CN110776458A - Fluorescent probe for detecting mitochondrial membrane potential and preparation method and application thereof - Google Patents
Fluorescent probe for detecting mitochondrial membrane potential and preparation method and application thereof Download PDFInfo
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- CN110776458A CN110776458A CN201911059578.3A CN201911059578A CN110776458A CN 110776458 A CN110776458 A CN 110776458A CN 201911059578 A CN201911059578 A CN 201911059578A CN 110776458 A CN110776458 A CN 110776458A
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- membrane potential
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- piperazine
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- 238000002360 preparation method Methods 0.000 title abstract description 4
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- UHBTWGXULSSHDL-UHFFFAOYSA-N CC1N(C)C2=CC=CC=C2C=C1.I Chemical compound CC1N(C)C2=CC=CC=C2C=C1.I UHBTWGXULSSHDL-UHFFFAOYSA-N 0.000 claims abstract description 11
- UOQXIWFBQSVDPP-UHFFFAOYSA-N 4-fluorobenzaldehyde Chemical compound FC1=CC=C(C=O)C=C1 UOQXIWFBQSVDPP-UHFFFAOYSA-N 0.000 claims abstract description 8
- 238000000746 purification Methods 0.000 claims abstract description 4
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- 238000006243 chemical reaction Methods 0.000 claims description 16
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- HUMNYLRZRPPJDN-UHFFFAOYSA-N benzaldehyde Chemical compound O=CC1=CC=CC=C1 HUMNYLRZRPPJDN-UHFFFAOYSA-N 0.000 claims description 14
- 239000000203 mixture Substances 0.000 claims description 10
- -1 piperazine-1, 4-diyl Chemical group 0.000 claims description 10
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- 238000000034 method Methods 0.000 claims description 8
- QNGNSVIICDLXHT-UHFFFAOYSA-N para-ethylbenzaldehyde Natural products CCC1=CC=C(C=O)C=C1 QNGNSVIICDLXHT-UHFFFAOYSA-N 0.000 claims description 7
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- 230000035484 reaction time Effects 0.000 claims description 6
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- 238000001953 recrystallisation Methods 0.000 claims description 3
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- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
- C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
- C07D215/12—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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Abstract
The invention belongs to the technical field of analytical chemistry, and provides a fluorescent probe for detecting mitochondrial membrane potential and a preparation method and application thereof. The structure formula of the mitochondrial membrane potential fluorescent probe is as follows:
can be obtained by reacting the reaction product of 4-fluorobenzaldehyde and piperazine with 1, 2-dimethyl-quinoline iodonium salt. The fluorescent probe provided by the invention has the characteristics of low biological toxicity, good membrane permeability, simple synthesis method and simple and convenient purification steps, and can be applied to cell imaging and detecting, marking or displaying mitochondrial membrane potential change.
Description
Technical Field
The invention belongs to the technical field of analytical chemistry, and particularly relates to a fluorescent probe for detecting mitochondrial membrane potential and application thereof.
Background
In the cycle process of the mitochondrial tricarboxylic acid, protons in the mitochondria are actively transported out of the mitochondria, so that the potential of the mitochondrial membrane with negative inside and positive outside reaches-160 mV to-180 mV. Mitochondrial membrane potential provides energy for the aerobic respiration process, catalyzing its decomposition of compounds with high stability. In addition, the mitochondrial membrane potential is closely related to the state of the cell, and the level of the mitochondrial membrane potential can accurately reflect the state of health of the cell. Therefore, the real-time observation of changes in mitochondrial membrane potential has important physiological, pathological, and pharmacological implications.
