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.

Schiff Base Complexes and their Assemblies on Surfaces

Schiff bases and their transition metal complexes are of significant current interest even though they have been prepared for decades. They have been used in various applications such as catalysis, corrosion protection, and molecular sensors. In this study, N-aryl Schiff base ketimine ligands as well as numerous new, differently substituted salen and salophen-type ligands and their cobalt(II), copper(II), iron(II), manganese(II), and nickel(II) complexes were synthesised. New solid state structures of the above compounds and the dioxygen coordination properties of cobalt(II) complexes and catalytic properties of three synthesised binuclear complexes were examined. The prepared complexes were applied in the formation of self-assembled layers on a polycrystalline gold surface and liquid-graphite interface. The effect of metal ion and ligand structure on the as-formed patterns was studied. When studying gold surfaces, a unique thiol-assisted dissolution of elemental gold was observed and a new thin gold foil preparation method was introduced. In the summary, synthesis, structures, and properties of Schiff base ligands and their transition metal complexes are described in detail and the applications of these reviewed. Assemblies of other complexes on a liquid-graphite interface and on a gold surface are also presented, and the surface characterisation methods and surfaces employed are described.

Enzymes with radical tendencies: the PFL family

The first glycyl radical in an enzyme was described 20 years ago and since then the family of glycyl radical enzymes (GREs) has expanded to include enzymes catalysing five chemically distinct reactions. The type enzymes of the family, anaerobic ribonucleotide reductase (RNRIII) and pyruvate formate lyase (PFL) had been studied long before it was known that they are GREs. Spectroscopic measurements on the radical and an observation that exposure to oxygen irreversibly inactivates the enzymes by cleavage of the protein proved that the radical is located on a particular glycine residue, close to the C-terminus of the protein. Both anaerobic RNRIII and PFL, are important for many anaerobic and facultative anaerobic bacteria as RNRIII is responsible for the synthesis of DNA precursors and PFL catalyses a key metabolic reaction in glycolysis. The crystal structures of both were solved in 1999 and they revealed that, although the enzymes do not share significant sequence identity, they share a similar structure - the radical site and residues necessary for catalysis are buried inside a ten stranded $\ualpha $/$\ubeta $-barrel. GREs are synthesised in an inactive form and are post-translationally activated by an activating enzyme which uses S-adenosyl methionine and an iron-sulphur cluster to generate the radical. One of the goals of this thesis work was to crystallise the activating enzyme of PFL. This task is challenging as, like GREs, the activating component is inactivated by oxygen. The experiments were therefore carried out in an oxygen free atmosphere. This is the first report of a crystalline GRE activating enzyme. Recently several new GREs have been characterised, all sharing sequence similarity to PFL but not to RNRIII. Also, the genome sequencing projects have identified many PFL-like GREs of unknown function, usually annotated as PFLs. In the present thesis I describe the grouping of these PFL family enzymes based on the sequence similarity and analyse the conservation patterns when compared to the structure of E. coli PFL. Based on this information an activation route is proposed. I also report a crystal structure of one of the PFL-like enzymes with unknown function, PFL2 from Archaeoglobus fulgidus. As A. fulgidus is a hyperthermophilic organism, possible mechanisms stabilising the structure are discussed. The organisation of an active site of PFL2 suggests that the enzyme may be a dehydratase. Keywords: glycyl radical, enzyme, pyruvate formate lyase, x-ray crystallography, bioinformatics

