Fluorescent probe for distinguishing and detecting mercaptan and monitoring Cys/GSH metabolism and preparation method of fluorescent probe

A fluorescent probe and thiol technology, which is applied in the field of fluorescent probes and achieves the effects of convenient operation, simple detection means and simple synthesis method

Inactive Publication Date: 2020-09-29
SHANXI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are few studies on the metabolites of Cys / GSH

Method used

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  • Fluorescent probe for distinguishing and detecting mercaptan and monitoring Cys/GSH metabolism and preparation method of fluorescent probe
  • Fluorescent probe for distinguishing and detecting mercaptan and monitoring Cys/GSH metabolism and preparation method of fluorescent probe
  • Fluorescent probe for distinguishing and detecting mercaptan and monitoring Cys/GSH metabolism and preparation method of fluorescent probe

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Preparation and Characterization of CM-O-AC:

[0042] (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 32...

Embodiment 2

[0045] A PBS buffer solution with a pH of 7.4 and a concentration of 10 mM was prepared, and a 2 mM CM-O-AC fluorescent probe stock solution was prepared with dimethyl sulfoxide (DMSO); 2 mM Cys / Hcy and GSH solutions were prepared with distilled water. Add 2 mL of PBS buffer (pH=7.4) and 10 μL of fluorescent probe stock solution into a fluorescent cuvette, measure the fluorescence spectrum of the probe on a spectrofluorometer, and then gradually add different volumes of Cys / Hcy and GSH solutions, Measure its fluorescence spectrum on a fluorescence spectrophotometer. After adding Cys and Hcy, the probe has two new fluorescence emission peaks at 480nm and 625nm (excitation is 380nm). With the addition of Cys / Hcy, the fluorescence intensity gradually increases until basically no until the change occurs ( Figure 4 a,b); After adding GSH, the probe only had a new fluorescence emission peak at 625nm (excitation at 380nm) ( Figure 4 c). When the excitation was 450nm, the probe ad...

Embodiment 3

[0047] For Cys / Hcy, take the concentration of Cys / Hcy as the abscissa, and the fluorescence intensity of the probe at 480nm as the ordinate, draw the graph and perform linear fitting, and the regression equation of the probe is: y=11.659x+ 41.367 (y=11.008x+68.748), linear correlation coefficient R 2 =0.9950(R 2 =0.9969), the detection limit was 0.10 μM (0.02 μM). GSH takes the fluorescence intensity of the probe at 545nm as the ordinate, draws a graph and performs linear fitting, and the regression equation of the probe is: y=5.785x+19.507, and the linear correlation coefficient R 2 =0.9964, the detection limit was 0.04 μM. (See Figure 5 )

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Abstract

The invention provides a fluorescent probe for distinguishing and detecting mercaptan and monitoring Cys/GSH metabolism as well as a preparation method and application of the fluorescent probe. The Chinese name of fluorescent probe is 10-(diethylamino)-3-((7-(diethylamino)-3-formyl-2-oxo-2H-chromen-4-yl) oxy)-5, 6-dihydrobenzo [c] xanthene-12-onium perchlorate, the English name is 10-(diethylamino)-3-((7-(diethylamino)-3-formyl-2-oxo-2H-chromen-4-yl)oxy)-5,6-dihydrobenzo[c]xanthen-12- ium perchlorate,, which is named as CM-O-AC. The invention provides a red fluorescent probe with multiple binding sites, good water solubility and large Stokes shift. The probe displays three different emission channels for distinguishing and detecting Cys/Hcy and GSH, and is successfully applied to HeLa celland living body imaging. In addition, the probe can realize recognition of the Cys/GSH metabolite SO2 at a cellular level, and realizes reversibility of a fluorescence signal under the action of H2O2. The probe can be used as a visual biological metabolism tool to be applied to physiological and pathological processes. The detection process is simple, sensitive and rapid, and the detection resultis accurate.

Description

technical field [0001] The invention relates to a fluorescent probe, in particular to a fluorescent probe for distinguishing and detecting thiols and monitoring Cys / GSH metabolism, a preparation method and application thereof. Background technique [0002] The small molecule thiols cysteine ​​(Cys), homocysteine ​​(Hcy) and glutathione (GSH) containing sulfhydryl groups play a vital role in many physiological and pathological processes. Hcy is an intermediate product in the process of methionine metabolism to produce Cys. Hcy is mainly converted into cysteine ​​through cystathionine through transsulfuration in cells. Under aerobic conditions, Cys is catalyzed by cysteine ​​peroxidase (CDO) to generate cysteine ​​sulfinic acid, which is further activated by cysteine ​​sulfinic acid decarboxylase (CSD) and aspartate aminotransferase (AAT) catalyzed to generate neurotransmitter taurine and gas signal molecule sulfur dioxide respectively. GSH is one of the main antioxidant su...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D311/78C09K11/06G01N21/64
CPCC07D311/78C09K11/06G01N21/6402C09K2211/1088
Inventor 黄永飞阴彩霞霍方俊张永斌
Owner SHANXI UNIV
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