Nitroxyl (Hno) and Carbonylnitrenes

Home , Hypochlorous acid, Hyponitrous acid, Imidogen, Nitroxyl, Sodium bicarbonate

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

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

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.

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

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

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 (HONS to HOSN) ...... 89

4.5.2 Isomerization of HOSN to HNSO ...... 91

4.6 Conclusions ...... 92

4.7 Experimental Methods ...... 93

4.7.1 General Methods...... 93

4.7.2 Membrane Inlet Design and Methods ...... 94

4.7.3 Detection of H2S from NaSH ...... 94

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4.7.4 Detection of HSNO from the Reaction of Acidified Nitrite with NaSH ...... 95

4.7.5 MIMS Experiments in the Presence of TCEP ...... 95

4.7.6 TCEP 31P NMR Experiments ...... 95

4.7.7 Computational Methods ...... 96

4.8 References ...... 97

4.9 Supporting Information Chapter 4 ...... 102

4.9.1 Optimized Geometries and Energies ...... 102

4.9.2 Supporting Figures ...... 129

Chapter 5: Chemistry and Reactivity of Carbonylnitrenes ...... 131

5.1 Nitrene Background ...... 131

5.2 Nitrene Substituent Effects...... 134

5.3 References ...... 139

Chapter 6: Nanosecond Time-Resolved Infrared (TRIR) Studies of Oxycarbonylnitrenes

...... 143

6.1 Introduction ...... 143

6.3 Time-Resolved IR Studies of Ethoxycarbonylnitrene (2) ...... 152

6.4 Time-Resolved IR Studies of t-Butyloxycarbonylnitrene (4) ...... 159

6.5 Conclusions ...... 168

6.6 Experimental Methods ...... 169

6.6.1 General Methods...... 169

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6.6.2 Procedure for the Synthesis of N-Ethoxycarbonyl Dibenzothiophene

Sulfilimine (6)...... 170

6.6.3 Procedure for the Synthesis of N-t-Butyloxycarbonyl Dibenzothiophene

Sulfilimine (7)...... 171

6.6.4 Steady-State Photolysis of Oxycarbonylnitrene Precursors 6 and 7 ...... 171

6.6.5 Time-Resolved IR Methods...... 171

6.6.6 Computational Methods ...... 172

6.7 References ...... 173

6.8 Supporting Information Chapter 5 ...... 180

6.8.1 Optimized Geometries and Energies ...... 180

6.8.2 Calculated Rotation Barriers ...... 223

6.8.3 Compound Characterization: 1H and 13C NMR Spectra of Final Compounds

...... 227

Chapter 7: The Singlet-Triplet Splittings of Benzoylnitrene, Acetylnitrene, and

Trifluoroacetylnitrene ...... 235

7.1 Introduction ...... 235

7.2 Experimental and Computational Analysis of Singlet-Triplet Splittings...... 237

7.3 Comparison with Solution Reactivity ...... 243

7.4 Conclusions ...... 245

7.5 Experimental ...... 245

7.5.1 Synthesis and Characterization ...... 245

7.5.2 Spectroscopic Measurements ...... 247

7.5.3 Computational Analysis ...... 248

7.6 References ...... 253

Chapter 8: Miscellaneous Work...... 258

8.1 Photochemical Precursors to HNO ...... 258

8.1.1 Photochemistry of N, N’, N”-Trihydroxyisocyanuric acid (THICA) ...... 258

8.1.2 Development of o-Quinonemethide-Based Photoprecursors to HNO ...... 264

8.2 Investigation of the Solution Chemistry of Persulfides (RSSH) ...... 267

8.3 References ...... 275

Curriculum Vitae ...... 282

List of Schemes

Scheme 1.1. Nitrogen oxidation states of various nitrogen oxides ...... 1

Scheme 1.2. Dimerization of HNO to produce hyponitrous acid, which dehydrates to ultimately produce N2O and H2O...... 2

Scheme 1.3. HNO and NO producing pathways of (a-b) Angeli’s salt and (c-e) Piloty’s acid and its derivatives...... 3

Scheme 1.4. Reactivity of HNO with (a) with thiols (RSH) to produce either disulfide

(excess thiol) or the rearranged sulfinamide, (b) with phosphines to form the phosphine oxide and aza-ylide products, and (c) ferric-heme systems to produce the corresponding iron-nitrosyl...... 5

Scheme 1.5. Potential HNO producing pathways generated from (a) oxidation of N- hydroxy-L-arginine (NOHA), (b) peroxidation of hydroxylamine, and (c) reaction of thiol with S-nitrosothiol...... 9

Scheme 2.1. Reactivity of HNO with (a) itself to ultimately produce N2O and H2O, (b) with thiols (RSH) to produce either disulfide (excess thiol) or the rearranged sulfinamide,

Scheme 2.2. Oxidation of N-hydroxy-L-arginine to produce HNO and cyanamide derivative via a nitroso intermediate ...... 32

Scheme 2.3. Reaction of HOCl with amino acids to produce CO2 and an aldehyde...... 33

Scheme 2.4. Oxidation-mediated HNO producing pathways for NH2OH, acetohydroxamic acid , and hydroxyurea ...... 34

Scheme 3.1. Potential HNO producing pathways generated from (a) reaction of thiol with

S-nitrosothiol, (b) peroxidation of hydroxylamine, and (c) HOCl-mediated oxidation of

N-hydroxy-L-arginine (NOHA)...... 49

Scheme 3.2. Oxidation of (a) L-arginine, (b) L-glutamine, and (c) L-asparagine to produce HNO and L-citrulline, L-glutamic acid, and L-aspartic acid, respectively...... 50

Scheme 3.3. Potential reactivity of the intermediate acylnitrosos of (a) NHG and (b)

NHA...... 59

Scheme 3.4. Non-HNO producing reactions between (a) hydroxylamine and HNO, acetohydroxamic acid (AHA) and an acylnitroso via attack from the nitrogen (b) or oxygen (c), and AHA and HNO via attack from the nitrogen (d) or oxygen (c)...... 61

Scheme 3.5. Decomposition of N-substituted hydroxamic acid with a pyrazalone leaving group (PY) to produce the byproduct (BY) and acylnitroso that can either be trapped by

(a) water to produce HNO and the corresponding carboxylic acid, or (b) acetohydroxamic acid resulting in non-HNO producing trapped products...... 62

- Scheme 4.1. Possible reactions of H2S/HS with O2 in aqueous solution including further

- reactivity and equilibration of polysulfides (HS (n+1)) where n = 1-9...... 78

Scheme 4.2. Reactivity of tris-(2-carboxyethyl) phosphine (TCEP) with a disulfide

(RSSR) or per/polysulfide (RSSH) in aqueous solution to produce TCEP-oxide

(TCEP=O) and TCEP-sulfide (TCEP=S), respectively...... 81

Scheme 4.3. Relevant isomers of HSNO with their expected pKa values...... 87

Scheme 4.4. Deprotonation of the SN(H)O (Y-isomer) and HSNO to produce the same –

SNO anion...... 88

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Scheme 4.5. Equilibria between HONS and HOSN involving a three-membered ring transition state.32 ...... 89

Scheme 4.6. (a) Rearrangement of the putative N-hydroxysulfenamide (RSNHOH) to sulfinamide (RS(O)NH2). (b) Proposed rearrangement of HONS to HOSN...... 89

Scheme 5.1. Reactivity of singlet and triplet nitrenes to produce one isomer or a mixture of isomers, respectively...... 133

Scheme 5.2. Reactivity of methylnitrene to form methyleneimine...... 134

Scheme 5. 3. Reactivity observed upon photolysis of benzoylnitrene precursor...... 135

Scheme 6.1. The photochemistry of ethoxycarbonylazide (1) to generate the singlet ethoxycarbonylnitrene (2s) which can either react with an alkene to produce a stereospecific product or intersystem cross to triplet ethoxycarbonylnitrene (2t) which will react with an alkene in a non-stereospecific manner...... 144

Scheme 6.2. Photolysis of t-butyloxycarbonylazide (3) to produce t- butyloxycarbonylnitrene (4) that predominantly gives 5,5-dimethyl-2-oxazolidinone (5) via an intramolecular C-H insertion reaction...... 144

Scheme 6.3. Reactivity observed upon photolysis of sulfilimine 6 in acetonitrile...... 156

Scheme 6.4. Reactivity observed upon photolysis of sulfilimine 6 in cyclohexane...... 159

Scheme 6.5. Reactivity observed upon photolysis of 7 in acetonitrile, dichloromethane, and Freon-113...... 162

Scheme 6.6. Production of 5 from either (a) direct reaction with singlet and triplet nitrenes 4s and 4t, or (b) solely through the singlet via thermal repopulation...... 168

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Scheme 7.1. Dibenzothiophene (DBT) Sulfilimine-based precursors to carbonylnitrenes.

...... 236

Scheme 8.1. Isomerization of oxynitrenes (RON) to the corresponding nitroso (RNO) species...... 258

Scheme 8.2. Thermolysis and potential photochemistry of N,N’,N’’- trihydroxyisocyanuric acid (THICA)...... 258

Scheme 8.3. Potential products formed via THICA photolysis...... 261

Scheme 8.4. Products resulting from the photogeneration of benzyloxynitrene in the presence of argon (Ar) or dioxygen (O2)...... 262

Scheme 8.5. Potential reactivity of the presumed benzyloxynitrene intermediate resulting from photolysis of Bn-THICA...... 263

Scheme 8.6. (a) Mechanism responsible for the photorelease of X from o- naphthoquinone methide (oNQM) precursors. (b) Potential HNO precursor (6) reactivity upon photolysis in aqueous solution...... 265

Scheme 8.7. Proposed synthetic methods for the formation of target compound 6...... 266

Scheme 8.8. (a) Generation and (b) further reactivity of persulfides (RSSH)...... 268

Scheme 8.9. Proposed synthesis of potential precursors 23 and 24...... 269

Scheme 8.10. Persulfide precursor scaffold and release mechanism at neutral pH...... 271

Scheme 8.11. Reaction of persulfide (RSSH) with HNO to initially produce the intermediate RSSNHOH. RSSNHOH can react with, (a) RSSH to generate RSSSSR and hydroxylamine (NH2OH), (b) RSSH to generate disulfide (RSSR) and HNS, (c) RSSH to generate RSSR and HONS, or (d) rearrange to form sulfiniamide like structure

(RSS(O)NH2)...... 272

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Scheme 8.12. Modification of peptide cysteine residues with methoxycarbonylsulfenyl chloride to generate protected persulfides that can be released upon exposure to amine nucleophiles...... 274

List of Figures

Figure 1.1. Common HNO precursors including, Angeli’s salt (AS), N-hydroxybenzene sulfonfamide (Piloty’s acid, PA), acyloxy nitroso compounds (AcON), bisacylated hydroxylamines (HA), pyrazolones derivatives, and acyl nitroso compounds (AN)...... 4

Figure 2.1. Schematic representation of MIMS sample cells and membrane probes...... 24

Figure 2.2. The HNO donors, AS, PA, 2-BrPA, and 2-MSPA, and the NO donor,

DEA/NO...... 25

Figure 2.3. MIMS signals observed at m/z 30, 31, and 44 following the addition of 50

µM AS to an argon-purged 0.1 M PBS solution containing 100 µM DTPA at pH 7.4 and

37 °C...... 25

Figure 2.4. MIMS signals observed at (a) m/z 30 and (b) m/z 31 following the addition of

100 µM DEA/NO to an argon-purged 0.1 M PBS solution containing 100 µM DTPA at pH 7.4 and 37 °C, and at (c) m/z 44, 30, and (d) 31 following the addition of 100 µL of

N2O (g) to an argon-purged 0.1 M PBS solution containing 100 µM DTPA at pH 7.4 and

37 °C...... 27

Figure 3.1. Potential endogenous HNO generating scaffolds previously explored...... 51

Figure 3.2. MIMS signals observed at (a) m/z 30, m/z 31, and m/z 44 following injection of NHG (500 µM, 0 min), then H2O2 (100 µM, 1 min), and finally metMb (5 µM, 2 min) into a solution of 0.1 M pH 7.4 PBS containing 100 µM DTPA at 37 °C. (b) Zoomed in version of the m/z 31 signal from experiment (a). (c) Comparison of the m/z 30 signals observed from reaction (a) with (open circles) and without (solid circles) a liquid nitrogen trap...... 54

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Figure 3.3. MIMS signals observed at m/z 30 following injection of GLN (500 µM), then either 100 µM H2O2 (red circles) or 2.5 mM H2O2 (red line), followed by metMb (5 µM) into a solution of 0.1 M pH 7.4 PBS containing 100 µM DTPA at 37 °C...... 55

Figure 3.4. Structures of L-glutamine (GLN), 5-N-hydroxy-L-glutamine (NHG), and 5-

N-hydroxy-L-asparagine (NHA)...... 56

Figure 3.5. MIMS signals observed at m/z 30 following injection of NHA (500 µM, 0 min), then H2O2 (100 µM, 1 min), and finally metMb (5 µM, 2 min) into a solution of 0.1

M pH 7.4 PBS containing 100 µM DTPA at 37 °C with (open circles) and without (solid circles) a liquid nitrogen trap...... 56

Figure 3.6. MIMS signals observed following the injection of NHG (500 µM, blue),

NHA (500 µM, red), or NH2OH (500 µM, black) at 0 min, followed by H2O2 (100 µM,

1.5 min), and finally hemin (5 µM, 3 min) into a solution of 0.1 M pH 7.4 PBS containing 100 µM DTPA at 37 °C...... 58

Figure 4.1. (a) MIMS spectrum, with 0.1 amu increments, observed following injection of NaSH into 0.1 M pH 7.4 PBS with metal chelator DTPA (100 µM) 20 °C. (b) MIMS observed intensity at m/z 34 and m/z 33 as a function of time following 100 µM NaSH injection at t = 0 min into 0.1 M pH 7.4 PBS with 100 µM DTPA at 20 °C. (c) Plot of signal intensity versus initial concentration of NaSH. Inset: m/z 34 signal detected by

MIMS following injection of 250 nM NaSH...... 76

Figure 4.2. MIMS observed intensity at m/z 63 as a function of time following injection of 10 mM of the pre-incubated reaction mixture (see experimental section), of (blue) sodium nitrite and NaSH in 0.2 M HCl or (red) NaSH alone, into 0.1 M pH 7.4 PBS with metal chelator DTPA (100 µM) at 20 °C...... 77

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Figure 4.3. MIMS signals observed at m/z 34 (red), 33 (blue), 48 (green), 64 (black), 65

(orange), and 66 (purple) following the injection of 500 µM NaSH into 0.1 M pH 7.4

PBS containing 100 µM DTPA at 21 °C...... 79

Figure 4.4. MIMS signals observed at m/z 34 (red), 33 (blue), 48 (green), and 64 (black) following the injection of 500 µM NaSH (+), Na2S2 (●), or Na2S4 (○) into 0.1 M pH 7.4

PBS containing 100 µM DTPA at 21 °C...... 82

Figure 4.5. 31P NMR spectra resulting from 700 mM TCEP (red), NaSH (70 mM) in the presence of TCEP (700 mM, blue), and Na2S2 (70 mM) in the presence of TCEP (700 mM, black). All samples were incubated for 30 min at 21 °C in 2 M pH 5 acetic acid buffer containing 10% D2O...... 84

Figure 4.6. MIMS signals observed at m/z 34 and 64 upon the addition of NaSH (350

µM) into a solution of 0.1 M pH 7.4 PBS with 100 µM DTPA at 21 °C; initially purged with either argon (red and blue) or dioxygen (green and black)...... 85

Figure 4.7. Plots of the average maximum signal intensity observed at m/z 34 (blue), 33

(red), 64 (black), and 48 (green), upon the consecutive injections (1-7) of 250 µL H2S (g) into the same solution of 0.1 M pH 7.4 PBS with 100 µM DTPA at 21 °C. Consecutive injections occurred after plateau of the signals...... 86

Figure 4.8. B3LYP/6-31G(d) calculated energies and barriers, relative to A, for the transformation of A to C, with explicit water molecules, in the gas phase (red) and with an SM8 solvation model for aqueous solvation (blue)...... 91

Figure 4.9. B3LYP/6-31G(d) calculated energies and barriers, relative to D, for the transformation of D to E, with explicit water molecules, in the gas phase (red) and with an SM8 solvation model for aqueous solvation (blue)...... 92

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Figure 5.1. Electronic configurations of (a) imigoden (NH) and (b) methylene (CH2). 132

Figure 5.2. O-C-N bond angle comparison between triplet and singlet carbonylnitrenes.

...... 135

Figure 5.3. Relevant resonance structures for carbonyl- and oxycarbonylnitrenes...... 136

Figure 6.1. Relevant resonance structures for carbonyl- and oxycarbonylnitrenes...... 146

Figure 6.2. Sulfilimine-based photoprecursors to oxycarbonylnitrenes 2 and 4...... 147

Figure 6.3. B3LYP/6-31G(d) calculated ΔEST values (ΔEST = ES - ET) and energies of the syn- and anti-forms, including zero-point vibrational energy correction, of (a) ethoxycarbonylnitrene (2) and (b) t-butoxycarbonylnitrene (4). Values in brackets include the 7 kcal/mol correction to the B3LYP/6-31G(d) values.28 ...... 148

Figure 6.4. B3LYP/6-31G(d) calculated geometries and energies of both syn- and anti- forms of (a) ethoxycarbonylnitrene and (b) t-butoxycarbonylnitrene. Energies shown in parentheses include zero-point vibrational energy correction. Values in brackets include the 7 kcal/mol correction to the B3LYP/6-31G(d) values...... 151

Figure 6.5. TRIR difference spectra averaged over the time scales indicated following

266 nm laser photolysis of sulfilimine 6 (3 mM) in argon-saturated acetonitrile. Blue and black bars reflect B3LYP/6-31G(d) calculated frequencies and intensities of ylide 8 and triplet ethoxycarbonylnitrene (2t), respectively...... 153

Figure 6.6. Kinetic traces observed following 266 nm laser photolysis of sulfilimine 6 in argon-saturated acetonitrile at (a) 1640 cm-1 from -1 to 9 μs, and (b) 1160 cm-1 from -1 to

9 μs, (c) 1690 cm-1 from -1 to 9 μs, and (d) 1690 cm-1 from -100 to 900 μs. Black curves are the calculated best fit to a single- or double-exponential function...... 155

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Figure 6.7. TRIR difference spectra averaged over the time scales indicated following

266 nm laser photolysis of sulfilimine 6 (3 mM) in argon-saturated cyclohexane. Black bar reflect B3LYP/6-31G(d) calculated frequency of triplet ethoxycarbonylnitrene (2t).

...... 157

Figure 6.8. Kinetic traces observed following 266 nm laser photolysis of sulfilimine 6 in argon-saturated cyclohexane at (a) 1640 cm-1 from -1 to 9 μs, (b) 1728 cm-1 from -1 to 9

μs, and (c) 2180 cm-1 from -5 to 45 μs. Black curves are the calculated best fit to a single- or double-exponential function...... 158

Figure 6.9. TRIR difference spectra averaged over the time scales indicated following

266 nm laser photolysis of sulfilimine 7 (3 mM) in argon-saturated acetonitrile. The black bar reflects the B3LYP/6-31G(d) calculated frequency of triplet t- butyloxycarbonylnitrene (4t)...... 160

Figure 6.10. Kinetic traces observed following 266 nm laser photolysis of sulfilimine 7 in argon-saturated acetonitrile at (a) 1640 cm-1 from -0.4 to 3.6 μs, (b) 1762 cm-1 from -

0.4 to 3.6 μs, and (c) 2180 cm-1 from -5 to 45 μs. Black curves are the calculated best fit to a single- or double-exponential function...... 161

Figure 6.11. TRIR difference spectra averaged over the time scales indicated following

266 nm laser photolysis of sulfilimine 7 (3 mM) in argon-saturated dichloromethane.

The black bar reflects the B3LYP/6-31G(d) calculated frequency of triplet t- butyloxycarbonylnitrene (4t)...... 163

Figure 6.12. Kinetic traces observed following 266 nm laser photolysis of sulfilimine 7 in argon-saturated dichloromethane at (a) 1638 cm-1 from -0.4 to 3.6 μs, (b) 1752 cm-1

xx from -0.4 to 3.6 μs, and (c) 2175 cm-1 from -5 to 45 μs. Black curves are the calculated best fit to a single- or double-exponential function...... 164

Figure 6.13. TRIR difference spectra averaged over the time scales indicated following

266 nm laser photolysis of sulfilimine 7 (3 mM) in argon-saturated Freon-113. The black bar reflects the B3LYP/6-31G(d) calculated frequency of triplet t- butyloxycarbonylnitrene (4t)...... 165

Figure 6.14. Kinetic traces observed following 266 nm laser photolysis of sulfilimine 7 in argon-saturated Freon-113 at (a) 1640 cm-1 from -0.4 to 3.6 μs, (b) 1764 cm-1 from -0.4 to 3.6 μs, and (c) 2180 cm-1 from -5 to 45 μs. Black curves are the calculated best fit to a single- or double-exponential function...... 166

Figure 7.1. Singlet carbonylnitrene and its oxazirine-like resonance contributor...... 236

Figure 7.2. Anion photoelectron spectra of (a) benzoylnitrene, (b) acetylnitrene, and (c) trifluoroacetylnitrene anions. The vertical sticks represent the calculated vertical detachment transitions...... 239

Figure 7.3. NRMOL calculated bond lengths (Å) and angles for the doublet anion and the neutral singlet and triplet spin states of benzoyl- (2a), acetyl- (2b), and trifluroacetylnitrene (2c)...... 241

...... 242

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Figure 8.1. Experimental setup for MIMS detection of photochemically produced gasses.

