Alzheimer’s disease (AD) is a very complex disorder and the most common form of dementia. The two pathological hallmarks of AD are extracellular amyloid-β (Aβ) plaques in cerebral cortex, and intraneuronal neurofibrillary tangles. In the early stages of the disease it can be difficult to accurately diagnose AD, as it is difficult to distinguish from normal signs of aging. There is thus a need for sensitive non-invasive tools, able to detect pathophysiological biomarker changes. One such approach is molecular imaging of Aβ plaque load in brain, using PET (positron emission tomography) ligands. We have developed and characterized two novel Aβ plaque neuroimaging PET ligands, AZD2184 and AZD4694. The 2-pyridylbenzothiazole derivate AZD2184, is a 11C-labeled PET ligand with a higher signal-to-background ratio compared to the widely used PET ligand PIB, a 11C-labeled phenylbenzothiazole based tool. This makes it possible to detect smaller changes in Aβ plaque deposition load, and therefore theoretically, also earlier diagnosis. A drawback with 11C-labeled PET ligands is the relatively short half-life. To meet the need for PET ligands with a longer half-life, we developed the pyridylbenzofuran derivate [18F]AZD4694. Although development of fluorinated radioligands is challenging due to the lipophilic nature of aromatic fluorine, we successfully developed a 18F-labeled PET ligand with a signal-to-background ratio matching PIB, the most widely used 11C-labeled PET ligand in clinical use. 3H-labeled derivates of AZD2184, AZD4694, and PIB, showed lower binding specificity towards Aβ plaques containing ApoE. The ApoE genotype per se did not significantly affect ligand binding, instead, the amount of ApoE incorporated to the Aβ plaques appears to be of importance for the binding characteristics of these amyloid PET ligands. Beta-secretase 1 (BACE1) mediates the first step in the processing of amyloid precursor protein (APP) to Aβ peptides, making BACE1 inhibition an attractive therapeutic target in AD. We developed and characterized three novel BACE1 inhibitors, AZD3839, AZ-4217, and AZD3293. AZD3839 and AZ-4217 contains an amidine group which interacts with the catalytic aspartases Asp-32 and Asp-228 of BACE1, effectively inhibiting the enzyme. All three compounds are potent and selective inhibitors of human BACE1, with in vitro potency demonstrated in several cellular models, including primary cortical neurons. All three compound exhibited dose- and time-dependent lowering of plasma, brain, and cerebrospinal fluid Aβ levels in several species, and two of the compounds (AZD3839 and AZD3293) were progressed into clinical trials. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Submitted.</p>

The main objective of this dissertation is the synthesis and study of modified surface systems for the development of bioactive platforms and their use in specific biotechnological and biomedical applications. This work has led to various biological template development projects; all in attempts to provide new surfaces and probes in nanotechnology. These projects focus mainly on protein modified surface platforms, liposome based spherical platforms, and carbon nanotubes based magnetic platforms. The planar platforms include gold, silicon and aluminum oxide surfaces. Spherical surfaces such as liposomes and nanoparticles were also studied, and finally, surface modification was extended to carbon nanotubes and magnetic nanoparticles. In this dissertation, the planar surface work focuses on demonstrating the behavior of proteins at interfaces in terms of conformation, stability and activity (e.g., of avidin, trypsin and antibodies) using fluorescence microscopy. Different ligands were attached chemically on the surfaces to incorporate hydrophobic hydrophilic and charged characteristics. A chelating agent (iminodiacetic acid, IDA), an affinity ligand (biotin), and reactive groups (amino and carboxylic groups) were covalently incorporated onto the surfaces. Proteins including myoglobin, cytochrome C, avidin, trypsin and immunoglobulin G (IgG) were used in this study. The results show that proteins and ligands were successfully attached to different surfaces. Protein adsorption studies illustrate