A series of fluorescein and rosamine derivatives have been prepared and their spectroscopical properties analyzed to determine their usefulness as donor and/or acceptors in "through-bond" energy transfer systems. Such new systems have been tailored to possess higher quantum yields, increased water solubility and higher pH independence. Some of these compounds have also been designed with handles for bioconjugation for use in intracellular imaging. The syntheses of most of these compounds have been optimized to afford higher yields in a multigram scale. Single molecule studies on a fluorescein/rosamine cassette are also reported.

Water-soluble fluorescent probes with emission in the 600-800 nm region have significant potential in biological applications such as cell imaging. Most fluorescent probes however suffer from limited fluorescence brightness in aqueous media due to aggregation and self-quenching. Their photostability in animal models for an extended period of time is also a concern. One way of improving their photophysical properties is to encapsulate them in a protective matrix to form fluorescent nanoparticles. We have synthesized a set of six through-bond energy transfer cassettes which emit in the 600-800 nm region with Fluorescein or BODIPY as donor and benzophenoxazine dye Nile Red or cyanine dye Cy5 as acceptor. Their photophysical properties in organic and aqueous media were evaluated. Some of these cassettes were encapsulated in silica or calcium phosphate nanoparticles (20 nm in diameter) to improve their solubility and photophysical properties in aqueous media. We also synthesized some water-soluble benzophenoxazine based fluorophores and the impact of different water-soluble groups on their emission characteristics in aqueous media was studied. Selected fluorophores were used for in vitro cellular imaging studies.

Thesis (Ph. D.)--University of Oregon, 2001. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 151-157). Also available for download via the World Wide Web; free to University of Oregon users.

One third of proteins in nature have been revealed as metalloproteins, whereas most of them remain uncharacterized, probably due to the lack of robust methods especially for tracking metalloproteins within the living context. Fluorescent labeling is capable to detect biomolecules with molecular resolution in living cells. Tracking metal-binding proteins in living cells by fluorescence could provide invaluable information in understanding their localization and potential functions in the native environment. A synthetic molecular probe NTA-AC was designed and synthesized to track metal-associated proteins in living cells upon chelation with metal ions. The fluorescent probe consists of a small molecular fluorophores, a metal-chelating moiety to direct the metal-chelated probe to the protein targets, and a photo-active crosslinker. Metal being chelated could help further explore potential binding targets and direct the fluorescent agent to the appropriate region, then subsequently covalent linkage to targets could be generated through photo-activation. NTA-AC was therefore chelated with different metals to examine its binding preference to different proteins. The Ni2+-chelating probe was applied to track Ni2+-binding proteins as an example to validate its applicability. Ni2+-NTA-AC preferentially binds to histidine-rich peptides and proteins thus verified its binding specificity. The Ni2+-chelated probe was further exploited to light up over-expressed histidine-rich proteins in Escherichia coli cells to validate its membrane permeability and binding specificity. In addition, the probe was applied to label His-tagged proteins expressed in tobacco plant cells to further evaluate its applicability in detecting and localizing the protein targets in eukaryotic cells. Afterwards, Ni2+-NTA-AC was exploited to track Ni2+-binding proteins in living Helicobacter pylori cells and incorporated with gel electrophoresis and mass spectrometry for protein identification. Many proteins identified are correlated to Ni2+-association and thus validating the applicability of the probe. Bi3+-chelated NTA-AC was therefore used to mine potential targets in H. pylori. Intense fluorescence was observed within H. pylori cells thus indicating the effectiveness of the fluorescent labeling. Protein separation and identification was therefore initiated to trace potential targets, while finding that some of the Bi 3+-coordinated proteins participate in various functioning pathways of the pathogens. The effects of colloidal bismuth subcitrate (CBS) on pH buffering and redox defense systems were therefore determined and verified, confirming that respective proteins could be potential therapeutic targets of the drug. Cr3+-NTA-AC was further applied to human Hep G2 cell line to determine Cr3+-binding targets in mammalian cells. Their localization on mitochondria was revealed, implying the potential effects of Cr3+ on mitochondria. Further confirmation of protein targets was performed through protein separation and identification. Proteins identified could be positively correlated to mitochondrial functions and thus revealing that Cr3+ might exert its effect at mitochondria. Addition of Cr3+ to Hep G2 could prevent mitochondrial fragmentation induced by hyperglycemia, which thus suggests the possible therapeutic function of Cr3+. The extensive application of NTA-AC in tracking Ni2+-, Bi3+- and Cr3+-associated proteins has validated the effectiveness of such strategy in detecting and localizing metalloproteins within the living context and thus could be extended to investigate other metalloproteomes. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

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Bibliography: leaves 177-181. / v, 181 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Chemistry, 1999

Thesis (M.S.)--Ohio University, August, 1980. / Title from PDF t.p.

Thesis (Ph. D.)--University of Adelaide, Dept. of Chemistry, 1999. / Includes bibliographical references (leaves 177-181).

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references. Also available in print.