In recent years the pharmaceutical industry has seen a rise in the number of drug compounds with low aqueous solubility, and consequently poor oral bioavailablility. One potential solution to this problem is to formulate such compounds as solid dispersions, whereby the drug is dispersed in a carrier matrix in the solid state. In this thesis, the hypothesis that a number of drug-drug and drug-polymer intermolecular interactions influence the physical stability and dissolution performance of solid dispersions is considered. The aim is to use correlations between drug molecular structure and solid dispersion performance to develop a platform to rapidly assess whether drug compounds will have favourable properties when formulated as a solid dispersion. Amorphous felodipine/copovidone solid dispersions are used as a model system to develop a suitable testing regime with regards to physical stability and dissolution performance. A laser light scattering technique developed in this work shows that morphological changes in felodipine/copovidone films exposed to water are due to polymer swelling. A combination of dissolution testing methodologies is also used to suggest a mechanism for the dissolution of bulk solid dispersion samples. Contributions of individual functional groups in the felodipine analogues to the physical stability and dissolution performance of their amorphous solid dispersions are assessed. Blocking of the felodipine amine hydrogen-bond-donor with an N-methyl, and the removal of chlorine substituents are both shown to reduce the physical stability of the solid dispersions. Correlations between molecular descriptors and data from the above experiments show that drug compounds are more likely to crystallise from solid dispersions with copovidone if they have a low log P, low relative molecular mass and low polarizability. Such correlations can form the basis of a screening method for the molecular design of analogous drug compounds likely to form high-performance solid dispersions with copovidone.

Surface chemical modifications are presented for supporting primary neurons in culture. The initial substrates for culture were glass and gold. The surface modifications were based on self assembled monolayer (SAM) approaches. Glass surfaces were initially modified by silanisation with either 3-aminopropyltrimethoxysilane (APTMS) or 3-aminopropyldimethylethoxysilane (APDES), to present amino-terminated surfaces. Gold surfaces were initially modified by thiol SAMs of either 11-amino-1-undecanethiol (AUT) or a peptide fragment of laminin (PA22-2), to present an amino- or peptide-terminated surface respectively. The amine-terminated surfaces of both glass and gold were subject to further modification. A heterobifunctional linker, containing a polyethylene glycol (PEG) spacer, was used to couple the peptide PA22-2 to the amino-terminated surfaces. Surface modifications were characterised using WCA, XPS and ToF-SIMS. The heterobifunctional linker bound homogeneously across the AUT SAM surface, however the linker was not distributed evenly on either of the amino silanisations of glass. Primary neurons from dissociated embryonic rat hippocampi were cultured on the modified glass and gold surfaces. The cell viability was measured during a 3 week long culture using calcein and ethidium homodimer fluorescence. Neuronal cell cultures were viable on all the gold surface modifications. The only viable glass surface was a control surface of adsorbed poly-l-lysine (PLL) on glass. Cell viability on the AUT and the Peptide-PEG-AUT modified gold surfaces was equivalent to the PLL coated glass. Inclusion of the PEG linker reduced protein adsorption from the media to the peptide modified gold surface, allowing cells to recognise the peptide rather than an adsorbed protein layer and improving their viability. The presented gold surface modifications provide suitable substrates for neural cultures which can be used in existing applications for investigating neural activity, such as; multi-electrode arrays, micro-fluidics devices, and surface plasmon resonance.