activity decrease by using fluorescence intensity. After attachment on hydrophobic functionalized surfaces. Along the same line, experiments were conducted on the comparison of silicon dioxide and gold-coated surfaces with immobilized enzymes, small molecules, and polymers for potential use as biosensors. Silicon dioxide wafers were prepared via silanization with 3-aminopropyl triethoxysilane (APTES) followed by glutaraldehyde activation and, finally, protein and/or small ligand attachment. Gold-coated surfaces were utilized for immobilizations using 16-mercaptohexadecanoic acid (MHA) which forms self-assembled monolayers (SAMs) on gold surfaces followed by covalently attachment of proteins. The activity of trypsin immobilized onto these surfaces was also measured. The silicon dioxide wafers when modified first with NH₂-PEG-NH₂ allowed for trypsin a relatively higher activity with about 11% greater activity than when attached on gold surfaces and 84% higher activity than on bare silicon surfaces. Furthermore, the bimolecular silicon dioxide surfaces were shown to be much more stable than the gold surfaces. The silicon dioxide surfaces with an immobilized reversible inhibitor, p-aminobenzamidine (PAB), show to very effectively bind proteins from solution compared to gold surfaces. Liposome were studied because their versatility and vast implications in bio-sensing and drug-delivery potential. In this work, liposomes were prepared with the phospholipids 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and cholesterol. The amino groups of DMPE were then modified with ligands that included iminodiacetic acid (IDA), and PEG. These functionalized liposomes were used to prepare dispersed gold “nano-dots” on their surface. These novel functional liposomes, with chelating ligands and polymers can be used to bind biomolecules and active compounds (nanoparticles of gold, quantum dots, drugs) with long stability. The results show that we can successfully manufacture functional liposomes and form gold nanoshells on their external surface. These two types of systems can be used as drug delivery, and as imaging systems. Their characterization and potential use in biomedical applications as contrast agents seems quite promising once complexity and stability of these gold nanoshells is elucidated. The modification and preparation of functional-carbon nanotubes was investigated with the chemical hetero-junction analysis between magnetic nanoparticles coated poly-acrylic acid (PAA) and multi-wall carbon nanotubes (MWCNTs). Magnetic nanoparticles were covalently attached to open-ended nanotubes. Initial evidence suggests that short functionalized multi-wall nanotubes can be continuously connected at their terminal ends for build-up of relatively large nanostructures based on serial configurations. It is shown that magnetic carbon nanotubes systems exhibit defined arrangements due to the influence of magnetic fields. Indeed, linear arrays of carbon nanotubes inter-connected through magnetic nanoparticles were prone to be manipulated in the presence of a magnet device. A potential application of these magnetic nanostructures was shown by successfully manipulating agarose beads in buffer solution as a model system. These results suggest that the use of continuously connected magnetic nanostructures with non-modified sidewall surfaces will find potential applications in the areas of bio-sensing, force transduction and cancer screening-manipulation among others.

Les complexes de la famille des ansa-chromocènes sont relativement peu nombreux, mais ils ont tout de même démontré des réactivités intéressantes comme la possibilité de coordonner une molécule de monoxyde de carbone au centre métallique sans être sous pression constante de gaz, ce qui n’est pas le cas pour l’homologue chromocène. L’ansa-chromocène le plus surprenant est sans doute le Me2Si(C5Me4)2Cr, car il est le seul qui ne comporte pas de ligand autre que celui de type ansa. Cependant, ce composé a été obtenu sans que le mécanisme de la réaction ne soit compris et prouvé, seul un mécanisme proposé a été publié. Au cours de cette étude, le mécanisme proposé a tout d’abord été infirmé grâce à de nombreuses expériences qui ont mené à