CN111410652B - Preparation of mitochondrion targeting type near-infrared fluorescent probe with aggregation-induced emission effect - Google Patents

Abstract

The invention discloses the preparation of a mitochondria-targeted near-infrared fluorescent probe with aggregation-induced luminescence effect. The fluorescent probe is composed of a hemicyanine part with near-infrared excitation and emission and a TPE part with aggregation-induced luminescence molecule. The probe has weak near-infrared fluorescence in benign solvents, and with the addition of poor solvent water, the fluorescence gradually increases, showing obvious AIE effect; and with the increase of pH, the fluorescence also gradually strengthens, showing a strong pH dependent. The probe is simple to prepare, has good photostability and biocompatibility, performs excitation and emission in the near-infrared region, has little biological photodamage and low background interference, and is a promising candidate for the development of near-infrared fluorescent probes with aggregation-induced luminescence. and application opens up a new way of thinking.

Description

Preparation of a mitochondria-targeted near-infrared fluorescent probe with aggregation-induced luminescence effect

technical field

The invention belongs to the technical field of fluorescent biosensors, in particular to the preparation of a class of mitochondria-targeted near-infrared fluorescent probes with aggregation-induced luminescence effect.

Background technique

Mitochondria are the sites of cellular oxidative phosphorylation, have their own genetic material and genetic system, and are semi-autonomous organelles. In addition to providing energy for cells, it also participates in life processes such as cell differentiation, cell information transmission and apoptosis, and has the ability to regulate cell growth. Changes in mitochondrial morphology and pH are associated with certain diseases, such as Parkinson's disease, Alzheimer's disease and cancer. Therefore, visualization of mitochondria is crucial for mitochondrial research and disease diagnosis.

Bio-optical imaging has been widely used in medical biological research due to the advantages of mature detection instruments, high sensitivity, high contrast, high resolution, intuitive imaging, fast imaging speed and non-destructive testing. Fluorescent Imaging technology is an important means of research in the field of biomedicine, which can be used to study the location and concentration of target molecules. resolution, etc. Fluorescent probe molecules have been widely studied and applied in molecular, ion detection and cell imaging technologies due to their advantages of simple synthesis, high sensitivity, good selectivity, short response time, and direct observation. The near-infrared (NIR) fluorescent dye labeling technology can perform real-time and continuous detection of target cells through fluorescent dye labeling due to its low tissue autofluorescence and strong tissue penetration ability. Imaging has broad application prospects, and is now gradually applied to the research of cell labeling and tracing.

Due to the ACQ effect, traditional small-molecule dyes have poor photostability and strong photobleaching, which are not conducive to long-range tracking. In 2001, Tang Benzhong et al. found that compounds such as Thiillo derivatives do not emit light in solution state, but emit strong fluorescence when aggregated. This phenomenon is called aggregation-induced emission (AIE) effect.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a fluorescent probe with aggregation-induced luminescence properties, which can be located in cell mitochondria, excited and emitted in the near-infrared region, with little damage to cells and little background interference. The present invention provides a mitochondria-targeted near-infrared fluorescent probe with aggregation-induced luminescence properties, the structural formula of which is shown in FIG. 1 .

The fluorescent probe of the present invention is composed of hemicyanine with mitochondrial targeting and near-infrared characteristics and aggregation-induced luminescent molecules. Due to the action of hydroxyl groups and TPE, with the increase of alkalinity, the aggregation effect and fluorescence become stronger and stronger.

The synthetic steps of the fluorescent probe provided by the present invention are shown in FIG. 1 .

The preparation method of the fluorescent probe provided by the present invention includes the following steps:

1) under nitrogen protection, with anhydrous DMF as solvent, IR-780 and 5-bromo-resorcinol were reacted at 80° C. for 4 hours under the effect of triethylamine to prepare the intermediate product mCy-Br.

2) Under nitrogen protection, using THF/H2O as solvent, mCy-Br and 4-(1,2,2-tristyryl)-phenylboronic acid pinacol ester under the action of K2CO3, refluxed for 24 hours to prepare Final product fluorescent probe.

