@phdthesis{Khayenko2023,
  author    = {Khayenko, Vladimir},
  title     = {Functional peptide-based probes for the visualization of inhibitory synapses},
  doi       = {10.25972/OPUS-32043},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-320438},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Short functional peptidic probes can maximize the potential of high-end microscopy techniques and multiplex imaging assays and provide new insights into normal and aberrant molecular, cellular and tissue function. Particularly, the visualization of inhibitory synapses requires protocol tailoring for different sample types and imaging techniques and relies either on genetic manipulation or on antibodies that underperform in tissue immunofluorescence. Starting from an endogenous activity-related ligand of gephyrin, a universal marker of the inhibitory post-synapse, I developed a short peptidic multivalent binder with exceptional affinity and selectivity to gephyrin. By tailoring fluorophores to the binder, I have obtained Sylite, a probe for the visualization of inhibitory synapses, with an outstanding signal-to-background ratio, that bests the "gold standard" gephyrin antibodies both in selectivity and in tissue immunofluorescence. In tissue Sylite benefits from simplified handling, provides robust synaptic labeling in record-short time and, unlike antibodies, is not affected by staining artefacts. In super-resolution microscopy Sylite precisely localizes the post-synapse and enables accurate pre- to post-synapse measurements. Combined with complimentary tracing techniques Sylite reveals inhibitory connectivity and profiles inhibitory inputs and synapse sizes of excitatory and inhibitory neurons in the periaqueductal gray brain region. Lastly, upon probe optimization for live cell application and with the help of novel thiol-reactive cell penetrating peptide I have visualized inhibitory synapses in living neurons. Taken together, my work provided a versatile probe for conventional and super-resolution microscopy and a workflow for the development and application of similar compact functional synthetic probes.},
  subject      = {Fluoreszenzsonde},
  language  = {en}
}
@phdthesis{Nordblom2023,
  author    = {Nordblom, Noah Frieder},
  title     = {Synthese und Evaluation von Gephyrinsonden f{\"u}r hochaufl{\"o}sende Mikroskopieverfahren},
  doi       = {10.25972/OPUS-30230},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-302300},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {This decade saw the development of new high-end light microscopy approaches. These technologies are increasingly used to expand our understanding of cellular function and the molecular mechanisms of life and disease. The precision of state-of-the-art super resolution microscopy is limited by the properties of the applied fluorescent label. Here I describe the synthesis and evaluation of new functional fluorescent probes that specifically stain gephyrin, universal marker of the neuronal inhibitory post-synapse. Selected probe precursor peptides were synthesised using solid phase peptide synthesis and conjugated with selected super resolution capable fluorescent dyes. Identity and purity were defined using chromatography and mass spectrometric methods. To probe the target specificity of the resulting probe variants in cellular context, a high-throughput assay was established. The established semi-automated and parallel workflow was used for the evaluation of three selected probes by defining their co-localization with the expressed fluorescent target protein. My work provided NN1Dc and established the probe as a visualisation tool for essentially background-free visualisation of the synaptic marker protein gephyrin in a cellular context. Furthermore, NN1DA became part of a toolbox for studying the inhibitory synapse ultrastructure and brain connectivity and turned out useful for the development of a label-free, high-throughput protein interaction quantification assay.},
  subject      = {Fluoreszenzmikroskopie},
  language  = {en}
}
@phdthesis{Nagl2022,
  author    = {Nagl, Patrick Alexander},
  title     = {Chemistry meets Cancer Immunotherapy: Synthesis and Characterization of Hapten-like Compounds for Selective Immunotherapy},
