In dieser Arbeit wird über die Entwicklung von neuartigen PET-Tracern für die In-vivo-Bildgebung von Cystein-Cathepsinen, Lysyloxidasen und Transglutaminase 2 als tumorassoziierte Matrix-modifizierende Enzyme berichtet. Dies beinhaltet im Einzelnen die Identifikation von Leitverbindungen, die Synthese und biochemische Charakterisierung von Analoga, die Etablierung von Methoden für deren Radiomarkierung sowie radiopharmakologische Untersuchungen auf molekularer, zellulärer und organismischer Ebene. In Kapitel 1 dieser Habilitationsschrift wird zunächst auf die Bedeutung der extrazellulären Matrix für die Tumorprogression eingegangen, wobei auch der Entwicklungsstatus Matrix-gerichteter Bildgebungssonden gestreift wird. Da die Hemmung der genannten Enzyme über die bildgebende funktionelle Diagnostik von Tumoren hinaus großes Potential im Hinblick auf die Pharmakotherapie neoplastischer Erkrankungen aufweist, wurde am Schluss von Kapitel 1 ebenso die generelle Bedeutung der PET- und SPECT-Bildgebung für den Prozess der Arzneistoffentwicklung dargelegt, da eine weitere Motivation dieser Arbeit in der Entwicklung von Sonden zur bildgebungsgestützten Therapie lag. Im Kapitel 2 wird eine Übersicht über strukturelle und funktionelle Aspekte der aufgeführten Matrix-modifizierenden Enzyme unter besonderer Berücksichtigung ihrer jeweiligen Funktion im Tumorgeschehen gegeben. Daran anschließend wird in den Kapiteln 3 und 4 der Stand zur Entwicklung von Inhibitoren bzw. Bildgebungssonden für diese Enzyme im Überblick dargestellt. Die Ergebnisse der Arbeit werden im Kapitel 5 präsentiert, wobei die sich Gliederung dieses Kapitels an den publizierten Arbeiten orientiert, die in diese kumulative Habilitationsschrift Eingang gefunden haben. Es handelt sich dabei im Wesentlichen um eine Kurzdarstellung der veröffentlichten Originalartikel, die durch weiterführende Aspekte ergänzt wurden, um den Bezug zwischen den einzelnen Arbeiten herzustellen. Kapitel 6 gibt eine kurze Gesamtzusammenfassung der Arbeit, an das Literaturverzeichnis in Kapitel 7 schließt sich mit Kapitel 8 die kumulative Zusammenstellung der zum Thema der Arbeit vom Autor veröffentlichten Zeitschriftenartikel an.:Vorbemerkungen und Zielstellung 1 1. Einführung 3 1.1. Bedeutung der extrazellulären Matrix für die Tumorprogression 3 1.2. Radiomarkierte Sonden zur Bildgebung der tumorassoziierten extrazellulären Matrix 14 1.3. Bedeutung der radiotracerbasierten Bildgebung in der Wirkstoffentwicklung 19 2. Strukturelle und biochemische Aspekte von Matrix-modifizierenden Enzymen: Cystein-Cathepsine, Lysyloxidasen und Transglutaminase 2 24 2.1. Cystein-Cathepsine 24 2.2. Funktionen von Cystein-Cathepsinen in der Tumorprogression 27 2.3. Lysyloxidasen 32 2.4. Funktionen von Lysyloxidasen in der Tumorprogression 36 2.5. Transglutaminase 2 42 2.6. Funktionen der Transglutaminase 2 in der Tumorprogression 46 3. Stand der Entwicklung von Inhibitoren der betrachteten Matrix-modifizierenden Enzyme 50 3.1. Inhibitoren von Cystein-Cathepsinen 50 3.2. Inhibitoren von Lysyloxidasen 61 3.3. Inhibitoren der Transglutaminase 2 64 4. Stand der Entwicklung von Bildgebungssonden für die betrachteten Matrix-modifizierenden Enzyme 70 4.1. Sonden für Cystein-Cathepsine 70 4.2. Sonden für Lysyloxidasen 74 4.3. Sonden für die Transglutaminase 2 76 5. Eigene Arbeiten zur Entwicklung von Radiotracern einschließlich der Identifikation, Synthese und Evaluierung geeigneter Liganden zur Bildgebung der vorgestellten Targetklassen 80 5.1. Entwicklung zu Cystein-Cathepsine 80 5.1.1. Auswahl der Leitverbindungen 80 5.1.2. Synthese und radiopharmakologische Charakterisierung eines 18F-markierten Azadipeptidnitrils 81 5.1.3. Synthese und radiopharmakologische Charakterisierung eines 11C-markierten Azadipeptidnitrils 87 5.1.4. Cyanohydrazide als potentielle chemoselektive Markierungsbausteine 91 5.1.5. Zusammenfassung und Ausblick 94 5.2. Lysyloxidasen 95 5.2.1. Auswahl der Leitverbindungen 95 5.2.2. Entwicklung einer Methode zur regioselektiven Markierung von Peptiden mit Fluor-18 97 5.2.3. Synthese und Konformationsanalyse eines N-Telopeptid-abgeleiteten Cyclopeptids 103 5.2.4. Radiopharmakologische Charakterisierung von N-Telopeptid-abgeleiteten Peptiden im Melanom-Xenograft-Mausmodell 109 5.2.5. Radiopharmakologische Charakterisierung eines N-Telopeptid-abgeleiteten Peptides in murinen Mammakarzinommodellen 114 5.2.6. Zusammenfassung und Ausblick 118 5.3. Transglutaminase 2 121 5.3.1. Auswahl der Leitverbindungen 121 5.3.2. Entwicklung von Assaymethoden und Synthese der dafür benötigten Substratverbindungen 123 5.3.3. Synthese und in-vitro-pharmakologische Charakterisierung von Nε-Acryloyllysinpiperaziden als irreversible Inhibitoren 137 5.3.4. 18F-Markierung und radiopharmakologische Charakterisierung eines Nε-Acryloyllysinpiperazids als aktivitätsbasierte Sonde 152 5.3.5. Zusammenfassung und Ausblick 167 6. Zusammenfassung und Schlussfolgerungen / Summary and Conclusions 171 7. Literaturverzeichnis 177 8. Kumulative Zusammenstellung der publizierten Arbeiten 243 8.1. Veröffentlichte Arbeiten zur Entwicklung Cystein-Cathepsin-gerichteter Radiotracer und Cyanohydraziden als potentielle Markierungsbausteine 243 8.1.1. Übersichtsartikel: “Cysteine cathepsins: their role in tumor progression and recent trends in the development of imaging probes” 243 8.1.2. Originalartikel: “Synthesis and Radiopharmacological Characterisation of a Fluorine-18-Labelled Azadipeptide Nitrile as a Potential PET Tracer for in vivo Imaging of Cysteine Cathepsins” 280 8.1.3. Originalartikel: “Synthesis and Radiopharmacological Characterisation of an 11C‐labelled azadipeptide nitrile as potential PET tracer for imaging of cysteine cathepsins” 304 8.1.4. Originalartikel: “Synthesis and X-ray Crystal Structure of N’-Cyano-N,N’-dimethyl-4-nitrobenzohydrazide” 319 8.2. Veröffentlichte Arbeiten zur Entwicklung Lysyloxidase-gerichteter Radiotracer und zur selektiven 18F-Markierung Lysin-enthaltender Peptide 328 8.2.1. Originalartikel: “Site-selective radiolabeling of peptides by 18F-fluorobenzoylation with [18F]SFB in solution and on solid phase: a comparative study” 328 8.2.2. Originalartikel: “Synthesis, 18F-labelling and radiopharmacological characterisation of the