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Thermostable Nps As Biocatalysts for the Synthesis of Purine Nucleoside

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Thermostable Nps As Biocatalysts for the Synthesis of Purine Nucleoside Thermostable Nucleoside Phosphorylases as Biocatalysts for the Synthesis of Purine Nucleoside Analogues Characterisation, immobilization and synthesis Xinrui Zhou, Berlin 2014 Thermostable Nucleoside Phosphorylases as Biocatalysts for the Synthesis of Purine Nucleoside Analogues Characterisation, immobilization and synthesis vorgelegt von M. Sc. Xinrui Zhou aus Kunming (China) von der Fakultät III – Prozesswissenschaften der Technischen Universität Berlin zur Erlangung des akademischen Grades Doktor der Naturwissenschaften – Dr. rer.nat. – genehmigte Dissertation Promotionsausschuss: Vorsitzender Prof. Dr. Roland Lauster Gutachter Prof. Dr. Peter Neubauer Gutachter Prof. Dr. Igor A. Mikhailopulo Gutachter Dr. habil. Roland Wohlgemuth Gutachter Prof. Dr. Vera Meyer Tag der wissenschaftlichen Aussprache: 14.02.2014 Berlin 2014 D83 The present work was performed from January 2011 to December 2013 in the Laboratory of Bioprocess Engineering at the Department of Biotechnology and at the Department of Chemistry, Technische Universität Berlin under the supervision of Prof. Dr. Peter Neubauer (Technische Universität Berlin) and Prof. Dr. Mikhailopulo (National Academy of Sciences of Belarus). When I consider Your heavens, the work of Your fingers, The moon and the stars, which You have ordained, What is man that You are mindful of him, and the son of man that You visit him? I will praise You, O Lord, with my whole heart; I will tell of all Your marvellous works. I will be glad and rejoice in You; I will sing praise to Your name, O Most High. Bible [Psalm 8:3-4, 9:1-2] Abstract i Abstract Nucleosides represent one class of fundamental building blocks of life systems. Their analogues are extensively used as therapeutic agents for cancer and viral diseases; as precursors of oligonucleotides for therapeutic or diagnostic use; and as molecular tools in research. However, many biologically active nucleosides are difficult to synthesize chemically, which hinders biological trials and studies as well as the application of these compounds. The chemo- enzymatic synthesis in comparison to the pure chemical synthesis offers great benefits as a simple, efficient and “green” technology in view of simplicity, costs and yield. The focus of this study was on the application of novel nucleoside phosphorylases (NPs) for the biocatalytic synthesis of nucleoside analogues. The applied enzymes originate from thermo- philic microorganisms (Deinococcus geothermalis, Geobacillus thermoglucosidasius, Thermus thermphilus, and Aeropyrum pernix). Five thermostable NPs (TtPyNP, GtPyNP, DgPNP, GtPNP and ApMTAP) were successfully recombinantly expressed in soluble and active form in the Gram-negative bacterium Escherichia coli, and were characterised with respect to temperature optimum, stability, substrate specificity, kinetic behaviour, pH optimum and in view of their similarity by sequence alignments. The results demonstrate their promising biocatalytic properties, especially for TtPyNP and GtPNP, which displayed particularly high catalytic activity towards natural substrates (3-6 times higher than the commonly used enzymes from E. coli) under the experimental conditions. Furthermore, they showed high promiscuity, i.e. they were able to catalyse a broad range of modified substrates. Both enzymes accept nucleosides modified in the sugar moiety, e.g. 2’- or 3’-NH2, 2’-F (ribo- or arabino-) and 2’-OH (arabino); GtPNP recognizes 2,6-Cl or F substituted purine. In coupled reactions with TtPyNP and GtPNP as catalysts various modified purine nucleosides were successfully synthesized. For the synthesis of purine nucleoside analogues, TtPyNP and GtPNP were successfully immobilized on magnetic microsphere beads with high residual enzyme activity, high enzyme loading, and further enhanced enzyme stability. The application of the immobilized enzymes in the synthesis of 2,6-dichloropurine riboside and 6-chloro-2-fluoropurine riboside (6C2FP-R) resulted in product yields of 78.5 % and 85.5 % (HPLC), the latter molecule was isolated and purified ( >98 %) by silica chromatography (normal-phase column) and the compound’s structure was confirmed. These results reveal the great practical potential of the studied biocatalysts. Hence, it is conceivable to produce a number of nucleoside analogues by the described method, which appears more efficient than other synthetic routes described in literature so far. ii Zusammenfassung Zusammenfassung Nukleoside sind bedeutende Bausteine lebender Systeme. Chemisch modifizierte Nukleoside sind hochwirksame Pharmaka für die Behandlung von Krebs und viralen Erkrankungen. Sie sind wichtige Werkzeuge in der Zellforschung, u.a. sind sie