CN110724524B

CN110724524B - Fluorescent probe for detecting polarity in cells and preparation method and application thereof

Info

Publication number: CN110724524B
Application number: CN201911211678.3A
Authority: CN
Inventors: 林伟英; 郭丁一; 刘闯; 田明刚
Original assignee: University of Jinan
Current assignee: University of Jinan
Priority date: 2019-12-02
Filing date: 2019-12-02
Publication date: 2021-02-19
Anticipated expiration: 2039-12-02
Also published as: CN110724524A

Abstract

The invention provides a fluorescent probe for detecting intracellular polarity by targeting mitochondria, which comprises the following components in percentage by weight:

. The probe can be used for detecting the polarity in cells. The polarity-sensitive PTT sensitizer prepared by the invention is used for researching the change of polarity in cells in the process of photo-induced cell death. The probe targets mitochondria in living cells. After a period of laser irradiation, the cell viability of the probe-preincubated Hepg2 live cells was significantly reduced. The probe successfully revealed down-regulation of mitochondrial polarity during light-induced cell death.

Description

Fluorescent probe for detecting polarity in cells and preparation method and application thereof

Technical Field

The invention belongs to the field of organic small molecule fluorescent probes, and particularly relates to a fluorescent probe for detecting polarity change in cells by targeting mitochondria and application thereof.

Background

Polarity has been regarded as an extremely important chemical parameter in the fields of chemistry and chemical engineering, and since its appearance, it has attracted the attention of most scholars, and there has been intensive research on the influence of polarity parameters in chemical reactions. With the continuing understanding of polarity by scholars, it is found that various vital activities in the biological microenvironment are also affected by changes in polarity. The metabolism of cells is always accompanied by the change of internal environment polarity, which is necessary for the processes of cell division, directed growth and movement, and the like, so that the morphology and functions of cells are directionally differentiated. If the polarity in the cell is abnormally changed, certain physiological or pathological phenomena can appear, so that the detection of the polarity change in the cell is important for monitoring different cell states, and can help people to deeply understand physiological processes and pathological processes.

Many key issues facing cell biology relate to the location and concentration of chemical substances, from molecular signaling to metabolites to exogenous toxins. Fluorescence molecular imaging is an indispensable tool in the fields of biology and life sciences, and fluorescence probes are very sensitive to specific analytes, so that the understanding of biological systems is changed. The fluorescent probe has the advantages of high sensitivity, strong selectivity, high response speed, simple operation and the like, and becomes an important tool for monitoring the microenvironment in the cell.

Mitochondria play an important role in maintaining a variety of physiological functions of the human body as an important organelle in eukaryotic cells. The essential role of mitochondria is to generate energy through oxidative phosphorylation and lipid oxidation. In addition to its well-known function as a power plant, mitochondria are also considered to be a neural center that controls certain aspects of the cell in some sense. Mitochondrial polarity is an important characteristic of organelles and has a large influence on cellular activities. Mitochondrial polarity also affects many processes in the cell's vital activities, such as protein trafficking and interactions, enzyme activity and stability, maintenance of cell function and cellular homeostasis, among others. Therefore, it is very important to accurately monitor the polarity change of mitochondria.

Disclosure of Invention

Aiming at the problem that physiological change research in the photoinduced cell death process is lacked at present, the invention provides the fluorescent probe for quickly detecting the polarity in the cell by targeting mitochondria, which has the advantages of high response speed and strong anti-interference capability.

Another object of the present invention is to provide an application of the above fluorescent probe in detecting polarity in biological cells.

In order to achieve the purpose, the invention adopts the following technical scheme.

A fluorescent probe for detecting intracellular polarity by targeting mitochondria has a chemical name of (4- ((9- (diethylamino) -5-oxo-5H-benzo [ a ] benzoxazine-2-yl) oxy) butyl) triphenylphosphine and a chemical structural formula shown in a formula (I):

formula (I).

