CN110194766B - Double-channel two-photon fluorescence polarity probe and preparation method and application thereof - Google Patents
Double-channel two-photon fluorescence polarity probe and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a dual-channel two-photon fluorescence polarity probe and a preparation method and application thereof, wherein the dual-channel two-photon fluorescence polarity probe has the following structure:
the two-photon fluorescence polar probe molecule of the invention shows specific response to polarity in a system with other interference factors. The cytotoxicity test shows that the probe has little toxic or side effect on cells, and the two-photon confocal fluorescence microscopic imaging experiment shows that the probe has good permeability on HeLa cells, can effectively position mitochondria in the cells (the positioning coefficient is 0.95), and is suitable for dual-channel two-photon fluorescence imaging and quantitative detection of polarity in the mitochondria of the cells.
Description
Technical Field
The invention relates to a dual-channel two-photon fluorescence polarity probe, a preparation method and application thereof, which are used for realizing the quantitative detection of the polarity in cell mitochondria by dual-channel two-photon fluorescence imaging and have the advantages of specific selectivity, high sensitivity and low biological toxicity.
Background
Mitochondria are very important organelles in eukaryotic cells, for example, the production of Adenosine Triphosphate (ATP) to supply energy to physiological processes, and thus it is called an energy source spring of cells. In addition, mitochondria are involved in many physiological processes such as providing energy for the biosynthesis of macromolecules and maintaining intracellular Ca2+Horizontal steady state, etc. Mitochondria are closely related to microenvironment parameters such as polarity, viscosity, pH, etc. And mitochondrial polarity is particularly important, which can affect mitochondrial activity. Among them, mitochondrial dysfunction may cause a series of diseases such as neurodegenerative diseases and the like. Mitochondrial dysfunction can lead to apoptosis. More importantly, mitochondrial polarity changes upon mitochondrial dysfunction, and thus apoptosis can be further monitored by detecting polarity changes.
Apoptosis, one of programmed death processes, plays an important role in biological processes, and thus detection of apoptosis is of great importance for biological research. Current methods for detecting apoptosis are based on traditional methods, such as detecting caspase protein activity, etc. However, these methods are costly, long cycle and poor sensitivity, and they are costly and do not meet the requirements for assessing apoptosis. To date, fluorescent probe assay methods for assessing apoptosis are still rare, and therefore, development of fluorescent probes with excellent performance for detecting apoptosis is required as soon as possible.
In the last decade, fluorescent probe methods have been widely used to monitor biological processes, and are based mainly on the fact that fluorescent probe technology is inexpensive, simple to use, high in sensitivity, and fast in response. Although many fluorescent probes have been developed, since there are few probes for detecting mitochondrial polarity, it is very urgent to develop mitochondrial polarity probes having excellent performance.
Polarity plays a very important role in biological processes. For example, polarity may modulate the permeability of organelle membranes. In addition, abnormal changes in polarity have a strong link with certain diseases. The current main mechanism for detecting polarity is based on an ICT mechanism, and the mechanism has higher sensitivity to environmental polarity, so that an accurate result is provided in the process of detecting polarity, and the mechanism has important significance for the research of biological environment. In addition, the polarity of the apoptosis process is greatly changed, so that the apoptosis can be detected through the polarity, and a good idea is provided for researching the apoptosis.
Disclosure of Invention
The invention aims to provide a double-channel two-photon fluorescence polarity probe and a preparation method and application thereof, and aims to solve the technical problem that a ratio type two-photon fluorescence polarity probe structure capable of positioning mitochondria is obtained through molecular design so as to realize double-channel two-photon fluorescence imaging detection of polarity change in mitochondria in an apoptosis process, and the double-channel two-photon fluorescence polarity probe has the advantages of single selectivity, high sensitivity and double-channel detection, and a cytotoxicity test shows that the fluorescence probe has almost no toxic effect on cells.