Vorstufen für die Herstellung von Oligonukleotiden für theapeutische und diagnostische Anwendungen. Allerdings ist die chemische Synthese von modifizierten Nukleosiden in den meisten Fällen kompliziert und mit niedrigen Ausbeuten verbunden, wodurch ihr Einsatz limitiert ist. Die chemo-enzymatische Synthese von Nukleosidanaloga bietet im Vergleich zur traditionellen chemischen Synthese als eine einfache und „grüne“ Technologie entscheidende Vorteile in Bezug auf Aufwand, Reaktionsausbeuten und Kosten. In der vorliegenden Arbeit liegt der Fokus auf neu isolierten Nukleosidphosphorylasen von verschiedenen thermophilen Mikroorganismen (Deinococcus geothermalis, Geobacillus thermoglucosidasius, Thermus thermophilus, Aeropyrum pernix). Fünf thermostabile Nukleosidphosphorylasen (TtPyNP, GtPyNP, DgPNP, GtPNP and ApMTAP) wurden rekombinant in dem Gram-negativen Bakterium Escherichia coli exprimiert, aufgereinigt und umfassend in Bezug auf die Temperatur- und pH Optima, Stabilität, Substratspezifität, ihr kinetisches Verhalten charaktersisiert, sowie ihre phylogenetische Sequenzähnlichkeit analysiert. Diese Charakteristika demonstrieren die vorteilhaften biokatalytischen Eigenschaften dieser Biokatalysatoren, welches besonders für die Enzyme TtPyNP und GtPNP zutrifft. Unter Reaktionsbedinungen, die für die biokatalytische Syntheses interessant sind, zeichnen sich diese Enzyme nicht nur durch eine hohe katalytische Aktivität gegenüber natürlichen Substraten aus, sondern auch durch ein hohes Maß an Promiskuität, d.h. eine große Anzahl modifizierter Verbindungen als Substrate nutzen können. Beide Enzyme akzeptieren Zucker, die z.B. in folgenden Positionen modifiziert sind: 2’- oder 3’-NH2, 2’-F (ribo- oder arabino-) und 2’-OH (arabino). GtPNP reagiert auch mit 2,6-Cl- oder F-substituierten Purinen. Zusammen können TtPyNP und GtPNP die Synthese verschiedener modifizierter Purinnukleoside katalysieren. Für die Anwendung wurden beide Enzyme auf magnetischen mikrospherischen Trägern immobilisiert. Die Immobilisate zeigten eine hohe Aktivität und eine hohe katalytische Dichte, sowie eine erhöhte Enzymstabilität. Bei der Anwendung dieser immobilisierten Biolkatalysatoren für die Synthese von Zusammenfassung iii 2,6-Dichloropurine Ribosid (2,6CP-R) und 6-Chloro-2-Fluoropurine Ribosid (6C2FP-R) wurden Produktausbeuten von 78.5 % bzw. 85.5 % erreicht. 6C2FP-R wurde auf >98% über eine Standard Silizium-Chromatographiesäule aufgereinigt und die Struktur verifiziert. Diese Ergebnisse zeigen das große praktische Potenzial der hier untersuchten Biokatalysatoren. Es ist daher naheliegend, dass einige biologisch wichtige Nucleosid durch das in dieser Arbeit beschriebene Verfahren effizienter als wie bisher in der Literatur beschrieben, hergestellt werden können. iv Acknowledgements Acknowledgements I am indebted to so many people for their encouragement and direct or indirect support which was essential for the successful completion of this research work. I am aware of that my words are so limit to express my deep gratitude and respect for all of them. I would like to express my greatest appreciation to my supervisor Professor Peter Neubauer. He offered me chance to work on my PhD in Germany and gave me this fascinating project. His brilliant ideas, endless energy, and his enthusiasm for biotechnology in both academic and industrial areas, greatly promotes the project progress. His constant encouragement, patient guidance and, especially during my thesis writing his kindness, gentleness and full support have touched me so much. I would like to express my deepest gratitude to my second supervisor Professor Igor A. Mikhailopulo, although we are separated by great distances, his unreserved long-last support, valuable and constructive suggestions come along with every move of the project. He also gave much attention and time to read, correct and make detailed comments on my reports and manuscripts. I cannot thank you more. I am grateful to my thesis committee members: Dr. Roland Wohlgemuth from Sigma-Aldrich, Professor Vera Meyer and Professor Roland Lauster from TU Berlin for their precious time and effort. Especially I am thankful to Dr. Roland Wohlgemuth for his great interests in this project and for our fruitful discussions through telephone conferences. His kind donation of the valuable intermediates a-D-Ribose-1-phosphate and 2’-deoxyl-a-D-ribose-1-phosphate allow us to investigate the reverse reactions in more details. I am very thankful to Kathleen Szeker for working closely with me in the first one and half years on this topic: we planning experiments together, discussing results together and sharing so many exciting moments together (e.g. watching the product peak appearing on the HPLC). I will not forget these happy memories. I am very appreciative towards my colleagues Jian Li and Jennifer Jaitzig for helping me on the LC/MS
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