Over the past few decades, amorphous solid dispersions (ASDs) have been of great interest to pharmaceutical scientists to address bioavailability issues associated with poorly water-soluble drugs. ASDs consist of an active pharmaceutical ingredient (API) that is typically dispersed in an inert polymeric matrix. Despite promising advantages, a major concern that has resulted in limited marketed formulations is the physical instability of these complex formulations. Physical instability is often manifested as phase heterogeneity, where the drug and carrier migrate and generate distinct phases, which can be a prelude to recrystallization. One important factor that dictates the physical stability of ASDs is the spatial distribution of API in the polymeric matrix. It is generally agreed that intimate mixing of the drug and polymer is necessary to achieve maximum stabilization, and thus understanding the factors controlling phase mixing and nano-domain structure of ASDs is crucial to rational formulation design. The focus of this thesis work is to better understand the factors involved in phase mixing on the nanometric level and get insights on the role of excipients on overall stabilization of these systems. The central hypothesis of this research is that an intimately mixed ASD will have better physical stability as compared to a partially homogeneous or a non-homogeneous system. Our approach is to probe and correlate phase homogeneity and intermolecular drug-excipient interactions to better understand the physical stability of ASDs primarily using solid-state nuclear magnetic resonance (SSNMR) spectroscopy and other solid-state characterization tools. A detailed investigation was carried out to understand the role of hydrogen bonding on compositional homogeneity on different model systems. A comprehensive characterization of ternary ASDs in terms of molecular interactions and physical stability was studied. Finally, long-term physical stability studies were conducted in order to understand the impact of different grades of a cellulosic polymer on phase homogeneity for two sets of samples prepared via different methods. Overall, through this research an attempt has been made to address some relevant questions pertaining to nano-phase heterogeneity in ASDs and provide a molecular level understanding of these complex systems to enable rational formulation design.

Concrete, due to its low cost, durability and fire resistance, is one of the world’s most widely used construction materials. Concrete is typically reinforced with steel bars and welded wire mesh. Since the cost of steel is increasing and steel corrosion is a significant contributor to structural failure, it is advantageous to find an alternative replacement reinforcement material which can not only replace the steel, but also resist corrosion. Over the past few decades, polymeric fibres have been used as concrete reinforcement. The chemical bond between the polymeric fibre and the cementitious matrix is an important factor in the fibre’s performance as a concrete reinforcement. Despite the great importance of the chemical bonding at the polymeric fibre/concrete interface, the chemical bonding at the interface is not well understood. To investigate the chemical interactions between polymeric materials and concrete, model systems of polymeric powder/white cement and polymeric fibre/white cement were chosen, where white cement was chosen for its suitability for nuclear magnetic resonance (NMR) experiments. The chemical interactions between poly(ethylenevinyl acetate) (EVA), poly(ether imide) (PEI), and poly(vinylidene fluoride) (PVDF) polymeric powders were studied via 13C NMR spectroscopy. It was found that EVA admixture undergoes hydrolysis in a cementitious matrix and follows a pseudo-second order kinetics model up to 32 days of cement hydration. PEI was also found to undergo hydrolysis at the imide functional group in a cementitious matrix. PVDF powder undergoes dehydrofluorination in the cementitious environment, producing a brown coloured polymer which is a result of conjugation of the polymer backbone. The interfacial transition zone between fluoropolymeric powder/white cement and steel and polymeric fibres (high density polyethylene/polypropylene, poly(vinyl alcohol), PEI, PVDF, and Nylon 6.6) was studied at short range using 19F, 27Al, and 43Ca NMR spectroscopy and at long range using the scanning electron microscopy/energy dispersive spectroscopy method. It was concluded that the chemistry of polymeric fibres themselves can alter the surrounding interfacial transition zone such that the calcium silicate hydrate favours a tobermorite or jennite-like structure, which could contribute to a strong or weak interface.

