BINDING OF HYDROGEN SULFIDE TO BIOLOGICALLY RELEVANT SCAFFOLDS: METAL SYSTEMS AND NON-COVALENT BINDING by MATTHEW DAVID HARTLE A DISSERTATION Presented to the Department of Chemistry and Biochemistry and the Graduate School of the University of Oregon in partial fulfillment of the requirements for the degree of Doctor of Philosophy December 2016 DISSERTATION APPROVAL PAGE Student: Matthew David Hartle Title: Binding of Hydrogen Sulfide to Biologically Relevant Scaffolds: Metal Systems and Non-covalent Binding This dissertation has been accepted and approved in partial fulfillment of the requirements for the Doctor of Philosophy degree in the Department of Chemistry and Biochemistry by: Victora DeRose Chairperson Michael Pluth Advisor Darren Johnson Core Member Bruce Branchaud Core Member Benjamin Young Institutional Representative and Scott L. Pratt Dean of the Graduate School Original approval signatures are on file with the University of Oregon Graduate School. Degree awarded December 2016 ii © 2016 Matthew David Hartle This work is licensed under a Creative Commons Attribution-SareAlike (United States) License. iii DISSERTATION ABSTRACT Matthew David Hartle Doctor of Philosophy Department of Chemistry and Biochemistry December 2016 Title: Binding of Hydrogen Sulfide to Biologically Relevant Scaffolds: Metal Systems and Non-Covalent Binding Hydrogen Sulfide (H2S) is an important biologically produced gasotransmitter along with carbon monoxide (CO) and nitric oxide (NO). Unlike CO and NO, the bioinorganic chemistry of H2S reactivity with biologically relevant metal centers remains underinvestigated. To address this gap, several model bio(in)organic complexes were used to understand the ligation and reaction chemistry of H2S, including phthalocyanine, protoporphyrin IX, tetraphenyl porphyrin, and a pyridine diimine zinc complex. In addition to being a reactive gasotransmitter, the hydrosulfide anion (HS–) has been found to be an important biological anion. Studies with readily available cobalt and zinc phthalocyanines in organic solution illustrated the importance of protonation state in the ligation and redox chemistry of H2S and highlighted the need for an organic-soluble source of HS–. To address this need, we developed a simple method to prepare tetrabutylammonium hydrosulfide (NBu4SH). Using NBu4SH, we expanded the knowledge of H2S reaction chemistry to encompass a significantly larger set of biologically relevant metals beyond iron using the protoporphyrin IX scaffold, revealing three principle reaction pathways: binding, no response, or reduction and binding. iv Iron in biology is of particular importance given its role in oxygen transport in hemoglobin. The swamp-dwelling bivalve L. Pectinata hemoglobin 1 (Hb1) transports H2S, via ligation to heme, to symbiotic bacteria. The stabilization of H2S in Hb1 is believed to be from one of the following: a protected pocket, hydrogen bonding with a proximal glutamate residue, or a complex combination of these or other factors. By using Collman's "Picket-Fence" porphyrin to isolate the protected pocket model, we determined that a protected pocket alone as insufficient to account for H2S stabilization on Hb1. This realization led to an examination of hydrogen bonding in the secondary coordination sphere of a zinc complex. Finally, we explored the role of HS– as a biologically relevant anion using a bis(ethynylaniline) supramolecular receptor. We determined that rather than covalently modifying the receptor molecule, HS– was bound in the pocket, similar to bacterial anion transport channel. This dissertation includes previously published co-authored material. v CURRICULUM VITAE NAME OF AUTHOR: Matthew David Hartle GRADUATE AND UNDERGRADUATE SCHOOLS ATTENDED: University of Oregon, Eugene, OR Colorado School of Mines, Golden, CO DEGREES AWARDED: Doctor of Philosophy, Chemistry, 2016, University of Oregon Bachelor of Science, Chemistry, 2012, Colorado School of Mines AREAS OF SPECIAL INTEREST: Space and Planetary Science and Engineering, Colorado School of Mines PROFESSIONAL EXPERIENCE: Biomass Compositional Analyst, National Renewable Energy Laboratory National Bioenergy Center, Golden, CO, 07/2011-08/2012 RPP Intern, National Renewable Energy Laboratory Integrated Applications Center, Golden, CO, 07/2011-08/2011 Physical Sciences Technician, United States Naval Observatory Alternate Master Clock, Schriever Air Force Base, CO, 06/2009-08/2009 GRANTS, AWARDS, AND HONORS: Graduate Student Award for Excellence in the Teaching of Chemistry, University of Oregon, 2016 Graduate Student Award for Excellence in the Teaching of Chemistry, University of Oregon, 2013 Outstanding Chemistry Service Award, Colorado School of Mines, 2012 Outstanding Achievement Award in Analytical Chemistry, Colorado School of Mines, 2011 vi Eagle Scout, Boy Scouts of America, 2008 Colorado School of Mines Medal of Achievement in Math and Science, Colorado School of Mines, 2007 PUBLICATIONS: Matthew D. Hartle, Samantha K. Sommer, Stephen R. Dietrich, Michael D. Pluth. “Chemically Reversible Reactions of Hydrogen Sulfide with Metal Phthalocyanines” Inorg. Chem. 2014, 53(15), 7800-7802. (Cover Article) Michael D. Pluth, Shannon W. Boettcher, George V. Nazin, Ann L. Greenaway, Matthew D. Hartle. “Collaboration and Near-Peer Mentoring as a Platform for Sustainable Science Education Outreach” J. Chem. Educ. 2015. 