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UC San Diego UC San Diego Electronic Theses and Dissertations UC San Diego UC San Diego Electronic Theses and Dissertations Title Design, synthesis and application of novel fluorescent nucleosides Permalink https://escholarship.org/uc/item/5w84v342 Author Greco, Nicholas Joseph Publication Date 2008 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, SAN DIEGO Design, synthesis and application of novel fluorescent nucleosides. A Dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Chemistry By Nicholas Joseph Greco Committee in charge: Professor Yitzhak Tor, Chair Professor Simpson Joseph Professor David Kleinfeld Professor Charles L. Perrin Professor Jerry Yang 2008 Copyright Nicholas Joseph Greco, 2008 All rights reserved The dissertation of Nicholas Joseph Greco is approved, and is acceptable in quality and form for publication on microfilm: ________________________________________ ________________________________________ ________________________________________ ________________________________________ ________________________________________ Chair University of California, San Diego 2008 iii DEDICATION to those whom without I would be nothing, but with I am everything my parents, Joseph and Barbara my brother, Anthony iv EPIGRAPH “There is a time in every man's education when he arrives at the conviction that envy is ignorance; that imitation is suicide.” Ralph Waldo Emerson v TABLE OF CONTENTS SIGNATURE PAGE .........................................................................................................................iii DEDICATION................................................................................................................................... iv EPIGRAPH ....................................................................................................................................... v TABLE OF CONTENTS.................................................................................................................. vi LIST OF FIGURES.......................................................................................................................... ix LIST OF SCHEMES .......................................................................................................................xiii LIST OF TABLES ...........................................................................................................................xiv ACKNOWLEDGEMENTS.............................................................................................................. xv VITA ...............................................................................................................................................xvii ABSTRACT...................................................................................................................................xviii Chapter 1. Nucleic acids: The need for fluorescent nucleosides.................................................. 1 1.1 History of nucleic acids ................................................................................................1 1.2 Primary structure of nucleic acids ...............................................................................2 1.3 Watson-Crick base pairing...........................................................................................4 1.4 Secondary structure of nucleic acids ..........................................................................5 1.5 Biological roles of the 3 major polymorphs...............................................................11 1.6 Using fluorescence spectroscopy to probe DNA .....................................................12 1.7 Applications of fluorescent nucleosides....................................................................12 1.8 Major families of fluorescent nucleosides.................................................................13 1.9 Aims of the thesis .......................................................................................................33 1.10 References................................................................................................................34 Chapter 2. Design, synthesis and application of novel fluorescent nucleosides .......................51 2.1 Fluorescent nucleoside design principles.................................................................51 2.2 Inspiration ...................................................................................................................53 vi 2.3 Synthesis of thymidine analogues.............................................................................54 2.4 Isomorphicity of modified nucleosides ......................................................................55 2.5 Photophysical properties of thymidine analogues....................................................56 2.6 Comparison to 2-aminiopurine ..................................................................................58 2.7 Fluorescence quenching............................................................................................60 2.8 Furanyl cytidine, adenosine and guanosine analogues...........................................61 2.9 Synthesis of furanyl cytidine analogue......................................................................62 2.10 Photophysical properties of furanyl cytidine analogue ..........................................63 2.11 Synthesis of furanyl adenosine and guanosine analogues ...................................64 2.12 Photophysical properties of furanyl adenosine and guanosine analogues ..........65 2.13 Effect of temperature on the emission of furanyl decorated nucleosides.............66 2.14 Photophysical evaluation of furanyl decorated nucleosides..................................67 2.15 Phosphoramidite synthesis of furanyl thymidine nucleoside.................................69 2.16 Site specific incorporation of furanyl thymidine nucleoside...................................69 2.17 Characterizing the furanyl modified oligonucleotide ..............................................72 2.18 Stability of modified duplexes ..................................................................................75 2.19 Absorption spectra of furanyl dT containing oligonucleotides...............................77 2.20 Steady state emission spectra of furanyl dT containing oligonucleotides ............77 2.21 Investigation of additional duplexes ........................................................................79 2.22 Thermal denaturation of additional duplexes .........................................................80 2.23 Photophysical properties of additional duplexes....................................................82 2.24 Effects of temperature on the photophysical properties of furanyl containing duplexes..................................................................................................................85 2.25 Enzymatic incorporation of furanyl dT analogue....................................................87 2.26 Site specific incorporation of furanyl dC analogue.................................................88 2.27 Improving the photophysical properties of furanyl dT analogue ...........................92 vii 2.28 Concluding remarks .................................................................................................96 2.29 Acknowledgements ..................................................................................................97 Appendix - Experimental information ..............................................................................97 2.30 References..............................................................................................................122 Chapter 3. Exploring the polarity of DNA grooves .....................................................................128 3.1 Introduction ...............................................................................................................128 3.2 Expression of polarity...............................................................................................130 3.3 Probe design.............................................................................................................132 3.4 Polarity reference scale ...........................................................................................141 3.5 Polarity of DNA grooves using furanyl dT analogue ..............................................145 3.6 Polarity conversion graphs ......................................................................................151 3.7 Conclusion and future plans ....................................................................................154 3.8 Acknowledgements ..................................................................................................160 Appendix - Experimental information ............................................................................160 3.9 References................................................................................................................168 viii LIST OF FIGURES Figure 1.1 Structures of nucleotides ...............................................................................................3 Figure 1.2 Sugar puckering in nucleic acids...................................................................................4 Figure 1.3 Hydrogen bonding pattern of DNA bases.....................................................................5 Figure 1.4 Major and minor grooves of
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