The invention discloses a dual-channel two-photon fluorescence polar probe, which is abbreviated as Mito-PF, takes carbazole as a matrix, and has the following structural formula:
the preparation method of the dual-channel two-photon fluorescence polarity probe comprises the following steps:
step 1: the compound 9-ethyl-6-iodo-9H-carbazole-3-carbaldehyde (1.0g, 2.86mmol), 4-fluoroacetylene (0.41g, 3.44mmol), triphenylphosphine palladium dichloride (9.65mg, 0.014mmol), cuprous iodide (5.24mg, 0.027mmol) and triethylamine (2ml) were added to a reactor and stirred at 30 ℃ under anhydrous and oxygen-free conditions for 12 hours; cooling the mixture to room temperature, filtering the precipitate and concentrating to obtain a crude product; the crude product was purified by column chromatography (petroleum/dichloromethane ═ 4: 1 as eluent) to afford intermediate 1, 0.80g, in 82% yield.
Step 2: compound 3-benzyl-2-methylbenzothiazole bromide (0.28g, 0.9mmol) and intermediate 1(0.3g, 0.9mmol) were mixed in 50mL of anhydrous ethanol and the mixed solution was refluxed under nitrogen until TLC showed completion of the starting material reaction (about 12 hours), and after the mixture was cooled to room temperature, the solvent was removed by rotary evaporator; the resulting crude product was washed with 30mL of saturated brine, then extracted with DCM (3 × 50mL) and the solvent was evaporated under vacuum and the crude product was purified by column chromatography (dichloromethane/methanol ═ 50: 1 as eluent) to give the desired product Mito-PF, 0.35g, 62% yield.
The synthetic process of the two-photon fluorescent probe Mito-PF comprises the following steps:
the dual-channel two-photon fluorescence polarity probe is used as a detection reagent when the polarity change in mitochondria in living cells is quantitatively detected for non-treatment or diagnosis. The detection method comprises the following steps:
the two-photon fluorescent probe is dissolved in DMSO to prepare 2mM mother liquor, 15 mu L of the mother liquor is taken to be respectively put in 3mL of solvents with different polarities, and ultraviolet spectrograms of the probe Mito-PF in different solvents are obtained. Under the excitation of 360nm wavelength, the fluorescence intensity at 436nm gradually decreases along with the increase of the polarity of the test system, and the fluorescence intensity hardly changes at 589 nm. And fluorescence intensity (I)436nm/I589nm) And Δ f, indicating that Mito-PF can be used for ratio-detecting the polarity of common solutions. To further verify the response characteristics of the probe Mito-PF to polarity, the absorption and fluorescence spectra of Mito-PF were measured in a range of polarities with different ratios of water and tetrahydrofuran. When the polarity of the solvent was increased from 10% water (Δ f ≈ 0.2556) to 80% water (Δ f ≈ 0.3103), only a slight change in the absorption maximum was observed, consistent with the results of measuring the polarity of different solvents. In contrast, when the polarity (Δ f) of the solution was decreased from 0.3103 (80% water) to 0.2556 (10% water), the fluorescence intensity of Mito-PF at 436nm increased by 3.6-fold, while the red emission at 589nm gave almost no response. The above results also show fluorescence intensity I436nm/I589nmThere is a good linear correlation with Δ f, which indicates that Mito-PF is highly sensitive to solvent polarity. To exploreThe optical stability of the needle Mito-PF in HeLa cells is a very important experiment, because the mitochondrial membrane potential is destroyed in the process of apoptosis, which affects the positioning performance of the probe, and thus, the real-time monitoring of the apoptosis process is concerned. Reported articles indicate that 3-chlorophenylhydrazone (CCCP) is a membrane potential breaker that can break mitochondrial membrane potential. The addition of CCCP experiments demonstrated that probe Mito-PF was not dependent on membrane potential for localization. In addition, etoposide (etoposide) is tested to induce the HeLa cell apoptosis process by using a probe Mito-PF, and the mitochondrial polarity in cells is gradually reduced along with the deep apoptosis.
