Kaiserslautern - Fachbereich Chemie
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The Griffith-Ley oxidation of alcohols to aldehydes and ketones is performed with either RuCl3 ⋅ (H2O)x or a highly stable, well-defined ruthenium catalyst and with cheap trimethylamine N-oxide (TMAO) as the oxygen source. The use of n-heptane as the solvent, which forms a second phase with TMAO and a part of the alcohol, allows the reactions to be performed with a minimum amount of catalyst. This results in high local concentrations and thus to very rapid conversions. Detailed quantum chemical calculations suggest, that the Griffith-Ley oxidation not necessarily requires high oxidation states of ruthenium but can also proceed with RuII/RuIV species.
Starting from [(η5-cyclopentadienyl)(η6-phenyl)iron(II)]imidazole, dicationic imidazolium salts were prepared by N-alkylation. Reaction of these compounds with basic metal precursors such as mesityl copper(I) or palladium(II) acetate led to mono respectively dicationic transition metal NHC complexes (NHC=N-heterocyclic carbene). Transmetalation using the copper(I) complexes opened up the access to NHC gold(I) compounds. PEPPSI-type NHC complexes of palladium(II) and platinum(II) were prepared by offering a neutral pyridine ligand to the transition metal center. A rhodium(I) NHC complex was accessible by deprotonation of the dicationic imidazolium precursor and subsequent treatment with [(COD)Rh(μ2-Cl)]2 (COD=1,5-cyclooctadiene). The new NHC complexes were investigated by means of NMR spectroscopy, mass spectrometry as well as single crystal X-ray structure analysis. Both, the palladium(II) containing PEPPSI-type and the gold(I) complex, were investigated for their catalytic properties in typical model reactions such as cyclization reactions, Suzuki coupling and cyanation. In addition, a selenium adduct was synthesized in order to study the electronic properties of the underlying ligand backbone. Based on the chemical shift in the 77Se NMR spectrum, it is evident that these NHC ligands possess rather poor π-acidity.
Die vorliegende Arbeit befasst sich mit der Untersuchung von Absorptionseigenschaften und elektronischer Kurzzeit-Dynamik von organischen Farbstoffmolekülen und supramolekularen Photokatalysatoren in der Gasphase. Dabei wurde erstmals sehr intensiv ein eine relativ unbekannte experimentelle Methode eingesetzt, nämlich die zeitaufgelöste, pump-probe (Anregung-Abfrage) Photofragmentations-Spektroskopie. Die Kombination eines kommerziellen Quadrupol Ionenfallen Massenspektrometers mit einem Femtosekunden Lasersystem erlaubt es die intrinsischen, elektronischen Eigenschaften molekularer, ionischer Systeme abzubilden. Neben Populationsdynamik angeregter Zustände wurden erstmals Schwingungs- und Rotationswellenpaket-Dynamik mit dieser Methode beobachtet und dokumentiert.
Im ersten Teil der Arbeit werden die Ergebnisse der Untersuchungen an einigen ausgewählten Fluoresecein-Derivaten und eines Carbocyanin-Farbstoffes präsentiert. Obwohl diese Modellsysteme zunächst nur dem Zweck dienen sollten die Möglichkeiten des experimentellen Aufbaus zu evaluieren, ergaben die Untersuchungen weiterhin tiefgreifende Einblicke in die elektronische Struktur isolierter organischer Farbstoffe, die bis heute in Literatur nicht dokumentiert worden sind.
Der zweite Teil befasst sich mit der Untersuchung an drei supramolekularen, ionischen Systemen zur photokatalytischen Wasserstofferzeugung. Dabei dienten wieder zwei der Systeme dem Zweck den experimentellen Aufbau zu evaluieren. Neben der elektronischen Populationsdynamik wurde mittels polarisationsabhängiger Messungen weitere Einblicke in den Elektronentransferprozess erhalten – ein Kernpunkt in der Wirkweise supramolekularer Katalysatoren. Die neugewonnen Erkenntnisse wurden schließlich verwendet um einen neuartigen Katalysator zu untersuchen. Dabei stellte sich heraus, dass die Labilität der Ligandensphäre am katalytischen Metallzentrum Untersuchungen am intakten System in Lösung stark beeinträchtigt und somit nur aussagekräftige Ergebnisse mittels einer Gasphasen Methode, einer wie der hier verwendeten, erhalten werden können.