C-terminal 30mer of Clostridium perfringens enterotoxin as a potential claudin-targeting peptide” 350 8.2.3. Originalartikel: “Cyclopeptides containing the DEKS motif as conformationally restricted collagen telopeptide analogues: synthesis and conformational analysis” 388 8.2.4. Originalartikel: “ Evaluation of Fluorine-18-Labeled α1(I)-N-Telopeptide Analogs as Substrate-Based Radiotracers for PET Imaging of Melanoma-Associated Lysyl Oxidase” 428 8.2.5. Originalartikel: “Targeting lysyl oxidase for molecular imaging in breast cancer” 453 8.3. Veröffentlichte Arbeiten zur Entwicklung von TGase 2-gerichteten Radiotracern sowie von Substratverbindungen und Assaymethoden für dieses Enzym 470 8.3.1. Übersichtsartikel: “Tissue transglutaminase: An emerging target for therapy and imaging” 470 8.3.2. Originalartikel: ”Synthesis and Kinetic Characterisation of Water‐Soluble Fluorogenic Acyl Donors for Transglutaminase 2“ 487 8.3.3. Originalartikel: “Solution-phase synthesis of the fluorogenic TGase 2 acyl donor Z-Glu(HMC)-Gly-OH and its use for inhibitor and amine substrate characterisation” 543 8.3.4. Originalartikel: “A fluorescence anisotropy-based assay for determining the activity of tissue transglutaminase” 562 8.3.5. Originalartikel: “Nε-Acryloyllysine Piperazides as Irreversible Inhibitors of Transglutaminase 2: Synthesis, Structure–Activity Relationships, and Pharmacokinetic Profiling” 589 / This work reports on the development of novel PET tracers for in vivo imaging of cysteine cathepsins, lysyl oxidases and transglutaminase 2 as tumour-associated matrix-modifying enzymes. Specifically, this includes the identification of lead compounds, the synthesis and biochemical characterisation of analogues, the establishment of methods for their radiolabelling, and radiopharmacological studies at the molecular, cellular and organismal levels. Chapter 1 of this habilitation thesis first discusses the importance of the extracellular matrix for tumour progression: In addition, the development status of matrix-directed imaging probes is reviewed. Since the inhibition of the aforementioned enzymes has great potential beyond the imaging functional diagnosis of tumours with regard to the pharmacotherapy of neoplastic diseases, the general importance of PET and SPECT imaging for the drug development process was also outlined at the end of chapter 1, since a further motivation of this thesis was provided by the potential use of probes for imaging-assisted therapy. In chapter 2, an overview of structural and functional aspects of the listed matrix-modifying enzymes is given with particular reference to their respective function in tumour processes. This is followed by an overview of the status of the development of inhibitors and imaging probes for these enzymes in chapters 3 and 4. The results of the work are presented in chapter 5, whereby the structure of this chapter is oriented towards the published work that has found its way into this cumulative habilitation thesis. This chapter is essentially a brief presentation of the original published articles, supplemented by further aspects to establish the relationship between the individual papers. Chapter 6 provides a brief overall summary of the thesis, and the bibliography in Chapter 7 is followed by Chapter 8, which provides a cumulative compilation of the journal articles published by the author on the topic of the thesis.:Vorbemerkungen und Zielstellung 1 1. Einführung 3 1.1. Bedeutung der extrazellulären Matrix für die Tumorprogression 3 1.2. Radiomarkierte Sonden zur Bildgebung der tumorassoziierten extrazellulären Matrix 14 1.3. Bedeutung der radiotracerbasierten Bildgebung in der Wirkstoffentwicklung 19 2. Strukturelle und biochemische Aspekte von Matrix-modifizierenden Enzymen: Cystein-Cathepsine, Lysyloxidasen und Transglutaminase 2 24 2.1. Cystein-Cathepsine 24 2.2. Funktionen von Cystein-Cathepsinen in der Tumorprogression 27 2.3. Lysyloxidasen 32 2.4. Funktionen von Lysyloxidasen in der Tumorprogression 36 2.5. Transglutaminase 2 42 2.6. Funktionen der Transglutaminase 2 in der Tumorprogression 46 3. Stand der Entwicklung von Inhibitoren der betrachteten Matrix-modifizierenden Enzyme 50 3.1. Inhibitoren von Cystein-Cathepsinen 50 3.2. Inhibitoren von Lysyloxidasen 61 3.3. Inhibitoren der Transglutaminase 2 64 4. Stand der Entwicklung von Bildgebungssonden für die betrachteten Matrix-modifizierenden Enzyme 70 4.1. Sonden für Cystein-Cathepsine 70 4.2. Sonden für Lysyloxidasen 74 4.3. Sonden für die Transglutaminase 2 76 5. Eigene Arbeiten zur Entwicklung von Radiotracern einschließlich der Identifikation, Synthese und Evaluierung geeigneter Liganden zur Bildgebung der vorgestellten Targetklassen 80 5.1. Entwicklung zu Cystein-Cathepsine 80 5.1.1. Auswahl der Leitverbindungen 80 5.1.2. Synthese und radiopharmakologische Charakterisierung eines 18F-markierten Azadipeptidnitrils 81 5.1.3. Synthese und radiopharmakologische Charakterisierung eines 11C-markierten Azadipeptidnitrils 87 5.1.4. Cyanohydrazide als potentielle chemoselektive Markierungsbausteine 91 5.1.5. Zusammenfassung und Ausblick 94 5.2. Lysyloxidasen 95 5.2.1. Auswahl der Leitverbindungen 95 5.2.2. Entwicklung einer Methode zur regioselektiven Markierung von Peptiden mit Fluor-18 97 5.2.3. Synthese und Konformationsanalyse eines N-Telopeptid-abgeleiteten Cyclopeptids 103 5.2.4. Radiopharmakologische Charakterisierung von N-Telopeptid-abgeleiteten Peptiden im Melanom-Xenograft-Mausmodell 109 5.2.5. Radiopharmakologische Charakterisierung eines N-Telopeptid-abgeleiteten Peptides in murinen Mammakarzinommodellen 114 5.2.6. Zusammenfassung und Ausblick 118 5.3. Transglutaminase 2 121 5.3.1. Auswahl der Leitverbindungen 121 5.3.2. Entwicklung von Assaymethoden und Synthese der dafür benötigten Substratverbindungen 123 5.3.3. Synthese und in-vitro-pharmakologische Charakterisierung von Nε-Acryloyllysinpiperaziden als irreversible Inhibitoren 137 5.3.4. 