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Synthesis of Natural Modified Nucleosides and Their Mass Spectrometric Quantification in Cells and Tissue

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Synthesis of Natural Modified Nucleosides and Their Mass Spectrometric Quantification in Cells and Tissue Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München Synthesis of natural modified nucleosides and their mass spectrometric quantification in cells and tissue Synthese von natürlichen modifizierten Nukleosiden und deren massenspektrometrische Quantifizierung in Zellen und Gewebe Daniel Globisch aus Kaiserslautern 2011 Erklärung Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung der LMU München vom 29. Januar 1998 von Prof. Dr. T. Carell betreut. Ehrenwörtliche Versicherung Diese Dissertation wurde selbstständig, ohne unerlaubte Hilfe erarbeitet. München, den 02.02.2011 Daniel Globisch Dissertation eingereicht am: 03.02.2011 1. Gutachter: Prof. Dr. T. Carell 2. Gutachter: Prof. Dr. S. A. Sieber Mündliche Prüfung am: 11.03.2011 The Truth Is Out There Danksagung An erster Stelle danke ich meinem Betreuer Prof. Dr. Thomas Carell für die interessante Themenstellung, die exzellenten Arbeitsbedingungen und die große wissenschaftliche Freiheit. Seine Motivationsstärke, aufbauenden Ratschläge und Begeisterung für die Bioorganik haben entscheidend zum Erfolg dieser Arbeit und meinem Interesse an der Forschung beigetragen. Vielen Dank für die hervorragende Unterstützung während meiner gesamten Promotionszeit. Den Mitgliedern der Prüfungskommission danke ich für ihre Bemühungen und den zeitlichen Aufwand bei der Evaluierung der Arbeit. Prof. Dr. Stephan A. Sieber danke ich recht herzlich für die Übernahme des Koreferats. Darüberhinaus bin ich ihm für die Hilfe bei allen massenspektrometrischen Fragestellungen zu großem Dank verpflichtet, wobei sich neben anregenden Diskussionen auch eine hervorragende und unkomplizierte Kooperation ergab. Prof. Dr. Martin Biel danke ich für die erfolgreiche Kooperation. Frau Slava Gärtner und Frau Sabine Voß danke ich für die hervorragende Hilfsbereitschaft bei allen bürokratischen und organisatorischen Fragen. Den Mitarbeitern der NMR- Spektroskopie und Massenspektrometrie an der LMU danke ich für die schnelle Aufnahme von NMR- und Massenspektren. Meinen Kooperationspartnern Dr. Stylianos Michalakis und Susanne Koch bei Prof. Dr. Martin Biel und Kerstin Kurz bei Prof. Dr. Stephan A. Sieber danke ich für eine unkomplizierte und angenehme Zusammenarbeit. Meinen arbeitskreisinternen Kooperationspartnern Tim Gehrke, Ines Thoma und Christian Trindler danke ich für den Einblick in andere interessante Themengebiete des Arbeitskreises Carell. Meinen Bachelorstudenten Ines Thoma und Ellen Broda, meinem Forschungspraktikanten Florian Huber und meiner amerikanischen Austauschstudentin Emma Cating danke ich für ihre hohe Einsatzbereitschaft und Motivation beim Bearbeiten der Projekte. Dem gesamten Arbeitskreis Carell danke ich für vier wunderschöne Jahre in denen ich viele bayrische Traditionen kennenlernen und viele großartige außeruniversitäre Ereignisse erleben durfte, wobei ich viele Freunde gewonnen habe. Christian Deiml, Martin Münzel, Tobias Brückl, Thomas Reißner, Christian Trindler und Andreas Glas danke ich für die lustigen Stadionbesuche, Championsleague-Abende im Fürstenegger und Junta-Abende. Ich wünsche euch allen viel Erfolg bei euren Projekten und Doktorarbeiten. Bedanken möchte ich mich auch beim gesamten tRNA und DNA Quantifizierungsteam mit denen viele Projekte erfolgreich bearbeitet und hunderte bis schätzungsweise tausende Proben analysiert werden konnten. Den größten Anteil hierbei hatten Tobias Brückl, Martin Münzel, Mirko Wagner, Dr. Markus Müller und Veronika Reiter. Ein besonderer Dank gilt Mirko Wagner für das Engagement in „seinem“ Zellkulturlabor. Hilfreiche Diskussionen und entscheidende Anregungen bekam ich neben den bereits genannten Personen von Dr. Sabine Schneider, Tim Gehrke, Christian Deiml, Christian Trindler, Thomas Reißner, Ines Thoma und Dr. David Pearson. Martin Münzel, Dr. Sabine Schneider, Dr. Markus Müller, Tim Gehrke und Mirko Wagner danke ich ganz besonders für das sehr gute und gewissenhafte Korrekturlesen dieser Arbeit. Mit Andreas Glas, Tobias Brückl und Martin Münzel danke ich meinen engsten Begleitern, die darüber hinaus zu echten Freunden geworden sind. Mit Andreas Glas verbrachte ich 3 Jahre in einer Box und konnte von ihm lernen wie man mit bayrischer Gelassenheit zum Erfolg kommen kann. Neben wissenschaftlichen Diskussionen konnte ich mich mit Ihm zu jeder Zeit über die aktuellsten Sportereignisse unterhalten. Die produktiven Diskussionen mit Tobias Brückl in den vergangenen vier Jahren werden für mich immer unvergessen bleiben. Zusammen starteten wir das Quantifizierungsprojekt und zerbrachen uns die Köpfe teilweise bis tief in die Nacht über das erfolgreiche