This thesis is concerned with coherent effects in high resolution mid-infrared gas phase spectroscopy using quantum cascade lasers (QCLs). An introductory chapter explains the importance of QCLs as radiation sources in the mid-infrared region of the spectrum and goes on to detail their development and structure. A discussion of coherent effects in spectroscopy follows, leading into the second chapter which discusses the theories relevant to the experimental sections of the thesis. In chapter 2 the theory underpinning direct and velocity selective, Doppler-free spectroscopy is discussed and a density matrix formalism is followed to derive the equations of motion that govern coherent excitation effects in two-level systems. In the final part of the chapter this treatment is extended to three-level systems. The equations derived in this chapter form the basis of quantitative interpretations of the phenomena observed in experimental data and presented in the remainder of the thesis. In chapter 3 the characterisation of a high power, narrow linewidth QCL is carried out. This laser is then used to perform direct and sub-Doppler resolution spectroscopy on NO, demonstrating non-linear absorption at high laser intensities and providing a measurement of the laser linewidth in the limit of slow frequency tuning. As the slow tuning rate increases, evidence of coherent transient effects is presented and density matrix theory used to model this behaviour. The data presented include the first observations of asymmetric Lamb dips and the onset of rapid passage oscillations from a Lamb dip. Pump-probe experiments on NO, utilising two cw QCLs are presented in chapter 4. The high level of velocity selection afforded by QCL excitation leads to coherent transient signals at far lower probe scan rates than previously reported. The effect of altering both the scan rate and the gas pressure and the importance of hyperfine structure are presented. A radio frequency noise source applied to one of the lasers is shown to broaden the laser linewidth, leading to rapid dephasing. A two-colour polarisation spectroscopy experiment is also presented which allows the measurement of both the absorption and the Doppler-free dispersion signals and the three-level density matrix formalism presented at the end of chapter 2 used to model the non-linear response of the system. The final chapter details the use of an acousto-optic modulator to create a pulse of mid-IR light using a cw QCL and the application of this to time resolved pump-probe spectroscopy. This capability suggests the prospect of achieving coherent population transfer by stimulated Raman adiabatic passage (STIRAP) using two such pulses. Simulations based on a simple three-level model and including Zeeman coherences are presented, which take the measured properties of the lasers used in this thesis as inputs to predict the potential population transfer achievable in NO as well as providing useful information about the angular momentum polarisation of the excited molecules. An experimental realisation of STIRAP would require the lasers to be stabilised, and so the final part of the chapter details experimental attempts to achieve stabilisation of an external cavity QCL, and suggests future avenues for improved implementation.

Organophosphorus-mediated reactions are important tools in organic chemistry and are used in the synthesis of highly desirable drug targets, such as morphine.1 A major drawback of traditional phosphorus-mediated reactions is the formation of stoichiometric amounts of phosphine oxide by-products; this renders the product purification difficult and reduces the atom efficiency of these transformations. For these reasons, catalytic variants become desirable; there being two potential strategies to achieve the catalysis; i) redox-driven and ii) redox-neutral.2-4 The redox-driven catalytic cycle requires a reductant for the turnover whilst the redox-neutral system uses a sacrificial reagent to directly turn over the phosphine oxide to the active phosphorus (V) reagent. This thesis will report upon a new class of Hydroxylmethyl-Aryl phosphine 1 and phosphine oxide 3 catalyst for use in a redox-driven catalytic reaction; the Staudinger reduction Scheme 1, and routes to a potential redox-neutral catalytic Mitsunobu reaction Scheme 2.

Absorption and emission spectra from the lowest energy transition in BODIPY have been simulated in the gas and water phase using a quantum mechanics/molecular mechanics approach, with DFT and the maximum overlap method (MOM). A post-SCF spin-purification to MOM yields transition energies in agreement with experimental data. Spectral bands were simulated using structures from ab initio molecular dynamics simulations, in which the solvent water molecules are treated classically and DFT is used for BODIPY. The resulting spectra are consistent with experimental data, and demonstrate how absorption and emission spectra in solution can be simulated using a quantum mechanical treatment of the solute. The electronic structure and photoinduced electron transfer (PET) processes in a fluorescent K+ sensor have been studied using DFT and TDDFT to rationalise its function. Absorption and emission energies of the fluorophore-localised intense excitation are more accurately described using MOM than TDDFT. Analysis of molecular orbital energies from DFT calculations in different phases cannot account for the sensors function. It is necessary to consider the relative energies of the electronic states. The inclusion of implicit solvent lowers the energy of the charge transfer state making a reductive PET possible in the absence of K+, while no such process is possible when the sensor is bound to K+. Binding within the ethene–argon and formaldehyde–methane complexes in ground and electronically excited states is studied with equations of motion coupled-cluster theory (EOM-CCSD), MP2 theory and dispersion-corrected DFT (DFT-D). MP2/MOM potential energy curves are in good agreement with EOM-CCSD calculations for the Rydberg and valence states studied. B3LYP-D3 calculations are in agreement with EOM-CCSD for ground and valence excited states, however for Rydberg states significant deviation is observed for a variety of DFT-D methods. Varying D2 dispersion parameters results in closer agreement with EOM-CCSD for Rydberg states.

