Compound, compounding method thereof, kit prepared from compound and application of compound in detecting glutathione
The technology of a compound and a kit is applied in the application field of glutathione detection, which can solve the problems of autofluorescence and biological damage, and limit the application, and achieve the effects of easy promotion and application, high sensitivity and simple operation.
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Embodiment 1
[0052] 7-(2-acylthiophene)-3H-phenoxazin-3-one preparation example
[0053] During the preparation of 7-(2-acylthiophene)-3H-phenoxazin-3-one, under the condition that catalyst exists, 7-hydroxyphenoxazinone sodium salt is mixed with 2-thiophenoyl chloride to react, 7-(2-Acylthiophene)-3H-phenoxazin-3-one is thus obtained. Wherein, the catalyst is selected from at least one of organic bases and inorganic bases; the organic base is selected from at least one of triethylamine and pyridine; the inorganic base is selected from potassium carbonate, sodium hydroxide, sodium carbonate and at least one of sodium bicarbonate; the molar ratio of the 7-hydroxyphenoxazinone sodium salt, 2-thienoyl chloride and catalyst is 0.5: (1~5): (0.5~5), 1: 2-10: 1-10
[0054] Preferably 1:(2~4):(1~4), more preferably 1:4:4; the reaction temperature of described reaction is 0 ℃~60 ℃, is preferably 50 ℃, and the reaction time is 1~24 hours; The above reaction is carried out in an organic solvent; t...
Embodiment 2
[0066] Spectral properties of the compound shown in formula I (hereinafter referred to as reagent 1) reacted with different concentrations of glutathione
[0067] Weigh 3.24 mg of Reagent 1 and make 10 mL of dimethyl sulfoxide solution as mother solution (1 mM).
[0068] In a 10mL graduated cylinder, add 5mL of PBS buffer solution (10mM, pH 7.4) and 50μL of probe stock solution, mix well and add the required glutathione sample solution. Finally, dilute the solution to 10 mL with a certain amount of PBS and mix well. After reacting at 37°C for 100 min, the UV-visible absorption spectrum and fluorescence emission spectrum were measured. When measuring the fluorescence emission spectrum, de-excite at 550nm; the slit width for excitation and emission is 10nm; the voltage is 400V.
[0069] figure 2 Fluorescence spectrum of reagent 1 reacting with 0-20 μM glutathione.
[0070] figure 2 The results show that reagent 1 in the present invention has the following characteristics:...
Embodiment 3
[0075] Reaction of reagent 1 (i.e. compound represented by formula I) with various amino acids (selectivity study)
[0076] Various substances were added to the 10μM reagent 1 solution: homocysteine (1mM), cysteine (1mM), serine (1mM), leucine (1mM), glutamic acid (1mM), amino acid (1 mM), alanine (1 mM), aspartic acid (1 mM), tyrosine (1 mM), histidine (1 mM). After reacting at 37°C for 100 min, the fluorescence emission spectrum was measured. When measuring the fluorescence emission spectrum, de-excite at 550nm; the slit width for excitation and emission is 10nm; the voltage is 400V.
[0077] 100 μl of the reagent 1 stock solution (1 mM) was added to 10 mL of the solution in which the above substances were mixed, and glutathione was added to make the final concentration 100 μM.
[0078] image 3 Fluorescence emission spectra obtained when reagent 1 (10 μM) was mixed with various amino acids. The numbers in the figure represent (1) probe, (2) homocysteine, (3) cystein...
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