  doi       = {10.25972/OPUS-21138},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-211385},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Chimeric antigen receptors (CARs) are able to specifically direct T cells to tumor antigens and therapy with anti-CD19 CARs has already cured cancer patients with B-cell lymphomas who have undergone long-term therapy non-successful. Despite this impressive result, the therapy is currently only approved as a last treatment option for blood cancers due to its life-threatening deficiencies. For patient safety and to enable additional application such as the treatment of solid tumors, CAR-T cells must be controllable, e. g. by chemically programmable CARs (cpCARs) regulated by hapten-like compounds. This thesis reports the synthesis and characterization of such hapten-like compounds. In the first step, seven different warheads with two different spacers were bound to biotin in order to find a suitable warhead for programming the cpCAR. In a second step, synthetic routes for the three pharmacophores folate, c(RGD), and an RGD peptidomimetic were developed. The routes allow the modification of the pharmacophores with one of the warheads from the first step. CuAAC was chosen as a bioorthogonal approach to link pharmacophores and warheads. In total, three different pharmacophores were modified with the 1,3-diketone motif of compound 21 leading to 112, 113 and 128. Activation of the T-cell signaling cascade was tested after binding of these hapten-like compounds to the cpCAR in the presence of suitable target structures. For 112, only a slight, non-significant, activation of the T-cell signaling cascade was observed, whereas for 113 and 128, a significant activation of the T-cell signaling cascade was observed. The poor solubility of the folate compounds led to alternative strategies. Folic acid was exchanged by pteroic acid and the bifunctional, linear compounds were enlarged to trifunctional dendrimers. Besides the reported regioisomer in 112, a second one, which was not reported to date, occurred by the cyclization of the linear RGD pentapeptide leading to 113. After the reported synthesis of an RGD peptidomimetic analogous to 128 could not be reproduced, a new synthetic route was developed. It also consists of 17 steps, but reduces the number of linear steps from 13 to 10. Moreover, the developed route contains an asymmetric hydrogenation step and is, compared to the published one, more flexible by the use of the copper-catalyzed azide-alkyne cycloaddition (CuAAC). In addition, an unknown reaction was observed. Instead of the formation of a Schiff base in the reductive amination of 129, an insertion of propargylamine occurred forming 131. The reaction is almost quantitative and in high purity. After requiring no purification, it could be predestined for industrial purposes, such as the synthesis of N-functionalized 1,2-dihydroquinolines or as a building block with various orthogonal functional groups. Besides the sulfonamide 16, the diketone (21, 27, 31) and lactam compounds (39 - 41), experiments on adapter molecules with further warheads were performed. In the synthesis of a proadapter approach, in which the warhead is formed only after the retro-aldol reaction catalyzed by the mAb, 6 of 10 steps were successfully performed. A newly developed synthesis to keto-sulfonyl and keto-sulfoxide compounds could not be completed but was performed on a small scale to the point of keto-sulfonyl and keto-sulfoxide. Furthermore, a universal synthesis route was designed to allow the introduction of the warhead at the end of the synthesis by acylation. Thus, after 5 shared steps, 3 of them in quantitative yield, different warheads may be introduced. Moreover, this also facilitates the purification and the analysis of the compounds by the absence of tautomerism or labile groups. However, the acylation experiments were not successful with either the acid cyanide or the Weinreb amide. In summary, this thesis has proven that the 1,3-diketone motif is a suitable warhead for programming the cpCAR, which was developed by Hudecek et al. (unpublished data). The hapten-like compounds 112, 113 and 128 simultaneously bind to integrin \${\alpha}_v{\beta}_3\$ and the cpCAR activating the T-cell signaling cascade. The modular synthesis strategy and the use of the bioorthogonal CuAAC allow straightforward access to these valuable immunotherapeutics but revealed the need for an additional purification step to remove copper ions.},
  subject      = {Organische Synthese},
  language  = {en}
}
@phdthesis{Wich2009,
  author    = {Wich, Peter Richard},
  title     = {Multifunctional Oligopeptides as an Artificial Toolkit for Molecular Recognition Events},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-38108},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2009},
  abstract  = {The main focus of this thesis was the synthesis and analysis of multifunctional oligopeptides. The study of their non-covalent interactions with various counterparts revealed interesting new results, leading to both methodological and application related progress. The first project of this thesis concentrated on the in-depth analysis of the peptide receptor CBS-Lys-Lys-Phe-NH2 to acquire a better understanding of its binding mode upon complexation with a substrate. In this context it was possible to develop—in cooperation with the group of Prof. Sebastian Schl{\"u}cker—a direct and label free spectroscopic detection of immobilized compounds which are often found in combinatorial libraries. This new screening method utilizes the advantages of the surface enhanced Raman spectroscopy and allowed for the first