O estudo de propriedades de transporte das fases vítreas do sistema LixTh1-xF4-3x por RMN do 7Li e do 19F mostrou que tanto os ions Li+ quanto F- são condutores. O lítio é móvel para todas as temperaturas estudadas e atua como cátion modificador do retículo, enquanto que o flúor é condutor apenas acima de 373K. Por espectroscopia vibracional ficou evidenciada a variação do número de coordenação do Th (de 8,5 a 9,5) em função da composição. No sistema LixU1-xF4-3x verificou-se que o átomo de urânio assume coordenação 8, independentemente da composição. O paramagnetismo dos vidros de urânio, determinado por medidas magnéticas e de RPE, é devido ao paramagnetismo inerente ao átomo de urânio isolado impedindo o estudo destas amostras por RMN. A determinação da estrutura cristalina da fase Li3ThF7 utilizando difração de raios-X por monocristal mostrou que este material cristaliza-se no grupo espacial P4/ncc do sistema tetragonal; a=6,200(1)&#197, c=12,937(2)&#197, Z=4 moléculas/cela unitária. A estrutura é formada por camadas de poliedros (ThF9) ,com quatro vértices comuns no plano ab. Os átomos de lítio localizam-se entre estas cama das ligando os poliedros [ThF9) ao longo do eixo c. Há uma desordem dos átomos de lítio que ocupam os sítios 8f e 16g na proporção 1:3. O efeito quadrupolar observado por RMN do 7Li na fase cristalina, acima de 333K, pode ser explicado pela troca de íons lítio entre sítios não equivalentes que modulam os gradientes de campo elétrico a que cada núcleo individual esta submetido. Cálculos dos componentes principais do tensor gradiente de campo elétrico, para os diferentes sítios ocupados pelo lítio, indicam a ionocovalência da ligação Th-F e o processo de difusão planar deste íon entre as camadas de poliedros de tório. O processo de difusão planar do lítio na fase cristalina Li3ThF7 comparado ao da amostra vítrea de mesma composição Li0,75Th0,25F1,75, explica o valor ligeiramente inferior da energia de ativação do lítio medido para a amostra cristalina. No cristal há periodicidade das camadas de lítio facilitando a mobilidade o que não acontece no vidro onde o encadeamento dos poliedros é distorcido a curta distância. O estudo cristaloquímico da fase policristalina LiZnF3 exibiu três tipos de estrutura possíveis: coríndon, ilmenita e LiTaO3, cujos grupos espaciais são indistinguíveis por difração de raios-X em policristais. Cálculos de segundo momento teórico comparados ao experimental observado por RMN continua do 19F, permitiram descartar a hipótese estrutural tipo LiTaO3. A desconvolução do espéctro de ressonância do 7Li, evidenciando o mascaramento dos satélites quadrupolares do lítio, possibilitou o cálculo do momento quadrupolar. Este efeito permitiu eliminar a estrutura tipo coríndon e mostrou que este é o primeiro fluoreto sintetizado com estrutura ilmenita. / The 7Li and 19F NMR study of transport properties of the vitreous phases in the system LixTh1-xF4-3x shows that both Li+ and F- are ionic carriers. In all the temperature range studied lithium cations are mobile and behave as a network modifier, meanwhile fluorine anions are mobile above 373K. The variation of the thorium coordination number (from 8.5 to 9.5) with composition is followed by vibration spectroscopy. It has been verified that, in the system LixU1-xF4-3x the coordination number of uranium (equal to 8) does not depends on the composition. The paramagnetism of uranium glasses determined by EPR and magnetic measurements, due to the paramagnetism of isolated uranium atom, forbids NMR studies on these samples. Crystal structure determination of Li3ThF7 has been performed on single crystal by X-ray diffraction: tetragonal system, space group P4/ncc; a= 6.200(1)&#197, c=12.937(2)&#197, Z=4. The structure is characterized by layers of ThF9 polyhedra sharing 4 corners in ab plane. The lithium atoms are localized between these layers and bridge ThF9 polyhedra along c-axis. There is a 1:3 disorder of lithium atoms in the sites 8f and 16g. The rapid exchange of the lithium ions between non equivalent sites modulates the electric field gradients seen by individual lithium and can explain the behavior of the 7Li quadrupolar effect observed (above 333K) in the crystalline phase. Calculations of the main components of the electric field gradient tensor according to the different sites of lithium atoms indicate a part of covalence in the Th-F bond and support the lithium planar diffusion between thorium polyhedra layers. The comparison between the lithium planar diffusion in the crystalline phase Li3ThF7, and in the vitreous composition Li0,75Th0,25F1,75 allows an explanation of the slightly weaker activation energy observed in the crystal. The periodicity of the Li+ layers in the crystal facilities this mobility hindered at the contrary by the short range distorted chains of thorium polyhedral in the glass. An X-ray study of the polycrystalline phase LiZnF3 does not permit to choose between the three possible structures: corindon, ilmenite or LiTaO3 type. A comparison between experimental and theoretical 19F second moment leads to eliminate the structural hypothesis of LiTaO3 type. The deconvolution of the 7Li resonance spectrum shows a first order quadrupolar effect of the lithium and allows the calculation of the quadrupolar splitting. This effect in contradiction with a corindon type structure, is in good agreement with the ilmenite type, showing the first fluoride synthesized with an ilmenite structure.