The two-photon fluorescence polar probe molecule shows specific response to polarity in a system with other interference factors. The cytotoxicity test shows that the probe has little toxic or side effect on cells, and the two-photon confocal fluorescence microscopic imaging experiment shows that the probe has good permeability on HeLa cells, can effectively position mitochondria in the cells (the positioning coefficients are 0.95 respectively), is suitable for dual-channel two-photon fluorescence imaging and quantitative detection of polarity in the mitochondria of the cells, and can monitor apoptosis in real time by detecting polarity change of the mitochondria.
Drawings
FIG. 1 is a graph showing UV absorption spectra of 10. mu.M probe in different polar organic solvents (a); (b) a fluorescence emission spectrum; (c) fluorescence intensity (I)436nm/I589nm) And Δ f.
FIG. 2 is a diagram showing (a) ultraviolet absorption spectra of 10. mu.M probe in different volume ratios of water/tetrahydrofuran mixed solvent; (b) a fluorescence emission spectrum; (c) fluorescence intensity (I)436nm/I589nm) And Δ f.
FIG. 3 is a graph of fluorescence emission from 10 μ M probes in tetrahydrofuran and methanol/glycerol mixed systems of varying viscosities.
FIG. 4 is a graph of pH stability of 10 μ M probe at different water/tetrahydrofuran ratios.
FIG. 5 is a sectional view showing (a) effective two-photon absorption of a 0.1mM probe in different volume ratios of a water/tetrahydrofuran mixed solvent; (b) relative two-photon fluorescence intensity (I)out) And input power (I)in) Graph of logarithmic relationship of (c).
FIG. 6 is a graph of HeLa cell viability at different concentrations (0. mu.M, 10. mu.M, 20. mu.M, 30. mu.M) of probe molecules.
FIG. 7 is a confocal fluorescence imaging image of location validation of mitochondria in HeLa cells co-stained with 10 μ M probe and 1 μ M mitoterrker green (MTG).
FIG. 8 is a graph of two experiments with and without human CCCP to explore the optical stability of the probe Mito-PF.
FIG. 9 is a confocal fluorescence image of 10 μ M probe induced apoptosis of HeLa cells at 50 μ M etoposide.
Detailed Description
The invention is further illustrated by the following examples.
Example 1: synthesis of fluorescent probe molecule Mito-PF
Compound 3-benzyl-2-methylbenzothiazole bromide (0.28g, 0.9mmol) and compound 1(0.3g, 0.9mmol) were mixed in 50mL of anhydrous ethanol and the mixed solution was refluxed under nitrogen until TLC showed completion of the raw material reaction (about 12 hours), and after the mixture was cooled to room temperature, the solvent was removed by rotary evaporator; the resulting crude product was washed with 30mL of saturated brine, then extracted with DCM (3 × 50mL) and the solvent was evaporated under vacuum and the crude product was purified by column chromatography (dichloromethane/methanol ═ 50: 1 as eluent) to give the desired product Mito-PF, 0.35g, 62% yield.
1H NMR(400MHz,DMSO)δ8.95(d,J＝7.1Hz,1H),8.51(d,J＝15.4Hz,1H),8.44(s,1H),8.40–8.36(m,1H),8.18(m,J＝14.4,10.1Hz,2H),8.12–8.09(m,1H),7.84–7.71(m,5H),7.66–7.60(m,2H),7.38(t,J＝13.9,7.1Hz,5H),7.29(t,J＝8.9Hz,2H),6.26(s,2H),4.55–4.50(m,2H),1.36(t,J＝6.8Hz,3H).13C NMR(150MHz,DMSO)δ173.37,152.05,143.28,141.69,140.58,134.43,134.00,133.94,130.50,129.93,129.68,129.41,129.03,128.71,128.33,127.50,126.20,124.97,124.46,123.03,122.92,117.23,116.61,116.46,114.16,111.21,111.07,110.54,90.50,87.55,51.72,38.18,14.36.
