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

Preparation of mitochondrion targeting type near-infrared fluorescent probe with aggregation-induced emission effect Download PDF

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CN111410652B
CN111410652B CN201910010982.5A CN201910010982A CN111410652B CN 111410652 B CN111410652 B CN 111410652B CN 201910010982 A CN201910010982 A CN 201910010982A CN 111410652 B CN111410652 B CN 111410652B
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fluorescent probe
aggregation
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CN111410652A (en
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孟萌
赵秀杰
郗日沫
陈韵
王建宁
曹彦梅
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Abstract

The invention discloses a preparation method of a mitochondrion targeted near-infrared fluorescent probe with aggregation-induced emission effect. The probe has weak near-infrared fluorescence in a benign solvent, and the fluorescence is gradually enhanced along with the addition of poor solvent water, so that an obvious AIE effect is displayed; and the fluorescence is gradually enhanced with the increase of pH, and the stronger pH dependence is shown. The probe is simple to prepare, has good light stability and biocompatibility, is excited and emitted in a near-infrared region, has small biological light damage and small background interference, and opens up a new idea for developing a near-infrared fluorescent probe with aggregation-induced emission and application.

Description

Preparation of mitochondrion targeted near-infrared fluorescent probe with aggregation-induced emission effect
Technical Field
The invention belongs to the technical field of fluorescence biosensors, and particularly relates to preparation of a mitochondrion targeted near-infrared fluorescence probe with aggregation-induced emission effect.
Background
Mitochondria are the site of oxidative phosphorylation of cells, possess own genetic material and genetic system, and belong to semi-autonomous organelles. In addition to supplying energy to cells, it is also involved in life processes such as cell differentiation, cell information transmission and apoptosis, and possesses the ability to regulate cell growth. Changes in mitochondrial morphology and changes in pH are associated with certain diseases such as: parkinson's disease, Alzheimer's disease and cancer. Therefore, visualization of mitochondria is important for studies of mitochondria and diagnosis of diseases.
The bio-optical imaging is widely applied to medical biological research due to the advantages of mature development of detection instruments, high sensitivity, high contrast, high resolution, direct imaging, high imaging speed, nondestructive detection and the like. Fluorescence Imaging (fluorescence Imaging) is an important means for research in the biomedical field, and can be used for researching the position, concentration and the like of a target molecule. Fluorescent probe molecules are widely researched and applied in molecular, ion detection and cell imaging technologies due to the advantages of simple synthesis, high sensitivity, good selectivity, short response time, direct observation and the like. Due to the characteristics of low tissue autofluorescence and strong tissue penetration capability, the near-infrared (NIR) fluorescent dye labeling technology can be used for real-time and continuous detection of target cells through fluorescent dye labeling, has a wide application prospect in the aspect of small animal living body imaging, and is gradually applied to cell labeling tracing research at present.
The traditional small molecular dye has poor light stability and strong photobleaching property due to the ACQ effect, and is not beneficial to long-range tracking. In 2001, Tang Benzhen et al found that compounds such as a thia derivative do not emit light in a solution state, but emit intense fluorescence upon aggregation, and this phenomenon is called aggregation-induced emission (AIE) effect.
Disclosure of Invention
The invention aims to provide a fluorescent probe with aggregation-induced emission characteristics, which can be positioned in cell mitochondria, excited and emitted in a near infrared region, and has small damage to cells and small background interference. The invention provides a mitochondrion targeting near-infrared fluorescent probe with aggregation-induced emission characteristics, and the structural formula of the mitochondrion targeting near-infrared fluorescent probe is shown in figure 1.
The fluorescent probe is composed of hemicyanine and aggregation-induced emission molecules with mitochondrial targeting and near-infrared characteristics, and due to the action of hydroxyl and TPE, the aggregation effect and fluorescence become stronger with the increase of alkalinity.
The synthetic steps of the fluorescent probe provided by the invention are shown in figure 1.
The invention provides a preparation method of a fluorescent probe, which comprises the following steps:
1) under the protection of nitrogen, IR-780 and 5-bromo-m-diphenol react for 4 hours at 80 ℃ under the action of triethylamine by taking anhydrous DMF as a solvent to prepare an intermediate product mCy-Br.
2) Under the protection of nitrogen, THF/H2O is used as a solvent, mCy-Br and 4- (1,2, 2-tristyryl) -phenylboronic acid pinacol ester are subjected to reflux reaction for 24 hours under the action of K2CO3, and the final product fluorescent probe is prepared.
The invention also provides application of the fluorescent probe in positioning the mitochondria of the cell.
Compared with the prior art, the invention has the following beneficial effects:
the mitochondrial positioning fluorescent probe has the following characteristics: (1) has AIE effect, good light stability and photobleaching resistance; (2) the probe is excited and emitted in a near infrared region, so that the damage to cells is small, and the background interference is small; (3) the probe targets mitochondria and can detect the pH response at mitochondria; (4) the probe can trace the long distance of mitochondria. The good characteristics of the probe indicate that the probe has great application value in the aspect of mitochondrial research.
