–C 30 Aryl, substituted C6–C 30 Aryl, C3–C 30 heteroaryl, substituted C3–C 30 Heteroaryl, phosphinyl, substituted phosphinyl, boryl, substituted boryl, silyl, substituted silyl, halogen, amino, substituted amino, and optionally heteroatom groups selected from the following: CO, O, S, SO, SO2, NR. a -N=, =N-;

[0010] R 2a Selected from C6–C 30 Aryl, substituted C6–C 30 Aryl, C3–C 30 heteroaryl, substituted C3–C 30 heteroaryl, C1–C 10 alkenyl, C1–C 10 Substituted alkenyl groups;

[0011] R 3a Hydrogen, deuterium, C1–C 30 Alkyl, substituted C1–C 30 Alkyl, C1–C 30 Alkenyl, substituted C1–C 30 alkenyl, C1–C 30 Alkyne group, substituted C1–C 30 alkynyl group, C6–C 30 Aryl, substituted C6–C 30 Aryl, C3–C 30 heteroaryl, substituted C3–C 30 heteroaryl, C1–C 30 cycloalkyl, substituted C1–C 30cycloalkyl, C1–C 30 Heterocyclic alkyl, substituted C1–C 30 Heterocyclic alkyl, phosphinyl, halogen, silyl, boronyl, germanium, arsenic, selenium, and optionally heteroatom groups selected from the following: CO, O, S, SO, SO2, NR are inserted at any position. a -N=, =N-;

[0012] R a It is independently selected from H, alkyl or aryl.

[0013] Alternatively, replace C6–C 30 Aryl, substituted C3–C 30 The substituents in heteroaryl groups are selected from alkyl, alkenyl, aldehyde, halogen, haloalkyl, ester-intercalated alkyl, alkoxy, alkylthio, and substituted amino groups.

[0014] Alternatively, replace C6–C 30 Aryl, substituted C3–C 30 The substituents in heteroaryl groups are selected from C1–C 30 Alkyl, C1–C 30 alkenyl, C1–C 30 Aldehyde, halogen, halogenated C1–C 30 C1–C with alkyl or ester insertion 30 Alkyl, C1–C 30 Alkoxy, C1–C 30 Amino groups substituted with alkylthio or phenyl groups.

[0015] Optionally, R' is selected from C6–C 30 Aryl, C6–C 10 alkyl-substituted C6–C 30 Aryl, halogen-substituted C6–C 10 alkyl-substituted C6–C 30 Aryl, C3–C 30 heteroaryl, C6–C 10 alkyl-substituted C3–C 30 Heteroaryl, halogen-substituted C6–C 10 Alkyl-substituted C3–C 30 Heteroaryl groups; preferably, R' is selected from C6–C 30 Aryl, C3–C 30 Mixed aromatic compounds.

[0016] Optionally, R 1a R 4a Independently selected from C6–C substituted with hydrogen, aryl, or alkyl. 30 Aryl, "S-atom-intercalated alkyl" substituted C6–C 30Aryl, "O-atom inserted alkyl" substituted C6–C 30 Aryl and halogen-substituted C6–C 30 aryl, aryl-substituted C6–C 30 Aryl, halogen-substituted alkyl, C6–C 30 Aryl, "aryl-substituted amino" substituted C6–C 30 Aryl, "ester-intercalated alkyl" substituted C6–C 30 Aryl, C3–C 30 Mixed aromatic compounds.

[0017] Optionally, R 1a Independently selected from phenyl, tert-butyl-substituted phenyl, CH3S-substituted phenyl, CH3O-substituted phenyl, naphthyl, phenyl-substituted phenyl, at least one methyl-substituted phenyl, CF3O-substituted phenyl, Br-substituted phenyl, "diphenyl-substituted amino"-substituted phenyl, "ester-intercalated methyl"-substituted phenyl, dibenzothiophene.

[0018] Optionally, R 2a Selected from C6–C 30 Aryl, "ester-intercalated alkyl" substituted C6–C 30 Aryl and alkyl substituted C6–C 30 Aryl, "oxygen-intercalated alkyl" substituted C6–C 30 aryl, aryl-substituted C6–C 30 Aryl, C3–C 30 C6–C substituted with heteroaryl and aldehyde groups 30 Aryl and halogen-substituted C6–C 30 Aryl and alkenyl substituted C6–C 30 Aryl.

[0019] Optionally, R 2a R5 is the same or different; R 2a R5 is independently selected from phenyl, naphthyl, phenyl substituted with "ester-intercalated methyl", propyl-substituted phenyl, phenanthryl, phenyl substituted with "oxygen-intercalated methyl", biphenyl, thiophene, phenyl substituted with formaldehyde, phenyl substituted with Cl, and phenyl substituted with vinyl.

[0020] Optionally, R 3a Selected from hydrogen atoms, deuterium atoms, and C6–C 30 Aryl, "ester-intercalated alkyl" substituted C6–C 30 aryl, at least one alkyl-substituted C6–C 30 Aryl.

[0021] Optionally, R 3aSelected from hydrogen atom, deuterium atom, phenyl, phenyl substituted with "ester-intercalated methyl", phenyl substituted with at least one methyl.

