Ion Spectroscopy Studies of DNA Cation Radicals Generated from Electron Transfer and Collision-Induced Dissociations Shu R. Huang A dissertation Submitted in partial fulfillment of the Requirements for the degree of Doctor of Philosophy University of Washington 2021 Reading Committee: Frantšek Tureček, Chair Matthew F. Bush Robert E. Synovec Program Authorized to Offer Degree: Department of Chemistry University of Washington © Copyright 2021 Shu R. Huang University of Washington Abstract Ion Spectroscopy Studies of DNA Cation Radicals Generated from Electron Transfer and Collision-Induced Dissociations Shu R. Huang Chair of the Supervisory Committee: Professor František Tureček Department of Chemistry This dissertation reports the UV/Vis spectroscopic investigations of transient DNA cation radical intermediates generated from collision-induced dissociation and electron transfer dissociation. To generate cation radicals using collision-induced dissociation, doubly-charged ternary Cu(II) complexes with auxiliary ligand 2,2’;6’,2’’-terpyridine and a DNA nucleobase were first formed via electrospray ionization and upon collision-induced dissociation, the ternary Cu(II) complexes undergo intramolecular redox reaction and oxidize the nucleobase to generate the DNA cation radical. To generate the cation radical using electron transfer dissociation, a doubly- charged single-strand DNA sequence was first generated via electrospray ionization and upon electron-transfer dissociation, the one electron charge-reduction will generate the DNA cation radical containing an extra hydrogen adduct. These two methods approximate the mechanism on how these DNA cation radicals are suspected to be generated in nature via ionization. Briefly, the first method of utilizing the collision-induced dissociation with ternary Cu(II) complexes represents the direct ionization of DNA. The second method of utilizing electron- transfer dissociation on multiply-charged DNA represents the indirect ionization of DNA. This dissertation covers five projects involving these radical species. The first project discusses the collision-induced dissociation of the ternary Cu(II)- 2,2’;6’,2’’-terpyridine-adenine complex to oxidize the adenine nucleobase. This spectroscopy investigation reveals the canonical 9-methyladenine cation radical is the dominant tautomer upon direct ionization of the nucleobase. Our high-level ab initio calculations also reveal the more stable 9-methylene-(1H)-adenine cation radical; unfortunately, this lower energy tautomer was not observed in the experimental action spectrum. As a follow-up, the second project is a targeted synthesis to generate the 9-methylene-(1H)-adenine tautomer. This elusive tautomer, generated in the gas-phase, also exhibited interesting gas-phase chemistry, where the primary dissociation proceeded with a hydrogen loss and a rearrangement to yield a protonated aminopteridine ion as the product. With deuterium labeling, reaction energetics and reaction kinetics, we propose an indirect mechanism for this rearrangement. The third and fourth projects report the generation of the hachimoji DNA cation radicals from collision-induced dissociation of the Cu(II) ternary complexes. This investigation revealed that the canonical tautomers are formed upon intramolecular electron transfer for the two synthetic purine nucleobases, isoguanine and 5-aza-7-deazaguanine. The two synthetic pyrimidine nucleobases, however, offer different gas-phase chemistry from each other. Based on the action spectrum and our ab initio calculations on the 6-amino-5-nitro-(1H)-pyrid-2-one cation radical, we concluded that the canonical structure is the dominant form produced upon electron transfer. For 1-methylcytosine, oxidation was accompanied by isomerization of the canonical tautomer to form 1-methylene-2-hydroxy-4-aminopyrimidine cation radical as the dominant ion, as revealed by the action spectrum, our density functional theory calculations, and deuterium exchange experiments. The last project discusses the one-electron transfer to a doubly-charged single strand DNA, GATC, to generate the cation radical moiety. The experimental spectrum and our density functional theory calculations reveal the cation radical isomerizes to a new 7,8-H- dihydroguanine cation radical. Acknowledgement First, I would like to thank my research advisor František Tureček for his years of support and guidance during my time here at the university. You showed me what it means to be a good teacher, a good mentor. More importantly, you have showed me what dedication to the craft means, what it means to produce consistent, high-quality science and lastly, the never- ending pursuit of knowledge. Thank you for that. Next, I would like to thank my undergraduate mentors, and now friends, Peter T. Palmer and Cadapakam “CJ” Venkatramani, for taking a chance on me as an undergrad and the help along the way. Thank you. Lastly, I would like to thank the members of the Tureček lab for your support, help, and guidance along the way. Thank you to the department and the university for providing me an environment to continue to learn and become better at my craft. And the NSF for funding throughout my time here. i Dedication To my sister, my family members, and my tight-knit group of friends in San Francisco, CA. ii Table of Content Abstract........................................................................................................................................ i Chapter 1—Introduction ............................................................................................................. 1 1.1 DNA Ionization Damage ....................................................................................................... 1 1.2 A Brief Introduction to Mass Spectrometry ........................................................................... 2 1.3. Electrospray Ionization to Generate Biomolecular Ions ....................................................... 3 1.4. The Quadrupole and the Quadrupole Ion Trap Mass Analyzers .......................................... 3 1.5. Tandem Mass Spectrometry ............................................................................................... 5 1.6. Collision-Induced Dissociation and CID of Cu(II) Complexes .............................................. 7 1.7. Electron Transfer Dissociation and Applied to Doubly-Charged DNA .................................. 8 1.8. Ultraviolet-Visible Light Photodissociation and UVPD Action Spectroscopy ........................ 9 1.9. Tandem MS DNA Fragmentation Pattern ...........................................................................12 1.10. Ion Detection ....................................................................................................................12 1.11. References .......................................................................................................................13 Chapter 2—Ground and Excited States of Gas-Phase DNA Nucleobase Cation-Radicals. A UV- Vis Photodissociation Action Spectroscopy and Computational Study of Adenine and 9- Methyladenine...........................................................................................................................15 2.1. Abstract ..............................................................................................................................15 2.2. Introduction ........................................................................................................................16 2.3. Experimental ......................................................................................................................18 2.3.1. Materials ......................................................................................................................18 2.3.2. Methods .......................................................................................................................18 iii 2.3.3. Calculations .................................................................................................................19 2.4. Results & Discussion .........................................................................................................21 2.4.1. Cation-Radical Generation ...........................................................................................21 2.4.2. UVPD Action Spectra ..................................................................................................22 2.4.3. Adenine Ion Structures and Spectra Assignment .........................................................23 2.4.4. 9-Methyladenine Ion Structures and Spectra Assignment ............................................24 2.4.5. Ion Dissociation Energies ............................................................................................25 2.4.6. Comparison of Adenine with Other DNA Nucleobase Cation Radicals.........................27 2.5. Conclusions .......................................................................................................................28 2.6. References .........................................................................................................................28 2.7. Appendix ............................................................................................................................36 Chapter 3—The Elusive Noncanonical Isomers of Ionized 9-Methyladenine and 2’- Deoxyadenosine .......................................................................................................................45 3.1.