CN111253935A - Two-photon fluorescent probe for detecting polarity and viscosity through two channels and preparation method and application thereof - Google Patents

Abstract

The invention discloses a dual-channel two-photon fluorescent probe for detecting polarity and viscosity, a preparation method and application thereof, which can simultaneously monitor the real-time changes of polarity and viscosity in cell mitochondria in two different channels, and its structural formula is as follows:

The two-photon fluorescent probe molecule of the present invention can utilize two different fluorescence bands to simultaneously perform fluorescence response to polarity (short wavelength, 410 nm) and viscosity (long wavelength, 580 nm). In addition, the cytotoxicity test shows that the probe has almost no toxic side effects on cells, and the two-photon confocal fluorescence microscopy imaging experiment shows that the probe has good permeability to HeLa cells and can effectively locate mitochondria in cells (the localization coefficient is 0.97). , for two-channel two-photon fluorescence imaging and detection of polarity and viscosity within cellular mitochondria.

Description

A two-photon fluorescent probe for detecting polarity and viscosity with two channels and its preparation method and use

technical field

The present invention relates to a two-photon fluorescent probe capable of detecting polarity and viscosity with two channels, and a preparation method and application thereof, so as to realize two-channel two-photon imaging and simultaneously detect the changes of polarity and viscosity in cell mitochondria. It has the advantages of excellent two-photon absorption performance, high membrane permeability, low cytotoxicity, high photostability and biocompatibility.

Background technique

Cellular and subcellular organelle viscosity is an important microenvironmental parameter that exerts biological functions by affecting the interaction and transmission of biomolecules and chemical signals within living cells. Abnormal changes in cell viscosity are closely related to diabetes, infarction, hypertension and other diseases. The polarity of a cell is a feedback of a series of complex mechanisms that establish and maintain the function of specific domains. Many cellular processes involving spatial arrangement and protein composition, such as differentiation, local membrane growth, activation of immune responses, directed cell migration, and vectorial transport of molecules across cellular layers, can lead to changes in polarity and development. Therefore, abnormal changes in polarity are closely related to disorders and diseases.

Mitochondria, as the energy source of cells, are important organelles in cells and play a key role in cellular metabolism, including providing metabolic energy through oxidative phosphorylation, generating reactive oxygen species (ROS), regulating Ca ²⁺ homeostasis, and triggering cellular Apoptotic cell signaling platform. Notably, the unique functions of mitochondria are related to maintaining the homeostasis of their parameters and microenvironment (eg pH, viscosity, polarity, temperature, etc.).

Small molecule fluorescent probes have been widely used in fluorescence imaging due to their high sensitivity, real-time detection, and rapid non-destructive analysis. Measuring the subcellular activity of living cells with fluorescent probes is a powerful means to gather information about specific subcellular microenvironments. At present, there are many fluorescent probes for single detection of mitochondrial polarity or viscosity, but two-photon fluorescent probes that can simultaneously detect mitochondrial polarity and viscosity have not been reported.

Apoptosis, the most common form of programmed cell death, is the key to regulating cellular homeostasis and maintaining tissue homeostasis. Defective apoptosis is the main cause of various pathological processes such as autoimmune diseases and cancer. Therefore, an effective therapeutic strategy for the treatment of these diseases, especially cancer, is to selectively induce cell death by modulating apoptotic pathways. The detection and monitoring of apoptosis is of great importance not only in many cell biology and clinical studies, but also in the therapeutic evaluation of apoptosis-targeted therapy. From the existing literature reports, apoptosis can cause changes in the intracellular mitochondrial microenvironment such as polarity, viscosity and pH. However, most fluorescent probes can only detect changes in one of the microenvironmental parameters, which will hinder our deeper understanding of the dynamic connection between the microenvironments during apoptosis. Therefore, it is very urgent to develop two-photon fluorescent probes with excellent dual-channel detection of polarity and viscosity, and to understand mitochondrial-related cellular events by monitoring the changes of mitochondrial viscosity and polarity.

