INVESTIGATION OF REACTIVE INTERMEDIATES: NITROXYL (HNO) AND CARBONYLNITRENES by Tyler A. Chavez A dissertation submitted to the Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland February 2016 © 2016 Tyler A. Chavez All rights reserved Abstract Membrane inlet mass spectrometry (MIMS) is a well-established method used to detect gases dissolved in solution through the use of a semipermeable hydrophobic membrane that allows the dissolved gases, but not the liquid phase, to enter a mass spectrometer. Interest in the unique biological activity of azanone (nitroxyl, HNO) has highlighted the need for new sensitive and direct detection methods. Recently, MIMS has been shown to be a viable method for HNO detection with nanomolar sensitivity under physiologically relevant conditions (Chapter 2). In addition, this technique has been used to explore potential biological pathways to HNO production (Chapter 3). Nitrenes are reactive intermediates containing neutral, monovalent nitrogen atoms. In contrast to alky- and arylnitrenes, carbonylnitrenes are typically ground state singlets. In joint synthesis, anion photoelectron spectroscopic, and computational work we studied the three nitrenes, benzoylnitrene, acetylnitrene, and trifluoroacetylnitrene, with the purpose of determining the singlet-triplet splitting (ΔEST = ES – ET) in each case (Chapter 7). Further, triplet ethoxycarbonylnitrene and triplet t-butyloxycarbonylnitrene have been observed following photolysis of sulfilimine precursors by time-resolved infrared (TRIR) spectroscopy (Chapter 6). The observed growth kinetics of nitrene products suggest a contribution from both the triplet and singlet nitrene, with the contribution from the singlet becoming more prevalent in polar solvents. Advisor: Professor John P. Toscano Readers: Professor Kenneth D. Karlin Professor Christopher Falzone ii Dedication In honor of all those who loved and supported me through this journey iii Acknowledgments I look back fondly on my time as a graduate student at Johns Hopkins University. My journey to JHU would not have been possible without the inspiration of my undergraduate advisor, Dr. Jon M. Fukuto. I must also thank my graduate advisor Professor John P. Toscano for his support of my scientific curiosity and career development. I would also like to thank the Toscano lab members: Dr. Yonglin Liu, Dr. Art Sutton, Dr. Meredith Cline, Dr. Gizem Keceli, Dr. Daryl Guthrie, Christopher Bianco, and Saghar Nourian for all the lessons, scientific discussions, and friendships. I was fortunate to work with some great scientists outside of our lab on a variety of interesting collaborative projects. I would like to thank Dr. Nazarenno Paolocci for both serving as a member of my graduate board oral committee and collaborating on a variety of HNO related projects. Further, I would like to thank Dr. Kit Bowen, Dr. Allyson Buytendyk, Dr. Jacob Graham, and Dr. Mark Pederson for all of their work on the carbonylnitrene project. I could not have made it through this process without the love, patience, and support of my wife Justine. She has been my rock and the best teammate that anyone could ask for. This PhD is just as much hers as it is mine. I also have to thank my entire family, especially my mother Nancy, for her unconditional love and support. I have also been the beneficiary of an amazing set of in-laws who have been extremely supportive of Justine and I through this journey. iv Table of Contents Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... iii Acknowledgments.............................................................................................................. iv Table of Contents ................................................................................................................ v List of Schemes .................................................................................................................. xi List of Figures .................................................................................................................. xvi List of Tables ................................................................................................................. xxiii List of Supporting Figures ............................................................................................. xxiv List of Supporting Tables............................................................................................... xxvi Chapter 1: Fundamental Chemistry and Detection of Nitroxyl (HNO) .............................. 1 1.1 Therapeutic Potential................................................................................................. 1 1.2 Chemistry, Reactivity, and Detection of HNO ......................................................... 2 1.2.1 Fundamental Chemistry of HNO ........................................................................ 2 1.2.2 HNO Donor Molecules ....................................................................................... 2 1.2.3 HNO Reactivity .................................................................................................. 4 1.2.4 HNO Detection Methods .................................................................................... 6 1.3 Potential Endogenous Pathways to HNO Production ............................................... 7 1.4 References ............................................................................................................... 10 Chapter 2: Detection of HNO by Membrane Inlet Mass Spectrometry ............................ 22 v 2.1 Introduction ............................................................................................................. 22 2.2 Membrane Inlet Design and Methods. .................................................................... 23 2.3 Detection of HNO by MIMS ................................................................................... 24 2.4 Differentiating HNO and NO MIMS Signals ......................................................... 27 2.5 HNO donor comparison .......................................................................................... 29 2.6 Detection of HNO from HOCl mediated oxidation of N-hydroxyarginine (NOHA) ....................................................................................................................................... 30 2.7 Conclusions and future directions ........................................................................... 35 2.8 Experimental Methods ............................................................................................ 35 2.8.1 General Methods............................................................................................... 35 2.8.2 Gas Chromatographic (GC) Headspace Analysis of N2O ................................ 36 2.8.3 Membrane Inlet Design and Methods .............................................................. 37 2.9 References ............................................................................................................... 38 Chapter 3: Heme-mediated Peroxidation of 5-N-Hydroxy-L-glutamine (NHG) to form Nitroxyl (HNO) ................................................................................................................. 48 3.1 Introduction ............................................................................................................. 48 3.2 Utilizing MIMS to Probe Potential Endogenous Pathways to HNO Production .... 50 3.2 Examination of Substrate Specificity ...................................................................... 56 3.2 Examination of Enzyme Specificity ........................................................................ 57 3.3 Potential Reactivity of the Expected Acylnitroso Intermediate .............................. 58 vi 3.4 Conclusions ............................................................................................................. 62 3.5 Experimental Methods ............................................................................................ 63 3.5.1 General Methods............................................................................................... 63 3.5.2 Gas Chromatographic (GC) Headspace Analysis of N2O ................................ 63 3.5.3 Membrane Inlet Design and Methods .............................................................. 64 3.6 References ............................................................................................................... 65 Chapter 4: Application of Membrane Inlet Mass Spectrometry (MIMS) to the Study of Hydrogen Sulfide (H2S) and Thionitrous Acid (HSNO) .................................................. 73 4.1 Introduction ............................................................................................................. 73 4.2 Detection of H2S by MIMS ..................................................................................... 75 4.3 Detection of HSNO by MIMS ................................................................................ 76 4.4 Examination of Larger Mass Signals from H2S Donors ......................................... 78 4.5 HSNO Isomerization ............................................................................................... 86 4.5.1 Sulfinamide type rearrangement