Biological methylation is defined as the transfer of a methyl group from S-adenosyl-L-methionine(SAM) to one of a wide range of potential acceptors such as DNA, RNA, protein, hormones and neurotransmitters. Protein arginine methylation is a common post-translational modification facilitated by protein arginine methyltransferases(e.g. PRMTI). The roles of these enzymes in vivo are currently poorly understood. The focus of the project is design and synthesis of PRMT inhibitors with the ultimate goal of evaluating their activities in cells. Preliminary work toward the synthesis of S-adenosyl-trifluoromethyl-L-homocystein and adenosyl 5'-[2-(tert-butoxycarbonylamino)ethyl-trifluoro methyl] thiophenium is described. The ternary crystal structure of PRMTI in complex with S-adenoSyl-L-homocystein(eSAH) and an arginine containing peptide (PBD IOR8) was used to design a series of potential bisubstrate inhibitors of PRMTI. The prototypical SAM analogues bearing guanidine group were sought to replace the reactive sulfonium centre with nitrogen. Analogue synthesis proceeded via successive reductive arnination of Y-arnino-Y-deoxyadenosine and deprotection in good overall yields. An alkyne SAM analogue, 5'-[(S-3-amino-3-carboxypropyl)-propargylaminol-5'-deoxyadenosine was prepared, which underwent efficient Cu(1) catalysed Huisgen reaction to yield a triazole derived SAM analogue 5'-[(S-3-amino-3-carboxypropyl)-[I-(2-guanidinoethyl)-IH-1,2,3-triazol-4-yl]methyl-amino]-5'-deoxyadenosine. Preliminary biological evaluation of the compounds by collaborators Professor Steve Ward and Dr Richard Parry at the University of Bath, confirmed that 5'-[(S-3-amino-3-carboxypropyl)- 3-guanidinopropyl-amino]-5'-deoxyadenosine and 5-[(S-3-amino-3-carboxypropyl)-5-guanidinopentyl-amino]-5'-deoxyadenosine are potent inhibitors of PRMTI but not the lysine methyltransferase SET7. A related N-6 modified SAM analogue 5'-[(S-3-amino-3-carboxypropyl)-3-guanidinopropylamino]-5'-deoxy-N6-(lI-azido-3,6,9-trioxaundecane)-amino adenosine bearing an azide tether was developed with the aim of allowing facile introduction of biotin or fluorescent dyes, using either Staudinger ligation, or Cu(1) catalysed Huisgen reaction to provide compounds that can be used for affinity purification of the target protein or study of its localisation in cells respectively. Finally, progress toward a novel, rapid and enantioselective synthesis of the natural product (+)-sinefungin is reported. Key dihydropyridazine intermediates were generated from adenosyl 5'-propaldehyde, commercially available azodicarboxylate derivatives and ester substituted vinyltriphenylphosphonium salt by successful extension of methodology first reported by Ley and co-workers. Deprotection and ring opening of clihydropyridazine compounds was attempted, and unfortunately we were not able to generate (+)-sinefungin, although it is hoped that this route can be developed to achieve this in the future.