To date, fluorescence imaging is the most important tool for studying mitochondrial membrane potential changes. The mitochondria are small in size, and electrodes are difficult to be accurately embedded, so that the application of an electrochemical method in detecting the mitochondrial membrane potential is limited. In contrast, the fluorescence imaging method has the advantages of low damage to biological samples, capability of in-situ and dynamic observation and the like, and is an ideal tool for researching mitochondrial membrane potential. Currently, TMRM and JC-1 are fluorescent probes commonly used for studying mitochondrial membrane potential. TMRM is commonly used for calculating the size of mitochondrial membrane potential, however, the calculation process is tedious, and the application of TMRM in biological research is limited. JC-1 shows a J-aggregation state in mitochondria with high membrane potential and emits orange fluorescence; the monomer state is presented in mitochondria with low membrane potential, and yellow fluorescence is emitted; JC-1 can reflect the state of mitochondrial membrane potential by the change in fluorescence color and is a mitochondrial membrane potential probe commonly used in biological studies. However, the fluorescence wavelength shift of the probe to membrane potential response is only 70 nm, which limits its application in cell imaging. In recent years, several mitochondrial membrane potential probes have been modified based on JC-1 structure, but the fluorescence wavelength shift does not change much due to matrix limitations. There is a great need to develop a mitochondrial membrane potential fluorescent probe that can achieve subcellular organelle migration.
Disclosure of Invention
Aiming at the problems that the mitochondrial membrane potential probe in the prior art is single in type and can not realize subcellular organelle migration when the mitochondrial membrane potential changes, the invention provides the mitochondrial membrane potential probe which has good selectivity, high sensitivity, red emission and can realize subcellular organelle migration. .
The invention also aims to provide an application of the fluorescent probe in detecting the mitochondrial membrane potential.
In order to achieve the purpose, the invention adopts the following technical scheme.
A fluorescent probe for detecting mitochondrial membrane potential is named as 2,2'- (((1E,1' E) - (piperazine-1, 4-diylbis (4, 1-phenylene) bis (ethylene-2, 1-diyl)) bis (1-methylquinolin-1-ium) iodonium salt, BJI for short, and has a chemical structural formula shown as a formula (I):
formula (I).
The preparation method of the fluorescent probe comprises the following steps:
(1) heating 2-methylquinoline and methyl iodide in ethanol for reaction, and separating after the reaction to obtain 1, 2-dimethyl-quinoline iodonium salt (compound 1):
(2) dissolving 4-fluorobenzaldehyde and piperazine in a mixed solvent of water and 2-methoxyethanol, heating the mixture to reflux, adding a 2-methoxyethanol solution of 4-fluorobenzaldehyde, continuously refluxing the mixture, cooling to room temperature after the reaction is finished, pouring the mixture into water to obtain a light yellow precipitate, filtering the precipitate, and drying to obtain 4, 4' - (piperazine-1, 4-diyl) benzaldehyde (compound 2):
(3) dissolving 1, 2-dimethyl-quinoline iodonium salt and 4, 4' - (piperazine-1, 4-diyl) benzaldehyde in ethanol, stirring at room temperature in the presence of pyrrolidine, separating out a solid, filtering to obtain a crude product, and purifying to obtain a product, namely a mitochondrial membrane potential fluorescent probe (BJI):
in the step (1), the molar ratio of the 2-methylquinoline to the methyl iodide is 1: 1.2.
In the step (2), the molar ratio of the 4-fluorobenzaldehyde to the piperazine is 1: 2.
In the step (3), the molar ratio of the 1, 2-dimethyl-quinoline iodonium salt to the 4, 4' - (piperazine-1, 4-diyl) benzaldehyde is 2: 1; the mol ratio of the 1, 2-dimethyl-quinoline iodonium salt to the pyrrolidine is 1:1-9
In the step (1), the reaction temperature is 90 ℃ and the reaction time is 24-48 hours.
In the step (2), the reaction temperature is 100 ℃, and the reaction time is 24-48 h.
In the step (3), the reaction temperature is room temperature, the reaction time is 12-24h, and the purification method is recrystallization.
The application of the fluorescent probe in detecting, marking or displaying mitochondrial membrane potential change. Can be excited by adopting a single photon with the wavelength of 488nm, and the detection waveband is a red light waveband of 550-650 nm.