This thesis describes optical microscopical and spectroscopical studies of 2D materials, including graphite/graphene and multilayer/single layer MoS2, under strong charge transfer doping. Under this conceptually unifying umbrella lie many aspects of materials behaviors unique to each of the systems. The strong chemical doping results from intercalation and surface adsorption, and changes the electronic properties of the host 2D materials drastically. Associated with the significant electronic change, aspects such as mass transport, surface reaction, and phase transformation are covered in the following chapters. The first chapter introduces representative members of the 2D materials family, graphene and molybdenum disulphide (MoS2). It briefly reviews the history, discovery and unique properties of each materials class. The other part of the introduction focuses on the main methods utilized in the study of these materials. A concise survey of Raman spectroscopy and optical reflectance contrast spectroscopy will be presented. The second chapter investigates the intercalation process of Li into bulk graphite. This is a revisit of an extensively studied subject, with a new set of experiments and theories. Here we show that the daunting technical difficulties of disentangling complex electrochemical systems can be cleanly addressed with optical methods with well defined samples. Measuring and understanding the intrinsic transport of Li in graphite electrodes has been a difficult task. The challenge is well recognized to stem from a multitude of simultaneous electrochemical processes as well as systematic heterogeneities in the sample. We distinguish the Li intercalation process in graphite from all other processes, combining optical reflectance microscopy and Raman spectroscopy. The heterogeneity problem is circumvented by using lithography to tailor a single crystal into a defined geometry. We apply two levels of theoretical models to interpret the intrinsic information revealed in our data. Concentration dependent diffusion coefficients are measured, in agreement with theoretical results. The effects of sample geometry and electrode reaction kinetics on the overall intercalation are elucidated. The third chapter presents the study of lithiation on single and few layer graphene. Raman spectroscopy reveals a high doping level similar in strength to that of the bulk intercalated compound. The optical reflectance imaging, however, shows a different observation from the bulk case. We directly visualize the surface film formation and associated strong doping. The lithiation in single and few layer graphene progresses differently from the bulk graphite, since certain stages of the intercalation compound cannot be sustained by a single or few layer sample. The realization of strong charge transfer doping in lithiated single and few layer graphene could lead to discoveries of interesting physics. The direct visualization of surface film formation could have important implications in the design of electrochemical energy storage systems. The fourth chapter explores the structural effect of strong charge transfer doping in bulk and multilayer MoS2 with optical methods. MoS2, as a representative material of the transition metal dichalcogenide family, possesses different structural polymorphs. Strong charge transfer doping induces a structural phase change, which goes from the usual thermodynamically stable semiconducting 2H phase into the metallic 1T/1T' phase. The metallic 1T/1T' structure can remain a metastable phase without the stabilization of intercalants. We optically induce the 1T/1T' to 2H phase change and measure the temperature dependent kinetics of the structural phase transformation with in situ Raman spectroscopy. We demonstrate a photolithography technique, which efficiently patterns in-plane coherent heterojunctions between 1T/1T' and 2H MoS2. The fifth and final chapter describes the study of the structural change in single layer MoS2. More spectroscopic methods are employed for characterization, such as photoluminescence spectroscopy and second harmonic generation. The results indicate that the structural change occurs in single layer MoS2 after reaction with n-butyl lithium. The structural change can be reversed by thermal and laser annealing, similar to the case of bulk and multilayer MoS2. The annealed MoS2 exhibits reduced crystallinity. Future directions to further this work are outlined in the last section.

Homology modeling is integral to structure-based drug discovery. Robust homology modeling to atomic-level accuracy requires in the general case successful prediction of protein loops containing small segments of secondary structure. For loops identified to possess α-helical segments, an alternative dihedral library is employed composed of (phi,psi) angles commonly found in helices. Even with imperfect knowledge coming from sequence-based secondary structure, helix or hairpin embedded loops, up to 17 residues in length, are successfully predicted to median sub-angstrom RMSD. Having demonstrated success with these cases, performance costs for these and other similar long loop predictions will be discussed. Dramatic improvements in both speed and accuracy are possible through the development of a Cβ-based scoring function, applicable to hydrophobic residues, that can be applied as early as half-loop buildup. With this scoring function, up to a 30-fold reduction in the cost to produce competitive sub-2 A loops are observed. Through the use of this scoring function, an efficient method will be presented to achieve ultra-high resolution buildup that restrains combinatorial explosion and offers an alternative to the current approach to full-loop buildup. This novel method is designed to be inherently suitable for homology model refinement.

