Kaiserslautern - Fachbereich Chemie
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The consumption of red meat is associated with an increased risk for colorectal cancer (CRC). Multiple lines of evidence suggest
that heme iron as abundant constituent of red meat is responsible for its carcinogenic potential. However, the underlying
mechanisms are not fully understood and particularly the role of intestinal inflammation has not been investigated. To address
this important issue, we analyzed the impact of heme iron (0.25 μmol/g diet) on the intestinal microbiota, gut inflammation
and colorectal tumor formation in mice. An iron-balanced diet with ferric citrate (0.25 μmol/g diet) was used as reference.
16S rRNA sequencing revealed that dietary heme reduced α-diversity and caused a persistent intestinal dysbiosis, with a
continuous increase in gram-negative Proteobacteria. This was linked to chronic gut inflammation and hyperproliferation of
the intestinal epithelium as attested by mini-endoscopy, histopathology and immunohistochemistry. Dietary heme triggered
the infiltration of myeloid cells into colorectal mucosa with an increased level of COX-2 positive cells. Furthermore, flow
cytometry-based phenotyping demonstrated an increased number of T cells and B cells in the lamina propria following heme
intake, while γδ-T cells were reduced in the intraepithelial compartment. Dietary heme iron catalyzed formation of fecal
N-nitroso compounds and was genotoxic in intestinal epithelial cells, yet suppressed intestinal apoptosis as evidenced by
confocal microscopy and western blot analysis. Finally, a chemically induced CRC mouse model showed persistent intestinal
dysbiosis, chronic gut inflammation and increased colorectal tumorigenesis following heme iron intake. Altogether, this study
unveiled intestinal inflammation as important driver in heme iron-associated colorectal carcinogenesis.
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.
1,2-unsaturated pyrrolizidine alkaloids (PAs) are natural plant constituents comprising more than 600 different structures. A major source of human exposure is thought to be cross-contamination of food, feed and phytomedicines with PA plants. In humans, laboratory and farm animals, certain PAs exert pronounced liver toxicity and can induce malignant liver tumors in rodents. Here, we investigated the cytotoxicity and genotoxicity of eleven PAs belonging to different structural classes. Although all PAs were negative in the fluctuation Ames test in Salmonella, they were cytotoxic and induced micronuclei in human HepG2 hepatoblastoma cells over-expressing human cytochrome P450 3A4. Lasiocarpine and cyclic diesters except monocrotaline were the most potent congeners both in cytotoxicity and micronucleus assays with concentrations below 3 μM inducing a doubling in micronuclei counts. Other open di-esters and all monoesters exhibited weaker or much weaker geno- and cytotoxicity. The findings were in agreement with recently suggested interim Relative Potency (iREP) factors with the exceptions of europine and monocrotaline. A more detailed micronuclei analysis at low concentrations of lasiocarpine, retrorsine or senecionine indicated that pronounced hypolinearity of the concentration–response curves was evident for retrorsine and senecionine but not for lasiocarpine. Our findings show that the genotoxic and cytotoxic potencies of PAs in a human hepatic cell line vary in a structure-dependent manner. Both the low potency of monoesters and the shape of prototype concentration–response relationships warrant a substance- and structure-specific approach in the risk assessment of PAs.
The weight of evidence pro/contra classifying the process-related food contaminant (PRC) acrylamide (AA) as a genotoxic carcinogen is reviewed. Current dietary AA exposure estimates reflect margins of exposure (MOEs) < 500. Several arguments support the view that AA may not act as a genotoxic carcinogen, especially not at consumer-relevant exposure levels: Biotransformation of AA into genotoxic glycidamide (GA) in primary rat hepatocytes is markedly slower than detoxifying coupling to glutathione (GS). Repeated feeding of rats with AA containing foods, bringing about uptake of 100 µg/kg/day of AA, resulted in dose x time-related buildup of AA-hemoglobin (Hb) adducts, whereas GA-Hb adducts remained within the background. Since hepatic oxidative biotransformation of AA into GA was proven by simultaneous urinary mercapturic acid monitoring it can be concluded that at this nutritional intake level any GA formed in the liver from AA is quantitatively coupled to GS to be excreted as mercapturic acid in urine. In an oral single dose–response study in rats, AA induced DNA N7-GA-Gua adducts dose-dependently in the high dose range (> 100 µg/kg b w). At variance, in the dose range below 100 µg/kg b.w. down to levels of average consumers exposure, DNA N7 -Gua lesions were found only sporadically, without dose dependence, and at levels close to the lower bound of similar human background DNA N7-Gua lesions. No DNA damage was detected by the comet assay within this low dose range. GA is a very weak mutagen, known to predominantly induce DNA N7-GA-Gua adducts, especially in the lower dose range. There is consensus that DNA N7-GA-Gua adducts exhibit rather low mutagenic potency. The low mutagenic potential of GA has further been evidenced by comparison to preactivated forms of other process-related contaminants, such as N-Nitroso compounds or polycyclic aromatic hydrocarbons, potent food borne mutagens/carcinogens. Toxicogenomic studies provide no evidence supporting a genotoxic mode of action (MOA), rather indicate effects on calcium signalling and cytoskeletal functions in rodent target organs. Rodent carcinogenicity studies show induction of strain- and species-specific neoplasms, with MOAs not considered likely predictive for human cancer risk. In summary, the overall evidence clearly argues for a nongenotoxic/nonmutagenic MOA underlying the neoplastic effects of AA in rodents. In consequence, a tolerable intake level (TDI) may be defined, guided by mechanistic elucidation of key adverse effects and supported by biomarker-based dosimetry in experimental systems and humans.
Purpose
We investigated the cytosolic and membrane-associated contents of polyphenols after 4 hours of incubation (50 μM of each polyphenol) in the colon carcinoma cell line T84 using a novel, rapid, and convenient method based on permeabilization of the cell membrane using digitonin. The colon carcinoma cell line was used to investigate the intestinal uptake of polyphenols present in apple products.
Recent Findings
The results showed that hydroxycinnamic acids (caffeic and 5-caffeoylquinic acid) were only detected in the cytosolic fractions. In contrast, 0.3 to 8.2% of the initial concentrations (50 μM) of the flavonoids phloretin, quercetin, phloretin 2′-O-glucoside, and quercetin 3-O-rhamnoside were found in the membrane-associated fractions. In the cytosolic fractions, 0.2–2.9% of these compounds were detected, corresponding to 25 to 40% of the total cell-associated (cytosolic plus membrane-associated fractions) polyphenol content.
Summary
Our results showed that after uptake, polyphenols were present in the cytosolic fraction of the cells as well as associated with the cell membrane. The presented method provides a useful in vitro tool for determining biologically active compounds in cellular fractions.
Heme oxygenase-1 (HO-1) is an enzyme located at the endoplasmic reticulum, which is responsible for the degradation of cellular heme into ferrous iron, carbon monoxide and biliverdin-IXa. In addition to this main function, the enzyme is involved in many other homeostatic, toxic and cancer-related mechanisms. In this review, we first summarize the importance of HO-1 in
physiology and pathophysiology with a focus on the digestive system. We then detail its structure and function, followed by a section on the regulatory mechanisms that control HO-1 expression and activity. Moreover, HO-2 as important further HO isoform is discussed, highlighting the similarities
and differences with regard to HO-1. Subsequently, we describe the direct and indirect cytoprotective functions of HO-1 and its breakdown products carbon monoxide and biliverdin-IXa, but also highlight possible pro-inflammatory effects. Finally, we address the role of HO-1 in cancer with a particular
focus on colorectal cancer. Here, relevant pathways and mechanisms are presented, through which HO-1 impacts tumor induction and tumor progression. These include oxidative stress and DNA damage, ferroptosis, cell cycle progression and apoptosis as well as migration, proliferation, and
epithelial-mesenchymal transition.
The present thesis reports on studies of atomically precise, size-selected tantalum
cluster ions \(Ta_n^±\) under cryogenic conditions in a FT-ICR mass spectrometer with respect to surface adsorbate interactions at the fundamental level, focusing on \(N_2\) and \(H_2\) adsorption and activation. The wealth of results presented here is the result of systematic studies that have revealed valuable kinetic, spectroscopic, and quantum chemical information, which together paint a comprehensive picture of the elementary adsorption steps and mechanisms in detail.
The \(N_2\) and \(H_2\) adsorption processes to \(Ta_n^+\) clusters exhibit dependencies on cluster size n and on adsorbate load. In terms of \(N_2\) adsorption, there is evidence for spontaneous \(N_2\) activation and cleavage by \(Ta_2^+\) - \(Ta_4^+\), while it appears to be suppressedby \(Ta_5^+\) - \(Ta_8^+\). The activation and cleavage of \(N_2\) molecules proceeds across
surmountable barriers and along much-involved multidimensional reaction paths.
Underlying reaction processes and involved intermediates are elucidated. Two different processes are characteristic of \(H_2\) adsorption: There are fast adsoprtion processes without competing desorption reactions at low \(H_2\) loadings, indicating dissociative adsorption processes, followed by slow adsorption reactions accompanied by multiple desorption reactions at high \(H_2\) loadings, indicating molecular \(H_2\) adsorption. The threshold is the completion of the first adsorbate shell. The \(N_2\) adsorption study of \(Ta_n^-\) clusters revealed that the \(N_2\) adsorption ability of anionic tantalum clusters depends strongly on cluster size n. The cluster size n = 9 is the minimum size for \(N_2\) adsorption onto \(Ta_n^-\) clusters to yield stable and detectable cluster adsorbate species \([Ta_n(N_2)_m]^-\).
Toxicology, the study of the adverse effects of chemicals and physical agents on living organisms, is a critical process in chemical and drug development. The low throughput, high costs, limited predictivity and ethical concerns related to traditional animal-based toxicity studies render them impractical to assess the growing number and complexity of both existing and new compounds and their formulations. These factors together with the increasing implementation of more demanding regulations, evidence the current need to develop innovative, reliable, cost effective and high throughput toxicological methods.
The use of metabolomics in vitro presents the powerful combination of a human relevant system with a multiparametric approach that allows assessing multiple endpoints in a single biological sample. Applying metabolomics in a cell-based system offers an alternative to both, the ethical concerns and relevance of animal testing and the restraining nature of single endpoint evaluations characteristic of conventional toxicological in vitro assays. However, there are still challenges that hamper the expansion of metabolomics beyond a research tool to a feasible and implementable technology for toxicology assessment.
The aim of this dissertation is to advance the applications of in vitro metabolomics in toxicology by addressing three major challenges that have limited its widespread implementation in the field. In chapter 2 the restrictive high cost and low throughput of in vitro metabolomics was addressed through the development, standardization and proof of concept of a high throughput targeted LC-MS/MS in vitro metabolomics platform for the characterization of hepatotoxicity. In chapter 3, the use of the developed in vitro metabolomics system was expanded beyond hazard identification, to its implementation for deriving dose- and time response metrics that were shown useful for Point of departure (PoD) estimations for human risk assessment. Finally, in chapter 4 in order to increase the reliance and confidence of using in vitro metabolomics data for risk assessment, the human relevance of the metabolomics in vitro assays was attempted to be improved by the implementation and evaluation of in vitro metabolomics in a hiPSCs-derived 3D liver organoid system.
The work developed here demonstrates the suitable of in vitro metabolomics for mechanistic-based hazard identification and risk assessment. By advancing the applications of metabolomics in toxicology, this work has significantly contributed to the aim of toxicology of the 21st century for a human-relevant non-animal toxicological testing, supporting the toxicology task of protecting human health and the environment.
This thesis describes the synthesis and extensive characterization of mononuclear
cis-(carboxylato)(hydroxo)iron(III) and cis-(carboxylato)(aqua)iron(II) complexes
among others and illuminates their capability to engage in hydrogen atom transfer
reactions via reactivity studies with suitable substrates. The employed carboxylates
include benzoate, p-nitrobenzoate, and p-methoxybenzoate. Additionally, the first
example for a solution-stable mononuclear cis-di(hydroxo)iron(III) complex is
presented, the extensive characterization of which aims to contribute to the
identification of spectroscopic markers and a better understanding of the role of the
carboxylate ligand in the above-mentioned complexes.
The cis-(carboxylato)(hydroxo/aqua)iron(III/II) complexes match the coordination
environment and the electronic properties of the active iron site in the resting state of
rabbit lipoxygenase as well as of the reaction intermediates postulated for the
enzymatic mechanism. In addition to being excellent structural and electronic models,
the cis-(carboxylato)(hydroxo)iron(III) complexes display reactivity in abstracting
hydrogen atoms from (weak) O–H and C–H bonds of suitable substrates, thus proving
themselves to be worthy functional model complexes for lipoxygenases. The findings
are supported with extensive structural, spectroscopic, spectrometric, magnetic, and
electrochemical investigations as well as with quantified thermodynamic and kinetic
parameters to allow for an adequate comparison between the derivatives with varying
carboxylate ligands and to other works. Moreover, the reactivity investigation for the
cis-(benzoato)(hydroxo)iron(III) (the first example found) was exemplary accompanied
by a thorough theoretical study (done by external cooperation partners), which
validates the experimental results and identifies an underlying concerted protoncoupled-electron-transfer (cPCET) mechanism for the
cis-(carboxylato)(hydroxo)iron(III) complexes – analogous to the one suggested for the
enzyme.
The synthesis and study of a functional structural model complex is extremely
challenging and rarely successful. Thus, this result alone represents a significant
scientific advancement for the field, as no such model for lipoxygenases had been
precedented prior to this project. The in-depth studies with derivatives of the initial cis-(benzoato)(hydroxo/aqua)iron(III/II) complexes further contribute to this
advancement by illuminating structure-function relations.
Malaria is still a big problem globally causing more than 400,000 deaths each year. Although very effective antimalarial drugs are on the market, their mode of action is still not fully understood. In the last years some patients showed an increased clearance time from Plasmodium falciparum malaria parasites after a therapy with the most effective antimalarial compound artemisinin. It was shown that mutations in the propeller domain of a protein called PfKelch13 are directly linked to this decreased susceptibility towards the drug. To gain insights into the protein function, I produced different mutants of a truncated version of this protein containing the BTB and propeller domain in high yield and purity in insect cells. I showed a positive correlation between the solubility of the recombinant protein and the artemisinin susceptibility. Prominent PfKelch13 mutants from the field with decreased artemisinin susceptibility (I543T, R539T, C580Y) were insoluble when recombinantly expressed suggesting that improper folding of PfKelch13 leads to this decrease in sensitivity. The mutation C580Y is the most frequent mutation in South East Asia. The substitution of this cysteine residue does not allow the formation of an intramolecular disulfide bond with cysteine C532 according to an existing crystal structure. Interestingly, substitution of these cysteines to serines did not show improper folding in insect cells, arguing that rather specific substitutions than the residue position itself are responsible for the alteration of drug sensitivity. To test the impact of the disulfide bond on artemisinin susceptibility, I generated stable transgenic parasites expressing the corresponding serine mutations. Neither C580S nor C532S showed decreased artemisinin susceptibility. Therefore, it could be excluded that the formation of the intramolecular disulfide bond has an influence on artemisinin susceptibility. We further asked, if the protein abundance has an influence on the sensitivity towards the drug. I successfully generated stable transgenic parasites expressing His8-PFKELCH13 fused to the glmS riboswitch. I showed that protein levels could be efficiently down-regulated by more than 90% resulting in a very low parasite susceptibility towards artemisinin. This strain offers a basis for future experiments in the understanding of the impact of PfKelch13 protein levels on biochemical pathways in malaria parasites.
Peroxiredoxins (Prxs) play an important role in protecting the cell from high amounts hydroperoxides. Among the five known Prxs in P. falciparum our group took PfAOP as a model enzyme to study the catalytic cycle. It has been shown that PfAOP reduces hydroperoxides like H2O2 or tBuOOH with fast kinetics, and that reduction of the protein is linked to the GSH/Grx system (Djuika et al. 2013). However, no direct kinetic data was available for the reductive half-reaction of PfAOP and GSH. In this thesis, I qualitatively showed that oxidized PfAOP can be glutathionylated and that in a next step glutathione can be transferred to PfGrx. I further determined the rate constants of the glutathionylation of PfAOP by stopped-flow measurements. Rate constants of around 10^5 M-1s-1 indicate a fast kinetic that is able to protect the protein from hyperoxidation and inactivation. Furthermore, I determined the activation energy, entropy and enthalpy for this reaction of 41.1 kJ/mol, -0.79 J/mol and 39.8 kJ/mol, respectively. Hence, the activation energy of the glutathionylation of oxidized PfAOP suggests the break of two to three hydrogen bonds and is rather temperature-independent.
Synergism of Lipoates and Established Anticancer Drugs in Cell and Mouse Models of Colorectal Cancer
(2021)
Das kolorektale Karzinom (KRK) ist eine der am häufigsten auftretenden Krebsentitäten und zeigt aktuell eine erhöhte Inzidenz und Mortalität bei Erwachsenen unter 50 Jahren in Europa und den USA auf. Zumeist im fortgeschrittenen Stadium diagnostiziert, ist die 5-Jahres-Überlebensrate des KRKs immer noch gering. Daher besteht eine Notwendigkeit neuer Therapieansätze und Angriffspunkte für Wirkstoffkandidaten, wenngleich Standardtherapien mit den Zytostatika 5-Fluorouracil (5 FU) oder Irinotecan (IT) und Biologika existieren. Einen solchen Angriffspunkt könnte der Energiemetabolismus darstellen, der für Krebszellen charakteristische Veränderungen aufweist. Das Lipoat CPI 613 ist ein Derivat der natürlich vorkommenden α Liponsäure (LA) und gehört aufgrund seiner einzigartigen Hemmung des veränderten Energiemetabolismus in Krebszellen als Vorreiter zu einer neuen Klasse von Wirkstoffsubstanzen. CPI-613 erwies sich bereits als Inhibitor mitochondrieller Multienzymkomplexe wie der Pyruvatdehydrogenase und der α Ketoglutaratdehydrogenase. Diese Wirkung wurde vorrangig in Krebszellen beschrieben.
Der Fokus dieser Arbeit lag zunächst auf der Untersuchung zellulärer Antworten auf die Behandlung von KRK-Zellen mit dem Lipoat CPI 613 und schlossen neben Effekten auf die mitochondrielle Integrität und Funktion der oxidativen Phosphorylierung auch die Endpunkte Zelltod, DNA-Schädigung und Autophagie ein. Hierfür wurde ein Panel an KRK-Zellen und auch nicht maligne transformierte humane Kolonepithelzellen (HCEC) untersucht. Darüber hinaus wurde in isolierten murinen Mitochondrien die Wirkungsweise von CPI-613 geprüft. Weiterhin sollte ein möglicher Synergismus durch eine Kombinationsbehandlung von Lipoaten wie CPI-613 oder dessen Muttersubstanz LA und Standardchemotherapeutika in der KRK-Behandlung wie 5-FU und IT charakterisiert werden. Nach Identifizierung der aussichtsreichsten Kombination in vitro folgten Studien zur Wirksamkeitssteigerung der Kombinationsbehandlung im Vergleich zur Einzelbehandlung in vivo sowie eine Beurteilung zu möglichen hämatotoxischen Nebeneffekten. Zu diesem Zwecke wurden sowohl das Xenograft-Modell in immundefizienten Mäusen (BALB/c nu/nu) als auch das Azoxymethan (AOM)/Dextran Natriumsulfat (DSS)-Model zur chemischen Induktion von KRK-Tumoren in C57BL/6-Mäusen genutzt.
Es konnte gezeigt werden, dass CPI-613 sowohl in isolierten Mitochondrien als auch in KRK-Zelllinien zu einer Reduktion des mitochondriellen Membranpotentials neben einer vermehrten Bildung von reaktiven Sauerstoffspezies führte. Dies ging mit einer deutlichen Verminderung der zellulären Atmung einher und äußerte sich in KRK-Zelllinien zudem in einer Reduktion der Mitochondrien-Anzahl. Während kein Zellzyklusarrest durch die Behandlung mit CPI 613 ausgelöst wurde, konnte in einem Panel von diversen KRK-Zelllinien Zelltod nachgewiesen werden. Dies war mit gleicher Potenz in den verschiedenen Zelllinien unabhängig des p53- und MSS/MSS-Status zu beobachten. Dabei wurden verschiedene und teils redundante Zelltodmechanismen wie Apoptose, Nekroptose und Caspase-unabhängigem Zelltod nach CPI-613 Behandlung nachgewiesen. Als Folge einer Behandlung mit CPI-613 kam es des Weiteren zu einer gesteigerten Autophagie-Rate in KRK-Zellen. Analysen zum genotoxischen Potential von CPI-613 ergaben keine Hinweise auf DNA-Schädigungen. Verschiedene Kombinationen von Lipoaten und Standardchemotherapeutika wurden in vitro auf deren Synergismus charakterisiert. Neben einer synergistischen Wirkung von CPI-613 in Kombination mit IT in KRK-Zellkulturmodellen konnte ebenfalls ein positiver Effekt in Mausmodellen des KRKs verzeichnet werden. Während CPI-613 bereits alleine in Xenograft-Modellen zu einer Reduktion des Tumorwachstums und somit zu einer verlängerten Überlebenszeit und damit einem Therapieerfolg führte, verstärkten sich diese Effekte deutlich in der Kombinationsbehandlung mit IT. In chemisch-induzierten Tumoren hingegen zeigte vor allem IT einen therapeutischen Effekt, welcher ebenfalls in der Kombination mit CPI-613 zu verzeichnen war. Eine Monotherapie mit CPI 613 führte in diesem Modell zu keinem signifikanten Therapieerfolg. Der Synergismus in vitro und in vivo gründet vornehmlich auf einer gesteigerten Zelltodrate, der Depletion von p53 sowie einer Reduktion der Autophagierate und nicht auf einer erhöhten DNA-Schädigung. Ein hämatotoxisches Nebenwirkungsprofil von CPI-613 wurde hier im Allgemeinen nicht beobachtet.
Zusammenfassend wurde in dieser Arbeit demonstriert, dass CPI-613 im Kontext von KRK den veränderten Energiemetabolismus angreift und zum Zelltod führt. Darüber hinaus konnte eine Genotoxizität von CPI-613 ausgeschlossen werden. Eine Kombination aus CPI-613 und IT führte in Xenograft- und chemisch-induzierten KRK-Tumoren zu einer gesteigerten Wirksamkeit in Bezug auf die Hemmung des Tumorwachstums und der Überlebenszeit. Die Befunde in Zellkultur- und Maus-Modellen des KRKs identifizierten CPI-613 als vielversprechenden Therapiebaustein für die Behandlung des KRKs.
Compared to canonical model organisms, the genetic toolbox of Kinetoplastid parasites have a considerable gap in the transgenic techniques available. The implementation of the CRISPR/Cas9 technology is poised to transform the way we perform genetic manipulations and offers a new and exciting horizon for molecular parasitology. In this study, we use the Kinetoplastid parasite Leishmania tarentolae as a model organism. This unicellular eukaryote is an attractive model for both basic and applied research. Understanding Leishmania’s basic biology is valuable to underpin differences to the host that might help to treat infectious diseases. Furthermore, it also provides new examples of non-conserved mechanisms that will help to understand the fundamental principles of the biology of eukaryotes and their evolution. In this work, the CRISPR/Cas9 system was used to study mitochondrial protein import.
Here I show the efficacy of CRISPR/Cas9 to generate knockout and knockin mutants. Proof- of- concept gene PF16 was used to generate knockout immotile parasites and knockin fluorescent mutants fused with mCherry. The APRT gene was also knocked out showing resistance to APP.
In addition, I generated endogenous mutants of a constituent of the mitochondrial import machineries, the sulfhydryl oxidoreductase Erv. I showed that the KISS domain and cysteine 17 are dispensable for survival dismissing that their functions correlate with the essential operation/s of Erv. I report that the ERV gene and the intervening sequences of its shuttle pair cysteines are refractory to ablation and modification, respectively, indicating that they are essential for survival. I also generated Erv interactomes using full-length and mutant (ErvΔKISS) baits showing candidates with hitherto unknown functions that might be related to Erv function.
I also tested the glmS riboswitch and generate endogenous mutants with CRISPR/Cas9. We asked if it was possible in Leishmania to obtain knockdown mutants with this technique. The evidence of this study indicates that the system is inefficient in provoking a knockdown phenotype for the genes characterized.
An alternative negative marker was also developed in this work. I propose the APRT gene as a novel and efficient counter-selectable marker as compared to the current yFCU and TK genes. The implementation of this system could lead to first shuffling experiments that are not feasible in Leishmania further highlighting the value of this model organism.
Despite their “weak nature” London dispersion interactions are omnipresent and of fundamental importance for many aspects of chemistry and biology and have often been underestimated in the description of intra- and intermolecular interactions. In this thesis, London dispersion is investigated in the gas phase with molecular beam experiments and quantum chemistry. The focus of this work lies in the investigation of London dispersion in the electronic ground state and the electronically excited state. For the electronic ground state, dispersion-bound dimers of triphenylmethane derivatives were analyzed. Depending on the dispersion energy donor, a tail-to-tail (TPM), head-to-tail (iPrTPM) or head-to-head (tBuTPM) arrangement can be assumed for the minimum structure. The tBuTPM dimer exhibits an exceptionally small C-H·· H-C contact which is stabilized by strong London dispersion interactions which was quantified by energy composition analysis. For the characterization of the dimer, the calculation of anharmonic frequencies was of high importance and was also validated with literature data. The second system, the chromone-MeOH balance represents an ideal molecular balance with two competing docking sites at the carbonyl oxygen. The experimental results are compared to theoretical predictions obtained from (TD)DFT-, DLPNO-CCSD(T) and SAPT-calculations to study the balance between electrostatics, induction and dispersion interaction in the S0 and T1 state. The chromone-solvent system was identified as an ideal system for studying London dispersion in multiple electonic states. Furthermore, candidates for derivivatives of chromone were analyzed with quantum chemical methods in the electronic ground and electronically excited state in an attempt to identify suitable candidates for further experiments. The 6-methylchromone shows promising behavior in stabilizing the inside pocket regardless of the electronic state and was analyzed in more detail with a variety of methods. Similar analysis of 2-CF3chromone and the 2-CF3, 6-methylchromone showed no special effect of a substitution in 2-position or possible cooperative effects.
Pyrrolizidine alkaloids (PA) are secondary plant metabolites occurring in a great many plant species worldwide, known to exhibit hepatotoxic, genotoxic and carcinogenic properties after metabolic activation. In recent years, contamination of food, feed and herbal medicines with PA has become an increasing problem. The concept of interim relative potency factors (iREP) proposed by Merz and Schrenk in 2016 was a new approach for risk assessment of PA. While existing approaches of risk assessment assumed equivalent toxic potency for all PA congeners, the approach of Merz and Schrenk considered the structural features of individual PA congeners based on existing data from the literature. In order to generate further data on the structure-specific toxicity of PA, congeners of different structural classes were investigated in different in vitro test systems.
In vitro cytotoxicity was investigated in primary rat hepatocytes, HepG2 C9 cells (overespressing human CYP3A4) and naïve HepG2 cells. Overall, it could be observed that lasiocarpine and the cyclic di-esters (except monocrotaline) showed much stronger cytotoxic effects in comparison to the tested mono-esters in both, primary rat hepatocytes and in HepG2 C9 cells. Primary hepatocytes were the most sensitive cells investigating cytotoxicity of different PA congeners, followed by the HepG2 C9 cells. This is confirmed by markedly higher metabolism rates for all investigated PA in primary rat hepatocytes determined in the metabolism experiments. In naïve HepG2 cells no cytotoxic effects could be observed. The influence of cytochrome P450 (CYP) on the formation of toxic metabolites seem to play a crucial role. This assumption could be beared using ketoconazole as CYP inhibitor and testing various pre-incubation times in primary rat hepatocytes. CYP activity was measured using 7-Benzoxyresorufin-O-Dealkylase (BROD) assay in primary rat hepatocytes and in HepG2 C9 cells. Glutahione (GSH) depletion using buthionine sulfoximine (BSO) showed slight stronger cytotoxic effects for several PA, but not for all tested.