SUMMARY OF THE INVENTION

The invention aims to provide a two-photon fluorescent probe capable of detecting polarity and viscosity with two channels, a preparation method and application thereof, and the technical problem to be solved is to obtain a suitable fluorescent probe structure through molecular design, which has two-photon absorption Excellent performance, low cytotoxicity, good membrane permeability and biocompatibility, high selectivity and photostability, to achieve dual-channel two-photon fluorescence imaging to simultaneously detect real-time changes in polarity and viscosity during apoptosis and dual-color imaging .

The two-photon fluorescent probe of the present invention, abbreviated as Mito-PV, takes carbazole as the parent body, and its structural formula is as follows:

The preparation method of the two-photon fluorescent probe of the present invention comprises the following steps:

Step 1: In a nitrogen atmosphere, 1-iodohexane (1.56 g, 7.38 mmol) was added to 2-methylbenzothiazole (1.0 g, 6.71 mmol) under stirring at 60 °C, and the crude product was obtained after cooling to room temperature; The crude product was purified by column chromatography (dichloromethane:methanol = 4:1 as eluent) to give 1.50 g (62%) of intermediate 1-3-hexyl-2-methylbenzothiazole iodide salt ;

Step 2: Compound 9-ethyl-6-iodo-9H-carbazole-3-carbaldehyde (1.0 g, 2.86 mmol), 4-fluorophenylacetylene (0.41 g, 3.44 mmol), triphenylphosphonium dichloride Palladium (9.65 mg, 0.014 mmol), cuprous iodide (5.24 mg, 0.027 mmol) and triethylamine (2 ml) were stirred at 30 °C under anhydrous and oxygen-free conditions for 12 hours; the mixture was cooled to room temperature, the precipitate was filtered and Concentration gave crude product; the crude material was purified by column chromatography (petroleum/dichloromethane = 4:1 as eluent) to give intermediate 3 - 3-(4-fluorophenylethynyl)-6-aldehyde -N-Ethylcarbazole, 0.80 g, 82% yield.

Step 3: Intermediate 1 (0.32 g, 0.9 mmol) and Intermediate 3 (0.3 g, 0.9 mmol) were mixed in 50 mL of absolute ethanol, refluxed under nitrogen for 12 hours, then cooled and removed by rotary evaporation solvent; the obtained residue was washed with 30 mL of saturated brine, then extracted with dichloromethane (3×50 mL), and the solvent was evaporated under vacuum; the residue was purified by column chromatography (dichloromethane:methanol=50:1 as eluent) to give 0.36 g (59%) of Mito-PV.

The synthetic route of the two-photon fluorescent probe Mito-PV of the present invention is as follows:

The application of the two-photon fluorescent probe of the present invention is to be used as a detection reagent in the dual-channel detection of the polarity and viscosity of mitochondria in cells. The detection method is as follows:

The two-photon fluorescent probe of the present invention was dissolved in DMSO to prepare a 2mM mother solution, and 15μL of each mother solution was taken into 3mL of solvents of different polarities to obtain the UV spectra of the probe Mito-PV in different solvents. Under the excitation of 360nm wavelength, its fluorescence intensity at 410nm gradually weakened with the increase of the polarity of the test system. At the same time, it was found that the probe Mito-PV only had a strong fluorescence emission peak at 580 nm in the glycerol solvent with high viscosity. This shows that Mito-PV can be used to detect the polarity and viscosity of common solutions. To further verify the dual-response properties of the probe Mito-PV to polarity and viscosity, the absorption and fluorescence spectra of Mito-PV were firstly measured in mixed solvents with different ratios of water and 1,4-dioxane. When the polarity of the solvent increases from 10% water (polarity parameter Δf ≈ 0.229) to 70% water (Δf ≈ 0.304), only a small change can be seen in the absorption maximum, which is different from the measured solvent. Polarity results are consistent. Conversely, when the polarity (Δf) of the solution was decreased from 0.304 (70% water) to 0.229 (10% water), the fluorescence intensity of Mito-PV at 410 nm increased 11-fold. Moreover, the fluorescence intensity of Mito-PV at 410 nm has a good linear relationship with Δf, confirming that Mito-PV has a good fluorescence response to polarity. Next, the fluorescence emission spectra of Mito-PV were measured in mixed solvents of methanol and glycerol with different ratios. With the gradual increase of glycerol content in the mixed system (viscosity increased from 1.0 to 300 cp), the fluorescence intensity of Mito-PV at 580 nm increased 15-fold, and there was a good linear relationship between log I _{580 nm} and log η. This indicates that Mito-PV also has a good fluorescence response to the viscosity of the solvent. In addition, the changes of polarity and viscosity of etoposide-induced apoptosis in HeLa cells were tested using this probe Mito-PV. With the apoptosis of cells, the short-wave blue channel fluorescence increased, indicating that the polarity decreased. Small, long-wavelength red channel fluorescence enhancement, indicating increased viscosity.