l’élaboration d’un nouveau mécanisme. Par la suite, la réactivité du Me2Si(C5Me4)2Cr a été approfondie en le faisant réagir avec divers réactifs. Aucun produit d’addition oxydante n’a été isolé, mais la réaction avec l’isonitrile forme un complexe asymétrique avec deux isonitriles coordonnés. La détermination du moment magnétique du composé Me2Si(C5Me4)2Cr confirme la présence de deux électrons non-pairés à la température de la pièce et évoque la possibilité d’une transition à S=2 à température plus élevée. La synthèse de nouveaux complexes de type ansachromocène insaturé a été tentée avec d’autres ligands ansa, et la réaction avec [C2H4(C9H6)2]Li2 mène à un complexe dimérique avec des ligands indényles pontés. / Only a few ansa-chromocenes complexes are known but some of them have shown interesting reactivities such as the ability to coordinate a carbon monoxide molecule to the chromium center without being under continuous gas pressure, which is not the case for the chromocene analogue. The most surprising ansa-chromocene is without doubt Me2Si(C5Me4)2Cr, because it is the only example of an ansa-chromocene lacking additional ligands. However, the compound was obtained by accident and the mechanism of its formation was neither well established nor understood. Only a tentative mechanism was published. During the course of the present study, the proposed mechanism has been disproved by a series of experiments that led to the elaboration of a new mechanism. The reactivity of Me2Si(C5Me4)2Cr has been studied by reactions with various compounds. No oxidative addition product was isolated, but the reaction with isonitrile yielded an asymmetric complex with two isonitriles coordinated to chronium. The determination of the magnetic moment of this complex confirmed two unpaired electrons at room temperature and indicated the possibility of an S=2 transition at higher temperature. The synthesis of new unsaturated ansa-chromocene complexes has been attempted with other ansa ligands, and the reaction with [C2H4(C9H6)2]Li2 led to a dimeric complex with bridged indenyl ligands.

Im Rahmen dieser Arbeit wurden Silaanaloga bereits bekannter organischer Riechstoffe und deren Derivate synthetisiert und auf ihre olfaktorischen Eigenschaften untersucht. Die erhaltenen Riechstoffe zählen zur Klasse der polycyclischen moschusartigen Riechstoffe bzw. zu den acyclischen holzig-ambraartigen Riechstoffen Des weiteren wurden penta- und hexakoordinierte Silicium-Komplexe dargestellt und strukturell charakterisiert, die unter anderem Silicium-Iod-, Silicium-Selen- und Silicium-Tellur-Bindungen aufweisen. Durch NMR-Untersuchungen im Festkörper und in Lösung konnten Erkenntnisse über die dynamische Umlagerung dieser Komplexe gewonnen werden. / In the course of this dissertation sila analogues of known organic odorants and their derivatives were synthesized and studied for their olfactory properties. The obtained odorants belong to the group of polycyclic musk odorants and acyclic woody-ambery odorants, respectively. Furthermore, penta- and hexacoordinate silicon complexes were synthesized and structurally characterized, amongst them were compounds with Si-I, Si-Se, and Si-Te bonds. NMR studies in the solid state and in solution gave information about the dynamic rearrangement of these complexes.

The design of ligands is one of the most important and simultaneously challenging fields of research in modern inorganic chemistry. The aim is to synthesise ligands that can serve as coordination units for a broad variety of metal fragments and different purposes. The ligands have to be very flexible concerning their donating behaviour and geometrical prerequisites in order to correspond to the required metal fragments. / Ziel der vorliegenden Arbeit war die Synthese eines Januskopfliganden der zweiten Generation und die Untersuchung seiner Reaktivität und seines Koordinationsverhalten. Als Zielverbindung wurde Di(benzothiazol-2-yl)phosphan (1) gewählt (Schema 7.1). Neben harten Koordinationsstellen enthalten die heteroaromatischen Substituenten dieses Liganden zusätzlich weiche, die das System im Vergleich zu z. B. Di(pyrid-2-yl)phosphan im Bezug auf mögliche koordinierte Metallfragmente flexibler machen sollten.