In contrast to the negative results of mutagenicity in ames fluctuation assay using Salmonella strains TA98 and TA100 with and without metabolic activation by S9 mix, all tested PA congeners showed micronuclei induction in the HepG2 C9 cell line. Again, laisocarpine and the cyclic di-esters (except monocrotaline) were the most potent ones. In conclusion, the data from cytotoxicity and genototoxicity experiments from the tested PA congeners confirm published iREP factors with a few exceptions, in particular for monocrotaline or echimidine.
Additionally, metabolism of six selected PA was studied in primary rat hepatocytes and HepG2 C9 cells. Genrally, it was found that almost all tested cyclic and open-chained di-esters (except retrorsine) showed much higher metabolism rates in both cell types, in comparison to the mono-esters, for which only low metabolism rates could be measured. The same was observed for the quantified amounts of reactive metabolites in the supernatants of both cell types. In general, also these data bear the results from cytotoxicity and genotoxicity experiments and help to better understand the complex metabolism and the structure-specific toxicity of different PA congeners.
This thesis is separated into seven distinct research projects on mono and multinuclear transition metal complexes as trapped ions in gas phase, as well as one chapter on focusing on the development of a new ion source to enable access to catalytic processes via coadsorption.
ElectroSpray Ionization (ESI) transfers ions from solution to gas phase for mass spectrometric investigations, allowing a broad variety of experimental methods to obtain fundamental insights into the molecular properties of isolated complexes devoid of solvent, crystal lattice, bulk, or supporting surface effects.
Collision Induced Dissociation (CID) researches molecular fragmentation mechanisms and their relative gas phase stabilities at room temperature. Laser experiments such as InfraRed (Multiple) Photon dissociation and Ultraviolet Photon dissociation offer information on the bonding motifs, resulting in molecular structures and their electronic ground states. When quantum chemical calculations utilizing Density Functional Theory (DFT) and Time Dependent Density Functional Theory (TD-DFT) are combined with monitored spectra, a better and deeper understanding of the structural properties and electronics of transition metal complexes is possible.
X-ray magnetic circular dichroism (XMCD) is a technique that analyzes the magnetic properties of isolated and trapped ions at cryogenic temperatures inside an externally applied magnetic field using high brilliant polarized X-ray photons in conjunction with a mass spectrometer. The element selective technique, combined with sum analysis, allows for the decomposition of the total magnetic moments in their spin and orbital magnetic moments in various metal centers. A determination of the magnetic couplings between distinct metal centers in multinuclear complexes is possible via Broken symmetry approach in combination with X-ray Magnetic Circular Dichroism (XMCD).
Qualitative NMR spectroscopic and quantitative calorimetric binding studies were performed to characterize the interaction of nontoxic mimics of the V-type nerve agent VX (O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate) and the Novichok nerve agent A-234 (ethyl (1-(diethylamino)ethylidene)phosphoramidofluoridate) with a series of receptors in 100 mM aqueous phosphate buffer at pH 7.4 and 37°C. These investigations provided information about the preferred geometry with which the nerve agent mimics are included into the receptor cavities and about the stability of the complexes formed. According to the results, the positively charged VX mimic prefers to bind to cation receptors such as sulfonated calixarenes and an acyclic cucurbituril but does not noticeably interact with cyclodextrins. While binding to the acyclic cucurbituril is stronger than that to calixarenes, the mode of inclusion into the sulfonatocalix[4]arene cavity is better suited for the development of scavengers that bind and detoxify V-type nerve agents. The neutral Novichok mimic, on the other hand, only interacts with the acyclic cucurbituril with a strength required for scavenger development. These binding studies thus provided guidelines for the further development of nerve agent scavengers.
Within toxicology, reproductive toxicology is a highly relevant and socially particularly sensitive field.
It encompasses all toxicological processes within the reproductive cycle and therefore includes many effects and modes of action. This makes the assessment of reproductive toxicity very challenging despite the established in vivo studies. In addition, the in vivo studies are very demanding both in terms of their conduct and interpretation, and there is scope for decision-making on both aspects. As a result, the interpretation of study results may vary from laboratory to laboratory. For the final classification, the assessment of relevance for men is decisive. The problem here is that relatively little is known about the species differences between men and the
usual test animals (rat and rabbit). The rabbit in particular has hardly been researched in molecular biology. The aim of the dissertation was to develop approaches for a better assessment of
reproductive toxicity, with two different foci: The first aim was to investigate species differences, focusing on the expression of xenobiotic transporters during ontogeny. Xenobiotic transporters, of the superfamily of ATP-binding cassette transporters (ABC) or solute carriers (SLC), are known to transport exogenous substances in
addition to their endogenous substrates and therefore play an important role in the absorption, distribution and excretion of xenobiotics. Species differences in kinetics can in turn have a major
impact on toxic effects. In the study, the expression of 20 xenobiotic transporters during ontogeny was investigated at the mRNA level in the liver, kidney and placenta of rats and rabbits and compared with that of men. This revealed major differences in the expression of the transporters between the species. However, further studies on the functionality and activity of the xenobiotic transporters are needed to fully assess the kinetic impact of the observed species differences. Overall, the study provides a valid starting point for further systematic investigations of species differences at the protein level. Furthermore, it provides previously unavailable data on the expression of xenobiotic transporters during ontogeny in rabbits, which is an important step in the molecular biological study of this species.
The second part focused on investigating the predictive power of in silico models for reproductive
toxicology in relation to pesticides. Both the commercial and the freely available models did not
perform adequately in the evaluation. Three reasons could be identified for this: 1. many pesticides
are outside the chemical space of the models, 2. different definition/assessment of reproductive
toxicity and 3. problems in detecting similarity between molecules. To solve these problems, an
extension of the databases on reproductive toxicity in relation to pesticides, respecting a uniform
nomenclature, is needed. Furthermore, endpoint-specific models should be developed which, in
addition to the usual structure-based fingerprints, use descriptors for, for example, biological
activity.
Overall, the dissertation shows how essential it is to further research the modes of action of
reproductive toxicity. This knowledge is necessary to correctly assess in vivo studies and their
relevance to men, as well as to improve the predictive power of in silico models by incorporating
this information.
Wine is a complex chemical mixture that is bound to change over time. Most wines are
produced for consumption within months. Some premium wines are meant to be maturated for
several years or even decades after bottling. The post-bottling evolution and the longevity of a
wine depends on its initial chemical composition and the storage conditions. Temperature,
exposure to light and the closure type are often mentioned as the most important storage
influences. Especially elevated temperature is known to cause accelerated aging reactions in
wine. Refrigerated wine storage cabinets promise to be the best storage option without the
need of a wine cellar. They are available in different sizes and fit in every household. However,
the influence of vibrations and low-interval temperature fluctuations caused by compressors
are parameters that have been neglected in literature. The aim of this thesis was to investigate
if vibrations and low-interval temperature fluctuations, which occur in refrigerated wine storage
cabinets, have an influence on the post-bottling evolution of a wine. The influence of both
parameters was studied separately from each other.
The impact of vibration on oxidation and gas uptake from the headspace of a wine bottle into
the wine was investigated using a model wine with saturated O2 and different headspace
volumes. The study revealed that vibration promotes the dissolution of O2 from the headspace
of bottle into the wine resulting in a faster SO2 consumption. Furthermore, it was shown that
horizontal bottle position accelerated the O2 uptake significantly. It was concluded that the
increased surface size between headspace and wine accelerates the O2 dissolution in wine.
Also, bigger headspace volumes caused an accelerated O2 uptake into the wine. An
experiment without any headspace volume revealed that the factors vibration and bottle
position did not accelerate the O2 consumption in wine. This proves that vibration and bottle
position accelerate only the dissolution of O2 in wine, but not the chemical reaction of O2 with
wine constituents.
The influence of vibration on the volatile profile of wine was investigated using Riesling
sparkling and still wines sealed with different closures that were subjected to vibration for six
months. Vibration caused no CO2 losses, SO2 and color changes in all wines indicating that
vibration caused by compressors has no impact on the gas permeability of the used closures.
However, vibration affected the volatile profile of sparkling wine and Riesling still wine sealed
with a screw cap. Similar to the model wine study described earlier, it was shown that the
equilibrium of volatile substances between the wine and the headspace in a bottle was
influenced by vibration. The gas-liquid-equilibrium of some volatile compounds was shifted
towards wine, while others were shifted towards headspace. As a result of this, the
concentration of volatile compounds in wine is changed. Besides this indirect influence of
vibration, the results of this study also suggested that specific degradation and formation
reactions of volatile compounds are directly influenced by vibration. These multiple effects of
2
vibration most likely explain why increasing vibration intensities could not be proportionally
related to the observed volatile changes. The investigation of different wine styles revealed
that the impact of vibration depends strongly on the initial composition of wine, age, and
packaging conditions. Especially, headspace volume, closure type and CO2 pressure are likely
to influence the equilibrium of volatile substances between the wine and the headspace in a
bottle.
Another study investigated the impact of low-interval temperature fluctuations on the volatile
profile of wine. For this purpose, a Riesling wine was stored for two years under different
temperature fluctuation patterns caused by compressors. Additionally, a model wine with nine
volatile substances with known concentrations was stored for eight months under the same
fluctuation patterns. The low-interval temperature fluctuations were compared to the mean
value of the temperature fluctuations. Chemical and sensory analysis revealed that that lowinterval temperature fluctuations accelerate wine aging reactions like ester hydrolysis and
monoterpene degradation. Even small temperature amplitudes showed a significant impact on
wine aging. The observed effect was explained by the Arrhenius equation which states that
reaction rates exponentially increase with rising temperatures. A pump effect of air through the
closure was initially assumed but not observed in this study. Small deviations in wine
temperature, such as those caused by door openings of a refrigerator were found to be
negligible. It was concluded that low-interval temperature fluctuations can accelerate wine
aging reactions. The amplitude of the temperature fluctuations should be as small as possible
during bottle storage of wine.
This thesis showed that both parameters, vibration, and low-interval temperature fluctuations,
have been proven to influence the evolution of wine during bottle storage. Regarding storage
conditions in a refrigerated wine storage cabinet, those parameters should be monitored. Wine
connoisseurs should therefore consider good wine cabinets, since some manufacturers
emphasize on the importance to minimize vibrations and temperature fluctuations in their
devices. The development of technology should be advanced to reduce both vibration and
temperature fluctuations in refrigerated wine storage cabinets. Future research should focus
on specific wine compounds in model systems and realistic vibration conditions to reveal the
relationship between vibration intensities and reaction rates. The impact of low-interval
temperature fluctuations on wine compositional changes should be investigated considering
horizontal and vertical bottle positions. The calculated acceleration factors due to temperature
fluctuations have to be verified by isotherm storage conditions at higher temperatures.
Hordatines are a characteristic class of secondary metabolites found in barley which have
been reported to be present in barley malt, beer and, recently, brewer ́s spent grain (BSG). However,
little is known about their biological activities such as antioxidative effects in beer or antifungal
activity as their main task within the plants. We conducted an in vitro investigation of the activity
of hordatines isolated from BSG towards enzymes of glucose metabolism. Hordatine-rich fractions
from BSG were prepared by solid-liquid extraction (SLE) with 60% acetone followed by purification
and fractionation. The fractions were characterised and investigated for their in vitro inhibitory
potential on α-glucosidase and glycogen phosphorylase α (GPα). Both enzymes are relevant within
the human glucose metabolism regarding the digestion of carbohydrates as well as the liberation of
glucose from the liver. In total, 10 hordatine-rich fractions varying in the composition of different
hordatines were separated and analysed by mass spectrometry. Hordatine A, B and C, as well as
hydroxylated aglycons and many glycosides, were detected in the fractions. The total hordatine
content was analysed by HPLC-DAD using a semi-quantitative approach and ranged from 60.7 ± 3.1
to 259.6 ± 6.1 μg p-coumaric acid equivalents/mg fraction. Regarding the biological activity of
fractions, no inhibitory effect on GPα was observed, whereas an inhibitory effect on α-glucosidase
was detected (IC50 values: 77.5 ± 6.5–194.1 ± 2.6 μg/mL). Overall, the results confirmed that
hordatines are present in BSG in relatively high amounts and provided evidence that they are potent
inhibitors of α-glucosidase. Further research is needed to confirm these results and identify the active
hordatine structure.
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.
This thesis comprises investigations on the interaction of \(N_2\) adsorbate molecules with size-selected iron metal clusters under cryo conditions. All investigations were performed at the customized fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer FRITZ. This setup serves a laser vaporization (LVAP) source to generate the investigated iron cluster ions. Cryo kinetic studies investigate the stepwise \(N_2\) adsorption on size selected \(Fe_n^+\) clusters under well-defined isothermal conditions. The adsorption behavior and the adsorption limits lead to information about the cluster structure and its reactivity. By coupling a tunable IR laser in the ICR cell, it is possible to perform cryo infrared photon dissociation (IR-PD) spectroscopy experiments. This provides information on binding motifs of the \(N_2\) adsorbates and the cluster structure. Combining both methods with quantum chemical calculations via density functional theory (DFT) substantiates the experimental results and deepens the fundamental insights into the cluster structure, their reactivity, and the metal-adsorbate bonding.
Die vorliegende Arbeit befasst sich mit der Untersuchung von (insbesondere neutralen) kalten, isolierten Molekülen, Aggregaten und Metallkomplexen in der Gasphase mittels UV- und kombinierter IR/UV-Laserspektroskopie im Molekularstrahl. Die Dissertation setzt sich im Wesentlichen aus drei Teilprojekten zusammen. Im ersten Teil wurden erste spektroskopische Untersuchungen in Kombination mit einer neu etablierten Laserdesorptionsquelle durchgeführt. Hierbei wurden zunächst die Desorptionstarget-Vorbereitung und die Expansionsbedingungen der Molekularstrahlquelle entscheidend optimiert. Trotz dieser Anpassungen waren die Ionensignalfluktuationen immer noch zu ausgeprägt um aussagekräftige kombinierte IR/UV-Experimente zu ermöglichen. Daraufhin wurde eine so genannte „Referenzsignal-Korrektur“ eingeführt. Mithilfe dieser Vorgehensweise konnten erste IR/R2PI-Spektren mit dem neuen Laserdesorptionsaufbau gemessen werden. Nach erfolgreichen IR/UV Experimenten an rein organischen Molekülen wurde der Fokus auf die spektroskopische Untersuchung von isolierten neutralen Kontaktionenpaaren (CIPs) gelegt. Hierbei standen insbesondere die Alkali-Ionenpaare (von \( Li^+ \) bis \( Cs^+ \) ) des para-Aminobenzoats (\( M^+ PABA^− \)) im Vordergrund, wobei in allen Experimenten eindeutige Resonanzverschiebungen in Abhängigkeit der Größe des koordinierenden Alkaliions festgestellt wurden. Dabei sind die spektralen Shifts auf elektronische Effekte zurückzuführen, die durch das Coulomb-Potential des Metallions hervorgerufen werden. Weiterhin wurde der neutrale OLED-relevante Metallkomplex Tris(8-hydroxychinolinato)aluminium (\( Alq_3 \)) ebenfalls erfolgreich desorbiert und in intakter Form im Flugzeitmassenspektrometer nachgewiesen. Im zweiten Teil der Arbeit wurden isolierte Chromon-Methanol-Cluster in Bezug auf nichtkovalente Wechselwirkungen analysiert. Bei diesem System liegen zwei nahezu isoenergetische Isomere vor, die sich strukturell durch unterschiedliche CH···O-Kontakte unterscheiden. Chromon besitzt die Eigenschaft nach elektronischer Anregung in die Triplet-Mannigfaltigkeit überzugehen, sodass an diesem Beispiel erstmalig ein neutraler Cluster in einem elektronisch angeregten Triplet-Zustand spektroskopisch untersucht werden konnte. Interessanterweise kommt es im T\(_1 \)-Zustand zu einem Verlust der Planarität des 4-Pyronrings, wodurch sich der energetische Abstand zwischen den beiden Minimumstrukturen vergrößert. Schlussendlich ist dieser energetische Effekt auf unterschiedliche elektrostatische und induktive Wechselwirkungen, jedoch kaum auf Dispersionseffekte zurückzuführen. Zusätzlich wurden Untersuchungen der Aggregation von Methanol an die geschützte Aminosäure AcTyr(Me)OMe durchgeführt, wobei ebenfalls potenzielle Clustergeometrien zugeordnet werden konnten. Im letzten Teil der Arbeit standen die in der Natur allgegenwärtigen Metall−Peptid-Wechselwirkungen im Fokus. In dem Zusammenhang wurde (mittels Dichtefunktionaltheorie) eine tiefgründige strukturelle Analyse der Aggregation eines monovalenten Aluminiumions an die geschützte Aminosäure AcTrpOMe ausgeführt. Hierbei wurde für das energetisch klar stabilste Isomer ein spezielles, energetisch ausgesprochen stabiles Strukturmotif gefunden, bei dem das Aluminiumion in die NH-Bindung des Indol-Substituenten insertiert ist. Aufgrund einer hohen (berechneten) Isomerisierungsbarriere kann ein derartiges Bindungsmotiv nicht im kalten Molekularstrahl gebildet werden, durchaus aber im Plasma einer Thermo-Ablationsquelle, wie sie im entsprechenden Molekularstrahlexperiment verwendet wurde. Weitere quantenchemische Untersuchungen haben ergeben, dass dieser Strukturtyp nur für bestimmte monovalente Metalle (z.B. \( Ti^+ \) oder \( Al^+ \) ) bevorzugt wird.
Oxidative folding of proteins in the mitochondrial intermembrane space of Leishmania tarentolae
(2022)
Mitochondrial genes encode for a few proteins. Thus, the majority of proteins has to be imported to the organelles, which is only possible in the unfolded state. The subsequent folding guarantees functionality. One of the proteins responsible for folding in the intermembrane space is Mia40, which is known in opisthokonts. No ortholog for Mia40 is known in kinetoplastida such as Leishmania tarentolae. First, already known candidates for Mia40 orthologs were investigated. In previous work Mic20 had been identified in Trypanosomes.1–4 Gene editing cassettes to knock-out or modify the gene LtaPh_3313851, which encodes the Mic20 ortholog, could not be inserted homozygously. Thus, the gene is assumed to be essential. Another protein that plays an important role in mitochondrial protein import is Erv, a known interactor of Mia40. Erv is also found in Leishmania. Two proteins of so far unknown function had been identified as potential interactors of Erv and could be candidates for Mia40 orthologs.5 Potential knock-out strains of one protein-encoding gene each were investigated. The knock-out of LtaP32.0380 was assumed to be complete and the gene dispensable. The knock-out cassette for LtaP07.0980 could be shown to be inserted heterozygously, which could indicate the essentiality of the gene. To identify new candidates for Mia40 orthologs in Leishmania tarentolae, potential substrates6 of the Mia40/Erv pathway were used as baits in the present work. Gene editing via CRISPR/Cas9 included attempts to insert knock-out or tagging cassettes to five different genes. Homozygous insertion succeeded for the C-terminal His8-tagging cassettes for LtaP19.1110, and for the N-terminal His8-tagging cassettes for LtaP25.1620 and LtaP09.1390. No homozygous gene editing could be observed for LtaP35.0210. The knock-out of LtaP04.0060 was assumed to be complete. The presence of the N-terminal His8-tagged substrate 4 (LtaP09.1390) could be shown in cell lysates. The correct position of tagged substrate 4 in the cell was confirmed. Further cell lysates were purified in pull-downs on Ni-NTA to obtain tagged substrate 4 with its interaction partners. The presence of tagged proteins in the eluates could be confirmed. To identify interacting proteins, mass spectrometry analysis was performed. In further experiments, DTT and TMAD were used to alter the redox conditions in the cells before lysis and purification. The evaluation of the data included the comparison of the proteins identified in different experiments and the comparison with potential interactors of Erv.5,7 Also, properties of Mia40 that might be conserved were considered. Two characteristic motifs of known Mia40 orthologs are a CPC and a twin CX9C motif. Thus, proteins with these or similar motifs were specifically searched for. Different candidates for Mia40 orthologs were identified and discussed.
Pyrrolizidine alkaloids are naturally occurring secondary plant metabolites mainly found in plant families of Asteraceae, Boraginaceae, and Fabaceae. Chemically, PAs consist of a pyrrolizidine core bearing hydroxyl groups, the so-called necine base, and mono- or dicarboxylic necine acids bound to the pyrrolizidine core via ester linkages. 1,2-unsaturated PAs are hepatotoxic, genotoxic, and carcinogenic due to the highly reactive pyrrolic metabolites formed by cytochrome P450 monooxygenases (CYPs) primarily in the liver. The presence of PAs as frequent contaminants in the wide variety of food and feed products would be a concern for public health.
Due to the inadequate data, the risk assessment of PAs was mainly approached using the two most toxic potent congeners, i.e., lasiocarpine and riddelliine. However, the toxic potencies of individual PA congeners differentiated widely between the congeners probably related to their structural features. The risk of PA-containing products is indeed overestimated, and a comprehensive risk assessment should take these differences into account.
After analyzing the data of many PAs, Merz and Schrenk derived interim Relative Potency (iREP) factors to present the differences in their toxicity between the sub-groups concerning their structural features. But since this concept was derived from an inadequate database, it was found that the relative toxicity of individual congeners cannot be entirely reliably evaluated. My work aimed to achieve more comprehensive congener-specific in vitro toxicological data and estimate the structure-related characteristics for refining this concept. For this purpose, ten congeners, lasiocarpine, monocrotaline, retrorsine, senecionine, seneciphylline, echimidine, europine, heliotrine, indicine, and lycopsamine, were determined in a series of in vitro test systems with different endpoints to quantify their cytotoxicity, genotoxicity, and mutagenicity.
Cytotoxicity was assessed using the Alamar blue assay. A clear structure dependence could be demonstrated in primary rat hepatocytes and HepG2 (CYP3A4) cells. On the contrary, in HepG2 cells, none of the selected PAs exhibited cytotoxic effects, probably due to the lack of CYPs. The role of CYP450 enzymes in metabolic activation was further confirmed using an inhibition assay and the activity of CYP450 enzymes was measured by a kinetic assay analyzing 7-benzyloxyresorufin-O-dealkylation (BROD). Furthermore, utilizing a glutathione-reductase-DTNB recycling assay indicated that glutathione might not play a critical role in PA-induced cytotoxicity. A micronucleus test was used for determining the PA-induced clastogenic genotoxicity. All selected PA congeners exhibited a concentration-dependent manner in the HepG2 (CYP3A4) cells. The relative potencies of PA congeners estimated from Alamar blue assay and micronucleus assay are generally consistent with the following ranking: lasiocarpine > senecionine > seneciphylline ≥ retrorsine > heliotrine (?) echimidine ≥ europine ≈ indicine ≈ lycopsamine ≈ monocrotaline. Compared to the iREP reported by Merz and Schrenk, monocrotaline exhibited considerably lower toxic potency. However, echimidine was more toxic than expected. On the other hand, mutagenicity was measured in Ames fluctuation assay with Salmonella typhimurium strains TA98 and TA100. None of the selected PA congeners up to 300 µM showed mutagenic effects despite metabolic activation with S9-mix.
This thesis reports about the investigation of di- and trinuclear coinage metal (Cu, Ag, Au) phosphine complexes with different anion adducts. Several mass spectrometric methods were utilized to investigate the complexes in gas phase without disturbing influences e.g. by the solvent. Electrospray Ionization (ESI) enabled the transfer of ions into the gas phase. In order to determine the fragmentation pathways and relative gas phase stabilities of these ions, Collision Induced Dissociation (CID) was used. The binding motifs and structures of the complexes were assigned by the help of Infrared (Multiple) Photon Dissociation (IR-(M)PD) at cryo (40 K, N2-tagged) and room temperature (300 K). Electron Transfer Dissociation/Reduction (ETD/R) was used to reduce the dicationic complexes to monocationic complexes. A tunable OPO/OPA laser system and the FELIX free-electron laser were used as IR laser sources. All experimental findings were supported by Density Functional Theory (DFT) calculation. In the first part of this thesis, the binding motifs and fragmentation behavior of the dinuclear coinage metal phosphine complexes with formate adduct were determined. Two different binding motifs were found and a stronger Cu-formate binding than in the case of Ag-formate. The dynamic bonding of hydrogen oxalate to phosphine ligand stabilized complexes were investigated in the second part. Several different binding motifs were determined. IR induced isomeric interconversions were found for the Ag complex whereas in case of the Cu complex a stiff hydrogen oxalate coordination seems to suppress such conversions. In the last part of this thesis, the ETD/R method was utilized to unravel the influence of oxidation states on the hydride and deuteride vibration modes of the trinuclear coinage metal complexes as well as the O2 adduct complexes and fragments with less complexity via IR-MPD and the FELIX free-electron laser. Unfortunately, an unambiguous assignment for the hydride and deuteride vibration modes is only possible for the fragments with less complexity.
In dieser Arbeit wurden photoaktive Übergangsmetallkomplexe mit häufig vorkommenden Metallen wie Chrom, Vanadium und Kupfer untersucht. Hierbei wurden ausgewählte Exemplare mit besonders interessanten photophysikalischen und photochemischen Eigenschaften in Bezug auf praktische Anwendungen spektroskopisch charakterisiert. Über statische und insbesondere zeitaufgelöste FTIR- und Lumineszenzspektroskopie wurde ein tieferes Verständnis der Dynamik nach Lichtanregung erzielt. Das Hauptziel dieser Forschung besteht darin seltene und teure Elemente wie Ruthenium und Iridium gegen häufigere Metalle zu ersetzen.
In diesem Zusammenhang wurden mononukleare, oktaedrische Chrom(III)- und Vanadium(III)-Komplexe mit Polypyridylliganden, die im Arbeitskreis von Prof. Dr. Katja Heinze synthetisiert wurden, spektroskopisch charakterisiert. Diese Systeme zeigen vielversprechende Lumineszenzeigenschaften mit einer roten bzw. nahinfraroten Phosphoreszenz, wobei bei tiefen Temperaturen besonders hohe Quantenausbeuten und längere Lebensdauern beobachtet werden konnten.
Außerdem wurden einkernige Chrom(0)-, Molybdän(0)- und Wolfram(0)-Komplexe spektroskopisch charakterisiert, die allesamt im Arbeitskreis von Prof. Dr. Biprajit Sarkar synthetisiert wurden. Es sind mononukleare Komplexe mit Pyridyl-Carben-Liganden und Carbonyl-Coliganden mit einer dualen Phosphoreszenz (Emissionsbande im roten und nahinfraroten Bereich), wobei sich die niederenergetische Bande interessanterweise bis 1300 nm erstreckt. Außerdem zeigen die drei Komplexe bei intensiver Bestrahlung mit sichtbarem oder UV-Licht in organischer Lösung eine photochemische Reaktivität.
Als weitere vielversprechende Luminophore (sichtbare Emission) wurden Kupfer(I)-Komplexe analysiert, die für organische Leuchtdioden relevant sind. Einerseits wurden zweikernige Systeme mit einer zentralen Cu2I2-Einheit untersucht, die sich durch eine Fluorierung an den Phosphin-Hilfsliganden von den Derivaten aus Vorarbeiten unterscheiden. Die Systeme wurden im Arbeitskreis von Prof. Dr. Stefan Bräse zur Verbesserung der Löslichkeit im Vergleich zu unfluorierten Derivaten entwickelt. Die spektroskopischen Befunde dieser Arbeit zeigen, dass insbesondere die Einführung von Trifluormethylgruppen nicht nur die Löslichkeit, sondern auch die Stabilität verbessert. Andererseits wurden vierkernige Komplexe mit näherungsweise oktaedrischen Cu4X4-Clustern (X = I, Br, Cl) charakterisiert, wobei sich teilweise eine stark thermochrome Lumineszenz mit zwei klar separierten roten bzw. blauen Phosphoreszenzbanden ergab. Der Ursprung dieser Thermochromie konnte erstmalig auf experimentellem Weg den starken strukturellen Veränderung innerhalb des Cu4X4-Clusters zugeordnet werden.
