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Xanthene derivative for detecting ONOO<-> as well as synthesis method and application of xanthene derivative

A synthesis method and derivative technology, applied in the field of fluorescent probes, can solve problems such as interference, and achieve the effects of low detection limit, sensitive response, and superior selectivity

Pending Publication Date: 2020-06-30
QUFU NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these fluorescent probes are effective in the quantitative detection of ONOO - The process is easily disturbed by various factors, such as different background probe concentrations, different cell microenvironments, etc.
Therefore, the design and development can detect ONOO in situ - Concentration of fluorescent probes remains a challenge

Method used

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  • Xanthene derivative for detecting ONOO&lt;-&gt; as well as synthesis method and application of xanthene derivative
  • Xanthene derivative for detecting ONOO&lt;-&gt; as well as synthesis method and application of xanthene derivative
  • Xanthene derivative for detecting ONOO&lt;-&gt; as well as synthesis method and application of xanthene derivative

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] One for detecting ONOO - The xanthene derivatives are characterized in that 9-(2-carboxyphenyl)-6(diethylamino)-1,2,3,4-tetrahydroxanthene is used as a fluorescent group to activate The C=C double bond is a reactive group; the xanthene derivative is preferably 9-(2-carboxyphenyl)-6(diethylamino)-4(4-benzylidene)-1,2 ,3,4-Tetrahydroxanthene, its chemical structure is:

[0062]

[0063] The present invention also includes a method for detecting ONOO - The synthetic method of the xanthene derivative, comprises the following steps:

[0064] (1) Add concentrated sulfuric acid to the flask, stir in an ice-water bath for 5 minutes, then add cyclohexanone, stir for 5 minutes at 0°C, and finally add 2-[(4-diethylamino)-2-hydroxybenzoyl] Benzoic acid, wherein the molar ratio of 2-[(4-diethylamino)-2 hydroxybenzoyl]benzoic acid and cyclohexanone is 1:2, react at (70-90)°C for 2h, after the reaction is completed, wait for the solution Cool to room temperature, pour into ice ...

Embodiment 2

[0068] f 494 Molecular Probes for ONOO - The responsive fluorescence spectroscopic properties of

[0069] (1) Prepare PBS buffered saline solution with pH=7.40 and a concentration of 10mM; prepare ONOO with a concentration of 1mM - the H 2 The standard solution of O, the DMSO solution of the probe that concentration is 1mM;

[0070] (2) First add 10uL of probe solution to a 5mL Ep tube, add 1mL of 10mM PBS buffered saline solution, then add 1mL of ethanol, and mix well. Take 0μL, 10μL, 20μL, 30μL, 40μL, 50μL, 60μL, 70uL, 80μL, 90μL and 100μL respectively, with a concentration of 1mM ONOO - A total of 11 parts of the solution were added to the Ep tubes.

[0071] (3) After reacting for 30 minutes, measure the fluorescence intensity by a fluorescence spectrophotometer, obtain the ratio of fluorescence intensity, and detect ONOO - The excitation wavelength of the fluorescence intensity is 425nm; get F 494 Fluorescence titration spectra of molecular probes, see Figure 7 . ...

Embodiment 3

[0074] Probes for ONOO - response time

[0075] (1) Prepare PBS buffered saline solution with pH=7.40 and a concentration of 10mM; prepare ONOO with a concentration of 1mM- H 2 The standard solution of O, the DMSO solution of the probe that concentration is 1mM;

[0076] (2) First add 10uL of probe solution to a 5mL Ep tube, add 1mL of 10mM PBS buffered saline solution, then add 1mL of ethanol, and mix well. Take 0 μL, 50 μL and 100 μL of ONOO at a concentration of 1 mM - A total of 3 parts of the solution were added to the Ep tube.

[0077] (3) After reacting for 30 minutes, measure the fluorescence intensity by a fluorescence spectrophotometer, obtain the ratio of fluorescence intensity, and detect ONOO - The excitation wavelength of the fluorescence intensity is 425 nm.

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Abstract

The invention relates to the technical field of fluorescent probes, in particular to a xanthene derivative for detecting ONOO<-> as well as a synthesis method and application of the xanthene derivative. 9-(2-carboxyphenyl)-6 (diethylamino)-1, 2, 3, 4-tetrahydroxanthene is used as a fluorescent group, and activated C = C double bonds are used as reaction active groups; the xanthene derivative is 9-(2-carboxyphenyl)-6 (diethylamino)-4 (4-benzylidene)-1, 2, 3, 4-tetrahydroxanthene. The xanthene derivative has excellent selectivity on ONOO <->; an obvious fluorescence signal can be read out; the response is sensitive, short response time to ONOO <-> is within several seconds, and detection limit is as low as (3.2 nm / L), which is far lower than the content of the two substances normally existing in cells, so that excessive ONOO <-> can be found in time, and in-situ detection can be carried out on the concentration of endogenous and exogenous ONOO <-> in living cells.

Description

technical field [0001] The invention relates to the technical field of fluorescent probes, in particular to a method for detecting ONOO - Xanthene derivatives and their synthesis methods and applications. Background technique [0002] Peroxynitrite (ONOO - ) is a strong oxidant and nucleophile with a short half-life, which belongs to the highly reactive reactive oxygen species (HROS). ONOO - It is composed of nitric oxide radicals (·NO) and superoxide radicals (·O 2 - ) is produced in the form of free diffusion, and the diffusion rate is (~1×1010M-1s-1). ONOO - It is mainly produced in macrophages, endothelial cells, platelets, leukocytes and neurons and plays an important role in the field of biochemistry. [0003] ONOO - It can directly oxidize / nitrify proteins, esters or nucleic acids in cells, destroy cell structure and function, and eventually lead to cell death. On the other hand, cells can also utilize the produced ONOO - to resist the invasion of microorgan...

Claims

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Application Information

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IPC IPC(8): C07D311/82C09K11/06G01N21/64G01N21/78
CPCC07D311/82C09K11/06G01N21/6428G01N21/6456G01N21/78C09K2211/1088
Inventor 尤进茂陆姣李赞谭江坤孙志伟常芮
Owner QUFU NORMAL UNIV