Many proteins are unstable in solution and must be formulated in the solid state. This has led to an increase in the use of lyophilized dosage forms. Lyophilization is a complicated processing method consisting of three major steps: freezing, primary drying, and secondary drying. This can lead to several formulation stability challenges including changes in ionization within the matrix, phase separation of the protein drug from added stabilizers, sufficient mobility within the system for movement of reactive species and protein side chains, and crystallization of excipients upon storage. Solid-State Nuclear Magnetic Resonance Spectroscopy (SSNMR) is used to characterize many important properties of lyophilized formulations including crystalline vs. amorphous content, polymorphic form, ionization profile, interaction between formulation components with domain sizes, and mobility within the cake matrix. In order to study ionization changes in lyophilized solids, SSNMR and UV/Vis Diffuse Reflectance spectroscopy were used. 13C-labeled fumaric, succinic, and butyric acids were added to formulations at various pH levels, and were used to quantify change in the ionization of the matrix by monitoring the ionization ratios of the carboxylic acid peaks using SSNMR. pH indicators were also added to the formulations and their ionization ratio was determined using UV/Visible Diffuse Reflectance Spectroscopy. The ionization profile in the solid state was compared with that in solution before lyophilization. A rank ordering of ionization shift was made in pharmaceutically relevant buffers. SSNMR proton relaxation times (1H T1 and 1H T1rho) for each formulation component can be compared to determine homogeneity within the lyophilized matrix. The concept of spin diffusion is used in order to determine the length scale on which the components are either homogeneous or phase separated. The domain size is typically 20-50 nm or 2-10 nm for 1H T1 and 1H T1rho, respectively. PVP and dextran polymers were phase separated on both domains for physical mixtures and lyophilized mixtures. BSA and lysozyme were both lyophilized with formulations containing sucrose, trehalose, or mannitol as the stabilizer. Mannitol crystallized, and the relaxation times showed phase separation. Sucrose and trehalose both formed homogeneous systems at both length scales when formulated in a 1:1 ratio with BSA or lysozyme. Aspartame was shown to be phase separated from trehalose. The SSNMR proton relaxation times were also used to measure the local mobility in the lyophilized matrix, as a timescale of picoseconds to nanoseconds is associated with the 1H T1 relaxation time. Mobility was monitored in formulations containing a fixed amount of sucrose and mannitol, but with a variable amount of an IgG2 protein. The 1H T1 relaxation times decreased as protein content increased. The formulations with the highest relaxation time (lowest mobility), was the most stable in accelerated temperature conditions as monitored by size exclusion chromatography and capillary isoelectric focusing. This method can be used to rank order the most stable formulations at time-zero. Anti-plasticization was also studied by formulating sorbitol in various ratios with trehalose. The 1H T1 relaxation times increased with increasing sorbitol content, while the glass transition temperature decreased. Sorbitol and trehalose glasses were also exposed to different temperature storage conditions. Sorbitol appears to promote aging, as the formulations with higher sorbitol content showed larger increases in proton relaxation time.