Außerdem sind Kupfer(I)-Komplexe vielversprechende Kandidaten zur Verwendung als Photosensibilisatoren. Bei einem vom Arbeitskreis von Dr. Michael Karnahl zu Verfügung gestellten Kupfer(I)-Einkerner mit einem Liganden mit ausgedehntem 𝜋-System ergab sich ein langlebiger, nicht-strahlender Triplett-Zustand. In einem verwandten Projekt wurden ein- und zweikernige Kupfer(I)-Komplexe untersucht, die im Arbeitskreis von Dr. Claudia Bizzarri synthetisiert wurden. Der Fokus lag hierbei auf dem Einfluss einer Dimerisierung (kovalente Verbindung zweier mononuklearer Komplexe) oder einer Protonierung eines Liganden auf die photophysikalischen Eigenschaften.
Metal-directed self-assembly with metals such as Pd(II) and Pt(II), that prefer a square
planar coordination geometry, has been showing remarkable potential to construct
supramolecular architectures such as helices, tubes, locks and cages. Some of these
complexes have also been shown to assemble by means of metal-metal interactions and,
more remarkably, the luminescent properties of certain complexes can also be used for
sensing. For instance, Pt(II) and Pd(II) complexes display strong phosphorescence which
is strongly reduced in the presence of oxygen. The work developed for this thesis is
divided into three main chapters dealing with the different properties of Pd(II) and Pt(II)
metal complexes.
Chapter I was mainly developed at Technische Universität Kaiserslautern with a short
partnership with the Kekulé-Institut für Organische Chemie und Biochemie at the
Universität Bonn and addressed the construction of coordination macrocycles and cages
from suitable Pd(II) sources and cyclopeptide-derived ligands. Cyclopeptide derived
hollow coordination compounds were obtained through Pd(II)-directed self-assembly.
Specifically, the treatment of the pyridine containing cyclopeptides CP1 and CP2 with
[Pd(en)(NO3)2] afforded the metallamacrocycle CP12Pd2 and the cage CP22Pd3. These
products were characterized by means of NMR spectroscopy and mass spectrometry. The
reaction between CP1 and [Pd(CH3CN)4](BF4)2] afforded, according to ESI-MS and NMR
measurements, a complex with the composition CP16Pd3 and the smaller cage CP14Pd2.
Binding studies indicated that CP12Pd2 incorporated different dicarboxylates, sodium 1,3-
benzenedisulfonate (BDS), and sodium 2,6-naphthalenedisulfonate (NDS) into its cavity.
In the case of BDS a 1:1 complex was formed that had a log Ka of 4.8 ± 0.2 in D2O/CD3OD,
1:1 (v/v). In the case of NDS, binding was slow on the NMR time-scale and involved the
binding of two guest molecules as confirmed by a crystal structure of the complex.
Based on these first examples of Pd(II)-containing cyclopeptide-derived coordination
compounds, future work should focus on the design of molecular architectures that can,
for example, be used as receptors for biorelevant guests.
In Chapter II, the aggregation abilities and photophysical properties of Pt(II) complexes
bearing tridentate-azolate-based ligands and cyclopeptides with peripheral pyridyl
moieties were investigated. This project was the result of a short-term secondment
developed at the Institut de Science et d'Ingénierie Supramoléculaires in Strasbourg.
Efforts were made at creating luminescent cyclopeptide-derived Pt(II) complexes by
coordinating CP1 or CP2 to suitable Pt(II)-containing precursors. The coordination of both
peptides to a known Pt(II) complex afforded insoluble products that could not be
characterized further. To circumvent these solubility issues, the synthesis of the
analogous cyclopeptide complexes containing more polar ligands was attempted.
Although mass spectrometry provided evidence for the formation of the target
complexes PtDeg-CP1 and PtDeg-CP2 in the crude reaction mixtures, the
products could not be isolated in pure form. The impure samples of PtDeg-CP1 and PtDegCP2 both exhibited orange emission.
Further work is necessary to improve the preparation of such complexes. Only then can
the characterization of their photophysical properties and self-assembling behavior be
addressed.
Chapter III was the result of a project executed at Micronit Micro Technologies B.V. in
Enschede, in which a microfluidic device with oxygen sensing abilities was produced from
nanoparticles containing Pt(II)-porphyrins. To this end, microfluidic devices containing
the Pt(II) complex PtTPTBPF incorporated in different polymeric matrices were
prepared and their oxygen sensing abilities characterized. It was shown that chips
containing the Pt(II) complex incorporated into OXNANO nanoparticles were highly
sensitive to oxygen, easy to fabricate, and allowed reliable oxygen quantification. Chips
made by using other polymeric matrices such as PDMS, Elastosil®E43 or polystyrene were
less suitable for the measurements.
The OXNANO-containing chips furthermore allowed measuring the oxygen consumption
of HUVEC cells in a biological assay even in repeated measurements. Future studies
should now involve using these chips for monitoring in real time small scale biological
processes.
The present thesis investigates the interaction of size selected transition metal cluster cations with \(N_2\) adsorbate molecules under cryogenic conditions. A tandem cryo ion trap mass spectrometer facilitates the recording of the adsorption kinetics of gases onto size selected transition metals clusters under isothermal cryo conditions. The combination with a tunable OPO/OPA laser system allows for the recording of Infrared-(Multiple) Photodissociation (IR-(M)PD) spectra of the resulting cluster adsorbate complexes. The comparison of the experimental results with DFT modelling allows for structural assignments and the evaluation of the clusters’ electronic properties. We apply the combination of these methods on rhodium, rhodium-iron alloy and tantalum clusters and present the results in seven research studies. The IR-(M)PD spectra of rhodium cluster adsorbate complexes indicate co-existing isomers and a spin quench with increasing number of adsorbed \(N_2\). Adsorption kinetics allowed to assign clusters with rough and smooth surfaces and to find features of adsorption reluctance. Calculated spin valley curves reveal a spin quench upon \(N_2\) adsorption onto the \(Rh_5^+\) cluster. Band shifts in the IR-(M)PD spectra of sequential \(N_2\) adsorption onto the \(Rh_6^+\) cluster are interpreted in terms of adsorbate induced charge dilution, which is supported by DFT calculations. Our combined approach reveals alloy like mixed rhodium iron clusters with adsorption site dependent \(N_2\) stretching vibrations, where \(N_2\) preferentially adsorbs onto rhodium sites. The \(Ta_4^+\) cluster facilitates the cleavage of the initial two \(N_2\) adsorbate molecules. By help of IR-(M)PD spectroscopy and DFT modelling we are able to assign a novel AEAS (across edge-above surface) mechanism and to assign an activated side-on intermediate of the third adsorbate molecule. The investigation sequential \(N_2\) adsorption onto the \(Ta_5^+\) cluster does not reveal any evidence for \(N_2\) activation. DFT modelling reveals low spin states.
With a yearly production of about 39 million tons, brewer’s spent grain (BSG) is the
most abundant brewing industry byproduct. Because it is rich in fiber and protein, it is commonly
used as cattle feed but could also be used within the human diet. Additionally, it contains many
bioactive substances such as hydroxycinnamic acids that are known to be antioxidants and potent
inhibitors of enzymes of glucose metabolism. Therefore, our study aim was to prepare different
extracts—A1-A7 (solid-liquid extraction with 60% acetone); HE1-HE6 (alkaline hydrolysis followed
by ethyl acetate extraction) and HA1-HA3 (60% acetone extraction of alkaline residue)—from various
BSGs which were characterized for their total phenolic (TPC) and total flavonoid (TFC) contents,
before conducting in vitro studies on their effects on the glucose metabolism enzymes α-amylase,
α-glucosidase, dipeptidyl peptidase IV (DPP IV), and glycogen phosphorylase α (GPα). Depending
on the extraction procedures, TPCs ranged from 20–350 μg gallic acid equivalents/mg extract
and TFCs were as high as 94 μg catechin equivalents/mg extract. Strong inhibition of glucose
metabolism enzymes was also observed: the IC50 values for α-glucosidase inhibition ranged from
67.4 ± 8.1 μg/mL to 268.1 ± 29.4 μg/mL, for DPP IV inhibition they ranged from 290.6 ± 97.4 to
778.4 ± 95.5 μg/mL and for GPα enzyme inhibition from 12.6 ± 1.1 to 261 ± 6 μg/mL. However, the
extracts did not strongly inhibit α-amylase. In general, the A extracts from solid-liquid extraction
with 60% acetone showed stronger inhibitory potential towards α-glucosidase and GPα than other
extracts whereby no correlation with TPC or TFC were observed. Additionally, DPP IV was mainly
inhibited by HE extracts but the effect was not of biological relevance. Our results show that BSG
is a potent source of α-glucosidase and GPα inhibitors, but further research is needed to identify
these bioactive compounds within BSG extracts focusing on extracts from solid-liquid extraction
with 60% acetone.
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.
The consumption of red meat is associated with an increased risk for colorectal cancer (CRC). Multiple lines of evidence suggest
that heme iron as abundant constituent of red meat is responsible for its carcinogenic potential. However, the underlying
mechanisms are not fully understood and particularly the role of intestinal inflammation has not been investigated. To address
this important issue, we analyzed the impact of heme iron (0.25 μmol/g diet) on the intestinal microbiota, gut inflammation
and colorectal tumor formation in mice. An iron-balanced diet with ferric citrate (0.25 μmol/g diet) was used as reference.
16S rRNA sequencing revealed that dietary heme reduced α-diversity and caused a persistent intestinal dysbiosis, with a
continuous increase in gram-negative Proteobacteria. This was linked to chronic gut inflammation and hyperproliferation of
the intestinal epithelium as attested by mini-endoscopy, histopathology and immunohistochemistry. Dietary heme triggered
the infiltration of myeloid cells into colorectal mucosa with an increased level of COX-2 positive cells. Furthermore, flow
cytometry-based phenotyping demonstrated an increased number of T cells and B cells in the lamina propria following heme
intake, while γδ-T cells were reduced in the intraepithelial compartment. Dietary heme iron catalyzed formation of fecal
N-nitroso compounds and was genotoxic in intestinal epithelial cells, yet suppressed intestinal apoptosis as evidenced by
confocal microscopy and western blot analysis. Finally, a chemically induced CRC mouse model showed persistent intestinal
dysbiosis, chronic gut inflammation and increased colorectal tumorigenesis following heme iron intake. Altogether, this study
unveiled intestinal inflammation as important driver in heme iron-associated colorectal carcinogenesis
The consumption of red meat is probably carcinogenic to humans and is associated with an increased risk to develop colorectal cancer (CRC). Red meat contains high amounts of heme iron, which is thought to play a causal role in tumor formation. In this study, we investigated the genotoxic and cytotoxic effects of heme iron (i.e., hemin) versus inorganic iron in human colonic epithelial cells (HCEC), human CRC cell lines and murine intestinal organoids. Hemin catalyzed the formation of reactive oxygen species (ROS) and induced oxidative DNA damage as well as DNA strand breaks in both HCEC and CRC cells. In contrast, inorganic iron hardly affected ROS levels and only slightly increased DNA damage. Hemin, but not inorganic iron, caused cell death and reduced cell viability. This occurred preferentially in
non-malignant HCEC, which was corroborated in intestinal organoids. Both hemin and inorganic iron were taken up into HCEC and CRC cells, however with differential kinetics and efficiency. Hemin caused stabilization and nuclear translocation of Nrf2, which induced heme oxygenase-1 (HO-1) and ferritin heavy chain (FtH). This was not observed after inorganic iron treatment. Chemical inhibition or genetic knockdown of HO-1 potentiated hemin-triggered ROS
generation and oxidative DNA damage preferentially in HCEC. Furthermore, HO-1 abrogation strongly augmented the cytotoxic effects of hemin in HCEC, revealing its pivotal function in colonocytes and highlighting the toxicity of free intracellular heme iron. Taken together, this study demonstrated that hemin, but not inorganic iron, induces ROS and DNA damage, resulting in a preferential cytotoxicity in non-malignant intestinal epithelial cells. Importantly, HO-1
conferred protection against the detrimental effects of hemin.
Abstract: Colorectal cancer (CRC) is a frequently occurring malignant disease with still low survival rates, highlighting the need for novel therapeutics. Merosesquiterpenes are secondary metabolites from marine sponges, which might be useful as antitumor agents. To address this issue, we made use of a compound library comprising 11 isolated merosesquiterpenes. The most cytotoxic compounds were smenospongine > ilimaquinone ≈ dactylospontriol as shown in different human CRC cell lines. Alkaline Comet assays and γH2AX immunofluorescence microscopy demonstrated DNA strand break formation in CRC cells. Western blot analysis revealed an activation of the DNA damage response with CHK1 phosphorylation, stabilization of p53 and p21, which occurred both in CRC cells with p53 knockout and in p53-mutated CRC cells. This resulted in cell cycle arrest followed by a strong increase in the subG1 population, indicative of apoptosis, and typical morphological alterations. In consistency, cell death measurements showed apoptosis following exposure to merosesquiterpenes. Gene expression studies and analysis of caspase cleavage revealed mitochondrial apoptosis via BAX, BIM and caspase-9 as main cell death pathway. Interestingly, the compounds were equally effective in p53-wildtype and p53-mutant CRC cells. Finally, the cytotoxic activity of the merosesquiterpenes was corroborated in intestinal tumor organoids, emphasizing their potential for CRC chemotherapy.
Subject of this thesis is the investigation of photo-induced ultrafast elementary processes within the electronic excited-state manifold in mass-selected multinuclear coinage metal complexes (CMCs) in the gas phase. The chief objective is to ascertain how the intramolecular deactivation dynamics and gas phase reactivity are influenced by so-called metallophilic interactions between multiple d^10/d^8 metal centers, which in turn give rise to metal-delocalized electronic transitions. To this end, suitable molecular precursor ions were transferred into the gas phase by electrospray ionization (ESI) and subsequently isolated, activated, and finally analyzed in a Paul-type quadrupole ion trap (QIT) mass spectrometer. The QIT is modified to accept UV/Vis/NIR irradiation from a femtosecond laser setup. By combining several ion trap-based ion activation techniques and electronic photodissociation (PD) spectroscopy, the fragmentation pathways, as well as intrinsic optical properties (electronic PD spectra) of ionic CMCs are explored. In addition, the unconventional time-resolved transient photodissociation (t-PD) method, based on a femtosecond pump-probe excitation scheme, was employed for the first time on CMC ions in isolation, in order to elucidate their intrinsic photodynamics. This thesis mainly comprises five publications, covering the mass spectrometric and laser spectroscopic characterization of multinuclear Ag(I), Au(I), and Pt(II) based ionic complexes.
We have investigated urine samples after coffee consumption using targeted and untargeted
approaches to identify furan and 2-methylfuran metabolites in urine samples by UPLC-qToF.
The aim was to establish a fast, robust, and time-saving method involving ultra-performance
liquid chromatography-quantitative time-of-flight tandem mass spectrometry (UPLC-qToF-MS/MS).
The developed method detected previously reported metabolites, such as Lys-BDA, and others that
had not been previously identified, or only detected in animal or in vitro studies. The developed
UPLC-qToF method detected previously reported metabolites, such as lysine-cis-2-butene-1,4-dial
(Lys-BDA) adducts, and others that had not been previously identified, or only detected in animal
and in vitro studies. In sum, the UPLC-qToF approach provides additional information that may be
valuable in future human or animal intervention studies.
Phosphodiesterases (PDEs) are essential enzymes for the regulation of pathways mediated
by cyclic adenosine monophosphate (cAMP). Secondary plant compounds like anthocyanins (ACs)
can inhibit PDE activity and, consequently, may be beneficial for lipid metabolism. This study
investigated 18 AC-rich juice extracts and pure reference compounds from red fruits for potential
inhibitory effects on PDE 3B activity. Extracts were obtained through adsorption on Amberlite® XAD
7 resin. Based on this screening, the chokeberry, blueberry, pomegranate, and cranberry extracts
were active, with half maximal inhibitory concentrations (IC50) ranging from 163 ± 3 µg/mL to
180 ± 3 µg/mL. The ACs in these extracts, peonidin-3-glucoside and cyanidin-3-arabinoside, were the
most active single compounds (IC50 = 56 ± 20 µg/mL, 108 ± 6 µg/mL). All extracts comprised high
amounts of phenolic compounds, as determined by the Folin–Ciocalteu assay, ranging from 39.8 ± 1.5
to 73.5 ± 4.8 g gallic acid equivalents (GAE)/100 g extract. Pomegranate and chokeberry extracts
exhibited the largest amounts of polyphenols (72.3 ± 0.7 g GAE/100 g, 70.6 ± 4.1 g GAE/100 g,
respectively). Overall, our results showed that fruit juice extracts and their ACs can inhibit PDE
activity. Any potential health benefits in vivo will be investigated in the future.
Red fruits and their juices are rich sources of polyphenols, especially anthocyanins.
Some studies have shown that such polyphenols can inhibit enzymes of the carbohydrate metabolism,
such as α-amylase and α-glucosidase, that indirectly regulate blood sugar levels. The presented
study examined the in vitro inhibitory activity against α-amylase and α-glucosidase of various
phenolic extracts prepared from direct juices, concentrates, and purees of nine different berries which
differ in their anthocyanin and copigment profile. Generally, the extracts with the highest phenolic
content—aronia (67.7 ± 3.2 g GAE/100 g; cyanidin 3-galactoside; chlorogenic acid), pomegranate
(65.7 ± 7.9 g GAE/100 g; cyanidin 3,5-diglucoside; punicalin), and red grape (59.6 ± 2.5 g GAE/100 g;
malvidin 3-glucoside; quercetin 3-glucuronide)—showed also one of the highest inhibitory activities
against α-amylase (326.9 ± 75.8 µg/mL; 789.7 ± 220.9 µg/mL; 646.1 ± 81.8 µg/mL) and α-glucosidase
(115.6 ± 32.5 µg/mL; 127.8 ± 20.1 µg/mL; 160.6 ± 68.4 µg/mL) and, partially, were even more potent
inhibitors than acarbose (441 ± 30 µg/mL; 1439 ± 85 µg/mL). Additionally, the investigation of single
anthocyanins and glycosylated flavonoids demonstrated a structure- and size-dependent inhibitory
activity. In the future in vivo studies are envisaged.
Winery by-products arise in high amounts during winemaking processes. Hence, recovery alternatives are of great interest. In this study, effects of extracts from winery by-products (Vitis vinifera L. cv. Riesling) on mitochondrial functions in human hepatocellular carcinoma (HepG2) cells were examined. Polyphenolic profiles of pomace (PE), stem (SE), vine leaf (VLE), and vine shoot extracts (VSE) were characterized by HPLC-UV/Vis-ESI-MS/MS. The extracts induced dose-dependent cytotoxic effects (PE > SE > VLE > VSE). VSE showed protective effects regarding modulation of tert-butyl hydroperoxide (TBH)-induced intracellular reactive oxygen species (ROS) levels. PE, SE and VLE increased the mitochondrial membrane potential (MMP), whereas VSE decreased it owing to mildly impaired mitochondrial respiration. Cells may try to compensate reduced respiration chain complex activities by increasing the mitochondrial mass, as indicated by enhanced citrate synthase activity and mRNA expression levels after VSE incubation. Thus, winery by-products represent interesting sources of bioactive compounds that exert positive or negative effects on mitochondrial functions.
Spin-crossover and valence tautomeric complexes are of tremendous interest in the field of molecular electronics, electronic storage devices and information processing. Herein, synthesis and characterization of the spin-crossover and valence tautomeric cobalt dioxolene complexes are reported. All the synthesized complexes contain N,N'-di-tert-butyl-2,11-diaza[3.3](2,6)pyridinophane (L-N4tBu2) as ancillary ligands. Only various types of co-ligands which are different dioxolene ligands, have been used. The mononuclear cobalt dioxolene complexes have been synthesized by using dideprotonated form of the dioxolene ligand 4,5-dichlorocatechol (H2DCCat) as co-ligands, and the cobalt bis(dioxolene) complexes have been synthesized by using dideprotonated form of the 3,3'-dihydroxy-diphenoquinone-(4,4') (H2(SQ-SQ)) as co-ligands.
Analytically pure samples of the complexes [Co(L-N4tBu2)(DCCat)] (1), [Co(L-N4tBu2)(DCCat)](BPh4) (2b), [Co2(L-N4tBu2)2(SQ-SQ)](BPh4)2.4 DMF (3b), [Co2(L-N4tBu2)2(Cat-SQ)](BF4)2.Et2O (3d), have been synthesized and characterized by X-ray crystallography, magnetic and electrochemical measurements. The complexes have been investigated by UV/Vis/NIR-, IR-, and NMR spectroscopic measurements.
The complex [Co(L-N4tBu2)(DCCat)] (1) shows temperature invariant high-spin cobalt(II) catecholate state. One-electron oxidation of 1 has yielded the complex [Co(L-N4tBu2)(DCCat)](BPh4) (2b). The solid state properties of 2b are best described by the low-spin cobalt(III) catecholate state, but the solution state properties of the complex 2b are best described by the valence tautomeric transition from the low-spin cobalt(III) catecholate to the low-spin cobalt(II) semiquinonate state.
For the cobalt bis(dioxolene) complexes, it is found that spin-crossover for the two cobalt(II) centers is accompanied by the electronic state changes of the coordinated bis(dioxolene) unit from singlet open-shell biradicaloid to singlet closed-shell quinonoid form in complex 3b. Approaching similar synthetic method to 3b, but performing the metathesis reaction with sodium tetrafluoroborate rather than sodium tetraphenylborate has resulted in the formation of the complex [Co2(L-N4tBu2)2(Cat-SQ)](BF4)2.Et2O (3d). The solid state properties of the complex are best described by the temperature induced valence tautomeric transition for the low-spin cobalt(III) center which is accompanied by the spin-crossover process for the cobalt(II) center. Thus, the electronic state of the complex 3d changes from LS-CoIII-Cat-SQ-CoII-LS to HS-CoII-(SQ-SQ)CS-CoII-HS state upon change in temperature.
Temperature-induced electronic configuration changes of the (SQ-SQ)CS2- ligands from open-shell biradicaloid to closed-shell quinonoid configurations are not observed for the nickel-, copper- and zinc bis(dioxolene) complexes 4a, 5a and 6b, respectively. For these complexes, the metal ions are bridged by (SQ-SQ)CS2- ligand and the paramagnetic metal ions are very weakly antiferromagnetically coupled.
Wine and alcoholic fermentations are complex and fascinating ecosystems. Wine aroma is shaped by the wine’s chemical compositions, in which both microbes and grape constituents play crucial roles. Activities of the microbial community impact the sensory properties of the final product, therefore, the characterisation of microbial diversity is essential in understanding and predicting sensory properties of wine. Characterisation has been challenging with traditional approaches, where microbes are isolated and therefore analyzed outside from their natural environment. This causes a bias in the observed microbial composition structure. In addition, true community interactions cannot be studied using isolates. Furthermore, the multiplex ties between wine chemical and sensory compositions remain evasive due to their multivariate and nonlinear nature. Therefore, the sensorial outcome arising from different microbial communities has remained inconclusive.
In this thesis, microbial diversity during Riesling wine fermentations is investigated with the aim to understand the roles of microbial communities during fermentations and their links to sensory properties. With the advancement of high-throughput tools based ‘omic methods, such as next-generation sequencing (NGS) technologies, it is now possible to study microbial communities and their functions without isolation by culturing. This developing field and its potential to wine community is reviewed in Chapter 1. The standardisation of methods remains challenging in the field. DNA extraction is a key step in capturing the microbial diversity in samples for generating NGS data, therefore, DNA extraction methods are evaluated in Chapter 2. In Chapter 3, machine learning is utilized in guiding raw data mining generated by the untargeted GC-MS analysis. This step is crucial in order to take full advantages of the large scope of data generated by ‘omic methods. These lay a solid foundation for Chapters 4 and 5 where microbial community structures and their outputs - chemical and sensory compositions are studied by using approaches and tools based on multiple ‘omics methods.
The results of this thesis show first that by using novel statistical approaches, it is possible to extract meaningful information from heterogeneous biological, chemical and sensorial data. Secondly, results suggest that the variation in wine aroma, might be related
to microbial interactions taking place not only inside a single community, but also the
IV
interactions between communities, such as vineyard and winery communities. Therefore, the true sensory expression of terroir might be masked by the interaction between two microbial communities, although more work is needed to uncover this potential relationship. Such potential interaction mechanisms were uncovered between non- Saccharomyces yeast and bacteria in this work and unexpected novel bacterial growth was observed during alcohol fermentation. This suggests new layers in understanding of wine fermentations. In the future, multi-omic approaches could be applied to identify biological pathways leading to specific wine aroma as well as investigate the effects upon specific winemaking conditions. These results are relevant not just for the wine industry, but also to other industries where complex microbial networks are important. As such, the approaches presented in this thesis might find widely use in the food industry.
Carotenoids are organic lipophilic tetraterpenes ubiquitously present in Nature and found across the three domains of life (Archaea, Bacteria and Eukaryotes). Their structure is characterized by an extensive conjugated double-bond system, which serves as a light-absorbing chromophore, hence determining its colour, and enables carotenoids to absorb energy from other molecules and to act as antioxidant agents. Humans obtain carotenoids mainly via the consumption of fruits and vegetables, and to a smaller extent from other food sources such as fish and eggs. The concentration of carotenoids in the human plasma and tissues has been positively associated with a lower incidence of several chronic diseases including, cancer, diabetes, macular degeneration and cardiovascular conditions, likely due to their antioxidant properties. However, an important aspect of carotenoids, namely β- and α-carotene and β-cryptoxanthin, in human health and development, is their potential to be converted by the body into Vitamin A.
Yet, bioavailability of carotenoids is relatively low (< 30%) and dependent, among others, on dietary factors, such as amount and type of dietary lipids and the presence of dietary fibres. One dietary factor that has been found to negatively impact carotenoid bioaccessibility and cellular uptake in vitro is high concentrations of divalent cations during simulated gastro-intestinal digestion. Nevertheless, the mechanism of action of divalent cations remains unclear. The goal of this thesis was to better understand how divalent cations act during digestion and modulate carotenoid bioavailability. In vitro trials of simulated gastro-intestinal digestion and cellular uptake were run to investigate how varying concentrations of calcium, magnesium and zinc affected the bioaccessibility of both pure carotenoids and carotenoids from food matrices. In order to validate or refute results obtained in vitro, a randomized and double blinded placebo controlled cross-over postprandial trial (24 male participants) was carried out, testing the effect of 3 supplementary calcium doses (0 mg, 500 mg and 1000 mg) on the bioavailability of carotenoids from a spinach based meal. In vitro trials showed that addition of the divalent cations significantly decreased the bioaccessibility of both pure carotenoids (P < 0.001) and those from food matrices (P < 0.01). This effect was dependent on the type of mineral and its concentration. Strongest effects were seen for increasing concentrations of calcium followed by magnesium and zinc. The addition of divalent cations also altered the physico-chemical properties, i.e. viscosity and surface tension, of the digestas. However, the extent of this effect varied according to the type of matrix. The effects on bioaccessibility and physico-chemical properties were accompanied by variations of the zeta-potential of the particles in solution. Taken together, results from the in vitro trials strongly suggested that divalent cations were able to bind bile salts and other surfactant agents, affecting their solubility. The observed i) decrease in macroviscosity, ii) increase in surface tension, and the iii) reduction of the zeta-potential of the digesta, confirmed the removal of surfactant agents from the system, most likely due to precipitation as a result of the lower solubility of the mineral-surfactant complexes. As such, micellarization of carotenoids was hindered, explaining their reduced bioaccessibility. As for the human trial, results showed that there was no significant influence of supplementation with either 500 or 1000 mg of supplemental calcium (in form of carbonate) on the bioavailability of a spinach based meal, as measured by the area-under curve of carotenoid concentrations in the plasma-triacylglycerol rich fraction, suggesting that the in vitro results are not supported in such an in vivo scenario, which may be explained by the initial low bioaccessibility of spinach carotenoids and the dissolution kinetics of the calcium pills. Further investigations are necessary to understand how divalent cations act during in vivo digestion and potentially interact with lipophilic nutrients and food constituents.
