京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第13178号 / 論農博第2857号 / 新制||農||1061(附属図書館) / 学位論文||H30||N5100(農学部図書室) / (主査)教授 小川 順, 教授 栗原 達夫, 教授 三上 文三 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

<p> This thesis presents the theoretical study of the mechanisms of gas-phase formamide hydrolysis using ab initio SCF-MO calculations. Four reaction paths were considered; (i) the reaction of formamide with OH- (ii) the acid catalyzed hydrolysis with protonation on the nitrogen atom (iii) the acid catalyzed hydrolysis with protonation on the oxygen (iv) the uncatalyzed hydrolysis. An unconstrained optimization of all parameters was performed on the transition state and equilibrium structures. The intrinsic reaction coordinate was then followed down from the transition state to the reactants and products. All of the molecular geometries were obtained using the 4-31G basis set and all wavefunctions and energies were calculated at the 6-31G** level of theory. The theory of atoms in molecules was applied to each reaction to study the mechanisms of structural change along the reaction coordinate. Molecular graphs were calculated at points along the reaction coordinate and these give a detailed pictorial outline of the entire reaction sequence. The Laplacian of the charge density successfully predicts the correct site of protonation and the enhanced reactivity of protonated formamide over that of neutral formamide. Both the acid catalyzed reaction with nitrogen protonation and the base enhanced hydrolysis reactions proceed without a barrier with respect to reactants and products. The acid catalyzed hydrolysis with protonation on the oxygen atom proceeds with a moderate activation barrier whereas the neutral hydrolysis involves the passage over a very high activation barrier. The two acid catalyzed reactions are competitive with the N-protonation mechanism being more favourable from a kinetic point of view while the O-protonation mechanism is thermodynamically more favourable.</p> / Thesis / Master of Science (MSc)

In order to achieve efficient therapeutic effect, it is important to understand the structure of biomaterials that are used in the therapeutic delivery system. This dissertation is dedicated towards understanding the hydrolysis pattern of plasmid DNA (pDNA) delivery vehicles comprised of poly(glycoamidoamine)s (PGAAs) under physiological conditions and effects of subtle changes in the chemical structure of the PGAAs on its biological performance. The unusual hydrolysis of the tartarate and galactarate based PGAAs was investigated by studying the hydrolysis of small model molecules which mimic the repeat unit of the respective polymers. In the case of galactarate and tartarate based molecules with terminal amines showed faster hydrolysis of the amide bonds. In addition for the tartarate based compounds, it was also found that it is necessary to have terminal amine functionality for the intramolecular hydrolysis to occur. The model compounds consists of two amide bonds and were designed symmetric, however amide bond on only one side of the tartarate moiety show underwent hydrolysis. Further studies show that one side of the amine assists the hydrolysis of the amide bond on the other side of the tartarate moiety. The degradation of poly(L-tartaramidopentaethylenetetramine) (<strong>T4</strong>) was also used to study the sustained release of pDNA from the layer-by-layer constructs of <strong>T4</strong>/pDNA. The thickness of the constructs was characterized by ellipsometry while the UV-visible spectroscopy was used to characterize the loading capacity of the constructs for pDNA. The indirect sustained release of pDNA under the physiological conditions with respect to time was characterized by the cellular uptake studies in HeLa cells. The increase in the uptake of the Cy5 labeled pDNA was seen at extended period of eleven days. The integrity of the sustained released pDNA for the transgene expression was characterized with an assay to see the expression of the green fluorescent protein (GFP) from the <strong>T4</strong>/GFP-pDNA layer-by-layer constructs. PGAAs show a very efficient delivery of the pDNA in a non-toxic manner. The chemical structure of the polymer can dictate the binding with pDNA and also the release of the pDNA form the polymer-pDNA complexes. In order to better understand the fundamentals of the nucleic acid delivery and to better design the nucleic acid delivery vehicles, subtle changes in the chemical structure of the PGAAs were designed and studied for the biological activity. The effect of charge type was investigated by designing and synthesizing guanidine based polymer series analogues to galactarate and tartarate based PGAAs (<strong>G1</strong> and <strong>T1</strong>) which incorporate secondary amines as the charge type on the polymer backbone. The guanidine based polymer series, poly(glycoamidoguanidine)s (PGAGs), show very non toxic behavior in HeLa cells at all the different polymer to pDNA ratio (<i>N/P</i> ratio) studied. Interestingly PGAGs are the only non-toxic guanidine containing polymers which are reported in the literature to the date. The cellular uptake of pDNA assisted from the PGAGs is a little higher than PGAAs compared although both the series of polymers show similar transgene expression. The transgene expression in case of PGAGs also imply the release of the polymer-pDNA complexes from the endosome. In another study of structure-bioactivity relationship based on the degree of polymerization (DP) of poly(galactaramidopentaethylenetetramine) (<strong>G4</strong>), it was found that the increase in the DP of <strong>G4</strong> increases the toxicity of the polymers in the HeLa cells. / Ph. D.