CN116178413A - Mitochondrial targeting fluorescent probe based on BODIPY, preparation method and application thereof - Google Patents
Mitochondrial targeting fluorescent probe based on BODIPY, preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a mitochondrial targeting fluorescent probe based on BODIPY, a preparation method and application thereof, and belongs to the technical field of optical analysis and detection. 2, 4-dimethylpyrrole reacts with p-chloromethyl benzaldehyde, then is oxidized with 2, 3-dichloro-5, 6-diyl-1, 4-benzoquinone (DDQ), finally is complexed with boron trifluoride diethyl ether under the condition of triethylamine to obtain compounds 1,2, 4-dimethylpyrrole and pyridine benzaldehyde, then is oxidized with 2, 3-dichloro-5, 6-diyl-1, 4-benzoquinone (DDQ), finally is complexed with boron trifluoride diethyl ether under the condition of triethylamine to obtain compound 2, and the compounds 1 and 2 are synthesized to obtain the mitochondrial targeting fluorescent probe. The invention has the advantages of good imaging effect, low toxicity, strong photo-bleaching resistance and the like.
Description
Technical Field
The invention belongs to the technical field of optical analysis and detection, and relates to a mitochondrial targeting fluorescent probe based on BODIPY, a preparation method and application thereof.
Background
Mitochondria are important energy supply workshops in cells, and play an indispensable role in the activities of cells, tissues, organs and whole life bodies, including metabolism of energy, generation of redox signals, regulation of calcium homeostasis, and control of apoptosis of cell pathways, etc. Research shows that the occurrence and development processes of various diseases are related to abnormal mitochondrial morphology and functions, such as Alzheimer disease, hypertension, hyperlipidemia, arteriosclerosis, cerebral hemorrhage, gastric ulcer, diabetes, gout, cancer and the like, so that mitochondrial imaging has important significance for diagnosis and treatment of subcellular level.
The conventional mitochondrial probes include rhodamine 123, JC-1, mitosacker series and the like, and although the mitochondrial probes are used for mitochondrial imaging as commercial probes, the mitochondrial probes still have various disadvantages of high cytotoxicity, weak photobleaching resistance, inapplicability to long-time mitochondrial fluorescence imaging and the like, so that a new mitochondrial fluorescence probe needs to be developed for real-time mitochondrial fluorescence imaging of living cells.
Disclosure of Invention
The invention aims to solve the problems existing in the prior art and provide a mitochondrial targeting fluorescent probe based on BODIPY, a preparation method and application thereof, and the technical problem to be solved by the invention is to provide the preparation method of the mitochondrial targeting fluorescent probe with good mitochondrial imaging effect, low toxicity and strong photobleaching resistance.
A mitochondrial targeting fluorescent probe based on BODIPY, characterized in that the mitochondrial targeting fluorescent probe is an aqueous solution of a compound having the structural formula:
the method for preparing the fluorescent probe with the mitochondrial targeting function comprises the following steps:
setting up
For compound 1, < ->
The fluorescent probe is compound 2, and the fluorescent probe is compound 3;
step one, compounds 1 and 2 were prepared, and the preparation reaction formulas of compounds 1 and 2 were as follows:
preparation of Compound 1:2, 4-dimethyl pyrrole reacts with p-chloromethyl benzaldehyde in the presence of trifluoroacetic acid in an inert gas atmosphere, then is oxidized with 2, 3-dichloro-5, 6-diyl-1, 4-benzoquinone (DDQ), and finally is complexed with boron trifluoride diethyl ether in the presence of triethylamine to obtain a compound 1.
Step two, preparing a compound 2: 2, 4-dimethylpyrrole reacts with pyridine benzaldehyde in the presence of trifluoroacetic acid in an inert gas atmosphere, then is oxidized with 2, 3-dichloro-5, 6-diyl-1, 4-benzoquinone (DDQ), and finally is complexed with boron trifluoride diethyl ether in the presence of triethylamine to obtain a compound 2;
step three, preparing a compound 3: compound 1 and compound 2 were dissolved in toluene and refluxed at 120 ℃ for 48 hours to give compound 3.
The reaction formula is as follows:
the inert gas in the first step and the second step is nitrogen.
In the first and second steps, the molar ratio of the pyridine tetra-formaldehyde or the p-chloromethyl benzaldehyde to the 2, 4-dimethyl pyrrole is 1:2.
the molar ratio of the compound 1 to the compound 2 is 1:1.
the fluorescent probe with the mitochondrial targeting function is applied to the field of biological labeling.
The fluorescent probe with the mitochondrial targeting function is used for live cell mitochondrial marking and imaging.
A method for detecting the morphology change of grains by utilizing the fluorescent probe with the mitochondrial targeting function detects the morphology change of grains by a confocal imaging technology.
The invention has the following advantages and beneficial effects:
(1) The mitochondrion targeting fluorescent probe prepared by the invention has the advantages of good water solubility, strong photobleaching resistance, low cytotoxicity and the like.
(2) The synthesis method has the advantages of mild reaction condition, simple process, low cost and high yield.