The gas phase infrared and fragmentation spectra of a systematic group of trimetallic oxo-centered
transition metal complexes are shown and discussed, with formate and acetate bridging ligands and
pyridine and water as axial ligands.
The stability of the complexes, as predicted by appropriate ab initio simulations, is demonstrated to
agree with collision induced dissociation (CID) measurements.
A broad range of DFT calculations are shown. They are used to simulate the geometry, the bonding
situation, relative stability and flexibility of the discussed complexes, and to specify the observed
trends. These simulations correctly predict the trends in the band splitting of the symmetric and
asymmetric carboxylate stretch modes, but fail to account for anharmonic effects observed specifically
in the mid IR range.
The infrared spectra of the different ligands are introduced in a brief literature review. Their changes
in different environments or different bonding situations are discussed and visualized, especially the
interplay between fundamental-, overtone-, and combination bands, as well as Fermi resonances
between them.
A new variation on the infrared multi photon dissociation (IRMPD) spectroscopy method is proposed
and evaluated. In addition to the commonly considered total fragment yield, the cumulative fragment
yield can be used to plot the wavelength dependent relative abundance of different fragmentation
products. This is shown to include valuable additional information on the excited chromophors, and
their coupling to specific fragmentation channels.
High quality homo- and heterometallic IRMPD spectra of oxo centered carboxylate complexes of
chromium and iron show the impacts of the influencing factors: the metal centers, the bridging ligands,
their carboxylate stretch modes and CH bend modes, and the terminal ligands.
In all four formate spectra, anharmonic effects are necessary to explain the observed spectra:
combination bands of both carboxylate stretch modes and a Fermi resonance of the fundamental of
the CH stretch mode, and a combination band of the asymmetric carboxylate stretch mode with the
CH bend mode of the formate bridging ligand.
For the water adduct species, partial hydrolysis is proposed to account for the changes in the observed
carboxylic stretch modes.
Appropriate experiments are suggested to verify the mode assignments that are not directly explained
by the ab initio calculations, the available experimental results or other means like deuteration
experiments.
Increasing costs due to the rising attrition of drug candidates in late developmental phases alongside post-marketing withdrawal of drugs challenge the pharmaceutical industry to further improve their current preclinical safety assessment strategies. One of the most common reasons for the termination of drug candidates is drug induced hepatotoxicity, which more often than not remains undetected in early developmental stages, thus emphasizing the necessity for improved and more predictive preclinical test systems. One reason for the very limited value of currently applied in vitro test systems for the detection of potential hepatotoxic liabilities is the lack of organotypic and tissue-specific physiology of hepatocytes cultured in ordinary monolayer culture formats.
The thesis at hand primarily deals with the evaluation of both two- and three-dimensional cell culture approaches with respect to their relative ability to predict the hepatotoxic potential of drug candidates in early developmental phases. First, different hepatic cell models, which are routinely used in pharmaceutical industry (primary human hepatocytes as well as the three cell lines HepG2, HepaRG and Upcyte hepatocytes), were investigated in conventional 2D monolayer culture with respect to their ability to detect hepatotoxic effects in simple cytotoxicity studies. Moreover, it could be shown that the global protein expression levels of all cell lines substantially differ from that of primary human hepatocytes, with the least pronounced difference in HepaRG cells.
The introduction of a third dimension through the cultivation of spheroids enables hepatocytes to recapitulate their typical native polarity and furthermore dramatically increases the contact surface of adjacent cells. These differences in cellular architecture have a positive influence on hepatocyte longevity and the expression of drug metabolizing enzymes and transporters, which could be proven via immunofluorescent (IF) staining for at least 14 days in PHH and at least 28 days in HepaRG spheroids, respectively. Additionally, the IF staining of three different phase III transporters (MDR1, MRP2 and BSEP) indicated a bile canalicular network in spheroids of both cell models. A dose-dependent inducibility of important cytochrome P450 isoenzymes in HepaRG spheroids could be shown on the protein level via IF for at least 14 days. CYP inducibility of HepaRG cells cultured in 2D and 3D was compared on the mRNA level for up to 14 days and inducibility was generally lower in 3D compared to 2D under the conditions of this study. In a comparative cytotoxicity study, both PHH and HepaRG spheroids as well as HepaRG monolayers have been treated with five hepatotoxic drugs for up to 14 days and viability was measured at three time points (days 3, 7 and 14). A clear time- and dose-dependent onset of the drug-induced hepatotoxic effects was observable in all conditions tested, indicated by a shift of the respective EC50 value towards lower doses by increasing exposure. The observed effects were most pronounced in PHH spheroids, thus indicating those as the most sensitive cell model in this study. Moreover, HepaRG cells were more sensitive in spheroid culture compared to monolayers, which suggests a potential application of spheroids as long-term test system for the detection of hepatotoxicities with slow onset. Finally, the basal protein expression levels of three antigens (CYP1A2, CYP3A4 and NAT 1/2) were analyzed via Western Blotting in HepaRG cells cultured in three different cell culture formats (2D, 3D and QV) in order to estimate the impact of the cell culture conditions on protein expression levels. In the QV system enables a pump-driven flow of cell culture media, which introduces both mechanical stimuli through shear and molecular stimuli through dynamic circulation to the monolayer. Those stimuli resulted in a clearly positive effect on the expression levels of the selected antigens by an increased expression level in comparison to both 2D and 3D. In contrast, HepaRG spheroids showed time-dependent differences with the overall highest levels at day 7.
The studies presented in this thesis delivered valuable information on the increased physiological relevance in dependence on the cell culture format: three-dimensionality as well as the circulation of media lead to a more differentiated phenotype in hepatic cell models. Those cell culture formats are applicable in preclinical drug development in order to obtain more relevant information at early developmental stages and thus help to create a more efficient drug development process. Nonetheless, further studies are necessary to thoroughly characterize, validate and standardize such novel cell culture approaches prior to their routine application in industry.
The N-containing heterocycles have received strong attention from the organic synthesis field because of their importance for pharmaceutical and material sciences. Nitrogen element plays an important role between inorganic salts and biomolecules, to search convenient methods combine C-N bond together become a hot topic in recent decades.
Since the early beginning of 20th century, transition-metal-catalyzed coupling reactions had been well-known and world widely spread in organic researchs, achieved abundant significant progress. In the other side, the less toxic and more challenging transition metal free coupling method remained further potential value.
With the evolution of amination reactions and oxidants, more and more effective, simplified, and atom economic organic synthesis methods will come soon. And these stories also drove me to think about investigating the novel cross-dehydrogenative-coupling amination methods development as the topics of my PhD research.
Thus, we selected the phenothiazine derivatives as the N-nucleophile reagents and the phenols as the C-nucleophile reagents. To achieve the transition metal-free CDC aminations of phenols with phenothiazines, we scanned the chemical toolbox and tested a series of both common and uncommon oxidants.
Firstly, we start the condition in the presence of cumene and O2. The proposed mechanism initiated by a Hock process, which would form in situ peroxo-species as initiator of the reaction. And the initial infra-red analysis predicted there is a strong O-H..N interaction.
In the second method, a series of iodines with different valance have been tested to achieve the C-N bond formation of phenols with phenothiazines. This time, a simplified and more efficient method had been developed, which also provides a wider scope of phenols. Several controlling experiments had been conducted for the plausible pathway research. Large-scale synthesis of target molecular was also successfully performed.
And then, we focus the research on the cross-coupling reaction of pre-oxidized(iminated) phenothiazine with ubiquitous phenols and indoles. In this task, we first regio-selectively synthesized the novel iminated phenothiazine derivatives with the traditional biocide and mild disinfectant, Chloramine T. Then the phenothiazinimine performed an ultra-simple condensation technique with phenol or indole coupling partners in a simplified condition. Parallel reactions were also performed to investigate the plausible pathway.
Benzene is a natural constituent of crude oil and a product of incomplete combustion of petrol
and has been classified as “carcinogenic to humans” by IARC in 1982 (IARC 1982). (E,E)-
Muconaldehyde has been postulated to be a microsomal metabolite of benzene in vitro
(Latriano et al. 1986). (E,E)-Muconaldehyde is hematotoxic in vivo and its role in the
hematotoxicity of benzene is unclear (Witz et al. 1985).
We intended to ascertain the presence of (E,E)-muconaldehyde in vivo by detection of a
protein conjugate deriving from (E,E)-muconaldehyde.
Therefore we improved the current synthetic access to (E,E)-muconaldehyde. (E,E)-
muconaldehyde was synthesized in three steps starting from with (E,E)-muconic acid in an
overall yield of 60 %.
Reaction of (E,E)-muconaldehyde with bovine serum albumin resulted in formation of a
conjugate which was converted upon addition of NaBH4 to a new species whose HPLC-
retention time, UV spectra, Q1 mass and MS2 spectra matched those of the crude reaction
product from one pot conversion of Ac-Lys-OMe with (E,E)-muconaldehyde in the presence
of NaBH4 and subsequent cleavage of protection groups.
Synthetic access to the presumed structure (S)-2-ammonio-6-(((E,E)-6-oxohexa-2,4-dien-1-
yl)amino)hexanoate (Lys(MUC-CHO)) was provided in eleven steps starting from (E,E)-
muconic acid and Lys(Z)-OtBu*HCl in 2 % overall yield. Additionally synthetic access to
(S)-2-ammonio-6-(((E,E)-6-hydroxyhexa-2,4-dien-1-yl)amino)hexanoate (Lys(MUC-OH))
and (S)-2-ammonio-6-((6-hydroxyhexyl)amino)hexanoate (IS) was provided.
With synthetic reference material at hand, the presumed structure Lys(MUC-OH) could be
identified from incubations of (E,E)-muconaldehyde with bovine serum albumin via HPLC-ESI+-
MS/MS.
Cytotoxicity analysis of (E,E)-muconaldehyde and Lys(MUC-CHO) in human promyelocytic
NB4 cells resulted in EC50 ≈ 1 μM for (E,E)-muconaldehyde. Lys(MUC-CHO) did not show
any additional cytotoxicity up to 10 μM.
B6C3F1 mice were exposed to 0, 400 and 800 mg/kg b.w. benzene to examine the formation
of Lys(MUC-OH) in vivo. After 24 h mice were sacrificed and serum albumin was isolated.
Analysis for Lys(MUC-OH) has not been performed in this work.
1,3-Diynes are frequently found as an important structural motif in natural products, pharmaceuticals and bioactive compounds, electronic and optical materials and supramolecular molecules. Copper and palladium complexes are widely used to prepare 1,3-diynes by homocoupling of terminal alkynes; albeit the potential of nickel complexes towards the same is essentially unexplored. Although a detailed study on the reported nickel-acetylene chemistry has not been carried out, a generalized mechanism featuring a nickel(II)/nickel(0) catalytic cycle has been proposed. In the present work, a detailed mechanistic aspect of the nickel-mediated homocoupling reaction of terminal alkynes is investigated through the isolation and/or characterization of key intermediates from both the stoichiometric and the catalytic reactions. A nickel(II) complex [Ni(L-N4Me2)(MeCN)2](ClO4)2 (1) containing a tetradentate N,N′-dimethyl-2,11-diaza[3.3](2,6)pyridinophane (L-N4Me2) as ligand was used as catalyst for homocoupling of terminal alkynes by employing oxygen as oxidant at room temperature. A series of dinuclear nickel(I) complexes bridged by a 1,3-diyne ligand have been isolated from stoichiometric reaction between [Ni(L-N4Me2)(MeCN)2](ClO4)2 (1) and lithium acetylides. The dinuclear nickel(I)-diyne complexes [{Ni(L-N4Me2)}2(RC4R)](ClO4)2 (2) were well characterized by X-ray crystal structures, various spectroscopic methods, SQUID and DFT calculation. The complexes not only represent as a key intermediate in aforesaid catalytic reaction, but also describe the first structurally characterized dinuclear nickel(I)-diyne complexes. In addition, radical trapping and low temperature UV-Vis-NIR experiments in the formation of the dinuclear nickel(I)-diyne confirm that the reactions occurring during the reduction of nickel(II) to nickel(I) and C-C bond formation of 1,3-diyne follow non-radical concerted mechanism. Furthermore, spectroscopic investigation on the reactivity of the dinuclear nickel(I)-diyne complex towards molecular oxygen confirmed the formation of a mononuclear nickel(I)-diyne species [Ni(L-N4Me2)(RC4R)]+ (4) and a mononuclear nickel(III)-peroxo species [Ni(L-N4Me2)(O2)]+ (5) which were converted to free 1,3-diyne and an unstable dinuclear nickel(II) species [{Ni(L-N4Me2)}2(O2)]2+ (6). A mononuclear nickel(I)-alkyne complex [Ni(L-N4Me2)(PhC2Ph)](ClO4).MeOH (3) and the mononuclear nickel(III)-peroxo species [Ni(L-N4Me2)(O2)]+ (5) were isolated/generated and characterized to confirm the formulation of aforementioned mononuclear nickel(I)-diyne and mononuclear nickel(III)-peroxo species. Spectroscopic experiments on the catalytic reaction mixture also confirm the presence of aforesaid intermediates. Results of both stoichiometric and catalytic reactions suggested an intriguing mechanism involving nickel(II)/nickel(I)/nickel(III) oxidation states in contrast to the reported nickel(II)/nickel(0) catalytic cycle. These findings are expected to open a new paradigm towards nickel-catalyzed organic transformations.
This thesis presents research studies on the fundamental interplay of diatomic molecules with transition metal compounds under cryogenic conditions. The utilized setup offers a multitude of opportunities to study isolated ions: The ions can either be generated by an ElectroSpray Ionization (ESI) source or a Laser VAPorization (LVAP) cluster ion source. The setup facilitates kinetic investigations of the ions with different reaction gases under well-defined isothermal conditions. Moreover it enables cryo InfraRed (Multiple) Photon Dissociation (IR-(M)PD) spectroscopy in combination with tunable OPO/OPA laser systems. In conjunction with density functional theory (DFT) modelling, the IR(M)-PD spectra allow for an assignment of geometric minimum structures. Furthermore DFT modelling helps to identify possible reaction pathways. Altogether the presented methods allow to gain fundamental insights into molecular structures and reactivity of the investigated systems.
The first part of this thesis focuses on the interplay of N2 with different transition metal clusters (Con+, Nin+, and Fen+) by cryo IR spectroscopy and cryo kinetics. In conjunction with DFT modelling the N2 coordination was elucidated (Con+), structures were assigned (Nin+), the concept of structure related surface adsorption behavior was introduced (Nin+), and the a first explanation for the inertness if Fe17+ was given (Fen+). Furthermore this thesis provides for a case study on the coadsorption of H2 and N2 on Ru8+ that elucidates the H migration on the Ru cluster. The last part of the thesis addresses the IR spectra of in vacuo generated [Hemin]+ complexes with N2, O2, and CO. Structures and spin states were assigned with the help of DFT modelling.
Chlorogenic acids (CGA) are phenolic compounds that form during the esterification of certain trans-cinnamic acids with (-)-quinic acid. According to several human intervention studies, they may have potential health benefits. Coffee is the main source of CGA in human nutrition, and is consumed either alone or in combination with a variety of foods. For this reason, the presented study aimed to clarify whether the simultaneous consumption of food, for example, a breakfast rich in carbohydrates, with instant coffee affects the absorption and bioavailability of CGA. The research specifically focused on how various food matrices, which are consumed at the same time as a coffee beverage, will influence kinetic parameters such as area under the curve (AUC), maximum plasma concentration (cmax), and time needed to reach maximum plasma concentration (tmax).
In a randomized crossover study, fourteen healthy participants consumed either pure instant coffee or coffee with a carbohydrate- or fat-rich meal. All of the subjects consumed the same quantity of CGA (3.1 mg CGA/kg body weight). Blood samples, collected at various time points up to 15 h after instant coffee consumption, were quantitatively analysed. Additionally, three urine collection intervals were chosen over a time period of 24h. High performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) was used to determine the CGA present, along with the concentrations of respective metabolites.
During a blind data review meeting, 20 of the 56 analysed plasma metabolites were chosen for further statistical analysis. A total of 36 metabolites were monitored in the urine samples. Similar as in the plasma samples, between-treatment differences, measured through AUC, Cmax, and tmax, of various CGA derived metabolites were to estimate. Each treatment was also analysed in terms of the correlation between the plasma AUC and urinary excretion of seven metabolites.
It is already known that inter-individual variations in CGA absorption depends on gut microbial degradation and affects the efficacy of these compounds. Microorganisms present in the gastrointestinal tract metabolise CGA to form dihydroferulic acid (DHFA) and dihydrocaffeic acid (DHCA) derivatives, which precede the subsequent formation of a wide range of metabolites. Therefore stool samples were collected from the participants within 12 h before the second study day. Subsequent an ex-vivo incubation of faecal samples with 5-O-caffeoylquinic acid (5-CQA), the main chlorogenic acid found in coffee was performed. An HPLC system connected to a CoulArray® detector was used to measure the concentrations of 5-CQA and its metabolites. Reduced concentrations of 5-CQA as well as the appearance of DHCA and caffeic acid (CA) in the gut microbiota medium, were monitored to calculate the inter-individual kinetics for each compound. In addition, these samples were analysed for microbiota content by an external laboratory (L&S, Bad Bocklet, Germany). These results were used to distinguish whether the decreased or increased content of a specific microorganism was related to an individual’s decreased or increased metabolic efficiency. Finally, we used to aforementioned results to evaluate if any correlation could be drawn between the plasma appearance, urinary excretion and ability of microorganisms to degrade 5-CQA.
Strong inter-individual variation was observed for AUC, Cmax and tmax. The AUC measured the quantity of CGA in plasma samples. We noted that pure instant coffee consumption resulted in slightly higher CGA bioavailability than instant coffee with the additional consumption of a meal. However, these differences were not statistically significant. Additionally, the metabolites were divided into groups, according to similarity and chemical properties. They were further classified into three groups according to their physical structure and predicated from the area of appearance: directly from coffee (quinics), after first degradation and metabolism (phenolics, all trans-cinammic acids and their sulfates and glucuronides) as well as colonic degradation and metabolism (colonics, all dihydro compounds). These respective metabolic classes showed significant differences in the AUC values of certain classes yet no significant between-treatment differences. Our results corroborated earlier studies in that the three caffeoylquinic acid (CQA) isomers were absorbed to a lower extent whereas all feruloylquinic acids (FQA) were detected in comparably high amounts in the plasma samples of the volunteers. However, the amount of these quinic acid conjugates in the plasma samples accounted for only 0,5% of the total amount of identified. In contrast, at least 8.7% of the investigated compounds were identified to be phenolics. Dihydro compounds, the so known colonics, were identified as the most common metabolites (90.8%). Additionally, dihydroferulic acid (DHFA), meta-dihydrocoumaric acid (mDHCoA), dihydrocaffeic acid-3-sulfate (DHCA3S) and dihydroisoferulic acid (DHiFA) were identified to account for 78% of the studied metabolites, and thus represent the most abundant compounds circulating in the plasma after coffee consumption.
Irrespective of treatment, the tmax value for early metabolites (quinic and phenolic compounds) was observed between 0 and 2 h after the ingestion of coffee and tmax value for late metabolites (colonic metabolites) was observed between 7 and 10 h. The amount of colonic metabolites had not returned to the baseline level 15 h after the ingestion of coffee. The co-ingestion of breakfast and coffee, when compared to the ingestion of coffee alone, significantly increased the Cmax values for all quinic and phenolic compounds, as well as two colonic metabolites (DHCA and DHiFA). These differences also revealed that the three treatments differed in terms of the kinetics of release. Thus, future studies should use an extended plasma collection time with shorter intervals (e.g. 2 h) to provide a full pharmacokinetic profile.
There were no statistically significant between-treatment differences in the urine samples collected 24 h after coffee ingestion. However, urine samples collected within six hours of the consumption of coffee alone or in combination with a fat-rich meal showed significantly higher CGA quantities than samples collected at the same time point for coffee ingested with a carbohydrate-rich. Strong inter-individual variability and the fact that only 14 healthy subjects participated in the study hindered the identification of any clear trend between the plasma concentrations of metabolites and their excretion in urine.
Four hours after the ex vivo incubation of 5-CQA with individual faecal samples the sum of 5-CQA, CA, and DHCA varied strongly between participants. These findings could result from binding effects of the phenolic compounds with faecal constituents, further degradation or metabolism, and/or the release of bound phenolic substances before the experiment started. We hypothesized that for participants with high plasma AUCs of dihydro compounds, their incubation samples show also high concentrations of CA and DHCA in the incubation medium after four hours. No significant correlation could be found.
This study and all of the outcomes were exploratory. Due to the limited number of participants, we could only investigate tendencies for how the co-ingestion of food affects the bioavailability of CGAs and their respective metabolites following coffee consumption. Therefore, the achieved results are only indicative. Despite this limitation, the data highlight that even though all three treatments had strong similarities in the total bioavailability of CGAs and metabolites from instant coffee, there were between-treatment differences in the kinetics of release. The co-ingestion of breakfast and coffee favoured a slow and continuous release of colonic metabolites while non-metabolized coffee components were observed in plasma within the first hour when coffee was ingested alone.
In conclusion, both a shift in gastrointestinal transit time and the plasma metabolite composition were observed when the ingestion of coffee alone or in combination with breakfast were compared. These results showed that breakfast consumption induces the retarded release of chlorogenic acid metabolites in humans. The data from our human intervention study suggest that the bioavailability of chlorogenic acids from coffee and their derivatives does not only depend on chemical structure, molecular size and active or passive transport ability, but is also influenced by inter-individual differences. Therefore, we strongly recommend that future studies include metabolism experiments that focus on microbiota genotypes and/or the genotyping of individual subjects. This type of research could be pivotal to elucidating whether, and how, genotype affects the metabolic profile after chlorogenic acid intake.
In the present work the concept of decarboxylative couplings and the strategy to use carboxylates as directing groups for C-H functionalizations have been decisively improved in three ways. These concepts emphasize the multifaceted nature of aromatic carboxylic acids as expedient starting materials in homogeneous catalysis to construct highly desirable molecular scaffolds in a straightforward fashion.
In the first project, the restriction of decarboxylative biaryl synthesis to exclusively couple aryl halides with ortho-substituted benzoic acids has been overcome by a holistic optimization of a Cu/Pd bimetallic catalyst system. Long ago postulated, this is now the proof that decarboxylative cross-couplings are not intrinsically limited to different decarboxylation propensities of benzoic acids or hampered by excess halides, accessing for the first time the entire spectrum of aromatic carboxylic acids as starting materials for the decarboxylative biaryl synthesis. The second project uses the carboxyl moiety as directing group for the ortho-arylation with aryl bromides and -chlorides catalyzed by comparatively inexpensive ruthenium. The carboxylic acid group remains untouched after the ortho-functionalization giving the possibility to a wealth of further diversifications via decarboxylative ipso-substitutions. Within the same project, a Cu/Ru bimetallic catalyst system was found to be able to switch the decarboxylative biaryl coupling from the ipso- to the ortho-position, complementing the Cu/Pd system developed in the first project. In a third project, a redox neutral C-C bond formation revealed the full synthetic potential of the carboxyl group. The COOH moiety acts as a classical directing group for the C-H hydroarylation of internal alkynes to form highly desirable 2-vinyl benzoic acids. With propargylic alcohols the hydroarylation is followed by an in situ esterification, showing that after easing the C-H cleavage, the directing group can be transformed into another functional group, thus, acting as a transformable directing group. Most importantly, a new fascinating reaction mode is activated by embedding the decarboxylation within the C-H functionalization event. This mode of action is capable to solve regioselectivity issues that inherently occur when dealing with carboxylates as directing groups. A so-called deciduous directing group is cast off simultaneously within the C-H functionalization event, resulting in an inherently monoselective pathway.
These methods were developed with the permanent goal of ensuring high sustainability. They do require neither pre-functionalized starting materials nor additional oxidants and provide access to a number of chemically relevant molecules from abundant, inexpensive and toxicologically innocuous educts.
Novel Pseudocyclopeptides Containing 1,4-Disubstituted 1,2,3-Triazole Subunits for Anion Recognition
(2017)
Anion recognition is one of the most rapidly growing areas in the field of Supramolecular Chemistry due to the vital role of anions in the environment, in biology and in industry. The development of new anion binding motifs that can also be combined with known ones in a novel receptor is a timely topic. In this context, we have synthesized three cyclic pseudopeptides 16, 17 and 18, containing conventional H-bond donors (amide) in combination with, respectively, triazole C–H or triazole C–I functions.
All three receptors were synthesized by using a combination of peptide and click chemistry. Structural studies show that all three pseudopeptides adopt conformations with the triazole C-H or C-I groups pointing into the cavity center to allow them to contribute to binding. Quantitative binding studies showed that the cyclic pseudohexapeptide 1 coordinates to oxoanions (sulfate, dihydrogenphosphate, and hydrogenpyrophosphate) with different binding strengths and complex stoichiometries in 2.5 vol% water/DMSO.
Anion selectivity of 16 significantly changes when the cavity size of this pseudopeptide is increased to obtain the larger analog 17. This pseudooctapeptide forms well defined complexes with protonated phosphate anions. The complexation involves sandwiching of a cyclic tetramer of dihydrogenphosphate or a dimer of dihydrogenpyrophosphate anions by two pseudopeptide rings. Both complexes were characterized structurally in the solid state. They are stable in solution (2.5 vol% water/DMSO) as result of the interaction between hydrogen bond donors of 17 and the oxygen atoms of the anionic aggregates. The complexes can also be transferred to the gas phase without decomposition.
Anion selectivity of 16 was further altered by introducing iodine atom in the C5 position of the 1,4-disubstituted 1,2,3-triazole units. The corresponding cyclic pseudohexapeptide 18 features a smaller cavity diameter than 17 as a result of the iodide atoms and was therefore found to only coordinate to smaller spherical anions such as chloride. It forms 1:1 complexes with chloride, bromide and iodide in 2.5 vol% water/DMSO. Among the halides, 18 has highest affinity for chloride followed by bromide and iodide. The same stability trend was also observed in the gas phase by ESI/MS.
Concluding, I prepared three new macrocyclic pseudopeptides during my PhD and characterized their complexes with anions in terms of structure and affinity. All of these pseudopeptides were shown to interact with phosphate-derived anions, which renders them unique among the anion receptors developed in the Kubik group before.
In the present work, the interaction of diatomic molecules with charged transition metal clusters and complexes was investigated. Temperature controlled isothermal kinetic studies served to elucidate the adsorption behavior of transition metal clusters. Infrared multiple photon dissociation (IR-MPD) experiments in conjunction with density functional theory (DFT) computations enabled the analysis of adsorbate induced changes on the structure and spin multiplicity of transition metal cores. A tandem cryo trap setup was used for the kinetic and spectroscopic investigations of the given compounds as isolated species in the gas phase. The presented investigations enabled insight into the metal-adsorbate bonding and provided cluster size and adsorbate coverage dependent information on cluster surface morphologies.