...... 259

Figure 8.2. MIMS signals observed at m/z 44, 31, and 28 upon photolysis of 1 mM

THICA in 0.1 M pH 7.4 PBS containing 100 µM DTPA...... 260

Figure 8.3. MIMS signals observed at m/z 44, 31, and 28 upon photolysis of 1 mM

THICA in 0.1 M pH 7.4 PBS containing 100 µM DTPA in the presence of a liquid nitrogen cold trap...... 261

Figure 8.4. TRIR difference spectra averaged over the time scales indicated following

266 nm laser photolysis of Bn-THICA (5 mM) in argon-saturated dichloromethane. ... 262

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List of Tables

Table 3.1. Substrate oxidation by the heme/H2O2 system...... 52

Table 4. 1. Comparison of the MIMS signals observed following the injection of 500 µM

NaSH, Na2S2, Na2S4, or H2S (g) into 0.1 M pH 7.4 PBS with 100 µM DTPA at 21 °C. All signal intensities are reported relative to the m/z 34 signal for each substrate...... 80

Table 4.2. Comparison of the MIMS signals observed following the injection of 500 µM

NaSH in the presence of various concentrations of TCEP into 0.1 M pH 7.4 PBS with

100 µM DTPA at 21 °C. All signal intensities are reported relative to the m/z 34 signal for each substrate. Samples were incubated for either a30 min, b160 min, or c24 hrs at 21

°C prior to injection into the MIMS. All signal intensities are reported relative to the m/z

34 signal for each substrate...... 82

Table 7.1. Experimental Observed Transitions and Calculated Values of benzoyl-, acetyl-

, trifluroacetyl-, phenyl-, and methylnitrene. All ΔEST values include zero-point energy corrections...... 240

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List of Supporting Figures

Figure S4.1. MIMS observed intensity at m/z 30 following injection of 250 µM at t = 0 min of the pre-incubated reaction mixture, of NaSH with S-nitrosoglutathione, into 0.1 M pH 7.4 PBS with 100 µM DTPA at 20 °C...... 129

Figure S4.2. 31P NMR spectra resulting from TCEP in basic solution (red), acidic solution (blue), and neutral solution (black). All samples were incubated for 30 min at 21

°C in aqueous solutions containing 10% D2O...... 129

Figure S4.3. 31P NMR spectra resulting from TCEP in basic solution alone (red) or in the presence of H2O2 (blue) to produce TCEP-oxide at 58 ppm. All samples were incubated for 30 min at 21 °C in aqueous solutions containing 10% D2O...... 130

Figure S4.4. B3LYP/6-31G(d) calculated energies and barriers, relative to F, for the transformation of F to H, in the gas phase...... 130

Figure S6.1. Energy profile for the rotation of C-O bond of singlet ethoxycarbonylnitrene

12...... 223

Figure S6.2. Energy profile for the rotation of C-O bond of triplet ethoxycarbonylnitrene

32...... 224

Figure S6.3. Energy profile for the rotation of C-O bond of singlet t-butoxycarbonylnitrene 14...... 225

Figure S6.4. Energy profile for the rotation of C-O bond of triplet t-butoxycarbonylnitrene 34...... 226

Figure S6.5. Kinetic traces observed at 1640 cm–1 following 266 nm laser photolysis of of 6 (3 mM) in argon-saturated acetonitrile (a) without or (b) with triethylsilane (TES) in

xxiv presence. The dotted curves are experimental data; the solid curves are best fits to a single-exponential function...... 226

1 Figure S6.6. H NMR (CDCl3) of oxycarbonylnitrene precursor 6...... 227

13 Figure S6.7. C NMR (CDCl3) of oxycarbonylnitrene precursor 6...... 228

1 Figure S6.8. H NMR (CDCl3) of oxycarbonylnitrene precursor 7...... 228

13 Figure S6.9. C NMR (CDCl3) of oxycarbonylnitrene precursor 7...... 229

1 Figure S6.10. H NMR (CD3CN) of oxycarbonylnitrene precursor 7...... 229

1 Figure S6.11. H NMR (CD2Cl2) of oxycarbonylnitrene precursor 7...... 230

1 Figure S6.12. H NMR (CD3CN) of oxycarbonylnitrene precursor 7 after 4 hour photolysis at 254nm in argon-purged CD3CN...... 231

1 Figure S6.13. H NMR (CD2Cl2) of oxycarbonylnitrene precursor 7 after 4 hour photolysis at 254nm in argon-purged CD2Cl2...... 232

1 Figure S6.14. H NMR (CD3CN) of oxycarbonylnitrene precursor 6 after 4 hour photolysis at 254nm in argon-purged CD3CN...... 233

1 Figure S6.15. H NMR (CD3CN) of oxycarbonylnitrene precursor 6 after 4 hour photolysis at 254nm in argon-purged CH3CN...... 234

xxv

List of Supporting Tables

Table S4.1. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for

[SN(H)OH]+...... 102

Table S4.2. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and intensities for [SN(H)OH]+...... 103

Table S4.3. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for

+ [SN(H)OH]  SN-OH2 TS...... 103

Table S4.4. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and

+ intensities for [SN(H)OH]  SN-OH2 TS...... 104

Table S4.5. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for

SN-OH2...... 104

Table S4.6. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and intensities for SN-OH2...... 105

Table S4.7. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for

+ SN-OH2 [HOSN-H] TS...... 105

Table S4.8. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and

+ intensities for SN-OH2 [HOSN-H] TS...... 106

Table S4.9. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for

[HOSN-H]+...... 106

Table S4.10. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and intensities for [HOSN-H]+...... 107

Table S4.11. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for

[HON(H)S-W]+...... 107

xxvi

Table S4.12. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and intensities for [HON(H)S-W]+...... 108

Table S4.13. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for

[HON(H)S-W]+  SN-2W TS...... 108

Table S4.14. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and intensities for [HON(H)S-W]+  SN-2W TS...... 109

Table S4.15. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for

SN-2W...... 109

Table S4.16. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and intensities for SN-2W...... 110

Table S4.17. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for

SN-2W  [HOSN(H)-W]+ TS...... 110

Table S4.18. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and intensities for SN-2W  [HOSN(H)-W]+ TS...... 111

Table S4.19. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for

[HOSN(H)-W]+...... 111

Table S4.20. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and intensities for [HOSN(H)-W]+...... 112

Table S4.21. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for

+ [HON(H)S-W] (H2O dielectric)...... 112

Table S4.22. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and

+ intensities for [HON(H)S-W] (H2O dielectric)...... 113

xxvii

Table S4.23. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for

+ [HON(H)S-W]  SN-2W TS (H2O dielectric)...... 113

Table S4.24. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and

+ intensities for [HON(H)S-W]  SN-2W TS (H2O dielectric)...... 114

Table S4.25. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for

SN-2W (H2O dielectric)...... 114

Table S4.26. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and intensities for SN-2W (H2O dielectric)...... 115

Table S4.27. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for

+ SN-2W  [HOSN(H)] TS (H2O dielectric)...... 115

Table S4.28. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and

+ intensities for SN-2W  [HOSN(H)] TS (H2O dielectric)...... 116

Table S4.29. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for

+ [HOSN(H)] (H2O dielectric)...... 116

Table S4.30. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and

+ intensities for [HOSN(H)] (H2O dielectric)...... 117

Table S4.31. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for

HOSN-W...... 117

Table S4.32. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and intensities for HOSN-W...... 118

Table S4.33. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for

HOSN-W  HNSO-W TS...... 119

xxviii

Table S4.34. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and intensities for HOSN-W  HNSO-W TS...... 120

Table S4.35. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for

HNSO-W ...... 121

Table S4.36. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and intensities for HNSO-W...... 122

Table S4.37. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for

HOSN-W (H2O dielectric)...... 123

Table S4.38. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and intensities for HOSN-W (H2O dielectric)...... 124

Table S4.39. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for

HOSN-W  HNSO-W TS (H2O dielectric)...... 125

Table S4.40. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and intensities for HOSN-W  HNSO-W TS (H2O dielectric)...... 126

Table S4.41. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for

HNSO-W (H2O dielectric)...... 127

Table S4.42. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and intensities for HNSO-W(H2O dielectric)...... 128

Table S6.1. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for the syn-singlet Ethoxycarbonylnitrene 2ssyn (Dipole moment = 4.55 debye) ...... 180

Table S6.2. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities for syn-singlet Ethoxycarbonylnitrene 2ssyn...... 181

xxix

Table S6.3. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for syn-triplet Ethoxycarbonylnitrene 2tsyn. (Dipole moment = 3.26 debye) ...... 182

Table S6.4. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities for syn-triplet Ethoxycarbonylnitrene 2tsyn...... 183

Table S6.5. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for the anti-singlet Ethoxycarbonylnitrene 2santi. (Dipole moment = 4.75 debye) ...... 184

Table S6.6. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities for anti-singlet Ethoxycarbonylnitrene 2santi...... 185

Table S6.7. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for anti-triplet Ethoxycarbonylnitrene 2tanti.. (Dipole moment = 3.77 debye) ...... 186

Table S6.8. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities for anti-triplet Ethoxycarbonylnitrene 2tanti...... 187

Table S6.9. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for syn-singlet t-butoxycarbonylnitrene 4ssyn. . (Dipole moment = 4.80 debye) ...... 188

Table S6.10. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities for syn-singlet t-butoxycarbonylnitrene 4ssyn...... 189

Table S6.11. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for syn-triplet t-butoxycarbonylnitrene 4tsyn. (Dipole moment = 3.50 debye) ...... 190

Table S6.12. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities for syn-triplet t-butoxycarbonylnitrene 4tsyn...... 191

Table S6.13. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for anti-singlet t-butoxycarbonylnitrene 4santi. (Dipole moment = 5.03 debye) ...... 192

xxx

Table S6.14. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities for anti-singlet t-butoxycarbonylnitrene 4santi...... 193

Table S6.15. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for anti-triplet t-butoxycarbonylnitrene 4tanti. (Dipole moment = 4.16 debye) ...... 194

Table S6.16. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities for anti-triplet t-butoxycarbonylnitrene 4tanti...... 195

Table S6.17. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for

1 the syn-singlet hydroxycarbonylnitrene HOC(O)Nsyn. (Dipole moment = 3.46 debye) 196

Table S6.18. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities

1 for syn-singlet hydroxycarbonylnitrene HOC(O)Nsyn...... 196

Table S6.19. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for

3 the syn-triplet hydroxycarbonylnitrene HOC(O)Nsyn. (Dipole moment = 2.29 debye) 197

Table S6.20. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities

3 for syn-triplet hydroxycarbonylnitrene HOC(O)Nsyn...... 197

Table S6.21. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for

1 the anti-singlet hydroxycarbonylnitrene HOC(O)Nanti. (Dipole moment = 3.76 debye)

...... 198

Table S6.22. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities

1 for anti-singlet hydroxycarbonylnitrene HOC(O)Nanti...... 198

Table S6.23. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for

3 the anti-triplet hydroxycarbonylnitrene HOC(O)Nanti. (Dipole moment = 2.91 debye) 199

Table S6.24. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities

3 for syn-triplet hydroxycarbonylnitrene HOC(O)Nsyn...... 199

xxxi

Table S6.25. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for

1 the singlet formylnitrene HC(O)N. (Dipole moment = 3.06 debye) ...... 200

Table S6.26. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities for singlet formylnitrene 1HC(O)N...... 200

Table S6.27. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for

3 the triplet formylnitrene HC(O)N. (Dipole moment = 1.81 debye) ...... 201

Table S6.28. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities for triplet formylnitrene 3HC(O)N...... 201

Table S6.29. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for

1 the singlet Ethoxycarbonylnitrene rotation transition state 2TS...... 202

Table S6.30. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities

1 for singlet Ethoxycarbonylnitrene rotation transition state 2TS...... 203

Table S6.31. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for

3 the triplet Ethoxycarbonylnitrene rotation transition state 2TS...... 204

Table S6.32. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities

3 for triplet Ethoxycarbonylnitrene rotation transition state 2TS...... 205

Table S6.33. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for

1 the singlet t-butoxycarbonylnitrene rotation transition state 4TS...... 206

Table S6.34. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities

1 for singlet t-butoxycarbonylnitrene rotation transition state 4TS...... 207

Table S6.35. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for

3 the triplet t-butoxycarbonylnitrene rotation transition state 4TS...... 208

xxxii

Table S6.36. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities

3 for triplet t-butoxycarbonylnitrene rotation transition state 4TS...... 209

Table S6. 37. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for the syn-ethoxycarbonylnitrene-acetonitrile ylide 9...... 210

Table S6.38. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities for the syn-ethoxycarbonylnitrene-acetonitrile ylide 9...... 211

Table S6. 39. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for the anti-ethoxycarbonylnitrene-acetonitrile ylide 9...... 212

Table S6.40. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities for the anti-ethoxycarbonylnitrene-acetonitrile ylide 9...... 213

Table S6.41. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for syn-5-ethoxycarbonyliminodibenzothiophene ...... 214

Table S6.42. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities or syn-5-ethoxycarbonyliminodibenzothiophene 6...... 215

Table S6.43. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for anti-5-ethoxycarbonyliminodibenzothiophene 6...... 216

Table S6.44. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities for anti-5-ethoxycarbonyliminodibenzothiophene 6...... 217

Table S6.45. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for syn-5-t-butoxycarbonyliminodibenzothiophene 7...... 218

Table S6.46. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities for syn-5-t-butoxycarbonyliminodibenzothiophene 7...... 219

xxxiii

Table S6. 47. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for anti-5-t-butoxycarbonyliminodibenzothiophene 7...... 220

Table S6.48. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities for anti-5-t-butoxycarbonyliminodibenzothiophene 7...... 221

xxxiv

Chapter 1: Fundamental Chemistry and Detection of Nitroxyl (HNO)

1.1 Therapeutic Potential

Nitrogen oxides have been investigated for centuries. Arguably one of the most prevalent nitrogen oxide to this point has been nitric oxide (NO) which was discovered to be endogenously generated resulting in in a Nobel prize in 1998.1–3 Nitroxyl (HNO), the one electron reduced and protonated congener of NO, has garnered much less attention relative to its other redox siblings (Scheme 1.1). However, in recent years interest in HNO has increased as it has been shown to be a potential therapeutic for heart failure.4–8 HNO has the unique ability to modulate both contractility and relaxation by modifying important cysteine residues on both the Ryanodine receptor 2 (RYR2) and the sarcoplasmic reticulum

Ca2+ ATPase (SERCA2a) regulator phospholamban (PLN).9,10 Further, HNO has been shown to make myofilaments more sensitive to Ca2+.11 Together, these HNO-derived effects result in enhancement of myocardial function, and therefore a potent treatment for heart failure. Even though much of the observed physiology of HNO has been explored there are still mechanistic elements missing to explain the observed physiological effects.

Scheme 1.1. Nitrogen oxidation states of various nitrogen oxides

1.2 Chemistry, Reactivity, and Detection of HNO

1.2.1 Fundamental Chemistry of HNO

HNO is a reactive intermediate with interesting chemical properties. Most notable is its reactivity with itself to produce hyponitrous acid (HON=NOH), which further

6 -1 -1 12,13 dehydrates to produce nitrous oxide (N2O) and water (k = 8 x 10 M s , Scheme 1.2).

The dimerization of HNO requires the use of donor molecules for its in situ generation.

Deprotonation of HNO is slow at neutral pH as the ground state singlet 1HNO must undergo a forbidden spin inversion to the ground state triplet 3NO- during this process.12 This unique

14 acid/base relationship made accurate determination of the pKa of HNO difficult. Initially,

15 the pKa of HNO was reported to be 4.7. Ultimately, the pKa of HNO was revised to be

11.4 making HNO, not NO-, the relevant species at physiological pH.12

Scheme 1.2. Dimerization of HNO to produce hyponitrous acid, which dehydrates to ultimately produce N2O and H2O.

1.2.2 HNO Donor Molecules

Due to dimerization, HNO must be generated in situ from donor molecules. The most common HNO donor is Angeli’s salt (AS) that was developed by Angelo Angeli in

1896.16 AS decomposes upon protonation under physiological pH to produce primarily

- 17 HNO and nitrite (NO2 , Scheme 1.3a). However, under acidic conditions AS becomes a

NO donor (Scheme 1.3b).18 Another common HNO donor is N-hydroxybenzene sulfonamide (Piloty’s acid, PA) which was also developed in 1896 by Oskar Piloty.19 PA

2 produces HNO under alkaline anaerobic conditions (Scheme 1.3c). However, under physiologically relevant conditions PA produces NO via oxidation to produce presumably the corresponding nitroxide (Scheme 1.3d).20 The Toscano lab and others have produced a variety of PA derivatives by making substitutions on the aromatic ring.21–23 These derivatives have led to the development of physiologically useful donors including 2- bromo-N-hydroxybenzenesulfonamide (2-BrPA, Scheme 1.3e) as well as a small molecule therapeutic that has shown enhancement of myocardial function in humans.24 The development of new donor molecules that exclusively release HNO under physiologically relevant conditions is an active area of research with a variety of new scaffolds and derivatives being explored (Figure 1.1).25–28

Scheme 1.3. HNO and NO producing pathways of (a-b) Angeli’s salt and (c-e) Piloty’s acid and its derivatives.

1.2.3 HNO Reactivity

Much of the physiology observed for HNO stems from its ability to react with thiols

(RSH, k = 105 – 106 M-1 s-1).5 NO, on the other hand, is quite unreactive with thiols under anaerobic conditions, but in the presence of oxygen undergoes rapid autoxidation to produce nitrogen dioxide (NO2) and/or dinitrogen trioxide (N2O3), both of which react rapidly with thiols.29–34 The reaction of HNO with thiols proceeds through an intermediate

N-hydroxysulfenamide (RSNHOH) which can either rearrange to the corresponding sulfinamide under low thiol concentrations, or in the presence of excess thiol react further to produce hydroxylamine (NH2OH) and the corresponding disulfide (RSSR, Scheme

1.4a).30,35,36

Scheme 1.4. Reactivity of HNO with (a) with thiols (RSH) to produce either disulfide (excess thiol) or the rearranged sulfinamide, (b) with phosphines to form the phosphine oxide and aza-ylide products, and (c) ferric-heme systems to produce the corresponding iron-nitrosyl.

Formation of the sulfinamide has been suggested to be a potential biomarker for

HNO as this modification is unique to other nitrogen oxides.37 Initially sulfinamide modification was thought to be irreversible, however, recent evidence has confirmed that the modification is reversible on a physiologically relevant timescale.38,39 Conversely, a majority of the reported HNO induced modifications of cysteine residues, in particular on cardiac myofilaments and RYR2, results in the formation of disulfides rather than sulfinamide.10,11 The identity of the modification is confirmed by facile reduction back to the free thiol by DTT on a timescale that is faster than sulfinamide reduction.

HNO is also capable of reacting with other nucleophiles such as phosphine to produce an aza-ylide and phosphine oxide (Scheme 1.4b).40,41 Phosphines are relatively unreactive towards NO both in the presence or absence of oxygen. However, in the

42–45 presence of excess NO, phosphines can react to ultimately produce N2O. Further,

HNO reacts with metal centers, most commonly ferric-heme, to produce metal-nitrosyl complexes (Scheme 1.4c). 46–51 HNO has also been shown to react with oxygen, however, due to the spin-forbidden nature of the reaction its relatively slow (k = 3 x 103 M-1 s-1).5,52

Until recently, the products of the reaction of HNO with O2 were not well understood. A recent report by Smulik et al., suggests that HNO and O2 react to form peroxynitrite

(ONOO-).53 Finally, HNO can also react with NO (k = 5.6 x 106 M-1 s-1) to produce

12,54 ultimately N2O and nitrite.

1.2.4 HNO Detection Methods

Detection methods for HNO have been explored for decades, but until recently, many techniques suffered from lack of sensitivity and the inability to distinguish HNO from other nitrogen oxides, especially NO. In recent years there have been multiple reports of new and improved HNO detection strategies. These strategies include thiol trapping,37,55–58 phosphine trapping,40,41 fluorescent methods,41,59–61 electrochemical methods,62 amongst others. The most commonly used method for the detection of HNO is gas chromatography (GC) headspace analysis to detect N2O, the product of HNO dimerization and dehydration. The N2O generated equilibrates between the liquid phase and gas phase which is determined by its partition coefficient and Henry’s law. Although this method can be extremely sensitive with proper instrumentation, detecting N2O can lead to misinterpretation of results, especially when other direct N2O producing pathways

8 are possible. For example, only N2O would be observed in the event a small amount NO was produced in the presence of HNO. However, additional experiments employing HNO specific traps (i.e., phosphines and thiols) can aid in confirming that the N2O observed is

HNO derived.