Die experimentellen Ergebnisse werden unterstützt durch quantenchemische Berechnungen von energetischen Minimum-Strukturen, den Strukturen von Übergangszuständen, sowie der Berechnung von Schwingungs- und UV/Vis-Absorptionsspektren mittels (zeitabhängiger) Dichtefunktionaltheorie (DFT & TD-DFT).
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a highly toxic and persistent organic pollutant, which is ubiquitously found in the environment. The prototype dioxin compound was classified as a human carcinogen by the International Agency for Research on Cancer. TCDD acts as a potent liver tumor promoter in rats, which is one of the major concerns related to TCDD exposure. There is extensive evidence, that TCDD exerts anti-estrogenic effects via arylhydrocarbon receptor (AhR)-mediated induction of cytochromes P450 and interferes with the estrogen receptor alpha (ERalpha)-mediated signaling pathway. The present work was conducted to shed light on the hypothesis that enhanced activation of estradiol metabolism by TCDD-induced enzymes, mainly CYP1A1 and CYP1B1, leads to oxidative DNA damage in liver cells. Furthermore, the possible modulation by 17beta-estradiol (E2) was investigated. The effects were examined using four different AhR-responsive species- and sex-specific liver cell models, rat H4II2 and human HepG2 hepatoma cell lines as well as rat primary hepatocytes from male and female Wistar rats. The effective induction of CYP1A1 and CYP1B1 by TCDD was demonstrated in all liver cell models. Basal and TCDD-induced expression of CYP1B1, which is a key enzyme in stimulating E2 metabolism via the more reactive formation of the genotoxic 4-hydroxyestradiol, was most pronounced in rat primary hepatocytes. CYP-dependent induction of reactive oxygen species (ROS) was only observed in rodent cells. E2 induced ROS only in primary rat hepatocytes, which was associated with a weak CYP1B1 mRNA induction. Thus, E2 itself was suggested to induce its own metabolism in primary rat hepatocytes, resulting in the redox cycling of catechol estradiol metabolites leading to ROS formation. In this study the role of TCDD and E2 on oxidative DNA damage was investigated for the first time in vitro in the comet assay using liver cells. Both TCDD and E2 were shown to induce oxidative DNA base modifications only in rat hepatocytes. Additionally, direct oxidative DNA-damaging effects of the two main E2 metabolites, 4-hydroxyestradiol and 2-hydroxyestradiol, were only observed in rat hepatocytes and revealed that E2 damaged the DNA to the same extent. However, the induction of oxidative DNA damage by E2 could not completely be explained by the metabolic conversion of E2 via CYP1A1 and CYP1B1 and has to be further investigated. The expression of low levels of endogenous ERalpha mRNA in primary rat hepatocytes and the lack of ERalpha in hepatoma cell lines were identified as crucial. Therefore, the effects of interference of ERalpha with AhR were examined in HepG2 cells, which were transiently transfected with ERalpha. The over-expression of ERalpha led to enhanced AhR-mediated transcriptional activity by E2, suggesting a possible regulation of E2 levels. In turn, TCDD reduced E2-mediated ERalpha signaling, confirming the anti-estrogenic action of TCDD. Such a modulation of the combined effects of TCDD with E2 was not observed in any of the other experiments. Thus, the role of low endogenous ERalpha levels has to be further investigated in transfection experiments using rat primary hepatocytes. Overall, rat primary hepatocyte culture turned out to be the more adaptive cell model to investigate metabolism in the liver, reflecting a more realistic situation of the liver tissue. Nevertheless, during this work a crosstalk between ERalpha and AhR was shown for the first time using human hepatoma cell line HepG2 by transiently transfecting ERalpha.
Due to their N-glycosidase activity, ribosome-inactivating proteins (RIPs) are attractive candidates as antitumor and antiviral agents in medical and biological research. In the present study, we have successfully cloned two different truncated gelonins into pET-28a(+) vectors and expressed intact recombinant gelonin (rGel), recombinant C-terminally truncated gelonin (rC3-gelonin) and recombinant N- and C-terminally truncated gelonin (rN34C3-gelonin). Biological experiments showed that all these recombinant gelonins have no inhibiting effect on MCF-7 cell lines. These data suggest that the truncated-gelonins are still having a specific structure that does not allow for internalization into cells. Further, truncation of gelonin leads to partial or complete loss of N-glycosidase as well as DNase activity compared to intact rGel. Our data suggest that C-and N-terminal amino acid residues are involved in the catalytic and cytotoxic activities of rGel. In addition, the intact gelonin should be selected as a toxin in the immunoconjugate rather than truncated gelonin.