Magnetic and Structural Characterization of Isolated Gaseous Ions by XMCD and IRMPD Spectroscopy
(2017)
This thesis comprises four independent research studies on the magnetic and structural characterization of isolated ions in the gas phase. The electrospray ionization (ESI) technique is used for the transfer of (multi-)metallic complexes and organic molecules from solution into the gas phase. The subsequent storage of molecular ions in ion traps allows for a variety of spectroscopic methods in order to investigate the intrinsic properties of the isolated species void of solvent, crystal lattice, bulk or supporting surface effects. The magnetic properties of metal complexes are elucidated by gas phase X-ray magnetic circular dichroism (XMCD) spectroscopy. The element selective technique in combination with sum rule analysis allows for a separate determination of spin and orbital magnetic moments at different metal centers. Structural investigations on isolated molecular ions in terms of coordination sphere, binding motifs and hydrogen bonds are conducted using infrared multiple photon dissociation (IRMPD) spectroscopy. A resonant two color IRMPD technique serves to increase fragmentation yields, overcome dissociation bottlenecks and reveal otherwise dark bands. Comparison of experimental IRMPD spectra with calculated harmonic absorption spectra by density functional theory (DFT) provides structural assignments for a profound understanding of intra- and intermolecular interactions.
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).
This thesis comprises several independent research studies on transition metal complexes as trapped ions in isolation. Electrospray Ionization (ESI) serves to transfer ions from solution into the gas phase for mass spectrometric investigations. Subsequently, a variety of experimental and theoretical methods provide fundamental insights into molecular properties of the isolated complexes: InfraRed (Multiple) Photon Dissociation (IR-(M)PD) spectroscopy provides information on binding motifs and molecular structures at cryo temperatures as well as at room temperature. Collision Induced Dissociation (CID) serves to elucidate molecular fragmentation pathways as well as relative stabilities of the complexes at room temperature. Quantum chemical calculations via Density Functional Theory (DFT) substantiate the experimental results and deepen the fundamental insights into the molecular properties of the complexes. Magnetic couplings between metal centers in oligonuclear complexes are investigated by Broken Symmetry DFT modelling and X Ray Magnetic Circular Dichroism (XMCD) spectroscopy.
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.
Redox-neutral decarboxylative coupling reactions have emerged as a powerful strategy for C-C bond formation. However, the existing reaction conditions possess limitations, such as the coupling of aryl halides restricted to ortho-substituted benzoic acids; alkenyl halides were not applicable in decarboxylative coupling reaction. Within this thesis, the developments of Pd/Cu bimetallic catalyst systems are presented to overcome the limitations.
In the first part of the PhD work, a customized bimetallic PdII/CuI catalyst system was successfully developed to facilitate the decarboxylative cross-coupling of non-ortho-substituted aromatic carboxylates with aryl chlorides. The restriction of decarboxylative cross-coupling reactions to ortho-substituted or heterocyclic carboxylate substrates was overcome by holistic optimization of this bimetallic Cu/Pd catalyst system. All kinds of benzoic acids regardless of their substitution pattern now can be applied in decarboxylative cross-coupling reaction. This confirms prediction by DFT studies that the previously observed limitation to certain activated carboxylates is not intrinsic. The catalyst system also presents higher performance in the coupling of ortho-substituted benzoates, giving much higher yields than those previously reported. ortho-Methyl benzoate and ortho-phenyl benzoate which have never before been converted in decarboxylative coupling reactions, gave reasonable yields. These together further confirm the superiority of the new protocol.
In the second part of the PhD work, arylalkenes syntheses via two different Pd/Cu bimetallic-catalyzed decarboxylative couplings have been developed. This part consists of two projects: 2a) decarboxylative coupling of alkenyl halides; 2b) decarboxylative Mizoroki-Heck coupling of aryl halides with α,β-unsaturated carboxylic acids.
In project 2a, widely available, inexpensive, bench-stable aromatic carboxylic acids are used as nucleophile precursors instead of expensive and sensitive organometallic reagents that are commonly used in previously reported transition-metal catalyzed cross-couplings of alkenyl halides. With this protocol, alkenyl halides for the first time are used in decarboxylative coupling reaction, allowing regiospecific synthesis of a broad range of (hetero)arylalkenes in high yields. Unwanted double bond isomerization, a common side reaction in the alternative Heck reactions especially in the coupling of cycloalkenes or aliphatic alkenes, did not take place in this decarboxylative coupling reaction. Polysubstituted alkenes that hard to access with Heck reaction are also produced in good yields. The reaction can easily be scaled up to gram scale. The synthetic utility of this reaction was also demonstrated by synthesizing an important intermediate of fungicidal compound in high yield within 2 steps.
In project 2b, a Cu/Pd bimetallic catalyzed decarboxylative Mizoroki-Heck coupling of aryl halides with α, β-unsaturated carboxylic acids was successfully developed in which the carboxylate group directs the arylation into its β-position before being tracelessly removed via protodecarboxylation. It opens up a convenient synthesis of unsymmetrical 1,1-disubstituted alkenes from widely available precursors. This reaction features good regioselectivity, which is complementary to that of traditional Heck reactions, and also presents excellent functional group tolerance. Moreover, a one-pot 3-step 1,1-diarylethylene synthesis from methyl acrylate was achieved, where solvent changes or isolation of intermediates are not required. This subproject presents an example of carboxylic acids utility in synthesizing valuable compounds which are hard to access via conventional methodologies.
This work introduces a promising concept for the preparation of new nano-sized receptors. Mixed monolayer protected gold nanoparticles (AuNPs) for low molecular weight compounds were prepared featuring functional groups on their surfaces. It has been shown that these AuNPs can engage in interactions with peptides in aqueous media. Quantitative binding information was obtained from DOSY-NMR titrations indicating that nanoparticles containing a combination of three orthogonal functional groups are more efficient in binding to dipeptides than mono or difunctionalised analogues. The strategy is highly modular and easily allows adapting the receptor selectivity to a
given substrate by varying the type, number, and ratio of binding sites on the nanoparticle
surface.
In this thesis, collision-induced dissociation (CID) studies serve to elucidate relative stabilities and to determine bond strengths within a given structure type of transition metal complexes. The infrared multi photon dissociation (IRMPD) spectroscopy combined with density functional theory (DFT) allow for structural analysis and provide insights into the coordination sphere of transition metal centers. The used combination of CID and IRMPD experiments is a powerful tool to obtain a detailed and comprehensive characterization and understanding of interactions between transition metals and organic ligands. The compounds’ spectrum comprises mono- or oligonuclear transition metal complexes containing iron, palladium, and ruthenium as well as lanthanide containing single molecule magnets (SMM). The presented investigations on the different transition metal complexes reveal manifold effects for each species leading to valuable results. A fundamental understanding of metal to ligand interactions is mandatory for the development of new and better organometallic complexes with catalytic, optical or magnetic properties.
Spin and orbital magnetic moments of isolated single molecule magnets and transition metal clusters
(2015)
In the present work, magnetic moments of isolated Single Molecule Magnets (SMMs) and transition
metal clusters were investigated. Gas phase X‐ray Magnetic Circular Dichroism (XMCD) in
combination with sum rule analysis served to separate the total magnetic moments of the
investigated species into their spin and orbital contributions. Two different mass spectrometry based
setups were used for the presented investigations on transition metal clusters (GAMBIT‐setup) and
on single molecule magnets (NanoClusterTrap). Both experiments were coupled to the UE52‐PGM
beamline at the BESSY II synchrotron facility (Helmholtz Zentrum Berlin) which provided the
necessary polarized X‐ray photons. The investigation of the given compounds as isolated molecules
in the gas phase enabled a determination of their intrinsic magnetic properties void of any influences
of e.g. a surrounding bulk or supporting surface
The aim of this work was to synthesize and characterize new bidentate N,N,P-ligands and their corresponding heterobimetallic complexes. These bidentate pyridylpyrimidine aminophosphine ligands were synthesized by ring closure of two different enaminones ( 3-(dimethylamino)-1-(pyridine-2-yl)-prop-2-en-1-one or 3-(dimethylamino)-1-(pyridine-2-yl)-but-2-en-1-one) with excess amount of guanidinium salts in the presence of base. The novel phosphine functionalized guanidinium salts were prepared from 2-(diphenylphosphinyl)ethylamine or 3-(diphenyl-phosphinyl)propylamine. These bidentate N,N,P-ligands contain hard and soft donor sites which allows the coordination of two different metal centers and bimetallic complexes. These bimetallic complexes can exhibit a unique behavior as a result of a cooperation between the two metal atoms. First, the gold(I) complexes of all these four different ligands were synthesized. The gold metal coordinates only to the phosphorus atom. It was proved by X-Ray crystallography technique and 31P NMR spectroscopy. Addition to the gold(I)-monometallic complexes, trans- coordinated rhodium complex of (2-amino)pyridylpyrimidine aminophosphine ligand was successfully prepared. The characterization of this complex was achieved by NMR and IR spectroscopy. Reacting the mono gold(I) complexes with the different metal salts like Pd(PhCN)2Cl2, ZnCl2, [Ru(p-cymene)Cl2] dimer gave the target heterobimetallic complexes. The second metal centers coordinated to the N,N donor site which was proved by the help of NMR spectroscopy and ESI-MS measurements. The Au(I) and Au-Zn complexes of N,N,P-ligands were examined as catalysts for the hydroamidation reaction of cyclohexene with p-toluenesulfonamide. They did not show activities under the tested conditions. Further studies are necessary to understand the catalytic activities and cooperativity between the two metal atoms. In addition, bi-and trimetallic complexes with the rhodium compound could be synthesized and tested in different organic transformations. Furthermore, the chiral hydroxyl[2.2]paracyclophane substituted with five different aminopyrimidines were accomplished. These aminopyrimidine ligands were synthesized by a cyclization reaction with hydroxyl[2.2]paracyclophane substituted enaminone and excess amount of corresponding guanidinium salts under basic conditions. In the last part of this work, kinetic studies of cyclopalladation reaction of the 2-(arylaminopyrimidin-4-yl)pyridine ligands with Pd(PhCN)2 These measurements were carried out by using UV-Vis spectroscopy. The spectral studies of cyclometallation step showed that the reaction fits a second order kinetics. In addition to this, a full kinetic investigation was performed at different temperatures and the activation parameters of complex formation were calculated.
Nitrogen element is preponderant in Nature. Found in its simplest form as diatomic gas in the air, as well as in elaborated molecules such as the double helix of DNA, this element is indisputably essential for life. Indeed, nitrogen is omnipresent in all metabolic pathways.
With the advent of green chemistry, researchers attempt to functionalize arenes without pre-functionalization of the later for the establishment of C-C bond formation. Why not C-N bond formation?
We investigated new oxidative amination reactions by cross-dehydrogenative-coupling. Concerned by atom economy and green processes, our objectives were: 1) to obviate pre-activation or pre-oxidation of both C-H coupling partner and N-aminating agent. 2) to avoid the use of chelating directing group.
We achieved C-N bond formation for some classes of amines. Thus, we will describe the reactivity of cyclic secondary amines: carbazole, in presence of catalytic amount of ruthenium (II) and copper (II) to build the challenging C-N bond between two carbazoles. The initial mechanistic experiments will be present and discuss.
Then, we will describe more challenging hetero-coupling formation between diarylamines and carbazoles. The new ruthenium (II)/ copper (II) catalytic system allowed forming the ortho-N-carbazolation of diarylamines. This reaction performed under mild conditions (O2 as terminal oxidant) displays an unusual intramolecular N-H••N interaction in the novel class of compounds.
Finally, we will present a surprising metal free C-N bond formation between the ubiquitous phenols and the phenothiazines. Initially conducted in the presence of transition metals (RuII/CuII), this reaction proved to be efficient with the only effect of cumene and O2. Those components suggest a mechanism initiated by a Hock process. An initial infra-red analysis might point out a strong intramolecular O-H••N interaction in the resulting products.
These first elements of reactivity, developed within the laboratory for “modern dehydrogenative amination reactions”, will be presented and discussed.
‘Dioxin-like’ (DL) compounds occur ubiquitously in the environment. Toxic responses associated with specific dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and polychlorinated biphenyls (PCBs) include dermal toxicity, immunotoxicity, liver toxicity, carcinogenicity, as well as adverse effects on reproduction, development, and endocrine functions. Most, if not all of these effects are believed to be due to interaction of these compounds with the aryl hydrocarbon receptor (AhR).
With tetrachlorodibenzo-p-dioxin (TCDD) as representatively most potent congener, a toxic equivalency factor (TEF) concept was employed, in which respective congeners were assigned to a certain TEF-value reflecting the compound’s toxicity relative to TCDD’s.
The EU-project ‘SYSTEQ’ aimed to develop, validate, and implement human systemic TEFs as indicators of toxicity for DL-congeners. Hence, the identification of novel quantifiable biomarkers of exposure was a major objective of the SYSTEQ project.
In order to approach to this objective, a mouse whole genome microarray analysis was applied using a set of seven individual congeners, termed the ‘core congeners’. These core congeners (TCDD, 1-PeCDD, 4-PeCDF, PCB 126, PCB 118, PCB 156, and the non dioxin-like PCB 153), which contribute to approximately 90% of toxic equivalents (TEQs) in the human food chain, were further tested in vivo as well as in vitro. The mouse whole genome microarray revealed a conserved list of differentially regulated genes and pathways associated with ‘dioxin-like’ effects.
A definite data-set of in vitro studies was supposed to function as a fundament for a probable establishment of novel TEFs. Thus, CYP1A induction measured by EROD activity, which represents a sensitive and yet best known marker for dioxin-like effects, was used to estimate potency and efficacy of selected congeners. For this study, primary rat hepatocytes and the rat hepatoma cell line H4IIE were used as well as the core congeners and an additional group of compounds of comparable relevance for the environment: 1,6-HxCDD, 1,4,6-HpCDD, TCDF, 1,4-HxCDF, 1,4,6-HpCDF, PCB 77, and PCB 105.
Besides, a human whole genome microarray experiment was applied in order to gain knowledge with respect to TCDD’s impact towards cells of the immune system. Hence, human primary blood mononuclear cells (PBMCs) were isolated from individuals and exposed to TCDD or to TCDD in combination with a stimulus (lipopolysaccharide (LPS), or phytohemagglutinin (PHA)). A few members of the AhR-gene batterie were found to be regulated, and minor data with respect to potential TCDD-mediated immunomodulatory effects were given. Still, obtained data in this regard was limited due to great inter-individual differences.
A positive affection of human health by nutrition is of high interest, especially for bioactive compounds which are consumed daily in high amounts. This is the case for chlorogenic acids (CGA) ingested by coffee. This molecule class is associated with several possible beneficial health effects observed in vitro that strongly depend on their bioavailability. So far factors influencing bioavailability of CGA such as dose, molecule structure and site of absorption haven´t been investigated sufficiently.
Therefore we performed an in vivo dose-response study with ileostomists, who consumed three different nutritional doses of CGA ingested as instant coffee (4,525 (HIGH); 2,219 (MEDIUM); 1,053 (LOW) μmol CGA). CGA concentrations were determined in ileal fluid, urine and plasma. Furthermore, we conducted an ex vivo study with pig jejunal mucosa using the Ussing chamber model to confirm the in vivo observations. Individual transfer rates of CGA from coffee were investigated, namely: caffeoylquinic acid (CQA), feruloylquinic acid (FQA), caffeic acid (CA), dicaffeoylquinic acid (diCQA) and QA at physiological concentrations (0.2–3.5 mM). Samples were analyzed by HPLC-DAD, -ESI-MS and -ESI-MS/MS.
About ⅔ of the ingested CGA by coffee consumption were available in the colon dose independent. Nevertheless, the results showed that the consumption of higher CGA doses leads to a faster ileal excretion. This corresponds to a plasma AUC0-8h for CGA and metabolites of 4,412 ± 751 nM*h0-8-1 (HIGH), 2,394 ± 637 nM*h0-8-1 (MEDIUM) and 1,782 ± 731 nM*h0-8-1 (LOW) respectively, and a renal excretion of 8.0 ± 4.9% (HIGH), 12.1 ± 6.7% (MEDIUM) and 14.6 ± 6.8% (LOW). Moreover interindividual differences in gastrointestinal transit times were related to differences in total CGA absorption. Thus the variety of patient´s physiology is a decisive bioavailability factor for CGA uptake. This is corroborated ex vivo by a direct proportional relationship of incubation time with absorbed CGA amount.
The consumption of high CGA doses influences the metabolism pattern as an increasing glucuronidation was observed with consumption of increasing CGA doses. However, the different CGA doses have only minor effects on the overall bioavailability which was confirmed ex vivo by a non-saturable passive diffusion of 5-CQA. Furthermore, we identified in the Ussing chamber an active efflux secretion for 5-CQA that decreases its bioavailability and the physicochemical properties of the CGA subgroups as an important bioavailability factor. Transferred amount in increasing order: diCQA, trace amounts; CQA ≈ 1%; CA ≈ 1.5%; FQA ≈ 2%; and QA ≈ 4%.
Altogether, the consumption of increasing CGA doses by coffee had a minor effect on oral bioavailability in ileostomists, such as a slightly increased glucuronidation. Thus, the consumption of high amounts of CGA from coffee in the daily diet is not limiting the CGA concentrations at the site of possible health effects in the human body. However, according to the patient´s physiology the interindividual gastrointestinal transit time which is possibly influenced by dose is influencing CGA bioavailability. Moreover, ex vivo CGA absorption is governed by diffusion as an absorption mechanism corroborating an unsaturable uptake in vivo and by the individual physicochemical properties of CGA.
ABSTRACT
"Spin and orbital contribution to the magnetic moment of transition metal clusters and complexes"
The spin and orbital contributions to the magnetic moments of isolated iron \(Fe_n^+\) \((7 ≤ n ≤ 18)\), cobalt \(Co_n^+\) \((8 ≤ n ≤ 22)\) and nickel \(Ni_n^+\) \((7 ≤ n ≤ 17)\) clusters were investigated. An experimental access to both contributions is possible by the application of x-ray magnetic circular dichroism (XMCD) spectroscopy. XMCD spectroscopy is based on x-ray absorption spectroscopy (XAS). It exploits the fact that for a magnetic sample the resonant absorption cross sections for negative and positive circular polarized x-rays differ for the transition from a spin orbit split ground state to the valence level. The resulting dichroic effects contain the information about the magnetism of the investigated sample. It can be extracted from the experimental spectrum via application of the so called sum rules. However, only the projections of the magnetic moments onto the quantization axis are experimentally accessible which corresponds to the magnetization of the sample.
We developed a method to apply XMCD spectroscopy to isolated clusters in the gas phase. A modified Fourier Transform Ion Cyclotron Resonance (FT-ICR) mass spectrometer was used to record the XA spectra in Total Ion Yield (TIY) mode, i.e. by recording the fragmentation intensity of the clusters in dependence of x-ray energy. The clusters can be considered to be a superparamagnetic ensemble. Thus, the magnetization follows a Langevin curve. The intrinsic magnetic moments can be calculated by Langevin correction of the experimental magnetic moments because the cluster temperature and the magnetic field are known.
The spin and the orbital magnetic moments are enhanced compared to the respective bulk values for all three investigated elements. The enhancement of the orbital contribution is more pronounced, by about a factor 3 - 4 compared to the bulk, than for the spin magnetic moment. However, if compared to the atomic value, both contributions are quenched. The orbital magnetic moment only amounts to about 10 - 15 % of the atomic value while the spin retains about 80 % of its atomic value. If the magnetic moments found for the clusters are put into perspective with respect to the atomic and bulk values by means of scaling laws, it becomes evident that both contributions follow different interpolations between the atomic and bulk value. The spin follows the well-known trend
\(n^{-1/3} = 1/(cluster radius)\) (n = number of atoms per cluster, assumption of a spherical particle). This trend relates to the ratio of surface to inner atoms in spherical particle. Hence, our interpretation is that the spin magnetic moment seems to follow the surface area of the cluster. On the other hand, the orbital magnetic moment follows \(1/n = 1/(cluster volume)\).
First XA spectra recorded with circularly polarized x-rays of a Single Molecule Magnet (SMM) \([Fe_4Ln_2(N_3)_4(Htea)_4(piv_6)]\) (Ln = Gd, Tb; \(H_3tea\) = triethanolamine, Hpiv = pivalic acid) are presented.
This thesis combines mass spectrometric studies on ionic dicarboxylic acids and transition metal cluster adsorbate complexes. IR-MPD spectra of protonated and deprotonated aliphatic and aromatic dicarboxylic acids provide insights in the nature of intramolecular hydrogen bonding. Investigations of their fragmentation behavior are supported by MP2 calculations. Prior work on cobalt transition metal clusters is extended to iron and nickel and three cobalt alloys have been studied.
Polychlorinated dibenzo-p-dioxins, dibenzofurans, and polychlorinated biphenyls are persistent environmental pollutants which ubiquitously occur as complex mixtures and accumulate in the food and feed chain due to their high lipophilic properties. Of the 419 possible congeners, only 29 share a common mechanism of action and cause similar effects, the so called dioxin-like compounds. Dioxin-like compounds evoke a broad spectrum of biochemical and toxic responses, i.e. enzyme induction, dermal toxicity, hepatotoxicity, immunotoxicity, carcinogenicity as well as adverse effects on reproduction, development, and the endocrine system in laboratory animals and in humans. Most, if not all, of the aforementioned responses, are mediated by the aryl hydrocarbon receptor. In the present work, the elicited biochemical effects of a selection of dioxin-like compounds and the non dioxin-like PCB 153 were examined in mouse (in vivo) and in human liver cell models (in vitro). Emphasis was given to the main contributors to the total toxic equivalents in human blood and tissues TCDD, 1-PnCDD, 4-PnCDF, PCB 118, PCB 126, and PCB 156, which likewise contribute about 90 % to the dioxin-like activity in the human food chain.
Three mouse in vivo studies were carried out aiming to characterize the alterations in hepatic gene expression as well as the induction of hepatic xenobiotic metabolizing enzymes after single oral dose. Based on the results obtained from mouse 3-day and 14-day studies, the seven test compounds can be categorized into three classes; the ones which are 'pure' AhR ligands (TCDD, 1-PnCDD, 4-PnCDF, and PCB 126) or solely CAR inducers (PCB 153), and the ones which are AhR/CAR mixed-type inducers (PCB 118, PCB 156). Moreover, the analysis of hepatic gene expression patterns after a single oral dose of either TCDD or PCB 153 revealed that the altered genes fundamentally differed. Profiling of significantly altered genes led to the conclusion that changes in gene expression were associated with different signalling pathways, in fact by AhR and CAR.
For investigating the role of the AhR in mediating biological responses, several experimental approaches were carried out, such as the analysis of blood plasma metabolites in Ahr knockout and wild-type mice. Genotype specifics and similarities were determined by HPLC-MS/MS analysis. Several plasma metabolites could be identified in both genotypes, but also differences were detected. Furthermore, an in vivo experiment was performed aiming to characterize AhR-dependent and -independent effects in female Ahr knockout and wild-type mice. For this purpose, mice received a single oral dose of TCDD and were killed 96 h later. Microarray analysis of mouse livers revealed that although the Ahr gene was knocked out in Ahr-/- mice, the quantity of affected genes were in the same order of magnitude as for Ahr+/+ mice, but the pattern of altered genes distinctly differed. In addition, the relative liver weights of TCDD-treated Ahr+/+ mice were significantly increased which led to the conclusion, that TCDD induced the development of hepatic steatosis in female Ahr wild-type.
The performed in vitro experiments aimed to characterize the effects elicited by selected DLCs and PCB 153 in human liver cell models by the use of HepG2 cells and primary human hepatocytes. In general, primary human hepatocytes were less responsive than HepG2 cells. This was not only observed in EC values derived from EROD assay, but also regarding microarray analysis in terms of differently regulated genes. In vitro REPs gained from both liver cell models widely confirmed the current TEFs, but some deviations occurred. The comparison of the TCDD-altered genes in both human cell types revealed that only a considerably small number of genes was in common up regulated by both human liver cell models, such as the established AhR-regulated highly inducible cytochrome P450s 1A1, 1A2, and 1B1 as well as other AhR target genes. Although the overlap was rather small, the TCDD-induced genes could be consistently associated with the broad spectrum of established dioxin-related biological responses. The gene expression pattern in primary human hepatocytes after treatment with selected DLCs (TCDD, 1-PnCDD, 4-PnCDF, and PCB 126) and PCB 153 was additionally characterized by microarray analysis. The highest response in terms of significantly altered genes was determined for TCDD, followed by 4-PnCDF, 1-PnCDD, and PCB 126, whereas exposure to PCB 153 did not evoke any significant changes in gene expression. The pattern of significantly altered genes was very homogenous among the four congeners. Genes associated with well-established DLC-related biological responses as well as novel dioxin-inducible target genes were identified, whereby an extensive overlap in terms of up regulated genes by all four DLCs occurred. In conclusion, the results from the in vitro experiments performed in primary human hepatocytes provided fundamental insight into the congeners' potencies and caused alterations in gene expression patterns. The obtained findings implicate that although the extent of enzyme inducibilities varied, the gene expression patterns are coincidental. Microarray analysis identified species-specific (mouse vs. human) as well as model-specific (in vitro vs. in vivo and transformed cells vs. untransformed cells) differences. In order to identify novel biomarkers for AhR activation due to treatment with dioxin-like compounds, five candidates were selected based on the microarray results i.e. ALDH3A1, TIPARP, HSD17B2, CD36, and AhRR. Eventually, ALDH3A1 turned out to be the most reliable and suitable marker for exposure to DLCs in both human liver cell models eliciting the highest mRNA inducibility among the five chosen candidates. In which way these species- and cell type-specific markers are involved in the dioxin-elicited toxic responses should be further characterized in vivo and in vitro.
The research presented in this PhD thesis is a contribution to the field of anion recognition in competitive aqueous solvent mixtures. Neutral anion receptors having a cage-type architecture have been developed on the basis of triply-linked bis(cyclopeptides) and their binding properties toward various inorganic anions have been studied.
The synthetic approaches chosen to assemble the targeted container molecules rely on dynamic chemistry under the template effects of anions such as sulfate and halides. As reversible reactions metal-ligand exchange and thiol-disulfide exchange were used. Disulfide exchange has previously provided singly- and doubly-linked bis(cyclopeptide) receptors whose anion affinities in 2:1 acetonitrile/water mixtures approached the nanomolar range. Metal-ligand interactions have so far not been used to assemble bis(cyclopeptides) in our group. The cyclopeptide building blocks required for both approaches, namely cyclic hexapeptides containing alternating 6-aminopicolinic acid and either (2S,4S)-4-cyanoproline or (2S,4S)-4-thioproline subunits could be synthesized successfully.
Self-assembly of the bis(cyclopeptide) held together by coordinative interactions has been attempted by treating the cyclopeptide trinitrile with square-planar palladium (II) complexes. The reaction was followed with different NMR spectroscopic techniques. Unfortunately, none of the experiments provides conclusive evidence that the targeted triply-linked cage was indeed formed.
Bis(cyclopeptides) containing three dithiol derived linkers between the cyclopeptide rings could be synthesizes successfully. Two complexes were isolated, albeit in small amounts, one containing linkers derived from 1,2-ethanedithiol and the other one from 1,3-benzenedithiol that contain a sulfate anion incorporated in the cavity between the cyclopeptide rings. Formation of triply-linked bis(cyclopeptides) containing different types of linkers could be achieved by performing the synthesis in the presence of different dithiols. Unfortunately, the two C3 symmetrical bis(cyclopeptides) containing a single linker type could not be isolated in analytically pure form so that only qualitative binding studies could be performed. Investigations in this context indicate extraordinary sulfate affinity for these bis(cyclopeptides). In particular, affinity of the receptor containing the 1,2-ethanedithiol linkers for sulfate anions is so high that is even able to dissolve barium sulfate under appropriate conditions and presumably exceeds the sulfate affinity of the doubly-linked bis(cyclopeptides). The sulfate anion present in the cavity of this bis(cyclopeptide) can be replaced by a large number of other anions, i.e. by selenate, perrhenate, nitrate, tetrafluoroborate, hexafluorophosphate and halides. None of these complexes proved to be as stable as the corresponding sulfate complex. In addition, 1H-NMR spectroscopic investigations provided information about the solution structure of the bis(cyclopeptide) anion complexes. Sulfate release from the cavity of the receptor is a slow process while exchange of other anions is significantly faster. Another interesting feature that has been observed for sulfate and selenate complexes of the 1,2-ethanedithiol-containing bis(cyclopeptide) is the very slow H/D rate with which protons on amide groups located inside the cavity of the cage are replaced by deuterium atoms in protic deuterated solvents. This effect in combination with the observation that the different deuterated bis(cyclopeptide) species exhibit individual amide NH signals in the 1H-NMR spectrum are indicative for well defined complex geometries with strong hydrogen-bonding interactions between the anion and the amide NH groups of the receptor. Following the H/D exchange rate in the presence of various salts indicated that anion exchange proceeds via the dissociated complex and not by direct replacement of one anion by another one.