The working principle of the fluorescent probe of the invention is as follows:
the fluorescent probe is a cationic salt type compound, a conjugate structure with proper size and strong electron-withdrawing groups ensure red light emission, and in addition, the specific structure of the probe enables the probe to be embedded with an RNA groove region so as to identify RNA. When the membrane potential of the mitochondria is higher, the probe is preferentially enriched on the mitochondria; when the mitochondrial membrane potential is decreased, the probe is detached from the mitochondria and migrates to the RNA.
The invention has the following advantages:
the fluorescent probe provided by the invention has the characteristics of low biotoxicity, good membrane permeability, simple synthesis method and simple and convenient purification steps, and can be successfully applied to cell imaging and can be used for distinguishing the change of mitochondrial membrane potential.
Drawings
FIG. 1 shows fluorescent probe BJI
1H NMR spectrum;
FIG. 2 shows a fluorescent probe BJI
13C NMR spectrum;
FIG. 3 is a photograph of the fluorescence of viable cells with probes BJI reduced in membrane potential by treatment with CCCP;
FIG. 4 is a selective spectroscopic test pattern of probe BJI;
FIG. 5 is a photograph of a co-localized fluorescence image of probe BJI and probe MTDR for live cell imaging;
FIG. 6 shows the results of the cytotoxicity test of probe BJI;
FIG. 7 fluorescence imaging of fixed cells by fluorescent probes BJI.
Detailed Description
The present invention will be further described with reference to the following examples and drawings, but the present invention is not limited to the following examples.
EXAMPLE 1 Synthesis of fluorescent Probe
(1) Synthesis of 1, 2-dimethyl-quinoline iodonium salt (compound 1):
adding 10 mL of ethanol into a round-bottom flask, then adding 1.1 mL of 2-methylquinoline, adding 0.5 mL of methyl iodide, heating to 60 ℃ for reaction for 30 hours, cooling the reaction system to room temperature after the reaction is finished, separating out a solid, filtering and washing by using ethanol to obtain the 1, 2-dimethyl-quinoline iodonium salt (compound 2) with the yield of 92%.
1H NMR (400 MHz, DMSO-d6) δ9.12 (d, J = 8.5 Hz, 1H), 8.60 (d, J = 9.0 Hz, 1H), 8.41 (dd, J = 8.2, 1.6Hz, 1H), 8.24 (ddd, J = 8.8, 7.0, 1.6 Hz, 1H), 8.14 (d, J = 8.5 Hz, 1H), 8.00(t, J = 7.6 Hz, 1H), 4.45 (s, 3H), 3.09 (s, 3H)。
(2) Synthesis of 4, 4' - (piperazine-1, 4-diyl) benzaldehyde (compound 2):
piperazine (1.0 g, 11.62 mmol) was dissolved in H
2O (18 mL) and 2-methoxyethanol (20 mL). The mixture was heated to reflux and then a solution of 4-fluorobenzaldehyde (0.63 mL, 5.81 mmol) in 2-methoxyethanol (5 mL) was added. The mixture was then refluxed for an additional 12 hours. After cooling to room temperature, the mixture is poured into H
2In O (50 mL), a pale yellow precipitate was obtained. The precipitate was filtered and dried to give 2 as a yellow solid in 93% yield.
1H NMR(400 MHz, DMSO-d6)δ9.74(s, 2H)，7.75 (d, J = 8.9 Hz, 4H)，7.08 (d，J = 8.9 Hz, 4H)，3.61 (s, 8H)。
(3) Synthesis of Probe BJI:
to a bottom flask with 5mL ethanol, 0.5g (1.75 mmol) of Compound 1 and 0.26g (0.875 mmol) of Compound 2 were added. The mixture was then stirred for 5 minutes, then 400 μ L pyrrolidine was added. The system was stirred at room temperature for 24 hours to complete the reaction and a dark brown solid precipitated. The final product was obtained by filtration and purified by recrystallization from ethanol in 56% yield.