The two-photon fluorescent probe of the present invention can simultaneously respond to changes in polarity and viscosity with two-color fluorescence. Cytotoxicity experiments also showed that the probe had low cytotoxicity. Two-photon confocal fluorescence microscopy imaging experiments showed that the probe had good permeability to HeLa cells and could effectively locate mitochondria in cells (localization coefficients were 0.97, respectively). It is suitable for two-photon fluorescence imaging of polarity and viscosity in cell mitochondria, and can detect real-time changes in polarity and viscosity during etoposide-induced apoptosis and dual-color imaging.

Description of drawings

Fig. 1 is the (left) ultraviolet absorption spectrum of 10 μM probe in different polar organic solvents; (right) fluorescence emission spectrum;

Figure 2 is the (left) UV absorption spectrum of 10 μM probe in mixed solvents of water/1,4-dioxane with different volume ratios; (right) fluorescence emission spectrum, inset: fluorescence intensity between I _410nm and Δf The linear relationship diagram of ;

Figure 3 is the (left) fluorescence emission spectrum of 10 μM probe in different volume ratios of methanol/glycerol mixed solvent; (right) the linear relationship between the fluorescence intensity I _580nm and Δf;

Figure 4 is the cross-sectional view of the effective two-photon absorption of the 0.1mM probe in mixed solvents (left) mixed solvents with different volume ratios of water/1,4-dioxane; (right) mixed solvents with different volume ratios of methanol/glycerol ;

Figure 5 is a graph of the viability of HeLa cells under the action of probe molecules at different concentrations (0 µM, 10 µM, 20 µM, 30 µM).

Figure 6 is a confocal fluorescence imaging image of 10 μM probe and 1 μM Mitotracker Deep red (MTDR) co-stained at the same time to verify mitochondrial localization in HeLa cells.

Fig. 7 is a confocal fluorescence imaging image of apoptosis of HeLa cells induced by 10 μM probe in 50 μM etoposide.

Detailed ways

The present invention will be further illustrated by the following examples.

Example 1: Synthesis of fluorescent probe molecule Mito-PV

Combine 3-hexyl-2-methylbenzothiazole iodide salt (0.32 g, 0.9 mmol) and 3-(4-fluorophenethynyl)-6-aldol-N-ethylcarbazole (0.3 g, 0.9 mmol) in 50 mL of anhydrous ethanol; the mixture was refluxed under nitrogen for 12 hours, then cooled, and the solvent was removed by rotary evaporation; the obtained residue was washed with 30 mL of saturated brine, then passed through dichloromethane (3×50 mL) ), and the solvent was evaporated under vacuum, and the residue was purified by column chromatography (dichloromethane:methanol=50:1 as eluent) to give 0.36 g (59%) of Mito-PV.

