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A kind of fluorescent probe and preparation method thereof for differentiating and detecting cys/hcy and gsh

A technology of fluorescent probe and synthesis method, applied in the field of fluorescent probe, can solve the problem of different detection of thiols rarely reported, and achieve the effects of simple synthesis method, obvious detection signal and convenient operation.

Active Publication Date: 2021-07-27
SHANXI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Distinct detection of thiols is rarely reported in model plants

Method used

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  • A kind of fluorescent probe and preparation method thereof for differentiating and detecting cys/hcy and gsh
  • A kind of fluorescent probe and preparation method thereof for differentiating and detecting cys/hcy and gsh
  • A kind of fluorescent probe and preparation method thereof for differentiating and detecting cys/hcy and gsh

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Experimental program
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Effect test

Embodiment 1

[0032] Preparation and characterization of NBD-O-1

[0033] (1) Preparation of Compound 1: In a 100 ml round bottom flask, 4-diethyl amino hydrarse (1.93 g, 10 mmol), 6-hydroxy-1-tetrahemethoelene (1.62 g, 10 mmol) and high Hichlorous acid (3 mL) is dissolved in 20 ml of acetic acid and the mixture was refluxed for 1.5 hours. After cooling to room temperature, the solution was poured into a mixture of ethyl acetate (15 mL) and petroleum ether (15 mL). The precipitate was filtered and washed with ethanol, then dried in vacuo to give a pure deep purple solid compound 1 (2.88 g, yield: 90%). 1 HNMR (600MHz, DMSO-D 6 Δ11.11 (S, 1H), 8.63 (S, 1H), 8.16 (D, J = 8.6 Hz, 1H), 7.91 (D, J = 9.3 Hz, 1H), 7.41 (D, J = 9.3 Hz, 1H), 7.27 (S, 1H), 6.95 (D, J = 8.6Hz, 1H), 6.87 (S, 1H), 3.67 (Q, J = 6.9 Hz, 4H), 3.01 (S, 4H), 1.24 ( T, J = 7.0 Hz, 6H). 13 C NMR (150MHz, DMSO-D 6 ) δ164.73, 164.31, 158.24, 155.23, 148.36, 146.21, 132.03, 129.40, 12.70, 117.99, 117.67, 117.66, 116.16, 96.98, 25.11,...

Embodiment 2

[0036] (1) The fluorescent probe stock solution of 2 mM NBD-O-1 was prepared with dimethyl sulfoxide (DMSO); 2 mm Cys / Hcy and GSH solution were formulated with distilled water.

[0037] (2) 2ML CH 3 CN / PBS buffer (V / V = ​​2 / 8, pH = 7.4) and 10 μl of fluorescent probe stock solution Add a fluorescence composite vendor to measure the fluorescence spectrum of the probe on a fluorescent spectrophotometer, and then gradually add different volumes. CYS / HCY and GSH solution, the fluorescence spectrum was measured on a fluorescence spectrophotometer, and two new fluorescence emission peaks were added at 550 nm and 625 nm. As Cys / Hcy added fluorescence intensity gradually increased. Until the basics do not change ( Figure 4 (A), (b)); after the addition of GSH, the probe only appears at 625 nm, and the new fluorescence emission peak ( Figure 4 In (c)), distinguishing detection can be realized.

[0038] (3) For Cys, the fluorescence intensity at the Cys concentration of 550 nm is ...

Embodiment 3

[0040] In different fluorescent polyvisses, 2 ml of CH is added separately 3 CN / PBS buffer (V / V = ​​2 / 8, pH = 7.4) and 10 μl of fluorescent probe reserve, and Cys / Hcy and GSH were added to other amino acids at 50 μm and 20 μm, and 10 equivalents. Aqueous solution, including ALA, ASP, ASN, ARG, GLY, GLU, GLN, HIS, IIE, LEU, LYS, MET, PHE, PRO, SER, TYR, THR, TRP AND VAL, so that its final concentration is 100 μm, then Fluorescence spectroscopy on fluorescence spectrophotometer, see the measurement results Image 6 . Experiments have shown that these amino acids do not interfere with the detection of CYS / Hcy and GSH.

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Abstract

The invention provides a fluorescent probe for distinguishing and detecting Cys / Hcy and GSH, its preparation method and application. The Chinese name of the fluorescent probe is 10‑(diethylamino)‑3‑((7‑nitrobenzo[c][1,2,5]oxadiazol‑4‑yl)oxy)‑ 5,6‑Dihydrobenzo[c]xanthene12ium, named NBD‑O‑1. The invention provides a method for the detection of Cys / Hcy and GSH by fluorescent probe NBD-O-1, which is prepared in PBS (pH 7.4, containing 20% ​​CH 3 CN, v / v) solution, the qualitative distinction of Cys / Hcy and GSH by ultraviolet and fluorescence spectrometers is not interfered by common amino acids. More importantly, the probe can be applied to the detection of Cys / Hcy and GSH in animals and plants, and the detection process is simple, sensitive and rapid, and the detection result is accurate.

Description

Technical field [0001] The present invention relates to a fluorescent probe, specifically belonging to a fluorescence probe for detecting CYS / Hcy and GSH and a preparation method thereof. Background technique [0002] Sulfhydryl groups have many useful properties on animals, plants and micro-life. With the evolution of the biological process, many amino acids are produced, including three specific small molecule thiol: cysteine ​​(Cys), homocysteine ​​(Hcy) and glutathione (GSH), which are present Animals, plants and microorganisms. The same type cysteine ​​(HCY) binding serine is formed by cystatic ether under the action of cystide ether B-synthase (CBs), and then split into cysteine ​​by cytyside ether G-lysis (CSE) ( Cys). Finally, glutathione (GSH) is formed of glutamate cysteine ​​ligase (GCl) and glutathotypeicide (GS). Many studies have shown that abnormal concentrations of three thiol may lead to a variety of human diseases, such as cancer, liver damage, skin disease an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D413/12C09K11/06G01N21/64
CPCC07D413/12C09K11/06C09K2211/1048C09K2211/1088G01N21/6428G01N21/6486
Inventor 黄永飞阴彩霞张永斌霍方俊
Owner SHANXI UNIV
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