A kind of hydroxyl radical ratiometric fluorescent probe and its preparation method and application

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: 2022-05-24
GUANGZHOU UNIVERSITY OF CHINESE MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • A kind of hydroxyl radical ratiometric fluorescent probe and its preparation method and application
  • A kind of hydroxyl radical ratiometric fluorescent probe and its preparation method and application
  • A kind of hydroxyl radical ratiometric fluorescent probe and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Synthesis of Fluorescent Probes

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

[0062]

[0063]

[0064] Wherein, the above-mentioned reaction conditions are: a.POBr 3 , DMF / DCM, 45℃; b.Pd(PPh 3 ) 4 , Na 2 CO 3 , toluene, 75℃, 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) Synthesis of compound 3 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),7.02–6.98(m...

Embodiment 2

[0080] Photophysical Properties of Example 2 Compound 4

[0081] The photophysical properties of compound 4 were tested, and the test results were as follows Figure 11 shown. where A is ACN / H 2 Fluorescence spectrum of compound 4 (20 μM) in O 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 corresponding UV-vis absorption spectrum of compound 4, OH was generated by the following conditions (H 2 O 2 50μM, EDTA-Fe 2+ 200 μM). Figure 11 The fluorescence spectrum in A shows that the fluorescence intensity of compound 4 is highly sensitive to changes in solvent hydrophobicity / hydrophilicity. Compound 4 shows weak fluorescence in pure water, but in acetonitrile (ACN) / H 2 The emission intensity of O mixed solvent varies with ACN and H 2 It increases with the increase of the O volume ratio. This property provides a potential possibility for the...

Embodiment 3

[0090] Example 3 Study on the 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 to compare with compound 4. • The reaction between OH and Coumarin-CA yielded the fluorescent product 7-hydroxycoumarin-3-carboxylic acid with an emission peak at 454 nm upon 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 absorption value at 610 nm, thus providing a simple method to monitor the reaction. Coumarin-CA (100 μM) or compound 4 (40 μM) was mixed with Fenton’s reagent EDTA-Fe2 during the test + (100μM) / H 2 O 2 (10 mM) in sodium borate buffer (pH 9.0). In the competition experiment, Coumarin-CA and compound 4 were premixed, and then the OH reaction was carried out. The reaction was performed in a 96-well plate, and the emission int...

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Abstract

The present invention is by rhodamine B (Rho) and N 2 O-type benzopyrrole boron complexes (Bobpy) were linked to construct a novel fluorescence resonance energy transfer (FRET) probe Rho‑Bob for ratiometric fluorescence detection and imaging of OH. Rho‑Bob exhibits environmental affinity / hydrophobicity sensitivity and excellent mitochondrial localization ability. Rho‑Bob has been successfully applied for ratiometric fluorescence imaging of intracellular OH. These OH can be produced by Fenton reaction or by intracellular drug activation. The Rho‑Bob probe has high selectivity and sensitivity to hydroxyl radicals, and the detection limit is as low as nanomolar level (680nM). The present invention uses Rho-Bob to observe for the first time the ·OH produced by artemisinin molecules in the mitochondria of cells, and to discover for the first time that endogenous hydroxyl radicals exist in the gastrointestinal tract (GI) of zebrafish under normal culture conditions. The present invention not only provides a practical probe for ·OH detection and imaging, but also provides an important idea for constructing novel ratiometric probes for other ROS.

Description

technical field [0001] The invention relates to a hydroxyl radical ratio fluorescent probe, a preparation method and application thereof, and belongs to the technical field of hydroxyl radical detection. Background technique [0002] Hydroxyl radical·OH is a highly reactive and oxidizing reactive oxygen radical, and is also considered to be one of the most harmful free radicals to living 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 cancer 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 radiotherapy of cancer. Hydroxyl radicals (·OH) can be formed under physiological and pathological cond...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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