| Part I:Intracellular pH is an important factor in cell biology and plays a critical role in many cellular events and is not uniformly distributed. For example, the cytoplasma is slightly alkaline at about pH7.2, whereas organelles, such as lysosomes and endosomes, have intracompartmental pHs of4.0-6.0. The acidic environments in lysosomes can activate degradative enzymes to facilitate the degradation of proteins in cellular metabolism. Increase of lysosomal pH will make these enzymes lose activity and cause cellular dysfunction. Therefore, monitoring pH changes inside living cells is important for exploring cellular functions and understanding physiological and pathological processes.Fluorescence techniques have been widely applied in the detection and fluorescence imaging of various ions and biological species in living cells due to their high selectivity, high sensitivity, simple operation and nondestructive analysis in cells. In general, there are two types of synthetic pH probes, one type for cytosol that work at a pH of6.8-7.4, and another type for the acidic organelles (for example, lysosomes) functioning in the pH range4.5-6.0. Although it is possible to find a large array of commercial probes for the former, the latter, especially for specific organelles such as lysosomes, is relatively few. The current specific lysosomal pH sensors, including the commercial LysoSensor probes, are usually designed based on PET mechanism. These probes consist of a fluorophore and an amino side chain. The PET process from amino side chain to fluorophore causes fluorescence quenching. The alkaline side chain can accumulate in acidic lysosomes and be protonated. The PET is inhibited, leading to an enhanced fluorescence signal. Unfortunately, these lysosomal probes can exhibit an "alkalizing effect" on lysosomes to give erroneous results because longer incubation with these probes can induce an increase in lysosomal pH. Therefore, the development of novel lysosomal probes which can avoid "alkalizing effect" and have high sensitivity and good membrane permeable would be of great significance and practical value.Rhodamine derivatives have excellent photophysical properties such as high fluorescence quantum yield, long absorption and emission wavelengths. Rhodamine lactam is sensitive to acidic pH and is suitable to study acidic organelles. Few rhodamine pH probes have been reported which can mark lysosomes in living cells and avoid "alkalizing effect". We designed and synthesized three rhodamine B-based probes with different pKa values, Rlyso-1, Rlyso-2, and Rlyso-3. All probes have high selectivity and sensitivity to acidic pH. The fluorescence intensity of Rlyso-1enhanced150-fold in acidic solution. Some metal ions can cause slight effect on the fluorescence intensity. Rlyso-2has higher quantum yield and better anti-interference ability. Rlyso-3is a dual function probe which can also detect Hg2+in weak base solution. All the probes have good membrane permeability and low cytotoxicity, and can avoid "alkalizing effect". They can be applied for fluorescence imaging in living cells and can be used as specific lysosomal pH probes.Part â…¡:Pyrazole derivatives have a wide range of biological activity and play an important role in the agrochemical and pharmaceutical industries. Based on our previous work, we synthesized several series of novel pyrazole derivatives. The structures of these compounds were confirmed by IR,1H NMR, HRMS and X-ray diffraction. We studied their optical properties and evaluated their biological activities.Chapter1, we reviewed the applications of pyrazole derivatives in anticancer, antimicrobial and materials.Chapter2, a series of novel2-pyrazolyl-5-aryl-1,3,4-oxadiazole derivatives were synthesized. Structural features were confirmed by IR,1H NMR, HRMS and X-ray diffraction, and the optical properties were investigated by UV-vis absorption and fluorescence spectroscopy in dichloromethane.Chapter3, a series of novel2-pyrazolyl-5-aryloxymethyl-1,3,4-oxadiazole derivatives were synthesized and characterized. Their UV spectra and fluorescence spectra were investigated in different solvents.Chapter4, we reported the one-pot synthesis of5-benzyl-2-phenylpyrazolo[1,5-a]pyrazin-4,6(5H,7H)-dione derivatives and evaluated their biological activities as anticancer agents. We found one compound that could inhibit the growth of H322cells in dosage-dependent manner through inducing apoptosis of cells.Chapter5, a series of novel N-aryl-3-aryl-l-arylmethyl-1H-pyrazole-5-carboxamide derivatives was synthesized by the reaction of3-aryl-l-arylmethyl-1H-pyrazole-5-carbonyl chloride with substituted aniline in good to excellent yields. Structures of the compounds were determined by IR,1H NMR and HRMS spectroscopy. The molecular structure was confirmed by the X-ray crystal analysis of one compound. These compounds were used to induce mouse osteoblast precursors MC3T3-E1into osteoblast and the induction was assessed by alkaline phosphatase (ALP) activity and the gene expression of bone sialoprotein (BSP). One effective compound which could induce osteogenesis was screened out.Chapter6, two series of novel pyrazole carboxamide derivatives containing piperazine moiety were synthesized and determined by IR,1H NMR and HRMS spectroscopy. Especially, the structure was confirmed by the X-ray crystal analysis of one compound. The antimicrobial activities in vitro of the compounds were evaluated. Preliminary study of the structure-activity relationship revealed that the substituent had significant effect on the antimicrobial activity. |
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