Diese Arbeit befasst sich mit dem bisher noch nicht untersuchten Mechanismus der Cyanophycinbiosynthese. Hierzu wurden verschiedene kurze Cyanophycinmoleküle chemisch synthetisiert, die als definierte Primer in in vitro Experimenten verwendet wurden. Die Verwendung dieser Primer ermöglichte erstmals die Richtung der Verlängerung des Cyanophycinmoleküls aufzuklären. Die durchgeführten Experimente zeigten, dass der Einbau der konstituierenden Aminosäuren sukzessiv vom Carboxyterminus aus erfolgt. Weiterhin wurde gezeigt, dass auch die nicht proteinogenen Aminosäuren Ornithin und Citrullin vom Enzym eingebaut werden. Mittels ortsgerichteter Mutagenese wurde zudem eine Zuordnung unterschiedlicher Abschnitte der Cyanophycin-Synthetase zu den verschiedenen vom Enzym katalysierten Teilreaktionen versucht. Mutationen im N-terminalen Bereich der Cyanophycin-Synthetase aus Anabaena variabilis ATCC 29413 führten dazu, dass Aspartat nicht mehr in Cyanophycin eingebaut wurde, eine Mutation im C-terminalen Bereich bewirkte, dass Arginin nicht mehr mit Cyanophycin verknüpft werden konnte. Als Reaktionsmechanismus wird für die Bindung beider Aminosäuren jeweils eine Phosphorylierung des C-terminalen Aspartatrestes von Cyanophycin als Acylphosphat vorgeschlagen, wobei die Phosphorylierung der beta-Carboxylgruppe mittels gamma-[³²P]-ATP nachgewiesen werden konnte, die Phosphorylierung der alpha-Carboxylgruppe jedoch nicht. Durch Vergleiche mit Enzymen ähnlicher Aminosäuresequenz und bekannter Raumstruktur wird eine mögliche Begründung für diese unterschiedlichen Befunde gegeben. / This work is occupied with the till now uninvestigated mechanism of the biosynthesis of cyanophycin. Therefore different short cyanophycin molecules were synthesized chemically, which were employed as defined primers for in vitro experiments. The usage of these primers made it possible to clear up the direction of the elongation of the cyanophycin molecule. Experiments showed that the incorporation of the constituent amino acids happens successively starting from the carboxy-terminus. Further it was shown that the nonproteinogenic amino-acids ornithine and citrulline are incorporated by the enzyme. Using site-directed mutagenesis an assignment between segments of the cyanophycin synthetase to different parts of the reactions catalyzed by the enzyme was carried out. Mutations in the N-terminal part of cyanophycin synthetase of Anabaena variabilis ATCC 29413 lead to the finding, that aspartate was not incorporated into cyanophycin anymore. A mutation in the C-terminal part resulted in the disability of the enzyme to incorporate arginine into cyanophycin. As reaction mechanism for the attachment of both of the amino acids a phosphorylation of the C-terminal aspartate as an acylphosphate was proposed. The phosphorylation of the beta-carboxylic-group could be shown by using gamma-[³²P]-ATP, the phosphorylation of the alpha-carboxylic group could not be shown. By comparison with enzymes that share a similar amino acid sequence and have a solved crystal structure a possible explanation for this finding is given.