In the second part, an immunotoxin composed of gelonin, a basic protein of 30 kDa isolated from the Indian plant Gelonium multiflorum and the cytotoxic drug MTX has been studied as a potential tool of gelonin delivery into the cytoplasm of cells. Results of many experiments showed that, on the average, about 5 molecules of MTX were coupled to one molecule of gelonin. The MTX-gelonin conjugate is able to reduce the viability of MCF-7 cell in a dose-dependent manner (ID50, 10 nM) as shown by MTT assay and significantly induce direct and oxidative DNA damage as shown by the alkaline comet assay. However, in-vitro translation toxicity MTX-gelonin conjugates have IC50, 50.5 ng/ml which is less toxic than that of gelonin alone IC50, 4.6 ng/ml. It can be concluded that the positive charge plays an important role in the N-glycosidase activity of gelonin. Furthermore, conjugation of MTX with gelonin through α- and γ- carboxyl groups leads to the partial loss of its anti-folate activity compared to free MTX. These results, taken together, indicate that conjugation of MTX to gelonin permits delivery of the gelonin into the cytoplasm of cancer cells and exerts a measurable toxic effect.
In the third part, we have isolated and characterized two ribosome-inactivating proteins (RIPs) type I, gelonin and GAP31, from seeds of Gelonium multiflorum. Both proteins exhibit RNA-N-glycosidase activity. The amino acid sequences of gelonin and GAP31 were identified by MALDI and ESI mass spectrometry. Gelonin and GAP31 peptides - obtained by proteolytic digestion (trypsin and Arg-C) - are consistent with the amino acid sequence published by Rosenblum and Huang, respectively. Further structural characterization of gelonin and GAP31 (tryptic and Arg-C peptide mapping) showed that the two RIPs have 96% similarity in their sequence. Thus, these two proteins are most probably isoforms arisen from the same gene by alternative splicing. The ESI-MS analysis of gelonin and GAP31 exhibited at least three different post-translational modified forms. A standard plant paucidomannosidic N-glycosylation pattern (GlcNAc2Man2-5Xyl0-1 and GlcNAc2Man6-12Fuc1-2Xyl0-2) was identified using electrospray ionization MS for gelonin on N196 and GAP31 on N189, respectively. Based on these results, both proteins are located in the vacuoles of Gelonium multiflorum seeds.
Wie Proteine sich innerhalb weniger Millisekunden korrekt falten können, ist eine der fundamentalen Fragen in der Biochemie. Ein beim Faltungsprozess durchlaufener Übergangszustand ist der molten globule Zustand (MG Zustand), der sich unter bestimmten Bedingungen stabilisieren und untersuchen lässt. In diesem Zustand ähnelt die Sekundärstruktur dem nativen Zustand, während die Tertiärstruktur eher dem vollständig entfalteten Zustand entspricht. In dieser Arbeit wurde der MG Zustand am Beispiel des Maltose bindenden Proteins (MBP) untersucht. Dazu wurde MBP bei pH 3,2 im MG-Zustand stabilisiert und dies mittels Fluoreszenz Spektroskopie bestätigt. Die Abstände zwischen definierten Aminosäuren im MG Zustand wurden durch Spinlabels, die an gezielt mutierten Cysteinpaaren angebracht wurden, mittels Elektronenspinresonanz (EPR) gemessen und mit den Abständen derselben Aminosäuren im nativen Zustand verglichen. Anhand von sieben verschiedenen Doppelmutanten wurde die periphere Struktur mittels gepulster EPR analysiert, zwei weitere Doppelmutanten dienten dazu, die Struktur der molekularen Bindungstasche von MBP mittels CW EPR zu untersuchen. Die Anwesenheit von Maltose führte im MG Zustand zu einer deutlichen Veränderung der Abstände bestimmter Spinlabels in der peripheren Struktur. Dies deutet darauf hin, dass MBP Maltose sogar im MG Zustand binden kann. Durch isotherme Titrationskalorimetrie (ITC) wurde diese Vermutung bestätigt: die Ergebnisse zeigen jedoch, dass der Bindungsprozess zwischen MBP und Maltose im MG Zustand mit 11 fach geringerer Bindungsenthalpie erfolgt wie im nativen Zustand. Die Abstände der Spinlabel Paare neben der Bindungstasche von MBP unterschieden sich im MG Zustand vom nativen Zustand weder mit noch ohne Maltose. Diese Ergebnisse weisen darauf hin, dass MBP im MG Zustand rund um die Bindungstasche bereits eine klar ausgebildete Tertiärstruktur besitzt. Um diese Befunde zu bestätigen, sollten nun Untersuchungen anhand weiterer Doppelmutanten und mittels empfindlicherer Messungen wie z.B. DQC durchgeführt werden.
