Molecular Interactions Chlorin Assemblies and NMR Separations

Photosynthesis is a chemical process in which the energy of the light quanta is transformed into chemical energy. Chlorophyll (Chl) molecules play a key role in photosynthesis; they function in the antennae systems and in the photosynthetic reaction center where the primary charge separation (CS) takes place. Bio-inspired mimicry of the CS is an essential unit in dye-sensitized solar cells. Aim of this study was to design and develop electron donor-acceptor (EDA) pairs from Chls and fullerenes (C60) or carbon nanotubes (CNT). The supramolecular approach was chosen, as long synthetic sequences required by the covalent approach lead to long reaction schemes and low yields. Here, a π-interaction between soluble CNTs and Chl was used in EDA construction. Also, a beta-face selective two-point bound Chl-C60 EDA was introduced. In addition, the photophysical properties of the supramolecular EDA dyads were analyzed. In organic chemistry, nuclear magnetic resonance (NMR) spectroscopy is the most vital analytical technique in use. Multi-dimensional NMR experiments have enabled a structural analysis of complex natural products and proteins. However, in mixture analysis NMR is still facing difficulties. In many cases overlapping signals can t be resolved even with the help of multi-dimensional experiments. In this work, an NMR tool based on simple host-guest chemistry between analytes and macromolecules was developed. Diffusion ordered NMR spectroscopy (DOSY) measures the mobilities of compounds in an NMR sample. In a liquid state NMR sample, each of the analytes has a characteristic diffusion coefficient, which is proportional to the size of the analyte. With normal DOSY experiment, provided that the diffusion coefficients of the analytes differ enough, individual spectra of analytes can be extracted. When similar sized analytes differ chemically, an additive can be introduced into the sample. Since macromolecules in a liquid state NMR sample can be considered practically stationary, even faint supramolecular interaction can change the diffusion coefficient of the analyte sufficiently for a successful resolution in DOSY. In this thesis, polyvinylpyrrolidone and polyethyleneglycol enhanced DOSY NMR techniques, which enable mixture analysis of similar in size but chemically differing natural products, are introduced.

Aspects and Applications of Pulse Sequence Design for Solution-State Nuclear Magnetic Resonance Spectroscopy

NMR spectroscopy enables the study of biomolecules from peptides and carbohydrates to proteins at atomic resolution. The technique uniquely allows for structure determination of molecules in solution-state. It also gives insights into dynamics and intermolecular interactions important for determining biological function. Detailed molecular information is entangled in the nuclear spin states. The information can be extracted by pulse sequences designed to measure the desired molecular parameters. Advancement of pulse sequence methodology therefore plays a key role in the development of biomolecular NMR spectroscopy. A range of novel pulse sequences for solution-state NMR spectroscopy are presented in this thesis. The pulse sequences are described in relation to the molecular information they provide. The pulse sequence experiments represent several advances in NMR spectroscopy with particular emphasis on applications for proteins. Some of the novel methods are focusing on methyl-containing amino acids which are pivotal for structure determination. Methyl-specific assignment schemes are introduced for increasing the size range of 13C,15N labeled proteins amenable to structure determination without resolving to more elaborate labeling schemes. Furthermore, cost-effective means are presented for monitoring amide and methyl correlations simultaneously. Residual dipolar couplings can be applied for structure refinement as well as for studying dynamics. Accurate methods for measuring residual dipolar couplings in small proteins are devised along with special techniques applicable when proteins require high pH or high temperature solvent conditions. Finally, a new technique is demonstrated to diminish strong-coupling induced artifacts in HMBC, a routine experiment for establishing long-range correlations in unlabeled molecules. The presented experiments facilitate structural studies of biomolecules by NMR spectroscopy.