O exossomo é um complexo multiproteico conservado evolutivamente de archaea a eucariotos superiores que desempenha funções celulares essenciais tais como: atividade exoribonucleolítica 3\'→5\', regulação dos níveis de mRNA, maturação de RNAs estruturais e controle de qualidade de RNAs durante os vários estágios do mecanismo de expressão gênica. Em Archaea, o exossomo é composto por até quatro subunidades diferentes, duas com domínios de RNase PH, aRrp41 e aRrp42, e duas com domínios de ligação a RNAs, aCsl4 e aRrp4. Três cópias das proteínas aRrp4 e/ou aCsl4 se associam com o núcleo hexamérico catalítico do anel de RNase PH e completam a formação do complexo. A proteína PaNip7 é um cofator de regulação do exossomo da archaea Pyrococcus abyssi e atua na inibição do complexo enzimático ligando-se simultaneamente ao exossomo e a RNAs. Neste projeto, a reconstituição in vitro do exossomo da archaea Pyrococcus abyssi formado pela proteína de topo PaCsl4 foi obtida. Para tanto foram realizadas análises de interação proteica usando as técnicas de cromatografia de afinidade, gel filtração e SDS-PAGE. Em adição à formação da isoforma PaCsl4-exossomo, um fragmento peptídico correspondente à região C-terminal da PaNip7 foi sintetizado pelo método da fase sólida, purificado por RP-HPLC e o purificado foi caracterizado por LC/ESI-MS almejando realizar futuros experimentos de interação com o exossomo. / The exosome is a multiprotein complex evolutionarily conserved from archaea to higher eukaryotes that performs essential cellular functions such as: 3\'→5\' exoribonucleolytic activity, regulation of mRNA levels, maturation of structural RNAs and quality control of RNAs during the various stages of the gene expression mechanism. In Archaea, the exosome is composed of up to four different subunits, two with RNase PH domains, aRrp41 and aRrp42, and two with RNAs binding domains, aCsl4 and aRrp4. Three copies of the aRrp4 and/or aCsl4 proteins associate with the hexameric catalytic core of the RNase PH ring and complete the formation of the complex. The PaNip7 protein is a regulating cofactor of the Pyrococcus abyssi archaeal exosome and acts in the inhibition of the enzyme complex by binding simultaneously to the exosome and RNAs. In this project, the reconstitution in vitro of the Pyrococcus abyssi archaeal exosome formed by the PaCsl4 top protein was achieved. To this end protein interaction analyses were performed using affinity chromatography, gel filtration and SDS-PAGE techniques. In addition to the formation of the PaCsl4-exosome isoform, a peptide fragment corresponding to the C-terminal region of PaNip7 was synthesized by solid-phase method, purified by RP-HPLC and the purified peptide was characterized by LC/ESI-MS aiming to perform future binding experiments with the exosome.

"Ghadiri reported the first synthetic peptide nanotubue in 1993, which has triggered extensive studies on peptide-based nanotubes and their potential application in molecular wires, catalysts and novel drug delivery vehicles. Our concerns focus on chromophore-modified cyclic peptides, which open a new way to design and synthesize novel nanoscale electronic or photonic devices, and are expected to provide the highly efficient electron and energy transfer that such devices require. This research concerned the design and synthesis of chiral a-amino acids with specific chromophores, including N-ethyl-3-carbazolylalanine and 9-anthrylalanine, and an 8-mer linear peptide (H-Aib-Car-Aib-Phe-Aib-Bpa-Aib-Phe-OH) and its corresponding cyclic peptide cyclo(Aib-Car-Aib-Phe-Aib-Bpa-Aib-Phe) that incorporate the N-ethyl-3-carbazolylalanine. This thesis describes the relevant background, synthetic strategies, experiments and results in detail. The carbazole derivatives were found to be very labile to strong acid, which might have caused self-condensation. In order to avoid the formation of acid-derived side-products, the Wittig-Horner reaction was used successfully in preparation of N-protected-3-(N'-ethyl-3-carbazolyl)-DL-alanine methyl ester. Dual enzymatic hydrolyses were developed to produce the chiral amino acids with high enantiomeric excess. ChiroCLEC-BL was used to selectively hydrolyze the N-acetyl-L-amino acid methyl ester, while amanoacylase was adopted to remove the acetyl group from the resulting N-acetyl-L-amino acid. Two model peptides were synthesized, a 4-mer peptide (H-Car-D-Ala-Bpa-D-Ala-OH) via the Boc-strategy, and an 8-mer peptide (H-Ala-D-Ala-Npa-D-MeAla-Ala-D-Ala-Bpa-D-Ala-OH) by the Fmoc-strategy. Eventually, the target linear peptide was synthesized via the Fmoc-strategy and then cyclized in solution."