18F-Markierung und radiopharmakologische Charakterisierung eines Nε-Acryloyllysinpiperazids als aktivitätsbasierte Sonde 152 5.3.5. Zusammenfassung und Ausblick 167 6. Zusammenfassung und Schlussfolgerungen / Summary and Conclusions 171 7. Literaturverzeichnis 177 8. Kumulative Zusammenstellung der publizierten Arbeiten 243 8.1. Veröffentlichte Arbeiten zur Entwicklung Cystein-Cathepsin-gerichteter Radiotracer und Cyanohydraziden als potentielle Markierungsbausteine 243 8.1.1. Übersichtsartikel: “Cysteine cathepsins: their role in tumor progression and recent trends in the development of imaging probes” 243 8.1.2. Originalartikel: “Synthesis and Radiopharmacological Characterisation of a Fluorine-18-Labelled Azadipeptide Nitrile as a Potential PET Tracer for in vivo Imaging of Cysteine Cathepsins” 280 8.1.3. Originalartikel: “Synthesis and Radiopharmacological Characterisation of an 11C‐labelled azadipeptide nitrile as potential PET tracer for imaging of cysteine cathepsins” 304 8.1.4. Originalartikel: “Synthesis and X-ray Crystal Structure of N’-Cyano-N,N’-dimethyl-4-nitrobenzohydrazide” 319 8.2. Veröffentlichte Arbeiten zur Entwicklung Lysyloxidase-gerichteter Radiotracer und zur selektiven 18F-Markierung Lysin-enthaltender Peptide 328 8.2.1. Originalartikel: “Site-selective radiolabeling of peptides by 18F-fluorobenzoylation with [18F]SFB in solution and on solid phase: a comparative study” 328 8.2.2. Originalartikel: “Synthesis, 18F-labelling and radiopharmacological characterisation of the C-terminal 30mer of Clostridium perfringens enterotoxin as a potential claudin-targeting peptide” 350 8.2.3. Originalartikel: “Cyclopeptides containing the DEKS motif as conformationally restricted collagen telopeptide analogues: synthesis and conformational analysis” 388 8.2.4. Originalartikel: “ Evaluation of Fluorine-18-Labeled α1(I)-N-Telopeptide Analogs as Substrate-Based Radiotracers for PET Imaging of Melanoma-Associated Lysyl Oxidase” 428 8.2.5. Originalartikel: “Targeting lysyl oxidase for molecular imaging in breast cancer” 453 8.3. Veröffentlichte Arbeiten zur Entwicklung von TGase 2-gerichteten Radiotracern sowie von Substratverbindungen und Assaymethoden für dieses Enzym 470 8.3.1. Übersichtsartikel: “Tissue transglutaminase: An emerging target for therapy and imaging” 470 8.3.2. Originalartikel: ”Synthesis and Kinetic Characterisation of Water‐Soluble Fluorogenic Acyl Donors for Transglutaminase 2“ 487 8.3.3. Originalartikel: “Solution-phase synthesis of the fluorogenic TGase 2 acyl donor Z-Glu(HMC)-Gly-OH and its use for inhibitor and amine substrate characterisation” 543 8.3.4. Originalartikel: “A fluorescence anisotropy-based assay for determining the activity of tissue transglutaminase” 562 8.3.5. Originalartikel: “Nε-Acryloyllysine Piperazides as Irreversible Inhibitors of Transglutaminase 2: Synthesis, Structure–Activity Relationships, and Pharmacokinetic Profiling” 589

Die vorliegende Arbeit beschäftigt sich mit der Stabilisierung von SFx- (x = 2-3) und SOyFz-Liganden (y, z = 1-2) in der Koordinationssphäre von Platinkomplexen, die den N-heterozyklischen Carben- (NHC)-Liganden 1,3-Bis(2,4,6-trimethylphenyl)-2-imidazolinyliden (IMes) besitzen. Die Synthese des SF3-Komplexes trans-[Pt(F)(SF3)(IMes)2] gelang durch Umsetzung von [Pt(IMes)2] mit SF4 oder SF6. Bei der SF6-Aktivierungsreaktion kam es zur Bildung von zusätzlichen fluorierten Nebenprodukten, welche durch den Vergleich mit den jeweiligen unabhängig synthetisierten Verbindungen charakterisiert wurden. Die starke Neigung zur Hydrolyse der SF3-Einheit bei Kontakt mit H2O führte zur Bildung von trans-[Pt(F)(SOF)(IMes)2]. Eine alternative Darstellung des S(=O)F-Komplexes konnte durch Umsatz von [Pt(IMes)2] mit SOF2 erreicht werden. Die S(=O)2F-Komplexe trans-[Pt(X)(SO2F)(IMes)2] (X = F, Cl) mit dem Schwefel in der formalen Oxidationsstufe IV wurden durch die Behandlung von [Pt(IMes)2] mit SO2F2 oder SO2ClF erhalten. Die Oxidation des S(=O)F- zum S(=O)2F-Liganden konnte durch Behandlung mit dem Oxygenierungsreagenz 3-Phenyl-2-(phenylsulfonyl)-oxaziridin (Oxaz) bewerkstelligt werden. Eine Oxidation und zugleich Fluorierung der S(=O)F-Einheit in trans-[Pt(F)(SOF)(IMes)2] wurde durch Behandlung mit XeF2 durchgeführt, wobei ein S(=O)F2-Ligand in trans-[Pt(F)(SOF2)(IMes)2]F(HF)n erhalten wurde. Reaktionen von trans-[Pt(F)(SF3)(IMes)2] mit der Lewis-Säure AsF5 oder einer HF-Quelle lässt die Generierung des SF2-Liganden als Teil der kationischen Komplexe trans-[Pt(F)(SF2)(IMes)2]X (X = F(HF)n-, As2F11-) zu. Eine vergleichbare Reaktivität wurde für trans-[Pt(F)(SO2F)(IMes)2] gefunden, wenn NaBArF4 (BArF4- = Tetrakis-[(3,5-trifluoromethyl)phenyl]borat) oder eine HF-Quelle eingesetzt wurden und infolge einer Fluoridabstraktion die Bildung eines SO2-Liganden in trans-[Pt(F)(SO2)(IMes)2]X (X = F(HF)n-, BArF4-) beobachtet wurde. / This work deals with the stabilisation of SFx- (x = 2-3) and SOyFz entities (y, z = 1-2) in the coordination sphere of platinum complexes bearing the N-heterocyclic carbene (NHC) ligand 1,3-bis(2,4,6-trimethylphenyl) 2-imidazolinylidine (IMes). The synthesis of the SF3 complex trans-[Pt(F)(SF3)(IMes)2] was achieved by converting [Pt(IMes)2] with SF4 or SF6. During the SF6 activation additional fluorinated by-products were formed that were characterised by the comparison with independently synthesised compounds. The strong tendency of the SF3 entity to hydrolyse in contact with H2O led to the formation of trans-[Pt(F)(SOF)(IMes)2]. An alternative synthesis of the S(=O)F complex was done by reaction of [Pt(IMes)2] with SOF2. The S(=O)2F complexes trans-[Pt(X)(SO2F)(IMes)2] (X = F, Cl) bearing sulfur ligands in the formal oxidation state IV were accessed by treatment of [Pt(IMes)2] with SO2F2 or SO2ClF. The oxidation of the S(=O)F to the S(=O)2F ligand was achieved by treatment with the oxygenating reagent 3-phenyl 2-(phenylsulfonyl) oxaziridine (Oxaz). An oxidation and fluorination of the S(=O)F group at the same time was done by treatment of trans-[Pt(F)(SOF)(IMes)2] with XeF2 to yield a S(=O)F2 ligand in trans-[Pt(F)(SOF2)(IMes)2]F(HF)n. Reactions of trans-[Pt(F)(SF3)(IMes)2] with the Lewis acid AsF5 or an HF source led to the generation of a SF2 ligand in trans-[Pt(F)(SF2)(IMes)2]X (X = F(HF)n-, As2F11-). A comparable reactivity was found for trans-[Pt(F)(SO2F)(IMes)2], when treated with NaBArF4 (BArF4- = tetrakis-[(3,5-trifluoromethyl)phenyl]borat) or an HF source to yield a SO2 ligand in trans-[Pt(F)(SO2)(IMes)2]X (X = F(HF)n-, BArF4-) by fluoride abstraction.

