CN111925316A

CN111925316A - Two-photon fluorescence polarity probe based on 4-fluorophenylethynyl and preparation method and application thereof

Info

Publication number: CN111925316A
Application number: CN202010836769.2A
Authority: CN
Inventors: 李鹏; 温帅帅; 任其凤; 陈晓华; 孟祥明
Original assignee: Anhui University
Current assignee: Anhui University
Priority date: 2020-08-19
Filing date: 2020-08-19
Publication date: 2020-11-13
Anticipated expiration: 2040-08-19
Also published as: CN111925316B

Abstract

The invention discloses a two-photon fluorescence polar probe based on 4-fluorophenylethynyl, a preparation method and application thereof, wherein the two-photon fluorescence polar probe based on 4-fluorophenylethynyl has the following structure:

the two-photon fluorescence polar probe molecule based on the 4-fluorophenylethynyl shows specific response to polarity in a system with other interference factors. The cytotoxicity test shows that the probe has lower biological toxicity to cells, and the two-photon confocal fluorescence microscopic imaging experiment shows that the probe has good light stability in the cells and is suitable for two-photon fluorescence imaging and detection of polarity in the cells.

Description

Two-photon fluorescence polarity probe based on 4-fluorophenylethynyl and preparation method and application thereof

Technical Field

The invention relates to a two-photon fluorescence polarity probe based on 4-fluorophenylethynyl, a preparation method and application thereof, which are used for realizing the detection of polarity in cells by two-photon fluorescence imaging and have the advantages of specificity in selectivity, high sensitivity and low biological toxicity.

Background

Polarity is an important component of the biological microenvironment, and the polarity of cells is feedback for cellular manipulation and regulatory mechanisms. It is these mechanisms that induce and maintain the function or function of subcellular organelles that result in a variety of physiological and pathological activities. Different physiological and pathological activities correspond to different cellular states. When the cell state changes, the polarity also changes to some extent. Therefore, the detection of cell polarity is essential for monitoring different cell states. 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.

Apoptosis, one of programmed death processes, plays an important role in biological processes, and detection of apoptosis is of great significance 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. 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. At present, many fluorescent probes have been developed, but since probes for detecting polarity are still in progress, it is necessary to develop a polar probe having excellent performance. In addition, the polarity of the apoptosis process can be greatly changed, so that the apoptosis can be evaluated through the polarity, and a good idea is provided for researching the apoptosis.

Disclosure of Invention

The invention aims to provide a two-photon fluorescence polar probe based on 4-fluorophenylethynyl and a preparation method and application thereof, and aims to solve the technical problem that a two-photon fluorescence probe structure capable of specifically detecting polarity is obtained through molecular design so as to realize the detection of polarity change in the apoptosis process of cells through two-photon fluorescence imaging, and the two-photon fluorescence probe has the advantages of specificity in selectivity and high sensitivity, and cytotoxicity tests show that the fluorescence probe has almost no toxic effect on the cells.

The invention relates to a two-photon fluorescence polarity probe based on 4-fluorophenylethynyl, which is abbreviated as POLAR-QF and has the following structural formula:

the invention relates to a preparation method of a two-photon fluorescence polar probe based on 4-fluorophenylethynyl, which comprises the following steps:

step 1: adding a compound 9-ethyl-6-iodine-9H-carbazole-3-formaldehyde (6g, 17.2mmol) into a dry three-necked bottle, adding DMF (60mL) for dissolving, dripping 3-5 drops of triethylamine (2-3mL) as a catalyst, adding malononitrile (1.36g, 20.6mmol), stirring at normal temperature for 1 hour, observing the temperature color change in the bottle, reacting for 2 hours, and observing the reaction condition by using a point plate. After 2 hours of reaction, 1L of H was added₂O stirred for 10-20min and extracted repeatedly with EA (ethyl acetate) until no product spots were observed on the plates. The crude product was obtained by rotary dry extraction and purified by column chromatography (n-hexane: ethyl acetate: dichloroethane: 10: 1: 3) to give compound 1 (dark red solid, 5.9g, yield 86.4%).