(3) The mitochondrial targeting fluorescent probe prepared by the invention can be used for biological imaging and mitochondrial marking, and is a good biomarker probe.
Drawings
FIG. 1 is a synthetic route diagram of the probe BL in example 1.
FIG. 2 is a nuclear magnetic resonance spectrum (hydrogen spectrum) of the compound 3 of the present invention.
FIG. 3 is a nuclear magnetic resonance spectrum (carbon spectrum) of the compound 3 of the present invention.
Fig. 4 is an LCMS diagram of compound 3 of the present invention.
FIG. 5 is a graph showing the absorption (left) and fluorescence (right) spectra of the probe BL in water in example 2.
FIG. 6 is a graph showing the results of MTT cytotoxicity test in PC-3 cells of the probe BL in example 3.
FIG. 7 is a graph showing the results of co-localized imaging of live cell mitochondria with probe BL and commercial mitochondrial dye Mito-Tracker red in example 4; wherein, fig. 7 (a) is an imaging diagram of BL versus live cell mitochondria; FIG. 7 (b) is a plot of Mito-Tracker red imaging of live cell mitochondria; FIG. 7 (c) is a superposition of images of BL and Mito-Tracker red on live cell mitochondria; fig. 7 (d) is the superposition of the transmission region of BL and the transmission region of Mito-Tracker red.
FIG. 8 is a graph showing the results of the photo-bleaching resistance test of the probe BL in example 5, wherein 1 to 14 represent the scanning numbers.
FIG. 9 is a graph showing the results of photo-bleaching resistance experiments of commercial probes Mito-Tracker red in example 5, wherein, 1 to 14 represent scan numbers.
Detailed Description
The technical scheme of the invention is further described in detail below with reference to specific embodiments. The procedures, conditions, reagents, experimental methods, etc. for carrying out the present invention are common knowledge and common knowledge in the art, except for those specifically mentioned below, and the present invention is not particularly limited.
The analytical instrument used was of the following specification type: nuclear magnetism (Bruker-400M, germany), ultraviolet spectrophotometer (UV-3010), fluorescence spectrophotometer (Rili, F-4500), laser confocal microscope (Olympus, FV 300), enzyme-labeled instrument (JC-MB 36), high performance liquid chromatograph (Agi lens 1220 Infinicity II).
Example 1: preparation of fluorescent organic molecules (BL) with mitochondrial targeting function
Example 2: photophysical properties of mitochondrial targeting fluorescent probes in water
The photophysical properties of BL were measured in normal solvent water, and the prepared BL was added to water with a final solution concentration of 10.0. Mu.m. After mixing uniformly and balancing at room temperature for 20 minutes, ultraviolet absorbance and fluorescence intensity were measured with a Hitachi U3010 absorbance spectrometer and a Hitachi F4500 fluorescence spectrometer. As shown in fig. 5 below, BL absorbs widely in the visible region in water, concentrates at 500nm, and the fluorescence emission peak concentrates at 510nm in water (λex=365 nm).
Example 3: cytotoxicity experiment of mitochondrial targeting fluorescent probe
Single cell suspension is prepared by culture solution containing 10% fetal calf serum, and 1000-10000 cells per well are inoculated into a 96-well plate, and the volume of each well is 100 μl. The administration was performed after culturing in a constant temperature incubator for 24 hours. The gradient of administration of compound 4 was set to 0, 20, 40, 60, 80, 100. Mu. Mol/l. After the administration according to the setting, the culture was continued in a constant temperature incubator for 24 hours. After 1 day of incubation, 20. Mu.l of MTT solution (5 mg/mL in PBS) was added to each well. Incubation was continued for 4 hours, the culture was terminated, the culture supernatant in the wells was removed by pipetting (centrifugation was required for suspension cells and then the culture supernatant in the wells was removed), 150. Mu.l of DMSO was added to each well, and shaking was performed for 10 minutes to allow the crystals to be sufficiently thawed. The light absorption value of each well was measured on an enzyme-linked immunosorbent assay (ELISA) by selecting a wavelength of 490nm, and the result was recorded. And drawing a cell growth curve graph by taking the administration concentration as an abscissa and the absorbance ratio as an ordinate. In FIG. 6, the cytotoxicity of the probe is shown, and the probe B-L has no obvious toxicity to normal cell lines at low concentration, which shows that the probe has low cytotoxicity and good biocompatibility. Example 4: co-localization experiments of mitochondria targeting fluorescent probe BL with commercial mitochondrial dye Mito-Tracker red on live cell mitochondria cells were cultured in modified Eagle's Medium (DMEM) at 37℃and 5% CO2 for 24 hours (10% fetal bovine serum and 1% penicillin and streptomycin added to DMEM). After cell attachment, the cells were treated with fresh medium containing 10.0. Mu.M BL and 10.0. Mu.M MitoTracker@RedCMXRos (MTR) for 0.5 hours. All cells were washed three times with PBS prior to imaging. Cell images were recorded using a confocal microscope (OLYMPUS, FLUOVIEW, FV3000, japan). Fluorescence of BL was captured using the green channel at 488nm and 25 ℃. The Red channel emission wavelength of Mi to-Tracker Red was measured to be 543nm at 25 ℃. Co-localization imaging results As shown in FIG. 7, the fluorescence image generated by BL overlapped with that obtained by MTR (FIG. 7), pearson correlation coefficient of 0.989, indicated that BL had good mitochondrial co-localization. As shown in fig. 7.D, the linear region of interest (ROI) fluorescence intensity profiles of BL and MTR changed simultaneously, indicating that BL can selectively accumulate in mitochondria of living cells. The mitochondrial localization experiment of BL shows that the organelle with prominent BL concentration in the cell is mitochondria, has mitochondrial targeting property and can be selectively accumulated in the cell mitochondria, and BL has good mitochondrial targeting property and subcellular imaging capability in living cells.