Characterisation and processing of amorphous binary mixtures with low glass transition temperature

The number of drug substances in formulation development in the pharmaceutical industry is increasing. Some of these are amorphous drugs and have glass transition below ambient temperature, and thus they are usually difficult to formulate and handle. One reason for this is the reduced viscosity, related to the stickiness of the drug, that makes them complicated to handle in unit operations. Thus, the aim in this thesis was to develop a new processing method for a sticky amorphous model material. Furthermore, model materials were characterised before and after formulation, using several characterisation methods, to understand more precisely the prerequisites for physical stability of amorphous state against crystallisation. The model materials used were monoclinic paracetamol and citric acid anhydrate. Amorphous materials were prepared by melt quenching or by ethanol evaporation methods. The melt blends were found to have slightly higher viscosity than the ethanol evaporated materials. However, melt produced materials crystallised more easily upon consecutive shearing than ethanol evaporated materials. The only material that did not crystallise during shearing was a 50/50 (w/w, %) blend regardless of the preparation method and it was physically stable at least two years in dry conditions. Shearing at varying temperatures was established to measure the physical stability of amorphous materials in processing and storage conditions. The actual physical stability of the blends was better than the pure amorphous materials at ambient temperature. Molecular mobility was not related to the physical stability of the amorphous blends, observed as crystallisation. Molecular mobility of the 50/50 blend derived from a spectral linewidth as a function of temperature using solid state NMR correlated better with the molecular mobility derived from a rheometer than that of differential scanning calorimetry data. Based on the results obtained, the effect of molecular interactions, thermodynamic driving force and miscibility of the blends are discussed as the key factors to stabilise the blends. The stickiness was found to be affected glass transition and viscosity. Ultrasound extrusion and cutting were successfully tested to increase the processability of sticky material. Furthermore, it was found to be possible to process the physically stable 50/50 blend in a supercooled liquid state instead of a glassy state. The method was not found to accelerate the crystallisation. This may open up new possibilities to process amorphous materials that are otherwise impossible to manufacture into solid dosage forms.

Understanding solid-state transformations during dehydration: new insights using vibrational spectroscopy and multivariate modelling

Many active pharmaceutical ingredients (APIs) have both anhydrate and hydrate forms. Due to the different physicochemical properties of solid forms, the changes in solid-state may result in therapeutic, pharmaceutical, legal and commercial problems. In order to obtain good solid dosage form quality and performance, there is a constant need to understand and control these phase transitions during manufacturing and storage. Thus it is important to detect and also quantify the possible transitions between the different forms. In recent years, vibrational spectroscopy has become an increasingly popular tool to characterise the solid-state forms and their phase transitions. It offers several advantages over other characterisation techniques including an ability to obtain molecular level information, minimal sample preparation, and the possibility of monitoring changes non-destructively in-line. Dehydration is the phase transition of hydrates which is frequently encountered during the dosage form production and storage. The aim of the present thesis was to investigate the dehydration behaviour of diverse pharmaceutical hydrates by near infrared (NIR), Raman and terahertz pulsed spectroscopic (TPS) monitoring together with multivariate data analysis. The goal was to reveal new perspectives for investigation of the dehydration at the molecular level. Solid-state transformations were monitored during dehydration of diverse hydrates on hot-stage. The results obtained from qualitative experiments were used to develop a method and perform the quantification of the solid-state forms during process induced dehydration in a fluidised bed dryer. Both in situ and in-line process monitoring and quantification was performed. This thesis demonstrated the utility of vibrational spectroscopy techniques and multivariate modelling to monitor and investigate dehydration behaviour in situ and during fluidised bed drying. All three spectroscopic methods proved complementary in the study of dehydration. NIR spectroscopy models could quantify the solid-state forms in the binary system, but were unable to quantify all the forms in the quaternary system. Raman spectroscopy models on the other hand could quantify all four solid-state forms that appeared upon isothermal dehydration. The speed of spectroscopic methods makes them applicable for monitoring dehydration and the quantification of multiple forms was performed during phase transition. Thus the solid-state structure information at the molecular level was directly obtained. TPS detected the intermolecular phonon modes and Raman spectroscopy detected mostly the changes in intramolecular vibrations. Both techniques revealed information about the crystal structure changes. NIR spectroscopy, on the other hand was more sensitive to water content and hydrogen bonding environment of water molecules. This study provides a basis for real time process monitoring using vibrational spectroscopy during pharmaceutical manufacturing.