A series of bicyclic peptides have been designed and synthesized to provide ammonium ion complexation sites via hydrogen bonding in a tetrahedral geometry. Molecular modeling dynamics and electrostatics studies indicate that target compounds 1d-6d may provide better selectivity for ammonium ions over potassium ions than the ammonium ionophore currently used for blood analysis applications, nonactin. Attempts to synthesize 1d, cyclo(L-Glu1�D-Val2�L-Ala3�D-Lys4�D-Val5�L-Val6)-cyclo-(1ã-4å), were unsuccessful due to poor solubility of the synthetic intermediates. This led to the design of 2d-6d in which specific amino acid residues were chosen to provide higher solubility. Compound 2d, cyclo(L-Glu1�D-Ala2�D-Ala3�L-Lys4�D-Ala5�L-Ala6)-cyclo-(1ã-4å), was successfully synthesized, but was also too insoluble for characterization or testing in an ion selective electrode (ISE) sensor format. Compound 6d, cyclo(L-Glu1�D-Leu2�Aib3�L-Lys4�D-Leu5�D-Ala6)-cyclo-(1ã-4å), was successfully synthesized and characterized. When 6d was incorporated into an ISE sensor and tested as an ammonium ionophore, results indicated that the bicyclic peptide lacked solubility in the ISE membrane. A 13C-NMR study has been initiated in order to evaluate selectivity of 6d for ammonium over potassium and sodium cations in solution. Preliminary results with the potassium ionophore valinomycin as a control have been completed.

The ability of incorporation non-canonical amino acids (ncAAs) using translation offers researchers the ability of extend the functionality of proteins and peptides for many applications including synthetic biology, biophysical and structural studies, and discovery of novel ligands. Here we describe the three projects where the addition of ncAAs to in vitro translation systems creates useful chemical biology techniques. In the first, a fluorinated histidine derivative is used to create a novel affinity tag that allows for the selective purification of peptides from a complex mixture of proteins. In the second, the high promiscuity of an editing-deficient valine-tRNA synthetase (ValRS T222P) is used to demonstrate ribosomal translation of 13 ncAAs including those with novel side chains, α,α disubstitutions, and cyclic β amino acids. Lastly, a couple of these amino acids are integrated into the powerful ligand discovery tool of mRNA display for the discovery of helical peptide ligands.

Die im Jahre 1963 von Robert Bruce Merrifield vorgestellte festphasengebundene Peptidsynthese beeinflusste in hohem Maße verschiedene Bereiche der Naturwissenschaften. Doch trotz zahlreicher neuer Entwicklungen hat sich in der das Prinzip der eingesetzten Trägermaterialien nicht grundlegend geändert. Geringfügig quervernetzte Polystyrol-Harze sind immer noch die meist verwendeten Trägermaterialien in der standardisierten Peptidsynthese. In dieser Arbeit wurden superparamagnetische Kern-Schale-Nanopartikel entwickelt und erstmals deren Einsatz als neues Trägermaterial für die Synthese von Peptiden, Peptid-Polymer-Konjugaten und Oligonucleotiden demonstriert. Unter Verwendung einer mikrowellenunterstützten Syntheseroute gelang es zunächst superparamagnetische Magnetitpartikel mit einem Durchmesser von durchschnittlich 6 nm darzustellen. Anschließend wurde ein neu entwickelter mikrowellenunterstützter Stöber-Prozess zur Herstellung von Magnetit-Silica-Kern-Schale-Nanopartikel angewendet, welche im dritten Schritt mit Aminopropyltrimethoxysilan funktionalisiert wurden. Es wurden hochpräzise, monodisperse Kern-Schale-Nanopartikel mit einem Durchmesser von durchschnittlich 69 nm und einem Beladungswert von 0.11 mmol/g erhalten, welche in durchgeführten Stabilitätstests hervorragende Ergebnisse zeigten und als neue Trägermaterialien für festphasengebundenen Synthesen getestet wurden. Die erforderliche Produktaufreinigung erfolgte durch ein externes Magnetfeld, durch welches die Partikel reversibel sedimentierbar sind. Erste Studien der Synthese einer 