Several innovative tandem mass spectrometric strategies have been developed for the structural determination and isomer differentiation of glycosides and oligosaccharides. Specifically, collisionally activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD) are used in conjunction with derivatization methods designed to exploit variations in binding energies or attach chromophores. These include metal complexation incorporating modified neutral auxiliary ligands and covalent derivatization involving site-specific reactions. The elucidation of flavonoid isomers is accomplished by electrospray ionization tandem mass spectrometry (ESI-MS/MS) via formation and CAD of metal/flavonoid complexes containing an auxiliary ligand. Addition of a metal salt and a suitable neutral auxiliary ligand to flavonoids in solution results in the formation of [M(II) (flavonoid-H) ligand]⁺ complexes by ESI which, upon collisional activated dissociation, often result in more distinctive fragmentation patterns than observed for conventional protonated or deprotonated flavonoids. We compare and explore the use of alternative pyridyl ligands, with electron-releasing substituents including 4,7-diphenyl-1,10-phenanthroline. Using this technique, three groups of flavonoid glycoside isomers are differentiated, including glycosides of apigenin, quercetin and luteolin. A tunable ESI-MS/MS strategy for differentiation of flavone and flavanone diglycoside isomers based on metal complexation with auxiliary ligands that have electron-withdrawing substituents is reported. A series of auxiliary ligands with electronwithdrawing substituents was synthesized in order to tailor the relative metal binding affinities of the ligands and thus directly influence the stabilities, and consequently the dissociation pathways, of the complexes. Upon collisionally activated dissociation, the complexes yield fragmentation patterns in which the abundances of key diagnostic ions are enhanced, thus facilitating isomer differentiation. A strategy for increasing the efficiency of IRMPD in a quadrupole ion trap (QIT) based on another metal complexation strategy is described. Two IR-active ligands (IRALs) that have an IR-active phosphonate functional groups were synthesized. The IR-active groups were therefore incorporated into the analyte complexes via metal complexation. We demonstrate this new IRMPD approach for the structural characterization of flavonoids. The fragment ions obtained by IRMPD are similar to those obtained by CAD and allow facile isomer differentiation of flavonoids. Fourier transform infrared absorption attenuated total reflectance (FTIR-ATR) and energyvariable CAD experiments indicate that the high IRMPD efficiencies stem from the very large IR absorptivities of the IR-active ligands. A simplified method for determining the sequence and branching of oligosaccharides using IRMPD in a QIT is described. An IR-active boronic acid (IRABA) reagent was synthesized and subsequently used to derivatize the oligosaccharides prior to IRMPD analysis. The IRABA ligand is designed to both enhance the efficiency of the derivatization reaction and to facilitate the photon absorption process. The resulting IRMPD spectra display oligosaccharide fragments that are formed from primarily one type of diagnostic cleavage, thus making sequencing straightforward. The presence of sequential fragment ions, a phenomenon of IRMPD, permit the comprehensive sequencing of the oligosaccharides studied in a single stage of activation. The approach is demonstrated for two series of oligosaccharides, the lacto-Nfucopentaoses (LNFPs) and the lacto-N-difucohexaoses (LNDFHs).

The optimisation of ligand-macromolecule interactions is fundamental to the design of therapeutic agents. The GRID method is a procedure for determining energetically favourable ligand binding sites on molecules of known structure using an empirical energy potential. In this thesis, it has been extended, tested, and then applied to the design of anti-influenza agents. In the GRID method, the energy of a hydrogen-bond is determined by a function which is dependent on the length of the hydrogen-bond, its orientation at the hydrogen-bond donor and acceptor atoms, and the chemical nature of these atoms. This function has been formulated in order to reproduce experimental observations of hydrogen-bond geometries. The reorientation of hydrogen atoms and lone-pair orbitals on the formation of hydrogen-bonds is calculated analytically. The experimentally observed water structures of crystals of four biological molecules have been used as model systems for testing the GRID method. It has been shown that the location of well-ordered waters can be predicted accurately. The ability of the GRID method to assist in the assignment of water sites during crystallographic refinement has been demonstrated. It has also been shown that waters in the active site of an enzyme may be both stabilized and displaced by a bound substrate. Ligands have been designed to block the highly conserved host cell receptor site of the influenza virus haemagglutinin in order to prevent the attachment of the virus to the host cells. The protein was mapped energetically by program GRID and specific ligand binding sites were identified. Ligands, which exploited these binding sites, were then designed using computer graphics and energy minimization techniques. Some of the designed ligands were peptides and these were synthesised and assayed. Preliminary results indicate that they may possess anti-influenza activity.