Membrane inlet (or introduction) mass spectrometry (MIMS) is a technique that has been in use since the early 1960’s. MIMS was first reported by Hoch and Kok as a method for the detection of hydrophobic gases dissolved in aqueous solution by mass spectrometry.63 Analytes partition from the liquid phase through a membrane and into the vapor phase via pervaporation.64 Pervaporation is a process that occurs in three stages:

First, the species of interest is adsorbed at the surface of the membrane, it then permeates through the membrane, and finally desorbs into vacuum where it is detected by mass spectrometry. Factors that influence MIMS sensitivity include the diffusability and solubility of the gas in the membrane material, membrane surface area, and membrane thickness. MIMS has been used in a wide variety of applications including the detection of nitric oxide (NO) in both aqueous and blood solutions.65,66 Recently, MIMS has been shown to be a viable method for HNO detection with nanomolar sensitivity under physiologically relevant conditions.67 In addition, this technique has been used to explore potential biological pathways to HNO production.68

1.3 Potential Endogenous Pathways to HNO Production

The profound effects of HNO on the cardiovascular system has led to interest in the potential endogenous generation of HNO. For the reasons outlined above, detection of endogenously produced HNO has been unsuccessful. However, researchers have explored a variety of HNO producing pathways that are physiologically relevant. The simplest method to produce HNO would be the conversion of endogenously produced NO.

However, H-N bond strengths (HNO BDE = ~50 kcal/mol)69 and unfavorable reduction potentials under physiological conditions (-0.68 V at pH 7, NHE)70 makes H-atom

7 abstraction and electron transfer unlikely mechanisms for HNO production under physiological conditions.

It is well established that NO is generated by nitric oxide synthase (NOS) mediated oxidation of arginine. However, in the absence of the tetrahydrobiopterin cofactor, HNO production is observed.71–76 It has also been shown that an intermediate in the endogenous production of NO, N-hydroxy-L-arginine (NOHA),77 is capable of being oxidized to produce HNO by a variety of oxidants including hypochlorus acid (HOCl, Scheme

68,78 1.5a). Hydroxylamine (NH2OH) has also been shown to be a potential precursor to

HNO production via heme-mediated peroxidation (Scheme 1.5b).79,80 In addition to oxidative mechanisms, HNO has been shown to be produced from the reaction of thiols with S-nitrosothiols (RSNO) resulting in the corresponding disulfide (Scheme 1.5c).30,81

Continued examination of potential endogenous HNO production pathways using improved detection methods will be important in validating the proposed endogenous generation and biochemical roles of HNO.

In the first section of this thesis we investigate the use of MIMS for the detection of HNO, as well as its ability to explore potential endogenous pathways to HNO production. Further, we extend our use of the MIMS system to the study of hydrogen sulfide (H2S) and related species.

Scheme 1.5. Potential HNO producing pathways generated from (a) oxidation of N- hydroxy-L-arginine (NOHA), (b) peroxidation of hydroxylamine, and (c) reaction of thiol with S-nitrosothiol.

1.4 References

(1) Murad, F. Discovery of Some of the Biological Effects of Nitric Oxide and Its

Role in Cell Signaling (Nobel Lecture). Angew. Chemie Int. Ed. 1999, 38, 1856–

1868.

(2) Ignarro, L. J. Nitric Oxide: A Unique Endogenous Signaling Molecule in Vascular

Biology (Nobel Lecture). Angew. Chemie Int. Ed. 1999, 38, 1882–1892.

(3) Furchgott, R. F. Endothelium-Derived Relaxing Factor: Discovery, Early Studies,

and Identifcation as Nitric Oxide (Nobel Lecture). Angew. Chemie Int. Ed. 1999,

38, 1870–1880.

(4) Paolocci, N.; Katori, T.; Champion, H. C.; St John, M. E.; Miranda, K. M.; Fukuto,

J. M.; Wink, D. A.; Kass, D. A. Positive Inotropic and Lusitropic Effects of

HNO/NO- in Failing Hearts: Independence from Beta-Adrenergic Signaling. Proc.

Natl. Acad. Sci. 2003, 100, 5537–5542.

(5) Miranda, K. M.; Paolocci, N.; Katori, T.; Thomas, D. D.; Ford, E.; Bartberger, M.

D.; Espey, M. G.; Kass, D. A.; Feelisch, M.; Fukuto, J. M.; Wink, D. A. A

Biochemical Rationale for the Discrete Behavior of Nitroxyl and Nitric Oxide in

the Cardiovascular System. Proc. Natl. Acad. Sci. 2003, 100, 9196–9201.

(6) Tocchetti, C. G.; Wang, W.; Froehlich, J. P.; Huke, S.; Aon, M. A.; Wilson, G. M.;

Di Benedetto, G.; O’Rourke, B.; Gao, W. D.; Wink, D. A.; Toscano, J. P.;

Zaccolo, M.; Bers, D. M.; Valdivia, H. H.; Cheng, H.; Kass, D. A.; Paolocci, N.

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Chapter 2: Detection of HNO by Membrane Inlet Mass Spectrometry

2.1 Introduction

Membrane inlet (or introduction) mass spectrometry (MIMS) is a technique that has been in use since the early 1960’s. MIMS was first reported by Hoch and Kok as a method for the detection of hydrophobic gases dissolved in aqueous solution by mass spectrometry.1 Analytes partition from the liquid phase through a membrane and into the vapor phase via pervaporation.2 Pervaporation is a process that occurs in three stages:

The MIMS technique has been used in a variety of applications including the monitoring of biological reactions and industrial processes.2,3 The application of MIMS for the detection of the small hydrophobic gas, nitric oxide (NO),3,4 suggested the possibility to use MIMS to detect the closely related gas, azanone (HNO, nitroxyl). HNO, the protonated one-electron reduced form of NO, has been recently recognized for its unique biological activity, especially as a potential therapeutic for heart failure.5–9

Detection of HNO is difficult due to its facile reactivity with itself to produce hyponitrous acid (HON=NOH, k = 8 x 106 M-1s-1), which subsequently dehydrates to form nitrous oxide

10 (N2O) (Scheme 2.1a). As a result of this reactivity, HNO must be generated in situ by the use of donor molecules. It is hoped that the development of the MIMS technique for

HNO detection and investigation of the aqueous chemistry of this potentially new gaseous signaling agent will ultimately shed light on its potential mechanisms of action in vivo.

Scheme 2.1. Reactivity of HNO with (a) itself to ultimately produce N2O and H2O, (b) with thiols (RSH) to produce either disulfide (excess thiol) or the rearranged sulfinamide, (c) or with phosphines to form the phosphine oxide and aza-ylide products.

2.2 Membrane Inlet Design and Methods.

The MIMS setup originally used for HNO detection,11 based on a design reported by Silverman and co-workers,3 consists of a membrane probe that includes a piece of silastic tubing attached to glass tubing at one end and sealed at the other by a glass bead

(Figure 2.1a). The glass tubing is attached via an ultra-torr Swagelok connection to an external vacuum chamber containing a dosing line that leads to a quadrupole mass spectrometer where ions are detected following electron ionization (EI). The membrane

23 probe is immersed in an aqueous solution in a sealable 4-mL glass sample cell fit with a sample injection port.

Recently, our laboratory has begun to use a Hiden HRP-40 MIMS system with a sample cell and membrane probe that have been optimized to detect gases dissolved in aqueous solution. The modified cell contains rotary blades with an imbedded magnet for efficient stirring past the silastic membrane. The sample cell is fit with an interior heating/cooling jacket for temperature regulation, as well as multiple ports for sample injection (Figure 2.1b). As described below, with this optimized setup we can reliably detect the HNO m/z 30 NO+ fragment ion signal at precursor concentrations as low as 25 nM. Given the first-order rate constant for HNO donor decomposition (t1/2 at 25 °C = ca.

-4 -1 15 minutes, kd = 7.7 x 10 s ) and the bimolecular rate constant for HNO dimerization, we estimate a detection limit of approximately 1 nM HNO.12

Figure 2.1. Schematic representation of MIMS sample cells and membrane probes.

2.3 Detection of HNO by MIMS

MIMS has been used to examine HNO production from a variety of donors, including disodium diazen-1-ium-1,2,2-triolate (Angeli’s salt, AS), N-

24 hydroxybenzenesulfonamide (Piloty’s acid, PA), and 2-bromo-N- hydroxybenzenesulfonamide (2-BrPA) (Figure 2.2). Typical MIMS traces observed at m/z

+ + 15 + + 30 (NO ), 31 (HNO / NO ), and 44 (N2O ) after addition of AS to an argon-purged phosphate buffered saline (PBS) at pH 7.4 containing the metal chelator, diethylenetriaminepentaacetic acid (DTPA) are shown in Figure 2.3. Aliquots of HNO donor solutions are injected after a flat baseline is established and ion current intensities are measured as a function of time.

Figure 2.2. The HNO donors, AS, PA, 2-BrPA, and 2-MSPA, and the NO donor, DEA/NO.

500

m/z 44 400 m/z 30

1.2

300 0.8

0.4 m/z 31

200 Ion Current (pA) 0.0

Ion Current (pA) 0 4 8 12 100 Time (min)

0 5 10 15 20 Time (min) Figure 2.3. MIMS signals observed at m/z 30, 31, and 44 following the addition of 50 µM AS to an argon-purged 0.1 M PBS solution containing 100 µM DTPA at pH 7.4 and 37 °C.

Although the m/z 44 signal is easily assigned to N2O, the ultimate product of HNO dimerization, assignments of the m/z 30 and 31 signals can be more complicated.

Depending on the donor, its concentration, and conditions of the MIMS experiment, the

25 m/z 30 signal can contain contributions from NO, HNO, and/or N2O. The EI mass spectra

+ of NO, HNO, and N2O all contain strong NO signals at m/z 30 due to either the parent ion

(NO) or major fragment ions (HNO and N2O). The HNO parent ion m/z 31 signal is very weak with an intensity less than 2.8% that of the m/z 30 fragment ion in the reported EI mass spectrum.13 Due to this weak intensity, the detection of HNO+ can also be complicated by natural abundance 15NO+. For example, small m/z 31 MIMS signals from the NO donor, DEA/NO, and from standard N2O gas can be observed (Figure 2.4). The intensity ratio of the m/z 31 to m/z 30 signals in each of these cases matches well with that expected from natural abundance 15N (0.4%). In light of the potential complications surrounding identification of the m/z 30 and 31 MIMS signals, additional experiments must be performed to determine the relative contributions of HNO, NO, and N2O to these signals.

350 (a) 1.4 (b) 300 1.2 250 1.0

200 0.8

150 0.6 100 m/z 30 0.4 m/z 31 0.2 Ion Current (pA) Ion Current 50 (pA) Ion Current

0 0.0

-2 0 2 4 6 8 10 12 -2 0 2 4 6 8 10 12 Time (min) Time (min)

2.0 2000 (c) (d)

1.5 1500

1.0 1000 m/z 44 m/z 30 0.5 m/z 31 500

Ion Current (pA) Ion Current

Ion Current (pA) Ion Current 0.0 0

-2 0 2 4 6 8 10 12 -2 0 2 4 6 8 10 12 Time (min) Time (min) Figure 2.4. MIMS signals observed at (a) m/z 30 and (b) m/z 31 following the addition of 100 µM DEA/NO to an argon-purged 0.1 M PBS solution containing 100 µM DTPA at pH 7.4 and 37 °C, and at (c) m/z 44, 30, and (d) 31 following the addition of 100 µL of N2O (g) to an argon-purged 0.1 M PBS solution containing 100 µM DTPA at pH 7.4 and 37 °C. 2.4 Differentiating HNO and NO MIMS Signals

HNO and NO can be differentiated by their reactivity with chemical traps. For example, HNO reacts readily with thiols (e.g., glutathione (GSH)) and phosphines (e.g., tris-(4,6-dimethylphenyl)phosphine-3,3′,3″-trisulfonic acid trisodium salt (TXPTS)) under both anaerobic and aerobic conditions (Scheme 2.1b,c).14,15 NO, on the other hand, is quite unreactive with thiols under anaerobic conditions, but in the presence of oxygen undergoes rapid autoxidation to produce nitrogen dioxide (NO2) and/or dinitrogen trioxide (N2O3), both of which react rapidly with thiols.14,16–20 In addition, NO is relatively unreactive with

27 phosphines, both in the presence or absence of oxygen. However, at high TXPTS

21–24 concentrations NO can react to produce ultimately N2O.

Consistent with the above reactivity, the m/z 30 signal generated from the NO donor, sodium 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA/NO, Figure 2.2) is unaffected by the addition of 250 µM GSH under anaerobic conditions; however, when the solution is purged with oxygen, the signal is diminished.11 In addition, the DEA/NO m/z

30 MIMS signal is unaffected by up to 250 µM TXPTS. In comparison, the m/z 30 signal produced from the HNO donor, 2-BrPA, is completely quenched by 250 µM GSH under anaerobic conditions or by 250 µM TXPTS under aerobic or anaerobic conditions.

Another method that has been used to distinguish MIMS signals originating from

25 NO from those due to HNO and/or N2O is the use of liquid nitrogen cold trap. Although the boiling point of HNO is unknown, it is likely higher than that of NO (bp = -152 °C at

1 atm). If this is the case, it should be possible to trap HNO and N2O (bp = -88 °C at 1 atm) selectively using an appropriate cold trap. Since the MIMS system is under vacuum, a liquid nitrogen (bp = -196 °C at 1 atm) cold trap was found to trap HNO (derived from

25 2-BrPA) and N2O gas effectively, but not NO (produced from DEA/NO). In addition, the m/z 31 signal (15NO+) from DEA/NO (Figure 2.4b) persists in the presence of a liquid nitrogen cold trap, indicating that this signal is not due to HNO.

The contribution of N2O to the m/z 30 MIMS signal observed following decomposition of HNO donors can be determined by analysis of the m/z 44 to m/z 30 signal intensity ratio. In MIMS experiments using relatively high donor concentrations (typically greater than 100 µM), dimerization is favored and primarily N2O is observed. Under these conditions the m/z 30 signal originates solely from N2O, and the 44:30 ratio is identical to

28 that of a standard N2O sample. However, at lower donor concentrations (typically less than

5 µM), the m/z 44 signal is not observed, and the m/z 30 signal is due to the NO+ fragment ion of HNO alone. Between these two extremes, the m/z 30 signal arises from contributions from both HNO and N2O. The relative contribution of these two species can be calculated

11 using the 44:30 ratio observed for a standard N2O sample.

2.5 HNO donor comparison

MIMS has been used to compare the HNO donors, AS, PA, and 2-BrPA.11 The m/z

30 MIMS signals produced from either 50 µM AS or 2-BrPA were examined as function of GSH and TXPTS concentration. For 2-BrPA, the m/z 30 signal was completely quenched with 50 µM and above GSH or TXPTS. In the case of AS, although the intensity of the m/z 30 signal was reduced in presence of either GSH or TXPTS, it was not completely quenched even at high concentrations of trap (100 – 250 µM). Since N2O is not observed at m/z 44 and all the HNO produced should be quenched at high trap concentrations, the residual m/z 30 signal was attributed to NO. The intensity of this residual signal arising from 50 µM AS was identical to that observed from 2 µM DEA/NO.

Because the NO-forming pathways of both DEA/NO and AS produce 2 moles of NO from each mole of precursor, this result indicates that approximately 2% of AS decomposes to

NO at pH 7.4. This minor production of NO would normally not be observed since HNO is an effective trap of NO,10 and is only revealed when the HNO is removed by a selective trap. The generation of a small amount of NO by AS at neutral pH is a possibility that has recently been considered.26

The decomposition of PA at physiological pH is slow, leaving it susceptible to oxidation, which ultimately leads to NO production.27,28 This oxidation requires both trace

29 metals and oxygen.29 The TXPTS quenching of the m/z 30 MIMS signal derived from PA was examined at pH 7.4 under aerobic conditions in the presence or absence of the metal chelator, DTPA.11 In the presence of DTPA, slow production of the m/z 30 signal is observed. Addition of TXPTS results in near complete quenching of this signal, indicating mostly HNO, but some NO production under these conditions. In absence of DTPA, the growth kinetics of the m/z 30 signal become much faster and the addition of TXPTS results in only modest quenching of the signal, indicating the production of more NO under these conditions. In contrast, analogous MIMS analysis of the decomposition of 2BrPA, which has a much shorter half-life (t1/2 = ca. 2 min at pH 7.4 and 37 °C), indicates that 2-BrPA is less susceptible to oxidation and HNO production remains the dominant pathway regardless of the presence of a metal chelator or oxygen.11

2.6 Detection of HNO from HOCl mediated oxidation of N- hydroxyarginine (NOHA)

The positive pharmacological profile of HNO has led to interest in elucidating potential endogenous pathways to HNO generation. Oxidation of hydroxylamine

(NH2OH), hydroxamic acids (RC(O)NHOH), and its derivatives (e.g., N- hydroxyguanidines) have been explored previously.30–36 Recent work by Donzelli et al. has investigated the oxidation of NH2OH by horseradish peroxidase (HRP) in the presence

34 III of hydrogen peroxide (H2O2). Generation of HNO is observed from a variety of Fe containing enzymes including HRP, the globins (metmyoglobin (metMb) and methemoglobin (metHb)), catalase, myeloperoxidase (MPO), lactoperoxidase (LPO), and hemin. HNO generation appears to be affected by the identity of the axial ligand, with histidine ligands producing the highest yields. Hydroxamic acid derivatives have their own

30 history in applications as metal chelators,37 and treatments for cancer.38 Similar to the

III results of hydroxylamine, oxidation of hydroxamic acids by H2O2/Fe systems have the ability to generate HNO.39–41 The amount of HNO that is able to avoid trapping in the enzyme pocket by the resulting FeIII formed is dependent on the enzyme.

N-hydroxy-L-arginine (NOHA) is an established biosynthetic intermediate involved in the endogenous production of NO from arginine by nitric oxide synthase

(NOS). NOS has also been shown to oxidize NOHA to HNO, rather than NO, in the absence of the biopterin cofactor.30,31,42–45 The detection of an iron nitrosyl species in NOS, rather than the typical FeIII resting state further suggests the generation of HNO.43,46

Recently, it has been suggested that that the biopterin radical intermediate oxidizes the

{FeNO}7 by one electron to produce {FeNO}6, which can then release NO. In the absence of the biopterin cofactor, HNO may be released from the {FeNO}7 species.47–49

It is well established that N-hydroxyguanadines have the ability to produce NO,

HNO, or both, from a variety of chemical oxidants.32,35,44,50 This process is presumed to involve a nitroso intermediate that leads to the production of the corresponding cyanamide derivative (Scheme 2.2). A biologically relevant oxidant potentially capable of oxidizing

NOHA to produce HNO is hypochlorous acid (HOCl). HOCl is a strong oxidant that is generated in vivo by MPO from the reaction of hydrogen peroxide (H2O2) with chloride ion. MPO is released by neutrophils, monocytes, macrophages, and has been proposed to play a role in cardiovascular disease.51–53 Activated neutrophils have been shown to produce HOCl at concentrations up to 100 µM in vitro, which may be a concentration that is attainable at inflammatory sites in vivo.54–56

Scheme 2.2. Oxidation of N-hydroxy-L-arginine to produce HNO and cyanamide derivative via a nitroso intermediate Investigation of the biologically relevant oxidation of NOHA has demonstrated that

MIMS is a viable technique for the study of possible endogenous pathways for HNO production.25 Consistent with the experiments performed by Donzelli et al., 500 µM

NH2OH or NOHA with 10 µM HRP and 100 µM H2O2 led to increases in MIMS signal intensities at m/z 30 and m/z 44 for NH2OH, but not for NOHA. The oxidation of NOHA by HOCl was initially monitored by GC headspace analysis to quantify the amount of N2O produced.25 NOHA (100 µM) was incubated with varying concentrations of HOCl at 37

°C in 0.1 M PBS at pH 7.4. Significant N2O production was observed only with excess

HOCl, and a ratio of HOCl to substrate 5:1 was chosen for subsequent experiments. This ratio of oxidant to substrate is potentially plausible in a biological setting since HOCl can be generated up to concentrations of 100 µM and NOHA has been observed at concentrations of 15 µM in blood samples.57,58

MIMS experiments following the injection of 500 µM HOCl into a PBS solution of 100 µM NOHA resulted in observation of signals at m/z 30 and m/z 44.25 The observed

44:30 intensity ratio was higher than that observed for authentic N2O. A portion of the m/z

44 signal was determined to be due to CO2 production that occurs via HOCl-mediated chlorination of the amine terminus to form ultimately the corresponding aldehyde and CO2

(Scheme 2.3). Previous work has shown that HOCl can oxidize amino acids to produce

59,60 CO2 and the corresponding aldehydes, and this was confirmed by MIMS experiments

25 with L-arginine (CO2 observed) and L-arginine methyl ester (no CO2 observed).

Scheme 2.3. Reaction of HOCl with amino acids to produce CO2 and an aldehyde.

To confirm that the N2O observed following HOCl oxidation of NOHA is due to

HNO dimerization rather than from a direct N2O-producing pathway, MIMS experiments were performed at lower concentrations (< 5 µM NOHA).25 Previous MIMS work has demonstrated that at HNO donor concentrations below 5 µM, N2O dimerization is not observed and the m/z 30 signal observed under these conditions is derived from HNO.

Reducing the concentration of NOHA to 2.5 µM or below results in the loss of the m/z 44 signal, while still maintaining the m/z 30 signal, corresponding to the direct detection of

HNO. The possibility that this signal could be due to the parent ion of NO, rather than

HNO fragmentation, was ruled out with the use of a liquid nitrogen cold trap. In the presence of this cold trap, a MIMS signal at m/z 30 is not observed, indicated that NO is not produced.25

In addition to NOHA, the HOCl oxidation of NH2OH, hydroxyurea, and acetohydroxamic acid were also examined.25 All of these species have been shown capable of producing HNO upon oxidation (Scheme 2.4).33,36 The oxidation of these substrates were monitored by MIMS and GC headspace analysis. MIMS experiments revealed the typical m/z 44 and m/z 30 signals for each substrate, albeit weaker ones for

33 acetohydroxamic acid. GC headspace analysis was consistent with these MIMS observations, showing significant production of N2O, relative to AS, from both NH2OH

(90%) and hydroxyurea (91 %), and relatively little from acetohydroxamic acid (8%)

Scheme 2.4. Oxidation-mediated HNO producing pathways for NH2OH, acetohydroxamic acid , and hydroxyurea The MPO-mediated HOCl oxidation of NOHA was examined to validate the biological relevance of this HNO producing pathway.25 For comparison, oxidation of both

NH2OH and NOHA (100 µM) was monitored in the presence of 15 or 75 nM MPO with excess chloride ion (143 mM) and 500 µM H2O2. HOCl generation was initiated by the addition of H2O2 to a solution of MPO and chloride ion in the presence of substrate. The reactions were examined by GC headspace analysis of N2O. With 15 nM MPO, only a small percentage of N2O (25%, relative to AS) is observed for the oxidation of NH2OH.