The research project presented in this thesis deals with the development of the alkylation of protected aldimines using organoaluminium and organozinc compounds as alkylating agents. To this end, efforts have been focused into the methylation reaction using trimethylaluminium and dimethylzinc. It was hoped to establish promising conditions using the methylate group and then to extend the catalytic system to other interesting nucleophiles. In the case of organoaluminium alkylation the reaction was extended to other nucleophiles, namely to the allyl and propargyl groups. The identificaton of suitable metal catalysts as well as diphosphine ligands was carried out by means of extensive high throughput screening. On the one hand [IrCl(COD)]2 proved to be very efficient when associated to AlMe3 or DABAL-Me3 in the non-enantioselective 1,2-addition reactions to aldimines (100% conversion in 3 h). On the other hand, [RhCl(COD)]BF4 was found to be able to efficiently catalyse the enantioselective 1,2-addition of Me2Zn to aldimine substrates (100% conversion in 3 h - up to 99% e.e.). A preliminary screening of a range of aldimines bearing different protecting groups aimed at selecting the most interesting substrate in terms of reactivity and ease of cleavage of the protecting group. Once this substrate had been identified, a range of derivatives was synthesised in order to appraise the scope of the newly developed reaction. The diphenylphosphinoyl (dpp) protecting group turned out to be the best activating group for aldimines tested within the framework of this study. In addition to being easy to introduce, the dpp group can also be removed easily under mild conditions. What is more, the presence of a phosphorus atom on this protecting group is a feature which was used to determine the enantiomeric excess by 31P NMR spectroscopy, thus providing a novel and efficient screening tool at disposal. In the course of this investigation, various issues were faced and tackled. One of them was the unexpected non-reproducibility taking place in the Rh-catalysed Me2Zn addition reaction; however, a deeper thinking of the reaction mechanism enabled us to solve this problem to eventually get a more robust catalytic system. Another one was the formation of a reduction product as a by-product of the Rh-catalysed Me2Zn addition reaction. Finally, several interesting attempts (Et2Zn addition, aliphatic imine synthesis), findings (effect of ligand bite angle) and hypotheses (testing of the BPM ligand) made during this study deserve to be studied further for improvement and optimisation.

This thesis describes the development and application of a rapid and sensitive electrospray ionization-mass spectrometry (ESI-MS) method to study the weak hydrophobic interactions seen in many Ub-Ub-binding domain (UBD) complexes. A range of UBDs has been screened against mono-Ub, di-Ub (Ub2) and tetra-Ub (Ub4). Affinities in the 2-200 J.lM range were found to be in excellent agreement with data obtained from other biophysical techniques. Insights into the UBD's preference for poly-Ub chain linkage and length are also provided by this methodology. Detection of a ternary complex involving Ub interacting simultaneously with two different UBDs demonstrated the co-existence of multisite interactions. A simple, clean and effective method for reducing charge states observed in ESI-MS without the use of any solution additives or instrumental modifications is also reported; with the charge reduction method ultimately promoting the investigation ofthe Ub-UBD interactions. Moreover, the development and application of a top-down proteomics approach to characterize the topology of an unanchored Ub dimer purified from rat skeletal muscle is also described in this thesis. This study has identified the topology of the Ub2 to be Lys48-linked. In addition, ESI-MS of endogenous Ub2 species has revealed the presence of cyclic Lys48-linked Ub2 and demonstrates for the first time that cyclisation of poly-Ub can also occur in vivo. Further to these studies, the inhibitory activity of small peptides against the complex formed by Ub with the ZnF domain of isopeptidase T (IsoT) is also investigated. Finally, the unusual effects of cation adduction upon the gas-phase conformation of three-helix bundle UBDs are revealed by ESI-IMS-MS and reported in this thesis.

