The objective of this thesis was to synthesize N2-modified guanosines (N2G) in order to introduce fluorescent and chelating ligands, such as diphenylamino, 2,2’-dipridylamino, 2-(2’-pyridyl)benzimidazolyl and p-pyrenylphenyl functionalities. Their photophysical properties were examined in order to gain further knowledge about the effect of guanine modification on its electronic structure. The impact of N2-modification was first studied in terms of self-assembly of the luminescent guanosines in solution and gas phase. Extensive NMR and ESI MS studies provided evidence that these N2-modified guanosines self-assemble exclusively into octamers with high-fidelity, in the presence of Group 1 and Group 2 metal ions. In addition, the first example of “empty” G-octamer (free of metal cations) was identified by ESI MS. Experimental results suggested that N2-substituents provide additional electronic and steric effects which drive the diastereoselectivity of self-assembly and provide additional stability. Hydrogen bonding of N2Gs with cytidine was monitored using fluorescence and NMR. In addition to GC base pair formation, the G-quartet-to-GC base pair structural transformation was studied using CD, fluorescence, and NMR spectroscopy. Due to the luminescent and chelating nature of some of the N2G derivatives, their interactions with a number of metal ions, such as Zn2+, Cd2+, Hg2+, La3+, Tb3+ and Eu3+ ions, were probed by using various spectroscopic methods. The overall optical response in the presence of metal ions was highly dependent on the nature of N2-substituent, and it varied from “turn-on” to “turn-off” response, clearly indicating that the modification at the N2-site of guanosine can be used to finely tune the optical response of these nucleosides. Finally, synthesis of a phosphorescent N2-arylguanosine containing the Ru2+ metal center was achieved and its photophysical and electrochemical properties were examined. / Thesis (Ph.D, Chemistry) -- Queen's University, 2009-09-22 16:02:20.934

Next generation electric dipole moment experiments require precise knowledge of the local magnetic fields in the experimental volume. Hyperpolarized xenon-129 has been proposed as a comagnetometer gas to be used in the neutron electric dipole moment experiment planned for TRIUMF. A flow through xenon polarizer was constructed and tested, and the hyperpolarized Xe-129 produced was transported to and characterized using a new AFP-NMR spectrometer. The polarization measured in the external AFP-NMR spectrometer was (12 ± 4)%. The longitudinal spin relaxation time T1 was found to be (77 ± 24) s in the experimental NMR volume, limited by leaks and field inhomogeneity. This represents good progress towards the eventual system for nEDM experiments where polarizations greater than 50% and T1, T2 relaxation times greater than 1000 s are expected.

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Next generation electric dipole moment experiments require precise knowledge of the local magnetic fields in the experimental volume. Hyperpolarized xenon-129 has been proposed as a comagnetometer gas to be used in the neutron electric dipole moment experiment planned for TRIUMF. A flow through xenon polarizer was constructed and tested, and the hyperpolarized Xe-129 produced was transported to and characterized using a new AFP-NMR spectrometer. The polarization measured in the external AFP-NMR spectrometer was (12 ± 4)%. The longitudinal spin relaxation time T1 was found to be (77 ± 24) s in the experimental NMR volume, limited by leaks and field inhomogeneity. This represents good progress towards the eventual system for nEDM experiments where polarizations greater than 50% and T1, T2 relaxation times greater than 1000 s are expected.

E. coli Citrate Synthase (CS) is a large hexameric protein with a molecular weight of 280kDa and belonging to the type II class of citrate synthases. The crystal structure of E. coli CS in the T (inactive) state is known. The structure shows that the backbone atoms of 42 residues (1-5, 266-297 and 330-335) have temperature factors about 9 times greater than the average as compared to the rest of the molecule. Residues 266-297, also referred to as the “Mobile loop”, are particularly of interest since they form a part of the active site and any rearrangement in the mobile loop can provide useful information about the R(active) state of the protein. In this study, Nuclear Magnetic Resonance (NMR) Spectroscopy has been used to study the flexible regions of E. coli CS in solution. The flexible residues have been assigned based on the amino acid type by 15N-specific amino acid labeling, while the residue type has been assigned by site-directed mutagenesis. Changes in the dynamics of the flexible residues, in response to the substrate binding, have been studied using both NMR and Fluorescence Spectroscopy. Also a method to use Mass Spectrometry for accessing the isotopic incorporation in the samples prepared for NMR spectroscopy has been described. The initial hypothesis in this study was that only the mobile loop residues which show significant high B-factors will contribute to the NMR spectrum. However in the NMR spectrum, in addition to the mobile loop, some uncharacterized flexible regions were also observed. We also found that some of the residues show signs of slow conformational exchange resulting in multiple signals in the NMR spectrum. In addition we see that the environment of some flexible residues is changed in the presence of substrates, a few residues were immobilized, but most remained mobile.

High-Level Waste (HLW), contains radioactive isotopes with half-lives upwards of millions of years. The long-term storage of HLW can be accomplished through vitrification of the mixture in a borosilicate melt, producing a chemically inert solid. Unfortunately, molybdates can precipitate from the glass melt as water- soluble oxides containing radionuclei. Part of this study is dedicated to the characterization and interpretation of the NMR parameters for a series of crystalline molybdate phases at ambient temperatures. 23Na, 95Mo and 133Cs MAS NMR data were correlated to the local structure and geometry, showing several key trends. From the ambient temperature studies, select crystalline phases were chosen for high-temperature experiments up to 700◦C. These experiments were used to characterize the behaviour of these phases at temperatures relevant to both the formation of solid waste forms and their long-term storage. With these spectral “fingerprints”, it is possible to monitor the phase separation behaviour at temperatures relevant to vitrification and long-term geological storage. For example, the conversion of CsNaMoO4·2H2O into Cs3Na(MoO4)2 and Na2MoO4 can be followed in situ through MAS NMR. Finally, a series of model nuclear waste glasses with varying amounts of Cs and Mo were studied, allowing direct observation of the crystallization process.