Under the same conditions, NOHA produces very little N2O (2%, relative to AS).

Increasing the concentration of MPO to 75 nM led to much larger yields of N2O from

NH2OH (73%, relative to AS); however, NOHA oxidation still produced only a small percentage of N2O (8%, relative to AS). These results differ from those using HOCl itself where both NH2OH and NOHA produce high yields of N2O. It was suggested that this

34 difference can be accounted for based on the slow production of HOCl from MPO and the reaction of N-hydroxyguanidines with HNO.25,61

2.7 Conclusions and future directions

The MIMS technique is a sensitive and selective detection method for HNO. It can be used to detect HNO under physiologically relevant conditions by both its parent ion

(HNO+, m/z 31) and its primary fragment ion (NO+, m/z 30), as well as its dimerization product, N2O (m/z 44). Reliable HNO detection down to precursor concentrations as low as 25 nM can be achieved. Distinguishing HNO from N2O and NO is possible by the use of chemical (thiols or phosphines) or physical (liquid nitrogen) traps. The versatility of this technique allows for investigation of possible HNO producing pathways that have the potential to be physiologically relevant.

Future MIMS work should involve exploring other potential chemical reactions that can produce HNO. For example, recent work has shown that H2S can react with S- nitrosothiols (RSNO) to generate thionitrous acid (HSNO), a potential precursor to HNO.62

HSNO has also been proposed to be an intermediate in the reaction of H2S and NO.

63 Further, H2S and NO have been shown to react to ultimately generate HNO. Exploration of these reaction by MIMS could shed light on potential mechanisms of HNO formation from biologically relevant reactions.

2.8 Experimental Methods

2.8.1 General Methods

Unless otherwise noted, materials were obtained from Aldrich Chemical Company,

Fisher Scientific, or Cambridge Isotope Laboratories and were used without further purification. N-hydroxy-L-arginine (NOHA) was purchased from Cayman Chemical.

Hydrogen peroxide (30 % v/v) was quantified by absorbance at 240 nm using a molar absorption coefficient of 39.4 M-1 cm-1.64 1H NMR and 13C NMR spectra were recorded on a Bruker Avance 400 MHz FT-NMR operating at 400 MHz and 100 MHz, respectively.

All resonances are reported in parts per million, and are referenced to residual CHCl3 (7.26 ppm, for 1H, 77.23 ppm for 13C). High-resolution mass spectra were obtained on a VG

Analytical VG-70S Magnetic Sector Mass Spectrometer operating in fast atom bombardment ionization mode. Masses were referenced to a 10% PEG-200 sample.

Ultraviolet-visible (UV-Vis) absorption spectra were obtained using a Hewlett Packard

8453 diode array spectrometer. Infrared (IR) absorption spectra were obtained using a

Bruker IFS 55 Fourier transform infrared spectrometer.

2.8.2 Gas Chromatographic (GC) Headspace Analysis of N2O

Gas chromatographic headspace analysis of N2O was performed using a Varian CP-

3800 gas chromatograph instrument equipped with a 1041 manual injector, electron capture detector, and a Restek packed column. Samples were prepared in 15 mL vials with volumes pre-measured for uniformity. Vials were filled with 5 mL PBS buffer, sealed with a rubber septum, and then purged with argon for 10 min. The vials were equilibrated at 37

36 was then sampled three successive times using a gastight syringe with a sample lock. HNO yields are reported relative to the HNO donor, 2-bromo-N-hydroxybenzenesulfonamide

(2BrPA), which by comparison to standard N2O samples we find provides quantitative production of HNO.

2.8.3 Membrane Inlet Design and Methods

The MIMS cell used is a Hiden HRP40 membrane inlet MS cell. The cell contains rotary blades with an imbedded magnet for efficient stirring across the silastic membrane.

The membrane is immersed in, unless stated otherwise, 20 mL of 0.1 M pH 7.4 phosphate buffered saline (PBS) containing 100 µM diethylene triamine pentaacetic acid (DTPA).

The sample cell is fitted with an interior heating/cooling jacket for temperature regulation, as well as multiple ports for sample injection. All samples were argon-purged prior to the experiments. Injections of samples are performed using gas-tight syringes after background signals stabilize. Mass spectra were obtained by electron impact (EI) ionization (70 eV) at an emission current of 1 mA; source pressures were approximately 5 x 10-7 – 1 x 10-6 Torr.

Relevant ion currents were measured after the system had reached a stable baseline.

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Meissner, J.; Noveanu, M.; Reiter, M.; Twerenbold, R.; Schaub, N.; Buser, A.;

Mueller, C. Use of Myeloperoxidase for Risk Stratification in Acute Heart Failure.

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ATPase Activity and Perturb Ca2+ Homeostasis in Human Coronary Artery

Endothelial Cells. Free Radic. Biol. Med. 2012, 52, 951–961.

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Protective Enzymes Superoxide Dismutase , Catalase and Glutathione Peroxidase.

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Chapter 3: Heme-mediated Peroxidation of 5-N- Hydroxy-L-glutamine (NHG) to form Nitroxyl (HNO)

3.1 Introduction

Nitroxyl (azanone, HNO) has become a molecule of interest in recent years as it has been shown to be involved in positive effects on the cardiovascular system, making it a potential therapeutic for heart failure.1–6 HNO is a reactive intermediate that rapidly (k =

8 x 106 M-1 s-1) reacts with itself to produce hyponitrous acid (HON=NOH) that further

7 dehydrates to form nitrous oxide (N2O). As a result of this reactivity, HNO must be produced in solution via donor molecules. Interest in the unique biological activity of HNO has highlighted the need for new sensitive and direct detection methods. Recently, new methods have been developed for the detection of HNO.8–17 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.18,19 MIMS has been used in a wide variety of applications including the detection of nitric oxide (NO) in both aqueous and blood solutions.20,21 Recently, MIMS has been shown to be a viable method for HNO detection with nanomolar sensitivity under physiologically relevant conditions.13

In addition, this technique has been used to explore potential biological pathways to HNO production.22

To this point HNO has yet to be observed in vivo, however, its potent effects on the cardiovascular system and its close relationship to NO suggests the potential for its endogenous generation. There have been multiple reports of possible HNO producing

48 pathways including, reaction of thiols with S-nitrosothiols to form HNO and the

23,24 25 corresponding disulfide, ferric-heme peroxidation of hydroxylamine (NH2OH), and oxidation of N-hydroxy-L-arginine (NOHA) by hypochlorus acid (HOCl) (Scheme 3.1).22

In addition, HNO can be produced from nitric oxide synthases (NOS) under conditions where the tetrahydrobiopterin cofactor is absent or in low concentrations.26–31

Scheme 3.1. Potential HNO producing pathways generated from (a) reaction of thiol with S-nitrosothiol, (b) peroxidation of hydroxylamine, and (c) HOCl-mediated oxidation of N- hydroxy-L-arginine (NOHA).

Another potential substrate for endogenous HNO production is glutamine (GLN).

Glutamine is one of the most abundant amino acids in plasma playing roles in several cellular processes.32–34 Recent reports have suggested that glutamine is linked to cardioprotection, however, the mechanism for the observed effects is not well understood.35–38 In a similar fashion to NOS-mediated oxidation of arginine, glutamine’s amide functional group has the potential to be oxidized to generate 5-N-hydroxy-L- glutamine (NHG, Scheme 3.2). NHG could then be further oxidized to produce an acylnitroso species can be hydrolyzed to produce HNO and the corresponding carboxylic acid.39 Therefore, we have examined the heme-mediated peroxidation of NHG to generate

HNO. To our knowledge, the oxidation of GLN or NHG as a potential pathway for endogenous HNO production has not yet been reported. HNO production via heme- mediated peroxidation is confirmed by both membrane inlet mass spectrometry (MIMS) and N2O analysis by gas chromatography (GC).

Scheme 3.2. Oxidation of (a) L-arginine, (b) L-glutamine, and (c) L-asparagine to produce HNO and L-citrulline, L-glutamic acid, and L-aspartic acid, respectively.

3.2 Utilizing MIMS to Probe Potential Endogenous Pathways to

HNO Production

Membrane inlet mass spectrometry (MIMS) is a method used to detect dissolved gases in solution through the use of a semipermeable hydrophobic membrane.18,19 We have previously demonstrated that the MIMS system has the ability to detect HNO generated either by substrate oxidation or decomposition of donor molecules.13,22 Hydroxylamine

25 22,27,40 22 (NH2OH), N-hydroxyguanadines, and hydroxamic acid scaffolds have been shown previously to serve as potential substrates for HNO production (Figure 3.1). In recent years, glutamine (GLN) has been shown to be cardioprotective, however, the chemistry responsible for the observed physiology has yet to be defined.37,38 Inspired by the ability of L-arginine to be oxidized to produce HNO, we explored glutamine oxidation as a potential endogenous pathway to HNO production (Scheme 3.2).

Figure 3.1. Potential endogenous HNO generating scaffolds previously explored.

Initial attempts at oxidizing GLN (500 µM) with hydrogen peroxide (H2O2, 100

µM) did not produced any observable HNO, or HNO-derived N2O by MIMS or GC headspace analysis, respectively. Further, with five equivalents of H2O2 (2.5 mM) we were unable to detect HNO signals. Similar attempts at oxidizing 5-N-hydroxy-L-glutamine

(NHG) (500 µM) with H2O2 (100 µM) did not produced any observable HNO or HNO derived N2O by MIMS or GC headspace analysis, respectively. Again, in the presence of five equivalents of H2O2 we were unable to detect HNO signals by MIMS. However, GC headspace analysis revealed small amounts (10%) of detectable HNO derived N2O at these concentrations (Table 3.1). As mentioned in Chapter 1, our detection limit of HNO (1 nM) under physiologically relevant conditions should be more than sufficient to detect the estimated 10 µM HNO produced during the course of this reaction. A slow rate of production and a small yield of detectable gases makes detection by MIMS difficult as the

51 gaseous species constantly permeate through the membrane, not allowing for accumulation and detection in a similar fashion to the GC headspace experiments.

Table 3.1. Substrate oxidation by the heme/H2O2 system.

All substrates (500 µM) were incubated in the presence of H2O2 (100 µM) and with the specified enzyme (5 µM) in 0.1 M pH 7.4 PBS containing 100 µM diethylene triamine pentaacetic acid (DTPA) at 37 °C overnight unless stated otherwise. HNO yields are reported relative to HNO donor, 2-bromo-N-hydroxybenzenesulfonamide (2BrPA), as a determined by GC headspace analysis of N2O (SEM ± 5%; n ≥ 3). Substrates (500 µM) b were incubated under the same conditions in the presence of H2O2 (2.5 mM) only. Experiment was performed under same conditions as outlined above with the exception of the use of 2.5 mM H2O2.

Interestingly, upon the addition of 1% metmyoglobin (metMb) to NHG (500 µM) and H2O2 (100 µM), we were able to successfully detect MIMS signals resulting from HNO at m/z 31 and HNO-derived N2O at m/z 44 and m/z 30 (Figure 3.2). It is important to note that nearly 50% of the m/z 31 signal observed here is a result of the natural abundance

15 + 15 NO that has fragmented from N2O. Nevertheless, detection of a m/z 31 signal beyond natural abundance 15N (0.4%) confirms HNO production. Similar to what was observed by

Donzelli et al.,25 in the presence of 200 µM glutathione no MIMS signals were observed, therefore providing further evidence for HNO production. As discussed previously, the m/z

30 (NO+) signal has the potential to also be due to NO production. However, the lack of

MIMS signals at m/z 30 in the presence of a liquid nitrogen cold trap confirmed NO (BP =

-152 °C, 1 atm) was not produced during the course of this reaction (Figure 3.1c). GC

52 headspace analysis of a reaction mixture at the same concentrations indicated a nearly fourfold increase in HNO derived N2O compared to H2O2 alone (Table 3.1). A similar phenomenon was observed previously by Donzelli et al. using nearly identical reaction

25 concentrations (500 µM substrate with 100 µM H2O2 and 5 µM heme). In their work, hydroxylamine produced a significant amount of HNO in the presence of both H2O2 and various ferric heme-containing enzymes. We were also able to detect MIMS signals under analogous conditions with the substitution of NH2OH as a substrate (data not shown).

(a) m/z 44 m/z 31 150 m/z 30

100

Ion Current (pA)

0 5 10 15 20 Time (min)

(b) 0.25 m/z 31

0.20

0.15

0.10

0.05

Ion Current (pA) Current Ion 0.00

0 5 10 15 20 Time (min)

30 m/z 30 (LN2) 25

Ion Current (pA) Current Ion 5

0 5 10 15 20 Time (min) Figure 3.2. MIMS signals observed at (a) m/z 30, m/z 31, and m/z 44 following injection of NHG (500 µM, 0 min), then H2O2 (100 µM, 1 min), and finally metMb (5 µM, 2 min) into a solution of 0.1 M pH 7.4 PBS containing 100 µM DTPA at 37 °C. (b) Zoomed in version of the m/z 31 signal from experiment (a). (c) Comparison of the m/z 30 signals observed from reaction (a) with (open circles) and without (solid circles) a liquid nitrogen trap.

Similar experiments performed with glutamine (GLN, 500 µM) in the presence of

H2O2 (100 µM) and metMb (5 µM) did not result in any detectable MIMS signals (Figure

3.3). This is not surprising as GLN would require a minimum of two equivalents of oxidant

54 to produce the expected acylnitroso species (Scheme 3.2). Similar experiments with GLN

(500 µM), using excess H2O2 (2.5 mM), and metMb (5 µM) produces modest MIMS signals at m/z 30 (Figure 3.3). The 5% HNO-derived N2O observed in the presence of five equivalents (2.5 mM) of H2O2 suggests that the initial oxidation of GLN to produce NHG is not as facile, potentially making GLN a non-ideal candidate for an oxidative precursor to HNO. However, NHG could be produced when NH2OH is substituted for ammonia

41–43 (NH3) in glutamine synthetase. The efficient substitution of NH2OH into the glutamine synthetase (GS) system has been utilized to develop a colorimetric assay for measurement of activity that results in NHG production.44 Although there has not been definitive evidence to support this process occurring in vivo, it is an interesting possibility given that

NH2OH substitutes well for the endogenous substrate (NH3) in GS, and the roles that hydroxylamine plays in vivo have yet to be fully determined. Further, HNO donor Angeli’s salt has been shown to have effects on glutamate levels by modulating GS activity suggesting that HNO could play a regulatory role in GS activity providing a potential feedback loop to NHG-mediated HNO production.45

14 m/z 30 (100 µM H2O2) 12 m/z 30 (2.5 mM H2O2) 10 8 6 4 2

Ion Current (pA) 0 -2

-2 0 2 4 6 8 10 Time (min) Figure 3.3. MIMS signals observed at m/z 30 following injection of GLN (500 µM), then either 100 µM H2O2 (red circles) or 2.5 mM H2O2 (red line), followed by metMb (5 µM) into a solution of 0.1 M pH 7.4 PBS containing 100 µM DTPA at 37 °C.

3.2 Examination of Substrate Specificity

Donzelli et al. demonstrated that the enzyme systems studied showed a level of substrate specificity for HNO production.25 Substitution of NOHA resulted in zero detectable HNO. We are also unable to detect any observable signals from NOHA under our conditions by MIMS or GC headspace analysis. Therefore, we wanted to explore in greater detail the substrate specificity of these systems. Experiments with 5-N-hydroxy-L- asparagine (NHA, 500 µM), the structural analog to NHG (Figure 3.4), in the presence of

H2O2 (100 µM) and metMb (5 µM) produce very small MIMS signals (Figure 3.5) and minimal (< 5%) HNO derived N2O quantified by GC headspace analysis (Table 3.1).

Again, in the presence of a liquid nitrogen trap the m/z 30 signal disappeared indicating that NO is not produced under these conditions (Figure 3.5).

Figure 3.4. Structures of L-glutamine (GLN), 5-N-hydroxy-L-glutamine (NHG), and 5-N- hydroxy-L-asparagine (NHA).

2.0 m/z 30

m/z 30 (LN2) 1.5

1.0

0.5

Ion Current (pA) 0.0

0 5 10 15 Time (min) Figure 3.5. MIMS signals observed at m/z 30 following injection of NHA (500 µM, 0 min), then H2O2 (100 µM, 1 min), and finally metMb (5 µM, 2 min) into a solution of 0.1 M pH 7.4 PBS containing 100 µM DTPA at 37 °C with (open circles) and without (solid circles) a liquid nitrogen trap.

The small signals observed were puzzling, as we did not anticipate the loss of one methylene to have significant effect on HNO production in this system. Surely, the reduction potential of NHA should nearly identical to that of NHG. Similar to the observations of Donzelli et al.,25 it is possible the enzyme used could play a role in the amount of HNO observed.

3.2 Examination of Enzyme Specificity

To test for enzyme specificity for the substrates NHG and NHA, we examined substrate (500 µM) oxidation by H2O2 (100 µM) in the presence of another heme enzyme, horseradish peroxidase (HRP, 5 µM). Similar to observations using metMb, NHG produced significant MIMS signals while NHA did not produce any detectable MIMS signals. GC headspace analysis shows a similar trend to that observed with metMb (Table

3.1). To confirm that the composition of the enzyme pocket is not resulting in specificity for NHG, we substituted hemin as a simple model for ferric porphyrin without an enzyme pocket. In the presence of 5 µM hemin we observe MIMS signals at m/z 30 from NHA,

NHG, and NH2OH (Figure 3.6). However, NHG and NH2OH produce significantly more

HNO-derived N2O compared to NHA (Table 3.1). Consistent with experiments using metMb, the signals observed by MIMS are not due to the production of NO as confirmed by identical experiments in the presence of a liquid nitrogen cold trap. Successful detection of HNO by MIMS, resulting from the hemin-mediated oxidation of NHA, still does not explain the difference in HNO production relative to NHG.

25 m/z 30 (NH2OH) m/z 30 (NHG) 20 m/z 30 (NHA)

Ion Current (pA) 5

0 5 10 15 Time (min) Figure 3.6. MIMS signals observed following the injection of NHG (500 µM, blue), NHA (500 µM, red), or NH2OH (500 µM, black) at 0 min, followed by H2O2 (100 µM, 1.5 min), and finally hemin (5 µM, 3 min) into a solution of 0.1 M pH 7.4 PBS containing 100 µM DTPA at 37 °C. 3.3 Potential Reactivity of the Expected Acylnitroso Intermediate

The expected intermediate involved in the heme-meditated oxidation of NHG and

NHA is an acylnitroso species (Scheme 3.2). Acylnitroso compounds are reactive intermediates known to be precursors to HNO following hydrolysis.39 They are also known to react similarly with primary amines in organic solvent (N-butylamine, k = 2.8 x 10-4 M-

1 s-1).46 Therefore, it is likely that the small amount of the neutral amine of NHG could react through an intramolecular cyclization mechanism to produce HNO and the corresponding pyrrolidone (Scheme 3.3a). NHA on the other hand, would be less prone to an analogous cyclization as this would require the formation of an unfavorable 4- membered ring (Scheme 3.3b). Any observed effect would be kinetic by nature, as the

NHA-derived acylnitroso would be expected to hydrolyze eventually in the presence of water to produce HNO and aspartic acid. Samples incubated for 2 hours or overnight produce different amounts of N2O by GC headspace analysis, however, the relative proportions are not significantly affected. These results indicate that the differences observed in HNO-derived N2O is not a direct result of the kinetic competition between

58 intramolecular cyclization and hydrolysis as the relative N2O production is not affected by incubation time.

Scheme 3.3. Potential reactivity of the intermediate acylnitrosos of (a) NHG and (b) NHA.

There remains the possibility that the excess NHG/NHA is reacting with the acylnitroso through a non-HNO producing pathway. In this case, the ability of NHG to produce significant amounts of HNO even in the presence of excess NHG is explained by its ability to cyclize to produce HNO. This intramolecular reaction could be faster than a second-order reaction with excess hydroxamic acid allowing for some HNO production prior to reaction between acylnitroso and NHG. In the case of NHA, the majority of acylnitroso produced would be trapped by excess NHA as it is less prone to cyclize. The

59 hydroxamic acid could also be trapping the HNO that is being generated at potentially different rates by either intramolecular cyclization of NHG, or hydrolysis of NHA. It is possible that HNO that is more rapidly generated, by the intramolecular cyclization in

NHG, is able to either permeate the MIMS membrane for detection or dimerize and dehydrate to produce N2O, avoiding being trapped by the hydroxamic acid. This mechanism would also account for the differences between observed HNO/N2O signals of

NHG and NHA.