Part A. Vibronic transitions of rhodium monoxide (Rh¹⁶O and Rh¹⁸O) were observed in the 380 to 700 nm region. Laser-induced fluorescence identified two ²[pi]r - X⁴Σ⁻ progressions with origins at {15 667, 15 976} and {15 874, 16 167} cm⁻¹. These progressions were labeled [15.8] ²[pi] - X⁴Σ⁻ and [16.0] ²[pi] - X⁴Σ⁻, respectively. Vibrational parameters were determined for the ground and excited states... Part B. Density functional and configuration interaction calculations on the lowest singlet and triplet potential energy surfaces of hydrogen, fluorine, and chlorine azide for the reactions XN₃ (~X¹A¹) -- NX(X³Σ; a¹Δ) + N₂ (X¹Σ⁺g) and XN₃ -- X(X²S; X²P₃/₂) + N₃(X²[pi]g) (X = H, F, Cl) show that the lowest energy dissociation pathway proceeds exothermically to NX(a) + N₂ . This surface is crossed on the bound singlet region by a dissociative triplet surface. Unimolecular decomposition rates for each pathway and the branching ratio support the experimental observations: HN₃ dissociates to ground state products while FN₃ and CIN₃ produce significant amounts of electronically excited NX. / Graduate

This thesis details the synthesis of molecular species designed for a variety of nanotechnological applications. The first chapter gives a brief introduction to nanotechnology and explains the concepts of supramolecular chemistry and self-assembly which are the basis on which this project is built. Special attention is given to non-covalent bonding interactions such as hydrogen-bonding and π- π interactions which drive the formation of complex architectures. The discussion is followed by many examples from the literature describing work that has been accomplished in various fields of supramolecular chemistry. The second chapter describes the synthesis, characterization and surface deposition studies of 1,4-diphenyl-1,3-butadiyne-3,3’-5,5’-tetracarboxylic acid (DPBDTC). Recent work on surface deposition studies of terphenyl-tetracarboxylic acid (TPTC) has shown the formation of a 2D network that resembles an aperiodic tiling structure that shows orientational order but no translational symmetry. DPBDTC was designed in order to improve the aperiodic characteristics of the network formed. An experimental exploration of the phase space of interacting random tilings was also possible using the network obtained from DPBDTC deposited from nonanoic acid along with networks obtained with different molecules and under different conditions. The third chapter focuses on the synthesis, characterization and surface deposition studies of a series of porphyrin molecules functionalised with isophthalic acid and ferrocene groups at the meso-positions. The isophthalic acid groups are incorporated to encourage intermolecular interactions while the ferrocene groups are designed as potential carriers of binary information in the form of the redox states of the iron center. The above molecules were designed in order to study their potential use in molecular memory systems. The series of molecules consisted of porphyrins having the acetylene spacer in various positions. The diethyl-ester protected derivatives of the carboxylic acids and their zinc and magnesium metal complexes were studied using spectroelectrochemistry in order to reveal the qualitative redox properties of these systems. The fourth chapter discusses the design, synthesis and characterization of a series of metallated homoleptic and heteroleptic dypyrrinato complexes, using the dipyrrin 5-ethynyl-ferrocenyl dipyrrin. These molecules were synthesized with the prospect of validating the role of such complexes as a part of a dye or an electrolyte in dye-sensitized solar cells. Electrochemical and spectroelectrochemical studies in solution were also performed.

This thesis describes the investigation of phase behaviour of binary and ternary mixtures at high pressure. The particular applications chosen to be explored in this phase behaviour investigation were supercritical fluid electrodeposition (SCFED) and carbon capture and storage (CCS). Chapter 1 introduces the phase behaviour of mixtures. Chapter 2 describes the equipment and analytical techniques used throughout this thesis including the high-pressure variable-volume view cell, electrical conductivity cell, high pressure FTIR cell, and high-pressure optical fiber phase analyser. Chapter 3 details the solubility and conductivity investigation of several supporting electrolytes in difluoromethane (CH2F2), which provided an electrochemical bath with sufficient conductivity for electrodeposition in supercritical fluids. The most effective supporting electrolyte amongst the eight ionic compounds tested was [N(nC4H9)4][Al(OC(CF3)3)4] which was found to give a moderate solubility and the highest conductivity (222 Scm2mol-1) in CH2F2. [N(nC4H9)4][Al(OC(CF3)3)4] was followed by [N(nC4H9)4][FAP] and [N(nCH3)4][FAP], , making