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Cys fluorescence sensor based on rhodamine B, preparing method and application

A fluorescent sensor and reaction technology, applied in the field of biochemistry, can solve the problems of interference, selective detection of Cys, not particularly good, long response time, etc., and achieve good photostability, convenient post-processing, and high quantum yield.

Inactive Publication Date: 2016-12-14
NANJING UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Literature 1 (Lin S, Yoon D, Ha D, et al. Caged rhodamine-based fluorescent probe for biothiol: Selective detection of cysteine ​​over homocysteine ​​and glutathionein water [J]. Sensors and Actuators B: Chemical, 2013, 188: 111-116. ) reported a method to synthesize a novel Cys sensor by condensation of vinyl benzoate lipid derivatives and fluorescein, with a yield of 8%. However, the product yield and selectivity are not particularly good, and the response too long
[0006] Document 2 (Murale D P, Kim H, Choi W S, et al.Extremely selective fluorescence detection of cysteine ​​or superoxide with aliphatic esterhydrolysis[J].RSC Advances,2014,4:46513-46516.) reported a fluorescein and acrylic acid Condensation, a method for synthesizing a new type of Cys sensor, the yield is 69%, but there is still the problem that Hcy interferes with the selective detection of Cys

Method used

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  • Cys fluorescence sensor based on rhodamine B, preparing method and application
  • Cys fluorescence sensor based on rhodamine B, preparing method and application
  • Cys fluorescence sensor based on rhodamine B, preparing method and application

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Experimental program
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Effect test

Embodiment 1

[0040] Synthesis of embodiment 1 fluorescent chemical sensor

[0041] 1. Synthesis of Compound 1

[0042] Rhodamine B (960mg, 2mmol) and ethylenediamine (1.3ml, 20mmol) were dissolved in absolute ethanol (40ml), the reaction temperature was controlled at 80°C, and the reaction time was 12h. After the reaction was completed, the solvent was removed under reduced pressure, and extracted , separated by a silica gel column to obtain a light yellow solid, compound 1 (880 mg, 92%). Compound 1 1 H NMR, 13 C NMR respectively as figure 1 , figure 2 shown.

[0043] 2. Synthesis of Compound 2

[0044] Add 97.02 mg (0.99 mmol) of maleic anhydride to 3 mL of chloroform containing 319.62 mg (0.66 mmol) of compound 1, and reflux for 20 hours. After the reaction, cool to room temperature, remove the solvent under reduced pressure, extract, and Silica gel column separation gave light yellow solid, compound 1, and light yellow solid, compound 2 (365 mg, 95%). Compound 1 1 HNMR, 13 C ...

Embodiment 2

[0047]Embodiment 2 ultraviolet selection performance test

[0048] The target compound has good solubility in methanol. It has been verified that the target compound can be dissolved in MeOH:HEPES (1mM, pH=7.4)=1:2 mixed solution, and 500ml of this solution is prepared as a stock solution (pH=7.2) .

[0049] Precisely configure the target compound as 1 x 10 -3 mol / LMeOH-H 2 O mixed solution (1 / 2, V / V), the concentration of amino acids GSH, Cys, Hcy, Lys, Gly, etc. is 5×10 -3 mol / L aqueous solution, and MeOH:HEPES (1mM, pH=7.4, 1 / 2, V / V) solution.

[0050] UV selectivity experiments such as Figure 7 As shown, take 3ml of the stock solution and place it in the liquid pool, add 30uL of the target compound, measure its initial absorbance, and then add 30uL of various amino acids prepared respectively, and measure its absorbance when it is stable. Observed Figure 7 It can be seen that the target compound has an obvious response effect to Cys, and a new peak appears at 492nm...

Embodiment 3

[0051] Embodiment 3 Fluorescence selection performance test

[0052] The target compound has good solubility in methanol. It has been verified that the target compound can be dissolved in MeOH:HEPES (1mM, pH=7.4)=1:2 mixed solution, and 500ml of this solution is prepared as a stock solution (pH=7.2) .

[0053] Precisely configure the target compound as 1 x 10 -3 mol / LMeOH-H 2 O mixed solution (1 / 2, V / V), the concentration of amino acids GSH, Cys, Hcy, Lys, Gly, etc. is 5×10 -3 mol / L aqueous solution, and MeOH:HEPES (1mM, pH=7.4, 1 / 2, V / V) solution.

[0054] Fluorescent selectivity experiments such as Figure 8 As shown, take 3ml of the stock solution and put it in the liquid pool, add 30uL of the target compound, measure its initial fluorescence intensity value, and then add 30uL of various prepared amino acids to measure its stable fluorescence intensity. Observed Figure 8 It can be seen that the target compound has an obvious response effect to Cys, and the fluorescen...

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Abstract

The invention discloses a Cys fluorescence sensor based on rhodamine B, a preparing method and an application. The preparing method includes the steps that the rhodamine B serves as a precursor, and a target product 3'-hydroxyl-3-carbonyl-3H-spiro[isobenzofuran-1,9'-xanthene]-6'-yl(E)-4-((2-(3',6'-di(diethylamide-3-carbonyl-spiro[isoindolinyl-1,9'-xanthene])-2-yl)ethyl)amino)-4-carbonyl butanol-2-crotonate is synthesized with two steps. According to the Cys fluorescence sensor, the preparing method and the application, Cys is detected through the target product, and it is found that the target product has a quite good detection effect on Cys; meanwhile, the raw materials are easy to get, the synthesis steps are simple, postprocessing is also quite convenient, and large-scale production can be easily achieved.

Description

technical field [0001] The invention belongs to the field of biochemistry, and in particular relates to the preparation and application of a Rhodamine B-based Cys fluorescence sensor. Background technique [0002] Cysteine ​​(Cys), or 2-amino-3-mercaptopropionic acid, plays a crucial role in regulating various functions of cells. Its main physiological function is to remove free radicals in the human body, and as an important antioxidant in the body, it protects the sulfhydryl groups in many molecules such as proteins and enzymes. When the content of Cys is abnormal, it will bring many health problems. For example: body edema, lethargy, liver damage, loss of muscle and fat, slow growth, skin lesions, etc. On the other hand, when the content of Cys is higher than the normal value, it can cause cancer, cardiovascular disease, and Alzheimer's disease. In view of the fact that Cys is so important to the human body, it is of great significance to study a method that can quickl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D519/00C09K11/06G01N21/64G01N21/33
CPCC07D519/00C09K11/06C09K2211/1029C09K2211/1088G01N21/33G01N21/6486
Inventor 包晓峰陈海浪吴小磊舒海李峰
Owner NANJING UNIV OF SCI & TECH
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