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Hydroxyl radical ratio type fluorescent probe as well as preparation method and application thereof

A technology of fluorescent probes and free radicals, applied in the direction of fluorescence/phosphorescence, chemical instruments and methods, luminescent materials, etc., can solve the problems of limited applications, achieve high selectivity and sensitivity, and excellent mitochondrial localization ability

Active Publication Date: 2020-04-28
GUANGZHOU UNIVERSITY OF CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the excitation and emission wavelengths of hTA are located in the ultraviolet and blue regions, respectively, which greatly limits its application in living organisms.

Method used

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  • Hydroxyl radical ratio type fluorescent probe as well as preparation method and application thereof
  • Hydroxyl radical ratio type fluorescent probe as well as preparation method and application thereof
  • Hydroxyl radical ratio type fluorescent probe as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Synthesis of embodiment 1 fluorescent probe

[0061] The synthetic route of the fluorescent probe is as follows:

[0062]

[0063]

[0064] Wherein, above-mentioned reaction condition is: a.POBr 3 , DMF / DCM, 45°C; b.Pd(PPh 3 ) 4 , Na 2 CO 3 , toluene, 75°C, EtOH / NaOH reflux; c. 4-dimethyl-3-ethylpyrrole, POCl 3 , DCM, 0° C., then 4-carboxyphenylboronic acid, DCM, reflux; d. EDC, HOBt, DMF, room temperature.

[0065] (1) Compound 3 was synthesized according to the reported method

[0066]

[0067] Compound 2: black solid (84%). 1 H NMR (400MHz, CDCl 3 )δ7.53–7.49(m,2H),7.26–7.22(m,1H),7.18–7.13(m,2H),3.62(s,3H),3.27(s,3H).ESI[M+H] + m / z calcd.for[C 11 h 12 BrN 2 ]251.01,found 251.00[M+H] + .Compound 2 1 H NMR spectrum as figure 2 shown.

[0068] Compound 3: yellow solid (75%). 1 H NMR (400MHz, d 6 -DMSO)δ13.39(s,1H),10.24(s,1H),9.91(s,1H),8.18(s,1H),7.82(d,J=8.4Hz,1H),7.62–7.60(m ,1H),7.38–7.30(m,2H),7.21–7.19(s,1H),7.09(d,J=8.0Hz,1H),7.02–6....

Embodiment 2

[0080] The photophysical property of embodiment 2 compound 4

[0081] The photophysical properties of compound 4 were tested, and the test results were as follows Figure 11 shown. Among them, A is ACN / H 2 O is the fluorescence spectrum of compound 4 (20 μM) in mixed solution; B is the fluorescence spectrum of compound 4 (20 μM) in ACN / PBS 2:1 solution before and after OH reaction, excited at 580 nm; C is before and after OH reaction, The UV-vis absorption spectrum of corresponding compound 4, OH is produced by the following conditions (H 2 o 2 50 μM, EDTA-Fe 2+ 200 μM). Figure 11 The fluorescence spectrum in A shows that the fluorescence intensity of compound 4 is highly sensitive to solvent hydrophobicity / hydrophilicity changes. Compound 4 has weak fluorescence in pure water, but in acetonitrile (ACN) / H 2 The emission intensity in mixed solvents of O varies with ACN and H 2 O volume ratio increases. This feature provides a potential possibility for the probe's memb...

Embodiment 3

[0090] Example 3 Reaction Kinetics of Compound 4 and OH

[0091] Coumarin-3-carboxylic acid (Coumarin-CA) is a commercial fluorescent probe for ·OH detection. The present invention selects Coumarin-CA and compound 4 for comparison. • The reaction between OH and Coumarin-CA produces the fluorescent product 7-hydroxycoumarin-3-carboxylic acid with an emission peak at 454 nm under excitation at 400 nm. Therefore, check the fluorescence intensity I 454 The reaction can then be monitored. Meanwhile, the reaction of OH with compound 4 can reduce its absorbance at 610 nm, thus providing a simple method to monitor the reaction. When testing, Coumarin-CA (100 μM) or compound 4 (40 μM) was mixed with Fenton’s reagent EDTA-Fe2 + (100μM) / H 2 o 2 (10 mM) in sodium borate buffer (pH 9.0). In the competition experiment, Coumarin-CA and compound 4 were mixed in advance, and then reacted with ·OH. The reaction was carried out in a 96-well plate, and the emission intensity I under excit...

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Abstract

The invention relates to a novel fluorescence resonance energy transfer (FRET) probe Rho-Bob, which is constructed by connecting rhodamine B (Rho) with an N2O type benzopyrrole boron complex (Bobpy),and is used for OH ratio type fluorescence detection and imaging. Rho-Bob shows the characteristic of being sensitive to the hydrophilicity / hydrophobicity of the environment, and has excellent mitochondrial localization capability. Rho-Bob is successfully applied to intracellular .OH ratio type fluorescence imaging. The .OH can be generated by a Fenton reaction, and can also be generated by activation of intracellular drugs. The Rho-Bob probe has high selectivity and sensitivity to hydroxyl radicals, and the detection limit is as low as a nanomole level (680nM). According to the invention, .OHgenerated by artemisinin molecules in cell mitochondria is observed for the first time by using Rho-Bob, and endogenous hydroxyl radicals are found to exist in zebra fish gastrointestinal tracts (GI)under normal culture conditions for the first time. The invention not only provides the practical probe for .OH detection and imaging, but also provides an important thought for constructing novel ratiometric probes of other ROS.

Description

technical field [0001] The invention relates to a hydroxyl radical ratio fluorescent probe and its preparation method and application, belonging to the technical field of hydroxyl radical detection. Background technique [0002] Hydroxyl radical · OH is an active oxygen radical with strong reactivity and oxidative properties, and is also considered to be one of the most harmful free radicals to organisms. Excess OH can cause oxidative damage to most biomolecules (including lipids, proteins, nucleic acids, etc.). For example, OH can affect the formation of disulfide bonds in proteins, leading to protein degradation and misfolding. The occurrence and development of many diseases such as nervous system diseases and cancers are usually accompanied by the generation of ·OH. Of course, OH radicals are not always harmful and useless. For example, OH is an important therapeutic ROS during radiation therapy of cancer. Hydroxyl radicals (·OH) can be formed under physiological and ...

Claims

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Application Information

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IPC IPC(8): C07F5/02C09K11/06G01N21/31G01N21/64
CPCC07F5/02C09K11/06G01N21/6428G01N21/314G01N21/6458C09K2211/1088C09K2211/1044C09K2211/1055
Inventor 刘芳邓涛王小娟
Owner GUANGZHOU UNIVERSITY OF CHINESE MEDICINE
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