Drawings
FIG. 1 is a synthetic route of the fluorescent probe of the present invention
FIG. 2 is a graph of the UV absorption of the fluorescent probe of the present invention in different pH solutions
FIG. 3 is a graph showing fluorescence emission of the fluorescent probe of the present invention at different pH solutions, Ex =660nm
FIG. 4 is a graph showing fluorescence emission of the fluorescent probe of the present invention in different THF/PBS solutions at pH 4, Ex =660nm
FIG. 5 is a graph showing fluorescence emission of the fluorescent probe of the present invention in different THF/PBS solutions at pH 5, Ex =660nm
FIG. 6 is a graph of fluorescence emission of fluorescent probes of the invention in different THF/PBS solutions at pH 6, Ex =660nm
FIG. 7 is a graph of fluorescence emission of fluorescent probes of the invention in different THF/PBS solutions at pH 7 with Ex =660nm
FIG. 8 is a diagram showing the localization of mitochondria in Hela cells by the fluorescent probe of the present invention
Detailed description of the preferred embodiment
The following will further explain the fluorescent probe of the present invention, its preparation method and its application in detail with reference to the accompanying drawings and examples.
Example 1:
synthesis of intermediate mCy-Br
The synthesis method is shown in figure 1.
The method comprises the following specific steps: taking a 10 mL dry two-mouth bottle with magnetons, adding IR-780 (50 mg, 0.0775 mmol) and 5-bromocatechol (27.0 mg, 0.143 mmol), performing vacuum-nitrogen replacement for 3 times, adding 2mL of anhydrous DMF and 0.1mL of anhydrous triethylamine into the reaction bottle under the protection of nitrogen, heating to 80 ℃, reacting for 4h, and performing vacuum drying to obtain a crude product. The crude product was isolated and purified by flash column chromatography (dichloromethane: methanol = 20/1, v/v) to yield the compound as a blue solid (yield about 62.9%). And (4) performing characterization through nuclear magnetism and mass spectrometry. 1H NMR (400 MHz, 298K, 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 (400 MHz, 298K, 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, 112.0, 102.9, 50.8,47.2, 30.0, 29.0, 28.5, 21.2, 11.7 hrms (esi): calculated values for C28H29BrNO2+ [ M ] +, 490.1376; actual value 490.1376.
Example 2:
synthesis of Probe
The synthesis method is shown in figure 1.
The method comprises the following specific steps: a reaction flask was charged with 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 displaced 3 times, added under nitrogen protection, added with 4ml THF, Pd (PPh3)4 (5 mg, 0.004 mmol), 1ml H2O, heated to reflux, extracted 4 times after 24 hours with dichloromethane, combined organic phase and concentrated to give crude product. The crude product was isolated and purified by flash column chromatography (dichloromethane: methanol = 20/1, v/v) to yield a blue solid compound (yield about 24%). And (4) performing characterization through nuclear magnetism and mass spectrometry. 1H NMR (400 MHz, 298K, CDCl3): Δ 8.58 (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, 298K, 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, 140.3,135.5, 131.4, 128.9, 127.8, 127.7,126.6, 126.3, 122.7, 122.1, 117.1, ESI.1, 110.1, 110.11, 11.9, 30.5, 9, 9.5, 9, 9.9, 9, 9.7, 9, 7, 9, 1, 9, 1, 9, 1, 9, 1, 9, 1, 9, 1, 9, 1, 9, 1, 9, 742.3680, and actual value 742.3681.
Example 3:
probe 10. mu.M 70% solutions of PBS/THF at different pH values were prepared and measured on a visible UV spectrophotometer, the results are shown in FIG. 2.
Example 4:
probes 10. mu.M 70% solutions in PBS/THF at different pH values were prepared and measured on a fluorescence spectrophotometer, the results are shown in FIG. 3.
Example 5:
solutions of probes 10 μ M pH =4 were prepared at different THF/PBS ratios, PBS 10%, 20%, 30%, 40%, 50%, 60%, 70%, respectively, and fluorescence was measured as shown in figure 4.
Example 6:
probes 10 μ M solutions with different THF/PBS ratios at pH =5 were prepared, PBS 10%, 20%, 30%, 40%, 50%, 60%, 70%, respectively, and fluorescence was measured as shown in fig. 5.
Example 7:
solutions of probe 10 μ M pH =6 were prepared at different THF/PBS ratios, PBS 10%, 20%, 30%, 40%, 50%, 60%, 70%, respectively, and fluorescence was measured as shown in figure 6.
Example 8:
solutions of probe 10 μ M pH =7 were prepared at different THF/PBS ratios, PBS 10%, 20%, 30%, 40%, 50%, 60%, 70%, respectively, and fluorescence was measured as shown in figure 7.
Example 9:
hela cells are inoculated into a 35mm confocal culture dish (2 x 105), after 24 hours of culture, culture solution with the probe concentration of 2.5 mu M is added, after 30 minutes of incubation, MitoTracker green (0.05 mu M) is added for 30 minutes of incubation, and after the culture solution is washed away by PBS, confocal imaging is carried out.

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):
Figure FDA0003743107000000011
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):
Figure FDA0003743107000000012
the structure of the raw material IR-780 is shown as (3):
Figure FDA0003743107000000013
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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CN112047977B (en) * 2020-08-20 2023-03-21 宁波大学 Mitochondrial targeting fluorescent probe and synthetic method and application thereof
CN112353755A (en) * 2020-12-04 2021-02-12 上海交通大学医学院附属第九人民医院 Double-target transdermal drug delivery nanogel and preparation method thereof
CN113476602B (en) * 2021-07-02 2023-04-25 南开大学 Preparation of novel high-photothermal conversion efficiency cyanine photosensitizer and self-targeting phototherapy of tumors

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