Electrostatic interactions such as halogen bonding and pnictogen bonding interactions have gained a lot of interest in the field of crystal engineering and pharmaceutical science. In the first part of this thesis, we expand our knowledge on anion coordinated halogen bonded cocrystals by looking at a series of cocrystals made from 3-iodoethynyl pyridine and 3-iodoethynylbenzoic acid. We utilize the power of mechanochemistry to create the new cocrystals made with phosphonium salts and use multinuclear solid-state nuclear magnetic resonance spectroscopy and X-ray diffraction and characterize them. We found that mechanochemistry is a fast and powerful tool to explore and synthesize new halogen bonded cocrystals and ³¹P solid-state NMR is a rapid way to identify the formation of a cocrystal. In the second part, we look at the versatility of the pnictogen atom, specifically antimony, as a pnictogen bond donor and a halogen bond acceptor. We evaluate these electrostatic interactions with nuclear quadrupolar resonance and found that nuclear quadrupole resonance is a strong spectroscopy tool to probe these types of electrostatic interactions.

This thesis presents analyses of hydrogen-bonded materials using solid-state nuclear magnetic resonance (NMR) spectroscopy. Proton dynamics were investigated in two classes of phosphate-based proton conductors: phosphate solid acids and tin pyrophosphates. These materials have the potential to be used as solid state proton conductors in fuel cells. Proton dynamics in phosphate solid acids were probed based on the attenuation of homonuclear dipolar coupling with increasing temperature. These studies showed that homonuclear dipolar recoupling NMR techniques can be employed in complex multi-spin systems. Additionally, two pathways for proton hopping in monoclinic RbH2PO4, a sample with two proton environments, were identified and quantified for the first time using a combination of dipolar recoupling and proton exchange NMR methods. Tin pyrophosphates, another class of solid-state proton conductor with analogous phosphate tetrahedral structure, were studied. Proton dynamics had to be analyzed via exchange-based NMR techniques as a result of low proton concentration in these materials. Proton mobility in tin pyrophosphate was found to increase with increased protonation. Furthermore, hydrogen bonding was investigated as a coordination mode in silicone boronic acid (SiBA) elastomers, potential materials for contact lens manufacture. As in the phosphate-based proton conductors, hydrogen bonding played an important role in the structure of the SiBA elastomers as one of the mechanisms through which these materials crosslink. In addition to hydrogen bonding, covalent bonding between boronic acids was found to occur at three- and four-coordinate boron centers. The purpose of this study was to determine the influence of boronic acid loading and packing density on crosslinking in SiBA elastomers. Boron coordination environments were investigated by 11B quadrupolar lineshape analysis. The incidence of four-coordinate dative bonding, a predictor of the stress-strain response in these materials, increased with boronic acid loading but was most heavily influenced by boronic acid packing density. / Thesis / Doctor of Philosophy (PhD) / Hydrogen bonds are intermolecular interactions that are significant in many structural (low crystal density in ice) and dynamic (enzymatic processes occurring under biological conditions) processes that are necessary to maintain life. In this thesis, solid-state nuclear magnetic resonance (NMR) spectroscopy is used to explore proton dynamics of hydrogen-bonded networks in various materials. Advanced NMR experiments that probe homo- and heteronuclear dipolar coupling interactions revealed possible pathways for proton transport in phosphate-based proton conducting materials. This study provided a better understanding of ion conducting mechanisms that can be used in intermediate-temperature fuel cell applications. Additionally, solid-state NMR was used in the identification of hydrogen bonding and other coordination modes in silicone boronate acids (SiBA), a class of elastomers with potential applications as contact lens. Boron coordination in SiBA elastomers was dependent on both boronic acid loading and boronic acid packing density.