Step 2: mixing compound 1(1.5g, 3.778mmol) and Pd₂(PPh₃)₂Cl₂(12.40mg 0.018mmol), CuI (6.98mg,0.036mol) as a catalyst in a Schlenk flask, repeatedly replacing the atmosphere inside the flask with a pump, introducing argon gas, allowing the reaction to proceed in a dry atmosphere, dissolving p-fluoroacetylene (0.544g, 4.53mmol) in THF (8mL), Et, and the like using a syringe₃Injecting N (2mL) solution into a reaction bottle, heating to 45 ℃ for reaction for 5 hours, observing the color deepening point plate in the bottle to observe that the raw materials are reacted completely, adding 800 mL of water after the reaction liquid is cooled to room temperature, stirring for 20-30min, repeatedly extracting for 3-4 times by using dichloroethane until no product point exists on the water layer plate, heating and concentrating to obtain 2.3g of crude product, and filtering by column chromatography (normal hexane: ethyl acetate: 7:1) to obtain dark redThe desired compound POLAR-QF (1.2g, 59.7% yield) is obtained as a colored solid.

The synthesis process of the two-photon fluorescent probe POLAR-QF based on 4-fluorophenylethynyl is as follows.

The invention discloses application of a two-photon fluorescence polar probe based on 4-fluorophenylethynyl, which aims at non-treatment or diagnosis and is used as a detection reagent for detecting polar change in living cells. The detection method comprises the following steps:

the probe POLAR-QF is dissolved in DMSO to prepare 2mM mother liquor, 15 mu L of the mother liquor is taken to be put in 3mL of solvents with different polarities respectively, and ultraviolet spectrograms of the probe POLAR-QF in different solvents are obtained. Which is described in_370max/I_370minThe fluorescence intensity gradually decreases with increasing polarity of the test system. And fluorescence intensity (I)_370max/I_370min) And Δ f, indicating that POLAR-QF can be used to detect the polarity of common solutions. To further verify the response characteristics of the probe POLAR-QF to polarity, the absorption and fluorescence spectra of POLAR-QF 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), a slight change was observed in the ultraviolet absorption spectrum, which is consistent with the results of measuring the polarity of different solvents. POLAR-QF at I when the polarity (Δ f) of the solution decreased from 0.3103 (80% water) to 0.2556 (10% water)_370max/I_370minThe fluorescence intensity of (A) was increased by 5.4 times. The above results also show fluorescence intensity I_370max/I_370minThere is a good linear correlation with Δ f, which indicates that POLAR-QF is highly sensitive to solvent polarity. At 90% tetrahydrofuran, the effective two-photon absorption cross-section of the probe appeared to be maximum at 780nm, 88 GM. We also explored the optical stability of the probe POLAR-QF in HeLa cells, which is a very important experiment because during apoptosis the intracellular environment is destroyed, which affects the photostability of the probe, and thus concerns whether the apoptosis can be monitored in real timeThe process of death. In addition, etoposide (etoposide) was tested to induce intracellular polarity changes during apoptosis in HeLa cells using probe POLAR-QF.

The probe molecule POLAR-QF shows specific response to polarity in a system coexisting 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 light stability in HeLa cells, is suitable for two-photon fluorescence imaging and in-situ detection of polarity in cells, and can be used for in-situ detection of the intracellular polarity change trend in the process of inducing apoptosis by etoposide (etoposide).

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)_370max/I_370min) 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)_370max/I_370min) And Δ f.

FIG. 3 is a graph of the pH stability of 10 μ M probe at different water/tetrahydrofuran ratios.

FIG. 4 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. 5 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. 6 is a confocal fluorescence imaging of 10 μ M probe incubation time in HeLa cells, investigating the photostability of probe Mito-PF in cells.

FIG. 7 is a confocal fluorescence image of 10 μ M probe induced apoptosis of HeLa cells at 50 μ M etoposide.

Detailed Description

The technical solution of the present invention is further illustrated by the following specific examples.