¹ H NMR (400 MHz, DMSO-d ₆ , ppm): δ 9.00 (s, 1H), 8.43 (dd, J=21.2, 13.9 Hz, 3H), 8.27 (t, J=9.0 Hz, 2H), 8.03 (d, J=15.6Hz, 1H), 7.87 (dd, J=8.1, 4.9Hz, 2H), 7.81-7.72 (m, 3H), 7.66 (dd, J=8.1, 5.7Hz, 2H), 7.31 ( t,J＝8.7Hz,2H),4.97(t,J＝7.0Hz,2H),4.56(m,J＝6.8Hz,2H), 1.88(dd,J＝15.0,7.7Hz,2H),1.46( dd, J=14.9, 7.2Hz, 2H), 1.40-1.24 (m, 7H), 0.84 (t, J=7.0 Hz, 3H). ¹³ C NMR (100MHz, DMSO, ppm): δ171.75, 160.61, 150.62, 142.64,141.22,140.07, 133.49,133.41,129.95,129.34,128.79,128.12,127.78,125.69,124.29,123.98,123.81,122.48, 122.41,119.20,116.55,116.14,115.92,113.58,110.64,110.46,110.08,90.04, 87.01, 48.58, 37.67, 30.76, 28.65, 25.42, 21.93, 13.85, 13.79.

Example 2: Spectral Test of Fluorescent Probe Molecules

The two-photon fluorescent probe of the present invention was dissolved in DMSO to obtain a 2mM mother solution, and 15 μL of each mother solution was taken in 3mL of solvents of different polarities to obtain the UV absorption spectra of the probe Mito-PV in different solvents (Figure 1 left). picture). Under the excitation of 360nm wavelength, its fluorescence intensity at 410nm gradually weakened with the increase of the polarity of the test system. Probe Mito-PV only has a strong fluorescence peak at 580nm in glycerol solvent with higher viscosity. This shows that Mito-PV can be used to detect the polarity and viscosity of common solutions (Figure 1 right). To further demonstrate the dual-response properties of the polarity and viscosity of the probe Mito-PV, the absorption and fluorescence spectra of Mito-PV were measured in mixed solvents with different ratios of water and 1,4-dioxane at 25 °C. (Figure 2 left, Figure 2 right). The fluorescence intensity of Mito-PV at 410 nm increased 11-fold when the polarity (Δf) of the solution decreased from 0.304 (70% water) to 0.229 (10% water). Moreover, the fluorescence intensity of Mito-PV at 410 nm has a good linear relationship with Δf (the inset of the right panel of Fig. 2). This indicates that Mito-PV can respond well to the change of solvent polarity. Next, the fluorescence spectra of Mito-PV were measured in mixed solvents of methanol and glycerol with different ratios (Fig. 2). With the gradual increase of glycerol content in the mixed system (viscosity increased from 1.0 to 300cp), the fluorescence intensity of Mito-PV at 580nm increased by 15 times (Fig. 2 left panel), and there was a good relationship between log I _580nm and log η. The linear relationship (right panel of Figure 2). This indicates that Mito-PV has a good fluorescence response to the viscosity of the solvent. The above results demonstrate that Mito-PV can utilize two distinct fluorescence bands for detecting the polarity and viscosity of common solutions.

Example 3: Two-photon performance test of fluorescent probe molecules

First, the effective two-photon absorption cross-sections of Mito-PV in different polar mixed solvents of water and 1,4-dioxane (with water contents of 10%, 40% and 70%, respectively) were tested. The effective two-photon absorption cross-section has a maximum at 720nm and gradually decreased from 88GM to 29GM with the increase of water content (left panel of Fig. 3). In different mixed solvents of methanol and glycerol (the contents of glycerol are 99%, 80% and 60%, respectively), the effective two-photon absorption cross-section of Mito-PF appeared the maximum at 840nm and gradually decreased from 96GM to 48GM with the decrease of glycerol content (Figure 3 right). The above demonstrates that Mito-PV is capable of two-photon bioimaging of intracellular polarity and viscosity.

Example 4: Cytotoxicity test

We performed cytotoxicity experiments using the MTT (5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) method. Mito-PV was added to live HeLa cells at various concentrations (0 μM, 10.0 μM, 20.0 μM, 30.0 μM) and tested after 24 hours. The results are shown in Figure 4. When the concentration of the probe reaches 30 μM, the cell viability still has About 90%, it shows that the fluorescent probe molecule of the present invention has no obvious toxic effect on cells and can be used for biological applications.