Student Number : 9006831D - PhD thesis - School of Chemistry - Faculty of Science / The ligand substitution reactions of aquacobalamin are fast and hence the usual inertness of the d6 Co(III) ion has been modified. It is well established that the reactions proceed through a dissociative interchange mechanism; however, previous ligand studies were performed in a KCl medium, which led to the formation of the more substitution-inert chloro complex. The kinetics of aquacobalamin were reinvestigated with the ligands N3–, NO2–, SCN–, S2O32–, OCN– and SeCN– in a NaNO3 medium. The reactions proceeded too rapidly for saturation kinetics to be observed and hence only the second-order rate constants could be obtained. These were corrected for pH and determined as a function of temperature, from which the activation parameters were determined. The donor atom of the ambidentate ligands were investigated and correlations were found between the Mulliken population on the donor atom, the energy of the highest occupied molecular orbital (HOMO) with σ symmetry, and Δ, the enthalpy of activation, and Δ, the entropy of activation, respectively. Good correlations occurred when the donor atoms were taken to be N for SCNII‡kHII‡kS– and NO2–; S for S2O32–; O for OCN– and Se for SeCN–. The effect that changing the environment of aquacobalamin has on its kinetics was observed by determining the rate constants for the reaction of pyridine with aquacobalamin in water and 70% ethanol. The rates were faster in water and the activation parameters obtained for the reaction of aquacobalamin with pyridine in 70% ethanol are larger than they are for the reaction in water. The larger ΔH‡ arises due to less bond formation between pyridine and Co in the transition state and ΔS‡ is larger because it is dominated by the freeing of the coordinated water i.e. bond breaking is the dominant process in the transition state. The effects of a bulkier ligand than water on the kinetics of aquacobalamin were investigated. The temperature dependence of the kinetics of the substitution of I– in iodocobalamin by imidazole, N3– and S2O32– was studied. Despite the increase in size of the departing ligand there is still nucleophilic participation of the incoming ligand in the transition state and hence the reaction still proceeds via an Id mechanism. In order to probe the cis-effect of the corrin in vitamin B12 derivatives, comparative studies were undertaken of the reactions of aquacobalamin and aqua-10-Xcobalamin, X = Cl, NO, NH2, where the H at C10 was replaced with an electron-donating (Cl, NH2) or electron-withdrawing (NO) group. Formation constants were obtained for aquacobalamin and aqua-10-chlorocobalamin for the substitution of coordinated H2O with various anions (N3–, NO2–, SCN–, S2O32–, OCN–, SeCN–) and neutral N-donor ligands (CH3NH3, pyridine, imidazole). The anionic ligands bind more strongly to aqua-10-chlorocobalamin than to aquacobalamin with log K values larger by between 0.10 and 0.63 (average 0.26) larger. The converse is true for the neutral N-donor ligands, where log K is smaller by between 0.17 and 0.3 (average 0.25). Semi-empirical molecular orbital (SEMO) calculations using the ZINDO/1 model on the hydroxo complexes show that charge density is delocalised from the axial donor atom to the metal and Cl. Thus the anionic ligands bind more strongly to aqua-10-chlorocobalamin because of the ability of the metal and the Cl at C10 to accept charge density from the ligand. The cobalt ion in aqua-10-chlorocobalamin is more electron rich than it is in aquacobalamin and so it is less likely to accept further electron density from a neutral axial donor ligand. This results in the stability being lower than that of aquacobalamin. The reaction kinetics of the substitution of H2O in aqua-10-chlorocobalamin were determined for the ligands N3– and pyridine. The reaction proceeds via a dissociative interchange mechanism since saturation was seen for pyridine and not for N3–. The activation parameters, ΔH‡ and ΔS‡, are lower for aqua-10-chlorocobalamin than aquacobalamin and hence it can be deduced that bond breaking between the coordinated water and the cobalt atom is more dominant in aquacobalamin. The rates of reaction are faster for aquacobalamin than they are for aqua-10-chlorocobalamin. SEMO calculations show that as the Co–O bond is stretched, the charge density on Co in aquacobalamin is always lower than that on aqua-10-chlorocobalamin, suggesting that aquacobalamin is a better electrophile towards the incoming ligand, thereby explaining the faster kinetics. Aqua-10-nitrosocobalamin was synthesised and characterised by FAB(MS), NMR and UV-vis spectroscopy. The strongly electron-withdrawing NO group has deactivated the metal ion towards ligand substitution, with neither 1.2 M pyridine nor 0.7 M N3– showing any spectroscopic evidence for the displacement of the axial H2O ligand. This provides further evidence that the electronic structure of the corrin ring can directly influence the ligand-binding properties of the metal. Aqua-10-aminocobalamin was synthesised from aqua-10-nitrosocobalamin but is unstable in solution. Hence, only a preliminary UV-vis study could be undertaken with the compound. This study shows that the shifts in the bands occur towards longer wavelengths than that of aqua-10-chlorocobalamin, suggesting that the amino group at the C10 position donates more electron density to the cobalt centre than the chloro group.