1H NMR (400 MHz, DMSO-
d 6) δ 8.90 (d,
J= 9.0 Hz, 2H), 8.52 (dd,
J=26.5, 9.0 Hz, 4H), 8.27 (d,
J= 8.9 Hz, 4H), 8.11 (s, 2H), 7.88 (d,
J= 7.4Hz, 6H), 7.69 (d,
J= 15.5 Hz, 2H), 7.14 (d,
J= 8.5 Hz, 4H), 4.50 (s, 6H),3.65 (s, 8H).
13C NMR (101 MHz, DMSO-
d 6) δ (ppm): 164.64, 147.88, 144.19,143.14, 129.44, 124.63, 123.24, 116.96, 114.20, 112.13, 106.39, 51.28, 45.67,27.29, 13.82, 8.83。
1H NMR and
13the C NMR spectrum is shown in FIGS. 1 and 2.
Example 2 response of fluorescent probes to different Membrane potentials
The density is 3 x 10
5HeLa cells/mL were seeded in sterilized 35 mm imaging dishes in CO
2Incubator (temperature 37 ℃, 5% CO)
2) Cells were allowed to adhere for more than 12 hours of culture. Then, a DMSO solution of the probe HJI obtained in example 1 was prepared as a stock solution at a concentration of 1 mM, the stock solution was added to the cell culture dish so that the final concentration was 5. mu.M, the culture was continued for 20 min, then the cell culture solution was aspirated, the cells were washed with PBS buffer 3 times, 1 mL of fresh medium was added, 10. mu.M of CCCP (which is an oxidative phosphorylation decoupling agent and is capable of lowering mitochondrial membrane potential) was added thereto for immediate imaging, and images were recorded at intervals of 0.5 min.
In the cell imaging experiment, the excitation wavelength is 488nm, and the detection band is the red light band 550-650 nm. The resulting fluorescence pictures are shown in FIG. 3. The probe HJI stains mitochondria when staining healthy cells with high membrane potential, and the probe migrates from mitochondria to nucleolar region as the membrane potential decreases.
Example 3 selectivity of fluorescent probes for different ions
DMSO stock solutions were prepared in the concentration of 5mM in the fluorescent probes prepared in example 1, and different amino acids (Ile, Arg, Ser, Asn, Gln, Glu, His, Ala, Hcy, N-Ace, Val, GSH) and NaCl, KNO were prepared
3、H
2O
2The PBS stock solution of (1), at a concentration of 100 mM. Then 5. mu.L of each probe stock was added to a 5mL volumetric flask, 10. mu.L of each different analyte was added to each volumetric flask, and finally 5mL was made up with PBS buffer. Then, fluorescence detection (excitation wavelength 500 nm) was carried out. The wavelength is plotted on the abscissa and the fluorescence intensity is plotted on the ordinate to obtain graph 4. As can be seen, there is no effect on probe fluorescence after addition of different analytes.
EXAMPLE 4 Co-localization of fluorescent probes with commercial probes
In the co-localization experiment, cells were stained with 200 nM MTDR for 30min, then 4 μ M BJI for 30min, and then cell culture fluid was aspirated, cells were washed 3 times with medium, and cell imaging was performed: collecting the fluorescence at 665-; the fluorescence signal of MTDR is collected by collecting 665-735 nm fluorescence with 647 nm as the excitation wavelength. The resulting fluorescence image is shown in FIG. 5. The counterstaining rate of both dyes was 89%, indicating that the probe stained mitochondria in living cells.