all of them to be satisfactory potential supporting electrolytes for SCFED. Chapter 4 describes the investigation of water solubility in CO2/N2 mixtures relevant to the CCS process. The scope of the investigation covers a wide pressure range and two levels of N2 (xN2= 0.05 and xN2= 0.10). This experimental study was conducted by using the FTIR technique as described in further detail in Chapter 2. It was found that the presence of N2 in CO2 lowered the solubility of H2O in supercritical CO2 with N2 compared to pure CO2. The solubility of water also decreases significantly when the concentration of N2 is increased from 5% to 10%. Chapter 5 further explores the role of phase behaviour in the application of CCS with the investigation of the phase envelope of the ternary mixtures of CO2 and permanent gases (Ar, N2, and H2). Three ternary mixtures were measured (90% CO2 + 5% N2 + 5% Ar, 98% CO2 + 1% N2 + 1% Ar, and 95% CO2 + 3% H2 + 2% Ar) by using the fiber optic reflectometer, as described in further detail in Chapter 2. The experimental data presented in this part also have been used to validate the equation of state for the CCS applications. It was found that the phase envelope of CO2 shifted to a higher pressure and the two-phase region become broader with the presence of permanent gases. Overall, both GERG-2004 and gSAFT provide a good agreement between the predicted and experimental data for all the ternary mixtures investigated, with the exception of the bubble-point line for the 3%H2 + 2%Ar + 95% CO2 mixture. Finally, Chapter 6 summarises the research that was conducted in this thesis. It also evaluates the progress made towards achieving the aims initially set-up in Chapter 1.

This dissertation describes new computational approaches at high resolution for practical structure-based drug discovery. It begins with a brief review of structure-based computational approaches for drug discovery in comparison with ligand-based ones, followed by a discussion of important applications in selecting drug-like compounds and predicting drug metabolites. Since three-dimensional target structures are crucial for structure-based drug discovery, a new methodology based on force fields for protein structure refinement was developed. This methodology employs the VSGB 2.0 energy model in combination with a robust protonation state assignment algorithm and efficient sampling strategies. High accuracy was obtained for predicting 2239 protein side chains and 115 14-20 residue loops. Given the precision and uniform robustness, this methodology is believed for the first time to be suitable to tackle practical problems in structure-based drug discovery. The VSGB 2.0 energy model was then applied in the development of a new accurate approach (IDSite) for predicting P450-mediated drug metabolism, a problem of great practical interest for drug discovery. IDSite is able to efficiently model induced-fit effects using flexible docking and constrained refinements. Sites of metabolism are determined based on the physical interactions between a P450 enzyme and the ligand. Preliminary tests with 56 compounds displayed both low false positive and low false negative rates, which demonstrate the high potential of IDSite to be used in metabolism tests for drug discovery. In conclusion, this dissertation presents new computational approaches at high resolution to problems related to structure-based drug discovery with unprecedented accuracy. Given such high accuracy, these approaches are very promising in addressing practical issues in pharmaceutical research and development, and in enhancing our capability in the search for new safe drugs.

This thesis describes spectroscopic studies of three different systems: silver nanoparticles, the dye molecule rhodamine 6G adsorbed on graphene, and the type IV pili and c-type cytochromes produced by the dissimilatory metal-reducing bacterium Geobacter sulfurreducens. Although these systems are quite different in some ways, they can all be considered examples of nanomaterials. A nanomaterial is generally defined as having at least one dimension below 100 nm in size. Silver nanoparticles, with sub-100 nm size in all dimensions, are examples of zero-dimensional nanomaterials. Graphene, a single atomic layer of carbon atoms, is the paradigmatic two-dimensional nanomaterial. And although bacterial cells are on the order of 1 µm in size, the type IV pili and multiheme c-type cytochromes produced by G. sulfurreducens can be considered to be one- and zero-dimensional nanomaterials respectively. A further connection between these systems is their strong interaction with visible light, allowing us to study them using similar spectroscopic tools. The first chapter of this thesis describes research on the plasmon-mediated photochemistry of silver nanoparticles. Silver nanoparticles support coherent electron