The performance of polymeric diphenylmethane diisocyanate (PMDI) resin is known to be highly dependent on the wood species. This species dependence may be due to differences in: cure chemistry, interphase morphology, or both of these factors. This study addresses aspects of the cure chemistry and interphase morphology of wood/PMDI bondlines; specifically these effects are compared using two woods: yellow-poplar and southern pine. In this study, the cure chemistry of wood-PMDI system was analyzed with solid state NMR (SSNMR) using wood samples cured with doubly labeled (15N,13C) PMDI resin. The kinetics of PMDI cure in the presence of wood was analyzed with differential scanning calorimetry. Thermogravimetric analysis was used to analyze the effect of resin impregnation on the degradation patterns of wood. The wood-PMDI bond morphology was probed with dynamic and static (creep) mechanical analyses in both dry and plasticized conditions. The effect of resin on wood polymer relaxations was quantitatively analyzed by both the time-temperature superposition principle and the Kohlrausch-Williams-Watts equation. The presence of a small but statistically significant species effect was observed on both the cure chemistry and bond morphology of wood-PMDI system at low cure temperatures. The cure of PMDI resin was found to be significantly faster in pine relative to corresponding poplar samples. Resin impregnation showed a significant species dependent effect on the wood mechanical properties; the resinated pine samples showed increase in compliance while the corresponding poplar samples became stiffer. The in situ lignin relaxation was studied with both dynamic and static modes, using plasticized wood samples. Results showed that the lignin relaxation was slightly affected by resin impregnation in both woods, but the effect was relatively larger in pine. Static experiments of dry wood samples showed a significant reduction in the interchain interactions of wood polymers in pine samples, exclusively. Investigation of plasticized pine samples, which focuses on the in situ lignin relaxations, showed only minor changes with resin impregnation. This led us to hypothesize that the large changes observed in dry samples, were due to the in situ amorphous polysaccharides. The wood-PMDI interactions were significantly reduced upon acetylation of wood. This study also discusses three new and highly sensitive methods for the analysis of wood-resin interactions. / Ph. D.

Nesta tese, a ressonância magnética nuclear do estado sólido (SSNMR) foi utilizada para estudar a composição química e estrutura dos componentes da parede celular de plantas. Visando contribuir no desenvolvimento de estratégias de despolimerização da biomassa, SSNMR foi inicialmente utilizada para estudar efeitos dos pré-tratamentos químicos e físicos, e da ação de enzimas sobre algumas biomassas. Os resultados mostraram que, em baixas concentrações, tratamentos ácidos são altamente efetivos na remoção das frações de hemicelulose, com pouco efeito nas frações de lignina e celulose. Já tratamentos alcalinos promovem eficiente deslignificação da biomassa, sendo que a mínima concentração da solução alcalina necessária para obter a máxima deslignificação depende do tipo de biomassa e da temperatura do tratamento. Os estudos por SSNMR foram correlacionados com estudos por outras técnicas, contribuindo para um entendimento mais profundo sobre o efeito dos pré-tratamentos e da hidrolise enzimática em diferentes biomassas. Outra parte da tese aborda a determinação da cristalinidade de celulose nativa (não extraída) de biomassa de bagaço de cana-de-açúcar. Utilizando a técnica de polarização cruzada em múltiplas etapas (Multi-CP) e um procedimento de subtração espectral, foi possível isolar os sinais de RMN da celulose nativa e a partir daí avaliar o índice de cristalinidade (CI). Esse método foi utilizado para avaliar o CI da celulose nativa de bagaço de cana-de-açúcar submetido à pré-tratamentos com H2SO4 e NaOH e os resultados não mostraram variações significativas do CI da celulose nas concentrações utilizadas, apesar do aumento da eficiência da hidrólise. Assim, ao contrário de muitos trabalhos encontrados na literatura, não parece que a cristalinidade da celulose seja um fator primordial no aumento de eficiência da hidrólise enzimática. Na parte final da tese, as interações intermoleculares entre os dois principais polissacarídeos da biomassa: celulose e xilano foram