[0022] Alternatively, the structural formula of the compound shown in Formula I is selected from the following compounds:

[0023]

[0024]

[0025] Optionally, a method for preparing 1,6-diyne compounds includes the following steps:

[0026] Reacting a starting material containing compounds of formulas IV and V with reaction I yields 1,6-diyne compounds.

[0027]

[0028] Optionally, the molar ratio of the compounds shown in Formula IV and Formula V is 1:1 to 5.

[0029] Optionally, reaction I is carried out in the presence of an acid reagent.

[0030] Optionally, the acid reagent includes p-toluenesulfonic acid, phenylsulfonic acid, p-nitrobenzenesulfonic acid, methanesulfonic acid, ferric chloride, and aluminum chloride;

[0031] Optionally, the temperature of reaction I is 25℃~100℃;

[0032] The reaction time for reaction I is 0.1 h to 48 h.

[0033] Optionally, the method for preparing the compound represented by Formula IV includes the following steps:

[0034] A starting material containing compounds of formulas VI and VII is reacted with reaction IV to obtain the compound of formula IV.

[0035]

[0036] Optionally, the molar ratio of the compounds shown in Formula VI and Formula VII is 1:1 to 5.

[0037] Optionally, reaction IV is carried out in the presence of a nucleophilic substitution reagent, which includes n-butyllithium, sec-butyllithium, tert-butyllithium, methyllithium, diisopropylaminolithium, and bis(trimethylsilylaminolithium).

[0038] Optionally, the temperature of reaction IV is -78℃ to 50℃; the reaction time is 0.1h to 24h.

[0039] Optionally, a culture dish containing the 1,6-diyne compound and mouse fibroblasts is incubated.

[0040] Optionally, the solution volume of the 1,6-diyne compound is 1 μL to 1000 μL.

[0041] Optionally, the solution concentration of the 1,6-diyne compound is 1 μM to 1000 μM.

[0042] Alternatively, the incubation conditions are as follows:

[0043] The temperature ranges from 5℃ to 50℃.

[0044] The time ranges from 0.1h to 48h.

[0045] Optionally, the 1,6-diyne compound is used in the preparation of mitochondrial fluorescent probes for mouse fibroblasts.

[0046] Optionally, the 1,6-diyne compound is used in the preparation of mitochondrial fluorescent probes for mouse mononuclear macrophage leukemia cells, HER2-breast cancer overexpressing cells, or human liver cancer cell lines.

[0047] According to a second aspect of this application, an application of a benzisoindole dimer compound as a mitochondrial fluorescent probe is provided.

[0048] Application of a benzo[i]isoindole dimer compound as a mitochondrial fluorescent probe;

[0049] The benzo[i]indole dimer is produced by the 1,6-diyne compound described in any one of the above-mentioned compounds during cell incubation.

[0050] Optionally, the benzo[i]isoindole dimer is selected from compounds having the structural formula described in Formula II:

[0051]

[0052] Optionally, R in Formula II 1 R 4 In the same formula I, R 4a ;

[0053] In formula II, R 7 R 8 In the same formula I, R 3a ;

[0054] In formula II, R 3 R 6 In the same formula I, R 2a ;

[0055] In formula II, R 1 R 2 R 3 R 4 R5 R 6 R 7 R 8 Meet one or more of the following conditions:

[0056] (i)R 1 R 4 same;

[0057] (ii)R 2 R 5 same;

[0058] (iii)R 3 R 6 same;

[0059] (iv)R 7 R 8 same.

[0060] Optionally, the excitation wavelength of the benzisoindole dimer compound is 220 nm to 1000 nm.

[0061] Optionally, the emission wavelength of the benzisoindole dimer compound is 400 nm to 800 nm.

[0062] Alternatively, the cell incubation conditions are as follows:

[0063] The temperature ranges from 5℃ to 50℃.

[0064] The time ranges from 0.1h to 48h.

[0065] According to a third aspect of this application, the application of 1,6-diyne compounds in detecting enzyme deficiencies in the respiratory chain is provided.

[0066] Application of a 1,6-diyne compound in the detection of enzyme deficiency in the respiratory chain.

[0067] Optionally, the NMR test results of the compounds represented by Formula I are shown in the table below:

[0068]

[0069]

[0070]

[0071] 1. Test of MP-1 probe molecule's effect on mitochondrial fluorescence in L292 cells:

[0072] Cell line: L929 cells (mouse fibroblasts).

[0073] Cell culture: Cells were cultured in a CO2 incubator (37°C, 5% CO2) in DMEM medium (penicillin (100 U / mL) and streptomycin (100 μg / mL)).

[0074] Cell imaging experiments: Cells were cultured in 15mm bottom-mounted cell culture dishes. Before labeling cells with the probe, the culture medium was removed, and the cells were washed twice with Hank's buffer to remove residual medium. 200 μL of probe MP-1 (10 μM) was added to the culture dish, and the cells were incubated in the dark for 30 minutes. The cells were washed twice with Hank's buffer to remove residual probe, and 1 mL of Hank's solution was added to the culture dish. Cells were observed under a 100x objective lens using a laser confocal microscope. The excitation wavelength was 543 nm, and the emission wavelength was 550-600 nm.