Contributions to the Theory of Finite-State Based Grammars

This dissertation is a theoretical study of finite-state based grammars used in natural language processing. The study is concerned with certain varieties of finite-state intersection grammars (FSIG) whose parsers define regular relations between surface strings and annotated surface strings. The study focuses on the following three aspects of FSIGs: (i) Computational complexity of grammars under limiting parameters In the study, the computational complexity in practical natural language processing is approached through performance-motivated parameters on structural complexity. Each parameter splits some grammars in the Chomsky hierarchy into an infinite set of subset approximations. When the approximations are regular, they seem to fall into the logarithmic-time hierarchyand the dot-depth hierarchy of star-free regular languages. This theoretical result is important and possibly relevant to grammar induction. (ii) Linguistically applicable structural representations Related to the linguistically applicable representations of syntactic entities, the study contains new bracketing schemes that cope with dependency links, left- and right branching, crossing dependencies and spurious ambiguity. New grammar representations that resemble the Chomsky-Schützenberger representation of context-free languages are presented in the study, and they include, in particular, representations for mildly context-sensitive non-projective dependency grammars whose performance-motivated approximations are linear time parseable. (iii) Compilation and simplification of linguistic constraints Efficient compilation methods for certain regular operations such as generalized restriction are presented. These include an elegant algorithm that has already been adopted as the approach in a proprietary finite-state tool. In addition to the compilation methods, an approach to on-the-fly simplifications of finite-state representations for parse forests is sketched. These findings are tightly coupled with each other under the theme of locality. I argue that the findings help us to develop better, linguistically oriented formalisms for finite-state parsing and to develop more efficient parsers for natural language processing. Avainsanat: syntactic parsing, finite-state automata, dependency grammar, first-order logic, linguistic performance, star-free regular approximations, mildly context-sensitive grammars

NMR Spectroscopy of Multi-domain Proteins : Immunoglobulin-like Domains of Human Filamin A

Proteins are complex biomacromolecules playing fundamental roles in the physiological processes of all living organisms. They function as structural units, enzymes, transporters, process regulators, and signal transducers. Defects in protein functions often derive from genetic mutations altering the protein structure, and impairment of essential protein functions manifests itself as pathological conditions. Proteins operate through interactions, and all protein functions depend on protein structure. In order to understand biological mechanisms at the molecular level, one has to know the structures of the proteins involved. This thesis covers structural and functional characterization of human filamins. Filamins are actin-binding and -bundling proteins that have numerous interaction partners. In addition to their actin-organizing functions, filamins are also known to have roles in cell adhesion and locomotion, and to participate in the logistics of cell membrane receptors, and in the coordination of intracellular signaling pathways. Filamin mutations in humans induce severe pathological conditions affecting the brain, bones, limbs, and the cardiovascular system. Filamins are large modular proteins composed of an N-terminal actin-binding domain and 24 consecutive immunoglobulin-like domains (IgFLNs). Nuclear magnetic resonance (NMR) spectroscopy is a versatile method of gaining insight into protein structure, dynamics and interactions. NMR spectroscopy was employed in this thesis to study the atomic structure and interaction mechanisms of C-terminal IgFLNs, which are known to house the majority of the filamin interaction sites. The structures of IgFLN single-domains 17 and 23 and IgFLN domain pairs 16-17 and 18-19 were determined using NMR spectroscopy. The structures of domain pairs 16 17 and 18 19 both revealed novel domain domain interaction modes of IgFLNs. NMR titrations were employed to characterize the interactions of filamins with glycoprotein Ibα, FilGAP, integrin β7 and dopamine receptors. Domain packing of IgFLN domain sextet 16 21 was further characterized using residual dipolar couplings and NMR relaxation analysis. This thesis demonstrates the versatility and potential of NMR spectroscopy in structural and functional studies of multi-domain proteins.

Computational studies on new chemical species in the gas-phase and the solid-state.