investigadas utilizando uma variedade de técnicas avançadas de RMN bidimensional. Neste trabalho, a arquitetura molecular de hastes de plantas de Arabidopsis Thaliana, sem nunca serem seca foi estudada. Utilizando a técnica refocused J-INADEQUATE (Increadible Natural Abundance Double Quantum Transfer Experiment via J coupling) observamos dois conjuntos de deslocamentos químicos distintos para o xilano, sendo um deles coincidente com aquele observado em solução. Em seguida, utilizamos experimentos SSNMR com o intuito de investigar se algum desses domínios de xilano estaria vinculado com a celulose. Experimentos CP-PDSD (Proton Driven Spin Diffusion detected via 13C through Cross-Polarization) demonstram a existência de proximidade espacial entre o novo domínio do xilano e o domínio da celulose. A comparação de resultados entre as amostras de padrão e o seu mutante deficiente em celulose (irx3) indicaram que o xilano com novo deslocamento químico é fortemente dependente da presença de celulose. A análise da mobilidade molecular pela técnica Dipolar Chemical Shift Correlation (DIPSHIFT), mostrou que as moléculas do novo domínio do xilano são altamente rígidas - uma característica partilhada com a celulose. Combinados, esses dados fornecem evidências de uma arquitetura molecular específica entre os dois polissacarídeos majoritários da parede celular. / Solid-state nuclear magnetic resonance (SSNMR) was used to study the chemical composition and structure of plant cell wall components. Aiming the development of depolymerization strategies, SSNMR was initially used to study the effects of chemical and physical pre-treatments, as well as the enzymatic action on the structure and composition of biomasses. The results showed that, at low concentrations, pre-treatments with acids are highly effective for removal of hemicellulose without significant effect on lignin and cellulose. In turn, the alkaline pre-treatment promotes efficient delignification of the biomass. The minimum concentration of the alkaline solution required to achieve the maximum delignification depends on the type of biomass and treatment temperature. SSNMR studies were correlated with studies using other techniques, contributing to an in-depth understanding of the effect of pre-treatments and enzymatic hydrolysis in different biomasses. Another part of the thesis discusses is the determination of native cellulose crystallinity (not extracted) of sugarcane bagasse biomasses. Using the cross-polarization technique in multiple blocks (Multi-CP) and a spectral subtraction approach, it was possible to isolate the NMR signals of the native cellulose and to evaluate the crystallinity index (CI). This method was used to accessof the CI of cellulose in sugarcane bagasse samples pre-treated with H2SO4 and NaOH. The results did not show significant variations of the cellulose CI, at the concentration used here, despite the increase in the hydrolysis efficiency. Thus, in contrast to some studies in the literature, it does not appear that the crystallinity of cellulose is a primary limiting factor concerning the enzymatic hydrolysis efficiency in biomasses. In the final part of this thesis, the intermolecular interactions between the two main polysaccharides of the plant cell wall, cellulose and xylan, were investigated using advanced two-dimensional NMR techniques. The molecular architecture of 13C labelled never-dried Arabidopsis Thaliana stems was studied. Using refocused J-INADEQUATE (Increadible Natural Abundance Double Quantum Transfer Experiment via J coupling) we observed two distinct chemical shifts in xylan, one of which coincides with that observed in solution. Next, we used SSNMR experiments toinvestigate the interaction between the novel xylan and cellulose domains. CP-PDSD (Proton Driven Spin Diffusion detected via 13C through Cross-Polarization) experiments demonstrated spatial proximity between the new xylan and cellulose domains. The same approach was used to study cellulose deficient (irx3) mutants and the comparison between the results indicate that the new xylan domain is cellulose-dependent. Dipolar Chemical Shift Correlation (DIPSHIFT) experiments were performed to analyse the molecular mobility of these polysaccharides showing that the novel xylan is highly rigid - a characteristic which is shared with cellulose. Combined, these data provide evidence for a specific molecular architecture between the two most common polysaccharides in plant cell walls.