[0075] 2. Test of MP-2 probe molecule's effect on mitochondrial fluorescence in L292 cells:

[0076] Cell line: L929 cells (mouse fibroblasts).

[0077] Cell culture: Cells were cultured in a CO2 incubator (37°C, 5% CO2) in DMEM medium (penicillin (100 U / mL) and streptomycin (100 μg / mL)).

[0078] Cell imaging experiments: Cells were cultured in 15mm bottom-mounted cell culture dishes. Before labeling cells with the probe, the culture medium was removed, and the cells were washed twice with Hank's buffer to remove residual medium. 200 μL of probe MP-2 (20 μM) was added to the culture dish, and the cells were incubated in the dark for 30 minutes. The cells were washed twice with Hank's buffer to remove residual probe, and 1 mL of Hank's solution was added to the culture dish. Cells were observed under a 100x objective lens using a laser confocal microscope. The excitation wavelength was 488 nm, and the emission wavelength was 500 nm–540 nm.

[0079] 3. Test of MP-3 probe molecule's effect on mitochondrial fluorescence in L292 cells:

[0080] Cell line: L929 cells (mouse fibroblasts).

[0081] Cell culture: Cells were cultured in a CO2 incubator (37°C, 5% CO2) in DMEM medium (penicillin (100 U / mL) and streptomycin (100 μg / mL)).

[0082] Cell imaging experiments: Cells were cultured in 15mm bottom-mounted cell culture dishes. Before labeling cells with the probe, the culture medium was removed, and the cells were washed twice with Hank's buffer to remove residual medium. 100 μL of probe MP-3 (50 μM) was added to the culture dish, and the cells were incubated in the dark for 30 minutes. The cells were washed twice with Hank's buffer to remove residual probe, and 1 mL of Hank's solution was added to the culture dish. Cells were observed under a 100x objective lens using a laser confocal microscope. The excitation wavelength was 488 nm, and the emission wavelength was 600 nm–650 nm.

[0083] 4. Test of MP-4 probe molecule's effect on mitochondrial fluorescence in L292 cells:

[0084] Cell line: L929 cells (mouse fibroblasts).

[0085] Cell culture: Cells were cultured in a CO2 incubator (37°C, 5% CO2) in DMEM medium (penicillin (100 U / mL) and streptomycin (100 μg / mL)).

[0086] Cell imaging experiments: Cells were cultured in 15mm bottom-mounted cell culture dishes. Before labeling cells with the probe, the culture medium was removed, and the cells were washed twice with Hank's buffer to remove residual medium. 20 μL of probe MP-4 (100 μM) was added to the culture dish, and the cells were incubated in the dark for 30 minutes. The cells were washed twice with Hank's buffer to remove residual probe, and 1 mL of Hank's solution was added to the culture dish. Cells were observed under a 100x objective lens using a laser confocal microscope. The excitation wavelength was 543 nm, and the emission wavelength was 550 nm–600 nm.

[0087] 5. MP-5 probe molecule fluorescence effect on L292 cell mitochondrial fluorescence:

[0088] Cell line: L929 cells (mouse fibroblasts).

[0089] Cell culture: Cells were cultured in a CO2 incubator (37°C, 5% CO2) in DMEM medium (penicillin (100 U / mL) and streptomycin (100 μg / mL)).

[0090] Cell imaging experiments: Cells were cultured in 15mm bottom-mounted cell culture dishes. Before labeling cells with the probe, the culture medium was removed, and the cells were washed twice with Hank's buffer to remove residual medium. 200 μL of probe MP-5 (10 μM) was added to the culture dish, and the cells were incubated in the dark for 30 minutes. The cells were washed twice with Hank's buffer to remove residual probe, and 1 mL of Hank's solution was added to the culture dish. Cells were observed under a 100x objective lens using a laser confocal microscope. The excitation wavelength was 543 nm, and the emission wavelength was 550 nm–600 nm.

[0091] 6. MP-6 probe molecule's effect on mitochondrial fluorescence in L292 cells:

[0092] Cell line: L929 cells (mouse fibroblasts).

[0093] Cell culture: Cells were cultured in a CO2 incubator (37°C, 5% CO2) in DMEM medium (penicillin (100 U / mL) and streptomycin (100 μg / mL)).

[0094] Cell imaging experiments: Cells were cultured in 15mm bottom-mounted cell culture dishes. Before labeling cells with the probe, the culture medium was removed, and the cells were washed twice with Hank's buffer to remove residual medium. 200 μL of probe MP-6 (10 μM) was added to the culture dish, and the cells were incubated in the dark for 30 minutes. The cells were washed twice with Hank's buffer to remove residual probe, and 1 mL of Hank's solution was added to the culture dish. Cells were observed under a 100x objective lens using a laser confocal microscope. The excitation wavelength was 543 nm, and the emission wavelength was 550 nm–600 nm.