Example 5: photo-bleaching resistance experiment of mitochondrial targeting fluorescent probe BL versus commercial probe Mi to-Tracker red
The BL photo-bleaching resistance test is specifically as follows: the cells used in the experiment were PC-3 cells, the digested cells were inoculated in a petri dish, incubated at 37℃under 5% CO2 for 24 hours to adhere to the cells, washed with PBS solution, incubated with BL (10. Mu.M) cell culture solution for 12 hours, washed with PBS solution three times to image, and subjected to a photo-bleaching experiment with a confocal laser microscope at a laser intensity of (65. Mu.W) and an excitation wavelength of (488 nm) for 50 seconds each time, and the experimental results are shown in FIG. 8.
The photo-bleaching resistance test of Mito-Tracker red is specifically as follows: the cells used in the experiment were PC-3 cells, the digested cells were inoculated in a petri dish, incubated at 37℃for 24 hours under 5% CO2 to adhere to the cells, the stale cell culture solution was washed with PBS solution, then the cells were further incubated with a cell culture solution containing Mito-Tracker red (10. Mu.M) for 30 minutes, washed with PBS solution three times to image, and then the photobleaching experiment was performed with a laser confocal microscope at a laser intensity of (65. Mu.W) and an excitation wavelength of (561 nm) for 50 seconds each time, and the experimental results were shown in FIG. 9.
The experimental results showed that the BL fluorescence signal was hardly lost after 200 seconds (4 times) of irradiation (FIG. 8). In contrast, fluorescence of the commercial probe MTR was reduced with the increase of the irradiation time, and could not be observed only after 700 seconds (graph). These results indicate that commercial probe MTR has poor photo-bleaching resistance under irradiation, while probe BL has good photo-bleaching resistance. Again, this demonstrates the biostability of BL. The results show that the probe BL can be applied to long-term imaging of living cells due to the excellent photobleaching resistance.
The specific embodiments described herein are offered by way of example only to illustrate the spirit of the invention. Those skilled in the art may make various modifications or additions to the described embodiments or substitutions thereof without departing from the spirit of the invention or exceeding the scope of the invention as defined in the accompanying claims.
Claims (7)
1. A mitochondrial targeting fluorescent probe based on BODIPY, characterized in that the mitochondrial targeting fluorescent probe has the following structural formula:
2. a method of synthesizing the fluorescent probe of claim 1, comprising the steps of:
setting up
For compound 1, < ->
The fluorescent probe is compound 2, and the fluorescent probe is compound 3;
step one, compound 1 is prepared: 2, 4-dimethyl pyrrole reacts with p-chloromethyl benzaldehyde in the presence of trifluoroacetic acid in an inert gas atmosphere, then is oxidized with 2, 3-dichloro-5, 6-diyl-1, 4-benzoquinone (DDQ), and finally is complexed with boron trifluoride diethyl ether in the presence of triethylamine to obtain a compound 1.
Step two, preparing a compound 2: 2, 4-dimethylpyrrole reacts with pyridine tetra-formaldehyde in the presence of trifluoroacetic acid in an inert gas atmosphere, then is oxidized with 2, 3-dichloro-5, 6-diyl-1, 4-benzoquinone (DDQ), and finally is complexed with boron trifluoride diethyl ether in the presence of triethylamine to obtain a compound 2;
step three, preparing a compound 3: compound 1 and compound 2 were dissolved in toluene and refluxed at 120 ℃ for 48 hours to give compound 3.
The inert gas in the first step and the second step is nitrogen.
3. The method for synthesizing a mitochondrial targeting fluorescent probe according to claim 2, wherein the molar ratio of the pyridine tetra-formaldehyde or the p-chloromethyl benzaldehyde to the 2, 4-dimethyl pyrrole in the first step and the second step is 1:2.
4. the method for synthesizing a mitochondrial targeting fluorescent probe according to claim 2, wherein the molar ratio of the compound 1 to the compound 2 is 1:1.
5. use of a mitochondrial targeting fluorescent probe according to any one of claims 1-4 in the field of biomarkers.
6. Use of a mitochondrial targeting fluorescent probe according to any one of claims 1-4 for live cell mitochondrial marking and imaging.
7. Use of a mitochondrial targeting fluorescent probe according to any one of claims 1 to 4 for detecting changes in morphology of particles, the changes in morphology of particles being detected by confocal imaging techniques.
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