There is intense activity in the area of theoretical chemistry of gold. It is now possible to predict new molecular species, and more recently, solids by combining relativistic methodology with isoelectronic thinking. In this thesis we predict a series of solid sheet-type crystals for Group-11 cyanides, MCN (M=Cu, Ag, Au), and Group-2 and 12 carbides MC2 (M=Be-Ba, Zn-Hg). The idea of sheets is then extended to nanostrips which can be bent to nanorings. The bending energies and deformation frequencies can be systematized by treating these molecules as an elastic bodies. In these species Au atoms act as an 'intermolecular glue'. Further suggested molecular species are the new uncongested aurocarbons, and the neutral Au_nHg_m clusters. Many of the suggested species are expected to be stabilized by aurophilic interactions. We also estimate the MP2 basis-set limit of the aurophilicity for the model compounds [ClAuPH_3]_2 and [P(AuPH_3)_4]^+. Beside investigating the size of the basis-set applied, our research confirms that the 19-VE TZVP+2f level, used a decade ago, already produced 74 % of the present aurophilic attraction energy for the [ClAuPH_3]_2 dimer. Likewise we verify the preferred C4v structure for the [P(AuPH_3)_4]^+ cation at the MP2 level. We also perform the first calculation on model aurophilic systems using the SCS-MP2 method and compare the results to high-accuracy CCSD(T) ones. The recently obtained high-resolution microwave spectra on MCN molecules (M=Cu, Ag, Au) provide an excellent testing ground for quantum chemistry. MP2 or CCSD(T) calculations, correlating all 19 valence electrons of Au and including BSSE and SO corrections, are able to give bond lengths to 0.6 pm, or better. Our calculated vibrational frequencies are expected to be better than the currently available experimental estimates. Qualitative evidence for multiple Au-C bonding in triatomic AuCN is also found.

Tailoring polyolefin properties by metallocene catalysis

This doctoral thesis deals with the syntheses of olefin homo- and copolymers using different kind of metallocene catalyst. Ethene, propene, 1-hexene, 1-hexadecene, vinylcyclohexane and phenylnorbornene were homo- or copolymerized with the catalysts. The unbridged benzyl substituted zirconium dichloride catalysts (1-4), ansa- bridged acenaphtyl substituted zirconium dichloride catalysts, ( 5, 6), rac- and meso-ethylene-bis(1-indenyl)zirconium dichlorides, (rac- and meso-8), rac-ethylene-bis(1-indenyl)hafnium dichloride, ( 12), bis(9-fluorenyl)hafnium dichloride (14 ) enantiomerically pure (R)- phenylethyl[(9-fluorenyl-1-indenyl)]ZrCl2, (11), 14 and asymmetric dimethylsilyl[(3-benzylindenyl-(2-methylbenzen[e]indenyl)] zirconium dichloride, (13), were prepared in our laboratory. Dimethylsilyl-bis(1-indenyl)zirconium dichloride, (9), isopropylidene(9-fluorenyl-cyclopentadienyl)zirconium dichloride, (10), and were obtained commercially. The solid-state structures of the catalysts rac- and meso-1 were determined by X-ray crystallography. Computational methods were used for the structure optimization of the catalyst rac- and meso-1 in order to compare the theoretical calculations with the experimental results. Polymerization experiments were conducted in a highly purified autoclave system using low pressures (< 5 bar) of gaseous monomers. The experiments were designed to attain the optimal catalytic activity and a uniform copolymer composition. The prepared homo- and copolymers were characterized by the gel permeation chromatography, GPC, differential scanning calorimetry, DSC, nuclear magnetic resonance, NMR, and Fourier transform infrared spectrometry, FTIR . Molar mass (Mw, Mn), molar mass distribution (Mw/Mn), tacticity, comonomer content, melting temperature, glass transition temperature, and end group structures and content were determined. A special attention was paid on the correlation of the polymer properties with the catalyst structures and polymerization conditions. An intramolecular phenyl coordination was found in phenyl substituted benzyl zirconocenes 1-3 explaining the decreased activity of the catalysts. Novel copolymers poly(propene-co-phenylnorbornene) and poly(propene co-vinylcyclohexane), were synthesized and high molar mass poly(ethene-co-1-hexene) and poly(ethene-co-1-hexadecene) copolymers with elastic properties were prepared. Activation of a hafnocene catalyst was studied with UV-Vis spectrometry and activation process for the synthesis of ultra high molar mass poly(1-hexene) was found out.