Molecular probe for detecting dihydrolipoyl succinyltransferase, and preparation method and use thereof
An oxadiazole, methylthio-based technology, applied in the field of chemical biology, can solve the problems of lack, the limitation of DLST function research, etc.
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Embodiment 1
[0094] Example 1: Preparation of Probes 1-6
[0095] (1) Take 0.20 mol of different substituted benzoic acids in a 250 mL round bottom flask, add 20 equivalents of methanol and 4 mL of 98% concentrated sulfuric acid therein, and then reflux for 4-6 h. After the methanol was distilled off under reduced pressure, the sample was mixed with silica gel and purified by column chromatography to obtain compound 1 with a yield of 96-99%.
[0096] (2) Take 0.15 mol of compound 1 in a 100 mL round bottom flask, add 2.5 times the equivalent of 80% hydrazine hydrate therein, and reflux for 2 h. After the system was cooled, a white solid was precipitated, washed with water and dried to obtain Compound 2 with a yield of 77-91%.
[0097] (3) Take 0.15mol of compound 2 in a 250mL round bottom flask, add 2 times the equivalent of KOH and 100mL of ethanol to it, stir and dissolve, then add dropwise 2 times the equivalent of CS 2 , Stirred at room temperature for 8h and then refluxed for 24-36h...
Embodiment 2
[0134] Embodiment 2: Probe 1-6 half inhibitory concentration (IC 50 )test
[0135] Make probes 1-6 into 5 corresponding concentrations of toxic liquid medium, take 5mL in a test tube, and use a microplate reader to measure the OD of the drug-containing sterile liquid medium 600 value, add 40μL containing Xanthomonas oryzae NB liquid medium (each liter medium contains 3g beef peptone, 5g peptone, 1g yeast powder, 10g glucose, pH 7.0-7.2), and then in 28℃, Cultivate on a shaking table at 180rpm for 36-48h, and use a microplate reader to measure the OD of each concentration of the bacterial solution 600 value.
[0136] Corrected OD value = OD value of bacteria-containing medium - OD value of sterile medium
[0137] Control effect% = (OD value of control medium bacterial solution after correction - OD value of toxic medium after correction) / OD value of control medium bacterial solution after correction × 100
[0138] Convert the inhibition rate data into a probability value ...
Embodiment 3
[0149] Embodiment 3: The labeling experiment of probe 1-6 to DLST in living cells
[0150] 1. In vivo labeling of probes 1-6 on rice bacterial blight (Xoo)
[0151] Take 2mL OD respectively 600 =0.6 Xoo bacteria solution, discard the supernatant after centrifugation, and then resuspend the cells with 0.99mL PBS (pH=7.2). 10 μL of probes 1-6 were added to make the final concentration 5 μM, and 10 μL of DMSO was added to the control group, and incubated at 25° C. for 2 h. Discard the supernatant after centrifugation, wash with PBS three times, add 100 μL PBS to resuspend the cells, and sonicate the cells in an ice bath. After centrifugation, dilute the protein to 1 μg / μL, take 45 μL supernatant, and then add biotin-N 3 , sodium ascorbate and BTTAA / CuSO 4 Mix the solution for the click reaction. Add 50 μL 2×loading buffer to the samples respectively, and boil at 95°C for 10 minutes. 20 μL samples were taken for SDS-PAGE, and then transferred to the membrane at a constant fl...
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