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.
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).
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.
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
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.
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.
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.
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.
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.
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.