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A Mitochondria-targeted Proportional Fluorescent Probe for Hypochlorous Acid Based on Fluorescence Resonance Energy Transfer Mechanism and Its Application

A fluorescence resonance energy, ratio fluorescent probe technology, applied in the ratio fluorescent probe and its application, the hypochlorous acid ratio fluorescent probe field, can solve the patent, limitation, and lack of structural diversity of the hypochlorous acid ratio fluorescent probe. issues of sex

Inactive Publication Date: 2019-03-26
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Chinese patents such as [201510794522], [201610323154], and [201610263094] involve the selective detection of hypochlorous acid, but they lack structural diversity and have limitations in application. So far, there is no patent for a hypochlorous acid ratio fluorescent probe targeting mitochondria

Method used

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  • A Mitochondria-targeted Proportional Fluorescent Probe for Hypochlorous Acid Based on Fluorescence Resonance Energy Transfer Mechanism and Its Application
  • A Mitochondria-targeted Proportional Fluorescent Probe for Hypochlorous Acid Based on Fluorescence Resonance Energy Transfer Mechanism and Its Application
  • A Mitochondria-targeted Proportional Fluorescent Probe for Hypochlorous Acid Based on Fluorescence Resonance Energy Transfer Mechanism and Its Application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] 4-(4-(7-(diethylamino)-2-oxo-2H-benzopyran-3-carbonyl)piperazinyl-1)benzaldehyde (1) was synthesized by a known method, and then the compound 1 is reacted with 3-methylbenzothiazole salt to obtain the mitochondrial-targeted hypochlorous acid ratio fluorescent probe based on the fluorescence resonance energy transfer mechanism of the present invention. The probe can undergo an oxidation reaction with hypochlorous acid to release a fluorescent 7-diethylaminocoumarin derivative 1.

[0026] The reaction formula is as follows:

[0027]

[0028] 4-(4-(7-(Diethylamino)-2-oxo-2H-benzopyran-3-carbonyl)piperazinyl-1)benzaldehyde (1,20mmol), 2-methyl Benzothiazole salt (2, 20 mmol) was refluxed in 50 mL of absolute ethanol, reacted for 3 hours, cooled to room temperature, concentrated, and subjected to silica gel column chromatography to obtain a red powder of the probe (3) of the present invention, melting point: 182 ° C, yield The rate is 87%.

[0029] H NMR spectrum deter...

Embodiment 2

[0033] The PBS / DMF buffer (pH=7.0, v / v=9:1, 10μM), quantitatively add 10μM KNO 3 ,Ba(NO 3 )2 ,AlCl 3 ,OCl - ,t-BuO ,H 2 o 2 ,O 2 - ,OONO - ,NO·, 1 o 2 ,t-BuOOH,NaNO 2 , Na 2 CO 3 ,NaCH 3 CO 2 , Na 2 S,NaSO 4 Fluorescence spectrophotometry and UV spectrophotometer tests were carried out after 3 hours of action, which showed that the probe had good selectivity to hypochlorous acid, and the comparison before and after adding hypochlorous acid showed ultraviolet absorption and fluorescence intensity of two emission wavelengths significant changes, see figure 2 , 3.

Embodiment 3

[0035] Intracellular fluorescence imaging:

[0036] RAW264.7 cells were first cultured with lipopolysaccharide (LPS, 1 μg / ml) for 12 h, then added phorbol ester (PMA, 1 μg / mL) in serum-free medium to continue incubation for 30 min, and then the probe (2 μM ) for 30 min. After washing the cells with PBS (pH 7.20) for 3 times, image them with a laser confocal microscope (LSM 700), collect fluorescence in the wavelength range of 405-550nm (blue light) and 560-700nm (red light), and obtain bright field images, fluorescence The image and the superimposed image of the two were then counted (*p Figure 4 .

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Abstract

The invention discloses a fluorescence resonance energy transfer (FRET) mechanism based ratio fluorescent probe for hypochlorous acid ratio in a targeting mitochondrion. The ratio fluorescent probe is characterized in that coumarin fluorophore is used as an energy donor of the ratio fluorescent probe, and styryl benzothiazole is used as an energy receptor, so as to form a fluorescence resonance energy transfer combination pair of which the chemical structural formula is shown as Formula (I). The invention also discloses application of the ratio florescent probe in detection of hypochlorous acid in PBS (Poly Butylenes Succinate) / DMF (Dimethyl Formamide) buffering liquid and biological cells. The experiment shows that the probe can selectively act on hypochlorous acid; the solution turns from orange red to be colorless and can be recognized by naked eye; the fluorescence turns from orange red to green, and the fluorescence is obvious in ratio response; the ratio can also be analyzed by ultraviolet absorbing and fluorospectrophotometry. With the adoption of the ratio fluorescent probe, the trace in-vitro pypocholoride can be detected with high sensitivity, and the ratio imaging in the cells can be performed; the ratio fluorescent probe is expected to play a role in the clinical medicine and industrial production and has important application value.

Description

technical field [0001] The invention relates to a proportional fluorescent probe and its application, in particular to a mitochondrial-targeted hypochlorous acid proportional fluorescent probe and its application based on the fluorescence resonance energy transfer (FRET) mechanism; it belongs to the organic small molecule fluorescent probe technology field. Background technique [0002] Hypochlorous acid is one of the important reactive oxygen species in various biological processes in organisms. Endogenous hypochlorous acid is mainly produced by hydrogen peroxide and chloride ions through oxidase catalysis. As a bactericide in living organisms, hypochlorous acid plays an important role in the immune system. Excess or deficiency of endogenous hypochlorous acid may lead to diseases, such as arthritis, arteriosclerosis, and tumors. Therefore, it is very important to realize rapid, sensitive and highly selective detection of hypochlorous acid in organisms. [0003] Mitochondr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D417/12C09K11/06G01N21/64G01N21/33
CPCC07D417/12C09K11/06C09K2211/1007C09K2211/1037C09K2211/1044C09K2211/1088G01N21/33G01N21/6486
Inventor 赵宝祥苗俊英武文丽赵璇
Owner SHANDONG UNIV
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