This thesis describes the development of practical and efficient computational approaches to the quantum dynamics of complex systems. Most of the work presented here relies on the generalized quantum master equation (GQME) formalism, which provides a simple equation of motion of reduced dimensionality for a set of dynamical quantities, e.g., nonequilibrium averages and equilibrium time correlation functions. The reduced dimensionality of the GQME comes at a cost: the introduction of the memory kernel, which accounts for the influence of all ``excluded'' degrees of freedom. Focusing first on the second-order perturbative treatment of the memory kernel known as Redfield theory, I present a collaborative effort to extend its applicability into highly non-Markovian regions via a mode freezing approach. In this method, a portion of bath modes characterized by low frequencies are treated as sources of static disorder and used to calculate modified Redfield dynamics. Application of the method to the spin-boson and FMO complex models indicates that the Redfield+frozen modes scheme consistently produces dynamics that are as good or better than bare Redfield dynamics. Next, we explore GQME approach coupled to the self-consistent solution of the memory kernel, which requires the calculation of auxiliary kernels. Previous implementations of the method had shown impressive boosts in efficiency and, when approximate methods were used to calculate the auxiliary kernels, accuracy over direct calculation of nonequilibrium averages. We show that this method, when formulated from the Mori perspective, is equally applicable to nonequilibrium averages and equilibrium correlation functions. In addition, we examine the dependence of the improvements afforded by the GQME framework on the choice projection operator and kernel closure. In particular, we demonstrate that improvements in efficiency, which rely on short memory lifetimes, are sensitively dependent on the choice of projection operator, and that the choice of kernel closure directly dictates the improvements in accuracy. In addition, we present evidence that indicates that the success of the GQME formalism when the auxiliary kernels are calculated via semi- and quasi-classical methods is largely due to the exact sampling of bath operators at t=0 required by the calculation of specific kernel closures. Next, we provide analytical arguments that delineate when the GQME framework coupled to the self-consistent solution of the memory kernel is likely to provide improvements in efficiency and accuracy. Finally, we present a path integral framework that can efficiently render the partially Wigner-transformed canonical density operator for systems coupled linearly to harmonic baths. This approach permits the direct calculation of any thermodynamic quantity and can be integrated into dynamical schemes like the Ehrenfest, surface hopping, or linearized semi-classical initial value representation methods to calculate equilibrium correlation functions.

