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    An Improved Synthesis of the Pacific Blue Fluorophore and Fluorescence-based Studies of Receptor-Ligand Interactions

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    Issue Date
    2016-12-31
    Author
    Lee, Molly Marie
    Publisher
    University of Kansas
    Format
    280 pages
    Type
    Dissertation
    Degree Level
    Ph.D.
    Discipline
    Medicinal Chemistry
    Rights
    Copyright held by the author.
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    Abstract
    Early-stage drug discovery and chemical biology projects often use fluorescence-based assays to obtain information about biological interactions and cellular processes. However, many of the best and brightest fluorophores suffer from major limitations such as very high cost and/or restrictive chemical properties that hinder their utility for studies of living biological systems. To create improved fluorescent molecular probes of receptor-ligand interactions and dynamic cellular processes, a major focus of this dissertation is on the bright coumarin-derived fluorophore Pacific Blue. Although Pacific Blue is commercially available, its high cost has restricted its utility as a building block for preparation of small molecule-derived and peptide-derived molecular probes. To overcome this limitation, we developed a new synthetic route that readily allows access to gram quantities of this fluorophore. This synthetic route is superior to previously published routes, and it can facilitate access to a wide variety of fluorescent ligands of receptors, biosensors, and related cellular probes. Quantification of receptor-ligand interactions is important for screening of both on-target and off-target affinities of chemical probes and drug candidates. These assays must be cost-effective, efficient, and high-throughput to keep up with fast-paced needs of drug discovery projects. Toward this end, we characterized a new FRET pair, comprising the endogenous fluorescent amino acid tryptophan and Pacific Blue. We demonstrate that these fluorophores allow quantification of interactions between small molecules and tryptophan-containing proteins in vitro. We also synthesized and evaluated Pacific Blue derivatives of paclitaxel (Taxol) as tools to label microtubules, detect cellular efflux by P-glycoprotein (P-gp), and potentially explore some of the paradoxical clinical responses associated with the parent anticancer drug. We also characterized two other new FRET pairs, Pacific Blue-Pennsylvania Green and Pacific Blue-Oregon Green, and investigated the stability of disulfide linkers both in vitro and in living cells as models of drug delivery systems. Finally, using an alternative detection platform of fluorescence polarization, we describe the development of methods for the characterization of inhibitors of a protein-protein interaction involved in iron homeostasis in the pathogenic bacterium Pseudomonas aeruginosa. This research extends the utility of Pacific Blue and related fluorophores as tools for studies of chemical biology and drug discovery.
    URI
    http://hdl.handle.net/1808/25773
    Collections
    • Medicinal Chemistry Dissertations and Theses [81]
    • Dissertations [4472]

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    785-864-8983
    KU Libraries
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    785-864-8983

    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
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    Contact KU ScholarWorks
    785-864-8983
    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    785-864-8983

    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    Image Credits
     

     

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