To become biologically active, most proteins need to fold into precise three dimensional

structures. It has been well established that all the folding information is contained

within the primary structure of a protein. However, the mechanisms utilized by proteins

to avoid sampling the extraordinarily large amount of possible conformations during

their folding process are just beginning to be understood. Molecular chaperones assist

the folding of newly synthesized and denatured proteins in acquiring their native state

in the crowded intracellular environment.

As a nascent chain leaves the ribosome, it is captured first by the upstream chaperones

and then possibly transferred to the downstream chaperonins. GroEL-GroES, the

Hsp-60 of E.coli, is one of the best studied chaperone systems. An appreciable amount

of data is available providing information regarding its structure and function. GroEL

encapsulates the substrate into the central cavity where folding occurs unimpaired by

aggregation and unwanted inter-molecular interactions. Nevertheless, many important

aspects of the GroEL mechanism remain to be addressed. Some of the open questions

we have addressed in this study include: In what conformation does a substrate protein

bind to the apical domains of GroEL; how is it that GroEL is able to accelerate the rate

of folding of certain proteins, and how do the conformational properties of the substrate

change as it undergoes repeated cycling.

By using ensemble FRET and Sp-FRET (Single Pair-Fluorescence Resonance Energy

Transfer), we have probed the conformation of the model substrate DM-MBP (Double

Mutant Maltose Binding Protein) during different stages of the functional cycle of

GroEL. With Sp-FRET coupled to PIE (Pulsed Interleaved Excitation), we have been

able to explore the heterogeneity of the GroEL bound substrate protein and observed a

bimodal conformational distribution. One of the two populations is as compact as the

native state, whereas the other is as extended as the unfolded protein in denaturant.

This unfolding is a local phenomena and can also be observed when the substrate is transferred from DnaK/J system (bacterial Hsp70) to GroEL, indicating the possibility

of the existence of this conformational heterogeneity in vivo as the protein follows the

cellular chaperone pathway.

Subsequent to GroEL binding, there is a transient expansion of the protein upon

binding of ATP to GroEL, followed by compaction when GroES triggers the encapsulation

of the protein inside the chaperonin cage. This transient expansion is however

found not to be a necessary event for the rate acceleration of DM-MBP folding, since

ADP-AlFx (transition state analogue of ATP hydrolysis) results in a much slower rate

of expansion, which does not cause a change in the folding rate.

Anisotropy measurements, probing the freedom of motion of different regions of the

GroEL bound protein, revealed that there is a segmental release of the substrate protein

from the GroEL surface upon binding of ATP and GroES. As a consequence, the

hydrophobic collapse of the protein upon encapsulation by GroES follows a step-wise

mechanism. In this process, less hydrophobic regions are released upon binding of ATP,

prior to more hydrophobic ones which are released only by GroES binding. Thus, the

order of Hydrophobic collapse is reversed as compared to spontaneous folding possibly

resulting in conformationally different folding intermediates.

Evidence that the folding pathway inside the cage differs from that of spontaneous

folding was obtained by observing the effect of external perturbations (e.g. mutations in

substrate protein and use of different solvent conditions) on the rate of spontaneous and

GroEL assisted folding reactions. These two folding reactions respond differently to the

introduced perturbations. Kinetic data obtained from ensemble FRET measurements

suggest that the conformation of refolding intermediate is altered by the GroEL cavity,

which leads to a fol

Following oxidative stress often two kinds of DNA-lesions can be found: 8-OxodG and the 2,6-Diamino-4-hydroxy-5-formamidopyrimidine (FaPydG). Both derive from the DNA-Nucleobase guanine. The FaPydG-lesion can exist both in the beta-configuration and the alpha configuration. To clarify biological questions concerning the basepairing and the encoding features of both forms it was necessary to synthesise of the alpha-anomer of the carbocyclic FaPydG-DNA-lesion. The synthesis of the beta-cFaPydG-lesion was already finished in 2005 in the Carell group. In a sixteen-step synthesis the alpha-lesion could be prepared in the form of its phosphoramidite component. Oligonucleotides could be prepared on solid support using automated DNA-synthesis.