[0095] 7. MP-7 probe molecule's effect on mitochondrial fluorescence in L292 cells:

[0096] Cell line: L929 cells (mouse fibroblasts).

[0097] Cell culture: Cells were cultured in a CO2 incubator (37°C, 5% CO2) in DMEM medium (penicillin (100 U / mL) and streptomycin (100 μg / mL)).

[0098] Cell imaging experiments: Cells were cultured in 15mm bottom-mounted cell culture dishes. Before labeling cells with the probe, the culture medium was removed, and the cells were washed twice with Hank's buffer to remove residual medium. 200 μL of probe MP-7 (10 μM) was added to the culture dish, and the cells were incubated in the dark for 30 minutes. The cells were washed twice with Hank's buffer to remove residual probe, and 1 mL of Hank's solution was added to the culture dish. Cells were observed under a 100x objective lens using a laser confocal microscope. The excitation wavelength was 543 nm, and the emission wavelength was 550 nm–600 nm.

[0099] 8. Test of MP-8 probe molecule's effect on mitochondrial fluorescence in L292 cells:

[0100] Cell line: L929 cells (mouse fibroblasts).

[0101] Cell culture: Cells were cultured in a CO2 incubator (37°C, 5% CO2) in DMEM medium (penicillin (100 U / mL) and streptomycin (100 μg / mL)).

[0102] Cell imaging experiments: Cells were cultured in 15mm bottom-mounted cell culture dishes. Before labeling cells with the probe, the culture medium was removed, and the cells were washed twice with Hank's buffer to remove residual medium. 200 μL of probe MP-8 (10 μM) was added to the culture dish, and the cells were incubated in the dark for 30 minutes. The cells were washed twice with Hank's buffer to remove residual probe, and 1 mL of Hank's solution was added to the culture dish. Cells were observed under a 100x objective lens using a laser confocal microscope. The excitation wavelength was 543 nm, and the emission wavelength was 550 nm–600 nm.

[0103] 9. Test of MP-9 probe molecule's effect on mitochondrial fluorescence in L292 cells:

[0104] Cell line: L929 cells (mouse fibroblasts).

[0105] Cell culture: Cells were cultured in a CO2 incubator (37°C, 5% CO2) in DMEM medium (penicillin (100 U / mL) and streptomycin (100 μg / mL)).

[0106] Cell imaging experiments: Cells were cultured in 15mm bottom-mounted cell culture dishes. Before labeling cells with the probe, the culture medium was removed, and the cells were washed twice with Hank's buffered saline to remove residual medium. 200 μL of probe MP-9 (10 μM) was added to the culture dish, and the cells were incubated in the dark for 30 minutes. The cells were washed twice with Hank's buffered saline to remove residual probe, and 1 mL of Hank's solution was added to the culture dish. Cells were observed under a 100x objective lens using a laser confocal microscope. The excitation wavelength was 543 nm, and the emission wavelength was 550 nm–600 nm.

[0107] 10. Test of MP-1 probe molecule co-localization fluorescence effect on L292 cell mitochondria:

[0108] Cell line: L929 cells (mouse fibroblasts).

[0109] Cell culture: Cells were cultured in a CO2 incubator (37°C, 5% CO2) in DMEM medium (penicillin (100 U / mL) and streptomycin (100 μg / mL)).

[0110] Cell imaging experiments: Cells were cultured in 15mm bottom-mounted cell culture dishes. Before labeling cells with the probe, the culture medium was removed, and the cells were washed twice with Hank's buffer to remove residual medium. 200 μL of probe MP-1 (10 μM) and 5 μM mitochondrial localization dye (MitoTracker Green) were added to the culture dish, and the cells were incubated in the dark for 30 minutes. The cells were washed twice with Hank's buffer to remove residual probe, and 1 mL of Hank's solution was added to the culture dish. Cells were observed under a 100x objective lens using a laser confocal microscope. The Pearson correlation coefficient was tested and calculated to be greater than 0.9, indicating that the probe molecule of this invention can effectively localize mitochondria.

[0111] 11. Test of MP-3 probe molecule co-localization fluorescence effect on L292 cell mitochondria:

[0112] Cell line: L929 cells (mouse fibroblasts).

[0113] Cell culture: Cells were cultured in a CO2 incubator (37°C, 5% CO2) in DMEM medium (penicillin (100 U / mL) and streptomycin (100 μg / mL)).

[0114] Cell imaging experiments: Cells were cultured in 15mm bottom-mounted cell culture dishes. Before labeling cells with the probe, the culture medium was removed, and the cells were washed twice with Hank's buffer to remove residual medium. 200 μL of probe MP-3 (10 μM) and 5 μM mitochondrial localization dye (MitoTracker Green) were added to the culture dish, and the cells were incubated in the dark for 30 minutes. The cells were washed twice with Hank's buffer to remove residual probe, and 1 mL of Hank's solution was added to the culture dish. Cells were observed under a 100x objective lens using a laser confocal microscope. The Pearson correlation coefficient was tested and calculated to be greater than 0.9, indicating that the probe molecules of this invention can effectively localize mitochondria.

