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Fluorescent probe for detecting tyrosinase as well as preparation method and application of fluorescent probe

A tyrosinase and fluorescent probe technology is applied in the field of detecting tyrosinase fluorescent probes and their preparation, and can solve the problem of poor tyrosine detection effect, high probe fluorescence background, small Stokes shift, etc. problem, to achieve the effect of strong anti-interference ability, low fluorescence background, and large Stokes shift

Pending Publication Date: 2022-05-20
WUHAN INSTITUTE OF TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to overcome above-mentioned technical deficiencies, propose a kind of detection tyrosinase fluorescent probe and its preparation method and application, solve the problem of high fluorescent background of probe, poor detection effect of probe to tyrosine in the prior art, Technical problems of small Stokes displacement and poor anti-interference ability

Method used

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  • Fluorescent probe for detecting tyrosinase as well as preparation method and application of fluorescent probe
  • Fluorescent probe for detecting tyrosinase as well as preparation method and application of fluorescent probe
  • Fluorescent probe for detecting tyrosinase as well as preparation method and application of fluorescent probe

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preparation example Construction

[0034] A second aspect of the present invention provides a method for preparing a fluorescent probe for detecting tyrosinase, comprising the following steps:

[0035] 7-Hydroxy-4-(trifluoromethyl)coumarin (HFC) and m-hydroxybenzyl bromide were refluxed under the effect of organic solvent and alkali, and then separated and purified to obtain the detection tyrosinase fluorescent probe (HFC -TYR). Concrete synthetic route is as follows:

[0036]

[0037] In the present invention, the mol ratio of 7-hydroxyl-4-(trifluoromethyl)coumarin, alkali and m-hydroxybenzyl bromide is 1:(2~4):(1~3), further 1:3: 2.

[0038] In the present invention, the organic solvent is at least one of acetonitrile, dimethylformamide (DMF), and anhydrous dichloromethane, and the base is at least one of anhydrous potassium carbonate, cesium carbonate, triethylamine, and pyridine.

[0039] In the present invention, the temperature of the reflux reaction is 40-70°C, further 60°C; the time of the reflux ...

Embodiment 1

[0046] A preparation method for detecting tyrosinase fluorescent probe, the specific steps are as follows:

[0047] Weigh HFC (150mg, 0.65mmol) and anhydrous potassium carbonate (269.52mg, 1.95mmol) in a 25mL round bottom flask, add 7mL of anhydrous acetonitrile to dissolve, stir at room temperature for 30 minutes, dissolve m-hydroxybenzyl bromide with 3mL of anhydrous acetonitrile (243.53mg, 1.30mmol), added dropwise, reflux at 60°C for 12 hours, stop the reaction after the reaction of the raw material HFC, cool to room temperature, add ethyl acetate and water for extraction, collect ethyl acetate layer samples, add Drying over sodium sulfate water, rotary evaporation, removal of solvent, to obtain the crude product, the crude product was subjected to silica gel column chromatography (CH 2 Cl 2 :PE=2:1) ​​to obtain a white solid, wash the solid with anhydrous ether, centrifuge, and separate the solid from the liquid. The resulting solid is HFC-TYR with higher purity.

[004...

Embodiment 3

[0055] Fluorescent spectrograms of the fluorescent probes obtained in Test Example 1 reacting with different concentrations of tyrosinase:

[0056] Add 1 μL of the stock solution of the above compound HFC-TYR, PBS buffer solution (999, 995.67, 992.33, 985.67, 979, 972.33, 965.67, 959, 945.67, 932.33, 919, 899, 879, 859, 832.33, 799 μL) and different volumes (0, 3.33, 6.67, 13.33, 20, 26.67, 33.33, 40, 53.33, 66.67, 80, 100, 120, 140, 166.67, 200 μL) of the above tyrosinase stock solutions, The corresponding working concentrations of tyrosinase are 0, 5, 10, 20, 30, 40, 50, 60, 80, 100, 120, 150, 180, 210, 250, 300 U / mL. After 12 hours of reaction, test Its fluorescence spectrum, such as Figure 5 As shown, the fluorescence intensity of the reaction system at 505 nm increases with the concentration of tyrosinase, indicating that the probe can detect different concentrations of tyrosinase.

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Abstract

The invention discloses a fluorescent probe for detecting tyrosinase as well as a preparation method and application of the fluorescent probe, and belongs to the technical field of chemical analysis and detection. The fluorescent probe for detecting tyrosinase has the following structural formula: the fluorescent probe prepared by the invention has the advantages of low fluorescent background, good chemical stability, good water solubility, high selectivity, large Stokes shift, strong anti-interference capability, high sensitivity, good detection effect and the like; the prepared fluorescent probe is in a fluorescence quenching state, and shows strong fluorescence after being combined with tyrosinase for recognition, and the fluorescence is enhanced by about 178 times; the fluorescent probe prepared by the invention not only can be used for in-vitro detection of tyrosinase activity, but also can be applied to screening of tyrosinase inhibitors, and has important significance on treatment of tyrosinase-related diseases; the fluorescent probe prepared by the invention can be used for detection and imaging of endogenous tyrosinase in cells and zebra fish, and is successfully used for detection and imaging of tyrosinase at cellular level and biological level.

Description

technical field [0001] The invention relates to the technical field of chemical analysis and detection, in particular to a fluorescent probe for detecting tyrosinase and its preparation method and application. Background technique [0002] Enzymes are important biological macromolecules and catalysts of human metabolism. It is of great significance to monitor the enzyme activity in biological samples in real time and accurately. Tyrosinase is a ubiquitous copper-containing oxidase that is widely found in plants, animals and fungi. Tyrosinase, which catalyzes the formation of melanin, is the rate-limiting enzyme of melanin production and is widely recognized as an important biomarker, whose metabolic abnormalities are associated with serious diseases, for example, overexpression of tyrosinase can lead to The generation of malignant melanoma, people who lack melanin will suffer from albinism or vitiligo. In addition, tyrosinase is related to mental diseases, such as Parkinson...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D311/16C09K11/06G01N21/64
CPCC07D311/16C09K11/06G01N21/6428G01N21/6486C09K2211/1007C09K2211/1088Y02P20/55
Inventor 孙琦郭芸李想罗晓刚
Owner WUHAN INSTITUTE OF TECHNOLOGY
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