The preparation method of the fluorescent probe comprises the following steps:

(1) and (3) heating the compound a and the compound b in DMF under the protection of nitrogen to react, and separating and purifying to obtain a compound c:

；

(2) under the protection of nitrogen, reacting the compound c with the compound d in potassium carbonate and DMF solution, separating and purifying to obtain a compound e:

；

(3) and heating the compound e and the compound f in acetonitrile under the protection of nitrogen for reaction, separating and purifying to obtain a compound g, namely the fluorescent probe:

。

in the step (1), the mass ratio of the compound a to the compound b is 1: 1;

in the step (1), the reaction temperature is 140 ℃, and the reaction time is 5 hours;

in the step (1), the separation and purification step comprises: and (3) extracting, separating and combining the reacted system with organic phases, and mixing the organic phases with petroleum ether: dichloromethane (V/V) =1:1 ratio developing solvent to remove the foregoing impurities, and then dichloromethane: methanol (V/V) =100:1 ratio developing solvent under normal pressure to obtain red product.

In the step (2), the mass ratio of the compound c to the compound d is 1: 5;

in the step (2), the reaction time is 12 h;

in the step (2), the separation and purification step comprises: adding dichloromethane and water into the reacted system, collecting an organic phase, distilling under reduced pressure to remove the solvent, and then carrying out column chromatography; the column chromatography eluent is dichloromethane: methanol (V/V) =130: 1.

In the step (3), the mass ratio of the compound e to the compound f is 1: 1;

in the step (3), the reaction temperature is 90 ℃, and the reaction time is 12 hours;

in the step (3), the separation and purification step comprises: distilling the reacted system under reduced pressure to remove the solvent, and then carrying out column chromatography; the column chromatography eluent is dichloromethane: methanol (V/V) =20: 1.

An application of the fluorescent probe in detecting the polarity in cells.

An application of the fluorescent probe in preparing a reagent for detecting polarity in cells.

The mechanism of the invention is as follows:

the fluorescent probe is constructed by connecting a polarization sensitive nile red and a mitochondrion targeted triphenylphosphine unit. According to the probe, positive charge groups such as triphenylphosphine and the like are introduced into a fluorophore, and as a mitochondrial membrane has high negative charge, lipophilic cation triphenylphosphine is preferentially enriched in mitochondria after entering cells to target the mitochondria; nile red, which is sensitive to polarity, is highly sensitive to environmental polarity. Therefore, the probe can detect the polarity change of the biological membrane, and experiments prove that the probe can be used for detecting the polarity change of the biological membrane. Mitochondrial polarity decline caused by cholesterol treatment was observed with the probe. Particularly, due to its high absorption rate and low quantum rate, a large amount of heat is generated under laser irradiation, thereby reducing the viability of cells. The probe successfully revealed that mitochondrial polarity was down-regulated during light-induced cell death. It is believed that probes can be a powerful tool in detecting mitochondrial polarity and PTT processes.

The invention has the following advantages:

the invention prepares the polarity-sensitive PTT sensitizer for the first time and is used for researching the change of polarity in cells in the process of photo-induced cell death. The probe targets mitochondria in living cells. After a period of laser irradiation, the cell viability of the probe-preincubated Hepg2 live cells was significantly reduced. The probe successfully revealed down-regulation of mitochondrial polarity during light-induced cell death.

Drawings

FIG. 1 shows a fluorescent probe NRTP¹H NMR spectrum;

FIG. 2 shows a fluorescent probe NRTP¹³A C NMR spectrum;

figure 3 is an imaging test of fluorescent probe NRTP in macrophages of different polarity, λ ex =561 nm;

FIG. 4 is a co-localization imaging application of fluorescent probe NRTP and mitochondrial crimson probe in living cells, wherein a-c is experimental living cell image Hepg2 cell incubation 5 μ M NRTP and d-f is 5 μ M NRTP and 200 nM MTDR co-incubation for 30 minutes. (a) Bright field image, (b, d) λ ex =561 nm, red channel, (e) λ ex = 647 nm, deep red channel, (c, f) merging image.

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 6-hydroxy nile red (c)

A50 mL pear-shaped flask was charged with reactant a (400 mg,2 mmol) and reactant b (350 mg, 2.1 mmol) dissolved in 5 mL of N-Dimethylformamide (DMF) in a dark brown-black solution under N₂Under protection, the reaction is carried out for 5h at 140 ℃, a new substance with red fluorescence is generated by TLC detection, and the combined organic phases are extracted and separated. Using petroleum ether: dichloromethane (V/V) =1:1 ratio developing solvent to remove the foregoing impurities, and then dichloromethane: methanol (V/V) =100:1 ratio developing solvent under normal pressure to obtain red product (c).

