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Lysosome targeted fluorescent dye based on nitrogen heterocyclic structure as well as preparation method and application of lysosome targeted fluorescent dye

A technology of fluorescent dyes and lysosomes, applied in the preparation of azo dyes, test samples, organic dyes, etc., to achieve the effects of simple synthesis, improved water solubility, and good photostability

Inactive Publication Date: 2019-04-26
HEFEI INSTITUTES OF PHYSICAL SCIENCE - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to solve the technical problems existing in the existing lysosome dyes, to provide a lysosome-targeted fluorescent dye based on nitrogen heterocyclic structure and its preparation method and application

Method used

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  • Lysosome targeted fluorescent dye based on nitrogen heterocyclic structure as well as preparation method and application of lysosome targeted fluorescent dye
  • Lysosome targeted fluorescent dye based on nitrogen heterocyclic structure as well as preparation method and application of lysosome targeted fluorescent dye
  • Lysosome targeted fluorescent dye based on nitrogen heterocyclic structure as well as preparation method and application of lysosome targeted fluorescent dye

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Synthesis of Dye Dansyl-1,4,7,10-tetraazacyclododecane (DS-C)

[0039]

[0040] Dissolve 638.7mg of 1,4,7,10-tetraazacyclododecane (3.71mmol) and 512.3mg of anhydrous potassium carbonate (0.371mmol) in 20mL of anhydrous acetonitrile, stir at room temperature for 30min; dissolve 250mg A solution of dansyl chloride (0.927 mmol) in 10 mL of anhydrous acetonitrile was added dropwise to the above reaction system, and the stirring reaction was continued at room temperature for 3 h. After the reaction was completed, the potassium carbonate powder was removed by suction filtration, and the acetonitrile was removed with a vacuum rotary evaporator. The obtained green sticky residue was purified by column chromatography to obtain the target product DS-C, and the eluent was CH 2 Cl 2 / CH 3 OH (V / V=10:1). 1 H NMR (400MHz, CDCl 3 ):δ8.56(d,1H), 8.38(d,1H),7.98(d,1H),7.52(m,2H),7.19(d,1H),3.62(t,4H),3.20(t, 4H),3.04(t,4H), 2.88-2.91(m,10H). 13 C NMR (100MHz, CDCl ...

Embodiment 2

[0041] Embodiment 2: the fluorescence spectrum of dyestuff DS-C in PBS buffer solution

[0042] Dissolve DS-C in ethanol to prepare a stock solution with a concentration of 10mmol / L. 2 μL DS-C was taken from the stock solution and added to 2 mL of PBS buffer solution (pH=7.4, 10 mM), so that the final concentration of the probe was 10 μM, and the fluorescence emission of the dye DS-C was measured at an excitation wavelength of 405 nm. And under the irradiation of 365nm ultraviolet lamp, take the fluorescent picture of dyestuff with digital camera. Fluorescence spectra and fluorescence photos of dyes such as figure 1 shown.

Embodiment 3

[0043] Example 3: Co-localization of the dye DS-C with the commercial lysosomal dye LysoTracker Deep Red in different cells

[0044] The lysosomal dye Deep Red was added to different types of cells respectively, at 37°C, 5% CO 2 After culturing for 30 min under the condition, wash once with medium or (PBS) to remove residual Deep Red. Subsequently, the fluorescent dye DS-C prepared in Example 1 was added to the above-mentioned cells to make the final concentration 5 μM. After culturing for 10 minutes, laser confocal imaging was performed. The imaging results are shown in figure 2 . It can be seen from the figure that the dye DS-C and Deep Red can overlap perfectly, indicating that the dye has the ability to specifically target lysosomes and is universal. The reason why the dye can specifically target the lysosome is that the nitrogen heterocyclic substituent is weakly basic, and the environment in the cell lysosome is weakly acidic, and the dye has the tendency to accumulat...

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Abstract

The invention discloses lysosome targeted fluorescent dye based on a nitrogen heterocyclic structure as well as a preparation method and an application of the lysosome targeted fluorescent dye. According to the lysosome targeted fluorescent dye based on the nitrogen heterocyclic structure, 5-(dimethylamino)-1-naphthalenesulfonic acid or 7-nitrobenzo-2-oxa-1,3-diazole is taken as a fluorescent parent, different types of nitrogen heterocyclic substituents are introduced to the molecular structure of the parent, and the structure of the molecular structure is shown in the description, wherein R is one selected from the groups shown in the description, and R1 is hydrogen or C1-5 alkyl and can be the same or different. The fluorescent dye adopts a one-step synthesis method, synthesis steps aresimple, reaction conditions are mild, and a product is easy to purify. The fluorescent dye not only can specifically target lysosomes of various cells, but also can label the lysosomes of the cells for a long time. Besides, the fluorescent dye can be used for monitoring formation of autophagic lysosomes in the cells and has broad application prospects in the field of biology.

Description

technical field [0001] The present invention relates to the field of synthesis and technical application, in particular to a lysosome-targeted fluorescent dye based on nitrogen heterocyclic structure and its preparation method and application. Background technique [0002] Lysosome is an important organelle in cells and plays a vital role in the normal life activities of cells: degradation and recycling of intracellular macromolecules, degradation of various intracellular and extracellular materials, and damage to organelles recycling, etc. Lysosomes are highly dynamic, and their shape and spatial distribution are constantly changing, and changes in their number and shape can often represent the state of life activity in cells. Lysosomal abnormalities usually cause a variety of diseases (such as gout, lysosomal storage disease or silicosis, etc.), and the disease process is often accompanied by changes in the number, size, shape, and structure of lysosomes. Therefore, the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C09K11/06C09B57/00G01N1/30
CPCC09B57/00C09K11/06C09K2211/1011C09K2211/1044C09K2211/1059C09K2211/1074G01N1/30
Inventor 张忠平杨林林王振洋赵君刘变化
Owner HEFEI INSTITUTES OF PHYSICAL SCIENCE - CHINESE ACAD OF SCI
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