Some of the synthesised DNA-Nucleotides contained the inserted FaPydG-lesion. Others showed an oxidation to alpha-c8-OxodG. This reaction is not known for natural lesion. Just a de-hydration to guanosine under harsh conditions is described. The observations can be ex-plained by formulation of a cyclic intermediate, which is possibly oxidized by the oxidation di-lution during the DNA-synthesis. If the sequence-dependent equilibrium between alpha cFaPydG and alpha c8-Hydro,hydroxydG is at the side of the closed form, an oxidation from this interme-diate directly to 8-OxodG is imaginable.

This assumption is proven by the fact that at the same time synthesised and completely identically treated DNA-strands of different sequence didn´t show oxidation products. This could be ascertained by high resolution ESI-FTICR-mesurement. With this analytical method even smallest variations are detectable in a reliable way, whereas the often used MALDI-TOF-mesurements not always show these variations.

Thermodynamic studies showed that alpha-cFaPydG doesn´t form a preferred DNA-basepair. All possible basepairs showed a strong destabilisation in comparison to the undamaged, cor-rect basepair. In vitro elongation experiments with S. cerevisiae polymerase Pol eta, G. stearothermophilus DNA-polymerase I and DinB from G. stearothermophilus showed that the alpha-lesion is a definitive block for all mentioned polymerases. Only by applying rigorous re-action conditions an elongation of the primer with BstPol I could be detected. This elongation showed a favored incorporation of dCTP, followed by an incorporation of dATP.

In the time to come experiments to avoid the unwanted oxidation of alpha-cFaPydG will be es-sential to promote the cocristallisation of the alpha-cFaPydG-oligomere with the Fpg-protein from Lactococcus lactis and the BstPol I.

In the second part of the thesis attempts were taken to prepare tetrafunctional amino acid derivatives, which were planned to be used in a PNA-template directed ligation reaction. Liga-tion takes place between two amino acids, so that for a ligation attempt with this method there is always needed a matching pair of amino acids.

This pair requires a connection of the aminoacid to the PNA over the side chain of the amino acid. The N- and C-terminus of the amino acid had to be unprotected for the ultimate ligation reaction. These termini must not react during the solid support PNA-synthesis. After ligation the template should be separated selectively. Thus, the following requirements for the pair of amino acids result: The amino acids have to possess functional side chains, which exist naturally after cleavage of the PNA-template. For the selective cleavage of the template a con-nection orthogonal to acid and base unstable protecting groups is necessary, because these are already needed for the solid support synthesis on the residual three termini of the compound. Imaginable orthogonal cleavable links are allylic compounds, which are cleavable with Pd(0), but also silicon based compounds, which are cleavable with fluoridions, would be appropriate.

In the area of the Pd(0)-cleavable link a retrosynthetic cut at this funtionality should be adequate. Here, e.g. a Horner-Wadsworth-Emmons

Übergangsmetall-Komplexe bzw. -Salze von Mangan, Rhodium, Iridium, Palladium und Kupfer wurden mit verschiedenen Aziridinderivaten umgesetzt. Dabei gelang es neutrale und kationische Mono-, Bis-, Tris- und Tetrakis-Aziridin-Komplexe darzustellen. Auch eine übergangsmetallvermittelte Aziridin-Dimerisierung konnte desöfteren beobachtet werden. Dabei entstanden N-(2-Aminoalkyl)aziridin-Komplexe. Durch die gelungene Freisetzung des formal dimerisierten Aziridinliganden, konnte eine übergangsmetallvermittelte Synthese von N-(2-Aminoalkyl)aziridinen beschrieben werden.

