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

A broad range of organocopper intermediates in different aggregation states were characterized by electrospray ionization (ESI) mass spectrometry, which provided valuable information on these fluxional species. To complement the mass spectrometric data, electrical conductivity measurements and theoretical calculations were employed. Tetrahydrofuran (THF) solutions of CuCN/(RLi)m stoichiometry (m = 0.5, 0.8, 1.0, and 2.0 and R = Me, Et, nBu, sBu, tBu, Ph) were analyzed by ESI mass spectrometry, and organocuprate anions were detected for all cases. The composition of these species showed clear dependence on the amount of RLi used. Thus, while cyanide-free Lin–1CunR2n– anions completely predominated for CuCN/(RLi)2 solutions, cyanide-containing Lin–1CunRn(CN)n– complexes prevailed for CuCN/(RLi)m reagents with m ≤ 1. Ligand mixing studies on LiCuMe2•LiCN and LiCuR2•LiCN systems (R = Et, nBu, sBu, tBu, Ph) revealed fast exchange equilibria operating in solution. When THF was substituted for the less polar diethyl ether (Et2O), no major new species were observed. However, the proportion of higher nuclearity anions was consistently greater in the latter solvent than in the former. Further experiments with 2-methyltetrahydrofuran (MeTHF), cyclopentyl methyl ether (CPME) and methyl tert-butyl ether (MTBE) solutions confirmed the suggestion that higher aggregation states are favored by lower polarity solvents. Additional conductivity experiments indicated that contact ion pairs strongly predominate for solutions in Et2O, whereas the more polar THF gives rise to larger amounts of solvent-separated ion pairs. Following the detection of organocuprate ions, their gas- and condensed-phase reactions were investigated. Collision-induced dissociation (CID) experiments were used to study intrinsic reactivities in the gas phase. Higher aggregates were found to break apart into fragments of lower nuclearity, whereas monomeric species decomposed by beta-H elimination when possible. In some CID spectra, the presence of hydroxyl-containing signals led to the conclusion that a reaction with background water inside the mass spectrometer was taking place. This bimolecular reaction was then studied in detail for many different systems. The results indicate that lithium centers seem to be a necessary (but not only) pre-requisite for hydrolysis. For example, no reaction was observed for monomeric CuMe2– anions, whereas the reactions of LiCu2Me4– and Li2Cu3Me6– were much faster. Following the successful characterization of organocuprates, their synthetically useful coupling reactions with alkyl halides were probed. ESI mass spectrometric experiments, supported by electrical conductivity measurements, indicated that LiCuMe2•LiCN reacts with a series of alkyl halides RX (R = Me, Et, nPr, nBu, PhCH2CH2, CH2=CHCH2, and CF3CH2CH2). The resulting Li+Me2CuR(CN)− intermediates then afford the observable Me3CuR− tetraalkylcuprate anions upon Me/CN exchanges with added MeLi. In contrast, the reactions of LiCuMe2•LiCN with neopentyl iodide and various aryl halides gave rise to halogen-copper exchanges. Concentration- and solvent-dependent studies suggested that lithium tetraalkylcuprates partly form Li+Me3CuR− contact ion pairs and presumably also triple ions LiMe6Cu2R2−. According to theoretical calculations, these triple ions consist of two square-planar Me3CuR− subunits binding to a central Li+ ion. Upon fragmentation in the gas phase, the Me3CuR− anions undergo reductive elimination, yielding both cross- (MeR) and homo-coupling products (Me2). The branching between these channels showed a marked dependence on the nature of R. The fragmentation of LiMe6Cu2R2− also affords both cross- and homo-coupling products, but strongly favors the former. This was rationalized by the preferential interaction of the central Li+ ion with two Me groups of each Me3CuR− subunit, which thereby block the homo-coupling channel. Finally, the reactivity of organocuprates in conjugate addition reactions was investigated, with cyano-substituted ethylenes C2Hn–4(CN)n, n = 1 – 4 as Michael acceptors. In the case of acrylonitrile, n = 1, polymerization was induced, but no reactive intermediates were detected. In contrast, the reaction with fumaronitrile, n = 2, permitted the detection of π-complexes in different aggregation states. The identities of the latter were confirmed by the release of intact fumaronitrile upon their fragmentation in the gas phase. The reactions with 1,1-dicyanoethylene, n = 2, did not halt at the stage of the π-complexes, but proceeded all the way to Michael adducts. In the case of tricyanoethylene, n = 3, dimeric polycyano carbanions were formed. For tetracyanoethylene, n = 4, the reaction instead leads to Cu(III) species, which undergo reductive eliminations. Thus, all intermediates commonly proposed for the conjugate addition of organocuprates to Michael acceptors were detected, providing strong evidence for the currently accepted mechanism.

