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Preparation method and application of ratio type fluorescent probe for peroxynitrite

A technology of peroxynitroso and fluorescent probes, applied in the field of fluorescent probes, can solve the problems of reducing probe sensitivity, interference, short analysis wavelength, etc., and achieve the effect of good spectral response performance

Active Publication Date: 2019-08-16
XIANGTAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, they have some problems: (1) These fluorescent probes have short analysis wavelengths, so they are easily interfered by autofluorescent signals generated by biomolecules in vivo, thereby reducing the sensitivity of the probes; (2) These fluorescent probes Most of them use the change of the intensity of a single fluorescence emission peak as the response signal, which is easily affected by the stability of the light source, the concentration of the probe and the factors of the instrument
However, no benzopyran-coumarin dye-based probes have been used to detect ONOO -

Method used

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  • Preparation method and application of ratio type fluorescent probe for peroxynitrite
  • Preparation method and application of ratio type fluorescent probe for peroxynitrite
  • Preparation method and application of ratio type fluorescent probe for peroxynitrite

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Synthesis of fluorescent probes

[0027] Synthetic route such as figure 1 . Synthesis of Coumarin 334: In a 100 mL round bottom flask, 8-hydroxyjulolidine-9-carbaldehyde (1.09 g, 5 mmol), ethyl acetoacetate (0.63 mL, 5 mmol) and piperidine (0.035 mL, 0.5mmol) was dissolved in 20.0mL dichloromethane, stirred and refluxed for 6h, stopped the reaction, and the solvent was removed by distillation under reduced pressure, and the thick product was subjected to column chromatography with dichloromethane eluent to obtain a dark yellow solid compound (0.85g, The yield is 60%), which is coumarin 334.

[0028] ONOO - Synthesis of fluorescent probes: In a 100 mL round bottom flask, coumarin 334 (0.42 g, 1.48 mmol) and 4-diethylamino ketoacid (0.46 g, 1.48 mmol) were dissolved in 8 mL of concentrated sulfuric acid, and the reaction mixture Stir at 90°C for 12h to stop the reaction, then cool the reaction mixture to room temperature, pour it into a beaker filled with 50g of ice w...

Embodiment 2

[0030] Fluorescent Probes and ONOO - Solution preparation

[0031] Preparation of probe solution: Weigh a certain amount of probe and dissolve it in methanol to make 1×10 -3 M probe solution. ONOO - Solution preparation: 0.70M H 2 o 2 solution, 0.60M HCl solution, 0.60M NaNO 2 The solution was mixed, and 1.5M NaOH solution was added rapidly, the excess H 2 o 2 Remove with manganese dioxide and store in a freezer at -20°C. Thaw before use, ONOO - The determination of concentration needs to measure the absorbance A of the solution at 302nm, and the calculation formula is: C ONOO -=A / 1.67 (mM).

Embodiment 3

[0033] Fluorescent Probes and ONOO - Determination of the fluorescence spectrum of the action

[0034] figure 2 for fluorescent probes with ONOO - The fluorescence spectrum of the action, the concentration of the fluorescent probe is 5 μM, ONOO - The concentrations are 0, 1.0, 2.0, 4.0, 6.0, 8.0, 10, 12, 14, 15 μM in turn. The excitation wavelength is fixed at 440nm, and the emission wavelength range is 450-820nm. The slit width is 5.0 nm / 5.0 nm, and the fluorescence measurement instrument used is a Hitachi F4600 fluorescence spectrophotometer. from figure 2 It can be seen that joining ONOO - Before, fluorescent probes had near-infrared (720nm) fluorescence emission peaks; adding ONOO - After that, a green emission peak appeared in the visible region (500nm). This is because the probe molecules are ONOO - Oxidation, resulting in cleavage, the smaller the conjugated structure, resulting in short-wavelength green fluorescence. And with ONOO - With the increase of th...

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Abstract

The invention relates to a preparation method and an application of a ratio type fluorescent probe for peroxynitrite (ONOO<->). The structural formula of the fluorescent probe is shown in the description. The invention provides the preparation method for synthesizing the fluorescent probe from 8-hydroxyjulolidine-9-carboxaldehyde, ethyl acetoacetate and 2-(4-diethylamino-2-hydroxybenzoyl)benzoicacid. The fluorescent probe is a near-infrared ratio type fluorescent probe for peroxynitrite. The fluorescent probe has a very high sensitivity to ONOO<->, and the fluorescence intensity ratio (F500 / F720) is enhanced by 72 times; the fluorescent probe has a very high selectivity to the ONOO<->, and is not interfered by other active oxygen, active nitrogen, active sulfur and biological thiols; and the fluorescent probe rapidly interacts with the ONOO<->, and the response time is within 7 min; and additionally, the fluorescent probe is applied to the detection of the content of peroxynitrite in living cells.

Description

technical field [0001] The invention belongs to the technical field of fluorescent probes, in particular to the preparation and application of peroxynitroso ratio fluorescent probes based on benzopyran-coumarin dyes. Background technique [0002] Peroxynitroso (ONOO - ) is produced by the superoxide anion radical (O 2 .-) reacts with nitric oxide (NO) to form a highly reactive oxygen species (C. H. Ischiropoulos, R. Radi, Nat. Rev. Drug Discovery, 2007, 6, 662-680). Recent studies have shown that ONOO - It can be used as a signaling molecule to participate in the regulation of many physiological activities such as cell growth, division, migration and apoptosis (B. Dickinson, C. Chang, Nat. Chem. Biol., 2011, 7, 504-511). And, thanks to ONOO - It has high oxidative properties to biomolecules, and has long been considered as a class of harmful molecules that damage DNA, proteins and other biomolecules and cause lipid peroxidation (T. Nagano, J. Clin. Biochem. Nutr., 2009...

Claims

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Application Information

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IPC IPC(8): C07D491/16C09K11/06G01N21/64
CPCC07D491/16C09K11/06C09K2211/1007C09K2211/1029C09K2211/1088G01N21/64G01N2021/6421
Inventor 李春艳江文丽
Owner XIANGTAN UNIV
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