These non-HNO producing reactions could mimic the known reaction between

47–49 NH2OH and HNO to produce ultimately N2 and H2O (Scheme 3.4a). In the case of acylnitroso trapping, the reaction could take place between the hydroxamic acid nitrogen and the acylnitroso nitrogen (Scheme 3.4b). However, this would require the presumably less nucleophilic site of AHA to attack. Reaction could also take place between the hydroxamic acid oxygen and acylnitroso nitrogen potentially producing nitrite, acetamide, and the corresponding carboxylic acid (Scheme 3.4c). Both mechanims would require avoiding reactivity at the carbonyl carbon. This is not entirely impossible as acylnitroso species are known to be trapped through reactivity at the nitrogen.50 Similarly, reactivity between HNO and AHA could proceed via nucleophilic attack of the hydroxamic acid nitrogen or oxygen on the nitrogen of HNO (Scheme 3.4d-e). Ultimately, this observed effect is a function of hydroxamic acid concentrations and are likely not relevant in a physiological setting.

To test this hypothesis, we incubated an N-substituted hydroxamic acid with a pyrazalone leaving group (100 µM, Scheme 3.5), a donor that releases the acylnitroso at physiological pH, in 0.1 M pH 7.4 PBS containing 100 µM DTPA with and without the addition of excess acetohydroxamic acid (AHA). The presence of excess AHA resulted in a significant reduction (75%) in the N2O observed by GC headspace analysis. This result confirms the possibility of hydroxamic acid trapping of either the acylnitroso or HNO that is generated accounting for the differences in HNO production by NHG and NHA. Further

61 product studies will be required to confirm which of the possible mechanisms is responsible for the observed difference in reactivity.

Scheme 3.5. Decomposition of N-substituted hydroxamic acid with a pyrazalone leaving group (PY) to produce the byproduct (BY) and acylnitroso that can either be trapped by (a) water to produce HNO and the corresponding carboxylic acid, or (b) acetohydroxamic acid resulting in non-HNO producing trapped products. 3.4 Conclusions

We have shown that both GLN and NHG have the potential to serve as endogenous precursors to HNO via heme-mediated peroxidation. The systems studied revealed unique

HNO production differences between NHG’s amino acid analog, NHA. Unexpectedly, compared with NHA, NHG produced significantly more HNO in every system tested.

Enzyme specificity did not appear to the reason for the observed difference in HNO production as experiments performed with hemin as the metal catalyst did not significantly alter the ratios of HNO production. The only significant difference between NHG and NHA in terms of reactivity is their potential to undergo an intramolecular cyclization with the anticipated acylnitroso intermediate. NHG and NHA would form 5- and 4-membered rings, respectively, via attack by the amine nitrogen. The 4-membered ring formation being less likely leaves the NHA derived acylnitroso vulnerable to further reactivity with excess NHA resulting in considerably less observable HNO. This is observed result is likely not

62 physiologically relevant. There are multiple potential non-HNO producing reactions that could account for the significant difference in HNO production between NHA and NHG.

Future product studies will be required to definitively establish the mechanism involved.

3.5 Experimental Methods

3.5.1 General Methods

Unless otherwise noted, materials were obtained from Aldrich Chemical Company,

Fisher Scientific, or Cambridge Isotope Laboratories and were used without further purification. N-hydroxy-L-arginine (NOHA) was purchased from Cayman Chemical.

Hydrogen peroxide (30 % v/v) was quantified by absorbance at 240 nm using a molar absorption coefficient of 39.4 M-1 cm-1.51 1H NMR and 13C NMR spectra were recorded on a Bruker Avance 400 MHz FT-NMR operating at 400 MHz and 100 MHz, respectively.

All resonances are reported in parts per million, and are referenced to residual CHCl3 (7.26 ppm, for 1H, 77.23 ppm for 13C). High-resolution mass spectra were obtained on a VG

Analytical VG-70S Magnetic Sector Mass Spectrometer operating in fast atom bombardment ionization mode. Masses were referenced to a 10% PEG-200 sample.

Ultraviolet-visible (UV-Vis) absorption spectra were obtained using a Hewlett Packard

8453 diode array spectrometer. Infrared (IR) absorption spectra were obtained using a

Bruker IFS 55 Fourier transform infrared spectrometer.

3.5.2 Gas Chromatographic (GC) Headspace Analysis of N2O

Gas chromatographic headspace analysis of N2O was performed using a Varian CP-

3800 gas chromatograph instrument equipped with a 1041 manual injector, electron

63 capture detector, and a Restek packed column. Samples were prepared in 15 mL vials with volumes pre-measured for uniformity. Vials were filled with 5 mL PBS buffer, sealed with a rubber septum, and then purged with argon for 10 min. The vials were equilibrated at 37

°C for 10 min in a block heater. An aliquot of an HNO donor solution or other relevant substrate species was then introduced into the thermally equilibrated headspace vial using a gastight syringe. Samples were then incubated overnight, unless otherwise stated, to ensure complete production and equilibration of N2O in the headspace. Headspace (60 μL) was then sampled three successive times using a gastight syringe with a sample lock. HNO yields are reported relative to the HNO donor, 2-bromo-N-hydroxybenzenesulfonamide

(2BrPA), which by comparison to standard N2O samples we find provides quantitative production of HNO.

3.5.3 Membrane Inlet Design and Methods

The MIMS cell used is a Hiden HRP40 membrane inlet MS cell. The cell contains rotary blades with an imbedded magnet for efficient stirring across the silastic membrane.

The membrane is immersed in, unless stated otherwise, 20 mL of 0.1 M pH 7.4 phosphate buffered saline (PBS) containing 100 µM diethylene triamine pentaacetic acid (DTPA).

Relevant ion currents were measured after the system had reached a stable baseline.

3.6 References

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Chapter 4: Application of Membrane Inlet Mass Spectrometry (MIMS) to the Study of Hydrogen Sulfide (H2S) and Thionitrous Acid (HSNO)

4.1 Introduction

Hydrogen sulfide (H2S), as with several other small molecule signaling agents (e.g., carbon monoxide (CO) and nitric oxide (NO)), was initially known for its inherent toxicity,1–3 but is now known to be endogenously generated and to serve many important

4–6 physiological roles. In mammals, H2S is generated by three enzymes, cystathionine-β- synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase

7 (3-MST). The physiological effects of H2S, including its impact on the cardiovascular system and inflammation,8 have been well documented; however, the chemical mechanisms responsible for these effects remain largely undefined. To investigate the chemistry of H2S, reliable detection methods are required and a number of techniques including fluorescence imaging, chemiluminescence, and colorimetric assays have been reported.9–11 Since membrane inlet mass spectrometry (MIMS) is a useful technique for the detection of small hydrophobic gases dissolved in aqueous solution with high

12–15 sensitivity and selectivity, we have applied this technique to detect H2S directly in aqueous solution.

Recent work by Filipovic et al. has suggested that HSNO is an important small molecule signaling agent.16 In these studies, HSNO was generated by either pulse radiolysis of Na2S and NaNO2 to generate HS and NO, from the reaction of acidified nitrite with NaSH, or from the reaction of S-nitrosoglutathione (GSNO) with NaSH. In addition,

Feelisch and co-workers have provided evidence for the production of nitrosopersulfide

(SSNO-) as a stable species derived from the reaction of S-nitrosothiols in the presence of

17 - excess H2S. Filipovic et al. acknowledged that SSNO production could be a result of polysulfides reaction with either acidified nitrite or GSNO; however, it was not reported under the conditions of their experiments. The pKa of HSNO has anticipated to be approximately 3.2, analogous to nitrous acid (HONO).18 Interestingly, Filipovic et al. were able to detect a mass corresponding to HSNO by ESI-TOF mass spectrometry at physiological pH, suggesting at a significant amount of the neutral species at pH 7.4. Their work also demonstrated that HSNO can permeate cell membranes and nitrosate cysteine residues. With the exception of a specific transport mechanism, this process would rely on diffusion of the neutral species across the cell membrane, requiring HSNO to be the predominant species at physiological pH.

If we compare HSNO with the structurally similar HONO, we would anticipate a pKa less than three, given that thiols are generally more acidic than alcohols. If the pKa of

HSNO is less than three, only a limited amount of HSNO (vs SNO-) would be present at physiological pH. Isomerization of HSNO to other neutral species could account for its ability to cross membranes. Isomers of HSNO (i.e., HONS, SN(H)O, HOSN, and HNSO) have been previously investigated by computational and matrix-isolation studies.19–21

Access to isomeric species may allow for an equilibrium mixture that would have an overall pKa higher than that anticipated for HSNO itself. Unraveling the potential contributions of HSNO isomers will be important to understand the mechanisms involved in HSNO mediated nitrosation across cellular membranes.

4.2 Detection of H2S by MIMS

We have examined H2S production from the donor molecule, sodium hydrogen

+ + sulfide (NaSH), by MIMS. MIMS signals are observed at m/z 34 (H2S ), m/z 33 (HS ),

+ + and m/z 32 (S /O2 ) (Figure 4.1a) and compare well with the reported mass spectrum of

22,23 H2S. Complications due to residual dioxygen results in high background intensity and unstable baseline for the m/z 32 signal, making reliable detection of this signal difficult.

As a result, our discussion will focus on the m/z 33 and m/z 34 signals. Upon injection of

100 µM NaSH into a solution of 0.1 M phosphate-buffered saline (PBS) at pH 7.4, we

+ observe an immediate increase in MIMS signal intensities for the parent H2S (m/z 34) and its primary fragment HS+ (m/z 33) (Figure 4.1b). The intensity of the m/z 34 signal is linearly dependent on the concentration of NaSH (Figure 4.1c). Under the current conditions of our experiment, we can detect H2S at concentrations as low as 250 nM NaSH.

24 Based on the pKa of H2S (pKa = 6.98 at 20 °C), at pH 7.4 nearly two-thirds of the H2S

- generated is expected to be in its anionic form (HS ). Consequently, the H2S concentration is approximately one-third that of the NaSH injected, and the observed detection limit of

H2S under the conditions of our experiment is approximately 85 nM.

(a) 500

400

300

200

Ion Current (pA) 100

30 31 32 33 34 35 36 m/z (b) 200

150

100

50 m/z 34 Ion Current (pA) m/z 33 0 0 5 10 15 20 Time (min) (c) 160 m/z 34 140

120 100 80 0.8 60 0.4 40 0.0

Ion Current (pA)

20 Ion Current (pA) 0 4 8 0 Time (min)

0 20 40 60 80 100 [NaSH] (M)

4.3 Detection of HSNO by MIMS

We have examined the generation of HSNO from the reaction of acidified nitrite with NaSH. We attempted to generate HSNO in a similar fashion to Filipovic et al.16 from the reaction of sodium nitrite (10 mM) with NaSH (10 mM) in acidic solution that was

76 subsequently injected into a solution of 0.1 M pH 7.4 PBS containing 100 µM DTPA.

Although we do observe very small MIMS signals corresponding to the parent ion (m/z 63,

HSNO+), these signals appear to be complicated by unknown species that are detected at the same mass from samples of NaSH itself (Figure 4.2). In the event the observed MIMS signal are indeed a result of HSNO, they are significantly smaller than those observed from

100 µM H2S (i.e., m/z 34, Figure 4.1b). We believe that reactivity of HSNO with multiple species in solution and homolysis of the weak S-N bond (29.4 kcal/mol)25 to generate NO and HS likely hinders its detection.21,26 Consistent with this expected reactivity, we observe NO (m/z 30) by MIMS via the reaction of NaSH (10 mM) with S- nitrosoglutathione (GSNO, 10 mM) (Figure S4.1). Feelisch and co-workers have also observed NO formation from the reaction of S-nitrosothiols with excess H2S, with the proposed intermediacy of SSNO-.17 HSSNO/SSNO- may play an important role in the chemistry of HSNO; however, its investigation by MIMS will require more detailed studies in the future.

0.5

0.4

0.3

0.2 + m/z 63 (H2S + H2NO2 ) 0.1 Ion Current (pA) m/z 63 (H2S alone) 0.0

0 5 10 15 20 Time (min) Figure 4.2. MIMS observed intensity at m/z 63 as a function of time following injection of 10 mM of the pre-incubated reaction mixture (see experimental section), of (blue) sodium nitrite and NaSH in 0.2 M HCl or (red) NaSH alone, into 0.1 M pH 7.4 PBS with metal chelator DTPA (100 µM) at 20 °C.

4.4 Examination of Larger Mass Signals from H2S Donors

After observing signals from NaSH at m/z 63, larger than H2S itself (m/z 34), we further investigated the identity of these larger mass signals. The initial hypothesis of the source of these signals was oxidation of H2S to produce a variety of species including polysulfides, thiosulfate, sulfate, etc. (Scheme 4.1).27,28 Upon injection of 500 µM of

NaSH into a MIMS cell containing 0.1 M pH 7.4 PBS with 100 µM DTPA we observed additional MIMS signals at m/z 48, 64, 65, and 66 (Figure 4.3). These masses were investigated as they have the potential to be parent ions or primary fragment ions of oxidized sulfur species (Scheme 4.1).

- Scheme 4.1. Possible reactions of H2S/HS with O2 in aqueous solution including further - reactivity and equilibration of polysulfides (HS (n+1)) where n = 1-9.

(a) (b) 250 800 200 600 150

400 100 m/z 64 200 m/z 34 50 Ion Current (pA) m/z 33 Ion Current (pA) m/z 48 0 0

-2 0 2 4 6 8 10 12 14 -2 0 2 4 6 8 10 12 14 Time (min) Time (min)

4 m/z 66 2 Ion Current (pA) m/z 65 0

-2 0 2 4 6 8 10 12 14 Time (min) Figure 4.3. MIMS signals observed at m/z 34 (red), 33 (blue), 48 (green), 64 (black), 65 (orange), and 66 (purple) following the injection of 500 µM NaSH into 0.1 M pH 7.4 PBS containing 100 µM DTPA at 21 °C.

Nagy et al. has reported that sodium salts of H2S (i.e., Na2S and NaSH) inherently

- 28 contain polysulfide contaminants (HS (n+1), n ≥ 1). Therefore, we investigated various H2S donors for their ability to produce pure H2S. Similar to what was found by Nagy et al., the amount of oxidized sulfur species was independent of the sodium salt source of H2S;

28 including anhydrous NaSH (Strem chemical and Alfa Aesar) and Na2S (Strem chemical).

Further, various handling and sample preparation methods were tested in attempt to determine the cause of these signals. Variables tested included stock solution pH (4-13), metal chelator (DTPA and Chelex 100), and inert storage of commercial samples.

However, none of the variables had a significant effect on the signals observed by MIMS.

Next, we turned to H2S gas, which presumably should be a pure source of H2S. However, the observed MIMS signals are nearly identical to NaSH-derived signals (Table 4.1).

Table 4. 1. Comparison of the MIMS signals observed following the injection of 500 µM NaSH, Na2S2, Na2S4, or H2S (g) into 0.1 M pH 7.4 PBS with 100 µM DTPA at 21 °C. All signal intensities are reported relative to the m/z 34 signal for each substrate.

Signals Monitored (m/z) 34 33 48 64 66 NaSH 1 0.38 0.13 0.28 0.013 Na2S2 1 0.38 0.13 0.28 0.013

Na2S4 1 0.38 0.11 0.22 0.011 H2S (g) 1 0.38 0.17 0.37 0.018

To probe the possibility that we are detecting polysulfide contaminants in our H2S donors, we examined the sodium salts of the polysulfides H2S2 and H2S4 using donors

Na2S2 and Na2S4, respectively. Interestingly, injection of NaSH, Na2S2, and Na2S4 produce nearly identical MIMS signals (Figure 4.4). These results suggested that upon injection of the donor into the MIMS cell a rapid equilibration between polysulfides in solution occured. Small amounts of polysulfide contaminants have the potential to start a chain reaction to produce an equilibrium mixture of polysulfides of various lengths (Scheme 4.1).

Therefore, we turned to phosphine nucleophiles as established traps for oxidized sulfur species.29,30 In the presence of disulfides (RSSR) tris-(2-carboxyethyl) phosphine (TCEP) reacts to produce two equivalents of thiol (RSH) and TCEP oxide (TCEP=O) (Scheme

4.2).29–31 In the presence of per- or polysulfides, TCEP reacts to produce the same two equivalents of thiol and TCEP sulfide (TCEP=S). (Scheme 4.2).30,31

Scheme 4.2. Reactivity of tris-(2-carboxyethyl) phosphine (TCEP) with a disulfide (RSSR) or per/polysulfide (RSSH) in aqueous solution to produce TCEP-oxide (TCEP=O) and TCEP-sulfide (TCEP=S), respectively.

Experiments performed with NaSH (500 µM) in the presence of excess TCEP (5 mM) did not result in significant differences in the observed MIMS signals (Table 4.2).

Again, a variety of conditions were tested in an attempt to improve trapping of oxidized sulfur species including, increasing the equivalents of TCEP (10 eq. – 50 eq.), changing the pH of the reactions (pH 4-8), and increasing reaction time between NaSH and TCEP prior to injecting the sample into the MIMS cell. However, none of these changes had any effect on the observed MIMS signals (Table 4.2). One explanation for this observation is that after trapping of the oxidized species by TCEP, the fully reduced H2S remaining reacts again with small amounts of residual oxygen in the sample cell, again restarting the equilibration of oxidized species. This process could continue until all the TCEP has been consumed. However, under such great excess of TCEP (50 eq.) this process is unlikely to explain the observations.

(a) (b)

400 1000

800 300

600 200 400 m/z 34 (NaSH) m/z 33 (NaSH) m/z 34 (Na S ) 2 2 100 m/z 33 (Na2S2) 200

Ion Current (pA) m/z 34 (Na S ) Ion Current (pA) 2 4 m/z 33 (Na2S4) 0 0

-2 0 2 4 6 8 10 12 14 -2 0 2 4 6 8 10 12 14 Time (min) Time (min) (c) (d) 140 300

120 250 100 200 80 150 60 m/z 48 (NaSH) m/z 64 (NaSH) 100 40 m/z 48 (Na2S2) m/z 64 (Na2S2) 50

Ion Current (pA) Current Ion 20 (pA) Current Ion m/z 48 (Na2S4) m/z 64 (Na2S4) 0 0

-2 0 2 4 6 8 10 12 14 -2 0 2 4 6 8 10 12 14 Time (min) Time (min) Figure 4.4. MIMS signals observed at m/z 34 (red), 33 (blue), 48 (green), and 64 (black) following the injection of 500 µM NaSH (+), Na2S2 (●), or Na2S4 (○) into 0.1 M pH 7.4 PBS containing 100 µM DTPA at 21 °C.

Table 4.2. Comparison of the MIMS signals observed following the injection of 500 µM NaSH in the presence of various concentrations of TCEP into 0.1 M pH 7.4 PBS with 100 µM DTPA at 21 °C. All signal intensities are reported relative to the m/z 34 signal for each substrate. Samples were incubated for either a30 min, b160 min, or c24 hrs at 21 °C prior to injection into the MIMS. All signal intensities are reported relative to the m/z 34 signal for each substrate.

Signals Monitored (m/z) 34 33 48 64 66 NaSH+TCEP(5 mM)a 1 0.38 0.32 0.70 0.033 NaSH+TCEP(25 mM)a 1 0.38 0.29 0.63 0.029 NaSH+TCEP(5 mM)b 1 0.38 0.28 0.61 0.028 NaSH+TCEP(5 mM)c 1 0.37 0.33 0.72 0.034

There is also the possibility that TCEP is not reacting with the species responsible for the observed MIMS signals. To confirm TCEP is indeed reacting with oxidized sulfur species in solution, we monitored reactions under similar conditions by 31P NMR. Control

82 experiments with TCEP alone indicated that there is a significant effect of pH on the observed 31P NMR signals. Under acidic conditions, TCEP-HCl is observed at 17 ppm, and in basic solution only TCEP itself is observed at -27 ppm (Figure S4.2). However, at neutral pH, the signals at -27 and 17 ppm significantly broaden (Figure 4.2). For this reason all NMR experiments were performed at pH 5 to afford sharp signals and facilitate reactivity between polysulfides and TCEP. In all pH solutions tested, a signal at 58 ppm is present. This signal is due to TCEP=O, which is confirmed by the addition of H2O2 to a sample of TCEP alone (Figure S4.3). Since the analogous MIMS experiments were not affected by pH, we believe the NMR experiments performed at pH 5 are an accurate comparison to our MIMS observations.

Reaction between NaSH (70 mM) and TCEP (700 mM) in 2M pH 5 acetic acid buffer containing 10% D2O, incubated for 30 minutes at 21 °C, produced a new signal at

52 ppm. The small chemical shift difference between the 52 and 58 ppm (TCEP=O) signals

30,31 is analogous to what is reported for other R3P=O/R3P=S chemical shift differences.

Therefore, we assign the 52 ppm signal to TCEP=S (Scheme 4.2). If there were quantitative trapping of sulfur a maximum 9:1 ratio of TCEP-HCl (17 ppm) to TCEP=S (52 ppm) is expected. The observed ratio translates to a ca. 9% yield of TCEP=S. By comparison, reaction between Na2S2 (70 mM) and TCEP (700 mM) in 2M pH 5 acetic acid buffer containing 10% D2O, incubated for 30 minutes at 21 °C, produced a much larger TCEP=S signal. This signal corresponds to ca. 85% yield of TCEP=S. These results confirm that

TCEP is indeed reacting with the oxidized sulfur species in solution. In addition, by 31P

NMR analysis, there is a significant difference in the amount of polysulfides from NaSH and Na2S2 that is not observed by MIMS. These results are not surprising, as it was initially

83 anticipated that Na2S2 would produce much more oxidized sulfur species compared to

NaSH. Nonetheless, this suggests there is further reactivity inherent to the MIMS system that is responsible for the signals observed.

