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ABSTRACT Title of Document: CYCLIC DINUCLEOTIDE ABSTRACT Title of Document: CYCLIC DINUCLEOTIDE POLYMORPHISM: APPLICATIONS IN CATALYSIS AND DETECTION. Benjamin Thompson Roembke, Doctor of Philosophy, 2015 Directed By: Dr. Herman O. Sintim, Department of Chemistry and Biochemistry Cyclic dinucleotides such as cyclic dimeric guanosine monophosphate (c-di-GMP,) cyclic dimeric adenosine monophosphate (c-di-AMP) and cyclic guanosine-adenosine monophosphate (cGAMP) are found in both prokaryotes and eukaryotes and are important in cell signaling processes. The work discussed herein aims at understanding the aggregative properties of these cyclic dinucleotides for interest in the biological implications of aggregation and for the purpose of various applications of these higher order structures. The applications include simple detection methods for these molecules as well as a unique G-quadruplex peroxidase that can be used for catalysis. To that end, a new fluorescent cyclic dinucleotide which incorporated the fluorescent purine analog, 2-aminopurine, was synthesized. This analog is capable of forming intercalated dimers with natural cyclic dinucleotides containing guanine which allows for a simple turn-off fluorescent method for these second messengers. Additionally, ligands containing four guanine bases were utilized to bind c-di-GMP and leading to a fluorescent turn-on fluorescent detection method. The c-di-GMP octamer was also studied for its ability to enhance the peroxidation ability of the iron- containing cofactor hemin. Its proficient ability to enhance catalysis suggests that DNA loops found in other G-quadruplex forming molecules are not necessary for catalytic enhancement of hemin. CYCLIC DINUCLEOTIDE POLYMORPHISM: APPLICATIONS FOR CATALYSIS AND DETECTION. By Benjamin Thompson Roembke Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, College Park, in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Organic Chemistry 2015 Advisory Committee: Professor Herman O. Sintim, Chair Professor Philip DeShong Professor Daniel Falvey Professor Jeffery Davis Professor Sergei Sukharev © Copyright by Benjamin Thompson Roembke 2015 Dedication It is with the greatest respect and humility that I dedicate this thesis to my beautiful wife, Leslie, and my parents, James and Holly. Their spiritual, emotional and educational advice has been invaluable during this undertaking. It is my hope that this small token of gratitude will help them understand how much they mean to me. ii Acknowledgements I would like to acknowledge my P.I., Dr. Herman O. Sintim, who provided the necessary facilities, funding (NSF CHEM 1307218) and advice to get me through this degree process. More importantly, I would like to thank Dr. Sintim for keeping faith in a lowly graduate student trying to find his way, when undoubtedly others in a similar position would have given up on him. I would also like to acknowledge my fellow graduate students, faculty and staff whose interesting and entertaining conversations kept my spirit burning and my ideas flowing. iii Table of Contents Dedication ..................................................................................................................... ii Acknowledgements ...................................................................................................... iii Table of Contents ......................................................................................................... iv List of Tables ............................................................................................................... vi List of Figures ............................................................................................................. vii List of Schemes .......................................................................................................... xiii List of Abbreviations ................................................................................................. xiv List of References ....................................................................................................... xv Chapter 1: Introduction ................................................................................................. 1 1.1: Nucleotide Messengers ..................................................................................... 1 1.2: C-di-GMP ......................................................................................................... 2 1.3: Polymorphism of c-di-GMP ............................................................................. 5 1.4: Polymorphism of c-di-GMP in vivo.................................................................. 7 1.5: Detection of c-di-GMP ................................................................................... 10 1.6: C-di-AMP ....................................................................................................... 13 1.7: Detection of c-di-AMP ................................................................................... 16 1.8: cGAMP and 2',3'-cGAMP .............................................................................. 17 1.9: New detection modalities................................................................................ 17 1.10: References ..................................................................................................... 18 Chapter 2: Octameric G8 c-di-GMP is an efficient peroxidase and this suggests that an open G-tetrad site can effectively enhance hemin peroxidation reactions ............. 24 2.1: Introduction ..................................................................................................... 24 2.2: Results and Discussion ................................................................................... 29 2.3: Conclusion ...................................................................................................... 37 2.4: Experimental ................................................................................................... 38 2.5: References ....................................................................................................... 42 Chapter 3: A cyclic dinucleotide containing 2-aminopurine is a general fluorescent sensor for c-di-GMP and 3',3'-cGAMP ....................................................................... 47 3.1: Introduction ..................................................................................................... 47 3.2: Results and Discussion ................................................................................... 49 3.3: Conclusion ...................................................................................................... 67 3.4: Experimental ................................................................................................... 68 3.5: References ....................................................................................................... 78 Chapter 4: Detection of c-di-GMP with the Smart Ligands PyroTASQ and NaphthoTASQ ............................................................................................................ 83 4.1: Introduction ..................................................................................................... 83 4.2: Results and Discussion ................................................................................... 88 4.3: Conclusion ...................................................................................................... 97 4.4: Experimental ................................................................................................... 98 4.5: Supporting Information................................................................................. 101 4.6: References ..................................................................................................... 101 Chapter 5: Conclusion and Future Perspectives ....................................................... 104 iv 5.1: Conclusions ................................................................................................... 104 5.2: References ..................................................................................................... 110 Appendix ................................................................................................................... 112 Bibliography ............................................................................................................. 115 v List of Tables Table 2.1. Kinetic parameters for peroxidation catalyzed by various G-quadruplex forming entities. [octameric c-di-GMP or DNA] = 10 µM, [Hemin] = 0.1 µM, [ABTS] = 2 mM, [H2O2] = 0.5, 1, 2, 3 and 4 mM, [NH4Cl] = 250 mM in addition to 10 mM KCl already present from c-di-GMP/DNA stock solution. Buffer: 50 mM Tris-HCl (pH = 7.9). Kinetic analysis for some assembled catalysts show high error. It is expected that a more precise kinetic analysis could be performed with stop flow instrumentation.. ......................................................................................................... 34 Table 2.2. Dissociation constants (Kd) of G-quadruplexes (octameric c-di-GMP and c-Myc) and hemin/analogs. Due to the insensitivity of UV-visible spectroscopy and high signal to noise ratio of the measurements made, high errors are observed for dissociation constants of hemin/analogs and G-quadruplexes.. .................................. 37 Table 2.3. Solvent gradient for purification of hemin analogs synthesized in this study. ..........................................................................................................................
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