oscillations, known as localized surface plasmons, at resonance frequencies that depend on the particle size and shape and the local dielectric environment. Nanoparticle absorption and scattering cross-sections are maximized at surface plasmon resonance frequencies, and the electromagnetic field is amplified near the particle surface. Plasmonic effects can enhance the photochemistry of silver particles alone or in conjunction with semiconductors according to several mechanisms. We study the photooxidation of citrate by silver nanoparticles in a photoelectrochemical cell, focusing on the wavelength-dependence of the reaction rate and the role of the semiconductor substrate. We find that the citrate photooxidation rate does not track the plasmon resonance of the silver nanoparticles but instead rises monotonically with photon energy. These results are discussed in terms of plasmonic enhancement mechanisms and a theoretical model describing hot carrier photochemistry. The second chapter explores the electronic absorption and resonance Raman scattering of the dye molecule rhodamine 6G (R6G) adsorbed on graphene. Graphene has been shown to quench the fluorescence of adsorbed molecules and quantum dots, and some previous studies have reported that the Raman scattering from molecules adsorbed on graphene is enhanced. We show that reflective contrast spectroscopy can be used to obtain the electronic absorption spectrum of R6G adsorbed on graphene, allowing us to estimate the surface concentration of the dye molecule. From these results we are able to calculate the absolute Raman scattering cross-section for R6G adsorbed on bilayer graphene. We find that there is no evidence of enhancement but instead that the cross-section is reduced by more than three-fold from its value in solution. We further show that a model incorporating electromagnetic interference effects can reproduce the observed dependence of the R6G Raman intensity on the number of graphene layers. The third and final chapter describes the preliminary results from studies of the dissimilatory metal-reducing bacterium Geobacter sulfurreducens. This anaerobic bacterium couples the oxidation of organic carbon sources to the reduction of iron oxides and other extracellular electron acceptors, a type of anaerobic respiration that necessitates an electron transport chain that can move electrons from the interior of the cell to the extracellular environment. The electron transport chain in G. sulfurreducens has not been completely characterized and two competing mechanisms for the charge transport have been proposed. The first holds that G. sulfurreducens produces type IV pili, protein filaments several nanometers in width, with intrinsic metallic-like conductivity. According to this mechanism, the conductive pili mediate electron transport to extracellular acceptors. The second proposed mechanism is that charge transport proceeds by electron hopping between the heme groups in the many c-type cytochromes produced by G. sulfurreducens. In this picture, the observed conductivity of the pili is due to hopping through associated cytochrome proteins. Our aim is to explore these alternative mechanisms for electron transport in G. sulfurreducens through electrical and optical studies. We report the work we have done thus far to culture and characterize G. sulfurreducens, and we show that preliminary micro-Raman studies of G. sulfurreducens cells confirm that we can detect the spectroscopic signature of c-type cytochrome proteins. Future directions for this ongoing work are briefly discussed.

The internal DNA of the Pf1 bacteriophage is known from its dimensions to be the most extended naturally occurring DNA. Understanding its conformation is critical to further insights about DNA stability and packing processes in Pf1 and similar filamentous phages, and is of broader interest to biophysical studies of DNA. Structural studies of the intact 36 MDa Pf1 bacteriophage by solid-state NMR have, from their inception, been remarkably ambitious undertakings due to the size of the system and its structural complexity. Assignment and structural characterization of the major coat protein have been aided by symmetry and abundance of signal, and have been remarkably successful. However, it is only with the advent of improvements in methodology that the DNA of Pf1 can be studied. Recent rapid advances in techniques such as dynamic nuclear polarization have greatly improved sensitivity and made solid-state NMR studies applicable to a broader range of biopolymers and biological assemblies. The first high-resolution NMR study of the Pf1 DNA is presented herein. Assignment of the 13C and 15N resonances of the DNA at the level of nucleotide type has revealed a number of unusual chemical shifts, at or beyond the edges of their respective ranges in available databases. These database