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Reduced glutathione fluorescent probe with pyrazoline as maternal body

A glutathione and fluorescent probe technology, applied in the field of fluorescent probes, can solve the problem of lack of specificity of reduced glutathione, and achieve the effects of improving high selectivity, high sensitivity, and enhancing membrane permeability

Inactive Publication Date: 2014-02-26
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The authorized patents [CN200710065564.3], [CN200810020777.9] and [CN201010558433.0] can realize the selection of active thiols in organisms, but lack the specificity of reduced glutathione
After searching, the application of the synthesized 2-(1,5-diphenyl-4,5-dihydro-1H-pyrazol-3-yl)phenylacrylate as a fluorescent probe for reduced glutathione is also No report

Method used

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  • Reduced glutathione fluorescent probe with pyrazoline as maternal body
  • Reduced glutathione fluorescent probe with pyrazoline as maternal body
  • Reduced glutathione fluorescent probe with pyrazoline as maternal body

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Synthesis of 2–(1,5-diphenyl-4,5-dihydro-1H-pyrazole-3-one from 1-(2-hydroxyphenyl)-3-phenylprop-2-en-1-one Base) phenyl acrylate reacts with reduced glutathione to generate the reaction process of pyrazoline glutathione derivative as shown in the following formula:

[0023]

[0024] Add chalcone (0.224g, 1.0mmol) (1), 0.162g phenylhydrazine (2) (1.5mmol), sodium hydroxide (0.12g, 3.0mmol) to 15mL of absolute ethanol, heat to reflux, continue React for about four hours, cool down after the reaction is complete, add 100mL of water to the residue, adjust the pH value to 5, continue to extract the water phase with dichloromethane three times, combine the organic phase, dry with anhydrous magnesium sulfate, petroleum ether: dichloromethane = 5:1 is the eluent, the light yellow solid (3) 0.144g in column chromatography, melting point: 167-168°C, yield: 45.8%.

[0025] H NMR spectrum determination: 1 H NMR (300MHz, CDCl 3 ):δ3.25(dd,1H,J=7.5,17.1Hz,4-H trans ),3.95(dd,...

Embodiment 2

[0034] Add 10 mL of prepared 2–(1,5-diphenyl-4,5-dihydro-1H-pyrazol-3-yl)phenylacrylate derivative to PBS / ethanol buffer solution with a micro-syringe Add 10 equivalents of Cys, Hcy, GSH, Arg, Asp, Glu, Gly, His, Lys, Ser, Thr, Trp, Tyr, Val, KNO to the solution of (20mM PBS, pH=7.4, 3:7, volume ratio) 3 , Ca(NO 3 ) 2 ,NaNO 3 ,Mg(NO 3 ) 2 , Zn(NO 3 ) 2 ,Fe(NO 3 ) 3 , the aqueous solution of hydrogen peroxide and glucose, after 12 hours of action, the fluorescence spectrophotometry test shows that 2–(1,5-diphenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl Acrylate derivatives have good selectivity to reduced glutathione, and the control before and after adding reduced glutathione shows a strong fluorescence enhancement effect.

[0035] see results figure 1 .

Embodiment 3

[0037] Intracellular fluorescence imaging test:

[0038] The experiment was divided into two groups, control group A and B. Control group A: HeLa cells were treated with 3.0 μM 2–(1,5-diphenyl-4,5-dihydro-1H-pyrazole at 37°C -3-yl) in the cell culture medium of phenyl acrylate derivatives and cultivated for 0.5 hours; Control group B: HeLa cells were soaked in cell culture medium containing 100mM N-ethylmaleimide for 8 hours, washed with PBS buffer solution After three times, culture in the cell culture medium added with 3.0μM 2-(1,5-diphenyl-4,5-dihydro-1H-pyrazol-3-yl)phenylacrylate derivative for 0.5 hours .

[0039] Fluorescence imaging showed that 2–(1,5-diphenyl-4,5-dihydro-1H-pyrazol-3-yl)phenylacrylate derivatives penetrated well into cells. In the control group A cells showed strong fluorescence. Control group B showed no fluorescence. See Figure 6 .

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Abstract

The invention discloses a reduced glutathione fluorescent probe with pyrazoline as a maternal body. The probe is 2-(1, 5-diphenyl-4, 5-dihydro-1H-pyrazole-3-yl)phenyl acrylate. The probe provided by the invention can selectively interact with reduced glutathione, has a fluorescence enhancement effect, can be used for analysis through fluorophotometry and can realize trace detection of reduced glutathione. With high sensitivity, the reduced glutathione fluorescent probe is suitable for wide popularization.

Description

technical field [0001] The invention relates to a fluorescent probe, in particular to a reduced glutathione fluorescent probe with pyrazoline as a parent. Background technique [0002] Pyrazoline derivatives have large molar extinction coefficients and high quantum yields, and are often modified for fluorescent probes and labeled biomolecules. In recent years, the use of fluorescence enhancement effect to detect reduced glutathione in living cells has been continuously developed. [0003] Glutathione is the most non-protein sulfhydryl group in cells and plays an important role in maintaining the physiological functions of cells, including intracellular redox reactions, foreign body metabolism, intracellular signal transduction and gene regulation. More specifically, glutathione is the most abundant small molecule biothiol (1-10mM) in cells, and there is a redox balance between reduced glutathione and oxidized glutathione. Studies have found that reduced glutathione plays a...

Claims

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Application Information

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IPC IPC(8): C07D231/06G01N21/64
CPCC07D231/06C09K11/06C09K2211/1044G01N21/6428
Inventor 赵宝祥苗俊英王胜清吴清华
Owner SHANDONG UNIV
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