Untersuchung öliger Suspensionen als parenterale Depotsysteme für rekombinante Proteine

Single-molecule methods play a growing role in materials science because they can reveal structural and dynamic features which are obscured by ensemble averaging in conventional spectroscopic techniques. In this work, such methods were used to study the dynamics of single dye molecules (guests) within different surrounding porous matrices (hosts) using wide-field microscopy and single-molecule tracking. A significant amount of tracking data was collected and sophisticated methods to analyse the data according to diffusion theory were developed. A method was established to directly correlate the diffusion information that is provided by single-molecule trajectories with the images of the porous host systems obtained by transmission electron microscopy (TEM). Furthermore, the results from single-molecule tracking experiments were compared with diffusion measurements using pulsed-field gradient NMR in the same samples.

The data presented in this thesis thus provide for the first time a detailed picture of the real mesoporous structure and its effects on the dynamic behavior of dye molecules at the nanometre to micron scale, e.g.~information about pore connectivity and accessibility. The methodology established here is expected to provide detailed insights into the dynamics of other important host-guest systems, such as bioactive molecules in porous materials for drug delivery or reactants in porous catalysts.

Messenger RNA localization occurs in the cytoplasm and allows temporal and spatial regulation of gene expression. In yeast, the localization of ASH1 mRNA to the tip of budding cells allows the asymmetric sorting of Ash1 protein, which has a key function in the regulation of mating-type switching. After cell division, asymmetric distribution of Ash1p restricts mating type switching to only the mother cell. The cytoplasmic transport of ASH1 mRNA to the bud tip depends on the myosin Myo4p, its adaptor She3p, and the specific RNA binding protein She2p. Three additional trans-acting factors Khd1p, Puf6p and Loc1p are involved in this process. All known RNA-binding proteins of ASH1 mRNA have revealed a nuclear connection, when following their cellular distribution by indirect immunofluorescence. Thus, an early step in the localization pathway might be the early recruitment of specific trans-acting factors to the mRNA already in the nucleus. The aim of this thesis was to investigate how nuclear key events such as early binding to localized transcripts and the subsequent assembly into a nuclear RNP can account for effective RNA localization. Following the route of She2p, it was possible to show nucleo-cytoplasmic shuttling of this RNA binding protein. Moreover, ASH1 mRNA and She2p were found accumulated within the nucleolus upon arrest of mRNA export. Interestingly, two additional trans-acting factors, Loc1 and Puf6p, both involved in ASH1 mRNA localization are also nucleolar proteins. Moreover, She2’s nuclear history seems to be important for an effective sorting of Ash1p. When restricting ASH1-She2p association to the cytoplasmic compartment artificially, the ASH1 mRNA was still localized but was prematurely translated during its transport. This suggests that nuclear RNP assembly has an influence on the later stages of cytoplasmic translational control. The nucleolus might represent the appropriate cellular compartment to provide the spatial framework for the assembly of localization competent RNPs since many RNPs are assembled in this region.

In dieser Dissertation wurden titankatalysierte Hydroaminierungsreaktionen von Alkinen und Alkenen durchgeführt. Außerdem wurde eine Methode für eine indirekte Hydratation von Alkinen mit anti-Markownikow-Regioselektivität entwickelt. Abschließend gelang es Hydroaminierungsreaktionen in der Synthese von biologisch aktiven Heterocyclen, wie Cumaronen, Indolen, Pyrrolen und Pyrrolidinen einzusetzen.

The study provides a comprehensive overview on different stabilization techniques for liposomal formulations. The selection of the appropriate technology for a particular formulation can thereby be based on several considerations. If free flowable particulate bulk material is desired the spraying-technologies are preferred over lyophilization. Another advantage of spraying-based technologies is the possibility to combine the liposome formation step and the drying step within the same process. It is a prerequisite for the selection of the stabilization technique that the integrity of the liposomes is preserved with the incorporated drug after the process. Spray-drying is related to a thermal stress for the formulation but only for a short time. For heat labile drugs processes with low process temperatures, e.g. freeze-drying, spray freeze-drying, inert-spray drying or supercritical drying are most adequate. However, for technologies with a freezing step a sufficient stabilization against freezing induced stress e.g. by the selection of appropriate cryoprotectors is needed.