(2) Synthesis of 2- (4-bromobutoxy) -9- (diethylamino) -5H-benzo [ a ] benzoxazin-5-one (e)

In a 50 mL pear-shaped flask, compound c (100 mg, 0.3 mmol), compound d (326 mg, 1.5 mmol) and potassium carbonate (208 mg, 1.5 mmol) were added and dissolved in 5 mL DMF, and the solution was bright red in N₂And reacting at room temperature for 12 h under protection. By TLC detection, a red fluorescent spot of slightly less polarity was generated. Dichloromethane and water were added to extract the product and the organic phase was collected and the solvent was distilled off under reduced pressure. The polarity of column chromatography separation is dichloromethane: methanol (V/V) =130:1 developing solvent under normal pressure to obtain red product (e) with 50% yield.

1H NMR (400 MHz, Chloroform-d) δ 8.24 (d, J = 8.7 Hz, 1H), 8.06 (d, J = 2.6 Hz, 1H), 7.62 (d, J = 9.1 Hz, 1H), 7.18 (dd, J = 8.7, 2.6 Hz, 1H), 6.68 (dd, J = 9.1, 2.7 Hz, 1H), 6.48 (d, J = 2.7 Hz, 1H), 6.32 (s, 1H), 4.24 (t, J = 5.9 Hz, 2H), 3.56 (t, J = 6.5 Hz, 2H), 3.49 (q, J = 7.1 Hz, 4H), 2.20 – 2.12 (m, 2H), 2.12 – 1.99 (m, 2H), 1.29 (t, J = 7.1 Hz, 6H).

(3) Synthesis of the Compound (4- ((9- (diethylamino) -5-oxo-5H-benzo [ a ] benzoxazin-2-yl) oxy) butyl) triphenylphosphine (g)

A50 mL pear-shaped flask was charged with compound e (100 mg, 0.2 mmol) and compound f (130 mg, 0.2 mmol) dissolved in 8 mL acetonitrile to give a bright red solution which was then placed under N₂Reacting for 12 h at 90 ℃ under protection. The solvent was removed by TLC and distilled off under reduced pressure. The polarity of column chromatography separation is dichloromethane: methanol (V/V) =20:1 developing solvent under normal pressure to obtain red product (e), NRTP for short, yield: 27 percent.

1H NMR (400 MHz, DMSO-d6) δ 8.05 (d, J = 8.7 Hz, 1H), 7.98 – 7.69 (m, 15H), 7.61 (d, J = 9.0 Hz, 1H), 7.21 (d, J = 8.9 Hz, 1H), 6.84 (d, J = 9.2 Hz, 1H), 6.66 (s, 1H), 6.20 (s, 1H), 4.26 (d, J = 6.0 Hz, 2H), 3.72 (d, J = 14.4 Hz, 2H), 3.51 (d, J = 7.2 Hz, 4H), 2.01 (t, J = 6.9 Hz, 3H), 1.78 (d, J = 10.8 Hz, 2H), 1.17 (t, J = 7.1 Hz, 6H), 13C NMR (101 MHz, MeOD) δ 183.18, 161.27, 152.25, 151.38, 146.62, 137.65, 134.92, 134.89, 133.78, 133.54, 133.44, 133.09, 130.81, 130.24, 130.11, 126.87, 124.76, 124.70, 118.92, 118.06, 117.30, 110.32, 106.20, 103.43, 95.64, 66.31, 44.76, 29.39, 29.23, 21.17, 20.66, 18.87, 11.64.

Example 2 cellular imaging of fluorescent Probe NRTP

(1) Cell culture, processing and staining

Hepatoma cells (Hepg 2) 5% CO at 37 ℃₂The incubator was supplemented with 10% FBS (fetal bovine serum) in H-DMEM (Dulbecco's modified Eagle's Medium, High Glucose). For cell imaging experiments, live Hepg2 cells were suspended in diluted medium at a cell concentration of 10000 cells/mL. 1mL of the cell suspension was added to a glass-bottomed dish and cultured for 24 hours to allow it to adhere, and then the cell culture solution was aspirated, and the cells were washed 3 times with the culture medium, followed by cell imaging experiments.

