Modified Nucleosides Part A: A Platform for the Chemical Tagging of Ribonucleic Acids for Analysis by Mass Spectrometry Part B: Base-Modified Thymidines Exhibiting Cytotoxicity towards Cancer Cells A Dissertation Submitted to the Graduate School of the University of Cincinnati in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy (PhD) In the Department of Chemistry of McMicken College of Arts and Sciences By: Kayla Borland Aug 2018 University of Cincinnati Cincinnati, Ohio Abstract This dissertation is focused on modified nucleosides. Part A focuses on method development for multiplex analysis of modified oligonucleosides while part B is a medicinal chemistry perspective of modified nucleosides as potential anti-cancer therapeutics. Part A uses mass spectrometry (MS) as an enabling technology for the characterization of post- transcriptionally modified nucleosides within ribonucleic acids (RNAs). These modified RNAs tend to be more challenging to completely characterize using conventional genomic-based sequencing technologies. As with many biological molecules, information relating to the presence or absence of a particular compound (i.e., qualitative measurement) is only one step in sample characterization. Additional useful information is found by performing quantitative measurements on the levels of the compound of interest in the sample. To unlock this information within RNA samples, previously reported duplex-based strategies to characterize modified RNAs in two different samples have been examined. Here is reported the use of poly adenosine polymerase (PAP), which – under optimized conditions – can add one 2’ azido modified nucleotide to the 3’-terminus of modified RNA. The addition of this azido-modified nucleotide can allow for the use of click chemistry to uniquely tag each sample. One sample is labeled with an unisotopically labeled alkyne while the other samples are reacted with an isotopically labeled alkyne. The two samples can easily be compared to one another based on doublet separate by difference in isotopic label mass. In part B modified nucleoside are synthesized because, current FDA-approved anti-cancer modified nucleosides elicit severe side effects warranting their improvement. Therefore, a series of compounds with a mechanism of action focused on inhibiting DNA replication was designed. Compound were inspired by the previous discovery that 5-(α-substituted-2-nitrobenzyloxy)methyluridine-5’-triphosphates terminate DNA synthesis. Thus, a library of thymidine analogs were synthesized and evaluated using a cell viability assay in MCF7 breast cancer cells, which were chosen because they had the greatest susceptibility to these nucleosides. The structure-activity relationship study lead to a compounds having α-tert-butyl-2-nitro-4-(phenyl)alkynylbenzyloxy; it caused 50% of MCF7 cell death at 9 ± 1 µM concentration. iii Acknowledgments This dissertation is dedicated to my family and my friends that have become like family for their continued support, prayers, and encouragement. I would like to acknowledge Dr. Limbach and Dr Merino for all their guidance in my journey to become a better scientist. iv Table of Contents Abstract ………………………………………………………………………………………....ii Acknowledgments …………………………………………………………………….….……iv List of Figures …………………………………………………………………………..……...ix List of Tables …………………………………………………………………………………xiv Part A 1 Chapter 1 Introduction .........................................................................................................17 1.1 Research goal ...............................................................................................................17 1.2 Introduction to Mass Spectrometry of Modified Ribonucleic Acids ...........................17 1.3 Stable Isotope Labeling Methods for RNA Mass Spectrometry ..................................20 1.4 18O labeling .................................................................................................................23 1.4.1 18O Labeling of RNA – Early Applications for Mass Spectrometry ...................25 1.4.2 18O Labeling of RNA for Modification Mapping by Mass Spectrometry ...........30 1.5 Future Outlook .............................................................................................................36 2 Chapter 2 Multiplexing .......................................................................................................38 2.1 Background ..................................................................................................................38 v 2.2 Experimental ................................................................................................................39 2.2.1 Materials: ..............................................................................................................39 2.2.2 Identification of Optimal azido-modified dNTP...................................................39 2.2.3 Optimization of PAP reactions .............................................................................39 2.2.4 Click reaction optimization ...................................................................................40 2.2.5 Calibration curve ...................................................................................................40 2.2.6 Model duplexes .....................................................................................................41 2.2.7 LC-MS ..................................................................................................................41 2.3 Results and Discussion .................................................................................................42 2.3.1 Identification of optimal azido-modified dNTP ...................................................42 2.3.2 Optimization of PAP extension ............................................................................43 2.3.3 Click reaction optimization ...................................................................................45 2.3.4 Model Duplexing Studies .....................................................................................47 2.3.5 Relative Quantification of Oligonucleotide Modification Levels ........................49 2.4 Conclusion ....................................................................................................................50 3 Chapter 3 Conclusions and Future Work ............................................................................52 3.1 Conclusions ..................................................................................................................52 3.2 Future work ..................................................................................................................52 3.3 Additional alkynes tags ................................................................................................53 3.4 T1 removal or inactivation ...........................................................................................58 3.5 PAP extension of oligonucleotide ................................................................................64 vi 3.6 T4 ligation ....................................................................................................................66 4 Chapter 4 Introduction ........................................................................................................70 4.1 Research goal ...............................................................................................................70 4.2 Introduction ..................................................................................................................70 4.3 Current nucleobase-modified antimetabolite chemotherapeutic drugs ........................72 4.3.1 Modified nucleobase drugs ...................................................................................72 4.3.2 Base-modified natural sugar nucleoside drugs .....................................................73 4.3.3 Base-modified unnatural sugar nucleoside drugs .................................................75 4.3.4 Development of novel nucleobase-modified antimetabolite drug candidates ......78 4.4 Conclusions and outlook ..............................................................................................87 5 Chapter 5 Base-Modified Thymidines Capable of Terminating DNA Synthesis as Novel Drug Candidates Showing Activity in Cancer Cells...................................................................88 5.1 Background ..................................................................................................................88 5.2 Chemical synthesis .......................................................................................................89 5.2.1 Materials ...............................................................................................................89 5.2.2 Chemical Synthesis ...............................................................................................90 5.2.3 Synthesized Compounds .......................................................................................90 5.3 Biochemistry experiments ..........................................................................................110 5.3.1 Cell cytotoxicity assay (MTT) ............................................................................110 5.3.2 DNA synthesis termination studies .....................................................................110 5.4 Results/discussion ......................................................................................................113