Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 04/06 Archiv

While a large variety of nucleophiles (N and sN) have been characterized according to eq 1.3, the number of E parameters for electrophiles, and specifically for different classes of Michael acceptors remains relatively small. A goal of this work was to include the synthetically important trans--nitrostyrenes (Chapter 2) and aliphatic nitroolefins (Chapter 3) into our reactivity scale, by measuring the rates of their reactions with stabilized carbanions. The reactions of (E)-1-ethoxy-2-nitroethene with pyridinium ylides, and of (E)-N,N-dimethyl-2-nitroethenamine with stabilized carbanions will be discussed in Chapter 4. Considerable effort has been spent on the development of organocatalytic asymmetric Michael additions. The reactions of nitrostyrenes with ketones or aldehydes have been used as model reactions for probing the efficiency and selectivity of new organocatalysts. The kinetic of the reaction of trans--nitrostyrene with cyclohexanone catalyzed by pyrrolidine and benzoic acid has been investigated, and the individual rate orders have been determined in order to provide insight into the catalytic cycle (Chapter 5). Diazo compounds are common and versatile reagents in organic synthesis despite their hazardous nature. Therefore, the rates of reactions of diazo compounds with Michael acceptors (Chapter 6) have been studied in order to elucidate the degree of concertedness of these 1,3-dipolar cycloadditions, and to investigate if they can be described by eq 1.3.

Intracellular protein delivery is offering numerous possibilities in research and in therapy. Aside gene therapy, protein delivery into living cells is one of the most promising tools for the treatment of various so far immedicable diseases including cancer. To develop a practicable protein delivery platform, a test system which allows easy control of successful intracellular delivery is needed. Therefore a test system based on two model proteins was established. A nuclear localization signal tagged EGFP molecule is enabling fast control of cellular uptake and endosomal release. The second model protein ß-galactosidase is evidencing that protein conformation is not irreversible disturbed by modification with the carrier molecules. Protein transduction technology is opening the door for a promising alternative to gene therapy, as it is lacking of the potential malignant side effects of gene therapy. The most limiting step in the development of a therapeutic drug remains the delivery process. In the last decade, many techniques to deliver proteins into living cells were developed. Although great efforts were made, so far no all-purpose technique is available that addresses all critical steps, like efficient uptake, endo-lysosomal escape, low toxicity, while maintaining enzymatic activity. Each method has got its limitation, for example cell type dependence. Among the so far used carriers, the most effective ones are cationic polymers like polyethylenimine. These carriers are lacking of precise structure and often show high toxicity, dependent on the molecular weight of the used polymer. In this thesis the properties of the three arm cationic oligomer 386, which was previously designed for siRNA delivery was investigated in regard of being applicable as a transduction carrier for protein delivery. This carrier molecule, in contrast to other cationic polymers used for protein delivery, is of precise structure, of low molecular weight and potentially degradable by proteases. The transduction oligomer was covalently bound to the protein by a bioreversible bond. Our results reveal that covalent coupling of the structure defined cationic oligomer 386 to a protein leads to a high efficient, serum insensitive and low toxic alternative to established protein transduction technologies. For a general all-purpose delivery system covalent coupling of the carrier to the cargo protein is indispensable. Protein delivery requires special properties to the linker molecule. Therefore in this work a new pH sensitive linker was developed which combines the advantages of click reactions with the implementation of a traceless cleavable bond between two conjugated molecules. Three different click chemistries were performed which all are compatible with the acid labile properties. A traceless cleavage may be a particularly important feature in protein transduction strategies, to maintain full bioactivity of enzymes and other proteins. The current example of 386 carrier-mediated cytosolic delivery and subsequent nuclear import of released nls-EGFP demonstrates the advantage of the traceless linker. To demonstrate that the modification does not irreversibly affect structure and biological activity of proteins, 386-AzMMMan-ßgalactosidase was delivered as a model enzyme. It exhibited cytosolic activity in the transduced cells far higher than without shuttle. Aside from these encouraging options for protein delivery and modification, the linker might have broader use in the design of novel programmed, acid labile and biodegradable drug delivery systems. Targeted therapeutics could, after delivery into acidic tumor areas or upon cellular uptake into endosomes, be dismantled from their outer shell including targeting ligands. Besides drug delivery, the linker may also be of interest for other applications, such as reversible labeling of various biological and also chemical molecules. The developed linking strategy and the presented concepts for transduction shuttles may help to get a step closer in the design of an all-purpose protein delivery platform, applicable on bench as on bedside.