No description available.

No description available.

This Thesis extends our fundamental knowledge of high-oxidation-state chemistry and in particular compounds of Xe(VIII), Xe(VI), and Ir(V). The crystal structure of XeVIIIO4 was obtained and provides important information on this fundamentally interesting endothermic and shock-sensitive compound. Macroscopic amounts of XeO3F2 have been prepared for the first time. Although the low-temperature Raman spectrum of solid XeO3F2 exhibits some frequency shifts and band splittings of the bending modes, the spectrum is similar to the Raman spectrum of the previously reported matrix-isolated compound. The crystal structures of decomposition and byproducts resulting from the syntheses of XeO3F2 have been obtained for [XeF5][HF2]∙XeOF4 and XeF2∙XeO2F2. The solid-state structure of xenon trioxide, XeO3, was reinvestigated by low-temperature single-crystal X-ray diffraction and shown to exhibit polymorphism that is dependent on crystallization conditions. The previously reported α-phase (orthorhombic, P212121) only forms upon evaporation of aqueous HF solutions of XeO3. In contrast, two new phases, β-XeO3 (rhombohedral, R3) and gamma-XeO3 (rhombohedral, R3c) have been obtained by slow evaporation of aqueous solutions of XeO3. The extended structures of all three phases result from Xe=O----Xe bridge interactions among XeO3 molecules that arise from the amphoteric donor-acceptor nature of XeO3. The Xe atom of the trigonal pyramidal XeO3-unit has three Xe---O secondary bonding interactions. The orthorhombic α-phase displays the greatest degree of variation among the contact distances and has a significantly higher density than the rhombohedral phases. The ambient-temperature Raman spectra of solid α- and gamma-XeO3 have also been obtained and assigned for the first time. Xenon trioxide interacts with CH3CN and CH3CH2CN to form O3XeNCCH3, O3Xe(NCCH3)2, O3XeNCCH2CH3, and O3Xe(NCCH2CH3)2. Their low-temperature single-crystal X-ray structures show that the xenon atoms are consistently coordinated to three electron-donor atoms which result in pseudo-octahedral environments around their xenon atoms. The adduct series provides the first examples of a neutral xenon oxide bound to nitrogen bases. Energy-minimized gas-phase geometries and vibrational frequencies were obtained for the model compounds O3Xe(NCCH3)n (n = 1−3) and O3Xe(NCCH3)n∙[O3Xe(NCCH3)2]2 (n = 1, 2). The natural bond orbital (NBO), quantum theory of atoms in molecules (QTAIM), electron localization function (ELF), and molecular electrostatic potential surface (MEPS) analyses were carried out to further probe the nature of the bonding in these adducts. Xenon trioxide forms adducts with the polytopic nitrogen base ligands: hexamine, DABCO, 2,2’-bipyridine, 1,10-phenanthroline, and 4,4’-bipyridine. The adducts were conveniently synthesized in aqueous or CH3CN solutions and are stable at room temperature. The crystal structures of hexamine∙2XeO3, hexamine∙XeO3∙H2O, 2,2’-bipyridine∙XeO3, 1,10-phenanthroline∙XeO3, and 4,4’-bipyridine∙XeO3 have been determined by low-temperature single-crystal X-ray diffraction. The structures consist of XeO3 molecules bridged by the ligands to form extended supramolecular networks with Xe---N bonds which range from 2.634(3) to 2.829(2) Å. Raman spectroscopy was used to characterize and probe the room-temperature stabilities of these adducts. The reaction of 1,4-diazabicyclo[2.2.2]octane (DABCO) with XeO3 in aqueous solutions yields thin, plate-shaped crystals which are severely twinned whereas the reaction of DABCO with XeO3 in the presence of HF forms [DABCOH]2[F2(XeO3)2]∙H2O and [DABCOH2][F][H2F3] which were also characterized by low-temperature X-ray crystallography and Raman spectroscopy. A reversible temperature-dependent phase transition occurred for [DABCOH]2[F2(XeO3)2]∙H2O. The structures of 2,2’-bipy∙XeO3 and 1,10-phen∙XeO3 provide the first examples of noble-gas chelates. The structure of hexamine∙XeO3∙H2O provides the first instance in which a noble-gas centre is coordinated by water. These compounds also represent the first examples of sp2- and sp3-hybridized N---Xe(VI) bonds and are rare examples of noble-gas compounds that are air-stable at ambient temperatures. Adducts between XeO3 and three molar equivalents of the nitrogen bases, pyridine and 4-dimethylaminopyridine (4-DMAP), have been synthesized and characterized. The crystal structures of (C5H5N)3XeO3, {(CH3)2)2NC5H4N}3XeO3∙H2O have been determined by low-temperature single-crystal X-ray diffraction. The reaction of hydrolyzed XeF6 in acetonitrile with pyridine or 4-DMAP afforded [C5H5NH]4[HF2]2[F2(XeO3)2] and [(CH3)2NC5H4NH][HF2]∙XeO3 which were characterized by low-temperature X-ray crystallography and Raman spectroscopy. The structures contain pyridinium cations that are hydrogen bonded to the fluoride coordinated to XeO3 and can be viewed as pyridinium fluoroxenates. The structure of (CH3)2NC5H5N∙XeO3∙H2O contains a water molecule that is hydrogen bonded to two oxygen atoms of two adjacent XeO3 molecules. The pyridine adduct, (C5H5N)3XeO3, was found to be relatively insensitive to shock, whereas the 4-DMAP adduct was extremely shock sensitive. The number of isolable compounds which contain different noble-gas−element bonds is limited for xenon and even more so for krypton. Examples of Xe−Cl bonds are rare and prior to this work, no definitive evidence for a Xe−Br bonded compound existed. The syntheses, isolation, and characterization of the first compounds to contain Xe−Br bonds ([N(C2H5)4]3[Br3(XeO3)3] and [N(CH3)4]4[Br4(XeO3)4]) and their chlorine analogues are described. The bromo- and chloroxenate salts are stable in the atmosphere at room temperature and were characterized in the solid state by Raman spectroscopy, low-temperature single-crystal X-ray diffraction, and in the gas phase by quantum-chemical calculations. They are the only known examples of cage anions that contain a noble-gas element. The Xe−Br and Xe−Cl bonds are weakly covalent and can be viewed as σ-hole interactions, similar to halogen bonds. Xenon trioxide reacts with alkali metal fluorides and chlorides to form a variety of room-temperature stable fluoro- and chloroxenate salts. The reaction of XeO3 with various ratios of KF in water afforded three new compounds. The crystal structures of α-K[F(XeO3)2], β-K[F(XeO3)2], α-K[FXeO3], K2[F2(XeO3)] have been determined. The reaction of XeO3 with aqueous CsF resulted in Cs3[F3(XeO3)2]. The XeVI−F bond lengths range from 2.3520(18) to 2.5927(17) Å. No stable product was isolated when [N(CH3)4]F was the fluoride source, but in the presence of HF, crystals of [N(CH3)4]3[HF2]2[H2F3]∙2XeO3 were obtained. The reaction of KCl with XeO3 in equimolar amounts resulted in the formation of K[ClXeO3] whereas the analogous reaction with CsCl yielded Cs3[Cl3(XeO3)4]. Attempts to synthesize Xe–P and Xe–S bonded compounds were unsuccessful and instead resulted in adducts between XeO3 and O-bases such as the phosphine oxide adduct, {(C6H5)3PO}2XeO3 and dimethylsulfoxide (DMSO) adduct {(CH3)2SO}3(XeO3)2. Although DMSO was found to be resistant to oxidation by XeO3, no significant Xe---S bonding interactions were observed. Acetone was found to be highly resistant to oxidation by XeO3 and forms {(CH3)2CO}3XeO3 at low temperatures. The reaction of pyridine-N-oxide yielded large crystals of (C5H5NO)3(XeO3)2 in which the structure contains short chains in contrast with ((CH3)2SO)3(XeO3)2 whose structure consists of discrete dimers. The reaction of XeO3 with the oxidatively resistant main-group oxide anion source, [N(CH3)4][OTeF5] in CH3CN solvent afforded [N(CH3)4][F5TeOXeO3(CH3CN)2]. Xenon trioxide reacts with potassium hydroxide to form the previously known K4[XeO6]∙2XeO3 salt which was characterized by Raman spectroscopy and low-temperature X-ray crystallography. The reaction of MgO with XeO3 yielded single crystals of [Mg(OH2)6]4[XeO6(XeO3)12O2]∙12H2O, which also contains perxenate-XeO3 interactions. Alkali metal carbonates also incorporate XeO3 into their crystal lattices. Raman spectra of M2[CO3(XeO3)n]∙xH2O (M = Na, K, Rb) were recorded and contain intense bands assigned to the XeO3 stretching modes and very weak bands assigned to the [CO3]2− modes. The reaction of dilute aqueous solutions of XeO3 with RbOH and atmospheric CO2 afforded single crystals of Rb2[CO3(XeO3)2]∙2H2O which were characterized by low-temperature X-ray crystallography. Attempts to incorporate XeO3 into other polyatomic anion salts such as KMnO4, NaClO3, and NaNO3 were unsuccessful. The reaction of IrO2 with XeF6 in aHF provided [Xe2F11][IrF6], whereas the reaction of IrO2 with KrF2 with ClF3 in anhydrous HF solvent provided [ClO2][Ir2F11] and [ClO2][(μ-OIrF4)3]. The structure of [(μ-OIrF4)3]− consists of a six membered Ir3O3 ring with four terminal fluorine atoms on each Ir atom. It was also found that ClF3 forms an adduct with [Xe2F11][HF2] in which the structural parameters of ClF3 are very similar to that of solid ClF3. The [ClO2][Ir2F11] salt provides the first structural information on the [Ir2F11]− anion and the [(μ-OIrF4)3]− anion represents the first isolated iridium oxide fluoride species. / Thesis / Doctor of Philosophy (PhD) / Xenon is a noble-gas element which is located in the far right-hand column of the periodic table and was previously thought to be chemically unreactive and incapable of forming compounds. In 1962, it was shown that xenon reacts with the most reactive compounds, such as elemental fluorine, but the resulting xenon compounds are themselves highly reactive. This Thesis extends the chemistry of some of the most unstable and chemically reactive xenon compounds that are currently known. One such compound, xenon trioxide, tends to easily detonate unless carefully handled. Methods of stabilizing xenon trioxide were developed and its behaviour with compounds which resulted in formation of new xenon compounds was studied. The molecular structures of these compounds were investigated in the solid with particular emphases on their chemical bonding. Iridium is one of the most chemically resistant metals known. Highly reactive xenon and krypton compounds were used synthesize new iridium compounds.

The development of analytical methods for emerging contaminants creates many unique challenges for analytical chemists. By their nature, emerging contaminants have inherent data gaps related to their environmental occurrence, fate, and impact. This dissertation is a compilation of three studies related to method development for the structural identification of emerging contaminants, the detection and quantification of chemicals used in unprecedented quantities and applications, and the extraction of compounds from complex matrices where the solvent-solute-matrix interactions are not completely understood. The three studies present analytical methods developed for emerging contaminants in complex matrices, including: fluorochemical surfactants in aqueous film-forming foams, oil dispersant surfactants in seawater, and fullerene nanomaterials in carbonaceous solids. Aqueous film-forming foams, used in military and commercial firefighting, represent environmentally-relevant commercial mixtures that contain a variety of fluorochemical surfactants. Combining the surfactant-selective ionization of fast atom bombardment mass spectrometry with high resolution mass spectrometry, chemical formulas for 11 different fluorochemical classes were identified. Then AFFF-related patents were used to determine the structures. Of the eleven classes of fluorochemicals, ten have little, if any, data on their environmental occurrence, fate, and potential impacts in the peer-reviewed literature. In addition, nine of the identified classes had either cationic or zwitterionic functionalities and are likely to have different transport properties compared to the well-studied anionic fluorochemicals, such as perfluorooctanoate. After the Deepwater Horizon oil spill in the summer of 2010, one of the emergency response methods for the mitigation of the oil's environmental impact was the use of unprecedented amounts of oil dispersant to break down the oil slick and encourage biodegradation. This event illustrated the need for rapid analytical method development in order to respond to the potential environmental disaster in a timely manner. Using large volume injection liquid chromatography with tandem mass spectrometry, an analytical method was developed for the trace analysis of the multiple dispersant surfactant classes and the potential degradation products of the primary surfactant. Limits of detection ranged from 49 ��� 3,000 ng/L. The method provided excellent recovery (86 ��� 119%) and precision (10 ��� 23% RSD), while also accommodating for the high salinity of seawater samples and analyte contamination. Despite the fact that fullerene nanomaterials have been studied for almost three decades, research is still being conducted to fully understand the environmental properties of these materials. Previous studies to extract fullerenes from environmental matrices have resulted in low efficiency, high variability, or the extraction efficiencies have gone unreported. Extraction by ultrasonication with toluene and 1-methylnaphthalene increased the recovery 5-fold of a spiked, isotopically-labeled C������ surrogate from carbon lampblack as compared to that of the conventional approach of extracting with 100% toluene. The study revealed the importance of evaluating experimental variables such as extraction solvent composition and volume, and sample mass, as they have a significant impact on the quantitative extraction of fullerenes from environmental matrices. / Graduation date: 2013 / Access restricted to the OSU Community at author's request from Aug. 15, 2012 - Aug. 15, 2013