(2) Confocal microscopy imaging

Viable Hepg2 cells were incubated using the fluorescent probe NRTP obtained in example 1, as shown in figure 3. Low-polarity macrophages (SM/CL) were constructed with Sphingomyelin (SM) and Cholesterol (CL), and high-polarity macrophages were constructed with Lecithin (LE). In addition, increasing the CL content of the solution can effectively reduce the polarity. Thus, different amounts of CL were mixed with LE (LE +10% CL, LE +40% CL) to construct unilamellar vesicles (GUV) of different polarity. These guv fluorescence images were acquired in the red and dark red channels, respectively, and are represented in red and green false colors, as shown in FIG. 3. In LE-built high polarity GUVs, strong emission of the red and deep red channels was found. The low polarity SM/CL GUVs show strong red emission and weak green fluorescence. Particularly, as the CL content in LE GUVs increases, the red fluorescence slightly changes, the deep red emission is obviously reduced, and the combined images have obvious change trend. The result shows that NRTP shows blue shift emission in a low polarity gradient sample, and can reflect the change of polarity in the gradient sample.

Example 3 Co-localization of fluorescent Probe NRTP with commercial Probe

To confirm the staining site of the fluorescent probe NRTP in the cells, co-localization staining imaging was performed using the commercially available mitochondrial dye mitochondrial deep red (MTDR) and the fluorescent probe NRTP obtained in example 1, respectively.

In the cell co-localization experiment, cells were stained with 200 nM MTDR for 30 min, then 5. mu.M NRTP for 30 min, and then cell culture fluid was aspirated away, and cells were washed 3 times with medium for cell imaging. Collecting the fluorescence at 570-620 nm by using 561 nm as an excitation wavelength to acquire the fluorescence signal of NRTP; the fluorescence signal of MTDR is collected by collecting 663-738 nm fluorescence with 647 nm as the excitation wavelength, and the obtained fluorescence image is shown in FIG. 4, in which live cell bright field imaging (FIG. 4 a), NRTP fluorescence imaging (FIG. 4 b), MTDR fluorescence imaging (FIG. 4 d) and MTDR and NRTP superposition image (FIG. 4 f) are sequentially performed from left to right. It can be seen that the cells reacted with the NRTP probe and then fluoresced red, and the cells reacted with the MTDR and then fluoresced green; the counterstaining rate of both dyes was 89%, indicating that the probe stained mitochondria in living cells.

Claims

1. A fluorescent probe for detecting intracellular polarity by targeting mitochondria has a chemical structural formula as follows:

。

2. a method for preparing the fluorescent probe of claim 1, comprising the steps of:

；

；

。

3. the method according to claim 2, wherein in step (1), the mass ratio of compound a to compound b is 1: 1; in the step (2), the mass ratio of the compound c to the compound d is 1: 5; in the step (3), the mass ratio of the compound e to the compound f is 1: 1.

4. The preparation method according to claim 2, wherein in the step (1), the reaction temperature is 140 ℃ and the reaction time is 5 h; in the step (2), the reaction time is 12 h; in the step (3), the reaction temperature is 90 ℃ and the reaction time is 12 h.

5. The preparation method according to claim 2, wherein in the step (1), the separation and purification step is: and (3) extracting, separating and combining the reacted system with organic phases, and mixing the organic phases with petroleum ether: developing solvent at dichloromethane V/V =1:1 ratio removed the previous impurities, followed by a subsequent separation with dichloromethane: the developing solvent with a methanol V/V =100:1 ratio gave a red product at normal pressure.

6. The preparation method according to claim 2, wherein in the step (2), the separation and purification step is: adding dichloromethane and water into the reacted system, collecting an organic phase, distilling under reduced pressure to remove the solvent, and then carrying out column chromatography; the column chromatography eluent is dichloromethane: methanol V/V =130: 1.

7. The preparation method according to claim 2, wherein in the step (3), the separation and purification step is: distilling the reacted system under reduced pressure to remove the solvent, and then carrying out column chromatography; the column chromatography eluent is dichloromethane: methanol V/V =20: 1.

8. Use of a fluorescent probe according to claim 1 for detecting polarity in a cell.

9. Use of a fluorescent probe according to claim 1 in the preparation of a reagent for detecting intracellular polarity.