The thesis aims at methodological developments in Nuclear Magnetic Resonance (NMR) and study of oriented samples like liquid crystals and single crystals and powder samples. Though methodological development in solid state NMR (ss-NMR) has gone far ahead, this work attempts to contribute some novel methods in this direction. The work presented here falls into two categories (i) methodological developments for obtaining information on anisotropic interactions and (ii) experiments which utilize the existing methodologies to study systems of interest under static condition and also under sample spinning at the Magic Angle. In the solid state, dipolar couplings play a crucial role. On the one hand these couplings could be used to transfer polarization from an abundant nucleus to a rare nucleus and increase the sensitivity of the rare nucleus. On the other hand, the measurement of dipolar couplings itself is crucial for extracting structural and dynamic information. A third aspect is that dipolar couplings could be used to obtain correlation, say between two different nuclear species or between the same kind of nuclei as in an exchange experiment. A major part of this thesis deals with all three aspects mentioned above. The thesis presents a new heteronuclear polarization transfer scheme which is devoid of some of the short comings of the existing and well-known polarization transfer schemes. This pulse sequence has been found to be useful in different contexts involving both spin ½ and spin 1 nucleus. The use of dipolar couplings for obtaining correlation in both static oriented systems and in powder samples has been illustrated. In the case of the powder sample, the study has been useful in obtaining useful orientation information. Finally, chemical shifts are known to be indicators of finer structural features of molecules in solution and solid state. 13 C MAS NMR studies have been exploited in understanding these structural features of short peptides containing prolines in the solid state and for comparing with their structures in solution. Chapter 1 covers the theoretical aspects required for the experimental work described in the thesis. A brief description of NMR has been followed by the explicit description of various interaction Hamiltonian’s in ss-NMR. Subsequently the experimental and the theoretical tools needed for ss-NMR study like Magic Angle Spinning (MAS), Cross-Polarization, Homo/Heteronuclear decoupling schemes have also been discussed. Chapter 2, describes a new heteronuclear polarization transfer scheme for oriented samples – named DAPT (Dipolar Assisted Polarization Transfer) and its application to different systems. DAPT uses a homonuclear decoupling sequence such as BLEW-12 for effecting heteronuclear polarization transfer. The chapter has been divided into five related parts. Section 2(A) starts with an introduction to the existing heteronuclear polarization transfer schemes. Subsequently the theoretical background of the new sequence is presented. Experimental implementation of the sequence in an oriented system, liquid crystal is presented and is compared with the well-known polarization transfer scheme, Hartmann-Hahn Cross Polarization (HH-CP). In 2(B) the implementation of the sequence as a local field spectroscopy for measuring heteronuclear dipolar couplings is presented. After initial discussion about local field spectroscopy and its relevance in ss-NMR, the improvements made in the earlier mentioned sequence along with its 2D implementation in a liquid crystal sample are described. A comparative study is also presented using DAPT with various other homonuclear decoupling sequences. Chapter 2(C) deals with the extension of DAPT to spin-1 systems. The difficulties in setting up the HH-CP in spin-1 systems are highlighted. Experimental demonstrations on a test sample of oriented CD3 I and also on a deuterated liquid crystal is described. The sequence has been incorporated as part of a 2D correlation experiment, where the F1 dimension provides the quadrupolar couplings of deuterium and the F2 the chemical shifts of the attached carbons. The comparison of the sequence with HH-CP, its merits and demerits are discussed and the potential applications are highlighted. Chapter 2(D) deals with the relatively less studied transition in 14N nucleus, known as the Overtone-Transition (OT). An introduction to OT and its relevance is provided in the beginning followed by the extension of DAPT in exciting and detecting OT. The experiments have been done on a single crystal of a model peptide, N-Acetyl-DL-Valine and are compared with the conventional method. Amide proton chemical shifts are also measured using DAPT in an indirect way. The advantages and the future application in studying OT are also discussed. Chapter 2(E) discusses the extension of DAPT to the single crystal of NAV and in identifying the molecules in the unit cell. The SLF spectrum of NAV is complicated due to the presence of two magnetically in-equivalent molecules in the unit cell and with pairs of splitting for each C - 1H and C - 1H pairs. The dipolar couplings are extracted from the experiment and with the aid of a MATLAB program and by incorporating the crystal coordinates, identification of C-1H and C-1H pairs belonging to a particular molecule have been carried out. Chapter 3 describes a novel and useful modification of the well-known Separated Local Field (SLF) sequence in solid state known as PISEMA (Polarization Spin Exchange at the Magic Angle). PISEMA is a popular technique for measuring heteronuclear dipolar couplings in oriented in oriented biological membranes and in liquid crystals. While it has several advantages such as a large dipolar scaling factor, narrow line-widths in the dipolar dimension and ease of setting up etc it suffers from a major