Hipóxia é definida como a baixa teor de oxigênio. Nos tumores a principal causa da hipóxia é a isquemia, que ocorre em função do rápido crescimento da massa tumoral e diminuição ou obstrução dos vasos sanguíneos que irrigam o interior dos tumores. Como a hipóxia é uma das causas do aumento da resistência à radioterapia de radiação e algumas formas de quimioterapia, a identificação de tumores com regiões de hipóxia é de elevada relevância e a utilização de radiofármacos tem sido muito promissora, por ser um método não invasivo e que podem mapear diferentes alterações fisiológicas associadas à hipóxia. Neste trabalho sintetizamos o ligante [[99mTc](O)2HL91], com rendimento final de síntese de 82,6% e preparamos o respectivo complexo de tecnécio, com eficiência de marcação maior que 97 %; também foi preparado o radiofármaco (18F)FAZA, com eficiência de marcação de 17,9% e pureza radioquímica, após purificação, maior que 86 %. Estudos de captação em células de melanoma murino B16F10, apresentaram taxa de captação de 0,73%, em condições de normóxia e de 8,5 % em condições de hipóxia para o [[99mTc](O)2HL91] , sobe as mesmas condições, de 0,73% e 0,98%, para o (18F)FAZA, respectivamente. Estudos de biodistribuição ex vivo mostraram taxa de captação em tumores da ordem de 4,3% para o [[99mTc](O)2HL91] e de 0,56% para o (18F)FAZA, a relação tumor/sangue foi de 2,6% e 2,5%, respectivamente. Para ambos os rins são a principal via de excreção. Análise, por autorradiografia, de cortes dos tumores mostraram claramente a concentração do [[99mTc](O)2HL91] em regiões de hipóxia/necrose. Imagem da distribuição dos radiofármacos em camundongos C57/Bl6, com tumores de células B16F10, utilizando sistema hibrido PET/SPECT/CT dedicado a pequenos animais, mostraram que a concentração do [[99mTc](O)2HL91] permitiu visualizar captação difusa em regiões do tumor, o mesmo foi observado para o (18F)FAZA, mas em uma taxa menor. Em conclusão, os resultados obtidos apresentam as possibilidades de preparação e utilização de dois radiofármacos, o [[99mTc](O)2HL91] e o (18F)FAZA, como agentes marcadores para hipóxia, utilizando as técnicas de SPECT e PET para imagem. Todavia, novos estudos deverão ser realizados para determinação da especificidade desses radiofármacos em diferentes linhagens tumorais / Hypoxia is a deficiency of oxygen in the cell. In tumors the primary cause of hypoxia is ischemia, which occurs due to the rapid growth of the tumor mass and reduction or blockage of the blood vessels decreasing nutrients and oxygen supply in more internal regions of the tumors. Once hypoxia is one cause of the increased resistance to radiation therapy and some forms of chemotherapy, their identification in tumors is highly relevant and use of radiopharmaceuticals has been very promising, because it is a noninvasive and can map different physiological changes associated with hypoxia. In this work, we synthesized the ligand [[99mTc](O)2HL91] given a final synthesis yield of 82.6% and prepared their technetium complex with the labeling efficiency greater than 97%, the (18F)FAZA radiopharmaceutical was also prepared with labeling yield of 17.9% and marking radiochemical purity higher of 86 %, after purification. Uptake studies in murine B16F10 melanoma cells showed uptake rate of 0.73% in normoxic conditions and 8.5% in hypoxic conditions, for [[99mTc](O)2HL91], and, under same conditions, 0.73% and 0.98%, for (18F)FAZA. Ex vivo biodistribution study showed uptake rate in tumors of approximately 4.3% for [[99mTc](O)2HL91] and 0.56% for (18F)FAZA, the tumor/blood ratio was 2.6% and 2.5% respectively. For both products the main route 16 of excretion was by the kidneys. Analysis by autoradiography of tumors sections clearly showed the concentration of [[99mTc](O)2HL91] in hypoxia/necrosis regions. The distribution of radiopharmaceuticals in C57/Bl6 mice implanted with tumor B16F10 cells, using dedicated small animals hybrid system PET/SPECT/C, permitted to observe the uptake of the [[99mTc](O)2HL91] in diffuses points in the tumor regions, the same was observed for the (18F)FAZA, but with lower intensity. In conclusion, the results obtained show possibilities for preparation and use of both radiopharmaceuticals, the [[99mTc](O)2HL91] and (18F)FAZA as agents for hypoxia marker, using the SPECT and PET image techniques. However, further studies should be conducted to determine the specificity of these radiopharmaceuticals in different tumor cell lines

A baixa oxigenação (hipóxia) altera drasticamente o metabolismo celular e a forma de produção de ATP, que em tumores pode estimular e permitir que as células desenvolvam mecanismos de escape, adaptação e resistência, contribuindo não só para um comportamento maligno e agressivo, mas também lhes conferindo resistência a tratamentos quimioterapêuticos e radioterapêuticos. A detecção de regiões de hipóxia em tumores pode ser realizada com diferentes radiofármacos. Neste trabalho preparamos e avaliamos o comportamento dos radiofármacos (18F)FAZA e [[99mTc]glucarato]- em células de melanoma murino B16F10, correlacionando dados bioquímicos e histopatológicos com a captação celular dos radiofármacos in vitro e com imagens em equipamento PET/SPECT/CT obtidas de camundongos C57Bl6 implantados com tumores. O (18F)FAZA foi obtido em rendimento de 17,9 % e pureza radioquímica de 86,72 %, enquanto que o rendimento e pureza radioquímica do [[99mTc]glucarato]- foi superior a 95 %, sendo que este complexo se liga à proteínas plasmáticas com taxa de aproximadamente 40 % e o complexo é desestabilizados pela mesmas, após 4 horas de incubação a 37 oC. O complexo também não é estável na presença de cisteína e histidina. A captação in vitro do [[99mTc]glucarato]- nas células foi da ordem de 0,1 % independente da condição e do tempo, enquanto que a captação de (18F)FAZA atingiu 0,9 % sob hipóxia e 0,2 % sob normóxia, nos primeiros 15 minutos de estudo. A biodistribuição ex vivo em camundongos apresentou taxa de captação por grama de tumor e razão tumor/sangue da ordem de 0,04 % e 1,49 para o [[99mTc]glucarato]- e de 0,34 % e 1,39 para o (18F)FAZA, em tempo de 1 hora. Imagem obtidas de camundongos, mostraram intensa captação da (18F)FDG no tumor, e tanto (18F)FAZA quanto [[99mTc]glucarato]- foram capazes de evidenciar regiões de hipóxia ou necrose, respectivamente, nos tumores, ainda que com baixa taxa de captação. Imagens autorradiográficas do [[99mTc]glucarato]- nos tumores excisados dos animais apresentaram distribuição homogênea no tumor, com algumas áreas de captação sugeridas como necróticas; tomando a autorradiografia como referência, o [[99mTc]glucarato]- não se concentrou na córtex renal, região reconhecidamente hipóxica. Assim, (18F)FAZA e [[99mTc]glucarato]- puderam ser preparados em nosso laboratório com qualidade suficiente para uso em pesquisa e demonstram potencial para utilização em novos estudos visando a detecção de regiões de hipóxia ou necrose, respectivamente / The low oxygen concentration, also named hypoxia, drastically alters cellular metabolism and the production form of ATP which, in tumors, can stimulate and allow cells to develop mechanisms for escape, adaptation and resistance, contributing not only to malignant and aggressive behavior, but also their conferring resistance to chemotherapeutic and radiotherapeutic treatments. The detection of