Example 1: synthesis of fluorescent probe molecule POLAR-QF

The compound 2- ((9-ethyl-6-iodo-9H-carbazol-3-yl) methylene) malononitrile (1.5g, 3.778mmol), Pd₂(PPh₃)₂Cl₂(12.40mg, 0.018mmol), CuI (6.98mg,0.036mol) as a catalyst in a Schlenk flask, repeatedly replacing the atmosphere inside the flask with a pump, introducing argon gas, allowing the reaction to proceed in a dry atmosphere, dissolving p-fluoroacetylene (0.544g, 4.53mmol) in THF (8ml), Et, and the like using a syringe₃Injecting an N (2ml) solution into a reaction bottle, heating to 45 ℃ for reaction for 5 hours, observing the color deepening in the bottle, observing the reaction of raw materials, cooling the reaction solution to room temperature, adding water, stirring, repeatedly extracting with dichloroethane until no product point exists on a water layer and a plate, heating and concentrating to obtain a crude product of 2.3g, and filtering by column chromatography (normal hexane: ethyl acetate: 7:1) to obtain a dark red solid target compound POLAR-QF (1.2g, yield of 59.7%).¹HNMR(400MHz,DMSO-d₆)8.74(d,J＝1.8Hz,1H),8.48(s,1H),8.32(m,1H),8.14(m,1H),7.86(d,J＝8.8Hz,1H),7.76(m,1H),7.69(m,1H),7.62(m,2H),7.25(t,J＝8.9Hz,2H),4.50(q,J＝7.1Hz,2H),1.32(t,J＝7.1Hz,3H).ESI-MS m/z:{[M+H]⁺}calcd,389.1328；found,389.1247.

Example 2: spectroscopic testing of fluorescent probe molecules

The probe POLAR-QF of the invention is dissolved in DMSO to prepare 2mM mother liquor, 15 mul of the mother liquor is respectively taken in 3mL of solvents with different polarities, and ultraviolet spectrums of the probe POLAR-QF in different solvents are obtained (figure 1 a). Which is described in_370max/I_370minThe fluorescence intensity gradually decreased with increasing polarity of the test system (FIG. 1 b). And fluorescence intensity (I)_370max/I_370min) And Δ f (FIG. 1c), indicating that POLAR-QF can be used for ratio-detecting the polarity of common solutions. To further verify the response characteristics of the probe POLAR-QF to polarity, the absorption and fluorescence spectra of POLAR-QF were measured in a range of polarities with different ratios of water and tetrahydrofuran (FIG. 2). When the polarity of the solvent is increased from 10% water (delta f is approximately equal to 0.2556) to 80% water (delta f is approximately equal to 0.3103)In the UV absorption spectrum, a slight change was observed, which was consistent with the results obtained with the polarity measurements of different solvents (FIG. 2 a). POLAR-QF at I when the polarity (Δ f) of the solution decreased from 0.3103 (80% water) to 0.2556 (10% water)_370max/I_370minThe fluorescence intensity increased by a factor of 5.4 (FIG. 2 b). The above results also show fluorescence intensity I_370max/I_370minThere is a good linear correlation with Δ f (FIG. 2c), indicating that POLAR-QF is highly sensitive to solvent polarity. To exclude the influence of pH, the pH stability was tested, and in the water/tetrahydrofuran system, the fluorescence intensity values of the probe POLAR-QF at 370nm hardly changed at different pH values (pH 6-9) at the same water content (water content of 80%), whereas the fluorescence intensity values of the emission peak at 370nm varied greatly at the same pH value (pH 7.0) at different water contents (50%, 80%). (FIG. 3) the results of this experiment show that the pH value has a small influence on the probe POLAR-QF. The probe POLAR-QF was also shown laterally to respond specifically to polarity in different pH environments.