1- The synthesis of unusually substituted halo-fluoroheterocycles has been achieved. 2,4,6-Tribromo-3,5-difluoropyridine and 4-bromo-2,3,5,6-tetrafluoropyridine were prepared from pentafluoropyridine, aluminium bromide and hydrogen bromide Reactions with lithium halides allowed the preparation of 4-iodo-2,3,5,6- tetrafluoropyridine, 4-iodo-2,6-dibromo-3,5-difluoropyridine, 4-iodo-2,3,5,6- tetrafluoropyridine and 4-chloro-2,6-dibromo-3,5-difluoropyridine.2- Reactions of 2,4,6-tribromo-3, 5-difluoropyridine with nucleophiles showed that selective substitution at the C-F centre can be achieved using hard (sodium ethoxide, phenoxide) nucleophiles.3- Lithium mediated reactions of 2,4,6-tribromo-3,5-difluoropyridine allowed selective functionalisation of the 4-position; lithium-halogen exchange occurred exclusively at this position giving a stable lithium derivative, which was successfully trapped with a range of electrophiles (trimethylsilyl chloride, acid chlorides).4- Palladium mediated couplings of 2,4,6-tribromo-3,5-difluoropyridine with a range of substituted phenylacetylenes was successful and the 2- and 6-positions were the most activated sites. Coupling with boronic acids gave the disubstituted or trisubstituted products; in the case of trisubstitution, all three positions (2-,4- and 6-) were activated towards coupling. 2,4,6-Tribromo-3,5-difluoropyridine formed a stable zinc derivative, which was coupled with iodoaromatics (iodobenzene, diiodobenzene).

19F NMR studies of proteins provide unique insight into biologically relevant phenomena such as conformational fluctuations, folding and unfolding, binding and catalysis. While there are many advantages to the use of 19F NMR, experimental challenges limit its widespread application. The focus of this thesis has been to address some of these limitations, including resonance assignment and perturbations arising from fluorine probes, and to develop more robust methods of studying protein topology by 19F NMR. 19F NMR experiments designed to measure local hydrophobicity and exposure were developed and evaluated in two systems, Fyn SH3 and calmodulin, labeled with 3-fluorotyrosine. Paramagnetic effects from dissolved oxygen, solvent isotope shifts from deuterium oxide, and 1H-19F NOEs were each sufficient in establishing relative solvent exposure, while the combination of effects from oxygen and deuterium oxide were able to delineate local hydrophobicity and solvent accessibility of 19F probes. Two NMR based resonance assignment protocols were developed using 13C, 15N-enriched 3-fluorotyrosine and 3-fluorophenylalanine, separately biosynthetically incorporated into calmodulin. In the first approach, isotopic enrichment facilitated two-dimensional heteronuclear experiments based on INEPT and COSY magnetization transfer schemes to correlate the fluorine nucleus to sidechain and backbone 1H, 13C, and 15N atoms, providing complete spectral assignment. The assignment of 3-fluorophenylalanine resonances was achieved using 19F-, and 15N-edited homonuclear NOE experiments to connect the fluorine nucleus to intraresidue and neighboring 1H and 15N resonances. While both strategies were successful, the NOE-based method was vulnerable to alternate relaxation mechanisms, including chemical shift anisotropy and chemical exchange. Structural perturbations arising from uniform incorporation of 3-fluorophenylalanine in calmodulin was thoroughly investigated using 19F and 1H-15N NMR spectroscopy, 15N spin relaxation and thermal denaturation via circular dichroism spectroscopy. While stability was unaffected, NMR experiments revealed increased protein plasticity, minor conformers and line broadening. The merit of fractional fluorine labeling in reducing such disruptions was demonstrated, and labeling levels of 60-75% provided an optimal balance between native-likeness and the usual advantages of 19F NMR in our system. The 19F NMR techniques developed here are broadly applicable and will expand the utility of 19F NMR in studies of protein systems.

E. coli Citrate Synthase (CS) is a large hexameric protein with a molecular weight of 280kDa and belonging to the type II class of citrate synthases. The crystal structure of E. coli CS in the T (inactive) state is known. The structure shows that the backbone atoms of 42 residues (1-5, 266-297 and 330-335) have temperature factors about 9 times greater than the average as compared to the rest of the molecule. Residues 266-297, also referred to as the “Mobile loop”, are particularly of interest since they form a part of the active site and any rearrangement in the mobile loop can provide useful information about the R(active) state of the protein. In this study, Nuclear Magnetic Resonance (NMR) Spectroscopy has been used to study the flexible regions of E. coli CS in solution. The flexible residues have been assigned based on the amino acid type by 15N-specific amino acid labeling, while the residue type has been assigned by site-directed mutagenesis. Changes in the dynamics of the flexible residues, in response to the substrate binding, have been studied using both NMR and Fluorescence Spectroscopy. Also a method to use Mass Spectrometry for accessing the isotopic incorporation in the samples prepared for NMR spectroscopy has been described. The initial hypothesis in this study was that only the mobile loop residues which show significant high B-factors will contribute to the NMR spectrum. However in the NMR spectrum, in addition to the mobile loop, some uncharacterized flexible regions were also observed. We also found that some of the residues show signs of slow conformational exchange resulting in multiple signals in the NMR spectrum. In addition we see that the environment of some flexible residues is changed in the presence of substrates, a few residues were immobilized, but most remained mobile.

Zugl.: Bremen, Univ., Diss., 2007