4-mer-Peptidsequenz zeigten Ausbeuten von über 70% und mit herausragender Reinheit von über 95%. Besonders eindrucksvolle Ergebnisse erzielten die Partikel bei der Synthese von Peptid-Polymer-Konjugaten, bei denen die Ligationsreaktionen mit vorher nicht dokumentierten Umsatzraten verliefen. Außerdem konnte die Anwendbarkeit bei der Synthese eines Trinucleotids nachgewiesen werden. / In 1963 Merrifield introduced the method of solid-phase supported synthesis and thus revolutionized peptide synthesis. In spite of several new developments, the main principle of established solid supports has not changed much. Still lightly cross linked poly(styrene) resins dominate the used supports. This work reports on surface amino functionalized, superparamagnetic nanoparticles with a protective silica shell to be applicable as colloidal supports for organic synthesis of peptides, peptide polymer conjugates and oligonucleotides. A microwave supported synthesis route lead to superparamagnetic magnetite particles with an average particle diameter of 6 nm. Subsequently a new developed microwave assisted Stöber process was used to build up magnetite-silica-core-shell-nanoparticles, which were functionalized in a third step with aminopropyltrimethoxysilane. Defined monodisperse core-shell nanoparticles were obtained with an average diameter of 69 nm and a concentration of free amino groups of 0.11 mmol/g, which showed excellent results in conducted stability tests and were used as new support materials for solid-supported syntheses. Convenient magnetic sedimentation proved to ensure ease of purification after each reaction step. Initial studies of a synthesis of a tetramer peptide sequence showed yields of more than 70% and an outstanding purity of more than 95%. The particles also showed impressive results in the synthesis of peptide-polymer conjugates, in which the ligation reactions proceeded conversion rates, which had not been published before. In addition, the applicability of the particles was demonstrated in the synthesis of a trinucleotide.

<p>Rheumatoid arthritis (RA) is an autoimmune disease affecting approximately 1% of the population in the western world. It is characterised by a tissue specific attack of cartilage in peripheral joints. Collagen induced arthritis (CIA) is one of the most commonly used animal models for (RA), with similar symptoms and histopathology. CIA is induced by immunisation of mice with type II collagen (CII), and the immunodominant part was previously found to be located between residues 256-270. This thesis describes the interaction between the MHC molecule, glycopeptide antigens from CII and the T cells that is essential in development of CIA. The glycopeptide properties for binding to the mouse MHC molecule Aq have been studied, as well as interaction points in the glycopeptide that are critical for stimulation of a T-cell response.</p><p>The thesis is based on five studies. In the first paper the minimal glycopeptide core, that is required for binding to the Aq molecule while still giving a full T cell response was determined. The second paper studied the roles of amino acid side-chains and a backbone amide bond as T-cell contact points. In the third paper the hydrogen bond donor-acceptor characteristics of the 4-OH galactose hydroxyl group of the glycopeptide was studied in detail. In the fourth paper we established a structure activity relationship (QSAR model) for (glyco)peptide binding to the Aq molecule. Finally, the stereochemical requirements for glycopeptide binding to the Aq molecule and for T-cell recognition was studied in the fifth paper.</p><p>The study was performed using collagen glycopeptide analogues, which were synthesised on solid phase. Amide bond and hydroxyl group isosteres were introduced for study of hydrogen bond donor-acceptor characteristics. Statistical methods were used to design a representative peptide test set and in establishing a QSAR model.</p><p>The results give a deeper understanding of the interactions involved in the ternary MHC-glycopeptide-T cell complex. This information contributes to research directed towards finding new treatments for RA.</p>