The observation that Na2S2 produces significantly more TCEP=S compared to

NaSH is not surprising from a chemical perspective. However, the stark differences from

84 our MIMS observations is puzzling. The only noteworthy difference between the NMR and MIMS experiments is the silastic membrane used in the MIMS system. It is unlikely there is any direct reaction between the sulfur samples and the membrane, however, it is possible that it is facilitating a reaction with residual dioxygen within the hydrophobic membrane. After this reaction takes place, the oxidized species could complete their permeation into vacuum for detection. To test the oxygen dependence of these larger mass signals we performed identical MIMS experiments under an atmosphere of dioxygen.

Under these conditions, we observe an increased in larger mass signals (i.e., m/z 64) and a decrease in the m/z 34 signal (Figure 4.6).

400

300

200

100

Ion Current (pA) m/z 34 (Ar) m/z 64 (Ar) m/z 34 (O2) m/z 64 (O2) 0

0 5 10 15 Time (min) Figure 4.6. MIMS signals observed at m/z 34 and 64 upon the addition of NaSH (350 µM) into a solution of 0.1 M pH 7.4 PBS with 100 µM DTPA at 21 °C; initially purged with either argon (red and blue) or dioxygen (green and black).

Additionally, consecutive injections of H2S (g) into the MIMS cell reveals a decrease in the average maximum intensity of larger mass signals while the intensities of m/z 33 and 34 increase as a function of injection (Figure 4.7). Each injection likely results in consumption of residual oxygen in our system, therefore, the sample is exposed to less

O2 upon each additional injection. This effect plateaus after 4 injections as we are likely reaching a competition between consumption of O2 and its permeation into our system.

Together these results suggest that the observed larger mass signals are dependent on oxygen concentration. As we are vulnerable to small amounts of residual oxygen penetrating our system, the potential for a membrane-facilitated oxidation process will be important in future MIMS studies.

(a)

2000

1500

1000

500 m/z 34 m/z 33

Avg. Max Intensity (pA) 0

0 1 2 3 4 5 6 7 Injection Number

(b)

400 m/z 64 m/z 48

300

200

100

Avg. Max Intensity (pA)

0 1 2 3 4 5 6 7 Injection Number Figure 4.7. Plots of the average maximum signal intensity observed at m/z 34 (blue), 33 (red), 64 (black), and 48 (green), upon the consecutive injections (1-7) of 250 µL H2S (g) into the same solution of 0.1 M pH 7.4 PBS with 100 µM DTPA at 21 °C. Consecutive injections occurred after plateau of the signals.

4.5 HSNO Isomerization

Although reliable detection of HSNO by our MIMS system was unsuccessful, its ability to cross a membrane, as was observed by Filipovic et al.,16 with an anticipated low pKa suggests other factors must be at play. Isomerization of HSNO to a variety of species has been investigated previously by both computational and matrix-isolation studies.19–21

HSNO isomers include HONS, HOSN, SN(H)O (Y-isomer), and HNSO, along with the corresponding cis and trans isomers where relevant (Scheme 4.3). Energies for transitions between these isomers have been previously investigated in the gas-phase by Bharatam et al.32 With the exception of rotational barriers between cis and trans isomers, all transitions between species involve barriers >25 kcal/mol in the gas phase. Recent work by

Timerghazin and co-workers, examined HSNO isomerization in an aqueous environment.25 Their work suggests that HSNO can isomerize to both HONS and the Y- isomer through a water-assisted proton transfer mechanism. This water-assisted mechanism brings barriers of isomerization from gas phase values of >25 kcal/mol to <10 kcal/mol, making these reactions kinetically feasible under physiologically relevant conditions

Scheme 4.3. Relevant isomers of HSNO with their expected pKa values.

The HONS isomer can be accessed from HSNO through a water-assisted proton transfer with a calculated barrier of 8 kcal/mol.25 This isomer is also be expected to have a pKa similar to that of HONO. Timerghazin and co-workers have shown the Y-isomer is

87 the most stable isomer in an aqueous environment by 3.1 kcal/mol.25 Since deprotonation of the Y-isomer generates the same anion involved in the HSNO acid/base equilibrium

(Scheme 4.4), we can approximate the pKa of the Y-isomer by comparing the reported energy difference between HSNO and the Y-isomer. Given this energy difference and pKa near 3 for HSNO, a Y-isomer pKa near 5 is estimated. Thus, neither HONS nor the Y- isomer can account for the anticipated higher overall pKa.

Scheme 4.4. Deprotonation of the SN(H)O (Y-isomer) and HSNO to produce the same – SNO anion.

The closest structural analog of HOSN with a known pKa is cyanic acid (HOCN),

33 which has a pKa of 3.5. Similar to HONS, the HOSN isomer does not appear to be an important factor in the equilibrium mixture as it pertains to the pKa. HNSO, on the other hand, likely has a considerably higher pKa relative to its other isomers. Although we are not aware of any structural comparison with an established pKa value, we assume that

HNSO, an amine with an electron withdrawing group substituent, would have a pKa significantly higher than 7.

HNSO is reported to be the most stable isomer in the gas phase,32 however the contribution of this isomer to HSNO chemistry will be dependent on the barrier(s) to its formation. A likely path to HNSO is the isomerization of HOSN. Therefore, generation of HOSN needs to be feasible (Scheme 4.3). The barrier from HONS to HOSN has been calculated by Bharatam et al. to be >90 kcal/mol in the gas phase. The reason for such a high barrier is likely due to a strained transition state structure involving a three membered ring (Scheme 4.5). A similarly high barrier (50.3 kcal/mol) was calculated by Timerghazin

88 and co-workers for generation of the cyclic isomer (cycl-SONH) from the Y-isomer, which was also suggested to involve a three-membered ring transition state (Scheme 4.3).

Timerghazin and co-workers conclude that the barrier to generate the cycl-SONH is too high to be kinetically feasible.

Scheme 4.5. Equilibria between HONS and HOSN involving a three-membered ring transition state.32

4.5.1 Sulfinamide type rearrangement (HONS to HOSN)

McCulla and co-workers recently reported a thorough computational investigation of the reaction between HNO and thiols.34 The reaction from a putative N- hydroxysulfenamide (RSNHOH) intermediate to the corresponding sulfinamide

(RS(O)NH2) product was shown to proceed through physiologically surmountable barriers.

With structural similarity between RSNHOH and HONS, it seems plausible that a

“sulfinamide type” rearrangement pathway may lead to the structurally analogous product,

HOSN (Scheme 4.6).

Scheme 4.6. (a) Rearrangement of the putative N-hydroxysulfenamide (RSNHOH) to sulfinamide (RS(O)NH2). (b) Proposed rearrangement of HONS to HOSN.

Our B3LYP/6-31G(d) calculated barriers for the two-step reaction from HONS to

HOSN (Scheme 4.6b) in the gas phase are 53.2 kcal/mol and 39.1 kcal/mol, respectively

(Figure S4.4). However, with the addition of explicit water molecules, the gas phase barriers for HONS to HOSN drop to 14.4 kcal/mol and 7.7 kcal/mol, respectively. Further, performing the calculation with an SM8 solvation model for aqueous environment drops the barriers to 10.5 kcal/mol and 1.2 kcal/mol, respectively (Figure 4.8). As observed by

Timerghazin and co-workers, addition of explicit water molecules to the reaction significantly lowers the barriers making them accessible under physiologically relevant conditions.

4.5.2 Isomerization of HOSN to HNSO

The isomerization of HOSN to HNSO has been investigated computationally by

Bharatam et al. in the gas phase.32 A gas phase barrier of >30 kcal/mol involving a proton transfer with a four-membered ring transition state is calculated. Analogous to the water- assisted proton transfer mechanisms described by Timerghazin and co-workers, addition of explicit water molecules is expected to lower the gas phase barriers. Indeed, when we calculate the barrier for the transition from HOSN to HNSO in the presence of explicit water molecules the barrier drops to 8.1 kcal/mol (Figure 4.9). Similar to the

91 transformation of HONS to HOSN, calculations performed with the SM8 solvation model for aqueous solvation decreases the barrier to 7.5 kcal/mol. With barriers to formation of less than 10 kcal/mol, we predict that HNSO is accessible under physiologically relevant conditions. Thus, the combination of these isomers, in particular the anticipated weakest acid HNSO, may account for anticipated higher pKa required for the permeation of membranes.

4.6 Conclusions

We have used MIMS to observe H2S with a detection limit of 85 nM under physiologically relevant conditions. We were unable to detect HSNO MIMS signals

92 reliably as a result of an unanticipated contribution NaSH alone. Upon investigation of larger mass signals it appears that we are observing an array of oxidized species that result from reaction of H2S with O2 that occurs within the membrane prior to full permeation into vacuum for detection. This observation is important in the interpretation of future applications of MIMS for the detection of H2S. Nonetheless, the reported ability for HSNO to cross membranes suggests a pKa that is inconsistent with that expected for HSNO itself.

Therefore, isomerization of HSNO to its corresponding isomers was examined. Water- assisted isomerization of HSNO to the Y-isomer, HONS, HOSN, and HNSO are calculated to be kinetically accessible under physiologically relevant conditions, with barriers <10 kcal/mol. We propose that these isomers are in equilibrium resulting in a higher pKa.

Based on our analysis, HNSO is the only isomer of HSNO that will be predominantly neutral at physiological pH. Nitrosation of cysteine residues would presumably involve

HSNO itself, or potentially the Y-isomer. HSNO and the Y-isomer have been previously investigated experimentally and computationally, respectively, to react with thiols.16,25

However, the potential biological reactivity of the other isomers remains to be determined.

The described work will offer a chemical perspective to a proposed biochemical mechanism of this potentially important signaling agent.

4.7 Experimental Methods

4.7.1 General Methods

Unless otherwise noted, materials were obtained from Aldrich Chemical Company,

Fisher Scientific, or Cambridge Isotope Laboratories and were used without further purification. Anhydrous sodium hydrogen sulfide was purchased from Strem Chemicals

Inc. 1H NMR and 13C NMR spectra were recorded on a Bruker Avance 400 MHz FT-

NMR operating at 400 MHz and 100 MHz, respectively. All resonances are reported in

1 parts per million, and are referenced to residual CHCl3 (7.26 ppm, for H, 77.23 ppm for

13C). High-resolution mass spectra were obtained on a VG Analytical VG-70S Magnetic

Sector Mass Spectrometer operating in fast atom bombardment ionization mode. Masses were referenced to a 10% PEG-200 sample. Ultraviolet-visible (UV-Vis) absorption spectra were obtained using a Hewlett Packard 8453 diode array spectrometer. Infrared

(IR) absorption spectra were obtained using a Bruker IFS 55 Fourier transform infrared spectrometer.

4.7.2 Membrane Inlet Design and Methods

The MIMS cell used is a Hiden HRP40 membrane inlet MS cell. The cell contains rotary blades with an imbedded magnet for efficient stirring across the silastic membrane.

The membrane is immersed in, unless stated otherwise, 20 mL of 0.1 M pH 7.4 phosphate buffered saline (PBS) containing 100 µM diethylene triamine pentaacetic acid (DTPA).

Relevant ion currents were measured after the system had reached a stable baseline.

4.7.3 Detection of H2S from NaSH

Samples of anhydrous NaSH were dissolved in argon-purged 0.1 M NaOH. The

NaSH solution was injected into an argon-purged 0.1 M pH 7.4 phosphate-buffered saline

(PBS) solution, with 100 μM of the metal chelator diethylenetriaminepentaacetic acid

(DTPA) at 21 °C with a gas-tight syringe. MIMS signals at m/z 33 and m/z 34 were monitored over time.

4.7.4 Detection of HSNO from the Reaction of Acidified Nitrite with NaSH

The reaction between acidified nitrite and NaSH was carried out in a separate sealed vial purged with argon. Equimolar amounts of NaNO2 and NaSH were incubated in 0.2 M

HCl for 10 minutes prior to injection into the MIMS cell (final concentration of 10 mM).

Following pre-incubation, these samples were immediately injected into solution of 0.1 M pH 7.4 PBS with 100 µM DTPA at 21 °C. MIMS signals at m/z 63, m/z 62, m/z 34, m/z 33, and m/z 30 were monitored over time after establishing a flat baseline.

4.7.5 MIMS Experiments in the Presence of TCEP

The reaction of TCEP with NaSH, H2S (g), Na2S2, or Na2S4 was carried out in a separate sealed vial purged with argon. Excess TCEP and sulfur species were incubated in buffered solutions for 30 minutes, unless otherwise stated, prior to injection into the MIMS cell. Following pre-incubation, these samples were immediately injected into solution of

0.1 M pH 7.4 PBS with 100 µM DTPA at 21 °C. MIMS signals were monitored over time after establishing a flat baseline.

4.7.6 TCEP 31P NMR Experiments

The reaction between TCEP and NaSH or Na2S2 was carried out in a separate sealed vial purged with argon. TCEP (700 mM) and sulfur species (70 mM) were incubated in 2

M pH 5 acetic acid buffer containing 10% D2O for 30 minutes at 21 °C prior to acquisition.

Following pre-incubation, these samples were immediately analyzed by 31P NMR.

Estimation of TCEP-sulfide (TCEP=S) yield were made by comparing the ratio of TCEP=S

95 to TCEP-HCl in the collected data relative to the maximum ratio possible if quantitative trapping occurred.

4.7.7 Computational Methods

Calculations were performed with Spartan 14.35 Geometries were fully optimized at the B3LYP level of theory with the 6-31G(d) basis set. Vibrational frequencies were also calculated to verify minimum energy structures (no imaginary frequencies) or transition states (one imaginary frequency) and to provide zero-point vibrational energy corrections.

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4.9 Supporting Information Chapter 4

4.9.1 Optimized Geometries and Energies

Table S4.1. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for [SN(H)OH]+.

B3LYP/6-31G* energy -528.964303 Hartrees Zero-point correction 0.027692596 Hartrees Thermal correction to energy 0.033338912 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z N 7 0.3935644 -0.4520881 0.0001668 S 16 -1.0731173 0.1148629 -0.0000343 H 1 0.6155341 -1.4607745 -0.0000378 O 8 1.5534697 0.2016073 -0.0001565 H 1 1.3716338 1.1747257 0.000671

102

Table S4.2. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and intensities for [SN(H)OH]+.

Frequency Intensity 465 11.27 545 83.69 875 185.32 926 24.01 1165 262.44 1308 94.79 1501 21.68 3255 156.48 3372 199.86

Table S4.3. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for + [SN(H)OH]  SN-OH2 TS.

B3LYP/6-31G* energy -528.871012 Hartrees Zero-point correction 0.023238091 Hartrees Thermal correction to energy 0.028648376 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z S 16 1.1337102 -0.1400224 0.0074507 N 7 -0.1958262 0.5497227 -0.0247676 O 8 -1.71843 -0.2124034 0.1115696 H 1 -1.782487 -0.8951857 -0.5998393 H 1 -1.2386527 0.9867118 -0.2385562

103

Table S4.4. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and + intensities for [SN(H)OH]  SN-OH2 TS.

Frequency Intensity -1786 164.93 253 72.12 289 41.47 543 273.01 905 49.07 998 142.83 1135 163.47 2241 286.33 3429 195.29

Table S4.5. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for SN-OH2.

B3LYP/6-31G* energy -528.970161 Hartrees Zero-point correction 0.027692596 Hartrees Thermal correction to energy 0.033338912 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z S 16 1.15988 -0.228774 -0.0035095 N 7 0.1273082 0.8013489 0.0125049 O 8 -1.8442697 -0.258718 -0.0112359 H 1 -2.3641534 0.1317251 -0.7396902 H 1 -2.330927 -0.0110385 0.7981941

104

Table S4.6. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and intensities for SN-OH2.

Frequency Intensity 172 33.62 190 77.82 247 36.33 431 15.55 545 206.07 1332 139.37 1623 80.14 3513 254.2 3617 126.75

Table S4.7. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for + SN-OH2 [HOSN-H] TS.

B3LYP/6-31G* energy -528.903754 Hartrees Zero-point correction 0.023602441 Hartrees Thermal correction to energy 0.028544091 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z S 16 0.384911 -0.536087 0.0002323 O 8 -1.268352 0.1804338 -0.0875777 H 1 -2.0164532 -0.0643826 0.5145229 H 1 -0.4488604 1.1918912 0.0150994 N 7 0.9219361 0.858059 0.0238974

105

Table S4.8. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and + intensities for SN-OH2 [HOSN-H] TS.

Frequency Intensity -1883 312.81 396 143.09 489 108.8 683 32.47 956 118.05 1174 29.18 1180 76.14 1686 191.71 3383 268.92

Table S4.9. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for [HOSN-H]+.

B3LYP/6-31G* energy -528.987532 Hartrees Zero-point correction 0.028965915 Hartrees Thermal correction to energy 0.033582027 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z S 16 0.0754326 -0.4638552 0.0000045 O 8 -1.2299383 0.4662736 -0.0001287 H 1 -2.0765891 -0.0413098 0.000757 N 7 1.3296072 0.3380947 -0.0000376 H 1 1.4019226 1.366141 0.000464

106

Table S4.10. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and intensities for [HOSN-H]+.

Frequency Intensity 365 47.11 423 200.95 790 42.11 804 153.85 839 228.15 1031 179.21 1234 4.21 3299 233.16 3420 349.39

Table S4.11. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for [HON(H)S-W]+.

B3LYP/6-31G* energy -605.418283 Hartrees Zero-point correction 0.055005683 Hartrees Thermal correction to energy 0.061687230 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z S 16 1.3014786 -0.7868882 -0.0009116 N 7 0.1487411 0.2854712 0.0012659 O 8 0.2904405 1.6108092 0.0013554 H 1 1.2554371 1.8267745 -0.000618 H 1 -0.9322863 0.039179 0.0017419 O 8 -2.3378655 -0.4400599 -0.0020757 H 1 -2.9095469 -0.2979724 -0.775123 H 1 -2.8990499 -0.3420637 0.7854859

107

Table S4.12. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and intensities for [HON(H)S-W]+.

Frequency Intensity Frequency Intensity 82 27.4 1147 260.61 90 2.86 1290 124.75 228 29.98 1329 114.54 276 54.56 1499 40.3 323 255.3 1636 31.71 469 91.57 2173 2917.99 501 12.83 3370 130.72 589 98.17 3579 76.49 924 116.52 3673 167.27

Table S4.13. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for [HON(H)S-W]+  SN-2W TS.

B3LYP/6-31G* energy -605.394669 Hartrees Zero-point correction 0.054276565 Hartrees Thermal correction to energy 0.061055731 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z S 16 1.7878143 0.246847 0.0145063 N 7 0.2246916 0.234735 -0.0690594 O 8 -0.4256899 -1.0143973 -0.0253074 H 1 0.2108608 -1.760365 0.107953 H 1 -1.8118311 1.1051764 0.0005035 O 8 -2.5049748 0.3996547 0.1096591 H 1 -1.9606616 -0.4662442 0.0796424 H 1 -3.1709209 0.4466768 -0.6115973

108

Table S4.14. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and intensities for [HON(H)S-W]+  SN-2W TS.

Frequency Intensity Frequency Intensity -145 6.56 1016 88.35 109 35.12 1123 424.46 282 112.62 1297 118.18 300 48.01 1590 96.54 331 117.74 1687 616.66 386 98.38 2818 1117.77 475 35.5 3306 311.8 529 118.79 3331 54.18 788 217.78 3504 397.6

Table S4.15. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for SN-2W.

B3LYP/6-31G* energy -605.431644 Hartrees Zero-point correction 0.052226517 Hartrees Thermal correction to energy 0.059787858 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z S 16 1.3930512 0.1797914 0.0021321 O 8 -0.5108232 1.1397578 -0.0812104 H 1 -0.6338071 1.9350274 0.4714803 N 7 1.3384529 -1.2649718 0.0108579 H 1 -1.3342169 0.5258834 0.0002121 O 8 -2.4452505 -0.540673 0.0096146 H 1 -3.0343894 -0.6674582 0.7711557 H 1 -3.0069862 -0.6079901 -0.7802008

109

Table S4.16. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and intensities for SN-2W.

Frequency Intensity Frequency Intensity 53 0.48 730 50.28 57 4.61 991 115.59 141 38.46 1381 26.65 225 25.12 1578 60.93 252 127.91 1647 35.52 318 93.12 2635 2358.49 354 148.51 3567 190.02 404 131.18 3583 58.99 416 72.03 3678 140.27

Table S4.17. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for SN-2W  [HOSN(H)-W]+ TS.

B3LYP/6-31G* energy -605.419974 Hartrees Zero-point correction 0.052843999 Hartrees Thermal correction to energy 0.059704674 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z S 16 1.1934323 -0.0192048 -0.008756 O 8 -0.074194 1.2505797 0.059708 H 1 0.2084695 2.1499396 -0.2057121 N 7 0.5143956 -1.3163605 0.0232937 H 1 -1.421561 0.5791173 -0.0419629 O 8 -2.0800554 -0.241085 -0.1037721 H 1 -1.4860808 -1.0440762 -0.0236745 H 1 -2.7625187 -0.2391373 0.6009029

110

Table S4.18. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and intensities for SN-2W  [HOSN(H)-W]+ TS.

Frequency Intensity Frequency Intensity -133 30.48 975 83.17 156 34.83 1163 369.15 228 106.09 1281 21.18 300 94.04 1562 92.23 355 85.71 1715 467.8 422 119.79 2329 1215.14 461 31.52 3165 425.94 523 133.07 3522 272.36 586 72.25 3525 232.65

Table S4.19. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for [HOSN(H)-W]+.