Employing site-directed spin labeling (SDSL), the structure of maltose-binding protein (MBP) had previously been studied in the native state by electron paramagnetic resonance (EPR) spectroscopy. Several spin-labeled double cysteine mutants were distributed all over the structure of this cysteine-free protein and revealed distance information between the nitroxide residues from double electron–electron resonance (DEER). The results were in good agreement with the known X-ray structure. We have now extended these studies to the molten globule (MG) state, a folding intermediate, which can be stabilized around pH 3 and that is characterized by secondary but hardly any tertiary structure. Instead of clearly defined distance features as found in the native state, several additional characteristics indicate that the MG structure of MBP contains different polypeptide chain and domain orientations. MBP is also known to bind its substrate maltose even in MG state although with lower affinity. Additionally, we have now created new mutants allowing for spin labeling at or near the active site. Our data confirm an already preformed ligand site structure in the MG explaining its substrate binding capability and thus most probably serving as a nucleation center for the final native structure.
Janus materials are anisotropic nano- and microarchitectures with two different faces consisting of distinguishable or opposite physicochemical properties. In parallel with the discovery of new methods for the fabrication of these materials, decisive progress has been made in their application, for example, in biological science, catalysis, pharmaceuticals, and, more recently, in battery technology. This Minireview systematically covers recent and significant achievements in the application of task-specific Janus nanomaterials as heterogeneous catalysts in various types of chemical reactions, including reduction, oxidative desulfurization and dye degradation, asymmetric catalysis, biomass transformation, cascade reactions, oxidation, transition-metal-catalyzed cross-coupling reactions, electro- and photocatalytic reactions, as well as gas-phase reactions. Finally, an outlook on possible future applications is given.
Colorectal cancer (CRC) is among the most frequent cancer entities worldwide. Multiple factors are causally associated with CRC development, such as genetic and epigenetic alterations,
inflammatory bowel disease, lifestyle and dietary factors. During malignant transformation,the cellular energy metabolism is reprogrammed in order to promote cancer cell growth and
proliferation. In this review, we first describe the main alterations of the energy metabolism found in CRC, revealing the critical impact of oncogenic signaling and driver mutations in key metabolic
enzymes. Then, the central role of mitochondria and the tricarboxylic acid (TCA) cycle in this process is highlighted, also considering the metabolic crosstalk between tumor and stromal cells in the
tumor microenvironment. The identified cancer-specific metabolic transformations provided new therapeutic targets for the development of small molecule inhibitors. Promising agents are in clinical
trials and are directed against enzymes of the TCA cycle, including isocitrate dehydrogenase, pyruvate dehydrogenase kinase, pyruvate dehydrogenase complex (PDC) and α-ketoglutarate dehydrogenase
(KGDH). Finally, we focus on the α-lipoic acid derivative CPI-613, an inhibitor of both PDC and KGDH, and delineate its anti-tumor effects for targeted therapy.
Work on the use of cyclic peptides or pseudopeptides as synthetic receptors started even before the field of supramolecular chemistry was firmly established. Research initially focused on the development of synthetic ionophores and involved the use of macrocycles with a repeating sequence of subunits along the ring to facilitate the correlation between structure, conformation, and binding properties. Later, nonnatural amino acids as building blocks were also considered. With growing research in this area, cyclopeptides and related macrocycles developed into an important and structurally diverse receptor family. This review provides an overview of these developments, starting from the early years. The presented systems are classified according to characteristic structural elements present along the ring. Wherever possible, structural aspects are correlated with binding properties to illustrate how natural or nonnatural amino acids affect binding properties.