Thesis advisor: Larry W. McLaughlin / There are two major goals for my project. The first is to create and characterize metal-ligand-DNA complexes that could be synthesized using traditional organic methods followed by solid phase techniques. The second is to demonstrate that these complexes with complementary DNA sequences could self-assemble into higher-ordered structures. In order to generate supramolecular DNA-metal structures such as cubic lattices, it is necessary to create an octahedral metal-ligand center tethering six DNA arms as a building block. The Iron/Ru (II) bis(2,2':6',2''terpyridine) derivatives were chosen because: (i) the complex is well known to present octahedral geometry; (ii) the coordination is very stable; and (iii) while previous work required the solid-phase synthesis of six DNA arms simultaneously--an inefficient process--by using terpyridine ligands we need only extend three arms at once. Thus, several terpyridine-linker compounds were synthesized via two different routes. A DNA 14mer was synthesized afterwards by "Reverse Coupling Protocol" on a solid phase synthesizer and the terpyridine was connected to it followed by elongation of the rest two DNA arms. The DNA-terpyridine complexes were evaluated by stepwise hybridization tests and gel electrophoresis with or without the assistance of radio labeling. In addition, the assembly of metal with the terpyridine-DNA complex was also characterized by adding different metal ions such as Iron (II) and Ru (II) to the complex. Various buffer conditions were applied in constructing those conjugates in order to help forming branched DNA-ligand-metal complexes with higher molecular weight. / Thesis (MS) — Boston College, 2010. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

*Please refer to dissertation for diagrams. / In addition, a precursor of tetraphenylene-based monodentate ligand ( S,S)-114 was also prepared, and the structures of five compounds, namely 89, 124, 125, 133 and 137 were examined by X-ray crystallographic analysis. These structural determinations were relevant in establishing regiochemistry and absolute stereochemistry.* / In the synthesis of enantiopure (S,R,S)-48 and (R,S,R)-48, two routes were successfully employed. One way was to follow the same pathway for the synthesis of racemic 48 by using enantiopure (S,S)- and (R,R)-1,8,9,16-teramethoxytetraphenylenes [(S,S)-89 and (R,R)- 89] as staring materials. Another way was by direct resolution of racemic 48 via derivatization into its two diastereomeric hexakis-(S)-camphorsulfonates 135 and 136. / In the synthesis of racemic 48, 3-nitrophenol (115) was employed as the starting material which upon a series of standard reactions provided 2,2'-diiodo-1,1'-biphenyl (119). Through sequential lithium-iodine exchange and Cu(II)-mediated oxidative cyclocoupling, 119 was converted to 1,8,9,16-tetramethoxytetraphenylene (89) . The key intermediate 1,8-dihydroxy-9,16-dimethoxytetraphenylene (87) was obtained by partial demethylation of 89. This intermediate was transformed to 1,4,5,8-tetrahydroxy-9,16-dimethoxytetraphenylene (126) by a quinone-hydroquinone strategy. Demethylation of 126 furnished the target compound 48. / This thesis describes the synthesis of 1,4,5,8,9,16-hexahydroxytetraphenylene (48)* in its racemic and enantiopure (S,R,S) and ( R,S,R) forms. Some essential background and previous works in this area are presented in the first chapter. / Wu Anhui. / "August 2006." / Adviser: Henry N. C. Wong. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1649. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 80-85). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