B3LYP/6-31G* energy -605.437067 Hartrees Zero-point correction 0.052526421 Hartrees Thermal correction to energy 0.059363330 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z S 16 1.1187912 -0.4483959 -0.0013443 O 8 1.2038954 1.1695574 0.0024698 H 1 2.1330787 1.4941787 0.0063879 N 7 -0.2738282 -0.9437026 -0.0010191 H 1 -3.1265638 0.2147339 0.7634988 O 8 -2.5361497 0.3541816 0.0034438 H 1 -1.2379016 -0.4003052 0.0008453 H 1 -3.0944404 0.2817322 -0.7893979

111

Table S4.20. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and intensities for [HOSN(H)-W]+.

Frequency Intensity Frequency Intensity 62 35.51 1009 86.54 69 9.61 1039 297.57 165 44.81 1256 87.18 285 89.18 1263 111.7 379 126.08 1624 9.38 406 142.21 2160 3320.54 452 128.67 3454 278.39 488 20.76 3570 68.78 790 132.99 3665 148.03

Table S4.21. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for + [HON(H)S-W] (H2O dielectric).

B3LYP/6-31G* energy -605.533046 Hartrees Zero-point correction 0.055398332 Hartrees Thermal correction to energy 0.062080755 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z S 16 1.0592232 -0.9951836 -0.0084128 N 7 0.2642896 0.3838617 -0.0046085 O 8 1.0178616 1.5065307 0.0110097 H 1 0.366771 2.2469057 0.0138651 H 1 -1.556834 0.2253276 0.0084378 O 8 -2.5361224 -0.0247705 0.0434301 H 1 -2.6228421 -0.8474004 0.5781318 H 1 -2.8386079 -0.2430084 -0.8690886

112

Table S4.22. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and + intensities for [HON(H)S-W] (H2O dielectric).

Frequency Intensity Frequency Intensity 74 113.42 970 679.77 90 6.74 1122 385.33 135 78.29 1384 66.73 176 3.68 1632 117.45 381 17.85 1668 94.54 485 89.91 2882 1808.03 505 88.56 3424 485.04 553 163.48 3441 236.71 936 32.14 3484 461.94

Table S4.23. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for + [HON(H)S-W]  SN-2W TS (H2O dielectric).

B3LYP/6-31G* energy -605.508951 Hartrees Zero-point correction 0.048013680 Hartrees Thermal correction to energy 0.055032687 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z S 16 -1.9154929 -0.1041355 0.1525391 N 7 -0.5379623 -0.4258455 -0.4004419 O 8 0.5888109 0.8246881 -0.2135724 H 1 0.3388358 1.3634498 0.5734014 H 1 2.7268151 -1.1561514 -0.2874917 O 8 2.6989254 -0.3307421 0.2356718 H 1 1.5939988 0.2870708 -0.0109045 H 1 3.4520828 0.2011492 -0.0893324

113

Table S4.24. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and + intensities for [HON(H)S-W]  SN-2W TS (H2O dielectric).

Frequency Intensity Frequency Intensity -643 5275.23 736 987.54 57 51.59 1036 1150.74 72 48.68 1145 88.1 96 22.62 1300 170.1 328 188.26 1577 197.44 407 529.55 1635 167.34 472 52.84 3101 193.93 522 75.26 3518 117.24 640 166.93 3588 279.26

Table S4.25. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for SN-2W (H2O dielectric).

B3LYP/6-31G* energy -605.543300 Hartrees Zero-point correction 0.051102350 Hartrees Thermal correction to energy 0.058312901 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z S 16 1.2942739 0.204771 0.2033492 O 8 -0.4736751 0.9760929 -0.3198033 H 1 -0.6169848 1.8442471 0.1206529 N 7 1.481123 -1.1545928 -0.2611831 H 1 -1.3240578 0.3506093 -0.1283883 O 8 -2.4200376 -0.5537799 0.1059763 H 1 -2.9152032 -0.3020569 0.9087069 H 1 -3.070295 -0.4654884 -0.6156604

114

Table S4.26. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and intensities for SN-2W (H2O dielectric).

Frequency Intensity Frequency Intensity 54 3.52 832 50.43 95 14.38 1189 111.07 115 86.82 1356 120.85 156 329.41 1554 121.52 277 62.39 1617 20.94 356 20.43 1880 4225.08 443 37 3475 416.76 464 221.93 3537 100.32 522 227.74 3612 219.89

Table S4.27. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for + SN-2W  [HOSN(H)] TS (H2O dielectric).

B3LYP/6-31G* energy -605.543388 Hartrees Zero-point correction 0.053164624 Hartrees Thermal correction to energy 0.060346571 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z S 16 1.1989674 -0.0492394 0.0086672 O 8 0.0691308 1.2387527 0.0047421 H 1 0.5102821 2.1157702 -0.0267301 N 7 0.5033523 -1.3461238 -0.0011238 H 1 -1.6353124 0.6480604 -0.0468154 O 8 -2.2187099 -0.167285 -0.0939413 H 1 -1.5846695 -0.9350243 -0.0470009 H 1 -2.8006124 -0.1898509 0.7033308

115

Table S4.28. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and + intensities for SN-2W  [HOSN(H)] TS (H2O dielectric).

Frequency Intensity Frequency Intensity -173 3.58 1004 135.65 84 92.61 1130 523.97 115 100.67 1259 45.07 257 128.42 1608 126.07 316 10.29 1664 403.89 360 148.56 3084 707.65 378 167.89 3213 525.91 418 46.64 3447 455.36 592 189.18 3474 398.33

Table S4.29. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for + [HOSN(H)] (H2O dielectric).

B3LYP/6-31G* energy -605.549586 Hartrees Zero-point correction 0.052298427 Hartrees Thermal correction to energy 0.059153236 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z S 16 1.1285758 -0.41927 0.0057536 O 8 1.0876966 1.1902671 0.0050712 H 1 2.0051239 1.5618792 0.0277535 N 7 -0.2284587 -1.0188362 -0.0134765 H 1 -2.4489344 1.2155496 0.0305427 O 8 -2.5522169 0.2531544 -0.0914246 H 1 -1.1755507 -0.4699898 -0.0179423 H 1 -3.1224779 -0.0146362 0.6527508

116

Table S4.30. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and + intensities for [HOSN(H)] (H2O dielectric).

Frequency Intensity Frequency Intensity 63 93.08 1056 201.07 136 2.88 1068 223.84 148 43.18 1154 154.78 250 113.64 1299 77.32 400 27.55 1598 88.66 408 108.88 2191 3567.09 441 293.2 3376 610.53 506 1.59 3539 74 787 231.87 3618 199.95

Table S4.31. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for HOSN-W.

B3LYP/6-31G* energy -605.109362 Hartrees Zero-point correction 0.043023621 Hartrees Thermal correction to energy 0.049506577 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z S 16 1.1204264 0.0037714 0.0112354 O 8 -2.1531422 0.1283083 -0.0917039 H 1 -2.6358347 0.2079502 0.7457375 H 1 -1.5448807 0.9002635 -0.10819 N 7 0.480741 1.3372783 -0.0033635 O 8 0.1186511 -1.3197111 -0.0028101 H 1 -0.835365 -0.9982829 -0.0376573

117

Table S4.32. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and intensities for HOSN-W.

Frequency Intensity 142 11.36 179 5.31 245 37.82 290 96.5 361 73.46 438 6.13 684 269.06 727 173.61 847 155.48 1223 3.54 1302 91.67 1649 71.33 3003 652.44 3396 253.66 3644 60.16

118

Table S4.33. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for HOSN-W  HNSO-W TS.

B3LYP/6-31G* energy -605.091808 Hartrees Zero-point correction 0.038429943 Hartrees Thermal correction to energy 0.044210442 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z S 16 1.0428705 0.0002599 -0.0127275 H 1 -1.1021347 0.7601021 0.0497887 O 8 -1.8916913 -0.0882934 0.0959128 H 1 -2.3965656 -0.1078025 -0.7362575 H 1 -1.0985077 -0.8878749 0.0546115 O 8 0.109683 1.2671667 0.004295 N 7 0.309621 -1.3142244 0.0048335

119

Table S4.34. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and intensities for HOSN-W  HNSO-W TS.

Frequency Intensity -1321 26.05 230 16.44 412 10.62 504 73.04 522 66.67 599 55.7 606 287.91 903 162.38 1157 18.62 1271 579.11 1352 843.9 1539 1568.97 1613 367.06 1894 370.65 3592 58.56

120

Table S4.35. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for HNSO-W

B3LYP/6-31G* energy -605.122301 Hartrees Zero-point correction 0.041940017 Hartrees Thermal correction to energy 0.048796008 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z O 8 1.3124224 -1.1820162 -0.0097823 S 16 1.231344 0.299708 0.0043391 N 7 -0.1246304 1.0120092 0.0031467 H 1 -0.9824637 0.4261297 -0.0101129 H 1 -3.2599348 0.0998917 -0.6961875 O 8 -2.7382131 -0.3525017 -0.0155945 H 1 -3.1803671 -0.12927 0.817863

121

Table S4.36. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and intensities for HNSO-W.

Frequency Intensity 18 73.82 60 9.59 120 1.43 175 5.19 256 298.37 319 4.01 461 23.5 956 149.31 983 56.45 1099 108.53 1196 131.01 1634 72.79 3120 532.46 3581 14.67 3693 47.35

122

Table S4.37. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for HOSN-W (H2O dielectric).

B3LYP/6-31G* energy -605.129570 Hartrees Zero-point correction 0.041940017 Hartrees Thermal correction to energy 0.048796008 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z S 16 -1.185197 0.1403456 0.0168951 O 8 2.3386044 -0.244622 -0.0918584 H 1 2.8508607 -0.1363071 0.7313967 H 1 1.9766858 -1.1470547 -0.0176518 N 7 -0.9047551 -1.3111803 -0.0076659 O 8 0.0973111 1.1868283 -0.0186756 H 1 0.981567 0.6784434 -0.0461328

123

Table S4.38. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and intensities for HOSN-W (H2O dielectric).

Frequency Intensity 34 33.32 121 16.04 170 96.91 254 79.77 385 178.53 411 40.32 488 99.32 718 281.46 898 162.39 1236 25.42 1302 147.09 1601 112.67 2748 1842.78 3531 49.3 3616 169.84

124

Table S4.39. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for HOSN-W  HNSO-W TS (H2O dielectric).

B3LYP/6-31G* energy -605.115930 Hartrees Zero-point correction 0.039878391 Hartrees Thermal correction to energy 0.045847959 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z S 16 1.0605573 0.0273983 -0.0124347 H 1 -1.2273172 0.6940851 0.0383328 O 8 -1.9550532 -0.1159123 0.0969059 H 1 -2.4924088 -0.1262946 -0.726523 H 1 -1.2647969 -0.891781 0.0418228 O 8 0.0959412 1.2646427 0.0051752 N 7 0.4126432 -1.3291752 0.0040962

125

Table S4.40. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and intensities for HOSN-W  HNSO-W TS (H2O dielectric).

Frequency Intensity -393 146 192 29.06 292 315.04 476 281.76 508 174.22 536 44.25 631 199.54 874 178.97 1151 66.06 1284 873.69 1430 376.71 1604 905.92 1868 289.67 2486 1508.82 3472 240.78

126

Table S4.41. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for HNSO-W (H2O dielectric).

B3LYP/6-31G* energy -605.147028 Hartrees Zero-point correction 0.041847578 Hartrees Thermal correction to energy 0.048552168 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z O 8 1.2246722 -1.2035573 -0.0044357 S 16 1.2156144 0.2872387 0.0001002 N 7 -0.1099963 1.0518501 0.0067996 H 1 -1.0029536 0.5044714 0.0065028 H 1 -3.1922582 0.0560541 -0.711364 O 8 -2.6436345 -0.3466807 -0.0133534 H 1 -3.1329447 -0.1173913 0.7979726

127

Table S4.42. B3LYP/6-31G* calculated IR frequencies (cm-1, scaled by 0.96) and intensities for HNSO-W(H2O dielectric).

Frequency Intensity 70 11.27 78 78.15 143 9.7 190 23.53 320 313.93 370 4.46 468 24.58 991 51.53 1002 147.9 1095 291.88 1176 147.45 1607 100.35 2958 1103.08 3540 50.2 3624 126.52

128

4.9.2 Supporting Figures

2500

2000

1500

1000

500

Ion Current (pA) m/z 30 0

-2 0 2 4 6 8 Time (min) Figure S4.1. MIMS observed intensity at m/z 30 following injection of 250 µM at t = 0 min of the pre-incubated reaction mixture, of NaSH with S-nitrosoglutathione, into 0.1 M pH 7.4 PBS with 100 µM DTPA at 20 °C.

Figure S4.2. 31P NMR spectra resulting from TCEP in basic solution (red), acidic solution (blue), and neutral solution (black). All samples were incubated for 30 min at 21 °C in aqueous solutions containing 10% D2O.

129

Figure S4.4. B3LYP/6-31G(d) calculated energies and barriers, relative to F, for the transformation of F to H, in the gas phase.

130

Chapter 5: Chemistry and Reactivity of Carbonylnitrenes

5.1 Nitrene Background

Nitrenes are reactive intermediates containing neutral, monovalent nitrogen atoms.1

Nitrenes are most commonly generated from the corresponding azide. However, there are other precursors including phenanthrene- and sulfilimine-based compounds that have been shown to generate the nitrenes photochemically.2–4 These compounds have the added advantage of being more stable for storage and handling compared to azide precursors.

Aliphatic and aromatic nitrenes typically favor triplet ground states by a wide margin. The nitrene nitrogen is sp hydridized with two degenerate p-orbitals that can be occupied in three electronic configurations. The parent nitrene (imidogen, NH) triplet state (3Σ’) is formed by the two valence electrons occupying each of the degenerate p-orbitals in a spin- parallel fashion (Figure 5.1a). The singlet states (1Δ) have two possibilities, the open shell or closed shell singlets, where the electrons are paired in two p-orbitals or in the same orbital, respectively (Figure 5.1a). The energetic difference between triplet and singlet nitrenes arises from the distribution and spin of the electrons in the p-orbital. In the triplet case, the electrons are spin parallel, and therefore, pay minimal energetic costs due to repulsion. Although the singlet states benefit from being spin paired, the energetic benefit does not outweigh the cost of electron-electron repulsion.

131

Figure 5.1. Electronic configurations of (a) imigoden (NH) and (b) methylene (CH2).

Aliphatic carbenes are also typically ground state triplets, however, usually by smaller margins. The carbene carbon is sp2 hybridized, resulting in two energetically different orbitals (sp2- and p-orbitals) for the valence electrons to occupy (Figure 5.1b).

Therefore, there is a higher energetic cost for carbenes to access the triplet state. Further, the singlet states of carbenes are not degenerate as both the open- and closed-shell species occupy orbitals of different energies.

132

Commonly, ground state electronics can be deciphered by reactivity with alkenes to generate either a mixture of isomers, representative of triplet reactivity, or stereospecific products that are indicative of singlet reactivity (Scheme 5.1). Rapid reactions of the singlet state and slow intersystem crossing rates have made direct detection of the triplet nitrene difficult. However, observation of triplet nitrenes has been successful by low temperature EPR, matrix-isolation, and time-resolved spectroscopy methods.2,3,5–13

Scheme 5.1. Reactivity of singlet and triplet nitrenes to produce one isomer or a mixture of isomers, respectively.

133

5.2 Nitrene Substituent Effects

The identity of the nitrene substituent has a significant effect on reactivity.

Alkylnitrenes like methylnitrene (CH3N) are ground state triplets by a large margin (31.2 kcal/mol);14 however, the triplet is difficult to detect due a low barrier of isomerization to methyleneimine (Scheme 5.2). Substitution of methyl for trifluromethyl slows the rate of isomerization, therefore allowing for isc to the triplet for observation by low temperature

UV-vis and EPR spectroscopy.15 Phenylnitrene (PhN) is another example that is a ground

16 state triplet, by a smaller margin of 18.5 kcal/mol. Similar to CH3N, rapid reaction of the initially generated singlet nitrene results in complicated product mixtures limiting further applications.12,17 A variety of other nitrenes have been explored including oxygen- substituted, sulfur-substituted, nitrogen-substituted, phosphorus-substituted nitrenes.2,18

Scheme 5.2. Reactivity of methylnitrene to form methyleneimine. In contrast to alkyl- and arylnitrenes, carbonylnitrenes (RC(O)N) have demonstrated primarily singlet reactivity.19 For example, photolysis of azide precursors in the presence of alkenes produced stereospecific products, indicative of singlet reactivity.19

Computational and matrix isolation studies has shown that the ground state of carbonylnitrenes is a closed-shell singlet. This state is stabilized by the interaction between the in-plane carbonyl oxygen lone pair and the empty in-plane orbital on the nitrene

134 nitrogen.10,19–22 This bonding interaction leads to an oxazirine-like structure for the singlet carbonylnitrene that has a much smaller O-C-N bond angle compared to the corresponding triplet carbonylnitrene (Figure 5.2). This significant geometry difference affords unique infrared (IR) signatures for singlet and triplet nitrenes.

Figure 5.2. O-C-N bond angle comparison between triplet and singlet carbonylnitrenes. Time-resolved infrared (TRIR) spectroscopy has been successful at detecting and probing reactivity of carbonylnitrenes in solution.4,10,23 Photolysis of the sulfilimine precursor produced signals representative of both singlet (1760 cm-1) and triplet (1488 cm-

1) benzoylnitrene along with the corresponding isocyanate. When the photolysis was performed in acetonitrile the singlet nitrene reacted with solvent resulting in the growth of an ylide, followed by cyclization to the oxadiazole (Scheme 5.3). TRIR spectroscopy was also applied to investigate other nitrenes including acetyl- and trifluroacetylnitrenes.4

Scheme 5. 3. Reactivity observed upon photolysis of benzoylnitrene precursor.

135

Oxycarbonylnitrenes, on the other hand, are computed to be ground state triplets since this oxazirene stabilization is now less important (Figure 5.3). The oxygen conjugation into the carbonyl stabilizes the triplet more than the corresponding closed-shell singlet state.22,24 Sherman and Jenks recently reported a thorough computational investigation of the effect of fluorination on the ground state multiplicity of acylnitrenes.25

They found that the electron withdrawing effect of fluorine substitution reduces the stabilization of the singlet configuration, which could also be important for the electronegative substituent of oxycarbonynitrenes. This effect is observed with the increasing of the calculated O-C-N bond angle that correlates with the increase in electron withdrawing magnitude of the substituent.

Figure 5.3. Relevant resonance structures for carbonyl- and oxycarbonylnitrenes. Although standard B3LYP/6-31G(d) calculations give optimized geometries for oxycarbonyl- and carbonylnitrenes in very good agreement with those calculated by higher levels of theory (CCSD(T) with complete basis set extrapolation or CBS-QB3

10,22,24 calculations), singlet-triplet energy gaps (ΔEST = ES – ET) are overestimated. For example, B3LYP/6-31G(d) calculations overestimate ΔEST by approximately 9 kcal/mol for formylnitrene (HC(O)N) and approximately 7 kcal/mol for carboxynitrene

(HOC(O)N).24 Despite the extensive study of nitrenes and the fundamental importance of

136 their ground state multiplicity and singlet-triplet splitting on reactivity, only three ΔEST values have been determined experimentally. Negative ion photoelectron spectroscopy has

26 27 been used to determine ΔEST for imidogen (NH), methylnitrene (CH3N), and phenylnitrene (PhN).16,28 Negative ions are generated under high-vacuum then irradiated to eject electrons. The measured kinetic energy (Ek) of the ejected electrons, in combination with the known photon energy (h), are used to determine the binding energy (EB) of each electron (h = Ek – EB). The difference in energy between the observed transitions, when the geometries of anions and neutrals as similar, represents the singlet-triplet energy gaps

(ΔEST = ES – ET, Scheme 5.4).

Scheme 5.4. ΔEST measured by negative ion photoelectron spectroscopy (PES).

137

In the carbonylnitrene section of this thesis we investigate the solution reactivity of oxycarbonylnitrenes by TRIR spectroscopy. We combine computational analysis with our observed results to interpret the observed chemistry of ethoxy- and t- butyloxycarbonynitrenes. Further, in collaboration with Bowen, Pederson, and co-workers

(JHU), we explore the application of negative ion PES in effort to obtain experimental

ΔEST values for benzoyl-, acetyl-, and trifluroacetylnitrene to corroborate our computational results.

138

5.3 References

(1) W. Lwowski. Nitrene. In Nitrenes; Lwowski, W., Ed.; John Wiley & Sons, Inc.,

New York, 1920; p 1.

(2) Wasylenko, W. a; Kebede, N.; Showalter, B. M.; Matsunaga, N.; Miceli, A. P.;

Liu, Y.; Ryzhkov, L. R.; Hadad, C. M.; Toscano, J. P. Generation of Oxynitrenes

and Confirmation of Their Triplet Ground States. J. Am. Chem. Soc. 2006, 128,

13142–13150.

(3) Desikan, V.; Liu, Y.; Toscano, J. P.; Jenks, W. S. Photochemistry of Sulfilimine-

Based Nitrene Precursors: Generation of Both Singlet and Triplet Benzoylnitrene.

J. Org. Chem. 2007, 72, 6848–6859.

(4) Desikan, V.; Liu, Y.; Toscano, J. P.; Jenks, W. S. Photochemistry of N- Acetyl-,

N- Trifluoroacetyl-, N- Mesyl-, and N- Tosyldibenzothiophene Sulfilimines. J.

