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Chemical Synthesis and Enzymatic Incorporation of Artificial Nucleotides

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Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München Chemical Synthesis and Enzymatic Incorporation of Artificial Nucleotides Chemische Synthese und enzymatischer Einbau von künstlichen Nukleotiden Sascha Serdjukow geboren in Haldensleben, Deutschland München, 2016 Erklärung Diese Dissertation wurde im Sinne von § 7 der Promotionsordnung vom 28. November 2011 von Herrn Prof. Dr. Thomas Carell betreut. Eidesstattliche Versicherung Diese Dissertation wurde eigenständig und ohne unerlaubte Hilfe erarbeitet. München, ....................... ........................................ Sascha Serdjukow Dissertation eingereicht: 18.01.2016 1. Gutachter: Prof. Dr. Thomas Carell 2. Gutachter: Prof. Dr. Anja Hoffmann-Röder Mündliche Prüfung: 18.04.2016 „Sei naiv und mach´ ein Experiment.“ Feodor Lynen „Auch eine Enttäuschung, wenn sie nur gründlich und endgültig ist, bedeutet einen Schritt vorwärts.“ Max Planck Publications M. Tomás-Gamasa, S. Serdjukow, M. Su, M. Müller, T. Carell, Angew. Chem. Int. Ed. 2015, 54, 796- 800. "Post-It" Type Connected DNA Created with a Reversible Covalent Cross-Link. S. Serdjukow, F. Kink, B. Steigenberger, M. Tomás-Gamasa, T. Carell, Chem. Commun., 2014, 50, 1861-1863. Synthesis of γ-labeled nucleoside 5′-triphosphates using click chemistry. M. Su, M. Tomás-Gamasa, S. Serdjukow, P. Mayer, T. Carell, Chem. Commun. 2014, 50, 409-411. Synthesis and properties of a Cu(II) complexing pyrazole ligandoside in DNA. Further Publications J. Gajewski, F. Buelens, S. Serdjukow, N. Cortina, H. Grubmueller, M. Grininger, Nat. Chem. Bio., manuscript under revision. Engineering fatty acid synthases (FAS) for directed polyketide production. H. Staudt, M.G. Hoesl, A. Dreuw, S. Serdjukow, D. Oesterhelt, N. Budisa, J. Wachtveitl, M. Grininger, Angew. Chem., Int. Ed. 2013, 32, 8463-8466. Directed manipulation of a flavoprotein photocycle. Conference Presentations Poster Presentation: “Enzymatic Incorporation of a Reversible Covalent DNA Cross-link”, CAS Conference Synthetic Biology, Munich, Germany, July 2015. Poster Presentation: “Enzymatic Incorporation of a Reversible Covalent DNA Cross-link”, VII. Nukleinsäurechemie-Treffen, Berlin, Germany, September 2015. Danksagungen Mein erster Dank gilt meinem Betreuer Prof. Dr. Thomas Carell für das Gewähren immenser akademischer Freiheit, wodurch die Neugier, zum Antrieb aller Bestrebungen werden konnte. Das damit verbundene Vertrauen an mich, diese Freiheit sinnvoll und gewissenhaft zu nutzen, habe ich immer als Wertschätzung empfunden. Für mich persönlich war die enorme Unterstützung und das Verständnis für die Schwierigkeiten, die mit einer Familiengründung verbunden sind, von unschätzbarem Wert. Bei Frau Prof. Dr. Anja Hoffmann-Röder bedanke ich mich für die Zweitkorrektur der Dissertation und den Mitgliedern der Prüfungskommission bin ich für die Begutachtung der Arbeit und der Teilnahme am Rigorosum zu Dank verpflichtet. Den deutschen Steuerzahlern möchte ich für die Finanzierung von akademischer Forschung danken. Die finanziellen Mittel tragen nicht nur zur Ausbildung von Wissenschaftlern bei, sondern leisten auch einen wichtigen Beitrag zur technologischen Weiterentwicklung der Gesellschaft und letztendlich der Menschheit. Die Gelegenheit durch ein Forschungsprojekt, einen winzigen Baustein zum Wissen der Menschheit hinzufügen zu dürfen, muss global betrachtet angesichts existenzieller Krisen als besonderes Privileg angesehen werden. Herrn Dr. Markus Müller danke ich für die Hilfe bei den Kristallisationsexperimenten und bei der Überwindung alltäglicher kleiner Hindernisse, die im Laboralltag auftreten. Für die gute Zusammenarbeit beim Projekt der künstlichen Basenpaare danke ich Dr. Maria Tomás- Gamasa und Meng Su. Den Kollegen aus dem Labor F4.017 verdanke ich abwechslungsreiche Musik, Hilfsbereitschaft und Diskussionen von wissenschaftlichen Fragestellungen. Dies und auch Unternehmungen außerhalb der Arbeit haben zu Verbundenheit und Freundschaft geführt. Den Mitgliedern der Arbeitsgruppe gebührt besonderer Dank für ihre Bereitschaft zum Teilen von Wissen und der Vermittlung von Fähigkeiten. Auch meinen Studenten Matthias K., Alexander N., Kerstin S., Lorenz K., Tobias B. und Patricia L. bin ich für ihr Engagement und ihre Neugier bei der Durchführung von Experimenten in Rahmen von Forschungspraktika und Bachelorarbeiten zu Dank verpflichtet. Für sorgfältiges Korrekturlesen und kritische Kommentare bin ich Edris Parsa, Ilka Sührer, Barbara Steigenberger, Thomas Wildenhof, Arne Schröder, Korbinian Brunner, Florian Kink, Dr. Iacovos Michaelides und Dr. Jakob Franke zu Dank verpflichtet. Meiner Freundin danke ich für die gemeinsame Zeit mit zahlreichen herrlichen