Example 2: spectroscopic testing of fluorescent probe molecules
Dissolving the fluorescent probe Mito-PF of the invention in DMSO to prepare 2mM mother liquor, and taking 15 mul of the mother liquor in 3mL of solvents with different polarities respectively to obtain ultraviolet spectrums of the probe Mito-PF in different solvents (figure 1 a). The value of fluorescence intensity at 436nm gradually decreased with increasing polarity of the solvent, and there was little change in fluorescence intensity at 589nm, providing a ratio response (fig. 1 b). And fluorescence intensity (I)436nm/I589nm) And Δ f (FIG. 1c), indicating that Mito-PF can be used for dual channel detection of polarity in common solutions. To further demonstrate the polar response characteristic of probe Mito-PF, the absorption and fluorescence spectra of Mito-PF were measured at 25 ℃ in polar ranges with different ratios of water and tetrahydrofuran (FIGS. 2a, 2 b). When the polarity (Δ f) of the solution was decreased from 0.3103 (80% water) to 0.2556 (10% water), the fluorescence intensity of Mito-PF at 436nm increased by 3.6-fold, while the red emission at 589nm was barely responsive. The above results also show fluorescence intensity I436nm/I589nmThere is a good linear correlation with Δ f (fig. 2c), which indicates that Mito-PF is highly sensitive to solvent polarity. The literature reports that THF and methanol have nearly the same viscosity (0.53cP vs. 0.60cP) but different polarities (0.21 vs. 0.31). The fluorescence intensity at 436nm of Mito-PF showed a large difference, while the red emission at 589nm showed only a small change. As the viscosity increased from 0.60cP to about 100cP, there was little change in fluorescence intensity at 436nm and 589nm (FIG. 3), indicating that probe Mito-PF was insensitive to viscosity changes. In order to exclude the influence of pH, the pH stability was tested, and the pH value was in the range of 6-9 in different water/tetrahydrofuran systems, the change of the fluorescence intensity value of the Mito-PF was not large (FIG. 4), and the experimental result shows that the influence of the pH value on the probe Mito-PF is small, and the probe Mito-PF is not sensitive to the change of the pH value. The results prove that the probe Mito-PF can specifically detect the polarity without being interfered by the external environment.
Example 3: two-photon performance testing of fluorescent probe molecules
In different mixed solvents of water and tetrahydrofuran (the content of tetrahydrofuran is 90%, 50% and 20%, respectively), the effective two-photon absorption cross section is maximum at 720nm and gradually decreases from 87GM to 41GM along with the decrease of the tetrahydrofuran content (FIG. 5 a). Two-photon excited fluorescence intensity of Mito-PF in different solvents was observed as a square of the input power (300-800mw) (FIG. 5 b). Mito-PF demonstrated the ability to be used for two-photon confocal fluorescence imaging of intracellular polarity.
Example 4: cytotoxicity test
We performed cytotoxicity experiments using the MTT (5-dimethylthiazol-2-yl-2, 5-diphenyltetrazolium bromide) method. Mito-PF was added to live HeLa cells at various concentrations (0. mu.M, 10.0. mu.M, 20.0. mu.M, 30.0. mu.M) and tested after 24 hours, as shown in FIG. 6, which shows that Mito-PF is very little bio-toxic and can be used biologically.
Example 5: cell localization assay
To investigate the mitochondrial localization performance of Mito-PF, which is very necessary to investigate mitochondrial polarity, we used the commercial dye Mitotracker green (MTG) here to perform co-localization studies with Mito-PF in HeLa cells. The results show that the red channel (. lamda.) of Mito-PFem＝560-600nm，λex720nm) and MTG (λ)em＝500-540nm，λex488nm) and the Pearson co-localization coefficient of Mito-PF with MTG was calculated to be 0.95 (fig. 7). These results indicate that Mito-PF can be well localized in the mitochondria of living cells.
