Date of Award

May 2021

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Chemistry

First Advisor

Alexander Arnold

Committee Members

James Cook, Terry Moore, Alan Schwabacher, Nicholas Silvaggi, Douglas Stafford

Keywords

Biological Probes, Coumarins, Fluorescence, Nuclear Receptors, Orcein Dye, Vitamin D Receptor

Abstract

PART I:The vitamin D receptor (VDR) is a ligand-dependent transcription factor and member of the nuclear hormone receptor superfamily. VDR is expressed in the epithelia of endocrine organs, digestive system, bronchi, kidneys, and thymus, as well as being present in leukocytes and bone cells. Cell proliferation, cell differentiation, and immunomodulation, along with calcium and phosphate homeostasis, are all processes regulated by the receptor. Within the cell, VDR can be membrane-bound or located in the nucleus. Nuclear localization of VDR transpires following the binding of vitamin D metabolites, the most active of which is 1α,25-dihydroxyvitamin D3 (calcitriol). Within the nucleus, interactions with coregulators and DNA occur to induce gene transcription. Universal tool compounds to further elucidate the expression and localization of VDR are currently unavailable. Therefore, development of a novel ligand that can easily be traced in vitro and in vivo is necessary to further probe the complete role VDR plays within the cell. Intrinsically fluorescent VDR ligands containing a coumarin scaffold were designed to mirror the structures of known VDR agonists. Understanding of the binding requirements for VDR ligand-binding pocket (VDR-LBP) affinity, in combination with molecular modeling using VDR crystal structures, has provided a foundation for rational compound design. Ten coumarin-containing ligands targeting VDR were synthesized to maintain an emission signal in the visible light range. Additionally, evaluation of the synthetic ligands in a luciferase-based transcription assay indicated their agonist and/or antagonistic properties towards VDR. Toxicity of the synthetic ligands under the transfected conditions is also reported. The development of a tight-binding, intrinsically fluorescent VDR ligand will not only provide a means to further investigate direct receptor-ligand interactions, but also allows for the development of a new in vitro, high-throughput screening assay that targets the VDR-LBP. Furthermore, and unlike immunohistochemistry, the use of an intrinsically fluorescent ligand in vivo could enable the physiological distribution of VDR to be determined in live cells and whole animals.

PART II:Orcein dye was used as a cheap fabric dye during the Middle Ages, but was repurposed as a histochemical stain near the end of the 19th century. Orcein dye can be used today to visualize altered stromal material, elastic and connective tissues, collagen, basement membrane, chromosomes, hepatitis B surface antigens, copper-associated protein, and hepatocellular carcinoma. Traditionally, orsellinic acid depsides were extracted from Roccella, Lecanora, and Varialaria lichens, which subsequently underwent hydrolysis, decarboxylation, and treatment with either urine or ammonia and air to produce the red-violet dye. In present day, direct production occurs from synthetic orcinol. A mixture of chemical structures results during the current manufacturing process, with batch to batch variation leading to inconsistent biological tissue staining. Isolation of the eight major chemical structures from orcein dye occurred in the 1950s, but it remains unclear which compound structures, or combination thereof, are responsible for staining. Isolated synthesis of the specific chemical components within orcein dye will aid in determining the structures responsible for optimal staining. Furthermore, isolated synthesis will provide consistency between product batches and diminish the variation currently observed during biological staining. Herein, the development of a scalable manufacturing process for α-hydroxy orcein, one of the major components of orcein dye, is described.

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