Kaiserslautern - Fachbereich Chemie
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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.
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.
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 \([Ta_n(N_2)_m]^-\).
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.
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.
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.
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).
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.
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.
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.