Wanderungen, der Geburt unseres Sohnes und vielen weiteren Dingen. Die Unterstützung und das gerechte Aufteilen von Arbeit und Verpflichtungen haben es uns ermöglicht, unser Kind, die jeweilige Promotion und eine erfüllte Partnerschaft unter einen Hut zu bringen. Meinem Sohn Martin möchte ich für sein fröhliches und lebhaftes Gemüt danken. Alle Anstrengungen und Entbehrungen kann er mit seinem Lächeln und seiner unendlichen Neugier wegwischen. Meinen Eltern bin ich dankbar für Ihren Glauben in meine Fähigkeiten und die Unterstützung während des Studiums, ohne die Vieles schwieriger bis unmöglich geworden wäre. Table of Contents I Table of Contents Summary .................................................................................................................................................. IV 1 Introduction ....................................................................................................................................... 1 1.1 DNA ................................................................................................................................................ 1 1.2 Nucleoside Chemistry .................................................................................................................... 2 1.3 Chemical (Tri)phosphorylation Methods .................................................................................... 4 1.4 Cu(I)-Catalyzed Alkyne-Azide Cycloaddition ............................................................................ 9 1.5 Application of Artificial Nucleosides and Nucleotides ............................................................. 13 1.5.1 Therapeutic Nucleosides and Nucleotides ............................................................................. 13 1.5.2 Unnatural Base Pairs – Alternative Hydrogen Bonding ........................................................ 14 1.5.3 Hydrophobic Unnatural Base Pairs ....................................................................................... 16 1.5.4 Metal-Base Pairs .................................................................................................................... 19 2 Aim of the Project ............................................................................................................................ 22 3 Results and Discussion .................................................................................................................... 24 Part I – Efforts Towards Faithful Transcription and Translation of a Metal Base Pair .................. 24 3.1 Synthesis of a Salicylaldehyde Ribonucleotide ......................................................................... 24 3.2 Synthesis of a Benzaldehyde Ribonucleotide ............................................................................ 28 3.3 Synthesis of a Salicylaldehyde Ribophosphoramidite .............................................................. 32 3.4 Transcription Experiments with STP and AldTP .................................................................... 38 3.5 Transcription Experiments using T7 RNA Polymerase Mutants ........................................... 43 Part II – A Covalent Base Pair ............................................................................................................... 47 3.6 Overview of the Synthesized Amine and Aldehyde Phosphoramidites .................................. 47 3.7 Melting Temperature Analysis of Strands Containing Amine and Aldehyde Bases ............. 49 3.8 Synthesis of a Salicylaldehyde and an Aromatic Amine Deoxyribonucleotide ...................... 55 3.9 Primer Extension Experiments with dSTP and dToTP ............................................................ 59 3.9.1 Primer Extensions with dSTP ................................................................................................ 59 3.9.2 Primer Extensions with dToTP .............................................................................................. 62 3.10 Co-crystallization of Bst Pol I with S:To Containing DNA ...................................................... 64 3.11 Chemical Efforts to Improve the Enzymatic Incorporation Efficiency.................................. 68 3.11.1 Synthesis of a Naphthalene Amine Base Triphosphate ......................................................... 68 3.11.2 Primer Extension Experiments with dNaaTP ........................................................................ 71 3.11.3 Design and Proposed Synthesis of Further Improved Covalent Base Pairs .......................... 77 Part III – Click Chemistry Labeling of Phosphate-modified Nucleotides .......................................... 79 3.12 Synthesis of -Labeled Nucleotides ...........................................................................................
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