The goal of this research is to identify a compound or family of compounds that would allow effective treatment of the influenza virus without unnecessary risks and side-effects. Influenza is a substantial problem in today’s society. Each year 36,000 people die in the United States due to influenza, or influenza related causes. Influenza is caused by two types of the virus, Type-A and Type-B. There are currently four FDA approved drugs to treat influenza—two are proton channel blockers and two are neuraminidase inhibitors. The goal of my research was to design a new drug that would allow physicians to effectively treat Type-A and Type-B influenza virus without having their patients endure unnecessary risks and side-effects. Density functional theory calculations were used to optimize the geometries of the ligands. Using sophisticated resources such as the Protein Data Bank and AutoDock, a library of twenty five ligands were docked into the N4 protein. Each docking was performed five times, resulting in one overall average docked energy. The averaged energies for each of the ligands were ranked from lowest to highest. Based upon a different study, one of my ligands were shown to have antiviral activity. From the docked energy for the ligand with confirmed antiviral activity, the results of the highest ranking ligand could be determined to have promising antiviral activity. The ligand shown below is the promising ligand, which will undergo further alterations and dockings to attempt to improve the antiviral activity. / Department of Chemistry

Src Homology 3 (SH3) domains are small protein- protein interaction domains that bind to proline-rich peptides, mediating a range of important biological processes. Because the deregulation of events involving SH3 domains forms the basis of many human diseases, the SH3 domains are appealing targets for the development of potential therapeutics. Previously in the field, no examples of entirely small-molecule ligands for the SH3 domains have been identified. However, in our research group, we have discovered a class of heterocyclic compounds that bind to the Tec SH3 domain at conserved residues in the proline-rich peptide binding site, with weak to moderate affinity. The highest affinity of these was 2- aminoquinoline (Kd = 125 mM). In this thesis, a range of approaches are described, that were intended to contribute towards development of higher affinity small-molecule ligands for the Tec SH3 domain. Preliminary experiments, involving testing a variety of compounds structurally related to 2- aminoquinoline, provided new structure activity information, and led to a better understanding of the 2-aminoquinoline/SH3 domain binding event. The major component of this thesis is a thorough investigation into the synthesis of a range of 2- aminoquinoline derivatives. N-Substituted- 2-aminoquinolines were synthesised, however these compounds bound the SH3 domain with slightly lower affinity than 2-aminoquinoline. 6- Substituted-2-aminoquinolines were subsequently prepared, and ligands were identified with up to six-fold improved affinity relative to 2-aminoquinoline, and enhanced selectivity for the Tec SH3 domain. The techniques used for the ligand binding studies were Nuclear Magnetic Resonance (NMR) chemical shift perturbation and Fluorescence Polarisation (FP) peptide displacement assays. As part of the ligand binding studies, it was intended that the 3D tructure of a 2- aminoquinoline ligand/SH3 complex would be obtained using NMR methods, provided that a ligand was identified that bound the SH3 domain in slow exchange on the NMR timescale. However, this goal was not fulfilled. Despite this, the work presented in this thesis provides a solid foundation for the development of potent 2-aminoquinoline ligands for SH3 domains, with engineered specificity. / Thesis (Ph.D.)--School of Molecular and Biomedical Science, 2005.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Thesis (M.S.)--Michigan State University. Dept. of Chemistry, 2006. / Title from PDF t.p. (viewed on June 19, 2009) Includes bibliographical references. Also issued in print.

Thesis (honors)--Georgia State University, 2007. / Title from file title page. Under the direction of Dabney White Dixon. Electronic text (88 p. : col. ill.) : digital, PDF file. Description based on contents viewed Sept. 30, 2008. Includes bibliographical references (p. 46-47).

Thesis (Ph. D.)--University of Missouri-Columbia, 1997. / Typescript. Vita. Includes bibliographical references (leaves 150-159). Also available on the Internet.

Thesis (M.S.)--University of North Carolina Wilmington, 2009. / Title from PDF title page (February 22, 2010) Includes bibliographical references (p. 103-105)

Thesis (Ph.D.)--Ohio University, June 2010. / Title from PDF t.p. Includes bibliographical references.