The nucleophilic reactivities of amines, hydrazines, and pyridines were quantified using benzhydrylium ions and quinone methides as reference electrophiles. The scope and limitations of the linear free energy relationship log k2 = sN(N+E) for predicting rates of the reactions of carbocations and acylation agents was studied.

The potential toxicity of nanoparticles currently raises many discussions in public and scientific life. The question whether nanoparticles are a threat to human health cannot be answered to complete satisfaction at the current state of knowledge. A versatile tool to investigate nanotoxicity are fluorescence microscopy and live-cell imaging as they provide excellent resolution and direct insight into cellular processes. In this work fluorescence based methods are used to investigate the influence of silica nanoparticles on human health, more precisely on the blood vessel system. At first, the synthesis and characterization of the following three different types of perylene labeled amorphous SiO 2 nanoparticle species is described: surface-labeled monodisperse particles, particles with a dye-containing silica core and a non-fluorescent silica shell and a surface-labeled nanoparticle network. The labeling of nanoparticles should not induce artificial cytotoxic effects when they are used for cytotoxicity assessment. This is achieved either by incorporating the dye into the nanoparticle’s structure or by covering only a minor surface fraction by dye molecules. The surface-labeled silica species are used to investigate nanotoxicity throughout this thesis. Another prerequisite for reliable dose-dependent nanotoxicity studies is the knowledge about the number of nanoparticles taken up by an individual cell. We therefore developed the Nano_In_Cell_3D ImageJ macro which is able to quantify nanoparticle uptake into cells. Nano_In_Cell_3D uses the fluorescence image of the cell membrane to segment the cell into an intracellular space, a transition region (e-membrane region) and an extracellular space. The number of present nanoparticles is calculated from the fluorescence intensity of each region. This custom-made method offers the possibility to quantify nanoparticles in the individual cellular regions. Nano_In_Cell_3D was validated by comparing the results to the well established quenching method. By using Nano_In_Cell_3D we could show that the cytotoxic impact of nanoparticles onto different cell lines correlates to their intracellular uptake. Primary human vascular endothelial cells (HUVEC) take up 310 nm silica nanoparticles more efficiently and are more sensitive to this nanoparticle species than cancer cells derived from the cervix carcinoma (HeLa). Upon nanoparticle contact, cellular viability of HUVEC is strongly reduced and membrane permeability increases leading to apoptosis. In contrast, HeLa cells show a considerable lower effect in both cellular viability and membrane permeability and do not show apoptosis. In consistence to these findings, HUVEC take up approximately 20 times more particles than HeLa cells within 4h. Interestingly nanoparticle uptake is clathrin mediated in both cell types. HUVEC grow in the blood vessel system under natural conditions and are therefore exposed to blood flow conditions. The latter can be simulated using a microfluidic system. We chose a microfluidic system based on the surface acoustic wave (SAW) technology which was characterized concerning fluid evaporation behavior, fluid temperature and flow velocities. Based on these results the system can be further improved to allow the assessment of nanotoxicity at blood flow conditions in a next step. The last part of the thesis focuses on interactions between silica nanoparticles and giant unilamellar vesicles (GUV)s. The latter serve as a simple model for the cell membrane. Nanoparticles in contact with the lipid membrane influence the morphological behavior of the vesicles during phase transition. In absence of nanoparticles, vesicles typically show extracellular budding processes. Nanoparticlesin contact with the cell membrane induce intravesicular budding of daughter vesicles, similar to endocytosis observed in living cells. Furthermore exocytosis processes were observed where daughter vesicles crossed the GUV membrane and were transferred from the intravesicular to the extravesicular space. These observations suggest that the fundamental mechanism of endocytosis can partly be explained by simple physical effects. In summary, this theses provides an experimental strategy to investigate the impact of nanoparticles onto human cells using SiO 2 nanoparticles as an example. Starting with the synthesis and characterization of nanoparticles it tackles the question how to quantify nanoparticle uptake into cells. Furthermore we could prove that cytotoxic effects can be correlated to nanoparticle uptake and were able to show that nanoparticles influence artificial membranes which is a first step to understand the basic mechanisms of nano-toxicity. The methodology developed in this thesis is expected to provide insight into cytotoxicity of a broad variety of different nanoparticle types.

