Fluorescent probe for distinguishing detection of Cys/Hcy and GSH and preparation method of fluorescent probe

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 effect of simple synthesis method, convenient operation and good metabolic pathway

Active Publication Date: 2019-09-06
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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  • Fluorescent probe for distinguishing detection of Cys/Hcy and GSH and preparation method of fluorescent probe
  • Fluorescent probe for distinguishing detection of Cys/Hcy and GSH and preparation method of fluorescent probe
  • Fluorescent probe for distinguishing detection of Cys/Hcy and GSH and preparation method of fluorescent probe

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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 100ml round bottom flask, 4-diethylamino salicylaldehyde (1.93g, 10mmol), 6-hydroxyl-1-tetralone (1.62g, 10mmol) and high Chloric acid (3ml) was dissolved in 20mL 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 (15ml) and petroleum ether (15ml). The precipitate was filtered and washed with ethanol, then dried in vacuo to obtain pure dark 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=8.6Hz,1H),7.91(d,J=9.3Hz,1H),7.41(d,J=9.3Hz, 1H), 7.27(s, 1H), 6.95(d, J=8.6Hz, 1H), 6.87(s, 1H), 3.67(q, J=6.9Hz, 4H), 3.01(s, 4H), 1.24( t,J=7.0Hz,6H). 13 C NMR (150MHz, DMSO-d 6 )δ164.73, 164.31, 158.24, 155.23, 148.36, 146.21, 132.03, 129.40, 120.70, 117.99, 117.67, 117.66, 116.16, 96.14, 45.71, 40.52, 26.98, 25.191.zI + calcd for 320...

Embodiment 2

[0036] (1) Prepare 2 mM NBD-O-1 fluorescent probe stock solution with dimethyl sulfoxide (DMSO); prepare 2 mM Cys / Hcy and GSH solutions with distilled water, respectively.

[0037] (2) 2mL CH 3 CN / PBS buffer solution (v / v=2 / 8, pH=7.4) and 10 μL of fluorescent probe stock solution were added to a fluorescent cuvette, and the fluorescence spectrum of the probe was measured on a fluorescence spectrophotometer, and then gradually added in different volumes Cys / Hcy and GSH solution, measured its fluorescence spectrum on a fluorescence spectrophotometer, after adding Cys / Hcy, the probe appeared two new fluorescence emission peaks at 550nm and 625nm, and the fluorescence intensity gradually increased with the addition of Cys / Hcy Until there is basically no change ( Figure 4 Middle (a), (b)); and after adding GSH, the probe only has a new fluorescence emission peak at 625nm ( Figure 4 Middle (c)), so distinguishing detection can be achieved.

[0038] (3) For Cys, take the Cys con...

Embodiment 3

[0040] In different fluorescent cuvettes, add 2mL CH 3 CN / PBS buffer (v / v=2 / 8, pH=7.4) and 10 μL fluorescent probe stock solution, then add Cys / Hcy and GSH respectively to make the final concentrations 50 μM and 20 μM, and 10 equivalents of other amino acids Aqueous solution, including Ala, Asp, Asn, Arg, Gly, Glu, Gln, His, IIe, Leu, Lys, Met, Phe, Pro, Ser, Tyr, Thr, Trp and Val, to make the final concentration of 100μM, and then in Measure the fluorescence spectrum on the fluorescence spectrophotometer, see the measurement results Figure 6 . Experiments have proved that these amino acids do not interfere with the differential detection of Cys / Hcy and GSH by the probe.

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Abstract

The invention provides a fluorescent probe for distinguishing detection of Cys/Hcy and GSH and a preparation method and application of the fluorescent probe. The English name of the fluorescent probeis 10-(diethylamino)-3-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)oxy)-5,6-dihydrobenzo[c]xanthen-12-ium, and the fluorescent probe is called as NBD-O-1. The invention provides a method for detecting theCys/Hcy and GSH by the fluorescent probe NBD-O-1. In a PBS solution with the pH of 7.4 and v/v of 2/8 and containing 20% CH3CN, the qualitative distinguishing detection of the Cys/Hcy and GSH is achieved through an ultraviolet and fluorescent spectrometer without the interference of common amino acids. More importantly, the probe can be applied to the detection of the Cys/Hcy and GSH in animals and plants, the detection process is convenient, sensitive and rapid, and a detection result is accurate.

Description

technical field [0001] The invention relates to a fluorescent probe, in particular to a fluorescent probe for distinguishing and detecting Cys / Hcy and GSH, a preparation method and application thereof. Background technique [0002] Mercapto has many useful properties for animals, plants and microscopic life. With the evolution of biological processes, many amino acids have been produced, including three specific small molecule thiols: cysteine ​​(Cys), homocysteine ​​(Hcy) and glutathione (GSH), which exist in animals, plants and microorganisms. Homocysteine ​​(Hcy) combines with serine to form cystathionine under the action of cystathionine b-synthase (CBS), which is then split into cysteine ​​( Cys). Ultimately, glutathione (GSH) is formed by glutamate cysteine ​​ligase (GCL) and glutathione synthase (GS). Many studies have shown that abnormal concentrations of the three thiols may lead to various human diseases such as cancer, liver damage, skin diseases and weakness,...

Claims

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Application Information

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Patent Type & Authority Applications(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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