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Die Stimulation des CD95-Todesrezeptors durch seinen natürlichen membranständigen Li-ganden CD95L führt zur kontextabhängigen Aktivierung von sowohl apoptotischen als auch nicht-apoptotischen Signalwegen. Durch Proteolyse wird aus dem membranständigen CD95L löslicher trimerer CD95L freigesetzt. Die Bindung von löslichem trimerem CD95L an CD95 ist nicht ausreichend, um die CD95-Signaltransduktion effizient zu stimulieren. Die Fähigkeit von löslichen CD95L-Trimeren CD95-vermittelte Signalwege robust zu aktivieren kann jedoch durch Oligomerisierung und artifizielle Immobilisierung an eine Oberfläche drastisch gesteigert werden. In dieser Arbeit wurde zunächst bestätigt, dass nur oligomere CD95L-Varianten, die z.B. durch Antikörpervernetzung von N-terminal getaggten rekombinanten CD95L-Varianten oder durch eine gentechnisch erzwungene Hexamerisierung von CD95L-Molekülen erhalten wur-den, in der Lage sind, effizient apoptotische und nicht-apoptotische Signalwege zu aktivieren. Ferner zeigte sich dann, dass die Bindung von löslichen CD95L-Trimeren nicht ausreichend ist, um die Translokation von CD95-Molekülen in detergenzunlösliche „Lipid Raft“- Membrandomänen zu stimulieren. Die „Lipid Raft“-Translokation ist ein zentrales Ereignis bei der CD95-Aktivierung und vor allem für die Induktion der Apoptose bedeutsam. Dabei ist ein selbstverstärkender Prozess aus Caspase-8-Aktivierung und „Lipid Raft“-Assoziation des CD95 von Bedeutung. Um die Interaktion von CD95 und CD95L mit Hilfe von hoch sensitiven zellulären Bindungs-studien analysieren zu können, wurden in dieser Arbeit desweiteren CD95L-Fusionsproteine entwickelt und hergestellt, an welche N-terminal eine Gaussia princeps Luziferase (GpL)- Reporterdomäne gekoppelt ist. So konnte mit den GpL-CD95L-Fusionsproteinen gezeigt werden, dass die Oligomerisierung von CD95L-Trimeren keinen Effekt auf die Ligandenbele-gung des CD95 hat. Dies spricht dafür, dass die höhere spezifische Aktivität von oligomeri-sierten CD95L-Trimeren nicht auf einer Aviditäts-vermittelten Zunahme der apparenten Affi-nität beruht, sondern dies deutet darauf hin, dass die sekundäre Aggregation von sich initial bildenden trimeren CD95L-CD95-Komplexen eine entscheidende Rolle in der CD95-Aktivierung spielt. Durch Scatchard-Analysen zeigte sich ferner, dass trimerer CD95L mit mindestens zwei zellulären Bindungsstellen unterschiedlicher Affinität interagiert. Bindungs-studien mit löslichen monomeren und trimeren GpL-CD95-Rezeptoren an membranständigen CD95L, als auch Inhibitionsstudien ergaben, dass trimerer CD95 weitaus besser an CD95L bindet. Dies legt nahe, dass es sich bei den zuvor beobachteten hoch- und niederaffinen Bindungsstellen für CD95L um monomere bzw. prä-assemblierte CD95-Moleküle handelt. Die GpL-CD95L-Fusionsproteine wurden auch genutzt, um die CD95-Translokation in „Lipid Rafts“ zu analysieren. So wurde trimerer GpL-CD95L als „Tracer“ zur Markierung von inaktiven CD95-Molekülen eingesetzt. Nach Aktivierung der übrigen freien CD95-Moleküle mit hoch aktivem hexameren Fc-CD95L konnte eine Zunahme der inaktiven GpL-CD95L-markierten Rezeptoren in „Lipid Rafts“ beobachtet werden. Offensichtlich stimulieren also aktivierte CD95-Moleküle in „trans“ die Ko-Translokation inaktiver CD95-Rezeptoren in „Lipid Rafts“. Dies bestätigte sich auch in Experimenten mit Transfektanten, die einen chimären CD40-CD95-Rezeptor exprimieren. Letzterer ist nach Stimulation mit CD40L in der Lage, intrazellu-läre CD95-vermittelte Signalwege zu aktivieren. Die Aktivierung von CD95-assoziierten Sig-nalwegen durch Stimulation von endogenem CD95 in CD40-CD95-Transfektanten resultierte nun in der Ko-Translokation von unstimulierten CD40-CD95-Rezeptoren in „Lipid Rafts“. Vice versa zeigte sich die Ko-Translokation von endogenem CD95 nach spezifischer Aktivierung des chimären CD40-CD95-Rezeptors. Schlussendlich erwiesen sich eine funktionsfähige Todesdomäne