The aim of this thesis was the investigation of the mitochondrial response mechanisms upon apoptotic stimuli. The specific objectives were the biophysical characterization of membrane dynamics and the specific roles of lipids in the context of apoptotic regulation occurring at the mitochondrion and its complex membrane systems. The BH4 domain is an anti-apoptotic specific domain of the Bcl-2 protein. Solid phase peptide synthesis was used to produce large amount of the peptide for biophysical studies. A protocol has been established and optimized, guarantying the required purity for biophysical studies. In detail the purification by high performance liquid chromatography and the characterisation via mass spectroscopy are described. The secondary structure of BH4 changes significantly in the presence of lipid vesicles as observed by infrared spectroscopy and circular dichroism. The BH4 peptide aggregates at the membrane surface and inserts slightly into the hydrophobic part of the membrane. Using nuclear magnetic resonance (NMR) and calorimetry techniques, it could even be shown that the BH4 domain modifies the dynamic and organization of the liposomes which mimic a mitochondrial surface. The second study was on the first helix of the pro-apoptotic protein Bax. This sequence called Bax-α1 has the function to address the cytosolic Bax protein to the mitochondrial membrane upon activation. Once again a protocol has been established for the synthesis and purification of this peptide. The aim was to elucidate the key role of cardiolipin, a mitochondria-specific phospholipid, in the interaction of Bax-α1 with the mitochondrial membrane system. The NMR and circular dichroism studies showed that Bax-α1 interacts with the membrane models only if they contain the cardiolipin, producing a strong electrostatic lock effect which is located at the membrane surface. Finally, a new NMR approach was developed which allows the investigation of the lipid response of isolated active mitochondria upon the presence of apoptotic stimuli. The goal was there to directly monitor lipid specific the occurring changes during these physiological activities.

Η Σωματοστατίνη (SRIF) είναι μια ορμόνη του υποθαλάμου. Ραδιοεπισημασμένα ανάλογα της χρησιμοποιούνται στη σπινθηρογραφική απεικόνιση όγκων που υπερεκφράζουν υποδοχείς της (στοχευμένη διάγνωση). Μειονεκτήματα της στοχευμένης διάγνωσης είναι η υψηλή και παρατεταμένη εντόπιση της ακτινοβολίας στους νεφρούς και η πρόσληψη των ραδιοπεπτιδίων από όργανα μη στόχους. Στόχος της παρούσας διατριβής είναι η ανάπτυξη νέων αναλόγων της SRIF τα οποία να πληρούν τις απαραίτητες προϋποθέσεις ώστε να καταστούν νέα διαγνωστικά μέσα. Αναπτύχθηκαν 33 νέα συνθετικά παράγωγα της SRIF. Σε πρώτη φάση συνετέθησαν απλά ανάλογα της SRIF. Ακολούθησε αξιολόγηση και σε αυτά τα οποία παρουσίαζαν ικανοποιητική συμπεριφορά σε σχέση με την συγγένεια τους με τον υποδοχέα και την πολικότητα τους συζεύχθηκε τετρααμινικός υποκαταστάτης για σύμπλεξη ραδιομετάλλου. Συνετέθησαν επίσης διμερή ανάλογα της SRIF και γλυκοπεπτιδικά ανάλογα. Τα πεπτίδια συντέθηκαν εφαρμόζοντας τεχνικές της Fmoc/tBu μεθοδολογίας. Ο συνδυασμός των μεθόδων που εφαρμόστηκαν οδήγησε στη λήψη των επιθυμητών δομών σε υψηλές αποδόσεις και καθαρότητα. Από τα αποτελέσματα των μελετών ανταγωνιστικής δέσμευσης τα οποία έγιναν σε μεμβράνες κυττάρων AR4-2J, αποδείχτηκε ότι όλα τα ανάλογα διαθέτουν πολύ υψηλές τιμές IC50. Ικανοποιήθηκε επίσης η απαίτηση για αύξηση της πολικότητας τους. Τα νέα ανάλογα πληρούν καταρχήν τις απαραίτητες προϋποθέσεις ώστε να καταστούν υποψήφια νέα διαγνωστικά μέσα και να βρουν κλινική εφαρμογή. Αναμένεται τα τελικά ραδιοπεπτίδια να οδηγούν σε μειωμένη πρόσληψη της ακτινοβολίας από τα όργανα μη στόχους και να εμφανίζουν αυξημένη απέκκριση από τους