comparisons, especially at key conformational reporter sites such as sugar C3' and C5', confirm important details of existing structural models, such as a C2'-endo/gauche sugar pucker, anti glycosidic angle, an overall lack of base pairing, and the presence of aromatic stacking. Specific protein-DNA contacts consistent with those predicted by models are also observed.Fragment-based ab initio chemical shift prediction methods are employed in efforts to derive additional information from the experimental chemical shifts. The Pf1 DNA is found to be most consistent with models of highly stretched P-DNA derived from DNA stretching experiments, in contrast to more conventional forms like A- or Z-DNA. Further, the goodness-of-fit of existing structural models as well as several novel models is assessed; it is found that one of the new models, "Hybrid/2XKM", created by combining recent highly refined DNA and coat protein models, best reproduces experimental chemical shift patterns, and should likely be used as a starting point for subsequent refinements. Similar methodology is applied to the selectivity filter of the S. lividans potassium ion channel KcsA, finding that changes in ion occupancy alone are insufficient to reproduce experimental chemical shift perturbations. Hydration is important to the environment of the Pf1 DNA, and to our ability to detect it. NMR investigation of water populations in Pf1 samples reveals that water is in contact with a number of buried protein residues and the internal DNA, making a strong case for the existence of a pool of "internal hydration water." Such a water population has great potential to further benefit solid-state NMR studies of the Pf1 bacteriophage. Also, a new tool to study, analyze, and predict the effects of crystal contacts on solid-state NMR spectra is presented, along with a discussion of isotopic labeling strategies to reduce spectral congestion and aid in the collection of structural restraints for complex biomolecular assemblies.

This dissertation presents multi-photon spectroscopic studies on molecule/metal interfaces and graphene. Two different aspects of these ultrathin molecular or atomic materials were investigated: (1) the electronic structure of molecule/metal interfaces and (2) nonlinear optical properties of graphene. For the case (1), two-photon photoemission (TPPE) using a femotosecond laser was employed to investigate occupied and unoccupied electronic states of molecule/metal interfaces. Here we selected two specific examples of interfaces, benzenethiols on Cu(111) and hexa-cata-hexabenzocoronene (HBC) on Cu(111), which are important model systems for an organic / electrode interface of organic semiconductor devices. Although the same copper substrate was used for all the experiments, the nature of interfaces was strongly affected by the interaction between molecular adlayers and metal substrate. Our TPPE measurements on two benzenthiol species, thiophenol and p-fluorothiophenol, on Cu(111) focus on the role of adsorbates in shifting surface polarization and effecting surface electron confinement. As the coverage of each molecule increases, their photoemission-measured work functions exhibit nearly identical behavior up to 0.4-0.5 ML, at which point their behavior diverges; this behavior can be fit to an interfacial bond model for the surface dipole. In addition, our results show the emergence of an interfacial electronic state 0.1-0.2 eV below the Fermi level. This electronic state is attributed to quantum-mechanical-confinement shifting of the Cu(111) surface state by the molecular adsorbates. Another TPPE study of ours was carried out on an organic semiconductor, HBC, deposited on Cu(111). An increase of HBC coverage continuously shifts the vacuum level of the Cu substrate until a coverage of 2 ML is reached. In the same range of coverage, the Shockley state and the image potential states are quenched while new unoccupied states develop. The momentum- and polarization-resolved photoemission spectra reveal that the new states are originated from a Cu image state. Electron localization is discussed with respect to the structural evolution of HBC. For the case (2), nonlinear optical scanning microscopy was designed to study third-harmonic generation (THG) from micron- scale graphene crystals on glass substrate. The polarization-, thickness-, and orientation- dependence of THG signals from the graphene were measured and compared to theoretical prediction using the nonlinear optical slab model of Bloembergen and Pershan. The results revealed in-plane isotropy and out-of-plane anisotropy of the THG signals and sub-quadratic dependence of the layer number. Due to the strong THG signal, background-free imaging of graphene crystal was carried out. This result implies the potential application of THG for imaging graphene on arbitrary substrates.