The present invention also provides the application of the fluorescent probe in localization of cell mitochondria.

Compared with the prior art, the beneficial effects of the present invention are:

The mitochondrial localization fluorescent probe of the present invention has the following characteristics: (1) it has AIE effect, good photostability and photobleaching resistance; (2) the probe is excited and emitted in the near-infrared region, and has little damage to cells. The background interference is small; (3) the probe targets mitochondria and can detect the pH response at the mitochondria; (4) the probe can track mitochondria over a long distance. The good properties of this probe indicate that it has great application value in mitochondrial research.

Description of drawings

Fig. 1 is the synthetic route of the fluorescent probe of the present invention

Fig. 2 is the ultraviolet absorption diagram of the fluorescent probe of the present invention under different pH solutions

Fig. 3 is the fluorescence emission diagram of the fluorescent probe of the present invention under different pH solutions, Ex=660nm

Figure 4 is the fluorescence emission diagram of the fluorescent probe of the present invention in pH 4 solution with different THF/PBS, Ex=660nm

Figure 5 is the fluorescence emission diagram of the fluorescent probe of the present invention in pH 5 solution with different THF/PBS, Ex=660nm

Figure 6 is the fluorescence emission diagram of the fluorescent probe of the present invention in pH 6 solution with different THF/PBS, Ex=660nm

Figure 7 is the fluorescence emission diagram of the fluorescent probe of the present invention in pH 7 solution with different THF/PBS, Ex=660nm

Fig. 8 is the localization diagram of the fluorescent probe of the present invention in mitochondria in Hela cells

How to specify

The specific implementation of the fluorescent probe of the present invention and its preparation method and application will be further described below with reference to the accompanying drawings and examples.

Example 1:

Synthesis of Intermediate mCy-Br

The synthesis method is shown in Figure 1.

Specific steps: take a dry 10 mL two-necked bottle equipped with a magnet, add IR-780 (50 mg, 0.0775 mmol) and 5-bromocatechol (27.0 mg, 0.143 mmol), and replace it with vacuum-nitrogen for 3 times, Under the condition of nitrogen protection, 2ml of anhydrous DMF and 0.1ml of anhydrous triethylamine were added to the reaction flask, heated to 80° C. to react for 4h, and drained under reduced pressure to obtain a crude product. The crude product was separated and purified by flash chromatography column (dichloromethane: methanol = 20/1, v/v) to obtain a blue solid compound (yield about 62.9%). Characterized by NMR and mass spectrometry. 1H NMR (400 MHz, 298 K, CDCl3): δ 8.53 (d, 1H), 7.46−7.25 (m, 6H), 7.15(s, 1H), 6.26 (d, 1H), 4.22 (t, 2H), 2.73 (t, 2H), 2.67 (t, 2H), 1.96−1.91 (m, 4H), 1.25 (s, 6H), 1.09 (t, 3H). 13C NMR (400MHz, 298 K, CD3OD): δ 177.3 , 162.6, 154.5, 141.6, 144.0, 133.4, 128.9, 127.1,126.6, 122.8, 121.9, 119.5, 115.5, 114.0, 102.9, 50.8,47.2, 30.0,29.0, 28.5, 11.7. HRMS (ESI): Calculated for C28H29BrNO2+ [M]+, 490.1376; Actual 490.1376.

Example 2:

Probe Synthesis

The synthesis method is shown in Figure 1.