92(4) 625-630. Michael D. Pluth, T. Spencer Bailey, Matthew D. Hammers, Matthew D. Hartle, Hillary A. Henthorn, Andrea K. Steiger. “Natural Products Containing Hydrogen Sulfide Releasing Moieties” Synlett. 2015, 26(19) 2633-2643. Matthew D. Hartle, Daniel J. Meininger. Lev N. Zakharov, Zachary J. Tonzetich, – Michael D. Pluth. “NBu4SH provides a convenient source of HS soluble in organic solution for H2S and anion-binding research” Dalton Trans. 2015, 44(46) 19782-19785. Matthew D. Hartle, Jim S. Prell, Michael D. Pluth. “Spectroscopic Investigations into the Binding of Hydrogen Sulfide to Synthetic Picket-Fence Porphyrins.” Dalton Trans. 2016, 45(11) 4843-4853. Matthew D. Hartle, John D. Gilbertson, Michael D. Pluth. “Stabilization of Zn(II) Hydrosulfide Complex Utilizing a Hydrogen-Bond Accepting Ligand.” Chem. Commun. 2016, 52(19) 7680-7682. Matthew D. Hartle, Michael D. Pluth. “A practical guide to working with H2S at the interface of chemistry and biology” Chem. Soc. Rev. 2016, DOI: 10.1039/C6CS00212A. Matthew D. Hartle, Ryan J. Hansen, Blakely W. Tresca, Samuel S. Prakel, Lev N. Zakharov, Michael D. Pluth, Michael M. Haley, Darren W. Johnson. “A Synthetic Supramolecular Receptor for Hydrosulfide Anion” Angew. Chemie. Int. Ed. 2016, 55(38) 11480-11484. Matthew D. Hartle, McKinna R. Tillotson, Michael D. Pluth. “Spectroscopic Investigation of the Reaction of Metallo-protoporphyrins with Hydrogen Sulfide” Journal of Inorganic Biochemistry. Submitted. vii ACKNOWLEDGMENTS Thank you to my Ph.D. advisor, Dr. Michael Pluth, for the excellent help during my time at the University of Oregon. From the first day on campus, Dr. Pluth has helped me define my goals, explore the different opportunities of chemistry, and serve as an excellent mentor and guide. Thank you, Dr. Victoria DeRose, my thesis chair, and authority for understanding the theoretical and practical application of EPR spectroscopy to metal systems. Thank you as well to Dr. Darren Johnson, a committee member who pushed me to look outside my research at entrepreneurship and understand how innovations in research could be applied to the market. I would also thank Dr. Bruce Branchaud, whose insights in committee meetings and classes lead to my thorough understanding of organic chemistry. Thank you, Dr. Benjamin Young, for participation in my dissertation committee. I would also like to thank Dr. Jim Prell for help with mass spectrometry experiments, Dr. Zack Tonzetich and Dr. Daniel Meinger for the assist in synthesizing NBu4SH, Dr. John Gilbertson and Maria Delgado for inspiration and material to study secondary coordination sphere interactions, Dr. Lev Zakharov for solving many crystal structures, and Dr. Michael Strain for the NMR instrumentation and allowing me to use liquid helium for the EPR. Finally, I would like to acknowledge Mrs. Suzanne Tibbits, my AP Chemistry teacher who first inspired me to pursue chemistry. I would especially like to thank the graduate students who have made my research path so memorable. Dr. Leticia Montoya, Dr. Samantha Sommer, Dr. Jackie McGrath, Dr. Matt Hammers, Dr. Spence Bailey, Hillary Henthorn, Dan Seidenkranz, Andrea Steiger, Matt Cerda, Ryan Hanson, and Annie Greenaway. viii I would like to acknowledge the contribution of my undergraduate mentees in particular. Sterling, McKinna, Sam, and Wyatt, your collective creativity, tenacity, and desire to understand the challenging research problems facing you inspired me to work harder and find the answers to many of the probing questions you asked. I must also emphatically thank my wife, who stood by me during the stressful times and supported me during the struggle. You are amazingly talented, and an inspiring mother. Thank you, my dear daughter, as well; your innocent exploration of the world was an unending source of joy. Thank you, mom and dad, for your support and teasing whenever I came close to "squishy" science. I would like to acknowledge the financial assistance from the National Institutes of Health, (R00-GM092970 to Dr. Pluth, R01-GM087398 to Dr Johnson and Dr. Haley), National Science Foundation (CHE-1454747 to Dr. Pluth, CHE-1255570 to Dr. Gilbertson),