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Preparation and applications of novel fluorescent probe capable of specifically recognizing cysteine

A technology of cysteine ​​and fluorescent probes, which is applied in the application field of fluorescent probes to detect cysteine ​​in vitro and in living cells, and can solve the problems of good penetration and large Stokes shift, etc. Achieve the effect of good selectivity, large Stokes displacement and low cost

Inactive Publication Date: 2017-07-14
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the structural similarity between cysteine ​​and homocysteine, it is difficult for this type of fluorescent probe to specifically distinguish cysteine ​​and homocysteine, making its application in biological samples or living bodies limited. Therefore, it is meaningful to develop probes that can specifically recognize cysteine ​​with good cell penetration, large Stokes shift, and near-infrared emission wavelengths.

Method used

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  • Preparation and applications of novel fluorescent probe capable of specifically recognizing cysteine
  • Preparation and applications of novel fluorescent probe capable of specifically recognizing cysteine
  • Preparation and applications of novel fluorescent probe capable of specifically recognizing cysteine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1: the synthesis of intermediate product

[0019] Dissolve 1,4-diethyl-1,2,3,4-tetrahydroquinoxaline salicylaldehyde (0.1172g, 0.5mmol) in 5mL of anhydrous dichloromethane, then add triethylamine (0.14ml , 1.0mmol) and acryloyl chloride (0.1358g, 1.5mmol), protected by argon, reacted for 1h under stirring at room temperature, quenched the reaction with distilled water, extracted with dichloromethane, dried over anhydrous sodium sulfate, and removed dichloromethane by rotary evaporation. Separation by column chromatography yielded a brown oil. Yield: 0.1261 g. Yield: 87.5%. 1 H NMR (500MHz, CDCl3)δ H 9.84(s,1H),6.97(s,1H),6.64(dd,J=17.3,1.2Hz,1H),6.37(dd,J=17.3,10.5Hz,2H),6.21(s,1H),6.08 -6.01(m,1H),3.51(t,2H),3.40-3.35(m,4H),3.24(t,2H),1.22-1.17(m,6H). 13 CNMR(125MHz, CDCl3)δ C 186.1, 164.9, 147.6, 142.0, 132.9, 132.3, 127.8, 116.5, 108.7, 102.5, 47.4, 45.8, 45.3, 44.6, 10.7, 10.0.

Embodiment 2

[0020] Embodiment 2: the synthesis of probe molecule

[0021] The product obtained in the previous step (0.0864g, 0.3mmol) was dissolved in 7mL of anhydrous dichloromethane, triethylamine (0.042mL, 0.3mmol) was added, and malononitrile (0.0198g, 0.3mmol) was added. , Stir at room temperature for 0.5h, quench the reaction with distilled water, extract with dichloromethane, dry over anhydrous sodium sulfate, spin evaporate the solvent, obtain the product by column chromatography, dry in vacuo overnight to obtain a red solid. Yield: 0.0108 g. Yield: 10%. The structure of the probe molecule is characterized as follows: 1 H NMR (500MHz, CDCl3)δ H 7.56(d, J=2.6Hz, 2H), 6.68(dd, J=17.3, 0.9Hz, 1H), 6.38(dd, J=17.3, 10.5Hz, 1H), 6.28(s, 1H), 6.14(dd ,J=10.5,0.9Hz,1H),3.60(t,2H),3.44(q,J=7.2,2H),3.39(q,J=7.2,2H),3.29(t,2H),1.24(t ,6H). 13 C NMR (125MHz, CDCl3)δ C 164.2, 149.8, 146.7, 143.4, 134.0, 132.6, 127.1, 116.6, 115.1, 112.2, 106.8, 102.5, 70.9, 47.7, 46.0, 45.6, 44.4, 10...

Embodiment 3

[0022] Embodiment 3: The present invention: the application of fluorescent probe

[0023] Dissolve the probe in the buffer solution (V 乙腈 / V PBS =2 / 8, pH=7.4) to prepare 1.0×10-5 mol / L solution, adding amino acids (Ala, Val, Try, Phe, His,, Iso, Ser, Asp, Lys, Arg, Gly, Met, Tyr, Glu, Thr) to the solution did not cause a change in fluorescence, adding Human amino acids (Cys, Hcy, GSH) cause fluorescence changes, and this fluorescent probe shows high sensitivity and high selectivity for cysteine ​​recognition. When cysteine ​​coexists with interfering substances (Ala, Val, Try, Phe, His,, Iso, Ser, Asp, Lys, Arg, Gly, Met, Tyr, Glu, Thr, Hcy, GSH), the probe does not Affected by interference factors, it shows a strong anti-interference ability. The probe molecule responds quickly to cysteine, and the change of fluorescence can be observed within 1 minute. The probe molecule can selectively recognize cysteine ​​in the range of pH 7 to 11, showing the application range of bio...

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Abstract

The present invention discloses a novel fluorescent probe capable of specifically recognizing cysteine, wherein the molecular structure formula is defined in the specification. According to the present invention, the fluorescent probe can distinguish the mercapto-containing cysteine from the mercapto-free amino acid region, can distinguish the cysteine from the homocystine and the glutathione having the similar structure, emits red light during cysteine detection, can reduce the background interference and the damage of light on tissue cells in biological applications, exhibits great Stokes shift, can reduce self-absorption so as to improve the sensitivity, can be used for the fluorescence sensing analysis of the cysteine in environments or biological samples, and has advantages of good selectivity to cysteine, high sensitivity, strong interference resistance, and good application prospect.

Description

technical field [0001] The present invention relates to the technical field of chemical analysis and detection, in particular to a method for preparing a novel fluorescent probe that can specifically recognize cysteine ​​and the application of the fluorescent probe in the detection of cysteine ​​in vitro and in living cells . Background technique [0002] Biothiols are important components of many proteins and small molecules, and play an important role in the process of cell life. Biothiols include cysteine ​​(Cysteine, Cys), homocysteine ​​(Homocysteine, Hcy) and glutathione (Glutataione, GSH) and so on. Cysteine ​​is not only the precursor of glutathione, acetyl coenzyme and taurine, but also the provider of sulfur ligands in the sulfur-iron complex of organisms. The lack of cysteine ​​in the human body will lead to slow growth, hair loss Symptoms such as depigmentation, edema, lethargy, liver function damage, muscle relaxation, and physical weakness. Abnormal concentr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D241/38C09K11/06G01N21/64
CPCC07D241/38C09K11/06C09K2211/1044G01N21/6428
Inventor 宋相志杨大雷刘兴江杨雷齐风佩
Owner CENT SOUTH UNIV
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