Example 6: Mito-PF stability experiments in mitochondria
In order to investigate the optical stability of the probe Mito-PF in HeLa cells, this is a very important experiment because during apoptosis the mitochondrial membrane potential is destroyed, which affects the localization performance of the probe, and thus it is relevant whether the apoptosis process can be monitored in real time. Reported articles indicate that 3-chlorophenylhydrazone (CCCP) is a membrane potential breaker that can break mitochondrial membrane potential. CCCP was added to one group of HeLa cells and not to the other group (FIG. 8). Fluorescence imaging was performed after 30 minutes. With and without CCCP, the green and red channels did not change in fluorescence imaging, and the mitochondrial green quotient staining and probe red overlap very well, demonstrating that probe Mito-PF does not rely on membrane potential for localization.
Example 7: confocal fluorescence imaging of apoptosis of cell mitochondria
Etoposide (etoposide) is a recognized apoptotic agent that is capable of causing apoptosis. From the literature, it is reported that apoptosis causes intracellular mitochondrial microenvironment changes, such as: polarity. Since mitochondrial polarity changes during apoptosis, polarity fluctuations can be detected to monitor apoptosis. Therefore, we performed some of the following experiments (fig. 9). Mito-PF (10. mu.M, 0.5 hr) was incubated in cells. Etoposide (50 μ M) was then added to the cells and imaging was performed every 10 min (a-E). Can be found in the blue channel (lambda) by imagingem420-460nm) gradually increased in fluorescence intensity. And in the red channel (lambda)em560-. From the analysis of the above data, we can clearly find that as the time of adding etoposide (etoposide) increases, i.e. apoptosis is deeper, the polarity of mitochondria in the cell gradually decreases (blue channel fluorescence increases, red channel fluorescence does not change). This suggests that apoptosis may cause a decrease in mitochondrial polarity within the cell. These data demonstrate that it is feasible to monitor apoptosis by intracellular mitochondrial polarity changes. The method provides a good method for monitoring apoptosis later and also provides a good idea for the application of fluorescent probes in biology later.
Claims (5)
1. A dual-channel two-photon fluorescence polarity probe is characterized in that the structural formula is as follows:
2. the preparation method of the dual-channel two-photon fluorescence polarity probe of claim 1, characterized by comprising the following steps:
step 1: adding 1.0g of a compound 9-ethyl-6-iodine-9H-carbazole-3-formaldehyde, 0.41g of 4-fluoroacetylene, 9.65mg of triphenylphosphine palladium dichloride, 5.24mg of cuprous iodide and 2ml of triethylamine into a reactor, and stirring for 12 hours at the temperature of 30 ℃ under anhydrous and oxygen-free conditions; cooling the mixture to room temperature, filtering the precipitate and concentrating to obtain a crude product; purifying the crude product by column chromatography to give intermediate 1;
the structural formula of the intermediate 1 is shown as follows:
step 2: mixing 0.28g of compound 3-benzyl-2-methylbenzothiazole bromide salt and 10.3 g of intermediate in anhydrous ethanol, refluxing the mixed solution under nitrogen until TLC shows that the raw materials are completely reacted, cooling the mixture to room temperature, and removing the solvent by a rotary evaporator; the resulting crude product was washed with saturated brine, then extracted with DCM and the solvent was evaporated under vacuum and the crude product was purified by column chromatography to give the desired product Mito-PF.
3. The method of claim 2, wherein:
in step 1, the crude product is purified by column chromatography using an eluent of petroleum: dichloromethane ═ 4: 1, v/v.
4. The method of claim 2, wherein:
in step 2, the eluent when the crude product was purified by column chromatography was dichloromethane: methanol 50: 1, v/v.
5. Use of the two-channel two-photon fluorescence polarity probe of claim 1, wherein:
the compound is used as a detection reagent for quantitatively detecting the polarity change in mitochondria in living cells for non-therapeutic or diagnostic purposes.
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