regions of hypoxia in tumors can be performed using different radiopharmaceuticals. In this work we prepared and evaluated the behavior of radiopharmaceuticals (18F)FAZA and [[99mTc]glucarate]- in B16F10 murine melanoma cells, biochemical and histopathological data correlating it with the radiopharmaceutical cellular uptake, both in vitro or by PET/SPECT/CT imaging obtained from C57Bl6 mice implanted with tumors. The (18F)FAZA was obtained in radiochemical yield of 17.8 % and radiochemical purity of 86.72 %, while the radiochemical yield and purity for [[99mTc]glucarate] - was higher to 95 %, and this complex binds to the plasma proteins at concentration of 40 %, however a the complex is unstable in presence of albumine after 4 hours, at 37 oC. The complex is unstable in the presence of cysteine and histidine, at 37 oC. The in vitro uptake of [[99mTc]glucarate]- in B16F10 cells was approximately 0.1% independently of experimental conditions, while (18F)FAZA reached 0.9%, under hypoxia, and 0.2%, under normoxia, the first 15 minutes of the study. The ex vivo biodistribution in mice showed uptake in tumor and tumor/blood ratio of the 0.04 % and 1.49 for [[99mTc]glucarate]- and 0.34 % and 1.39 for (18F)FAZA. Imaging obtained from mice showed intense uptake of (18F)FDG in the tumor, and both (18F)FAZA and [[99mTc]glucarate]- were able to show hypoxia or necrotic regions in the tumor. Autoradiographic imaging showed homogeneous distribution of [[99mTc]glucarate]- in the slices of tumor excised from animals; taking kidney autoradiography as a reference, the [[99mTc]glucarate]- was not concentrated in renal cortex, a region admittedly hypoxic. In conclusion (18F)FAZA and [[99mTc]glucarate]- could be prepared in our laboratory with sufficient quality for use in research and demonstrate potential for use in future studies aiming to detect regions of hypoxia and necrosis, respectively

RESUME: Le travail de recherche rapporté dans cette thèse concerne lamélioration de traceurs marqués au fluor-18 utilisés en routine clinique : la 2-[18F]fluoro-L-tyrosine et le 2-désoxy-2-[18F]fluoro-D-glucose. Les résultats relatifs à lacide aminé, de valeur confirmative pour les connaissances publiées antérieurement dans la littérature, consistent en une validation chez le rat qui entérine le potentiel de ce traceur pour létude de la vitesse de synthèse des protéines cérébrales in vivo. Les perspectives futures pour ce traceur sont dès lors lextension de son utilité dans le domaine de loncologie et son utilisation pour létude de phénomènes physiologiques neurologiques. Durant ce travail, des techniques décrites dans la littérature, mais non pratiquées au CRC ont fait lobjet dune implémentation et sont maintenant accessibles (modèle du rat vigile, méthodes de synthèse de polymères à empreinte moléculaire). La partie principale du travail concerne la récupération du [18F]fluorure et son utilisation pour le marquage nucléophile sans étape dévaporation. La synthèse du 2-désoxy-2-[18F]fluoro-D-glucose a servi de réaction témoin pour tester lapplicabilité des méthodes développées dans ce cadre. Deux stratégies différentes, lune utilisant des supports ioniques et des solvants protiques, lautre utilisant des supports non ioniques et des solvants non protiques, ont permis datteindre les buts fixés avec des rendements dincorporation du [18F]fluorure de même ordre de grandeur que ceux obtenus en radiochimie usuelle du fluor-18. La méthode utilisant les supports non ioniques a par ailleurs démontré sa grande généralité vis-à-vis de précurseurs divers, aliphatiques et aromatiques, dans des conditions de marquage diverses, notamment à température modérée. Les perspectives de ces méthodes nouvelles pour la fabrication des traceurs TEP tirent parti de la possibilité de les implanter dans un automate miniaturisé (milli- ou micro-réacteur), à visée synthétique ou analytique. Lefficacité et la simplicité des méthodes de récupération sans évaporation mises au point dans ce travail les destinent à être utilisées aussi bien en développement des traceurs quen synthèse de routine. Elles sont applicables aussi bien à léchelle des automates courants quà celle des futures applications microfluidiques. Par ailleurs, nous sommes persuadés de lintérêt des polymères à empreinte moléculaire dans le créneau des méthodes analytiques. Egalement applicables à des systèmes miniaturisés, ils devraient aider à la réalisation danalyses automatisées des produits finis et à une libération accélérée. Le gain de temps et les moindres pertes de principe actif conduiront alors à une meilleure disponibilité des traceurs TEP et à leur participation accrue aux objectifs de la médecine personnalisée. Nous pensons dès lors avoir ouvert quelques pistes de recherche prometteuses pour la mise en application de ses nouvelles technologies au domaine de la tomographie à émission de positon. / SUMMARY: The results reported in this work concern the improvement of 18-fluorine labelled radiopharmaceuticals used in routine clinical applications: 2-[18F]fluoro-L-tyrosine and 2-deoxy-2-[18F]fluoro-D-glucose. The study of the metabolism of non carrier added 2-[18F]fluoro-L-tyrosine in rats confirms that this tracer is rapidly and extensively incorporated into cerebral proteins and is therefore well suited to the assessment of Protein Synthesis Rate (PSR) in vivo by PET. A correction for the appearance of metabolites is advised for quantitative interpretation of the data. An improvement in the radiosynthesis is necessary to make 2-[18F]fluoro-L-tyrosine widely available for its application in oncology and to envisage the extended use of this tracer for the study of the protein synthesis in other physiological or pathological processes. The second chapter deals with use of molecular imprints in the PET radiochemistry. The molecularly imprinted polymers were synthetized, characterized and tested for the production of specific PET tracers and the plasma analysis of the parent metabolites. The third part of the work consisted in a development of new methods for the [18F]fluoride recovery in order to permit the labelling of different precursors through nucleophilic substitution without the evaporation step classically performed in 18-fluorine radiochemistry. The synthesis of 2-deoxy-2-[18F]fluoro-D-glucose has been used as a tool for the evaluation of the developed methods. Two strategies were considered to concentrate and recover the [18F]fluoride. The first one used ionic solid supports and protic solvents. The second one relied on the use of non ionic solid supports and non protic solvents. Both strategies led us to reach [18F]fluoride incorporation yields as high as in classical radiosyntheses with evaporation. Ionic liquids and tertiary alcohols were also evaluated in order to improve the tolerance of the [18F]fluoride nucleophilic substitution to water. The molecularly imprinted polymers and the new methods for the recovery of [18F]fluoride will now be tested for the implementation of PET tracers radiosynthesis and quality control into microchip devices.