This thesis reports on investigations on the structure and reactivity of dipeptide-alkali metal complexes, a series of ruthenium bearing catalysts, dysprosium based single molecule magnets and organometallic di-cobalt complexes. A variety of experimental and theoretical methods was used dependent on the problem: collision induced dissociation, hydrogen/deuterium exchange reactions, gas phase reactions with \(D_2\), infrared multiple-photon dissociation and the determination of minimum energy structures, IR absorption spectra, transition states and electronic transitions based on density functional theory.
A case study was carried out to explore the influence of alkali metal ions on the gas phase structure of the dipeptide Carnosine. CID experiments on protonated Carnosine and its alkali metal complexes in an ion trap resulted in different fragment pathways dependent on the size of the alkali metal. The complexation of small ions (\(Li^+\) and \(Na^+\)) promoted the cleavage of bonds in the molecules backbone under CID, while \(Rb^+\)- and \(Cs^+\)-Carnosine complexes underwent the exclusive loss of the alkali metal. CID breakdown curves reflected the different binding behavior of the alkali ions to Carnosine. Gas phase H/D exchange reactions with \(D_2O\) resulted in the exchange of several protons of the protonated dipeptide, while its alkali metal complexes underwent no exchange reactions. DFT derived energetical minimum isomers exhibited only charge solvated tridentate structures, whereas salt bridge as well as charge solvated binding motives are reported in literature on complexes of alkali metal ions and oligopeptides. This study was published in a similar version as a paper in Zeitschrift für Physikalische Chemie.
A combination of the four dipeptides Carnosine, Anserine, GlyHis and HisGly with alkali metal ions was investigated with the help of CID, IR-MPD spectroscopy and H/D exchange reactions with \(ND_3\). The aim of the survey was to elucidate the influence of the methyl-group at the histidine ring, of the peptide sequence and chain length on the binding motives of the alkali ions. The experimental results were compared to DFT derived minimum energetical isomers. A moderate accordance was found for DFT predicted IR absorptions to IR-MPD spectra. A systematic nomenclature was developed reflecting all binding motives of the four dipeptides to alkali ions. Carnosine complexes all alkali metal ions in an uniform motive. DFT derived energetical minimum isomers of the three other dipeptides showed strong conformational changes with increasing size of the alkali ion. The most favored binding motive of all peptides was the tridentate complexation of the alkali ion by a carboxylic and an amidic oxygen atom, while the electron donating nitrogen atom either belongs to the Histidine ring or the amine group. The ability to form hydrogen bonds in a certain binding motive is essential for the preference of the Histidine or amine nitrogen atom as an electron donor. The charge solvated binding motive is the most common within all found isomers. Several structures exhibited hydrogen bonded protons. Those can be interpretated as intermediates between the charge solvated and the salt bridge binding motive. CID breakdown curves of the cationic complexes of the dipeptides with \(K^+\), \(Rb^+\) and \(Cs^+\) resulted in a fair agreement of \(E^{50\%}_{com}\) values with DFT derived Gibbs free binding energies. CID led to multiple fragments of the \(Li^+\) and \(Na^+\) dipeptide complexes and to an insufficient correlation between the \(E^{50\%}_{com}\) values and metal-dipeptide free binding enthalpies. Gas phase H/D exchange reactions of the protonated dipeptides with \(ND_3\) resulted in the exchange of all labile protons with comparable relative partial rate constants. The assumption of coexisting single and double exchange reactions per single collision led to an enhancement in quality of the pseudo first order kinetic fits of the experimental derived data. The \(Li^+\), \(Na^+\) and \(K^+\) complexes of the dipeptides exhibited a reduction in the number of exchanged protons, significantly lower rate constants for H/D exchange and only single exchange reactions.
The complexation of the doubly charged cationic transition metal \(Zn^{2+}\) by deprotonated Carnosine led to crucial conformational changes with respect to the alkali metal complexes. Former DFT calculations on the gas phase structure of \([Carn-H,Zn^{II}]^+\) were now compared to IR-MPD spectra. IR-MPD spectra exhibited several of the DFT predicted IR absorptions while the overall agreement in the position of bands is only partially satisfactory. The complex \([Carn-H,Zn^{II}]^+\) was furthermore used in order to study the band dependent enhancement of fragmentation efficiency by application of a resonant 2-color IR-MPD pump/probe scheme. In literature, it is assumed that the slopes of linear fits to the log-log scale of experimental data (fragmentation efficiency vs. laser pulse energy) correlate to the number of photons needed for fragmentation. No reasonable number of photons for the fragmentation of the molecule was derived with this approach. However, it could be shown that the number of photons of the pump laser needed for fragmentation is reduced by the use of a second IR color. The change of the delay between the pump and probe laser pulse had an influence on the shape of the absorption bands. Irradiation with the probe laser pulse before the pump laser caused a heating of the molecule which resulted in a broadening of bands. No broadening was observed when the probe laser was applied simultaneously or after the pump laser. CID and IR-MPD fragmentation channels differed in their relative abundance. Furthermore, relative abundancies of fragments were specific to the excited vibrational motions. This study provides essential approaches for the further study of the mechanism of resonant 2-color IR-MPD spectroscopy.
Several ruthenium catalysts for transfer hydrogenation reactions were synthesized by L. Ghoochany (research group W. Thiel, TU Kaiserlautern). CID measurements on isotopic labeled species led to the following conclusion about the activation process of the catalyst: a nitrogen-ruthenium bond is broken, the pyrimidine ring of the substituted 2-R-4-(2-pyridinyl)pyrimidine ligand rotates about 160° and a carbon-ruthenium bond is formed under subsequent loss of a HCl (or DCl) molecule. The mass spectrometers CID amplitude was calibrated with a set of “thermometer ions”. CID breakdown curves were used for determination of \(E^{50\%}_{com}\) values of three differently substituted catalysts. Finally, activation energies were estimated by means of the calibration. The resulting activation energies showed a qualitative correlation to DFT derived activation energies. These results are part of a manuscript which was submitted to Chemistry – A European Journal and is currently in the review process. Further studies on this series of transition metal complexes included CID on ligand exchanged species, 1- and 2-color IR-MPD spectroscopy, gas phase reactions with \(D_2\) and DFT based modeling of the reaction coordinate of the \(D_2\) insertion. The exchange of the anionic chlorido ligand in solution led to three complexes with different fragmentation thresholds. CID derived activation amplitudes correspond well to the order predicted by the hard/soft acids/bases (HSAB) concept. 1-color IR-MPD experiments on two complexes showed only a few bands. Resonant 2-color IR-MPD increased the overall fragmentation efficiency and uncovered several dark bands. DFT derived IR absorption spectra correlate well to IR-MPD spectra while some bands are still not observable. Gas phase reactions with \(D_2\) showed an increase of the mass of the activated complex of +4 m/z. This was interpreted in terms of an incorporation of a \(D_2\) molecule under heterolytical cleavage of the \(D_2\) molecule and can be compared to a back reaction of the activation. The reaction coordinate of the \(D_2\) incorporation was modeled with DFT at the B3LYP/cc-pVTZ level of theory and different activation energies were derived dependent on the substituent. Reactions of three differently substituted complexes with \(D_2\) resulted in different relative partial rate constants. The comparison to rate constants derived from transition state theory showed a qualitative but not quantitative correlation to the experimental results. This study contributes to our ongoing work on the assignment and isolation of reaction intermediates in the gas phase.
A series of dysprosium based complexes was synthesized by A. Bhunia (research group P. W. Roesky, KIT) and studied within the collaborative research center SFB/TRR 88 “3MET”. We contributed to this work with ESI-MS, CID and experiments on H/D exchange reactions with \(ND_3\) in the gas phase. Those complexes consist of a central triple-charged dysprosium cation and two identical salen-type ligands which allow for a complexation of up to two transition metals. The monometallic dysprosium complex shows single molecule magnet (SMM) behavior in SQUID measurements, while the incorporation of two double-charged manganese cations leads to ferromagnetic behavior. The interaction of terminal amine groups with the manganese ions caused a hinderance of the exchange H/D exchange reaction with \(ND_3\) in the gas phase. Alternatively, the terminal amine groups of the monometallic dysprosium complex allow for the bond of two \(Ni^{2+}(tren)\) complexes. ESI-MS studies showed anionic as well as cationic complexes due to deprotonation or protonation in solution. CID studies led to fragmentation schemes which correlate quite well to the predicted structures of the complexes. These results are part of two publications in Inorganic Chemistry and Dalton Transactions. Further studies on this series of mono-, di- and trimetallic complexes are reported in this thesis. H/D exchange reactions with \(D_2O\) in solution yielded in an exchange of all labile protons for the cationic complexes. Anionic complexes underwent a partial or a complete exchange of labile protons. A comparison of 1- and 2-color IR-MPD spectra of anionic and cationic complexes as well as H/D exchanged species allowed for the assignment of vibrational bands. Furthermore, preferred protonation sites were derived by comparing the results of IR-MPD experiments and H/D exchange reactions in solution and in the gas phase. This study contributes to our ongoing work on the determination of magnetic properties of isolated ions in the gas phase at the Helmholtz-Zentrum Berlin.
The complex \([(^4CpCo)_2(\mu-C_2Ph_2)]\) (\(^4Cp\) = tetraisopropyl-cyclopentadiene) was synthesized by J. Becker (research group H. Sitzmann, TU Kaiserslautern). The cationic complex and several reaction products were characterized by ESI-MS. Some of the experimental data contributed to the diploma thesis of J. Becker. The cationic reaction products and the complex itself were subject of IR spectroscopic characterization. IR-MPD efficiency changed crucially with modification of the complex, yielding \([(^4CpCo)_2(\mu-C_2Ph_2)X]^+ (X=H, (H+CH_3CN), Cl, O)\). The contribution of various fragmentation channels to the overall fragmentation efficiency was studied in detail. An increase of photon flux resulted in a saturation of preferred \(C_2Ph_2\) loss, additional alkyl fragments out of the \(^4Cp\) rings arising. Several absorption bands were found in the mid- and near-IR region. A model system from literature was used to identify seemingly levels of DFT theory by reference to X-ray crystal structure data. The B3LYP and the B97D functional with cc-pVDZ and Stuttgart 1997 ECP basis sets were identified for calculations of the complex \([(^4CpCo)_2(\mu-C_2Ph_2)]^+\) and of its reaction products. An elongation of the Co-Co bond distance was observed for the cationic reaction products with \(Cl^-\) and \(O^{2-}\). Calculations with B3LYP and B97D resulted in different electronic ground states. We did not obtain a good agreement of calculated vibrational modes and recorded IR-MPD spectra. DFT predicted more absorption bands than observed, especially those corresponding to aliphatic symmetric \(CH_n (n=2, 3)\) and aromatic CH stretch motions. Future 2-color IR-MPD experiments might resolve currently prevailing discrepancies. TD-DFT calculations yielded several electronic transitions that do not correspond to the IR-MPD spectra. The chosen levels of theory for DFT and TD-DFT calculations does not seem to be appropriate. IR-MPD spectra have to be remeasured in order to normalize the spectra to photon flux. Furthermore, a different strategy has to be developed for ab initio calculations on the complexes under study.
A combination of various methods applied to isolated ions in the gas phase and in solution allowed for the study of their structure, binding energies and reactivity. 1- and 2-color IR-MPD spectroscopy combined with DFT predicted absorption spectra of different isomers enabled an assignment of vibrational bands and binding motives of the molecules. The derived results are important for further studies on the binding behavior of peptides and the reaction behavior of metal complexes.
This thesis combined gas phase mass spectrometric investigations of ionic transition metal clusters that are either homogeneous \((Nb_n^{+/-}, Co_n^{+/-})\) or heterogeneous \(([Co_nPt_m]^{+/-})\), of their organo metallic reaction products, and of organic molecules (aspartame and Asp-Phe) and their alkali metal ion adducts.At the Paris FEL facility CLIO a newly installed FT-ICR mass spectrometer has been modified by inclusion of an ion bender that allows for the usage of additional ion sources beyond the installed ESI source. The installation of an LVAP metal cluster source served to produce metal cluster adsorbate complex ions of the type \([Nb_n(C_6H_6)]^{+/-}\). IR-MPD of the complexes \([Nb_n(C_6H_6)]^{+/-} (n = 18, 19)\) resulted in \([Nb_n(C_6)]^{+/-} (n = 18, 19)\) fragments. Spectra are broad, possibly because of vibronic / electronic transitions. In Kaiserslautern the capabilities of the LVAP source were extended by adding a gas pick up unit. Complex gases containing C-H bonds otherwise break within the cluster forming plasma. More stable gases like CO seem to attach at least partially intact. Metal cluster production with argon tagged onto the cluster failed when introducing argon through the pick up source, but succeeded when using argon as expansion gas. A new mass spectrometer concept of an additional multipole collision cell for metal cluster adsorbate formation is currently under construction. Subsequent cooling shall achieve high resolution IR-MPD spectra of transition metal cluster adsorbate complexes.Prior work on reaction of transition metal clusters with benzene was extended by investigating the reaction with benzene and benzene-d6 of size selected cationic cobalt clusters \(Co_n^+\) and of anionic cobalt clusters \(Co_n^-\) in the size range \(n = 3 - 28\) and of bimetallic cobalt platinum clusters \([Co_nPt_m]^{+/-}\) in the size range \(n + m \le 8\). Dehydrogenation by cationic cobalt clusters \(Co_n^+\) is sparse, it is effective in small bimetallic clusters \([Co_nPt_m]^+ (n + m \le 3)\). Thus single platinum atoms promote benzene dehydrogenation while further cobalt atoms quench it. Dehydrogenation is ubiquitous in reactions of anionic cobalt clusters. Mixed triatomic clusters \([Co_2Pt_1]^-\) and \([Co_1Pt_2]^-\) are special in causing effective reactions and single dehydrogenation through some kind of cooperativity while \([Co_nPt_{1,2}]^- (n \ge 3)\) do not react at all. Kinetic isotope effects KIE(n) in total reaction rates are inverse and - in part - large, dehydrogenation isotope effects DIE(n) are normal. A multistep model of adsorption and stepwise dehydrogenation from the precursor adsorbate proves suitable to rationalize the found KIEs and DIEs in principle. Particular insights into the effects of charge and of cluster size are largely beyond this model. Some DFT calculations - though preliminary - lend strong support to the otherwise assumed structures and enthalpies. More insights into the cause of the found effects of charge, size and composition of both pure and mixed clusters shall arise from ongoing high level ab initio modeling (of especially the \(n + m = 3\) case for mixed clusters).The influence of the methylester group in the molecules aspartame (Asp-PheOMe) and Asp-Phe has been explored. Therefore, their protonated and deprotonated species and their complexes with alkali metal ions attached were investigated with different techniques utilizing mass spectrometry.Gas phase H-/D-exchange with \(ND_3\) has proven that in both molecules all acidic NH and OH binding motifs do exchange their hydrogen atom and that simultaneous multi exchange is present. Kinetic studies revealed that with alkali metal ions attached the speed of the first exchange step decreases with increasing ion size. The additional OH of the carboxylic COOHPhe group in Asp-Phe increases the exchange speed by a constant value. CID experiments yielded water and the protonated Asp-Phe anhydride as main fragments out of the protonated molecules, neutral Asp anhydride and \([Phe M]^+ / [PheOMe M]^+\) for \(Li^+\) and \(Na^+\) attached, and neutral aspartame / Asp-Phe and ionic \(M^+\) for \(K^+\), \(Rb^+\) and \(Cs^+\) attached. The threshold energy \(E_{CID}\), indicating ion stability, decreases with increasing ion size. For aspartame fragmentation occurs at lower \(E_{CID}\) values for complexes with \(H^+\), \(Li^+\) and \(Na^+\) than for the Asp-Phe analoga. Complexes with \(K^+\), \(Rb^+\) and \(Cs^+\) give the same \(E_{CID}\) value for aspartame and Asp-Phe. IR-MPD investigations lead to the same fragments as the CID experiments. In combination with quantum mechanical calculations a change in the preferred structure from charge-solvated, tridentate type for complexes with small alkali metal ions (\(Li^+\)) to salt-bridge type structure for large alkali metal ions (\(Cs^+\)) could be confirmed. Calculations thereby reveal nearly no structural differences between aspartame and Asp-Phe for cationized species. The deprotonation of the additional COOHPhe group in Asp-Phe is preferred against other acidic positions. A better experimental distinction between possible (calculated) structure types would arise from additional FEL IR-MPD measurements in the energy range of 600 to 1800 \(cm^{-1}\). The comparison of the \(E_{CID}\) values with calculated fragmentation energy values proves that not only for alkali metal complexes with \(K^+\), \(Rb^+\) and \(Cs^+\), but also for \(Li^+\) and \(Na^+\) the bond breaking of all metal atom bonds is part of the transition state. The lower \(E_{CID}\) values for aspartame with small cations may be explained in terms of internal energy. Aspartame is a larger molecule, possesses more internal energy and can be recognized as the larger heat bath. Less energy is needed for fragmentation, if the Phe part with the additional methylester group is involved in the fragmentation process.
Palladium-Catalyzed C–C Bond Formations via Activation of Carboxylic Acids and Their Derivatives
(2013)
Applications of carboxylic acids and their derivatives in transition metal-catalyzed cross-coupling reactions regio-selectively forming Csp3-Csp2, and Csp2-Csp2 bonds were explored in this thesis. Several important organic building blocks such as aryl acetates, diaryl acetates, imines, ketones, biaryls, styrenes and polysubstituted alkenes were successfully accessed from carboxylic acids and their derivatives by the means of C–H activation and decarboxylative cross-couplings.
An efficient and practical protocol for the synthesis of biologically important ethyl 2-arylacates through the dealkoxycarbonlative cross-coupling reaction between aryl halides and malonates was developed. Activation of the alpha-proton of alkyl esters by a copper catalyst allowed the deprotonation of esters even in the presence of mild bases, leading to a straightforward and efficient approach to alkyl alpha-diarylacetate from simple alkyl acetates and aryl halides.
The addition of a primary amine into the coupling reaction of alpha-oxocarboxylic acids and aryl halides led to an unprecedented low-temperature redox-neutral decarboxylative coupling process, providing a green and efficient method for the preparation of azomethines, in which all the three substituents can be independently varied. A minor modification of this protocol allowed us to easily access the corresponding ketones.
The decarboxylative coupling of robust aryl mesylates as well as polysubstituted alkenyl mesylates using our customized imidazolyl phosphine ligands was realized, further expanding the scope of carbon electrophiles in decarboxylative coupling reactions. Variation of the ligands led to two complementary protocols, providing the corresponding biaryls and polysubstituted olefins in high yields.
The use of a new class of pyrimidinyl phosphine ligands dramatically reduced the reaction temperatures of decarboxylative cross-coupling reactions between aromatic carboxylic acids and aryl or alkenyl triflates. The new catalyst system for the first time allowed the efficient decarboxylative biaryls synthesis at only 100 °C, representing a significant achievement in redox-neutral decarboxylative coupling reactions.
In this study, two outstanding subgroups of organic-inorganic hybrid materials have been investigated. The first part covers the design, synthesis, characterization and application of seven novel Metal Organic Frameworks (MOFs) containing functionalized biphenyl dicarboxylates as linkers. In the second part, the surface modification of the metal oxides ZrO2, TiO2 and Al2O3 using phosphonate derivates is reported.
Firstly three functionalized MOF structures; ZnBrBPDC, ZnNO2BPDC and ZnNH2BPDC were synthesised using 4,4´-biphenyldicarboxylic acid derivatives with different functional groups (-Br, -NO2, -NH2) Powder X-ray diffraction (PXRD) measurements indicated that the synthesised MOFs posses the interpenetrated IRMOF-9 structure with a cubic topology, which was also confirmed with single crystal X-ray measurements. The chemical structure of the MOF materials was further proved by solid state NMR and IR measurements. N2 adsorption measurements showed Type I isotherms for all three structures with large surface areas. TGA measurements of the evacuated samples were in good agreement with the elemental analysis data. The results proved that their thermal stability is between 325 °C - 450 °C.
Adsorption properties of these MOF structures were tested using light alkanes (CH4, C2H6, C3H8, and n-C4H10) at three different temperatures. For all adsorbents, the maximum uptakes were observed at 273 K. When the temperature was increased, the amount of the adsorbed gas decreased. All three MOFs showed strong affinities for n-butane. The lowest uptakes were observed for CH4.
The effect of functional groups on the IRMOF series was also examined by synthesizing amide functionalized biphenyl linkers. For this purpose, four different linkers containing amides with different alkyl chains (C1-C4) were synthesized and used for the synthesis of four new MOF structures ZnAcBPDC, ZnPrBPDC, ZnBuBPDC and ZnPeBPDC.
PXRD measurements of ZnAcBPDC indicated that the structure contains two different phases. PXRD patterns of ZnPrBPDC, ZnBuBPDC and ZnPeBPDC revealed non-interpenetrated structures which were further proved by single crystal X-ray measurements. The chemical structure of the MOF materials was further confirmed by X-ray spectoscopy, solid state NMR and IR measurements.
N2 adsorption measurements of the MOF structures were carried out using different activation methods. For all four MOFs, Type I isotherms were obtained. ZnAcBPDC showed the highest BET surface area. ZnAcBPDC and ZnBuBPDC were tested for their alkane, alkene and CO2 adsorption capacities.
In the second part of the work, the surface modification of three different metal oxides, ZrO2, TiO2 and Al2O3 was performed. For this purpose firstly three different fluorescent phosphonate derivatives containing thiophene units were synthesized from their halo derivatives in a four step synthesis and then used as coupling molecules for the surface modification. Nine different surfaces were obtained (38@TiO2, 39@TiO2, 40@TiO2, 38@Al2O3, 39@Al2O3, 40@Al2O3, 38@ZrO2, 39@ZrO2, 40@ZrO2).
All three modified metal oxide surfaces were characterized using elemental analysis, solid state NMR and IR spectroscopy. The BET surface areas of the materials were determined by N2 adsorption measurements. TGA was used to determine the stability of the surfaces. Maximum loadings were obtained for ZrO2 surfaces.
Due to the strong luminescence of the coupling molecules, the modified surfaces were checked for their light emission. All ZrO2 and Al2O3 surfaces showed fluorescence with exception of 40@Al2O3. On the other hand, for the modified TiO2 surfaces, no fluorescence could be observed.
The scientific aim of this work was to synthesize and characterize new bidentate and tridentate phosphine ligands , their corresponding palladium complexes and to examine their application as homogenous catalysts. Later on, a part of the obtained palladium catalysts was immobilized and used as heterogonous catalyst.
Pyrimidinyl functionalized diphenyl phosphine ligands were synthesized by ring closure of [2-(3-dimethylamino-1-oxoprop-2-en-yl)phenyl]diphenylphosphine with an excess of substituted guanidinium salts. Furthermore to increase the electron density at phosphorous centre the two aryl substituents on the phosphanyl group were exchanged against two alkyl substituents. Electron rich pyrimidinyl functionalized dialkyl phosphine ligands were synthesized from pyrimidinyl functionalized bromobenzene in a process involving lithiation followed by reaction with a chlorodialkylphosphine.
Starting from the new synthesized diaryl phosphine ligands, their corresponding palladium complexes were synthesized. I was able to show that slight changes at the amino group of [(2-aminopyrimidin-4-yl)aryl]phosphines lead to pronounced differences in the stability and catalytic activity of the corresponding palladium(II) complexes. Having a P,C coordination mode, the palladium complex can catalyze rapidly the Suzuki coupling reaction of phenylbronic acid with arylbromides even at room temperature with a low loading.
Using the NH2 group of the aminopyrimidine as a potential site for the introduction of an other substituent, bidentate and tridentate ligands containing phosphorous atoms connected to the aminopyrimidine group and their corresponding palladium complexes were synthesized and characterized.
Two ligands [2- and 4-(4-(2-amino)pyrimidinyl)phenyl]diphenylphosphine (containing NH2 group) functionalized with a ethoxysilane group were synthesized. The palladium complexes based on these ligands were prepared and immobilized on commercial silica and MCM-41. Using elemental analysis, FT-IR, solid state 31P, 13C and 29Si CP–MAS NMR spectroscopy, XRD and N2 adsorption the success of the immobilization was confirmed and the structure of the heterogenized catalyst was investigated.
The resulting heterogeneous catalysts were applied for the Suzuki reaction and exhibited excellent activity, selectivity and reusability.
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.
The scientific intention of this work was to synthesize and characterize new bidentate, tridentate and multidentate ligands and to apply them in heterogenous catalysis. For each type of the ligands, new methods of synthesis were developed. Starting from 1,1'-(pyridine-2,6-diyl)diethanone and dimethylpyridine-2,6-dicarboxylate different bispyrazolpyridines were
synthesized and novel ruthenium complexes of the type (L)(NNN)RuCl2 could be obtained. The complexes with L = triphenylphosphine turned out to be highly efficient
catalyst precursors for the transfer hydrogenation of aromatic ketones. Introduction of a butyl group in the 5-positions of the pyrazoles leads to a pronounced increase of catalytic activity.
To find a method for the synthesis of bispyrimidinepyridines, different reactants and condition were applied and it was found that these tridentate ligands can be obtained by mixing and grinding the tetraketone with guanidinium carbonate and silica, which plays the role of a catalyst in this ring closing reaction.
The bidentate 2-amino-4-(2-pyridinyl)pyrimidines were synthesized from different substrates according to the desired substituent on the pyrimidine ring.
Reacting these bidentate ligands with the ruthenium(II) precursor [(η6-cymene)Ru(Cl)(μ
2-Cl)]2 gave cationic ruthenium(II) complexes of the type [(η6-cymene)Ru(Cl)(adpm)]Cl (adpm = chelating 2-amino-4-(2-yridinyl)pyrimidine ligand). Stirring the freshly prepared complexes with either NaBPh4, NaBF4 or KPF6, the chloride anion was exchanged against other coordinating anions (BF4-, PF6-, BPh4-).Some of these ruthenium complexes have shown very special activities in the transfer hydrogenation of ketones by reacting them in the absence of the base. This led to detailed investigations on the mechanism of this reaction. According to the activities and with the help
of ESI-MS experiments and DFT calculations, a mechanism was proposed for the transfer hydrogenation of acetophenone in the absence of the base. It shows that in the absence of the base, a C-H bond activation at the pyrimidine ring should occur to activate the catalyst.
The palladium complexes of bidentate N,N ligands were examined in coupling reactions. As expected, they did not show very special activities.
Multidentate ligands, having pyrimidine groups as relatively soft donors for late transition metals and simultaneously possessing a binding position for a hard Lewis-acid, could be obtained using the new synthesized bidentate and tridentate ligands.
Mechanisms underlying the biological effects of coffee and its constituents are incompletely understood. Many effects have been attributed solely to caffeine, neglecting that coffee is a mixture of many chemical substances. Some authors suggest that the main mechanism of action of caffeine is to antagonize adenosine receptors (AR); a second effect is the inhibition of phosphodiesterases with the subsequent accumulation of cAMP and an intensification of the effects of catecholamines. Although the inhibition of phosphodiesterases may contribute to the actions of caffeine, there is growing evidence that most pharmacological effects of this xanthine result from antagonism of AR.