Example 3: two-photon performance testing of fluorescent probe molecules

POLAR-QF showed maximum effective two-photon absorption cross-section at 780nm in various mixed solvents of water and tetrahydrofuran (tetrahydrofuran content 90%, 50% and 20%, respectively) and gradually decreased from 88GM to 32GM as the tetrahydrofuran content decreased (FIG. 4 a). Two-photon excitation fluorescence intensity of POLAR-QF in different solvents was observed as squared with input power (300-800mw) (FIG. 4 b). POLAR-QF 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. POLAR-QF 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 after 24 hours, the results were tested, as shown in FIG. 5, and it was revealed that POLAR-QF had little biological toxicity and could be applied to a living organism.

Example 5: cell photostability test

Study of photostability of POLAR-QF in cells, whichThe polarity change in the apoptosis process is very necessary to be researched, and the apoptosis needs a certain time, which affects the light stability of the probe, so that the method is related to whether the apoptosis process can be monitored in real time for a long time. Our results show that the blue channel (. lamda.) of POLAR-QF_em420-460nm) was observed to generate fluorescence 10min after the addition, and the fluorescence intensity stabilized without much change by 40min (fig. 6). These results indicate that POLAR-QF can image well in living cells for long periods of time.

Example 6: confocal fluorescence imaging of apoptosis

Etoposide (etoposide) is a recognized apoptotic agent that is capable of causing apoptosis. From the literature, it is reported that apoptosis causes changes in the intracellular microenvironment, such as: polarity. Since the polarity changes during apoptosis, polarity fluctuations can be detected to assess apoptosis. Therefore, we performed the following experiment (fig. 7). POLAR-QF (10. mu.M, 0.5 hour) was incubated intracellularly. Etoposide (50 μ M) was then added to the cells for imaging. The fluorescence intensity enhancement in the blue channel can be found by imaging. This is consistent with the fluorescence data from in vitro tests. From the analysis of the above data, we can clearly find that with the addition of etoposide, i.e. the deep apoptosis, the polarity inside the cell decreases (blue channel fluorescence increases). This suggests that etoposide-induced apoptosis will cause a decrease in intracellular polarity. These data demonstrate that it is feasible to assess apoptosis by intracellular 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

1. A two-photon fluorescence polarity probe based on 4-fluoro phenylethynyl is characterized in that the structural formula is as follows:

2. the preparation method of the two-photon fluorescence polar probe based on 4-fluorophenylethynyl group in claim 1, which is characterized by comprising the following steps:

step 1: adding a compound 9-ethyl-6-iodine-9H-carbazole-3-formaldehyde into a dry three-necked bottle, adding DMF (dimethyl formamide) for dissolving, dropwise adding triethylamine serving as a catalyst, adding malononitrile, stirring at normal temperature, observing temperature and color change in the bottle, and spotting a plate to observe the reaction condition; extracting after the reaction is finished until no product spot can be observed on a water layer spot plate, performing spin-drying extraction to obtain a crude product, and purifying the crude product by using a column chromatography to obtain a compound 1;

step 2: compound 1, Pd₂(PPh₃)₂Cl₂And CuI as catalyst, placing into a Slek bottle, reacting under dry argon atmosphere, and dissolving p-fluoroacetylene in THF and Et with a syringe₃Injecting the N mixed solution into a reaction bottle, heating to 45 ℃ for reaction for 5 hours, observing the reaction of the raw materials by a color deepening spot plate in the bottle, adding water for stirring after the reaction liquid is cooled to room temperature, extracting by dichloroethane until no product spot exists on a water layer spot plate, heating and concentrating to obtain a crude product, and purifying by column chromatography to obtain a dark red solid target compound POLAR-QF.

3. The method of claim 2, wherein:

in step 1, the eluent used in purifying the crude product by column chromatography was n-hexane to ethyl acetate: dichloroethane 10: 1: 3, v/v.

4. The method of claim 2, wherein:

in step 2, the eluent used in purifying the crude product by column chromatography was n-hexane: ethyl acetate 7:1, v/v.

5. Use of a two-photon fluorescent polar probe based on 4-fluorophenylethynyl group of claim 1, wherein:

for non-therapeutic or diagnostic purposes, the compounds are used as detection reagents for detecting changes in polarity in living cells.