Photoinduced ultrafast isomerizations are fundamental reactions in photochemistry and photobiology. This thesis aims for an understanding of the generic forces driving these reactions and a theoretical approach is set up, able to handle realistic systems, whose fast relaxation is mediated by conical intersections. The main focus is on the development of strategies for the prediction and accelerated optimization of conical intersections and their application to artificial compounds with promising physicochemical properties for technical applications as molecular switches. All calculations are based on advanced quantum chemical methods and mixed quantum-classical dynamics. In the first part of this thesis the two-electron two-orbital theory by Michl and Bonacic-Koutecky used in its original formulation to rationalize the structure of conical intersections in charged polyene systems is extended by including the interactions of the active pair of electrons with the remaining closed-shell electrons that are present in any realistic system. A set of conditions, called resonance and heterosymmetry conditions, for the formation of conical intersections in multielectronic systems are derived and verified by calculations on the basic units ethylene, cis-butadiene and 1,3-cyclohexadiene at various geometries and functionalizational patterns. The quantitative results help to understand the role of geometrical deformations and substituent effects for the formation of conical intersections and to derive rules of thumb for their qualitative prediction in arbitrary polyenes. An extension of the rules of thumb to conical intersection seams is formulated. The strategy pursued is to divide the molecular system into basic units and into functional groups. Each unit and its intersection space are treated independently, thereby reducing the dimensionality of the search space compared to the complete molecule. Subsequently, the interconnectivity of the intersection spaces of the different units is determined and an initial guess for the complete seam is constructed. This guess is then fed into a quantum chemistry package to finalize the optimization. The strategy is demonstrated for two multi-functionalized systems, hemithioindigo-hemistilbene and trifluoromethyl-pyrrolylfulgide. In the second part of this thesis state-of-the-art quantum chemical calculations and time-resolved transient and infrared spectroscopy are used to reconstruct the complex multi-channel isomerization mechanisms of hemithioindigo-hemistilbene and trifluoromethyl-indolylfulgide. Both the cis-trans isomerization in hemithioindigo-hemistilbene and the electrocyclic ring closure/opening in indolylfulgide are characterized by a charge transfer in the excited state. The ability of each system to stabilize this charge transfer is essential for the returning to the ground state. The relaxation to the ground state through extended regions of the seam is found to be the decisive step determining the reaction speed and the quantum yield. In the last part of this thesis mixed quantum-classical dynamics simulations at multi-configurational perturbation theory (MS-CASPT2) level, using Tully's fewest switches surface hopping approach, are performed to study the ultrafast photoreactivity of 1,3-cyclohexadiene in the gas-phase. For this purpose a numerical routine for the efficient calculation of non-adiabatic couplings at MS-CASPT2 level is presented. The major part of the excited molecules are found to circumvent the 1B2/2A1 conical intersection and reach the conical intersection seam between the excited state and the ground state instantaneuosly. Time constants for the evolution of the wavepacket on the bright 1B2-state, the relaxation into the 2A1-state and the return to the ground state are extracted. It is demonstrated that the accessibility of the conical intersection seam depends on its energetic and spatial relation to the minimum energy path, as well as on the momentum which is accumulated during relaxation on the excited state potential energy surface.