One of the main objectives of this work was to investigate whether substances other than caffeine in coffee may influence the homeostasis of intracellular cyclic nucleotides in vitro and in vivo. The influence of selected coffee compounds, extracts and brews on key elements involved in the adenosine receptor-mediated signaling pathway have been investigated.
A further aim of this work was also to determine if coffee or some coffee constituents may have a stimulatory effect on the cellular heme oxygenase activity (HO-activity). Two coffee extracts, a slightly (AB1) and an intensively roasted coffee (AB2), were studied along with selected individual compounds. Caffeine and low substituted pyrazines showed no effect on the HO-activity, while NMP, pyrazines with a greater substitution pattern such as Tetramethylpyrazine (TMP) and 2-Ethyl-3,5(6)-dimethylpyrazine (2-E-3,5-DMP) and both coffee extracts significantly induced the HO-activity in liver hepatocellular carcinoma (HepG2), intestinal colo-rectal adenocarcinoma (Caco-2) and in some instances in monocytic leukemia (MM6) cells.
It was found that caffeine, theophylline, coffee extracts from conventional or functional coffees, pyrazines (2,3-DE-6-MP, 2-Isobutyl-3-methoxyP), 5-CQA and caffeic acid all significantly inhibited the basal cytoplasmatic PDE activity in lysates of lung tumour xenograft cells (LXFL529L) and human platelets. To a somewhat lesser extent, PDE inhibition was also found in experiments performed with paraxanthine and other pyrazines (2-E-3,5-DMP, TMP and 2-E-5-MP). Thus the degree of roasting has a considerable impact on constituents of influence for PDE activity. Caffeine, coffee polyphenols and some pyrazines and further, as yet unknown roasting products appear to represent the main modulating constituents.
In two coffee intervention studies, a short-term (8 weeks) and a long-term study (24 weeks), comprising 8 and 84 healthy volunteers respectively, we examined extracellular key elements of the adenosine pathway including plasma adenosine levels and adenosine deaminase activity. Additionally, we studied the intracellular cAMP concentration and the PDE activity in platelets as surrogate biomarkers of adipocytes.
Results of in vitro experiments had suggested that the concentrations of caffeine and coffee extracts required to obtain a half maximal inhibition were in the upper range of physiological conditions. Yet, it was demonstrated for the first time in vivo that moderate consumption of coffee can modulate the activity of platelet phosphodiesterases in humans in long and short term. In both studies, the first exposure to coffee showed a strong inhibition (p<0.001) of the PDE activity in the platelet lysates of the participants while the second coffee phase showed no or a slight effect when compared with the first coffee intervention.
In both studies a significant increase (p<0.001) in intraplatelet cAMP concentrations during the wash-out phase (after the first coffee phase) was observed. This response could be due to inhibition of the PDE activity in the previously phase extending in to the wash out phase. However, the behavior of cAMP in the following study phases cannot be easily explained. It may be hypothesized that this effect is attributable to adaptive effects to allow PDE inhibition. One possibility is the modulation of the expression of membrane-bound adenosine receptors in platelet precursors, which still have a nucleus. This may potentially influence adenylate cyclase activity in mature platelets. For the observed effects, in addition to caffeine other ingredients of coffee appear to play a role. The findings suggest that monitoring of cAMP homeostasis in platelets is not a useful surrogate biomarker for effects in other tissues.
Neither the activity of adenosine deaminase nor the adenosine concentrations in plasma were markedly modulated by the coffee consumption in both trials. This may reflect the fact that adenosine is subject to quick and effective enzymatic turnover by phosphorylation (adenosine kinase) or deamination (adenosine deaminase) allowing keep its concentration within a well balanced homeostasis. However, it is also well known, that considerable variability exists in the responses to coffee drinking. In part, such variability is due to caffeine tolerance, but there is also evidence for a genetic background.
Altogether the data reported here provide further evidence for the perception that coffee consumption is associated with beneficial health effects demonstrated for the cAMP enhancement in platelets, known to counteract platelet aggregation. The effects observed for the influence of cellular heme oxygenase (HO) are in line with the well documented antioxidative activity of coffee and its constituents.
Development of New Methods for the Synthesis of Aldehydes, Arenes and Trifluoromethylated Compounds
(2012)
In the 1st project, successful development of 2nd generation of a palladium catalyst for the selective hydrogenation of carboxylic acids to aldehydes was accomplished. This project was done in cooperation with Dipl. Chem. Thomas Fett from Boeringer Ingelheim, Austria. The new catalyst is highly effective for the conversion of diversely functionalized aromatic, heteroaromatic and aliphatic carboxylic acids to the corresponding aldehydes in the presence of pivalic anhydride at 5 bar hydrogen pressure, which was otherwise achieved either at 30 bar of hydrogen pressure or by using waste intensive hypophosphite bases as reducing agent. Our method has increased the synthetic importance of this valuable transformation. Selective hydrogenation of carboxylic acids to the corresponding aldehydes is now possible with industrial hydrogenation equipment as well as laboratory scale glass autoclaves. It might also convince the synthetic organic chemists to use this transformation for routine aldehyde synthesis in the laboratories.
In the 2nd project, a microwave assisted Cu-catalyzed protodecarboxylation of arenecarboxylic acids to arenes is achieved. This work was done in collaboration with Dipl. Chem. Filipe Manjolinho under the supervision of Dr. Nuria Rodríguez. In the presence of 1-5 mol% of inexpensive CuI/1,10-phenanthroline catalyst generated in situ under microwave radiations, diversely functionalized arenes and heteroarene carboxylic acids have been decarboxylated to the corresponding arenes in good yields at 190 °C in 5-15 min. The loss of volatile arenes with the release of CO2 is controled by the use of sealed high pressure resistant microwave vessels. These reactions are highly beneficial for parallel synthesis in drug discovery due to their short reaction time. Microwave technology will also help in the future to develop more effective catalysts for protodecarboxylation rections.
Based on the microwave assisted protodecarboxylation strategy, decarboxylative coupling of arenecarboxylic acids with aryl triflates and tosylates was also conducted under microwave radiation which provided higher yields of the corresponding biphenyls from deactivated substrates in short reaction time compared to the conventional heating.
In the 3rd project, crystalline, potassium (trifluoromethyl)trimethoxyborate was successfully applied for the synthesis of benzotrifluorides under the oxidative conditions. This project was done in cooperation with Dipl. Chem. Annette Buba. In the presence of Cu(OAc)2 and molecular oxygen, arylboronates were coupled with K+[CF3B(OMe)3] in DMSO at 60 °C. A variety of benzotriflurides was synthesized in good yields under the optimized reaction conditions. This protocol for the oxidative trifluoromethylation of arylboronates is the base for the development of decarboxylative trifluoromethylation reaction of arenecarboxylic acids.
The 4th project discloses the simple and straightforward synthesis of trifluoromethylated alcohols by nucleophilic addition of potassium (trifluoromethyl)trimethoxyborate to carbonyl compounds. This project was done in cooperation with Dr. Thomas Knauber and Dipl. Chem. Annette Buba. In the presence of K+[CF3B(OMe)3] in THF at 60 °C, diversely functionalized aldehydes and ketones were successfully converted into the corresponding trifluoromethylated alcohols.
The 3rd and 4th projects demonstrate the successful establishment of crystalline and shelf stable potassium (trifluoromethyl)trimethoxyborate as highly versatile CF3-source in nucleophilic trifluoromethylation reactions. These new protocols are characterized by their user-friendliness and broad applicability under mild reaction conditions, thus they are beneficial for late stage introduction of CF3-group into organic molecules.
The present PhD thesis is mainly focused on synthesis, characterization and catalytic application of functionalized triphenylphosphine (TPP) ligands and their complexes. We developed a simple and effective strategy to immobilize TPP: A methylester group attached to one of the phenyl rings of TPP allowes the derivatization of the ligand with 3-trimethoxysilylpropylamine, a typical silane coupling agent used for the covalent immobilization of organic compounds on silica surfaces. The resulting functionalized TPP was further coordinated to Pd, Rh and Ru precursors to achieve homogeneous complexes which can be tethered on silica by the post synthetic grafting method and co-condensation method. The obtained heterogeneous catalysts exhibited excellent activity, selectivity and reusability in Suzuki, hydrogenation and transfer hydrogenation reactions. In order to investigate the stability of the catalysts, different types of characterizations such as TEM, solid state NMR of the used catalysts as well as AAS of filtrate and leaching tests were carried out. The results prove the practicability and efficiency of our method. This strategy was further modified to generate an anionic side chain linked to the TPP core by simply replacing the trimethoxysilylpropylamine group by sodium(3-amino- 1-propanesulfonate), which allowes the immobilization on imidazolium modified SBA-15 through electrostatic interaction. The obtained material was further reacted with PdCl2(CNPh)2 and the resulting hybrid material was used for the hydrogenation of olefins allowing mild reaction conditions. The catalyst shows excellent activity, selectivity and stability and it can furthermore be reused for at least ten times without any loss of activity. TEM images of the used catalyst clearly show the absence of palladium nanoparticles, proving the high stability of the palladium compound. By AAS no palladium could be detected in the products and further leaching tests very- fied the reaction to be truly heterogeneous. This concept of non-covalent immobili- zation guarantees a tight bonding of the catalytically active species to the surface in combination with a high mobility, which should be favorable for other catalyses.
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.
A number of natural products are known that contain an enamide as a key structural feature. This functionality is a very important subunit in various biologically active products and pharmaceutical drug lead compounds. In addition, enamides serve as highly versatile synthetic intermediates, particularly in the pericyclic reaction, formation of heterocycles, cross-coupling and in asymmetric synthesis. As a result, several protocols have been devised for the preparation of enamides. Traditional syntheses include condensation of aldehydes and ketones with amides or from hydroxylamines and acetic anhydride, require harsh conditions and yield mixtures of E/Z products. Several metal catalyzed approaches have been also investigated, such as isomerization of N-allylamides and catalytic cross-coupling of amides with vinyl halides or pseudohalides. These protocols proceed under milder conditions but suffer from the limited availability of these starting materials. The research described in this dissertation focuses on efficient and atom-economic preparation of enamides and thioenamides, using readily available starting materials. We developed catalyst systems generated in situ from bis(2-methallyl)-cycloocta-1,5-diene-ruthenium(II), phosphines and Lewis acid or base, efficiently catalyze the addition of primary amides and thioamides to terminal alkynes with exclusive formation of the anti-Markovnikov products in high yield and stereoselectivity under mild reaction conditions. The generality of the newly developed methodologies is demonstrated by common functional group tolerance. Furthermore, Markovnikov products were formed via phosphine-catalyzed addition of cyclic amides to phenylacetylene derivatives. The hydroamidation protocol of primary amides was successfully used in the synthesis of naturally occurring compounds, such as alatamide, lansiumamide A, botryllamides C and E, and the key intermediate in the synthesis of aristolactam. In order to investigate the reaction mechanism, the addition of various amides and carboxylic acids to terminal alkynes was performed using deuterium labeled starting materials and followed by in situ NMR and GC-MS studies.
The main focus of this dissertation is the synthesis and characterization of more recent zeolites with different pore architectures. The unique shape-selective properties of the zeolites are important in various chemical processes and the new zeolites containing novel internal pore architectures are of high interest, since they could lead to further improvement of existing processes or open the way to new applications. This dissertation is organized in the following way: The first part is focused on the synthesis of selected recent zeolites with different pore architectures and their modification to the acidic and bifunctional forms. The second part comprises the characterization of the physicochemical properties of the prepared zeolites by selected physicochemical methods, viz. powder X-ray diffractometry (XRD), N2 adsorption, thermogravimetric analysis (TGA/DTA/MS), ultraviolet-visible (UV-Vis) spectroscopy, atomic absorption spectroscopy (AAS), infrared (IR) spectroscopy, scanning electron microscopy (SEM), 27Al and 29Si magic angle spinning nuclear magnetic resonance (MAS NMR) spectroscopy, temperature-programmed reduction (TPR), temperature-programmed desorption of pyridine (pyridine TPD) and adsorption experiments with hydrocarbon adsorptives. The third part of this work is devoted to the application of test reactions, i.e., the acid catalyzed disproportionation of ethylbenzene and the bifunctional hydroconversion of n-decane, to characterize the pore size and architecture of the prepared zeolites. They are known to be valuable tools for exploring the pore structure of zeolites. Finally, an additional test, viz. the competitive hydrogenation of 1-hexene and 2,4,4-trimethyl-1-pentene, has been applied to probe the location of noble metals in medium pore zeolite. The synthesis of the following zeolite molecular sieves was successfully performed in the frame of this thesis (they are ranked according to the largest window size in the respective structure): • 14-MR pores: UTD-1, CIT-5, SSZ-53 and IM-12 • 12-MR pores: ITQ-21 and MCM-68 • 10-MR pores: SSZ-35 and MCM-71 All of them were obtained as pure phase (except zeolite MCM-71 with a minor impurity phase that is hardly to avoid and also present in samples shown in the patent literature). The synthesis conditions are very critical with respect to the formation of the zeolite with a given structure. In this work, the recommended synthesis recipes are included. Among the 14-MR zeolites, the aluminosilicates UTD-1 (nSi/nAl = 28), CIT-5 (nSi/nAl = 116) and SSZ-53 (nSi/nAl = 55) with unidimensional extra-large pore opening formed from 14-MR rings exhibit promising catalytic properties with high thermal stability and they possess strong Brønsted-acid sites. By contrast, the germanosilicate IM-12 with a structure containing 14-MR channels intersecting with 12-MR channels is unstable toward moisture. It was found that UTD-1 and SSZ-53 zeolites are highly active catalysts for the acid catalyzed disproportionation of ethylbenzene and n-decane hydroconversion due to their high Brønsted acidity. To explore their pore structures, the applied two test reactions suggest that UTD-1, CIT-5 and SSZ-53 zeolites contain a very open pore system (12-MR or larger pore systems) because the product distributions are not hampered by too small pores. ITQ-21, a germanoaluminosilicate zeolite with a three-dimensional pore system and large spherical cages accessible through six 12-MR windows, can be synthesized with nSi/nAl ratios between 27 and >200. It possesses a large amount of Brønsted-acid sites. The aluminosilicate zeolite MCM-68 (nSi/nAl = 9) is an extremely active catalyst in the disproportionation of ethylbenzene and in the n-decane hydroconversion. This is due to the presence of a high density of strong Brønsted-acid sites in its structure. The disproportionation of ethylbenzene suggests that MCM-68 is a large pore (i.e., at least 12-MR) zeolite, in agreement with its crystallographic structure. In the hydroconversion of n-decane, the presence of tribranched and ethylbranched isomers and a high isopentane yield of 58 % in the hydrocracked products suggest the presence of large (12-MR) pores in its structure. By contrast, a relatively high value for CI* (modified constraint index) of 2.9 suggests the presence of medium (10-MR) pores in its structure. As a whole, the results are in-line with the crystallographic structure of MCM-68. SSZ-35, a 10-MR zeolite, can be synthesized in a broad range of nSi/nAl ratios between 11 and >500. This zeolite is interesting in terms of shape selectivity resulting from its unusual pore system having unidimensional channels alternating between 10-MR windows and large 18-MR cages. This thermally very stable zeolite contains both, strong Brønsted- and strong Lewis-acid sites. The disproportionation of ethylbenzene classifies SSZ-35 as a large pore zeolite. In the hydroconversion of n-decane, the suppression of bulky ethyloctanes and propylheptane clearly suggests the presence of 10-MR sections in the pore system. By contrast, the low CI* values of 1.2-2.3 and the high isopentane yields of 56-60 % in the hydrocracked products suggest that SSZ-35 also possesses larger intracystalline voids, i.e., the 18-MR cages. The results from the catalytic characterization are in good agreement with the crystallographic structure of zeolite SSZ-35. It was also found that the nSi/nAl ratio influences the crystallite size and therefore the external surface area. As a consequence, product selectivities are also influenced: The lowest nSi/nAl ratio or the smallest crystallite size sample produces larger amounts of the relatively bulky products. The formation of these products probably results from the higher conversion or they are preferentially formed on the external surface area of the catalyst. Zeolite MCM-71 (nSi/nAl = 8) possesses an extremely thermally stable structure and contains a high concentration of Brønsted-acid sites. Its structure allows for the separation of n-alkanes from branched alkanes by selective adsorption. MCM-71 exhibits unique shape-selective properties towards the product distribution in ethylbenzene disproportionation, which is different to those obtained in the medium pore SSZ-35 zeolite. All reaction parameters are fulfilled to classify MCM-71 as medium pore zeolite and this is in good agreement with its reported structure consisting of two-dimensional network of elliptical 10-MR channels and an orthogonal sinusoidal 8-MR channels. The competitive hydrogenation of 1-hexene and 2,4,4-trimethyl-1-pentene was exploited to probe that the major part of the noble metal is located inside the intracrystalline void volume of the medium pore zeolite SSZ-35.
Photochemical reactions are of great interest due to their importance in chemical and biological processes. Highly sensitive IR/UV double and triple resonance spectroscopy in molecular beam experiments in combination with ab initio and DFT calculations yields information on reaction coordinates and Intersystem Crossing (ISC) processes subsequent to photoexcitation. In general, molecular beam experiments enable the investigation of isolated, cold molecules without any influence of the environment. Furthermore, small aggregates can be analyzed in a supersonic jet by gradually adding solvent molecules like water. Conclusions concerning the interactions in solution can be derived by investigating and fully understanding small systems with a defined amount of solvent molecules. In this work the first applications of combined IR/UV spectroscopy on reactive isolated molecules and triplet states in molecular beams without using any messenger molecules are presented. Special focus was on excited state proton transfer reactions, which can also be described as keto enol tautomerisms. Various molecules such as 3-hydroxyflavone, 2-(2-naphthyl)-3-hydroxychromone and 2,5-dihydroxybenzoic acid have been investigated with regard to this question. In the case of 3-hydroxyflavone and 2-(2-naphthyl)-3-hydroxychromone, the IR spectra have been recorded subsequent to an excited state proton transfer. Furthermore the dihydrate of 3-hydroxyflavone has been analyzed concerning a possible proton transfer in the excited state: The proton transfer reaction along the water molecules (proton wire) has to be induced by raising the excitation energy. However, photoinduced reactions involve not only singlet but also triplet states. As an archetype molecule xanthone has been analysed. After excitation to the S2 state, ISC occurs into the triplet manifold leading to a population of the T1 state. The IR spectrum of the T1 state has been recorded for the first time using the UV/IR/UV technique without using any messenger molecules. Altogether it is shown that IR/UV double and triple resonance techniques are suitable tools to analyze reaction coordinates of photochemical processes.
The enamide moiety is an important substructure often encountered in biologically active compounds and synthetic drugs. Furthermore, enamides and their derivatives are versatile synthetic intermediates for polymerization, [4+2] cycloaddition, crosscoupling, Heck-olefinination, Halogenation, enantioselective addition or asymmetric hydrogenation. Traditional syntheses of this important substrate class involve rather harsh reaction conditions such as high temperatures and/or the use of strong bases. In continuation of our work on the addition of secondary amides to alkynes, we have developed a broadly applicable protocol for the catalytic addition of N-nucleophiles such as primary amides, imides and thioamides to terminal alkynes. The choice of ligands and additives determines the regiochemical outcome so that with two complementary catalyst systems, both the E-anti-Markovnikov products and the Z-anti-Markovnikov products can be synthesized highly regio- and stereoselectively.
It was recently reported that imatinib causes cell death in neonatal rat ventricular cardiomyocytes (NRVCM) by triggering endoplasmic reticulum (ER) stress and collapsed mitochondrial membrane potential. Retroviral gene transfer of an imatinib-resistant mutant c-Abl into NRVCM appeared to alleviate imatinib-induced cell death and it was concluded that the observed imatinib-induced cytotoxicity is mediated through direct interactions of imatinib with c-Abl. The imatinib effects were described as being specific for cardiomyocytes only, which are relevant also for the in vivo situation in man. [Kerkelä et al. 2006] The goal of the present study was to reproduce the published experiments and to further explore the dose-response relationship of imatinib-induced cell death in cardiomyocytes. Additional markers of toxicity were investigated. The following biochemical assays were applied: LDH release (membrane leakage marker), MTS-reduction (marker of mitochondrial integrity), ATP cellular contents (energy homoeostasis) and caspase 3/7 activity (apoptosis). The endoplasmatic reticulum (ER) stress markers eIF2α (elongation initiation factor 2α), XBP1 (X Box binding Protein 1), and CHOP (cAMP response element-binding transcription factor (C/EBP) homologous protein) were determined at the transcriptional and protein level. Online monitoring of cell attachment of, oxygen consumption and acidification of the medium by rat heart cells (H9c2) seated on chips (Bionas) allowed the determination of the onset and reversibility of cellular functions. Image analysis measured the spontaneous beating rates after imatinib treatment. The role of imatinib-induced reactive oxygen species was evaluated directly by 2’,7’-Dichlorofluorescein fluorescence and indirectly by means of interference experiments with antioxidants. The specificity of imatinib-induced effects were specific to cardiomyocytes was evaluated in fibroblasts derived from rat heart, lung and skin. The specific role of c-Abl in the imatinib-induced cellular toxicity was investigated by specific gene silencing of c-Abl in NRVCM. The results demonstrated that imatinib caused concentration-dependent cytotoxicity, apoptosis, and ER stress in heart, skin and lung fibroblasts, similar or stronger to those observed in cardiomyocytes. Similar to the results from cardiomyocytes, ER stress markers in fibroblasts were only increased at cytotoxic concentrations of imatinib. This effect was not reversible; also, reactive oxygen species did not participate in the mechanism of the imatinib-induced cytotoxicity in NRVCM. Small interfering RNA (siRNA)-mediated reduction of c-Abl mRNA levels by 51 % and c-Abl protein levels by 70 % had neither an effect on the spontaneous beating frequency of cardiomyocytes nor did it induce cytotoxicity, apoptosis, mitochondrial dysfunction or ER stress in NRVCM. Incubation of imatinib with c-Abl siRNA-transfected NRVCM suggested that reduced c-Abl protein levels did not rescue cardiomyocytes from imatinib-induced cytotoxicity. In conclusion, results from this study do not support a specific c-Abl-mediated mechanism of cytotoxicity in NRVCM.
A series of (oligo)phenthiazines, thiazolium salts and sulfonic acid functionalized organic/inorganic hybrid materials were synthesized. The organic groups were covalently bound on the inorganic surface through reactions of organosilane precursors with TEOS or with the silanol groups of material surface. These synthetic methods are called the co-condensation process and the post grafting. The structures and the textural parameters of the generated hybrid materials were characterized by XRD, N2 adsorption-desorption measurements, SEM and TEM. The incorporations of the organic groups were verified by elemental analysis, thermogravimetric analysis, FT-IR, UV-Vis, EPR, CV, as well as by 13C CP-MAS NMR and 29Si CP-MAS NMR spectroscopy. Introduction of various organic groups endow different phsysical, chemical properties to these hybrid materials. The (oligo)phenothiazines provide a group of novel redox acitive hybrid materials with special electronic and optic properties. The thiazolium salts modified materials were applied as heterogenized organo catalysts for the benzoin condensation and the cross-coupling of aldehydes with acylimines to yield a-amido ketones. The sulfonic acid containing materials can not only be used as Broensted acid catalysts, but also can serve as ion exchangable supports for further modifications and applications.
Colorectal cancer is the second most prevalent cancer form in both men and women in the Europe. In 2002, alimentary cancer (oesophagus, stomach, intestines) made up 26% of the annual incident cases of cancer amongst males in Europe, whereby about half of those were cancers of the colon and rectum (Eurostat 2002). Epidemiological evidence accumulating over the last decades indicates that besides a genetic disposition, diet plays a strong epigenetic role in the genesis of cancer. It is generally assumed that diet is causal for up to 80% of colorectal cancer (Bingham 2000). With the prospect of an approximated 50% rise in global cancer incidence over the first two decades of the 21st century, the World Health Organisation (WHO) has emphasized the need for an improvement in nutrition. Indeed there is increasing public health awareness with respect to nutrition. Today, living healthily is associated with less consumption of animal fats and red (processed) meat, moderate or no consumption of alcohol coupled with increased physical activity, and frequent intake of fruits, vegetables and whole grains (Bingham 1999; Johnson 2004). This idealogy partly stems from scientific epidemiological evidence supportive of an inverse correlation between the consumption of fruits and vegetables and the development cancer. Besides fibre and essential micro-nutrients like ascobate, folate, and tocopherols, the anti-carcinogenic properties of fruits and vegetables are generally thought to be rooted in the bioactivity of secondary plant components like flavonoids (Johnson 2004; Rice-Evans and Miller 1996; Rice-Evans 1995). Along with the increased public health awareness, has also come a burgeoning and lucrative dietary supplement industry, which markets products based on polyphenols and other potentially healthy compounds, sometimes with questionable promises of better health and increased longevity. These claims are based on accumulating in vitro and in vivo evidence indicating that flavonoids and polyphenols in fruits and vegetables can hinder proliferation, induce apoptosis of cancerous cells (Kern et al. 2005; Kumar et al. 2007; Thangapazham et al. 2007), act as antioxidants (Justino et al. 2006; Rice-Evans 1995) and influence cell signalling pathways (Marko et al. 2004; Joseph et al. 2007; Granado-Serrano et al. 2007), all of which are potential mechanisms proposed for their anti-carcinogenic activity. However, not only is the vast variety of supplements worrisome, but also problematic, is their easy accessibilty (just a click away on the internet) and the amount that can potentially be consumed. Such supplements are usually offered in pharmaceutical form (tablets, capsules, powder, concentrates) containing concentrations well beyond what is normally comsumable from the diet. For example, quercetin’s recommended intake is about 1g daily. However, estimates portend a possible daily increase of upto 1000 fold of the daily intake of quercetin (Hertog et al. 1995). Mindful of the concept of dose coined from the words of swiss scientist Paracelsus “What is it that is not poison? All things are poison and nothing is without poison. The right dose differentiates a poison and a remedy.” (“Alle Dinge sind Gift und nichts ist ohn’ Gift; allein die Dosis macht, dass ein Ding kein Gift ist”), it is thus conceivable that such high concentrations may not only reverse the acclaimed positive effects of flavonoids and polyphenols but also have negative effects thereby representing a health risk. The fact that direct evidence of the beneficial effects of flavonoids and polyphenols remains wanting, if not entirely lacking, coupled with the afore-mentioned marketing trend demands for a thorough examination of the possible adverse effects that may arise from increased consumption of flavonoids and polyphenols. The genesis and progression of cancer is usually accompanied by dysfunctional signalling of certain cell signalling pathways. Typical for colon carcinogenesis is the malfunctioning of the Wnt-signalling pathway, a pathway, which is crucial for the growth and development of normal colonocytes. The dysfunction of the Wnt-signalling pathway occurs in a manner that culminates in a proliferation stimulus of colonocytes, while differentiation is increasingly minimized. Hence, tumourigenesis is promoted. Interupting the proliferation stumuli by intervening in the actions of components of the Wnt-signalling pathway is one potential mechanism for the anti-carcinogenic action of flavonoids and polyphenols (Pahlke et al. 2006; Dashwood et al. 2002; Park et al. 2005). However, as previously hinted, the indulgence in the consumption of flavonoids and polyphenols based supplements could instead lead to a proliferation stimulus and provoke or promote carcinogenesis in normal cells or pre-cancerous cells respectively. The aim of this work was to
Within this thesis a series of molecular species has been studied, with focus on hydrogen bonded species and on (solvated) transition metal complexes. Experimental techniques such as FT-ICR-MS and IRMPD were combined with ab initio calculations for the determination of structure and reactivity of the aforementioned types of systems. On the basis of high level electronic structure calculations of neutral water clusters (H2O)n with n = 17-21 a transitional size regime has been determined, where a structural stabilization between all-surface and interior configurations alternates with the addition or removal of a single water molecule. Electronic structure calculations suggested that for n = 17 and 19 the interior configuration would be energetically more stable than the all-surface one. The gas phase infrared spectrum of the singly hydrated ammonium ion, NH4+(H2O), had previously been recorded by photodissociation spectroscopy of mass selected ions and interpreted by means of ab initio calculations. The present work provides additional information on the shape of the potential energy curves of NH4+(H2O) along the N-H distance on MP2/aug-cc-pVDZ level of theory yielding an anharmonic potential shape. Calculation of potential energy curves of the O-H mode of the intramolecular hydrogen bond of various dicarboxylic acids (oxalic to adipic acid) revealed that the shapes of the potentials directly correlate to the size of the system and the resulting ring strain The shape of the potential is also influenced by the charge of the system. Calculation of anharmonic frequencies based on the VPT2 approach lead to reasonable results in all systems with narrow potentials. IRMPD spectra of complexes in the gas phase have been recorded for a series of cationic vanadium oxide complexes when reacted with acetonitrile, methanol and ethanol. The experimental spectra are compared to calculated absorption spectra. The systematic DFT study identifies potential candidates for reductive nitrile coupling in cationic transition metal acetonitrile complexes. On the basis of the calculations, the formation of metallacyclic structures in group 3 through 7 complexes can be ruled out. Solvation of the transition metal cation by five acetonitrile ligands leads to a reductive nitrile coupling reaction in three types of complexes, namely those containing either niobium, tantalum or tungsten.