[0115] 12. Test of MP-1 probe molecule's fluorescence effect on mitochondria of other cells:

[0116] Cell lines: RAW 264.7 cell (mouse mononuclear macrophage leukemia cell); SKBR3 cell (HER2-overexpressing breast cancer cell); HuH-7 cell (human liver cancer cell line).

[0117] Cell culture: Cells were cultured in a CO2 incubator (37°C, 5% CO2) in DMEM medium (penicillin (100 U / mL) and streptomycin (100 μg / mL)).

[0118] Cell imaging experiments: We cultured the above three cell types in 15mm bottom cell culture dishes. Before labeling the cells with the probe, the culture medium was removed, and the cells were washed twice with Hank's buffer to remove residual culture medium. 200 μL of probe MP-1 (10 μM) was added to each cell culture dish, and the cells were incubated in the dark for 30 minutes. The cells were washed twice with Hank's buffer to remove residual probe, and 1 mL of Hank's solution was added to each culture dish. The cells were observed under a 100x objective lens using a laser confocal microscope. This experiment demonstrates that the fluorescent probe of this invention has good adaptability in most microenvironments.

[0119] 13. Cell silencing experiment 1

[0120] Cells were cultured in imaging dishes for 6 hours, then treated with cytc-290 (a designed silent siRNA) or non-targeted RNA for a certain period before live-cell imaging under a confocal laser scanning microscope. After 12 hours of cytc-290 treatment, Hoechst 33342 and MTG staining were good, but approximately 10% of the cells were not labeled with MP-1. After 18 hours of cytc-290 treatment, approximately 30% of the cells were not labeled with MP-1. In contrast, the control group treated with non-targeted RNA still showed good MP-1 staining. These results indicate that the probe MP-1 can effectively reflect respiratory chain activity and can be used to detect enzyme deficiencies in the respiratory chain.

[0121] 14. Cell silencing experiment 2

[0122] Cells were cultured in imaging dishes for 6 hours, then treated with cytc-436 (a designed silent siRNA) or non-targeted RNA for a certain period before live-cell imaging under a confocal laser scanning microscope. After 6 hours of cytc-436 treatment, Hoechst 33342 and MTG staining were good, but approximately 5% of cells could not be labeled with MP-1. In contrast, the control group treated with non-targeted RNA still showed good MP-1 staining. These results indicate that the MP-1 probe can effectively reflect respiratory chain activity and can be used to detect enzyme deficiencies in the respiratory chain.

[0123] The beneficial effects that this application can produce include:

[0124] 1) This application has discovered new uses for 1,6-diyne compounds and opened up a new field of application.

[0125] 2) The benzisoindole dimer compound of this application has different excitation wavelengths and emission wavelengths, which can meet the needs of different cellular mitochondrial fluorescent probes.

[0126] 3) The benzisoindole dimer compound of this application can be well localized to mitochondria as a probe molecule.

[0127] 4) The benzisoindole dimer compound of this application has good adaptability as a probe molecule in most microenvironments. Attached Figure Description

[0128] Figure 1 This is the mass spectrum of benzoindole dimer.

[0129] Figure 2 This is the mass spectrum of the cell lysis extract. Detailed Implementation

[0130] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments.

[0131] Unless otherwise specified, all raw materials used in the embodiments of this application were purchased through commercial channels.

[0132] The analysis method in the embodiments of this application is as follows:

[0133] Fluorescence analysis was performed using a Zeiss LSM 780 confocal fluorescence microscope.

[0134] Mass spectrometry analysis was performed using a Thermo Fisher Scientific LTQ FTICR-MS instrument.

[0135] Hank's buffer solution consisted of 1.26 mM CaCl2, 0.49 mM MgCl2, 0.41 mM MgSO4, 5.33 mM KCl, 0.44 mM KH2PO4, 4.17 mM NaHCO3, 138 mM NaCl, 0.34 mM Na2HPO4, and 5.55 mM glucose, with a pH of 7.4.

[0136] Silent siRNAs (cytc-290, cytc-436) were purchased from Suzhou GeneGene Co., Ltd.

[0137] Example 1

[0138] MP-1 probe molecule fluorescence effect on L292 cell mitochondrial fluorescence:

[0139] Cell line: L929 cells (mouse fibroblasts).

[0140] Cell culture: Cells were cultured in a CO2 incubator (37°C, 5% CO2) in DMEM medium (penicillin (100 U / mL) and streptomycin (100 μg / mL)).

[0141] Cell imaging experiments: Cells were cultured in 15mm bottom-mounted cell culture dishes. Before labeling cells with the probe, the culture medium was removed, and the cells were washed twice with Hank's buffer to remove residual medium. 200 μL of probe MP-1 (10 μM) was added to the culture dish, and the cells were incubated in the dark for 30 minutes. The cells were washed twice with Hank's buffer to remove residual probe, and 1 mL of Hank's solution was added to the culture dish. Cells were observed under a 100x objective lens using a laser confocal microscope. The excitation wavelength was 543 nm, and the emission wavelength was 550-600 nm.

[0142] Example 2

[0143] MP-2 probe molecule fluorescence effect on L292 cell mitochondrial fluorescence:

[0144] Cell line: L929 cells (mouse fibroblasts).