Specific steps: intermediate 1 (30 mg, 0.048 mmol), 4-(1,2,2-tristyryl)-phenylboronic acid pinacol ester (40 mg, 0.087 mmol), K2CO3 (0.338 g, 2.45 mmol), vacuum-nitrogen replaced 3 times, added under nitrogen protection, added 4ml THF, Pd(PPh3)4 (5 mg, 0.004 mmol), 1ml H2O, heated to reflux, added dichloromethane after 24 hours to extract 4 times, the organic phases were combined and concentrated to obtain crude product. The crude product was separated and purified by flash chromatography column (dichloromethane:methanol=20/1, v/v) to obtain a blue solid compound (yield about 24%). Characterized by NMR and mass spectrometry. 1H NMR (400 MHz, 298 K, CDCl3): δ8.58 (d, 1H), 7.52 (s, 1H), 7.37(d, 2H), 7.26 (s, 1H), 7.16−7.06 (m, 22H) , 6.96(s, 1H), 6.08 (d, 1H), 4.06 (t, 2H), 2.60-2.63 (t, 2H), 1.89−1.91 (m, 4H), 1.29 (s, 6H), 1.07 (t , 3H). 13C NMR(400 MHz, 298 K, CDCl3): δ 175.7, 162.9, 162.7, 158.5, 155.4, 144.7, 143.7, 143.4, 143.2, 141.9, 141.7, 141.5, 140.3, 128.95, 2 , 127.7,126.6, 126.3, 122.7, 122.1, 117.1, 114.4,113.1, 111.2, 110.4, 102.5, 100.9,50.6, 50.1,31.9, 30.0, 28.4, 22.7, 17. H. RMS (E4) Calculated C54H48NO2+ [M]+, 742.3680; actual value 742.3681.

Example 3:

The probes were prepared at 10 μM 70% PBS/THF solutions with different pH values and measured on a visible ultraviolet spectrophotometer. The results are shown in FIG. 2 .

Example 4:

10 μM 70% PBS/THF solutions of different pH values of the probe were prepared and measured on a fluorescence spectrophotometer. The results are shown in FIG. 3 .

Example 5:

Prepare probe 10 μM pH=4 solutions with different THF/PBS ratios, PBS accounts for 10%, 20%, 30%, 40%, 50%, 60%, 70%, respectively, and measure the fluorescence, the results are shown in Figure 4.

Example 6:

Prepare probe 10μM pH=5 solutions with different THF/PBS ratios, PBS accounts for 10%, 20%, 30%, 40%, 50%, 60%, 70%, respectively, and measure the fluorescence. The results are shown in Figure 5.

Example 7:

Prepare probe 10μM pH=6 solutions with different THF/PBS ratios, PBS accounts for 10%, 20%, 30%, 40%, 50%, 60%, 70%, respectively, and measure the fluorescence, the results are shown in Figure 6.

Example 8:

Prepare probe 10 μM pH=7 solutions with different THF/PBS ratios, PBS accounts for 10%, 20%, 30%, 40%, 50%, 60%, 70%, respectively, and measure the fluorescence. The results are shown in Figure 7.

Example 9:

Hela cells were seeded in a 35mm confocal culture dish (2*105), and after 24 hours of culture, a culture solution with a probe concentration of 2.5 μM was added, and after 30 minutes of incubation, MitoTracker green (0.05 μM) was added for a total of 30 minutes of incubation. Confocal imaging after washing off the culture medium.

Claims (3)

1. A gathering induced luminescence fluorescent probe based on a hemicyanine structure excited and emitted by near infrared light is characterized in that the structural formula is shown in a figure (1):

2. the method of claim 1, wherein the assay comprises the steps of:

under the protection of nitrogen, taking anhydrous DMF as a solvent, and reacting IR-780 and 5-bromo-m-diphenol for 4 hours at 80 ℃ under the action of triethylamine to prepare an intermediate product mCy-Br; under the protection of nitrogen, THF/H ₂ O is used as solvent, intermediate mCy-Br and 4- (1,2, 2-triphenylethylene) -phenylboronic acid pinacol ester are reacted in K ₂ CO ₃ And Pd (PPh) ₃ ) ₄ And (3) under the action of reflux reaction for 24 hours, preparing a final product fluorescent probe, wherein the structure of an intermediate product mCy-Br is shown as (2):

the structure of the raw material IR-780 is shown as (3):

3. the use of the fluorescent probe of claim 1 for intracellular mitochondrial imaging and pH detection for purposes other than diagnosis or treatment of disease.

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