Acidic zeolites like H-Y, H-ZSM-5, H-MCM-22 and H-MOR zeolites were found to be the selective adsorbents for the removal of thiophene from toluene or n-heptane as solvent. The competitive adsorption of toluene is found to influence the adsorption capacity for thiophene and is more predominant when high-alumina zeolites are used as adsorbents. This behaviour is also reflected by the results of the adsorption of thiophene on H-ZSM-5 zeolites with varied nSi/nAl ratios (viz. 13, 19 and 36) from toluene and n-heptane as solvents, respectively. UV-Vis spectroscopic results show that the oligomerization of thiophene leads to the formation of dimers and trimers on these zeolites. The oligomerization in acid zeolites is regarded to be dependent on the geometry of the pore system of the zeolites. The sulphur-containing compounds with more than one ring viz. benzothiophene, which are also present in substantial amounts in certain hydrocarbon fractions, are not adsorbed on H-ZSM-5 zeolites. This is obvious, as the diameter of the pore aperture of zeolite H-ZSM-5 is smaller than the molecular size of benzothiophene. Metal ion-exchanged FAU-type zeolites are found to be promising adsorbents for the removal of sulphur-containing compounds from model solutions. The introduction of Cu+-, Ni2+-, Ce3+-, La3+- and Y3+- ions into zeolite Na+-Y by aqueous ion-exchange substantially improves the adsorption capacity for thiophene from toluene or n-heptane as solvent. More than the absolute content of Cu+-ions, the presence of Cu+-ions at the sites exposed to supercages is believed to influence the adsorption of thiophene on Cu+-Y zeolite. It was shown experimentally for the case of Cu+-Y and Ce3+-Y that the supercages present in the FAU zeolite allow for an access of bulkier sulphur-containing compounds (viz. benzothiophene, dibenzothiophene and dimethyl dibenzothiophene). The presence of these bulkier compounds compete with thiophene and are preferentially adsorbed on Cu+-Y zeolite. IR spectroscopic results revealed that the adsorption of thiophene on Na+-Y, Cu+-Y and Ni2+-Y is primarily a result of the interaction of thiophene via pi-complexation between C=C double bond (of thiophene) and metal ions (in the zeolite framework). A different mode of interaction of thiophene with Ce3+-, La3+- and Y3+-metal ions was observed in the IR spectra of thiophene adsorbed on Ce3+-Y, La3+-Y and Y3+-Y zeolites, respectively. On these adsorbents, thiophene is believed to interact via a lone electron pair of the sulphur atom with metal ions present in the adsorbent (M-S interaction). The experimental results show that there is a large difference in the thiophene adsorption capacities of pi-complexation adsorbents (like Cu+-Y, Ni2+-Y) between the model solution with toluene as solvent and the model solution with n-heptane as solvent. The lower capacity of these zeolites for the adsorption of thiophene from toluene than from n-heptane as solvent is the clear indication of competition of toluene in interating with adsorbent in a way similar to thiophene. The difference in thiophene adsorption capacities is very low in the case of adsorbents Ce3+-Y, La3+-Y and Y3+-Y, which are believed to interact with thiophene predominantly by direct M3+-S bond (thiophene interacting with metal ion via a lone pair of electrons). TG-DTA analysis was used to study the regeneration behaviour of the adsorbents. Acid zeolites can be regenerated by simply heating at 400 °C in a flow of nitrogen whereas thiophene is chemically adsorbed on the metal ion. By contrast, it is not possible to regenerate by heating under idle inert gas flow. The only way to regenerate these adsorbents is to burn off the adsorbate, which eventually brings about an undesired emission of SOx. The exothermic peaks appeared at different temperatures in the heat flow profiles of Cu+-Y, Ce3+-Y, La3+-Y and Y3+-Y are also indicating that two different types of interaction are present as revealed by IR spectroscopy, too. One major difficulty in reducing the sulphur content in fuels to value below 10 ppm is the inability in removing alkyl dibenzothiophenes, viz. 4,6 dimethyl dibenzothiophene, by the existing catalytic hydrodesulphurization technique. Cu+-Y and Ce3+-Y were found in the present study to adsorb this compound from toluene to a certain extent. To meet the stringent regulations on sulphur content, selective adsorption by zeolites could be a valuable post-purification method after the catalytic hydrodesulphurization unit.
Uncoupling protein1 (UCP1) in brown adipose tissue was discovered earlier as the main uncoupling source of respiration. We describe the basic facts and a modest contribution of our group to the area of research on mitochondrial uncoupling proteins. After defining the terms uncoupling, leak, proton-mediated uncoupling, we discuss the assumption that due to its low abundance, uncoupling protein 2 (UCP2) can provide only mild uncoupling, i.e. can decrease the proton motive force by several mV only. A fatty acid cycling mechanism is described as a plausible explanation for the protonophoretic function of all uncoupling proteins together with our experiments supporting it. A speculation for the phylogenesis of all uncoupling proteins can be deduced by estimated UCP2 content in several tissues, and details of its activation are explained on the basis of our experiments. In the present study a solubilization and refolding method for UCP2 from inclusion bodies was developed and characterized. As it was known and also demonstrated from previous experiments on UCP1 that fatty acids are substrates, we used the same procedure to study the function of UCP2. Utilizing spin-labelled fatty acids (SLFA) for our experiments we demonstrated the binding of fatty acids to UCP2, and the competition of other natural fatty acids like oleic acid, palmitic acid, arachidonic acid and eicosatrienoic acid to the preformed complex emphasizes the presence of a fatty acid binding site for mitochondrial UCP2. The findings were observed by EPR spectroscopy where the highly immobilized spectra with presence of spin-labelled fatty acid eventually end up as free spin label spectra with a particular concentration of the natural fatty acid added to the UCP2 bound with spin-labelled fatty acid. This fits in significantly with the earlier findings of UCP1 and also leads to assumption of functional explanation about the physiological relevance between the uncoupling proteins functions. The present study, in which representative and sensitive parameters for EPR spectroscopy were established, at the same time describes the concentration effects of fatty acids upon the protein bound with spin-labelled fatty acids which are much of importance in comparison to physiological levels, being in the micromolar range (µM) as compared with milli molar (mM) as for UCP1 previously. In appropriate examples, different fatty acids are used and compared with competitors like alkylsulfonates also emphasizing the function of the protein. And the studies with the effect of nucleotides inhibition demonstrate that there exists a putative binding site for fatty acids. Much significance lies in demonstration with the spin-labelled-ATP studies where competition of ATP to the protein bound to spin-labelled ATP explains about the inhibition effect of nucleotides on the UCP2. So the present study applies different methods for the functional characterization of UCP2. The studies of natural fatty acids and alkylsulfonates with UCP2 bound to spin-labelled fatty acid, and study of nucleotide inhibition on UCP2 are closely related and give the much awaited answer to the question of functional similarities between UCP1 and UCP2. This supports the discussion of many groups which predict the functional similarity between these two proteins based upon sequence homology. Also many attempts have been reported in literature to explain the physiological functional relevance where by this present study can also be added to as we now suppose from the present conclusions of our experiments.
Sterisch anspruchsvolle Cyclopentadienyl-Liganden wurden zur Stabilisierung neuer Mono(cyclopentadienyl) Verbindungen der schweren Erdalkalimetalle eingesetzt und deren Funktionalisierbarkeit dieser Spezies wurde exemplarisch durch die Synthese neutraler Tripeldecker-Sandwichkomplexe demonstriert. Die dabei ausgebildeten Molekülstrukturen lassen sich mittels DFT-Rechnungen zuverlässig vorhersagen. In diesem Zusammenhang wurde ebenfalls der Cyclononatetraenyl-Ligand, dessen Komplexeigenschaften bisher nur unzureichend untersucht wurden, eingesetzt. Im Rahmen dieser Arbeit gelang die Synthese des Bis(cyclononatetraenyl)bariums, Ba(C9H9)2, und dessen spektroskopische Charakterisierung. DFT-Rechnungen sagen für diesen Komplex eine Metallocenstruktur mit nahezu parallelen Ringen und einem Ba-Ring Abstand von 2.37 Å voraus. Durch den Einsatz des Tetraisopropylcyclopentadienyl (4Cp) und Tri(tert.-butyl)cyclopentadienyl (Cp’)-Liganden gelang die Synthese von Bis- und Monocyclopentadienyl-Verbindungen der frühen und späten Lanthanoide. Besonders interessant in diesem Zusammenhang ist die erfolgreiche Darstellung des Azido-Clusters, [Na(dme)3]2[4Cp6Yb6(N3)14] (4Cp= (Me2CH)4C5H), der die unterschiedlichen Koordinationsmöglichkeiten des Azido-Liganden in einem einzigen Komplex vereint. Vergleichbare Komplexe waren in der Organolanthanoidchemie bisher unbekannt. Durch Substitution am Cyclopentadienyl-System lassen sich dessen elektronische und sterische Eigenschaften signifikant verändern. Die Auswirkungen dieser Effekte können sehr eindrucksvoll an Manganocen-Komplexen demonstriert werden, in denen sich der low- und high-spin Zustand energetisch nur sehr wenig unterscheiden. Der elektronische Grundzustand einer Reihe unterschiedlich substituierter Manganocen-Komplexe wurde mittels Festkörpermagnetismus, ESR, Röntgenstrukturanalyse, EXAFS und variabler Temperatur UV-Vis Spektroskopie bestimmt, und mit dem Substitutionsmuster am Cyclopentadienyl-System korreliert. Spin-Gleichgewichte ließen sich für [(Me3C)C5H4]2Mn, [(Me3C)2C5H3]2Mn und [(Me3C)(Me3Si)C5H3]2Mn nachweisen. Theoretische Rechnungen postulieren, dass Cerocen, Ce(C8H8)2, ein Beispiel für Moleküle mit gemischt-konfiguriertem Grundzustand sei, der durch 80 % [(Ce)f1e2u(cot)e2u3] und 20 % [(Ce)f0e2u(cot)e2u4] beschreiben werden könne. Obwohl dieses Molekül bereits seit 1976 bekannt ist, ist dessen elektronische Struktur bis heute sehr umstritten. Im Rahmen dieser Arbeit wurden neue Synthesekonzepte für diese Verbindung entwickelt und die elektronische Struktur mittels magnetischer Messungen im Festkörper, EXAFS und XANES Studien untersucht. Die dabei erhaltenen Daten sind in sehr guter Übereinstimmung mit den theoretischen Rechnungen und belegen die Bedeutung eines gemischt-konfigurierten Grundzustandes bei der Bindung in Organometallkomplexen der f-Block Metalle. Während in Cerocen nur ein temperaturunabhängiger Paramagnetismus (TIP) beobachtet werden kann, findet man eine starke Temperaturabhängigkeit der magnetischen Suszeptibilität in Ytterbium Systemen des Typs Cp’2Yb(bipy’) [Cp´ und bipy´ sind substituierte Cyclopentadienyl- oder 4,4’-substituierter 2,2’-Bipyridyl-Liganden]. Temperaturabhängige XANES-Experimenten belegen, dass auch in diesen Systemen ein gemischt-konfigurierter Grundzustand vorliegt, der durch [(Yb)f14(bipy)b1()0] und [(Yb)f13(bipy)b1()1] beschreiben werden kann. Der relative Anteil beider Wellenfunktionen zum Grundzustand wird durch Substitution am 2,2’-Bipyridyl- oder Cyclopentadienyl-System signifikant beeinflusst. Modelle, mit denen sich dieses Verhalten qualitativ beschreiben lässt, wurden im Rahmen dieser Arbeit entwickelt. Ein kinetisch stabilisiertes, adduktfreies Titanocen wurde unter Verwendung des Di(tert.-butyl)cyclopentadienyl Liganden hergestellt und dessen Reaktivität gegenüber kleinen Molekülen, z.B. CO, N2 und H2 untersucht. Im Rahmen der Reaktivitätsstudien wurden ebenfalls 2,2’-Bipyridyl Addukte an das Cp’2Ti Fragment synthetisiert und deren magnetische Eigenschaften erforscht. Durch Variationen am 2,2’-Bipyridyl System lässt sich das Singlet-Triplet Splitting in diesem System gezielt steuern.
Metallocenes containing diarylethene type photochromic switches are synthesized, characterized and tested in polyolefin catalysts. Propylene polymerizations using unbridged bis(2,3-dibenzo[b]thiophen-3-yl)cyclopenta[b]thien-3-yl)zirconium dichloride/MAO (80) treated with 254nm UV irradiation produced bimodal polymer distributions by GPC. This was due to an increase in the low molecular weight fractions when the closed form of the catalyst/photoswitch was made. Comparison with similarly structured catalyst without photoisomerization properties did not produce bimodal polymer under identical conditions. Propylene polymerizations made with dimethylsilyl[(1,5-dimethyl-3-phenylcyclopenta[b]thien-6-yl)][(2,3-dibenzothien-3-yl)cyclopenta[b]thien-6-yl)]zirconium dichloride/MAO (86) with 254nm UV irradiation caused a 3 fold increase in the polymer molecular weight. Polymers made with ethylene and ethylene/hexene using (80) after UV irradiation did not show differences in measured polymer properties. Polymerizations with ethylene/ hexene mixtures using (86) had increased activity and co-monomer (hexene) incorporation with UV irradiation.
The HMG-CoA reductase inhibitors SIM, LOV, ATV, PRA, FV and NKS were investigated for their effects on human SkMCs. We were able to demonstrate that statins can induce oxidative stress (ROS formation, GSH-depletion, TBARS), apoptosis (, caspase-3 activity, nuclear morphology) and necrosis (LDH-leakage) in hSkMCs. After incubation with statins, the sequence of cellular events starts by the increased formation of ROS (30 min) followed by caspase-3 activation (2-4 hours) and necrosis (LDH-leakage) and formation of condensed and fragmented nuclei after 24-72 hours. It was shown that, antioxidants (NAC, DTT, TPGS, M-2 and M-3) and the HMG-CoA reductase downstream metabolites (MVA, F, FPP, GG and GGPP) protected against statin-induced ROS formation, caspase-3 activation and partially from necrosis. The caspase-3 inhibitor Ac-DEVD-CHO rescues cells partially from necrosis. These results suggest that the statin-induced necrosis is HMG-CoA dependent and occurs secondary to apoptosis, which by decrease of ATP is driven into necrosis. The increase of ATP observed at low concentrations and early time points suggest an increased glycolytic activity. This was confirmed by increased PDK-4 gene expression and increased PFK2/F-2,6-BPase expression both activator of glycolysis. Glycolysis was also confirmed for some statins by increased cellular lactate concentations. The consequence of PDK-4 mediated pyruvate dehydrogenase inactivation is the metabolic switching from fatty acid to amino acid from proteins as energy source. The oxidative stress hypothesis was further supported by the induction of the FOXO3A transcription factor, which is involved in regulating MnSOD-2 expression in the mitochondrium. The mechanism by which statins produce ROS is still not resolved. There is an indirect evidence from our experiments as well as from the literature, that immediately after the statin treatment, intracellular Ca2+ is mobilized due to HMG-CoA reductase inhibition, which after mitochondrial uptake could lead to increased ROS formation.
Under physiological conditions oxygen is constantly being converted to reactive oxygen intermediates, in mitochondria, peroxisomes, cytochrome p450 systems, macrophages, neutrophils and in plasma membranes. These reactive oxygen species (ROS) are toxic and therefore alter cell integrity leading to cell damage. To protect itself against this toxic effect of ROS, living systems have developed defence systems that scavenge ROS formation. These systems include some enzymes, transporting proteins and small antioxidant molecules for instance vitamin C and E. This thesis describes a study on the antioxidant chemistry and activity of vitamin C in vivo and in vitro systems using ESR spectroscopy. Also, a new method was designed to label ascorbic acid with a fluorescent marker. Moreover, some important criteria were considered for the evaluation and quantification of ascorbyl radicals in human blood plasma using two types of ESR spectrometers.
Clusters bridge the gap between single atoms or molecules and the condensed phase and it is the challenge of cluster science to obtain a deeper understanding of the molecular foundation of the observed cluster specific properties/reactivities and their dependence on size. The electronic structure of hydrated magnesium monocations [Mg,nH2O]+, n<20, exhibits a strong cluster size dependency. With increasing number of H2O ligands the SOMO evolves from a quasi-valence state (n=3-5), in which the singly occupied molecular orbital (SOMO) is not yet detached from the metal atom and has distinct sp-hybrid character, to a contact ion pair state. For larger clusters (n=17,19) these ion pair states are best described as solvent separated ion pair states, which are formed by a hydrated dication and a hydrated electron. With growing cluster size the SOMO moves away from the magnesium ion to the cluster surface, where it is localized through mutual attractive interactions between the electron density and dangling H-atoms of H2O ligands forming "molecular tweezers" HO-H (e-) H-OH. In case of the hydrated aluminum monocations [Al,nH2O]+,n=20, different isomers of the formal stoichiometry [Al,20H2O]+ were investigated by using gradient-corrected DFT (BLYP) and three different basic structures for [Al,20H2O]+ were identified: (a) [AlI(H2O)20]+ with a threefold coordinated AlI; (b) [HAlIII(OH)(H2O)19]+ with a fourfold coordinated AlIII; (c) [HAlIII(OH)(H2O)19]+ with a fivefold coordinated AlIII. In ground state [AlI(H2O)20]+ (a) which contains aluminum in oxidation state +1 the 3s2 valence electrons remain located at the aluminium monocation. Different than for open shell magnesium monocations no electron transfer into the hydration shell is observed for closed shell AlI. However, clusters of type (a) are high energy isomers (DE»+190 kJ mol-1) and the activation barrier for reaction into cluster type (b) or (c) is only approximately 14 kJ mol-1. The performed ab initio calculations reveal that unlike in [Mg,nH2O]+, n=7-17, for which H atom eliminiation is found to be the result of an intracluster redoxreaction, in [Al,nH2O]+,n=20, H2 is formed in an intracluster acid-base reaction. In [Mg,nH2O]+, n>17, the magnesium dication was found to coexist with a hydrated electron in larger cluster sizes. This proves that intermolecular electron delocalization - previously almost exclusively studied in (H2O)n- and (NH3)n- clusters - can also be an important issue for water clusters doped with an open shell metal cation or a metal anion. Structures and stabilities of hydrated magnesium water cluster anions with the formal stoichiometry [Mg,nH2O]-, n=1-11, were investigated by application of various correlated ab initio methods (MP2, CCSD, CCSD(T)). Metal cations surely have high relevance in numerous biological processes, and as most biological processes take place in aqueous solution hydrated metal ions will be involved. However, in biological systems solvent molecules (i.e. water) compete with different solvated chelate ligands for coordination sites at the metal ion and the solvent and chelate ligands are in mutual interactions with each other and the metal ion. These interactions were investigated for the hydration of ZnII/carnosine complexes by application of FT-ICR-MS, gas-phase H/D exchange experiments and supporting ab initio calculations. In the last chapter of this work the Free Electron Laser IR Multi Photon Dissocition (FEL-IR-MPD) spectra of mass selected cationic niobium acetonitrile complexes with the formal stoichiometry [Nb,nCH3CN]+, n=4-5, in the spectral range 780 – 2500 cm-1 are reported. In case of n=4 the recorded vibrational bands are close to those of the free CH3CN molecule and the experimental spectra do not contain any evident indication of a potential reaction beyond complex formation. By comparison with B3LYP calculated IR absorption spectra the recorded spectra are assigned to high spin (quintet, S=2), planar [NbI(NCCH3)4]+. In [Nb,nCH3CN]+, n=5, new vibrational bands shifted away from those of the acetonitrile monomer are observed between 1300 – 1550 cm-1. These bands are evidence of a chemical modification due to an intramolecular reaction. Screening on the basis of B3LYP calculated IR absorption spectra allow for an assignment of the recorded spectra to the metallacyclic species [NbIII(NCCH3)3(N=C(CH3)C(CH3)=N)]+ (triplet, S=1), which has formed in a internal reductive nitrile coupling reaction from [NbI(NCCH3)5]+. Calculated reaction coordinates explain the experimentally observed differences in reactivity between ground state [NbI(NCCH3)4]+ and [NbI(NCCH3)5]+. The reductive nitrile coupling reaction is exothermic and accessible (Ea=49 kJ mol-1) only in [NbI(NCCH3)5]+, whereas in [NbI(NCCH3)4]+ the reaction is found to be endothermic and retarded by significantly higher activation barriers (Ea>116 kJ mol-1).
Lung cancer, mainly caused by tobacco smoke, is the leading cause of cancer mortality. Large efforts in prevention and cessation have reduced smoking rates in the U.S. and other countries. Nevertheless, since 1990, rates have remained constant and it is believed that most of those currently smoking (~25%) are addicted to nicotine, and therefore are unable to stop smoking. An alternative strategy to reduce lung cancer mortality is the development of chemopreventive mixtures used to reduce cancer risk. Before entering clinical trails, it is crucial to know the efficacy, toxicity and the molecular mechanism by which the active compounds prevent carcinogenesis. 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosonornicotine (NNN) and benzo[a]pyrene (B[a]P) are among the most carcinogenic compounds in tobacco smoke. All have been widely used as model carcinogens and their tumorigenic activities are well established. It is believed that formation of DNA adducts is a crucial step in carcinogenesis. NNK and NNN form 4-hydroxy-1-(3-pyridyl)-1-butanone releasing and methylating adducts, while B[a]P forms B[a]P-tetraol-releasing adducts. Different isothiocyanates (ITCs) are able to prevent NNK-, NNN- or B[a]P-induced tumor formation, but relative little is know about the mechanism of these preventive effects. In this thesis, the influence of different ITCs on adduct formation from NNK plus B[a]P and NNN were evaluated. Using an A/J mouse lung tumor model, it was first shown that the formation of HPB-releasing, O6-mG and B[a]P-tetraol-releasing adducts were not affected when NNK and B[a]P were given individually or in combination, of by gavage. Using the same model, the effects of different mixtures of PEITC and BITC, given by gavage or in the diet, on DNA adduct formation were evaluated. Dietary treatment with phenethyl isothiocyanate (PEITC) or PEITC plus benzyl isothiocyanate (BITC) reduced levels of HPB-releasing adducts by 40*50%. This is consistent with a previously shown 40% inhibition of tumor multiplicity for the same treatment. In the gavage treatments with ITCs it seemed that PEITC reduced HPB-releasing DNA adducts, while levels of BITC counteracted these effects. Levels of O6-mG were minimally affected by any of the treatments. Levels of B[a]P-tetraol releasing adducts were reduced by gavaged PEITC Summary Page XII and BITC, 120 h after the last carcinogen treatment, while dietary treatment had no effects. We then extended our investigation to F-344 rats by using a similar ITC treatment protocol as in the mouse model. NNK was given in the drinking water and B[a]P in diet. Dietary PEITC reduced the formation of HPB-releasing globin and DNA adducts in lung but not in liver, while levels of B[a]P-tetraol-releasing adducts were unaffected. Additionally, the effects of PEITC, 3-phenlypropyl isothiocyanate, and their N-acetylcystein conjugates in diet on adducts from NNN in drinking water were evaluated in rat esophageal DNA and globin. Using a protocol known to inhibit NNNinduced esophageal tumorigenesis, the levels of HPB-releasing adduct levels were unaffected by the ITCs treatment. The observations that dietary PEITC inhibited the formation of HPB-releasing DNA adducts only in mice where the control levels were above 1 fmol/µg DNA and adduct levels in rat lung were reduced to levels seen in liver, lead to the conclusion that in mice and rats, there are at least two activation pathway of NNK. One is PEITC-sensitive and responsible for the high adduct levels in lung and presumably also for higher carcinogenicity of NNK in lung. The other is PEITC-insensitive and responsible for the remaining adduct levels and tumorigenicity. In conclusion, our results demonstrated that the preventive mechanism by which ITCs inhibit carcinogenesis is only in part due to inhibition of DNA adduct formation and that other mechanisms are involved. There is a large body of evidence indicating that induction of apoptosis may be a mechanism by which ITCs prevent tumor formation, but further studies are required.
The development of recombinant DNA techniques opened a new era for protein production both in scientific research and industrial application. However, the purification of recombinant proteins is very often quite difficult and inefficient. Therefore, we tried to employ novel techniques for the expression and purification of three pharmacologically interesting proteins: the plant toxin gelonin; a fusion protein of gelonin and the extracellular domain of the subunit of the acetylcholine receptor (gelonin-AchR) and human neurotrophin 3 (hNT3). Recombinant gelonin, acetylcholine receptor a subunit and their fusion product, gelonin-AchR were constructed and expressed. The gelonin gene, a 753 bp polynucleotide was chemically synthesized by Ya-Wei Shi et al. and was kindly provided to us. The gene was first inserted into the vector pUC118 yielding pUC-gel. It was subsequently transferred into pET28a and pET-gel was expressed in E. coli. The product, gelonin was soluble and was purified in two steps showing a homogeneous band corresponding to 28 kD on SDS-PAGE. The expression of the extracellular domain of the -subunit of AchR always led to insoluble aggregates and even upon coexpression with the chaperonin GroESL, very small and hardly reproducible amounts of soluble material were formed, only. Therefore, recombinant AchR- gelonin was cloned and expressed in the same host. The corresponding fusion protein, gelonin-AchR, again formed aggregates and it had to be solubilized in 6 M Gu-HCl for further purification and refolding. The final product, however, was recognized by several monoclonal antibodies directed against the extracellular domain of the -subunit of AchR as well as a polyclonal serum against gelonin. Expression and purification of recombinant hNT3 was achieved by the use of a protein self-splicing system. Based on the reported hNT3 DNA sequence, a 380 bp fragment corresponding to a 14 kD protein was amplified from genomal DNA of human whole blood by PCR. The DNA fragment was cloned into the pTXB1 vector, which contains a DNA fragment of intein and chintin binding domain (CBD). A further construct, pJLA-hNT3, is temperature-inducible. Both constructs expressed the target protein, hNT3-intein-CBD in E. coli by the induction with IPTG or temperature, however, as aggregates. After denaturation and renaturation, the soluble fusion protein was slowly loaded on an affinity column of chitin beads. A 14 kD hNT3 could be isolated after cleavage with DTT either at 4 °C or 25 °C for 48 h. Based on nerve fiber out-growth of the dorsal root ganglia of chicken embryos, both, hNT-3-intein-CBD and hNT3 itself exhibit almost the same biological activity.