time a surface mapping of a single polystyrene bead for the identification of peptides in femtomolar concentrations. Hence, this method allows a very fast and sensitive detection of resin bound compounds. The development of this promising new approach set the starting point for future experiments to enable on-bead library screenings and to investigate the complex formation of immobilized compounds. After the comprehensive analysis of the basic structural features of small peptide receptors in the first part of this thesis, the second big block focused on its in vitro evaluation using biological relevant targets. Therefore, several different modifications of the initial peptide structures were synthesized. These modifications provided a molecular toolkit for the tailor made synthesis of structures individually designed for the respective target. The first tests addressed the interaction with Alzheimer's related amyloid fibrils. During these experiments, the successful SPPS syntheses of tri- and tetravalent systems were achieved. The comparison of the multivalent form with the corresponding monovalent version was then under special investigations. These concentrated mainly on the interaction with various bacteria strains, as well as with different parasites. To localize the compounds within the organisms, the synthesis of fluorescence labelled versions was achieved. In addition, several compounds were tested by the Institute for Molecular Infection Biology of the University of W{\"u}rzburg for their antibacterial activity. This thorough evaluation of the biological activity generated precious information about the influence of small structural changes in the peptide receptors. Especially the distinct influence of the multivalency effect and the acquired synthetic skills led to the development of an advanced non-covalent recognition event, as described in the final project of this thesis. The last part of this thesis discussed the development of a novel inhibitor for the serine protease beta-tryptase based on a tailor-made surface recognition event. It was possible to study and analyze the complex interaction with the unique structure of tryptase, that features a tetrameric frame and four catalytic cleavage sites buried deep inside of the hollow structure. However, the point of attack were not the four binding pockets, as mostly described in the literature, but rather the acidic areas around the cleavage sites and at the two circular openings. These should attract peptides with basic residues, which then can block the accessibility to the active sites. A combinatorial library of 216 tetravalent peptide compounds was synthesized to find the best structural composition for the non-covalent inhibition of beta-tryptase. For the screening of the library a new on-bead assay was applied. With this method a simultaneous readout of the total inhibition of all library members was possible, thus allowing a fast and direct investigation of the still resin bound inhibitors. Several additional experiments in solution unveiled the kinetics of the inhibition process. In conclusion, both mono- and multivalent inhibitors interact in a non-destructive and reversible way with the tryptase.},
  subject      = {Peptidsynthese},
  language  = {en}
}
@phdthesis{Machon2008,
  author    = {Machon, Uwe Rainer},
  title     = {Entwicklung von Cysteinproteaseinhibitoren - ein klassischer und ein kombinatorischer Ansatz zur Inhibitoroptimierung},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-33395},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2008},
  abstract  = {Ziel der Dissertation „Entwicklung von Cysteinproteaseinhibitoren - ein klassischer und ein kombinatorischer Ansatz zur Inhibitoroptimierung" war die Optimierung von neuen Inhibitoren von Falcipain-2 und Rhodesain als neue potentielle Wirkstoffe gegen Malaria bzw. die Schlafkrankheit {\"u}ber zwei verschiedene Methoden. Es handelt sich hierbei um einen klassischen und einen kombinatorischen Ansatz. Der klassische Ansatz basiert auf einer Struktur, deren Aktivit{\"a}t per Zufall entdeckt wurde. In Screenings von synthetisierten Strukturanaloga, gest{\"u}tzt durch virtuelle Docking-Experimente am aktiven Zentrum der Cysteinproteasen, wurden Struktur-Aktivit{\"a}ts-Beziehungen erarbeitet. Bei der kombinatorischen Methode wurde zun{\"a}chst ein peptidischer Inhibitor entworfen, der durch Festphasensynthese an einem geeigneten Harz synthetisiert wurde. Durch den kombinatorischen Einsatz von Aminos{\"a}uren konnte auf diese Weise unter enormer Zeitersparnis eine Bibliothek von 150 Inhibitoren synthetisiert werden. In einem Screening dieser harzgebundenen Inhibitoren wurden anschließend die potentesten Inhibitoren identifiziert. Die Aktivit{\"a}t der gefundenen Inhibitoren aus beiden Ans{\"a}tzen an den protozoischen Erregern wurde durch in-vitro-Experimente an Plasmodien und Trypanosomen untersucht. Beim klassischen Ansatz wurde eine neue Substanzklasse entwickelt, die