Das S100A4-Protein ist für die Manifestierung eines metastatischen Phänotyps bei vielen Tumorarten von enormer Bedeutung. Die Aufklärung der zugrunde liegenden Mechanismen und der Interaktionspartner von S100A4 stellt daher einen vielsprechenden Forschungsansatz dar, um neue Erkenntnisse über das Verhalten von Tumorzellen während des Metastasierungsprozesses zu erhalten. Darauf aufbauend können neue Ansatzpunkte für die Therapie metastasierender Krebserkrankungen gewonnen werden. In dieser Hinsicht ist das bisher einer Behandlung kaum zugängliche maligne Melanom als besonders aggressiver und frühzeitig metastasierender Tumor ein ideales Modell zur Aufklärung der zellulären und molekularen Prozesse, über die S100A4 seine Metastasen-fördernden Wirkungen ausübt. Das Ziel der vorliegenden Arbeit war die biochemische und radiopharmakologische Charakterisierung der S100A4-RAGE-Interaktion sowie die Untersuchung der Beteiligung von S100A4 an Prozessen der Metastasierungskaskade in vitro und in vivo. Dies erforderte die Herstellung von rekombinantem S100A4-Protein und die Generierung von stabil mit S100A4-transfizierten Melanomzellen, die damit eine heraufregulierte S100A4-Proteinbiosynthese aufweisen. Die Gewinnung von rekombinantem S100A4 in biologisch funktioneller Form unter Verwendung eines prokaryotischen Expressionssystems erfolgte mit einem Reinheitsgrad von ca. 92%. Das rekombinante S100A4-Protein wurde mit dem Aktivester N-Succinimidyl-4-[18F]fluorbenzoat radioaktiv markiert und charakterisiert. Es wurde die Interaktion zwischen S100A4 bzw. 18F-markiertem S100A4 und der löslichen RAGE-Isoform sRAGE mit einer moderaten Bindungsaffinität im µM-Bereich nachgewiesen. Des Weiteren erfolgte erstmals die Analyse der radiopharmakologischen Eigenschaften von 18F-S100A4 mittels Untersuchungen zur zellulären Assoziation sowie zur metabolischen Stabilität, Bioverteilung und zu In-vivo-Interaktionen mittels Kleintier-Positronen-Emissions-Tomographie in der Ratte. Die In-vitro-Experimente wurden an Endothelzellen (HAEC) und an stabil mit RAGE-transfizierten A375-, A375-mock bzw. nicht transfizierten A375-Melanomzellen durchgeführt. Die A375-hRAGE-Zellen zeigten eine deutlich heraufregulierte RAGE-Proteinbiosynthese während die Endothelzellen eine vergleichsweise geringe intrazelluläre RAGE-Proteinkonzentration aufwiesen. Bei den Melanomzellen kann aufgrund der höheren Assoziation von 18F-S100A4 an A375-hRAGE-Zellen auf eine selektive Bindung von 18F S100A4 an RAGE-Rezeptoren auf der Zelloberfläche geschlossen werden. Die Assoziation von 18F S100A4 an Endothelzellen war bei 37°C in Gegenwart von nicht markiertem rekombinantem S100A4 signifikant vermindert, dementsprechend findet eine spezifische Interaktion von 18F-S100A4 mit Zelloberflächenrezeptoren der Endothelzellen statt. Dieses Ergebnis und die insgesamt höhere Bindung von 18F S100A4 an Endothelzellen im Vergleich zur Assoziation an Melanomzellen lassen neben RAGE noch andere Rezeptoren wie z. B. internalisierende Scavenger-Rezeptoren vermuten. Die In-vivo-Stabilitätsuntersuchungen verdeutlichen einen proteolytischen Abbau von 18F S100A4, allerdings belegen das Vorhandensein von 67% intaktem 18F-S100A4-Protein nach einer Stunde, die Stabilität von 18F-S100A4 in vivo. Die Bioverteilungs- bzw. PET-Untersuchungen zeigen eine schnelle, innerhalb weniger Minuten stattfindende hohe Akkumulation in den Nieren und verdeutlichen somit die renale Ausscheidung von 18F S100A4. Die maßgeblichen Anreicherungen in Milz, Leber, Blut, Lunge und Nebennieren lassen Interaktionen mit Oberflächenrezeptoren dieser Gewebe erkennen. Die temporäre Retention von 18F-S100A4 in der Lunge, dem Hauptsyntheseorgan von RAGE, und die verminderte 18F-S100A4-Akkumulation in Gegenwart des spezifischen RAGE-Liganden glykLDL ist ein Hinweis dafür, dass S100A4 in vivo in der Lunge an RAGE bindet. Die Aktivitätsanreicherungen in Milz, Leber und Nebenniere deuten aufgrund der geringeren RAGE-Synthese in diesen Organen auf die Interaktion von 18F-S100A4 mit anderen Zelloberflächenrezeptoren z. B. aus der Familie der Scavenger-Rezeptoren hin. Die Beteiligung von S100A4 an Metastasierungsprozessen des malignen Melanoms wurde an stabil mit S100A4-transfizierten A375-Melanomzellen, die eine Heraufregulierung der humanen bzw. murinen S100A4-Proteinbiosynthese im Vergleich zu A375-mock- (Vektor-Kontrolle) und nicht-transfizierten A375-Zellen zeigen, untersucht. Die A375-hS100A4-Zellen sezernierten zudem eine signifikant höhere S100A4-Proteinkonzentration in das umgebende Zellkulturmedium im Vergleich zu den Kontrollen. In dieser Hinsicht konnte bei den A375-hS100A4-Zellen, vermutlich aufgrund der höheren extrazellulären S100A4-Konzentration, eine gesteigerte Proliferations-, Motilitäts-, Migrations- und Invasionsrate gegenüber den A375-mock- und A375-Zellen nachgewiesen werden. In diesem Zusammenhang stehen ebenso die gesteigerte RAGE-Proteinbiosynthese und die signifikant höhere Aktivität des Transkriptionsfaktors NF-κB bei A375-Zellen nach 24-stündiger Inkubation mit Kulturmedium der A375-hS100A4-Zellen. Demnach wirkt vermutlich das extrazelluläre S100A4-Protein als autokriner bzw. parakriner Regulator von RAGE und NF κB. Die subkutane Injektion der A375- und stabil transfizierten A375-Melanomzellen in Nacktmäuse führte zur Entwicklung subkutaner Tumore an der Injektionsstelle. Bereits zwei Wochen nach der Injektion etablierten die A375-hS100A4-Zellen die signifikant größeren Tumore im Vergleich zu den A375-mS100A4-, A375-mock und A375-Zellen. Nach Injektion der Zellen in die Schwanzvene der Nacktmäuse konnte keine Entwicklung von Metastasen im Tierkörper festgestellt werden. IN DER VORLIEGENDEN ARBEIT WURDE NACHGEWIESEN: • RAGE ist ein Rezeptor für das S100A4-Protein. Allerdings gibt es eindeutige Hinweise für weitere S100A4-Zielproteine an der Zelloberfläche. • Die bedeutende Rolle von extrazellulärem S100A4 bei wichtigen zellulären Metastasierungsprozessen sowie bei der Aktivierung von Signalproteinen wie NF-κB und RAGE beim malignen Melanom. Die weitere Aufklärung der S100A4-spezifischen Signalkaskaden und Rezeptoren bei metastasierenden Tumorerkrankungen sowie die Charakterisierung von S100A4 als klinischen Parameter bei Patienten mit malignem Melanom stellen hoch interessante Aspekte in der Krebsforschung dar.