Proton translocation coupled to electron transfer reactions in terminal oxidases

Terminal oxidases are the final proteins of the respiratory chain in eukaryotes and some bacteria. They catalyze most of the biological oxygen consumption on Earth done by aerobic organisms. During the catalytic reaction terminal oxidases reduce dioxygen to water and use the energy released in this process to maintain the electrochemical proton gradient by functioning as a redox-driven proton pump. This membrane gradient of protons is extremely important for cells as it is used for many cellular processes, such as transportation of substrates and ATP synthesis. Even though the structures of several terminal oxidases are known, they are not sufficient in themselves to explain the molecular mechanism of proton pumping. In this work we have applied a complex approach using a variety of different techniques to address the properties and the mechanism of proton translocation by the terminal oxidases. The combination of direct measurements of pH changes during catalytic turnover, time-resolved potentiometric electrometry and optical spectroscopy, made it possible to obtain valuable information about various aspects of oxidase functioning. We compared oxygen binding properties of terminal oxidases from the distinct heme-copper (CcO) and cytochrome bd families and found that cytochrome bd has a high affinity for oxygen, which is 3 orders of magnitude higher than that of CcO. Interestingly, the difference between CcO and cytochrome bd is not only in higher affinity of the latter to oxygen, but also in the way that each of these enzymes traps oxygen during catalysis. CcO traps oxygen kinetically - the molecule of bound dioxygen is rapidly reduced before it can dissociate. Alternatively, cytochrome bd employs an alternative mechanism of oxygen trapping - part of the redox energy is invested into tight oxygen binding, and the price paid for this is the lack of proton pumping. A single cycle of oxygen reduction to water is characterized by translocation of four protons across the membrane. Our results make it possible to assign the pumping steps to discrete transitions of the catalytic cycle and indicate that during in vivo turnover of the oxidase these four protons are transferred, one at a time, during the P→F, F→OH, Oh→Eh, and Eh→R transitions. At the same time, each individual proton translocation step in the catalytic cycle is not just a single reaction catalyzed by CcO, but rather a complicated sequence of interdependent electron and proton transfers. We assume that each single proton translocation cycle of CcO is assured by internal proton transfer from the conserved Glu-278 to an as yet unidentified pump site above the hemes. Delivery of a proton to the pump site serves as a driving reaction that forces the proton translocation cycle to continue.

Molecular mechanisms of bacteriophage phi6 RNA-dependent RNA polymerase and its utilization in biotechnology

Double-stranded RNA (dsRNA) viruses encode only a single protein species that contains RNA-dependent RNA polymerase (RdRP) motifs. This protein is a central component in the life cycle of a dsRNA virus, carrying out both RNA transcription and replication. The architecture of viral RdRPs resembles that of a 'cupped right hand' with fingers, palm and thumb domains. Those applying de novo initiation have additional structural features, including a flexible C-terminal domain that constitutes the priming platform. Moreover, viral RdRPs must be able to interact with the incoming 3'-terminus of the template and position it so that a productive binary complex is formed. Bacteriophage phi6 of the Cystoviridae family is to date one of the best studied dsRNA viruses. The purified recombinant phi6 RdRP is highly active in vitro and possesses both RNA replication and transcription activities. The extensive biochemical observations and the atomic level crystal structure of the phi6 RdRP provides an excellent platform for in-depth studies of RNA replication in vitro. In this thesis, targeted structure-based mutagenesis, enzymatic assays and molecular mapping of phi6 RdRP and its RNA were used to elucidate the formation of productive RNA-polymerase binary complexes. The positively charged rim of the template tunnel was shown to have a significant role in the engagement of highly structured ssRNA molecules, whereas specific interactions further down in the template tunnel promote ssRNA entry to the catalytic site. This work demonstrated that by aiding the formation of a stable binary complex with optimized RNA templates, the overall polymerization activity of the phi6 RdRP can be greatly enhanced. Furthermore, proteolyzed phi6 RdRPs that possess a nick in the polypeptide chain at the hinge region, which is part of the extended loop, were better suited for catalysis at higher temperatures whilst favouring back-primed initiation. The clipped C-terminus remains associated with the main body of the polymerase and the hinge region, although structurally disordered, is involved in the control of C-terminal domain displacement. The accumulated knowhow on bacteriophage phi6 was utilized in the development of two technologies for the production of dsRNA: (i) an in vitro system that combines the T7 RNA polymerase and the phi6 RdRP to generate dsRNA molecules of practically unlimited length, and (ii) an in vivo RNA replication system based on restricted infection with phi6 polymerase complexes in bacterial cells to produce virtually unlimited amounts of dsRNA. The pools of small interfering RNAs derived from dsRNA produced by these systems were validated and shown to efficiently decrease the expression of both exogenous and endogenous targets.