DISSERTATION Titel Synthesis and Application of Fluorinated Carbohydrates and other Bioactive Compounds angestrebter akademischer Titel Doktorin der Naturwissenschaften (Dr. rer. nat.) Verfasserin: DI Michaela Braitsch Matrikelnummer: 9726189 Dissertationsgebiet Chemie Betreuer: Univ. Prof. Dr. Walther Schmid Wien im Oktober 2009 für Nicolas 7. 12. 1989 – 21. 2. 2008 Erfolgsrezept Ich will das Geheimnis verraten, dass mich zum Ziel geführt hat. Meine Stärke liegt einzig und allein in meiner Beharrlichkeit. LOUIS PASTEUR DANKSAGUNG Die vorliegende Arbeit entstand im Zeitraum von Feburar 2005 bis Oktober 2009 am Institut für Organische Chemie der Universität Wien. An dieser Stelle möchte ich mich bei allen, die zum Entstehen und Gelingen dieser Arbeit beigetragen haben, recht herzlich bedanken: Meinem Betreuer Prof. Walther Schmid für die interessante Themenstellung, sowie seine Hilfe und Unterstützung über die gesamte Zeit. Meinen Arbeitskollegen Michael Fischer, Stefan Hader, Ralph Hollaus, Christoph Lentsch, Roman Lichtenecker, Michael Nagl, Norberth Neuwirth, ChrisTina Nowikow, Christoph Schmölzer, Helga Wolf für die freundschaftliche Zusammenarbeit und das kollegiale Arbeitsklima. Den fleißigen Bienchen Gerlinde Benesch, Martina Drescher und Jale Özgur fürs Organisieren des Laborhaushaltes. Der NMR‐Abteilung Hanspeter Kählig, Lothar Brecker und Susanne Felsinger fürs Messen von zahlreichen Spektren. Der HPLC‐ und MS‐Abteilung Sabine Schneider und Peter Unteregger. Meiner Familie, im speziellen meinen Eltern und meiner Schwester Cornelia sowie meinen Freunden für ihre Unterstützung und ihren Glauben an mich. Meinem Freund Lukas für seine unglaubliche Geduld über all die Zeit. TABLE OF CONTENT TABLE OF CONTENT TABLE OF CONTENT _________________________________________________________________ I SHORT SUMMARY _________________________________________________________________ III ZUSAMMENFASSUNG _______________________________________________________________ IV LIST OF ABBREVIATIONS _____________________________________________________________ V CHAPTER‐ A SYNTHESIS AND APPLICATION OF FLUORINATED MALTOSE DERIVATIVES ____ 1 Abstract _______________________________________________________________________ 2 Theoretical Background __________________________________________________________ 3 History of Fluorine ______________________________________________________________________ 3 Appearance ___________________________________________________________________________ 3 Fluorinated Organic Compounds __________________________________________________________ 4 Introduction of Fluorine into Organic Compounds_____________________________________________ 7 Nuclear Magnetic Resonance ____________________________________________________________ 11 Fluorine NMR ________________________________________________________________________ 11 Graphical Abstract _____________________________________________________________ 13 Strategy for the ‘Galacto‐Type’‐6‐F‐Maltose Derivative _______________________________________ 14 Results and Discussion __________________________________________________________ 15 Maltose Binding Protein ________________________________________________________________ 15 Syntheses of the Reporter System ________________________________________________________ 17 Synthesis of Fluorinated Maltose Derivatives _______________________________________________ 22 Binding Studies using the 2‐F‐Maltose Reporter System _______________________________________ 24 Relative Affinity Studies using the 2‐F‐Maltose Reporter System ________________________________ 25 Conclusion ___________________________________________________________________________ 27 Experimental Procedures ________________________________________________________ 28 General methods ______________________________________________________________________ 28 General procedures ____________________________________________________________________ 28 CHAPTER‐ B PRELIMINARY EXPERIMENTS REGARDING ENZYMATIC SYNTHESIS OF FLUORINATED DISACCHARIDES ________________________________________ 47 Abstract ______________________________________________________________________ 48 Theoretical Background _________________________________________________________ 49 Fluorine in organic chemistry ____________________________________________________________ 49 Enzyme‐catalyzed Glycoside Synthesis _____________________________________________________ 49 Enzymatic glycosylation using β‐Galactosidase ______________________________________________ 51 Graphical Abstract _____________________________________________________________ 54 Strategy for the Galactose Derivatives _____________________________________________________ 54 Strategy for the Glucose Derivatives ______________________________________________________ 55 Results and Discussion __________________________________________________________ 56 Synthesis of Donor Substrates ___________________________________________________________ 56 Synthesis of Acceptor Substrates _________________________________________________________ 57 I Mechanism of the DAST Reaction _________________________________________________________ 58 Page TABLE OF CONTENT Experimental Procedures ________________________________________________________ 60 General Methods _____________________________________________________________________ 60 General Procedures ___________________________________________________________________ 60 CHAPTER‐ C SYNTHESIS AND APPLICATION OF 5‐HYDROXY‐DICLOFENAC METABOLITE __ 71 Abstract ______________________________________________________________________ 72 Short Summary of Immunologic Tests ______________________________________________ 73 Theoretical Background __________________________________________________________ 74 Definition of Allergy ___________________________________________________________________ 74 Hypersensitivity ‐ disorders caused by immune responses _____________________________________ 74 Nature of allergens ____________________________________________________________________ 75 Immunotherapy ______________________________________________________________________ 76 Immediate hypersensitivity – most prevalent type of hypersensitivity disease _____________________ 76 Metabolism and potential immune reactions of diclofenac ____________________________________ 80 Graphical Abstract ______________________________________________________________ 83 First Dimethyl amide Approach __________________________________________________________ 83 Final Synthetic Strategy ________________________________________________________________ 84 Results and Discussion ___________________________________________________________ 85 Experimental Procedures ________________________________________________________ 88 General Methods _____________________________________________________________________ 88 General Procedures ___________________________________________________________________ 88 NMR SPECTRA APPENDIX OF SELECTED COMPOUNDS ________________________ 99 REFERENCES ___________________________________________________ 139 CURRICULUM VITAE 147 II Page SHORT SUMMARY SHORT SUMMARY This PhD Thesis describes the synthesis and application of various fluorinated carbohydrate derivatives and the synthesis of a Diclofenac metabolite to investigate a potential immunologic mechanism underlying Diclofenac‐hypersensitivity. The increasing interest in fluorinated organic compounds is due to the fact that these derivatives not only exhibit challenging biological and physico‐chemical properties, but also allow the application of high end NMR techniques as a result of the highly sensitive 19F nucleus. The development of a novel reporter system to study protein interactions via 19F‐NMR was established. This approach uses 2‐19F‐labeled maltose as a spy ligand to indirectly probe protein‐ protein interactions of proteins fused or tagged to maltose binding protein. For that purpose, a variety of stereoselective fluorinated carbohydrate derivatives was synthesized. (Chapter A) Synthetic efforts to 6‐deoxy‐6‐fluoro‐glucose and –galactose derivatives as potential substrates for enzymatic synthesis of disaccharides are described. (Chapter B) Diclofenac (VoltarenTM) is a member of nonsteroidal anti‐inflammatory drugs (NSAIDs) and has been used for its antipyretic, analgetic and anti‐inflammatory activities in the treatment of rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, and acute muscle pain. Although this drug has proven to be highly effective and secure, various adverse drug reactions have been reported in the past, including hepatotoxicity. There are several postulations of an involvement of IgE, but mechanistic evidence is still not available. To prove drug‐specific IgE mediated reactions against the nonsteroidal anti‐inflammatory drug Diclofenac, 5‐Hydroxy‐Diclofenac metabolites were synthesized. (Chapter C) III Page ZUSAMMENFASSUNG ZUSAMMENFASSUNG Die vorliegende Doktorarbeit beschäftigt sich sowohl mit der Synthese und Anwendung von fluorierten Kohlenhydraten als auch mit der Herstellung von Diclofenac Metaboliten für mechanistische Untersuchungen in der Allergieforschung. Das Interesse an fluorierten organischen Verbindungen ist in den letzten Jahren stark gestiegen. Diese Entwicklung wird einerseits auf die veränderten biologischen und physikalisch‐chemischen Eigenschaften zurückgeführt, andererseits ermöglicht der hoch empfindliche 19F Kern die Anwendung von speziellen NMR Techniken. Zur Untersuchung von Proteinbindungen mittels