[0145] Cell culture: Cells were cultured in a CO2 incubator (37°C, 5% CO2) in DMEM medium (penicillin (100 U / mL) and streptomycin (100 μg / mL)).

[0146] Cell imaging experiments: Cells were cultured in 15mm bottom-mounted cell culture dishes. Before labeling cells with the probe, the culture medium was removed, and the cells were washed twice with Hank's buffer to remove residual medium. 200 μL of probe MP-2 (20 μM) was added to the culture dish, and the cells were incubated in the dark for 30 minutes. The cells were washed twice with Hank's buffer to remove residual probe, and 1 mL of Hank's solution was added to the culture dish. Cells were observed under a 100x objective lens using a laser confocal microscope. The excitation wavelength was 488 nm, and the emission wavelength was 500 nm–540 nm.

[0147] Example 3

[0148] MP-3 probe molecule fluorescence effect on L292 cell mitochondrial fluorescence:

[0149] Cell line: L929 cells (mouse fibroblasts).

[0150] Cell culture: Cells were cultured in a CO2 incubator (37°C, 5% CO2) in DMEM medium (penicillin (100 U / mL) and streptomycin (100 μg / mL)).

[0151] Cell imaging experiments: Cells were cultured in 15mm bottom-mounted cell culture dishes. Before labeling cells with the probe, the culture medium was removed, and the cells were washed twice with Hank's buffer to remove residual medium. 100 μL of probe MP-3 (50 μM) was added to the culture dish, and the cells were incubated in the dark for 30 minutes. The cells were washed twice with Hank's buffer to remove residual probe, and 1 mL of Hank's solution was added to the culture dish. Cells were observed under a 100x objective lens using a laser confocal microscope. The excitation wavelength was 488 nm, and the emission wavelength was 600 nm–650 nm.

[0152] Example 4

[0153] MP-4 probe molecule fluorescence effect on L292 cell mitochondrial fluorescence:

[0154] Cell line: L929 cells (mouse fibroblasts).

[0155] Cell culture: Cells were cultured in a CO2 incubator (37°C, 5% CO2) in DMEM medium (penicillin (100 U / mL) and streptomycin (100 μg / mL)).

[0156] Cell imaging experiments: Cells were cultured in 15mm bottom-mounted cell culture dishes. Before labeling cells with the probe, the culture medium was removed, and the cells were washed twice with Hank's buffer to remove residual medium. 20 μL of probe MP-4 (100 μM) was added to the culture dish, and the cells were incubated in the dark for 30 minutes. The cells were washed twice with Hank's buffer to remove residual probe, and 1 mL of Hank's solution was added to the culture dish. Cells were observed under a 100x objective lens using a laser confocal microscope. The excitation wavelength was 543 nm, and the emission wavelength was 550 nm–600 nm.

[0157] Example 5

[0158] MP-5 probe molecule fluorescence effect on L292 cell mitochondrial fluorescence:

[0159] Cell line: L929 cells (mouse fibroblasts).

[0160] Cell culture: Cells were cultured in a CO2 incubator (37°C, 5% CO2) in DMEM medium (penicillin (100 U / mL) and streptomycin (100 μg / mL)).

[0161] Cell imaging experiments: Cells were cultured in 15mm bottom-mounted cell culture dishes. Before labeling cells with the probe, the culture medium was removed, and the cells were washed twice with Hank's buffer to remove residual medium. 200 μL of probe MP-5 (10 μM) was added to the culture dish, and the cells were incubated in the dark for 30 minutes. The cells were washed twice with Hank's buffer to remove residual probe, and 1 mL of Hank's solution was added to the culture dish. Cells were observed under a 100x objective lens using a laser confocal microscope. The excitation wavelength was 543 nm, and the emission wavelength was 550 nm–600 nm.

[0162] Example 6

[0163] MP-6 probe molecule's effect on L292 cell mitochondrial fluorescence:

[0164] Cell line: L929 cells (mouse fibroblasts).

[0165] Cell culture: Cells were cultured in a CO2 incubator (37°C, 5% CO2) in DMEM medium (penicillin (100 U / mL) and streptomycin (100 μg / mL)).

[0166] Cell imaging experiments: Cells were cultured in 15mm bottom-mounted cell culture dishes. Before labeling cells with the probe, the culture medium was removed, and the cells were washed twice with Hank's buffer to remove residual medium. 200 μL of probe MP-6 (10 μM) was added to the culture dish, and the cells were incubated in the dark for 30 minutes. The cells were washed twice with Hank's buffer to remove residual probe, and 1 mL of Hank's solution was added to the culture dish. Cells were observed under a 100x objective lens using a laser confocal microscope. The excitation wavelength was 543 nm, and the emission wavelength was 550 nm–600 nm.

[0167] Example 7

[0168] MP-7 probe molecule fluorescence effect on L292 cell mitochondrial fluorescence:

[0169] Cell line: L929 cells (mouse fibroblasts).