sehr gute Hemmeigenschaften an beiden Cysteinproteasen mit IC50-Werten im niedrigen mikromolaren Bereich zeigten. Außerdem besaßen sie eine hohe in-vitro-Aktivit{\"a}t gegen{\"u}ber den Erregern im gleichen Konzentrationsbereich. Einige der Inhibitoren zeigten keine Zytotoxizit{\"a}t an Makrophagen. Aus dem klassischen Ansatz konnten also hochaktive Substanzen mit geringer Zytotoxizit{\"a}t entwickelt werden, deren Einsatz als Wirkstoffe gegen Malaria oder der Schlafkrankheit denkbar ist. F{\"u}r den kombinatorischen Ansatz wurde zur Inhibitoroptimierung eine Screeningmethode f{\"u}r Falcipain-2 und Rhodesain direkt an einem geeigneten Harz zur Festphasensynthese entwickelt. Neu bei dieser Screeningmethode war es, dass erstmals ein quantitatives Screening einer Inhibitorbibliothek m{\"o}glich sein sollte, und nicht nur die besten Inhibitoren identifiziert werden k{\"o}nnen. Aus den Ergebnissen der Festphasenscreenings an beiden Proteasen wurden 14 besonders interessante Inhibitoren der Bibliothek ausgew{\"a}hlt und synthetisiert. Diese Verbindungsklasse zeigte ebenfalls sehr gute Ergebnisse an den isolierten Enzymen, in den mikrobiologischen Tests an den Erregern jedoch fielen alle Ergebnisse vergleichsweise schlechter aus. Die schlechte L{\"o}slichkeit, die Bioverf{\"u}gbarkeit und der Metabolismus durch den Erreger der peptidischen Inhibitoren schienen von großer Bedeutung zu sein. Der bearbeitete kombinatorische Ansatz lieferte eine neuartige Screeningmethode, die auch auf andere Targets anwendbar ist.},
  subject      = {Enzyminhibitor},
  language  = {de}
}
@phdthesis{Bickert2008,
  author    = {Bickert, Volker},
  title     = {Neue k{\"u}nstliche Guanidiniocarbonylpyrrol-Rezeptoren zur Komplexierung von Oxo-Anionen in Wasser},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-32460},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2008},
  abstract  = {Ziel der Dissertation „Neue k{\"u}nstliche Guanidiniocarbonylpyrrol-Rezeptoren zur Komplexierung von Oxo-Anionen in Wasser" war die Weiterentwicklung dieser Rezeptoren nach Schmuck f{\"u}r die Komplexierung insbesondere von Carboxylaten, um sie hinsichtlich Bindungsaffinit{\"a}t und Substratspezifit{\"a}t zu optimieren. Dazu wurde zun{\"a}chst die Synthese zweier wichtiger Grundbausteine in einzelnen Schritten vollst{\"a}ndig {\"u}berarbeitet, wobei ver{\"a}nderte Reaktionsbedingungen und Aufarbeitungsschritte zu gesteigerten Ausbeuten f{\"u}hrten. Dadurch ist es nun m{\"o}glich, diese Bausteine effizienter zu synthetisieren und im Multigramm-Maßstab f{\"u}r die Darstellung von Rezeptoren zur Oxo-Anionen-Erkennung einzusetzen. Weiterhin wurde die Verbesserung der Komplexierungseigenschaften gegen{\"u}ber Carboxylaten auf zwei Arten untersucht: zum einen durch das Anbringen eines zus{\"a}tzlichen Seitenarms an der Guanidinio-Einheit zur Bildung von Guanidiniocarbonylpyrrol-Tweezer-Rezeptoren, zum anderen durch das Einf{\"u}hren einer zweiten positiven Ladung neben der Carboxylat-Bindungsstelle (CBS) zur Darstellung biskationischer Guanidiniocarbonylpyrrol-Rezeptoren. Zur Darstellung von Tweezer-Rezeptoren wurde ein zus{\"a}tzlicher Seitenarm an der N'-Position der Guanidinio-Einheit angebracht. Die beiden Arme sollten ein Substrat pinzettenartig von zwei Seiten, mit der CBS als Kopfgruppe, komplexieren k{\"o}nnen. Durch zus{\"a}tzliche Wechselwirkungen des neuen Seitenarms sollte neben einer st{\"a}rkeren Komplexierung vor allem eine h{\"o}here Substratspezifit{\"a}t erzielt werden. Die experimentell ermittelten Bindungskonstanten lagen allerdings im Bereich der N'-unsubstituierten Rezeptoren. Somit stellen die Tweezer-Modifikationen daher keine Verbesserung der Guanidiniocarbonylpyrrol-Rezeptoren dar. In einem weiteren Projekt zur Rezeptor-Optimierung wurden, durch Einf{\"u}hrung einer zweiten positiven Ladung in Form einer terminalen Ammonium-Gruppe, biskationische Guanidiniocarbonylpyrrol-Rezeptoren erfolgreich synthetisiert. Die Komplexierungseigenschaften dieser biskationischen Rezeptoren wurden in Bindungsstudien vornehmlich mit Aminos{\"a}urecarboxylaten mittels UV- und Fluoreszenz-Spektroskopie, Massenspektrometrie, NMR-Spektroskopie, ITC und Molecular Modeling Berechnungen untersucht. Anhand der Substratspezifit{\"a}t der biskationischen Rezeptoren wurde deutlich, dass die Spacerl{\"a}nge, an der die zus{\"a}tzliche positive Ladung angebracht ist, eine entscheidende Rolle bei der Komplexierung spielte. Galten eigentlich starre, pr{\"a}organisierte, kurze Linker als vorteilhaft hinsichtlich der Entropie, so ist hier zu erkennen, dass l{\"a}ngere, flexiblere Linker zu einer besseren Komplexierung f{\"u}hren k{\"o}nnen, wenn geeignete zus{\"a}tzliche nichtkovalente Wechselwirkungen m{\"o}glich sind. Die biskationischen Rezeptoren stellen damit eine Optimierung des Carboxylat-Bindungsmotivs der Guanidiniocarbonylpyrrol-Rezeptoren nach Schmuck in der Anionen-Erkennung dar.},
  subject      = {Molekulare Erkennung},
  language  = {de}
}