Viruses are known to cause many diseases, from the common cold and cold sores to more serious diseases such as the Ebola virus disease and AIDS. Viruses have evolved different strategies to enter and infect cells. In order to infect a cell, viruses have to overcome the cell membrane barrier to deliver their genome to the site of replication. Enveloped viruses can either fuse directly at the plasma membrane or with an endosomal membrane after endocytic uptake. In this work, I studied the early steps in virus entry of herpes simplex virus 1 (HSV-1) and foamy virus (FV) by means of fluorescence microscopy. The virus particles contain two different labels, one located at the envelope and the other at the capsid so that fusion can be detected upon separation of the two colors in space. The virus preparations were optimized for a high dual-color virus yield and live-cell imaging experiments were performed with spinning-disk confocal microscopy in 3D to gain insights into the entry kinetics. In order to determine the time-scale when virus fusion occurs, the percentage of virions containing both envelope and capsid signals was evaluated over time. Virus particles that are taken up by endocytosis face an increasing proton concentration within maturing endosomes. However, the emission of some fluorescent proteins is known to be pH-dependent and the use of pH-sensitive fluorescent proteins, such as GFP, can result in critical artifacts in live-cell imaging. Therefore, experimental approaches are presented to circumvent this issue. To obtain dynamic information on virus fusion, single virus tracing experiments were performed with high time resolution to investigate individual fusion events in real-time. In the case of foamy virus, sixteen fusion events, visualized by color separation, were observed. Thereof, four fusion events were observed at the plasma membrane and twelve fused with an endosomal membrane after endocytic uptake. Moreover, an intermediate stage during the fusion process of foamy viruses was identified that lasted over minutes. This stage was characterized by an increase in the distance between the fluorescent envelope and capsid signals before the final color separation event. Hence, it was possible for the first time to visualize single fusion events of foamy virus in real-time and characterize the corresponding dynamics. The results provide new insights into the entry pathway and fusion process of this unconventional retrovirus.

Part I: Total Synthesis of Variecolortides A–C The variecolortides are structurally intriguing, fungal natural products that present a unique merger of three major streams of biosynthesis. They can be traced back to other known natural products, namely the viocristins and echinulins, and we wanted to prove if they could somehow come together to form the variecolortides. Thus, these natural products were prepared in short sequences and their critical linkage was subsequently investigated. It was found that an unprecedented hetero-Diels-Alder reaction effected this linkage and furnished the central spirocyclic core of the variecolortides. Ultimately, a highly convergent route to all three variecolortides (A–C) was developed. The natural products were each efficiently synthesized as racemates in seven steps or less (longest linear sequence), largely avoiding protecting group chemistry. Part II: Towards the Total Synthesis of Naphthomycin K and Divergolides C and D Ansamycins are an important class of natural products that often show potent antibacterial and antiviral activities. We have engaged in the total syntheses of several new aminonaphthoquinone ansamycins, including naphthomycin K and divergolides C and D. A unified approach to their naphthoquinone core was targeted and led to the development of a novel, cyano-substituted Danishefsky-type diene. The latter was used in the synthesis of cyano-substituted naphthoquinones, naphthalenes and other highly functionalized small molecules. Since the cyano-substituted naphthoquinones could not be further elaborated into the desired natural products, alternatively substituted bromonaphthalenes were instead synthesized over short sequences. In addition, several strategies towards the synthesis of an ansa chain fragment of divergolides C and D were developed. Part III: Synthesis of Photochromic Open-Channel Blockers Optical control of transmembrane ion channel function with high temporal and spatial precision opens the door for a better understanding of neuronal circuits and pharmacological associated questions. Several red-shifted derivatives of QAQ (quaternary ammonium- azobenzene-quaternary ammonium), a powerful doubly charged photochromic blocker, have been synthesized. They allow for remote control of Kv and Nav channel conductance with light and offer the opportunity to reversibly silence neuronal activity.