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Spin labeled fluorescent probe with single electron structure and application thereof

A technology of spin labeling and fluorescent probes, applied in fluorescence/phosphorescence, luminescent materials, organic chemistry, etc., can solve the problems of oxidative damage to the body, affect physiological functions, increase active oxygen, etc., and achieve low cytotoxicity and mild reaction conditions , good cell permeability

Inactive Publication Date: 2017-05-31
CHINA PETROLEUM & CHEM CORP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the one hand, if reactive oxygen species and reactive nitrogen are excessively produced in the body, it will lead to "oxidative stress". The generated reactive oxygen species and reactive nitrogen species exceed the tolerance of the body, which will cause oxidative damage to the body and affect normal physiological functions.
For example, long-term exposure to radiation will lead to a large increase in active oxygen, which will trigger free radical chain reactions and cause body damage

Method used

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  • Spin labeled fluorescent probe with single electron structure and application thereof
  • Spin labeled fluorescent probe with single electron structure and application thereof
  • Spin labeled fluorescent probe with single electron structure and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Synthesis of spin-labeled fluorescent probes

[0040] Proceed as follows:

[0041] (1) Rhodamine B (0.27mmol) and 4-hydroxyl-2,2,6,6,-tetramethyl-piperidine nitroxide radicals in equimolar amounts were added to the reaction solvent 10mL dichloromethane, and 4-Dimethylaminopyridine (0.027mmol) (as a catalyst), stirred for 20 minutes, then added N,N'-dicyclohexylcarbodiimide (0.27mmol) (as a dehydrating agent), and reacted at room temperature under the protection of argon 24h.

[0042] (2) The solvent was removed by rotary evaporation under reduced pressure, and the heating temperature was lower than 50°C.

[0043] (3) With dichloromethane / methanol=20 / 1 (v / v) as eluent, 200 mesh neutral alumina is as stationary phase, carry out column chromatographic separation; Spin labeled fluorescent probes.

Embodiment 2

[0044] Example 2: Detection experiment of intracellular hydroxyl radicals

[0045] The in vitro hydroxyl radical generation method adopts the classic Fenton system, that is, hydrogen peroxide is catalyzed by ferrous ion ([Fe 2+ ] / [H 2 o 2 ]=1:6), the hydroxyl group is captured by DMSO (1%) in acetonitrile to generate a relatively stable methyl radical, which combines with the probe to generate a stable product, which leads to the recovery of fluorescence to achieve the purpose of detection. In the hydroxyl radical detection reagent, the concentration of the probe is 7.5 μM, and the concentration of the hydroxyl radical is 0-100 μM. And the probe has good selectivity for hydroxyl radicals in the presence of DMSO. For the detection of hydroxyl free radicals produced by cells, rotenone was used to stimulate the cells, and a comparative study was carried out on liver cancer cells and normal cells. In the intracellular hydroxyl radical detection reagent, the cells were pretreat...

Embodiment 3

[0047] Example 3: Detection experiment of intracellular thiol free radicals

[0048] The in vitro glutathione free radical generation method uses horseradish peroxidase to oxidize phenol to generate phenoxy free radicals in the presence of hydrogen peroxide, and then oxidizes glutathione to generate glutathione free radicals, and then probes Capture glutathione free radicals to generate corresponding fluorescent derivatives. In vitro glutathione free radical detection reagent, probe 5 μM, with phenol 20 μM, glutathione 5 μM, hydrogen peroxide 5 μM and horseradish peroxidase 0.0625 U / mL in phosphate at 10 mM (pH 7.4) React in buffer solution for 10 min. For the detection of glutathione free radicals produced by cells, HL-60 cells rich in myeloperoxidase were selected. In the intracellular glutathione free radical detection reagent, the cells were pretreated with 2 μM probe for 10 min, and then mixed with 10 μM phenol After incubation with 2 μM hydrogen peroxide for 10 min, th...

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Abstract

The invention discloses a spin labeled fluorescent probe with single electron structure. The spin labeled fluorescent probe with single electron structure is prepared by the following method: (1) adding rhodamine B and 4-hydroxy-2, 2, 6, 6,-tetramethyl-piperidine nitrogen oxygen free radical into a reaction solvent dichloromethane, adding 4-dimethylaminopyridine to carry out reaction for 20min, then adding N, N'-dicyclohexyl carbodiimide, carrying out reaction at room temperature under argon protection for 24h; and (2) at the end of the reaction, removing the solvent, and subjecting the remaining materials to column chromatography separation: adopting dichloromethane / methanol=20 / 1 (v / v) as the eluant, taking 200-mesh neutral alumina as the stationary phase, collecting eluent, and removing the eluate. The spin labeled fluorescent probe with single electron structure provided by the invention has good cell permeability and low cytotoxicity, can be used for detection of intracellular hydroxyl radicals, mercapto free radicals and, cellular redox environment and oxidation resistance evaluation research of polyphenolic antioxidants.

Description

technical field [0001] The present invention relates to a spin-labeled fluorescent probe with a single-electron structure and its application, in particular to a method for the detection of hydroxyl radicals, mercapto radicals, and cellular redox environments and the evaluation of the antioxidant performance of polyphenol antioxidants Fluorescence analysis system. Background technique [0002] As we all know, organisms are extremely complex systems, and the balance of redox state in cells is extremely important to maintain normal physiological functions in cells of organisms. Studies have shown that changes in the cellular redox environment are closely related to a series of diseases, such as cancer, ischemia-reperfusion injury, and neurodegenerative diseases such as Alzheimer's disease. Therefore, monitoring the dynamic changes in the redox environment of cells can provide rich physiological and pathological information for clinical medicine and other fields. [0003] The...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C09K11/06C07D405/12G01N21/64
CPCC07D405/12C09K11/06C09K2211/1029C09K2211/1088G01N21/6404
Inventor 宋新旺张继超郭勇祝仰文郭兰磊于群潘斌林郭淑凤刘煜陈晓彦王帅王丽娟王红艳田志铭李青华
Owner CHINA PETROLEUM & CHEM CORP
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