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Compound for detecting cell autophagy enhancement as well as preparation method and application thereof

A compound and cell technology, applied in the fields of compounds containing group 3/13 elements of the periodic table, chemical instruments and methods, organic chemistry, etc., can solve the problem of inability to indicate enhanced autophagy, poor membrane permeability of metal complexes, etc. problems, to achieve the effect of high-throughput screening, excellent cell permeability, and simple operation

Inactive Publication Date: 2018-11-23
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are patent reports that metal complexes are used as markers of autophagy lysosomes, but the metal complexes themselves have poor membrane permeability, and after entering cells through the endocytic pathway, they are trapped by endosomes and cannot be released into cells, so only Can be transported to autolysosomes for eventual degradation, and does not respond to pH changes, so the detection of newly acidified organelles cannot quickly indicate the enhancement of autophagy [Ref: CN 104152529A]

Method used

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  • Compound for detecting cell autophagy enhancement as well as preparation method and application thereof
  • Compound for detecting cell autophagy enhancement as well as preparation method and application thereof
  • Compound for detecting cell autophagy enhancement as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1: the synthesis of compound SN1

[0045] Add (R1=R2=H) benzaldehyde (0.106g, 1mmol) and 2-cyano-2-(ethoxycarbonylvinyl)-1-H-pyrrole (0.380g, 2mmol) into the reaction vessel, dissolve in 50ml of anhydrous dichloromethane. Extract 0.5ml of trifluoroacetic acid (TFA) catalyst into the system with a syringe, put into a magnetic stirrer to stir under anhydrous and dark conditions, and monitor with a thin-layer chromatographic plate after reflux for 7 days until the described 2-cyano-2-(B Oxycarbonylvinyl)-1-H-pyrrole was completely consumed. Then 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ: 0.227g, 1mmol) was added to the reaction solution, stirred at room temperature for 30 minutes, and then triethylamine (TEA) was added After stirring 3ml evenly, add boron trifluoride-ether solution (BF 3 ·Et 2 (2) 6ml (mass percentage 48%), continue stirring and reflux reaction for 1 hour, obtain the blue solution with red fluorescence, point plate monitoring until the ...

Embodiment 2

[0046] Embodiment 2: pH titration experiment of SN1

[0047] Compound SN1 is a colorless to pale yellow solution in methanol, and its UV absorption spectrum is shown in image 3, there are two absorption peaks at 374 nm and 413 nm. TFA was added to the methanol solution of iso-BODIPY for pH titration, and an obvious color change from light yellow to red was observed during the titration from pH 7.0 to 2.12, accompanied by strong red fluorescence. When the pH value is below 6.0, the generation of fluorescence begins to be observed, and when the pH value changes from 7.0 to 4.1, the fluorescence emission intensity (λem=635nm) increases by more than 17 times. image 3 The inset in shows the change in absorption at 618 nm during the pH titration. A pKa value of 4.71 was obtained by using the Henderson-Hasselbach equation. The changes of ultraviolet absorption spectrum and fluorescence spectrum indicated that iso-BODIPY is suitable for pH fluorescent probe and may have the poten...

Embodiment 3

[0048] Example 3: Lysosome fluorescent labeling and real-time monitoring application of SN1

[0049] The KB cell line was provided by the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. KB cells were cultured in serum-free freezing medium (RPMI) 1640 supplemented with 10% fetal bovine serum (FBS). Place it in a carbon dioxide incubator at 37°C for 4 hours. Cells can be directly scanned and imaged without washing. The synthesized iso-BODIPY and labeled lysosomal fluorescent probe were further studied by 3D live cell imaging technology. Hela cells were loaded with 1 (15 μM) for 30 minutes and observed under a confocal microscope, then rapamycin (20 μM) was added under the microscope. For each field, a series of 10-20 images of successive focal planes, separated by 0.8 μm, were recorded and plotted against the vertical projection of each focal plane. Record fluorescence images at selected time points and ca...

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Abstract

The invention discloses a compound for detecting cell autophagy enhancement. The compound has a structure as shown in the description. The compound disclosed by the invention can be uniformly distributed in cells, and almost no any fluorescent light exists in cytoplasm or an autophagosome closely related to autophagy and an early endosome. Therefore, when the autophagosome or the early endosome ismature or acidified, strong red fluorescent light can be emitted by a material, and the strength of the fluorescent light and the acidification degree are in quantitative association; by detecting anacidifying process of an organelle in real time, the cell autophagy enhancement can be indirectly indicated. The invention discloses a preparation method of the compound.

Description

technical field [0001] The invention relates to the field of biological detection, and is a material for detecting autophagy enhancement by detecting organelle acidification in real time. Background technique [0002] Autophagy is an intracellular degradation process by which cells control cellular homeostasis and survival [Ref: Kroemer, G.; Marino, G.; Levine, B. Molecular cell 2010, 40, 280.]. Enhanced autophagy is beneficial in aging and various neurodegenerative disease models such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis [Ref: Tan, C.C.; Yu, J.T.; Tan , M.S.; Jiang, T.; Zhu, X.C.; Tan, L. Neurobiology of aging 2014, 35, 941; Wolfe, D.M.; Lee, J.H.; Kumar, A.; Lee, S.; Orenstein, S. J.; journal of neuroscience 2013, 37, 1949; Menzies, F. M.; Fleming, A.; Rubinsztein, D.C. Nature reviews. Neuroscience 2015, 16, 345.]. In such diseases, the enhancement of autophagy can enhance the clearance of aggregates caused ...

Claims

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Application Information

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IPC IPC(8): C07F5/02C09K11/06G01N21/64
CPCG01N21/6428C09K11/06C07F5/02C09K2211/1029
Inventor 沈珍刘汉壮宋文婷
Owner NANJING UNIV
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