Novel Fluorescent Probes For Hydrogen Sulfide And NAD(P)H:Quinone Oxidoreductase 1 Detection

Hydrogen sulfide serves as an indispensable gaseous molecule for cell signaling. It is involved in various vital physiological and pathological processes. The changes of H2S concentration in the body are related to many diseases, such as Alzheimer’s disease, down syndrome, diabetes, liver cirrhosis, chronic obstructive pulmonary disease, etc. Therefore, developing a fluorescence probe, which have lysosomes positioning function and can detect hydrogen sulfide in it, is particularly important for understanding biological function of H2S. NADH:quinone oxidoreductase 1 (NQO1) is a kind of flavin protease, which exists in various tissues and organs in mammals, but overexpressing in a variety of cancer tissue. Therefore, developing a kind of sensitive and specific NQO1 fluorescence probe, which can carry on the quantitative, real-time and visual detection of NQO1 in cells, is significant to study the expression level and space distribution of NQO1 within living cells.This work mainly covers the following two parts:1. A lysosome targeting coumarin-based fluorescent probe for H2S was prepared from 3-aminophenol, carbonochloridicacid ethylester, ethyl 4-chloroacetoacetate, morpholine, sodium azide, and its structure was characterized by 1H NMR,13C NMR and MS. Weak fluorescence intensity was detected when the probe solution was excited by 355 run UV light. After incubating with H2S 15 minutes, strong emission peak at 458 nm appeared under the same excitation wavelength (355 nm). And the fluorescence intensity at 458 nm is dependent on the concentration of H2S with the linear response range being 0-20 μmol/L. The detection limit of H2S is 37 nmol/L. Besides, the selective binding experiment for substrates indicated that the probe has good selectivity for hydrogen sulphide. On account of the fluorescent signal stabilities within the scope of the lysosome’s pH, probe shows the good identification characteristics of hydrogen sulfide in lysosomes of living cells. The experiment of Co-incubation with commercialized dye in living cells shows that the probe can target on the lysosome of living cells and detect H2S.2. A coumarin-based fluorescent probe for NAD(P)H:quinone oxidoreductase 1 was prepared from 7-Amino-4-methylcoumarin and quinone propionic acid, and its structure was characterized by 1H NMR, 13C NMR and MS. Weak fluorescence intensity was detected when the probe solution was excited by 355 nm UV light After incubating with NQO1 for 40 minutes, strong emission peak at 449 nm obviously arise with the same excitation wavelength (355 nm). The fluorescence intensity at 449 nm is dependent on the concentration of NQO1 with the linear response range being 5-150 ng/mL and the detection limit of NQO1 is 1.76 ng/mL. The probe has good selectivity for NQO1. Besides, probe remains stable optical properties in a range of pH (pH=6-10). The laser-scanning confocal experiment shows that this probe can apply to visual detcecting NQO1 in living cells.