Thioredoxin reductase fluorescent probe targeting mitochondria as well as preparation method and application thereof
A thioredoxin, mitochondrial technology, applied in the field of fluorescent probes, can solve the problems of low probe sensitivity and selectivity, insufficient for in vivo test research, easy fluorescence quenching, etc., and achieves good fluorescence imaging effect, Excellent inhibitory effect, high sensitivity effect
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Embodiment 1
[0055] The preparation of embodiment 1 fluorescent probe Cur-Fy
[0056] The preparation process of fluorescent probe Cur-Fy is as follows:
[0057]
[0058] Step 1: Dissolve acetylacetone (5g, 50mmol) in ethyl acetate under the protection of boron oxide (2.5g, 36mmol, 0.7eq.), and stir at 70°C for 30min. Add 5-methyl-2-furancarbaldehyde (1.1g, 10mmol), tributyl borate (2.3g, 10mmol) and continue stirring for 30min. Ethyl acetate (3mL) dissolved in n-butylamine (0.73g, 10mmol) was added dropwise ), the dropwise addition was completed and the temperature was raised to 100 degrees, and the reaction was carried out for 5 hours. Cool down to room temperature, add 1N HCl to adjust the pH to 3, extract with ethyl acetate, dry the organic layer with anhydrous sodium sulfate, spin-evaporate ethyl acetate, and purify the solid by column chromatography to obtain 1.5 g of the target compound intermediate A1 as a yellow solid. rate of 81%.
[0059] Intermediate A1: 1 H NMR (400MHz,...
Embodiment 2
[0066] The measurement of the ultraviolet absorption spectrum and the fluorescence emission spectrum of embodiment 2 fluorescent probe Cur-Fy
[0067] After testing the fluorescent probe Cur-Fy ultraviolet maximum characteristic absorption peak is at 440nm, and the fluorescence emission wavelength is 540nm, such as image 3 shown. In a further study, it was found that after adding thioredoxin reductase to react with the fluorescent probe, the fluorescence was significantly enhanced at 540nm, as Figure 4 shown.
[0068] (1) The assay method of fluorescent probe Cur-Fy ultraviolet absorption spectrum
[0069] Microplate reader, model: Synergy H1, BioTek, USA. Potassium phosphate buffer (PBS) preparation: sodium chloride (NaCl), 8g; potassium chloride (KCl), 0.2g; disodium hydrogen phosphate (NaCl) 2 HPO 4 ), 1.44g; Potassium dihydrogen phosphate (KH 2 PO 4), 0.24 g, adjusted to pH 7.4, constant volume 1L, autoclaved, stored at room temperature.
[0070] Experimental met...
Embodiment 3
[0074] Example 3. Determination of Cur-Fy inhibitory activity on thioredoxin reductase
[0075] Inhibition of thioredoxin reductase activity by fluorescent probe Cur-Fy was determined by DTNB method, and the inhibitory activity IC of Cur-Fy on thioredoxin reductase 50 is 2.48μM ( Figure 5 ), indicating that Cur-Fy has a better inhibitory effect on thioredoxin reductase.
[0076] Experimental method: Preparation of test buffer solution: Take 200uL 1M potassium phosphate solution, 0.2mL 500mM EDTA solution, 20uL 20mg / mL BSA, 10uL 48mM NADPH, add to 1.73mL double distilled water, and mix well. Take 35.5uL test buffer solution into a 384-well plate, add thioredoxin reductase (T9698, sigma) 0.3uL, 1uL Cur-Fy (final concentration is 0.3125, 0.625, 1.25, 2.5, 5, 10μM) and incubate for 5min, Finally, 3.2uL of 63mM DTNB was added, and the absorbance was read at 412nm, recorded every 10s for 2min, and the inhibition rate of the fluorescent probe on thioredoxin reductase was calculate...
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