Example 5 toxicity of fluorescent probes to cells
HeLa cells with a cell density of 8000 cells/mL were seeded into a part of wells of a 96-well plate, and the remaining wells were filled with PBS buffer under CO conditions as follows
2Incubating cells in an incubator: the experimental group was cell samples after 2 hours, 24 hours and 36 hours of incubation with a medium containing 5. mu.M BHI, the control group was cell-containing samples without dye, and the blank group was PBS buffer sample. After the incubation was complete, the cell culture was replaced with fresh medium and 10 μ L of MTT was added to each well and the cells were incubated for an additional 4 hours. After the incubation was completed, the medium was removed, 200 μ L of DMSO was added to each well, and it was shaken with a shaker for 10 min to dissolve formazan. Each well was tested at 570 nm using a microplate readerThe cell survival rate (survivvalrate) can be calculated by the following equation:
wherein A is
sampleAbsorbance for experimental group, A
cAbsorbance of control group, A
bAbsorbance of blank. Plotting the probe incubation time as abscissa and the cell viability as ordinate 6: the cell survival rate is still up to 90% after 36 h of staining, which indicates that the toxicity of the probe to the living cells is low.
EXAMPLE 6 imaging application of fluorescent probes in immobilized cells
The fluorescent probe DMSO stock prepared in example 1 was prepared at a concentration of 1 mM. Then 20. mu.L of the diluted solution was diluted with one mL of the medium to obtain a 20. mu.M diluted solution of the probe. The inoculated cells were treated with l mL paraformaldehyde for 30min, washed 3 times with PBS, and then washed with 0.5 mL of 5% Triton
TMTreating with X-100 for 3 min, incubating at room temperature in probe diluent for 30min, washing with PBS for 3 times, and placing the cells growing adherent to the surface of the slide glass; then, bright field imaging and fluorescence imaging (excitation wavelength 488nm, emission band 550-650 nm) were performed by fluorescence microscope, and the results are shown in FIG. 7: fluorescent probe BJI was able to stain the cytoplasm and nucleolus of fixed cells, emitting red fluorescence.
Claims (5)
1. A fluorescent probe for detecting mitochondrial membrane potential has a chemical structural formula as follows:
2. a method of preparing a fluorescent probe according to claim 1, comprising the steps of:
(1) heating 2-methylquinoline and methyl iodide in ethanol for reaction, and separating after the reaction to obtain 1, 2-dimethyl-quinoline iodonium salt:
；
(2) dissolving 4-fluorobenzaldehyde and piperazine in a mixed solvent of water and 2-methoxyethanol, heating the mixture to reflux, adding a 2-methoxyethanol solution of 4-fluorobenzaldehyde, continuously refluxing the mixture, cooling to room temperature after the reaction is finished, pouring the mixture into water to obtain a light yellow precipitate, filtering the precipitate, and drying to obtain 4, 4' - (piperazine-1, 4-diyl) benzaldehyde:
(3) dissolving 1, 2-dimethyl-quinoline iodonium salt and 4, 4' - (piperazine-1, 4-diyl) benzaldehyde in ethanol, stirring at room temperature in the presence of pyrrolidine, separating out solids, filtering to obtain a crude product, and purifying to obtain a product, namely a mitochondrial membrane potential fluorescent probe:
3. the process according to claim 2, wherein in the step (1), the molar ratio of 2-methylquinoline to methyl iodide is 1: 1.2;
in the step (2), the molar ratio of 4-fluorobenzaldehyde to piperazine is 1: 2;
in the step (3), the molar ratio of the 1, 2-dimethyl-quinoline iodonium salt to the 4, 4' - (piperazine-1, 4-diyl) benzaldehyde is 2: 1; the molar ratio of the 1, 2-dimethyl-quinoline iodonium salt to the pyrrolidine is 1: 1-9.
4. The production method according to claim 2, wherein in the step (1), the reaction temperature is 90 ℃ and the reaction time is 24 to 48 hours;
in the step (2), the reaction temperature is 100 ℃, and the reaction time is 24-48 h;
in the step (3), the reaction temperature is room temperature, and the reaction time is 12-24 h; the purification method is recrystallization.
5. Use of a fluorescent probe according to claim 1 for detecting, labeling or displaying changes in mitochondrial membrane potential.
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