νεφρούς. Από προκαταρτικά πειράματα βρέθηκε ότι τα πιο πάνω επετεύχθησαν. / Somatostatin is a hyrothalamic regulatory peptide hormone with a pan-antisecretory profile. A large variety of human tumors are expressing multple somatostatin receptors. Synthetic somatostatin analogues radiolabelled with a variety of metallic radionuclide’s are used for the diagnosis and staging of sstr positive tumors but there is the main problem of high accumulation of the radiopeptides on non target organs and the high renal uptake of the radioactivity which leads to nephrotoxicity. Aim of this study was the development of new SRIF analogues as candidate useful clinical tools for the diagnosis and staging of sstr positive tumors. There have been developed thirty three new SRIF analogues and a glycoaminoacid suitable for use in SPPS. First, new analogues of [Tyr3]Octreotate were developed and the best of them, these that satisfied the relation between polarity and binding affinity to the sstr2, were coupled with a tetraaminic chelator ligand for the future complexation of a radionuclide. In other approach peptide dimers were developed. New glycopeptides were also developed. All analogues were synthesized on the solid phase applying several techniques of Fmoc/tBu strategy in high yield and purity. The binding affinity of the new SRIF analogs was checked with experiments of competitive binding with membranic preparations of AR42J cells, positive on SRIF receptors. All the new analogues are able to bind the sstr2 with high affinities. The new analogues accomplish the pre-condition to become new and useful clinical tools after their radiolabelling in the field of peptide-receptor imaging. It is expected that the new molecules will be able to have increased renal excretion via the kidneys and low non target accumulation. From preliminary experiments was shown that the above were succeeded.

Rheumatoid arthritis (RA) is an autoimmune disease affecting approximately 1% of the population in the western world. It is characterised by a tissue specific attack of cartilage in peripheral joints. Collagen induced arthritis (CIA) is one of the most commonly used animal models for (RA), with similar symptoms and histopathology. CIA is induced by immunisation of mice with type II collagen (CII), and the immunodominant part was previously found to be located between residues 256-270. This thesis describes the interaction between the MHC molecule, glycopeptide antigens from CII and the T cells that is essential in development of CIA. The glycopeptide properties for binding to the mouse MHC molecule Aq have been studied, as well as interaction points in the glycopeptide that are critical for stimulation of a T-cell response. The thesis is based on five studies. In the first paper the minimal glycopeptide core, that is required for binding to the Aq molecule while still giving a full T cell response was determined. The second paper studied the roles of amino acid side-chains and a backbone amide bond as T-cell contact points. In the third paper the hydrogen bond donor-acceptor characteristics of the 4-OH galactose hydroxyl group of the glycopeptide was studied in detail. In the fourth paper we established a structure activity relationship (QSAR model) for (glyco)peptide binding to the Aq molecule. Finally, the stereochemical requirements for glycopeptide binding to the Aq molecule and for T-cell recognition was studied in the fifth paper. The study was performed using collagen glycopeptide analogues, which were synthesised on solid phase. Amide bond and hydroxyl group isosteres were introduced for study of hydrogen bond donor-acceptor characteristics. Statistical methods were used to design a representative peptide test set and in establishing a QSAR model. The results give a deeper understanding of the interactions involved in the ternary MHC-glycopeptide-T cell complex. This information contributes to research directed towards finding new treatments for RA.