Example 5: Cell Localization Assay

To investigate the mitochondrial localization properties of Mito-V, the commercial dye Mitotracker Deepred (MTDR) we used here was co-localized with Mito-PV in HeLa cells. The results show that the fluorescence images of the yellow channel (λ _em =500-600 nm, λ _ex =720 nm) of Mito-PV and MTDR (λ _em =655-755 nm, λ _ex =635 nm) overlap well, and the Pearson of Mito-PV and MTDR The colocalization coefficient was calculated to be 0.97 (Figure 5). These results suggest that Mito-PF can localize well in the mitochondria of living cells.

Example 6: Apoptosis confocal fluorescence imaging

Etoposide can induce apoptosis and is a recognized apoptotic agent. From the existing literature reports, apoptosis can cause changes in the intracellular mitochondrial microenvironment such as polarity, viscosity and pH. Therefore, we attempted to use Mito-PV to monitor real-time changes in mitochondrial polarity and viscosity during etoposide-induced apoptosis. 10 μM Mito-PV was added to the cells and incubated for 0.5 h. Etoposide (50 μM) was then added to the cells and imaging was performed every 15 minutes. Through imaging, it can be found that the fluorescence intensity in the blue channel (Blue Channel, λ _em =420-460 nm) gradually increases, indicating that the polarity is gradually decreasing. In the red channel (Red Channel, λ _em =500-600 nm, λ _ex =720 nm), the fluorescence intensity is also gradually increased, indicating that the viscosity is gradually increasing. Through the analysis of the above data, we can clearly find that as the time of adding etoposide increases, the apoptosis is deeper, and the polarity of mitochondria in the cell gradually decreases and the viscosity increases gradually. This suggests that etoposide-induced apoptosis can lead to a decrease in mitochondrial polarity and an increase in intracellular viscosity.

Claims (5)

1. A two-photon fluorescent probe for detecting polarity and viscosity by two channels is abbreviated as Mito-PV, and is characterized in that the structural formula is as follows:

2. a method for preparing the two-photon fluorescent probe according to claim 1, which comprises the steps of:

step 1: adding 1-iodohexane into 2-methylbenzothiazole under stirring at 60 ℃ in a nitrogen atmosphere, and cooling to room temperature to obtain a crude product; purifying the crude product by column chromatography to obtain intermediate 1-3-hexyl-2-methylbenzothiazole iodide salt;

step 2: stirring and reacting a compound 9-ethyl-6-iodine-9H-carbazole-3-formaldehyde, 4-fluoroacetylene, triphenylphosphine palladium dichloride, cuprous iodide and triethylamine for 12 hours at the temperature of 30 ℃ under anhydrous and oxygen-free conditions; cooling the mixture to room temperature, precipitating, filtering and concentrating to obtain a crude product; purifying the crude material by column chromatography to obtain an intermediate 3- (4-fluorophenylethynyl) -6-formyl-N-ethylcarbazole;

and step 3: mixing the intermediate 1 and the intermediate 3 in absolute ethyl alcohol, carrying out reflux reaction for 12 hours under the protection of nitrogen, then cooling, and removing the solvent through rotary evaporation; the residue obtained was washed with saturated brine, then extracted with dichloromethane and the solvent was evaporated under vacuum; the residue was purified by column chromatography to give the desired product Mito-PV.

3. The method of claim 2, wherein:

in step 1, the eluent for column chromatography purification is dichloromethane: methanol 4: 1, v/v.

4. The method of claim 2, wherein:

in step 3, the eluent for column chromatography purification is dichloromethane: methanol 50: 1, v/v.

5. Use of the two-photon fluorescent probe according to claim 1, wherein: the two-photon fluorescent probe is used as a detection reagent when detecting the polarity and viscosity in mitochondria in cells.

Images