Translation of an mRNA template into its corresponding protein is necessarily a highly accurate process. In all organisms, this translation is performed by the universally conserved macromolecular machine, the ribosome. However, the mechanisms through which the ribosome is able to regulate translation, and therefore ensure its fidelity, are not well understood. Often these types of mechanisms, which ensure molecular fidelity, utilize multiple, transient states over which cognate and non-cognate substrates are discriminated multiple times. However, such transient and/or rarely populated states are difficult to study by conventional, ensemble experimental techniques. In this thesis, single-molecule fluorescence resonance energy transfer (smFRET), which alleviates many of these limitations, is used in order to interrogate the dynamics of a translation factor, release factor 1 (RF1), and how they are organized to ensure accurate and efficient recognition of stop-codons during the termination stage of translation. In order to observe the dynamics of the RF1 binding and codon discrimination processes with smFRET, a relatively high concentration of fluorophore-labeled RF1 must be used in order to observe significant binding to sense-codons; however, such high concentrations are not accessible with traditional smFRET total internal fluorescence microscopy. Therefore, in Chapter 2 a novel approach to breaking this concentration barrier is presented, in which robustly-passivated gold-based nanoaperture arrays are developed to limit the excitation volume used in smFRET measurements of RF1. Unfortunately, as in the case of RF1 binding to sense-codon programmed ribosomes, many of the ribosomal dynamics that are in principal observable using smFRET are too fast to observe using current wide-field detectors. Therefore, Chapter 3 investigates the precision and accuracy with which transient conformational dynamics can be quantified using single-molecule techniques such as smFRET. As a case study, these approaches were used to analyze the dynamics of the GS1-GS2 equilibrium of the pretranslocation (PRE) ribosome--a situation where transient intermediate states that can be observed using single-particle cryo-electron microscopy are not seen using smFRET. In Chapter 4, a novel computational method is developed to address such temporally-limited single-molecule data, and in doing so, it is used to analyze the structural contributions of tRNA to ribosomal transition state energy barriers using temperature-dependent smFRET with temporal super-resolution. The temperature-dependence of reaction rate constants is governed by the underlying thermodynamic landscape of the molecular system. To investigate the energy landscape over which the PRE ribosome operates, temperature-dependent smFRET experiments were performed on PRE complexes containing different tRNAs. By investigating the relative temperature-dependence of the rate constants involved in the GS1 - GS2 equilibrium as a function of tRNA identity, nascent polypeptide chain presence, and A and P site occupation, relative thermodynamic contributions of the different structural elements were quantified. Unfortunately, this investigation was complicated by fast rate constants which approach the time resolution limitations of smFRET TIRF experiments, especially with the increased temperatures used in these experiments. Additionally, it is complicated by the heterogeneity within the ensemble of ribosomes that is created when some of the enzymatically-prepared ribosomal complexes fail to undergo, or undergo additional rounds of translation. To overcome these complications, a novel computational method to achieve temporal super-resolution. This method uses Bayesian inference for the analysis of sub-temporal resolution data (BIASD). By integrating this approach with a Bayesian variational mixture model, the fast dynamics of heterogenous populations can be accurately and precisely quantified. This then allowed the contributions of the structural differences that the various tRNA make to the underlying PRE complex energy landscape to be determined. The conformational dynamics that regulate the binding affinity and codon discrimination ability of RF1 are investigated in Chapter 5. During the elongation stage of translation, class I release factors compete with aminoacyl-tRNAs to interrogate the mRNA triplet-nucleotide codon that is located in the ribosomal aminoacyl-tRNA binding (A) site. To avoid deleterious effects, class I RFs must be able to accurately discriminate stop-codons from sense-codons, only triggering the termination stage of translation and catalyzing the release of the nascent polypeptide chain from the peptidyl-tRNA located in the ribosomal peptidyl-tRNA binding (P) site upon recognition of a stop-codon. Despite its importance for ensuring the accuracy of gene expression, the high fidelity mechanism through which class I RFs discriminate sense codons remains elusive. Using smFRET, the kinetics with which a fluorophore-labeled, bacterial RF1 binds to the A site of bacterial ribosomal release complexes carrying a fluorophore-labeled peptidyl-tRNA in the P site and either a stop-codon, or a sense-codon that differs from a stop-codon by a single nucleotide (i.e., a near-stop codon) programmed in the A-site are investigated. The results of these experiments, as well as analogous experiments performed using RF1 mutants or antibiotic inhibitors of RF1 function, reveal that RF1 binding affinity and codon discrimination occurs via a multistep process. Taken together with molecular dynamics simulations of wildtype and mutant RF1, these data demonstrate how the conformation dynamics of the switch loop modulate RF1 binding affinity and codon discrimination--enabling the elucidation of some of the molecular details through which class I RFs ensure the integrity of translation elongation and the fidelity of translation termination.

This thesis focuses on an important aspect of protein functionality – protein-protein interactions (PPI). Three physical chemistry techniques for or derived from protein-protein interaction investigation are discussed. First, in Chapter 2, we demonstrate a new fluorescent imaging technique that creates high-order nonlinear signals by harnessing the frustrated fluorescence resonance energy transfer (FRET) – energy transfer between certain proteins close in proximity which is commonly used in PPI studies. In Chapter 3, we combine fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET), two most commonly used approaches to monitor protein-protein interactions in vivo, to create a novel hybrid strategy, bioluminescence assisted switching and fluorescence imaging (BASFI), which integrates the advantages of FRET and BRET. We demonstrate BASFI with Dronpa-RLuc8 fusion constructs and drug-inducible intermolecular FKBP-FRB protein-protein interactions in live cells with high sensitivity, resolution, and specificity. Finally, in Chapter 4, we propose a systematic free energy perturbation (FEP) protocol to computationally calculate the binding affinities between proteins. We demonstrate our protocol with the gp120 envelope glycoprotein of HIV-1 and three broadly neutralizing antibodies (bNAbs) of the VRC01 class and analyze antibody residues’ contributions to the binding which further provides insights for antibody design.