Gelingen der Projekte. Die Zusammenarbeit mit ihm hätte nicht besser sein können und trug maßgeblich zum Erfolg unserer Doktorarbeiten bei. Neben wissenschaftlichen Themen fanden wir immer Zeit für private Gespräche und lustige Momente. Danke für diese Zeit! Nachdem Tobias und Andreas fertig wurden, ersetzte Martin Münzel beide in einer Person. Er übernahm den Abzug in meiner Box und wurde Projektpartner meines zweiten Projekts. Seine Zielstrebigkeit gepaart mit seiner sympathischen Art machten ihn zu einem perfekten Mitstreiter. Neben fruchtbaren wissenschaftlichen Diskussionen konnten wir auch immer über die verschiedensten Sportarten diskutieren und in der Freizeit zusammen Fußball spielen. Ständige Unterstützung erhielt ich von meinen Eltern Gabriele und Axel, meinem Bruder Julian Globisch und meinen Großeltern. Eure Hilfe während der Doktorarbeit und darüber hinaus war unheimlich wichtig für mich. Deshalb gehört Euch ein Dank von ganzem Herzen! Mein letzter und größter Dank geht an Patricia Burkert, die mich zu jeder Sekunde der letzten vier Jahre unterstützt und aufgebaut hat. Ich danke dir für die Geduld, wenn es des Öfteren ein bis zwei Stunden länger als versprochen gedauert hat. Eine bessere Unterstützung und ein größeres Verständnis für meine Freude an der Forschung kann ich mir nicht vorstellen. Parts of this thesis work were published or presented on conferences Publications 1) D. Globisch*, M. Münzel*, M. Müller, S. Michalakis, M. Wagner, S. Koch, T. Brückl, M. Biel, T. Carell. “Tissue Distribution of 5-Hydroxymethylcytosine and Search for Active Demethylation Intermediates” PloS One 2010, 5, e15367. 2) M. Münzel, D. Globisch, C. Trindler, T. Carell “Efficient Synthesis of 5-Hydroxymethylcytosine Containing DNA” Org. Lett. 2010, 12, 5671–5673. 3) M. Münzel*, D. Globisch*, T. Brückl, M. Wagner, V. Welzmiller, S. Michalakis, M. Müller, M. Biel, and T. Carell. “Quantification of the Sixth DNA-Base Hydroxymethyl-dC in the Brain” Angew. Chem. Int. Ed. 2010, 49, 5375–5377. (Selected as Hot Paper and Highlighted in Chem. Eng. News 2010, 88 (No 27), 24–25.) 4) T. Brückl*, D. Globisch*, M. Wagner, M. Müller, and T. Carell. “Parallel isotope based quantification of modified tRNA nucleosides” Angew. Chem. Int. Ed. 2009, 48, 7932–7934. (Selected as Hot Paper and Highlighted in Chem. Eng. News 2009, 87 (No 38), 35.) * These authors contributed equally to this work v Conference Presentations NAR award winner for Poster Presentation: 8th Nucleic Acid Conference (NACON VIII), Sheffield, England, 09/2010. “The Sixth DNA Base 5-Hydroxymethylcytosine in Mammalian Brain” Presented in: Nucl. Acids Res. 2010, 38, 7871–7875. Oral Presentation: 3rd EuChemMS Chemistry Congress, Nuremberg, Germany, 08/2010. “Quantification of the sixth DNA nucleoside 5-Hydroxymethylcytosine and modified tRNA nucleosides by HPLC-MS” Poster Presentation: Bayer Ph.D. Student Course 2010, Cologne, Germany, 07/2010. “Synthesis of Natural Modified Nucleosides for Quantitative HPLC-MS Analysis” Poster Presentation: Gordon Research Conference on Bioorganic Chemistry, Proctor Academy, Andover, NH, USA, 06/2009. “Quantitative Analysis of Modified tRNA Nucleosides” Poster Presentation: Synthesefest 2009 – A Celebration of Organic Chemistry in Munich, Munich, Germany, 03/2009. “Synthesis of Hypermodified Adenosine Derivatives Present in tRNA” Poster Presentation: EMBL Conference on Chemical Biology 2008, Heidelberg, Germany, 10/2008. “Synthesis of Hypermodified tRNA-Nucleosides” Poster Presentation: 3rd Nucleic Acid Chemical Biology Ph.D. Summer School, Odense, Denmark, 06/2007. “Synthetic approach to the natural tRNA modification Wybutosine (yW)” vi Other Publications 5) Y. M. Loksha, D. Globisch, R. Loddo, G. Collu, P. La Colla, E. B. Pedersen. “A Novel Synthetic Route for the Anti-HIV Drug MC-1220 and its Analogues” ChemMedChem 2010, 5, 1847–1849. 6) D. Globisch, N. Bomholt, V. V. Filichev, E. B. Pedersen. “Stability of Hoogsteen Type triplexes – Electrostatic attraction between duplex backbone and TFO using an intercalating conjugate” Helv. Chim. Acta 2008, 91, 805–818. 7) Y. M. Loksha, D. Globisch, E. B. Pedersen, P. La Colla, G. Collu, R. Loddo. “Synthesis and Anti-HIV-1 Evaluation of 1,5-Disubstituted Pyrimidine-2,4-diones” J. Heterocyclic Chem. 2008, 45, 1161–1166. Conference Article 1) E. B. Pedersen, A. M. A. Osman, D. Globisch, M. Paramasivam, S. Cogoi, N. Bomholt, P. T. Jørgensen, L. E. Xodo, V. V. Filichev. “Triplex glue by synthesizing conjugated flexible intercalators” Nucleic Acids Symp. Ser. 2008, 52, 37–38. vii viii Table of contents Table of contents ...................................................................................................................... ix 1. Summary ........................................................................................................................... 1 2. Zusammenfassung
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