und die Aktivierung der Caspase-8 als essentiell für die „Lipid Raft“-Assoziation von aktivierten CD95-Molekülen und auch für die durch diese Rezeptorspezies induzierte Ko-Translokation von inaktiven Rezeptoren in „Lipid Rafts“. / Membrane-bound CD95L activates the CD95 death receptor to induce context-dependent apoptotic and non-apoptotic signaling pathways. In contrast, soluble trimeric CD95L, which is released by proteolysis, is not sufficient to stimulate CD95-induced signaling. However, the ability of soluble CD95L trimers to activate robust CD95 mediated signaling pathways can be increased drastically by oligomerization and artificial immobilization on the cell surface. In this work, it has been confirmed that only the oligomeric CD95L-variants, produced by an-tibody crosslinking of N-terminal tagged recombinant CD95L-variants or by genetic engineer-ing-enforced formation of hexamers, are able to efficiently activate both apoptotic and non-apoptotic signaling pathways. Moreover, it has been shown that binding of soluble trimeric CD95L is not sufficient to stimulate translocation of CD95 molecules to the “lipid raft”-containing compartment of the cell membrane. This translocation of CD95 to “lipid rafts” is a pivotal event in CD95 activation and mainly meaningful, especially for induction of apoptosis. Thereby an auto-amplification-loop of caspase-8 activation and association of CD95 with “lipid rafts” is of importance. To analyze CD95-CD95L interactions, highly sensitive cellular binding studies using CD95L fusion proteins linked to the N-terminal Gaussia princeps luciferase (GpL) have been per-formed. With GpL-CD95L fusion proteins it has been demonstrated that oligomerization of CD95L trimers has no major effect on CD95 occupancy. Therefore higher specific activity of oligomerized CD95L trimers is not related to an avidity-driven increase in apparent affinity. This suggests that a process of secondary aggregation of the initially formed trimeric CD95L-CD95 complexes is crucial for CD95 activation. Furthermore, the data obtained from scat-chard analysis showed that trimeric CD95L interacts with at least two binding sites of different affinity. This was further examined by performing binding studies of soluble monomeric and trimeric GpL-CD95 receptors to membrane-bound CD95L and neutralization assays. It was observed that trimeric CD95 receptor can bind to CD95L much better. These results suggest that the high and low affinity binding sites concern to monomeric or rather pre-assembled CD95 molecules. Moreover, GpL-CD95L fusion proteins have been employed to analyze translocation of CD95 to “lipid rafts”. In these experiments, GpL-CD95L trimers were applied to “mark” inactive CD95 molecules. Upon activation of the remaining free CD95 molecules using highly active Fc-CD95L, an increased association of these inactive receptors with “lipid rafts” was observed. Apparently activated CD95 molecules stimulate in “trans” the co-translocation of inactive CD95 receptors to “lipid rafts”. This has also been confirmed in experiments with transfectants expressing chimeric CD40-CD95 receptors. These chimeric receptors are able to activate CD95-mediated signaling pathways after stimulation with CD40L. After stimulation of endogenous CD95 in CD40-CD95 transfectants the unstimulated chimeric CD40-CD95 receptors co-translocated to “lipid rafts”. Conversely, activation of CD95-associated pathways by specific stimulation of chimeric CD40-CD95 receptors resulted in co-translocation of the endogenous CD95. In conclusion, it has been shown that a functional death domain and caspase-8 activation turned out to be essential for both “lipid raft” association of signaling-active CD95 molecules and co-translocation of inactive CD95 receptors induced by active receptor species.