[0170] Cell culture: Cells were cultured in a CO2 incubator (37°C, 5% CO2) in DMEM medium (penicillin (100 U / mL) and streptomycin (100 μg / mL)).

[0171] Cell imaging experiments: Cells were cultured in 15mm bottom-mounted cell culture dishes. Before labeling cells with the probe, the culture medium was removed, and the cells were washed twice with Hank's buffer to remove residual medium. 200 μL of probe MP-7 (10 μM) was added to the culture dish, and the cells were incubated in the dark for 30 minutes. The cells were washed twice with Hank's buffer to remove residual probe, and 1 mL of Hank's solution was added to the culture dish. Cells were observed under a 100x objective lens using a laser confocal microscope. The excitation wavelength was 543 nm, and the emission wavelength was 550 nm–600 nm.

[0172] Example 8

[0173] MP-8 probe molecule's effect on L292 cell mitochondrial fluorescence:

[0174] Cell line: L929 cells (mouse fibroblasts).

[0175] Cell culture: Cells were cultured in a CO2 incubator (37°C, 5% CO2) in DMEM medium (penicillin (100 U / mL) and streptomycin (100 μg / mL)).

[0176] Cell imaging experiments: Cells were cultured in 15mm bottom-mounted cell culture dishes. Before labeling cells with the probe, the culture medium was removed, and the cells were washed twice with Hank's buffer to remove residual medium. 200 μL of probe MP-8 (10 μM) was added to the culture dish, and the cells were incubated in the dark for 30 minutes. The cells were washed twice with Hank's buffer to remove residual probe, and 1 mL of Hank's solution was added to the culture dish. Cells were observed under a 100x objective lens using a laser confocal microscope. The excitation wavelength was 543 nm, and the emission wavelength was 550 nm–600 nm.

[0177] Example 9

[0178] MP-9 probe molecule's effect on L292 cell mitochondrial fluorescence:

[0179] Cell line: L929 cells (mouse fibroblasts).

[0180] Cell culture: Cells were cultured in a CO2 incubator (37°C, 5% CO2) in DMEM medium (penicillin (100 U / mL) and streptomycin (100 μg / mL)).

[0181] Cell imaging experiments: Cells were cultured in 15mm bottom-mounted cell culture dishes. Before labeling cells with the probe, the culture medium was removed, and the cells were washed twice with Hank's buffered saline to remove residual medium. 200 μL of probe MP-9 (10 μM) was added to the culture dish, and the cells were incubated in the dark for 30 minutes. The cells were washed twice with Hank's buffered saline to remove residual probe, and 1 mL of Hank's solution was added to the culture dish. Cells were observed under a 100x objective lens using a laser confocal microscope. The excitation wavelength was 543 nm, and the emission wavelength was 550 nm–600 nm.

[0182] Example 10

[0183] MP-1 probe molecule colocalization fluorescence effect on L292 cell mitochondria:

[0184] Cell line: L929 cells (mouse fibroblasts).

[0185] Cell culture: Cells were cultured in a CO2 incubator (37°C, 5% CO2) in DMEM medium (penicillin (100 U / mL) and streptomycin (100 μg / mL)).

[0186] Cell imaging experiments: Cells were cultured in 15mm bottom-mounted cell culture dishes. Before labeling cells with the probe, the culture medium was removed, and the cells were washed twice with Hank's buffer to remove residual medium. 200 μL of probe MP-1 (10 μM) and 5 μM mitochondrial localization dye (MitoTracker Green) were added to the culture dish, and the cells were incubated in the dark for 30 minutes. The cells were washed twice with Hank's buffer to remove residual probe, and 1 mL of Hank's solution was added to the culture dish. Cells were observed under a 100x objective lens using a laser confocal microscope. The Pearson correlation coefficient was tested and calculated to be greater than 0.9, indicating that the probe molecule of this invention can effectively localize mitochondria.

[0187] Example 11

[0188] MP-3 probe molecule colocalization fluorescence effect on L292 cell mitochondria:

[0189] Cell line: L929 cells (mouse fibroblasts).

[0190] Cell culture: Cells were cultured in a CO2 incubator (37°C, 5% CO2) in DMEM medium (penicillin (100 U / mL) and streptomycin (100 μg / mL)).

[0191] Cell imaging experiments: Cells were cultured in 15mm bottom-mounted cell culture dishes. Before labeling cells with the probe, the culture medium was removed, and the cells were washed twice with Hank's buffer to remove residual medium. 200 μL of probe MP-3 (10 μM) and 5 μM mitochondrial localization dye (MitoTracker Green) were added to the culture dish, and the cells were incubated in the dark for 30 minutes. The cells were washed twice with Hank's buffer to remove residual probe, and 1 mL of Hank's solution was added to the culture dish. Cells were observed under a 100x objective lens using a laser confocal microscope. The Pearson correlation coefficient was tested and calculated to be greater than 0.9, indicating that the probe molecules of this invention can effectively localize mitochondria.

[0192] Example 12

[0193] MP-1 probe molecule fluorescence effect on mitochondria of other cells:

[0194] Cell lines: RAW 264.7 cell (mouse mononuclear macrophage leukemia cell); SKBR3 cell (HER2-overexpressing breast cancer cell); HuH-7 cell (human liver cancer cell line).