problem. The technique is highly sensitive to the proton off-sets which affect the measured dipolar couplings. In the present chapter the origin of this problem has been analyzed in detail and a solution has been proposed. The modification to the experiment has been implemented on a liquid crystal and the off-set independence of the new sequence has been demonstrated. Further studies on a more rigid system such as a solid single crystal has been used to verify the effect of the modification on homonuclear decoupling efficiency and the consequent effects on the line widths in addition to off-set independence. The advantages of the proposed method over the existing one in terms of line-width and robustness in measuring heteronuclear dipolar couplings are demonstrated. Chapter 4 presents a study of deuterium exchange on a di-peptide. Deuterium as well as carbon-13 NMR spectroscopy has been extensively used earlier on static powder samples for studying exchange phenomena. In the present study we have applied the methodology for obtaining relative N-D vector orientation in a di-peptide. The magnetization exchange between deuterium nuclei through the dipolar couplings between them has been monitored. The need to match the quadrupolar split energy levels of two different deuterium’s differently oriented in the magnetic field requires that the sample be spun slowly. Characteristic exchange powder patterns were obtained which were used to infer relative orientation information. Comparison with the crystal structure indicates that the magnetization is likely to be inter-molecular rather than intra-molecular. The chapter follows the following sequence. A brief description about the importance of exchange studies in ss-NMR is presented. A theoretical approach is followed by a discussion of the angular dependence of the frequencies and the visualization of the mutual re-orientation angles. The motivation of the project followed by the experimental techniques, especially the use of slow MAS ~ 100 Hz in exchange studies are also presented. Initial studies have been carried out on di-methyl sulphone to check the reproducibility of the earlier reported results and later the sequence is extended to amide proton deuterated di-glycine. The 2D exchange spectrum recorded under slow MAS is then discussed in the context of the crystal structure and possible amide deuteriums involved in the exchange process are inferred. Chapter 5 discusses the natural abundant 13C and 15N NMR spectroscopy in the solid state in designed tri-peptides containing prolines. Proline is a unique amino acid because of it torsion angle values and is responsible for the turns and the globularity of the proteins. The well studied SH3 domain protein often binds to short peptides containing prolines and hence these study gains importance. Three peptides containing prolines were taken up for study. For peptide (1), the conformation was observed as cis/trans in the solution state and for the other two peptides it was all trans. The X-ray studies showed that peptide (1) has two molecules in the unit cell with both cis conformation. This motivated us to look at the solid state spectra of the peptides. Chemical shifts are signatures of conformers and it was established from the chemical shift differences that there exist two molecules in the unit cell for peptide (1), both in cis conformation. The conformers for the other two peptides predicted by NMR chemical shifts also matched with those obtained from X-ray studies. This opens up the possibility of using simple NMR measurements in the solid state as tools for secondary structure determination in larger peptides and proteins.

The aim of this thesis was the investigation of the mitochondrial response mechanisms upon apoptotic stimuli. The specific objectives were the biophysical characterization of membrane dynamics and the specific roles of lipids in the context of apoptotic regulation occurring at the mitochondrion and its complex membrane systems. The BH4 domain is an anti-apoptotic specific domain of the Bcl-2 protein. Solid phase peptide synthesis was used to produce large amount of the peptide for biophysical studies. A protocol has been established and optimized, guarantying the required purity for biophysical studies. In detail the purification by high performance liquid chromatography and the characterisation via mass spectroscopy are described. The secondary structure of BH4 changes significantly in the presence of lipid vesicles as observed by infrared spectroscopy and circular dichroism. The BH4 peptide aggregates at the membrane surface and inserts slightly into the hydrophobic part of the membrane. Using nuclear magnetic resonance (NMR) and calorimetry techniques, it could even be shown that the BH4 domain modifies the dynamic and organization of the liposomes which mimic a mitochondrial surface. The second study was on the first helix of the pro-apoptotic protein Bax. This sequence called Bax-α1 has the function to address the cytosolic Bax protein to the mitochondrial membrane upon activation. Once again a protocol has been established for the synthesis and purification of this peptide. The aim was to elucidate the key role of cardiolipin, a mitochondria-specific phospholipid, in the interaction of Bax-α1 with the mitochondrial membrane system. The NMR and circular dichroism studies showed that Bax-α1 interacts with the membrane models only if they contain the cardiolipin, producing a strong electrostatic lock effect which is located at the membrane surface. Finally, a new NMR approach was developed which allows the investigation of the lipid response of isolated active mitochondria upon the presence of apoptotic stimuli. The goal was there to directly monitor lipid specific the occurring changes during these physiological activities.