[0195] Cell culture: Cells were cultured in a CO2 incubator (37°C, 5% CO2) in DMEM medium (penicillin (100 U / mL) and streptomycin (100 μg / mL)).

[0196] Cell imaging experiments: We cultured the above three cell types in 15mm bottom cell culture dishes. Before labeling the cells with the probe, the culture medium was removed, and the cells were washed twice with Hank's buffer to remove residual culture medium. 200 μL of probe MP-1 (10 μM) was added to each cell culture dish, and the cells were incubated in the dark for 30 minutes. The cells were washed twice with Hank's buffer to remove residual probe, and 1 mL of Hank's solution was added to each culture dish. The cells were observed under a 100x objective lens using a laser confocal microscope. This experiment demonstrates that the fluorescent probe of this invention has good adaptability in most microenvironments.

[0197] Example 13

[0198] Cell silencing experiment 1

[0199] Cells were cultured in imaging dishes for 6 hours, then treated with either cytc-290 (a designed silent siRNA) or non-targeted RNA. After a certain treatment time, live-cell imaging was performed under a confocal laser scanning microscope. After 12 hours of cytc-290 treatment, Hoechst 33342 and MTG staining were good, but approximately 10% of the cells were not labeled with MP-1. After 18 hours of cytc-290 treatment, approximately 30% of the cells were not labeled with MP-1. In contrast, the control group treated with non-targeted RNA still showed good MP-1 staining. These results indicate that the probe MP-1 can effectively reflect respiratory chain activity and can be used to detect enzyme deficiencies in the respiratory chain.

[0200] Example 14

[0201] Cell silencing experiment 2

[0202] Cells were cultured in imaging dishes for 6 hours, then treated with cytc-436 (a designed silent siRNA) or non-targeted RNA for a certain period before live-cell imaging under a confocal laser scanning microscope. After 6 hours of cytc-436 treatment, Hoechst 33342 and MTG staining were good, but approximately 5% of cells could not be labeled with MP-1. In contrast, the control group treated with non-targeted RNA still showed good MP-1 staining. These results indicate that the MP-1 probe can effectively reflect respiratory chain activity and can be used to detect enzyme deficiencies in the respiratory chain.

[0203] Mass spectrometry analysis

[0204] Take L292 cells from Example 1 above, remove all culture medium, add 8 mL of deionized water, and allow the cells to swell and rupture after 1 hour. Collect the lysate, sonicate at 20 kHz for 10 minutes, then vortex at 2500 rpm for 10 minutes, and finally extract with dichloromethane. Remove the organic phase and perform mass spectrometry analysis with 1 / 1000 trifluoroacetic acid.

[0205] Depend on Figure 1 The molecular weight of the benzoindole dimer was found to be 483.26 (C). 36 H 23 N2 + ),Depend on Figure 2 The molecular weight of the obtained cell lysate was 483.22. This experimental result indicates that the 1,6-diyne compound is converted into a mitochondrial probe benzoindole dimer in cells.

[0206] The above description is merely a few embodiments of this application and is not intended to limit this application in any way. Although this application discloses preferred embodiments as described above, it is not intended to limit this application. Any changes or modifications made by those skilled in the art without departing from the scope of the technical solution of this application using the disclosed technical content are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. Use of a 1,6-diyne compound in the preparation of a mitochondrial fluorescent probe, wherein the 1,6-diyne compound has a structural formula as shown in Formula I: wherein X is a nitrogen atom; R' is a phenyl group; and the compound of Formula I has a structural formula selected from the group consisting of: Formula I. Formula I; wherein The method for preparing a mitochondrial fluorescent probe comprises the following steps: incubating a culture dish containing the 1,6-diyne compound and mouse fibroblasts. R 1a for , One of them; R 4a It is hydrogen; R 2a For one of the following: R 3a is hydrogen.

2. Use according to claim 1, characterized in that, The volume of the solution of the 1,6-diyne compound is 1 μL to 1000 μL. 。 3. Use according to claim 1, characterized in that, The concentration of the solution of the 1,6-diyne compound is 1 μM to 1000 μM. The incubation conditions are as follows:

4. Use according to claim 3, characterized in that, The temperature is 5 ℃ to 50 ℃.

5. Use according to claim 3, characterized in that, The time is 0.1 h to 48 h.

6. Use according to claim 3, characterized in that, Use of the 1,6-diyne compound in the preparation of a mitochondrial fluorescent probe for mouse fibroblasts. Use of the 1,6-diyne compound in the preparation of a mitochondrial fluorescent probe for mouse monocyte macrophage leukemia cells or HER2-breast cancer overexpression cells or human hepatoma cell lines.

9. Use of a 1,6-diyne compound in the preparation of a reagent for detecting the absence of an enzyme in the respiratory chain, wherein the 1,6-diyne compound is selected from the group consisting of the 1,6-diyne compounds of any one of claims 1 to 8.

7. The use according to claim 1, characterized in that, ​ 8. The use according to claim 1, characterized in that, ​ ​

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