Org. Chem. 2008, 73, 4398–4414.

(5) Wasserman, E.; Smolinsky, G.; Yager, W. a. Electron Spin Resonance of Alkyl

Nitrenes. J. Am. Chem. Soc. 1964, 86, 3166–3167.

(6) Ferrante, R. F. Spectroscopy of Matrix-Isolated Methylnitrene. J. Chem. Phys.

1987, 86, 25.

(7) Leyva, E.; Platz, M. S.; Persy, G.; Wirz, J. Photochemistry of Phenyl Azide: The

Role of Singlet and Triplet Phenylnitrene as Transient Intermediates. J. Am. Chem.

Soc. 1986, 108, 3783–3790.

(8) Sigman, M. E.; Autrey, T.; Schuster, G. B. Aroylnitrenes with Singlet Ground

139

States: Photochemistry of Acetyl-Substituted Aroyl and Aryloxycarbonyl Azides.

J. Am. Chem. Soc. 1988, 110, 4297–4305.

(9) Desikan, V.; Liu, Y.; Toscano, J. P.; Jenks, W. S. Photochemistry of N- Acetyl-,

N- Trifluoroacetyl-, N- Mesyl-, and N- Tosyldibenzothiophene Sulfilimines. J.

Org. Chem. 2008, 73, 4398–4414.

(10) Pritchina, E. A.; Gritsan, N. P.; Maltsev, A.; Bally, T.; Autrey, T.; Liu, Y.; Wang,

Y.; Toscano, J. P. Matrix Isolation, Time-Resolved IR, and Computational Study

of the Photochemistry of Benzoyl Azide. Phys. Chem. Chem. Phys. 2003, 5, 1010–

1018.

(11) Buron, C.; Platz, M. S. Laser Flash Photolysis Study of Carboethoxynitrene. Org.

Lett. 2003, 5, 3383–3385.

(12) Borden, W. T.; Gritsan, N. P.; Hadad, C. M.; Karney, W. L.; Kemnitz, C. R.; Platz,

M. S. The Interplay of Theory and Experiment in the Study of Phenylnitrene. Acc.

Chem. Res. 2000, 33, 765–771.

(13) Gritsan, N. P.; Platz, M. S. Kinetics, Spectroscopy, and Computational Chemistry

of Arylnitrenes. Chem. Rev. 2006, 106, 3844–3867.

(14) Travers, M. J.; Cowles, D. C.; Clifford, E. P.; Ellison, G. B.; Engelking, P. C.

Photoelectron Spectroscopy of the CH3N− Ion. J. Chem. Phys. 1999, 111, 5349.

(15) Gritsan, N. P.; Likhotvorik, I.; Zhu, Z.; Platz, M. S. Observation of

Perfluoromethylnitrene in Cryogenic Matrixes. J. Phys. Chem. A 2001, 105, 3039–

3041.

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Spectroscopy of the Phenylnitrene Anion. J. Am. Chem. Soc. 1992, 114, 8699–

8701.

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1995, 28, 487–492.

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(19) Sigman, M. E.; Autrey, T.; Schuster, G. B. Aroylnitrenes with Singlet Ground

States: Photochemistry of Acetyl-Substituted Aroyl and Aryloxycarbonyl Azides.

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(20) Autrey, T.; Schuster, G. B. Are Aroylnitrenes Ground-State Singlets?

Photochemistry of β-Naphthoyl Azide. J. Am. Chem. Soc. 1987, 109 (19), 5814–

5820.

(21) Gritsan, N. P.; Pritchina, E. A. Are Aroylnitrenes Species with a Singlet Ground

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Methoxycarbonylnitrenes by Computational Methods and a Laser Flash Photolysis

Study of Benzoylnitrene. J. Org. Chem. 2004, 69, 8583–8593.

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Intercombination Energy Difference in NH. J. Chem. Phys. 1976, 65, 4323.

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142

Chapter 6: Nanosecond Time-Resolved Infrared (TRIR) Studies of Oxycarbonylnitrenes

6.1 Introduction

Oxycarbonylnitrenes (ROC(O)N) have a rich history in the development of our modern mechanistic understanding of nitrene chemistry.1 Seminal studies by Lwowski and co-workers on ethoxycarbonylazide (1) demonstrated ethoxycarbonylnitrene (2) undergoes intermolecular insertion reactions into C-H bonds and adds to double bonds with partial stereospecificity that suggested reaction from both singlet 2s and presumably lower energy triplet nitrene 2t (Scheme 6.1).2–5 Similar work in Germany also demonstrated that photochemical decomposition of t-butyloxycarbonylazide (3) produces t- butyloxycarbonylnitrene (4) that predominantly gives 5,5-dimethyl-2-oxazolidinone (5) via an intramolecular C-H insertion reaction in a variety of solvents (Scheme 6.2).6–8 More recent synthetic and spectroscopic studies on oxycarbonylnitrenes have confirmed the above reactivity and provided additional insight into these interesting reactive intermediates.9–12

143

Scheme 6.2. Photolysis of t-butyloxycarbonylazide (3) to produce t- butyloxycarbonylnitrene (4) that predominantly gives 5,5-dimethyl-2-oxazolidinone (5) via an intramolecular C-H insertion reaction.

Lwowski and co-workers were also the first to suggest that the classic photo-

Curtius rearrangement of acyl azides to isocyanates does not occur from the nitrene intermediate, but rather directly from the azide.13–15 Subsequent low-temperature matrix infrared and solution nanosecond time-resolved infrared (TRIR) spectroscopic studies confirmed that the isocyanate is not formed from a relaxed nitrene intermediate.16–18

Recently, ultrafast IR and UV-Vis studies of Platz and co-workers have provided spectroscopic evidence for this pathway, demonstrating directly that the first singlet excited state (S1) of the acyl azide is the isocyanate precursor.19–22

144

Low-temperature ESR spectroscopic studies confirmed the suspected triplet ground state for ethoxycarbonylnitrene and related species,23 consistent with the observed non- stereospecific trapping of alkenes. These observations are in contrast to those found for carbonylnitrenes (RC(O)N), which have been shown to be ground state singlets that are not detectable by ESR spectroscopy and show retention of configuration when adding to alkenes.10,24

The singlet and triplet state structures and energies of both oxycarbonyl- and carbonylnitrenes have been thoroughly investigated by computational methods, which provide insight into their different ground states.11,18,25–29 High-level theory indicates that carbonylnitrenes are closed-shell ground state singlets owing to a significant bonding interaction between a carbonyl oxygen lone pair and an empty orbital on the nitrene nitrogen.18,26–29 Indeed, computed structures of singlet carbonylnitrenes have significant cyclic oxazirene character (Figure 6.1). Oxycarbonylnitrenes, on the other hand, are computed to be ground state triplets since this oxazirene stabilization is now less important

(Figure 6.1) and oxygen conjugation into the carbonyl stabilizes the triplet more than the corresponding closed-shell singlet state.27,28 Inductive effects have also been suggested to play a role in reducing the stabilization of the singlet by limiting the carbonyl oxygen’s nucleophilicity/basicity, as well as by increasing the O-C-N bond angle in accord with

Bent’s rule.30–32

145

Figure 6.1. Relevant resonance structures for carbonyl- and oxycarbonylnitrenes. Although standard B3LYP/6-31G(d) calculations give optimized geometries for oxycarbonyl- and carbonylnitrenes in very good agreement with those calculated by higher levels of theory (CCSD(T) with complete basis set extrapolation or CBS-QB3

18,27,28 calculations), singlet-triplet energy gaps (ΔEST = ES – ET) are overestimated. For example, B3LYP/6-31G(d) calculations overestimate ΔEST by approximately 9 kcal/mol for formylnitrene (HC(O)N) and approximately 7 kcal/mol for carboxynitrene

(HOC(O)N).28

To gain additional insight into the chemistry and reactivity of oxycarbonylnitrenes, we are pleased to report herein nanosecond TRIR studies of ethoxycarbonylnitrene (2) and t-butyloxycarbonylnitrene (4) in a variety of solvents. Although azides are standard photoprecursors for nitrenes, recent work has demonstrated that these intermediates also can be efficiently generated from analogous sulﬁlimine precursors.29,33–36 Thus, we have utilized the sulfilimine photoprecursors 6 and 7 for these studies (Figure 6.2).

146

Figure 6.2. Sulfilimine-based photoprecursors to oxycarbonylnitrenes 2 and 4. 6.2 Computational Analysis of Ethoxy- and t- Butyloxycarbonylnitrenes

The B3LYP/6-31G(d) calculated ΔEST values of the syn- and anti-rotamers of ethoxycarbonylnitrene (2) are 12.8 and 8.8 kcal/mol, respectively, and 13.1 and 9.3 kcal/mol, respectively, with the inclusion of zero-point vibrational energy correction

(Figure 6.3a). Given that B3LYP/6-31G(d) calculations overestimates the ΔEST of

28 carboxynitrene (HOC(O)N) by 7 kcal/mol, we estimate that the ΔEST values of the syn- and anti-rotamers of ethoxycarbonylnitrene (2) are 6.1 and 2.3 kcal/mol, respectively

(Figure 6.3a). Similar to HOC(O)N,28 the syn- and anti-rotamers of singlet nitrene 2s are very close in energy, but the syn-rotamer of triplet nitrene 2t is 3.7 kcal/mol lower in energy than the corresponding anti-rotamer (Figure 6.3a). The barrier to rotation from syn- to anti- forms of 2s is calculated to be 6.1 kcal/mol, while that of triplet 2t is 9.4 kcal/mol.

In comparison, the B3LYP/6-31G(d) calculated ΔEST values of the syn- and anti- rotamers of t-butyloxycarbonylnitrene (4) were found to be 11.0 and 7.2 kcal/mol, respectively, and 11.4 and 7.8 kcal/mol, respectively, with the inclusion of zero-point vibrational energy correction. Taking into account the 7 kcal/mol B3LYP/6-31G(d)

28 correction, the ΔEST values of the syn- and anti-rotamers of t-butoxycarbonylnitrene are

147 estimated to be 4.4 and 0.8 kcal/mol respectively (Figure 6.3b). These ΔEST values match well with CBS-QB3 calculated values for methoxycabonylnitrene that have been previously reported.27

148

As with ethoxycarbonylnitrene, the syn- and anti-rotamers of singlet nitrene 4s are very close in energy, while the syn- and anti-rotamers of triplet nitrene 4t are separated by

3.2 kcal/mol in favor of the syn-form (Figure 6.3b). The B3LYP/6-31G(d) calculated barriers to rotation from syn- to anti-forms of 4s is calculated to be 4.7 kcal/mol, while that for triplet 4t is 7.5 kcal/mol (Supporting Information).

Analysis of the B3LYP/6-31G(d) calculated geometries of both ethoxy- and t- butoxycarbonylnitrene show a contribution from the oxazirine resonance form in the singlet states of both nitrenes. This effect is observed in the significantly smaller O-C-N bond angles of 91.5° and 91.1° in the singlet syn- and anti-ethoxycarbonylnitrenes (2s), respectively, relative to 120.4° and 117.8° for triplet syn- and anti-ethoxycarbonylnitrene

(2t), respectively (Figure 6.4a). The same effect is also observed for t- butoxycarbonylnitrenes with O-C-N bond angles of 89.9° for both the syn- and anti- forms of the singlet (4s), while the syn- and anti-triplets (4t) have calculated angles of 119.2° and

116.8°, respectively. However, calculated triplet ground states (positive ΔEST values) suggests a less important role of this resonance form compared to that in carbonylnitrenes.30–32

Significant differences in the geometries of the two nitrene spin states results in considerably different IR signatures. The singlet nitrenes are calculated to have strong vibrations at ca. 1750 cm-1, while the triplets are calculated to appear at ca. 1605 cm-1

(scaled by 0.96).37 Therefore, both singlet and triplet oxycarbonylnitrenes can be distinguished by their frequencies in solution if their lifetimes are sufficiently long enough.

149

Previous TRIR work on other carbonylnitrenes has shown that they can be detected in solution at frequencies similar to those calculated.29,33,38

150

151

6.3 Time-Resolved IR Studies of Ethoxycarbonylnitrene (2)

Upon 266 nm laser photolysis of N-ethoxycarbonyl dibenzothiophene sulfilimine

(6) in argon-saturated acetonitrile (CH3CN), the TRIR difference spectrum shown in Figure

6.5 is obtained. A transient species observed at 1640 cm-1, grows in within the time resolution of our experiment (50 ns, k ≥ 2.0 x 10-7 s-1) and decays with a first-order rate constant of 5.5 x 105 s-1. Photolysis in oxygen-saturated acetonitrile does not affect the rate of decay of this signal. Platz and Buron detected triplet ethoxycabonylnitrene (2t),

11 upon photolysis of carboethoxyazide (1), and did not observe reactivity with O2. We assign the observed 1640 cm-1 signal to triplet ethoxycarbonylnitrene (2t), which is a reasonable match with its calculated frequency (ca. 1605 cm-1). By comparison, the calculated IR frequency of singlet ethoxycarbonylnitrene (2s) is at 1752cm-1. Similar to previous work,11 the lifetime of the 1640 cm-1 signal is shortened in the presence of the H- atom donor triethylsilane (TES) further suggesting that the triplet nitrene is responsible for the signal observed (Supporting Information).

152

1.5 ylide 8 -1 1690 cm ylide 8 -1 1.0 triplet nitrene 2t 1160 cm -1 1640 cm

ylide 8 0.5 -1 1285 cm

0.0

Absorbance -0.5

 0.0 - 0.2 µs 0.2 - 0.4 µs 0.4 - 0.6 µs -1.0 0.6 - 0.8 µs 0.8 - 1.2 µs 1.2 - 2.0 µs -3 2.0 - 2.8 µs -1.5x10 2.8 - 3.6 µs

1800 1750 1700 1650 1300 1250 1200 1150 -1 Wavenumber (cm )

Figure 6.5. TRIR difference spectra averaged over the time scales indicated following 266 nm laser photolysis of sulfilimine 6 (3 mM) in argon-saturated acetonitrile. Blue and black bars reflect B3LYP/6-31G(d) calculated frequencies and intensities of ylide 8 and triplet ethoxycarbonylnitrene (2t), respectively.

Triplet nitrene 2t decays at the same rate as the growth of signals at 1690, 1285, and 1160 cm-1 (Figure 6.6). These bands match well with the B3LYP/6-31G(d) calculated frequencies of ylide 8 (Supporting Information). Therefore, we assign these signals to ylide 8, the product of reaction of the nitrene with acetonitrile (Scheme 3). The observed rate of ylide growth fits well to a biexponential function, indicative of kinetics comprised of a fast component, produced within the instrumental time resolution (k = 2 x 107 s-1), and a slower, resolvable component (k = 5.5 x 105 s-1). The slow component of the ylide growth matches the decay of the triplet nitrene, indicating that this portion of the observed kinetics is due to 2t, although carbonylnitrene reactivity with acetonitrile has typically been associated with the singlet. The fast component is likely the result of reactivity of the

153 singlet nitrene 2s, consistent with the reported growth of 2s within 10 ns in Freon-113 by

Platz and Buron.11 The ylide further decays with an observed first-order rate of 3.5 x 105 s-1 to produce oxadiazole 9 (Scheme 3), which is confirmed by 1H NMR spectroscopic analysis of the reaction (Supporting Information).

154

40 (a) triplet nitrene 2t -1 20 1640 cm 5 -1 kobs = 5.5 x 10 s 0

Intensity

 -20

-6 -40x10

0 2 4 6 8µs Time

-6 150x10 (b)

100 ylide 8 -1 1160 cm 50 7 -1 kobs1 = 2.0 x 10 s 5 -1 Intensity 0 kobs2 = 5.5 x 10 s

 Fast : Slow = 2 : 1 -50 0 2 4 6 8µs Time -6 300x10 (c) 200 ylide 8 -1 100 1690 cm 7 -1 Intensity kobs1 = 2.0 x 10 s 0 5 -1

 kobs2 = 4.7 x 10 s Fast : Slow = 1.1 : 1 -100 0 2 4 6 8µs Time -6 300x10 (d) ylide 8 -1 200 1690 cm 3 -1 kobs = 3.5 x 10 s

100

Intensity

 0

0 200 400 600 800µs Time

Figure 6.6. Kinetic traces observed following 266 nm laser photolysis of sulfilimine 6 in argon-saturated acetonitrile at (a) 1640 cm-1 from -1 to 9 μs, and (b) 1160 cm-1 from -1 to 9 μs, (c) 1690 cm-1 from -1 to 9 μs, and (d) 1690 cm-1 from -100 to 900 μs. Black curves are the calculated best fit to a single- or double-exponential function.

155

Scheme 6.3. Reactivity observed upon photolysis of sulfilimine 6 in acetonitrile.

Upon 266 nm laser photolysis of sulfilimine 6 in argon-saturated cyclohexane, the

TRIR difference spectrum shown in Figure 7 is observed. Analogous to TRIR experiments in acetonitrile, we detect 2t at 1640 cm-1(Figure 6.8a). Here, the product of reaction with solvent is presumably amide 11 (Scheme 6.4). The triplet nitrene decays (k = 7.1 x 105 s-

1) at the same rate as the growth of the slow component of amide 11, which is observed at

1728 cm-1, in excellent agreement with its reported frequency (1730 cm-1).39 We also observe a fast component for the growth of the 1728 cm-1 signal, suggesting reactivity from singlet nitrene 2s (Figure 6.8b). In addition, we observe a very weak positive band at 2180 cm-1, produced within the instrumental time resolution (Figure 6.8c). This species is assigned to ethoxyisocyanate (9, Scheme 6.4), which is in reasonable agreement with the reported literature frequency of 2204 cm-1. Since this species is formed within the instrumental time resolution (50 ns), the source of the isocyanate is either an excited state of precursor 6 or singlet nitrene 2s (Scheme 6.4).

156

0.0 - 0.2 µs -3 amide 10 0.2 - 0.4 µs 1.0x10 -1 0.4 - 0.6 µs 1728 cm 0.6 - 0.8 µs 0.8 - 1.2 µs triplet nitrene 2t 1.2 - 2.0 µs -1 1640 cm 2.0 - 2.8 µs 0.5 2.8 - 3.6 µs

Absorbance 0.0



-0.5 1800 1750 1700 1650 1600 1550 -1 Wavenumber (cm )

Figure 6.7. TRIR difference spectra averaged over the time scales indicated following 266 nm laser photolysis of sulfilimine 6 (3 mM) in argon-saturated cyclohexane. Black bar reflect B3LYP/6-31G(d) calculated frequency of triplet ethoxycarbonylnitrene (2t).

157

(a) triplet nitrene 2t -1 50 1640 cm 5 -1 kobs = 7.1 x 10 s 0

Intensity -50



-6 -100x10 0 2 4 6 8µs Time

-6 600x10 (b)

400 amide 10 -1 1728 cm 200 7 -1 kobs1 = 2 x 10 s

Intensity Intensity 5 -1 kobs2 = 6.8 x 10 s

 0 Fast : Slow = 2 : 1

0 2 4 6 8µs Time

-6 100x10 (c) 50

0 isocyanate 11

Intensity Intensity -50 -1

 2180 cm 7 -1 kobs > 2 x 10 s

0 10 20 30 40µs Time Figure 6.8. Kinetic traces observed following 266 nm laser photolysis of sulfilimine 6 in argon-saturated cyclohexane at (a) 1640 cm-1 from -1 to 9 μs, (b) 1728 cm-1 from -1 to 9 μs, and (c) 2180 cm-1 from -5 to 45 μs. Black curves are the calculated best fit to a single- or double-exponential function.

158

Scheme 6.4. Reactivity observed upon photolysis of sulfilimine 6 in cyclohexane.

6.4 Time-Resolved IR Studies of t-Butyloxycarbonylnitrene (4)

Upon 266 nm laser photolysis of sulfilimine 7 in argon-saturated acetonitrile, the

TRIR difference spectrum shown in Figure 6.9 is observed. Triplet nitrene 4t is detected at 1640 cm-1 (Figures 6.9, 6.10a); however, in contrast to the chemistry observed with nitrene 2 in acetonitrile, formation of the corresponding acetonitrile ylide is not observed.

Triplet nitrene 4t instead decays at the same observed rate as the growth of an intense signal at 1762 cm-1 (Figures 6.9, 6.10b). This 1762 cm-1 band matches reasonably well with the reported literature frequency of oxazolidinone 5 (1740 cm-1),40 formed via an intramolecular C-H insertion reaction (Scheme 5). 1H NMR product analysis following

254 nm Rayonet photolysis in both acetonitrile-d3 and dichloromethane-d2, confirms the production of 5 in ca. 90% yield (Supporting Information). The growth of oxazolidinone

5 displays biexponential kinetics (Figure 6.10b) with the fast component presumably due to production from singlet nitrene 4s. We also observe the production of t-butoxyisocyante

(12) at 2180 cm-1, which grows within the instrumental time-resolution (Figure 6.10c).

159

0.0 - 0.2 s -3 3x10 0.2 - 0.4 s 0.4 - 0.6 s oxazolidinone 5 0.6 - 0.8 s -1 0.8 - 1.2 s 2 1762 cm 1.2 - 2.0 s 2.0 - 2.8 s 2.8 - 3.6 s triplet nitrene 4t 1 -1 1640 cm

Absorbance

 0

-1

1850 1800 1750 1700 1650 1600 -1 Wavenumber (cm )

Figure 6.9. TRIR difference spectra averaged over the time scales indicated following 266 nm laser photolysis of sulfilimine 7 (3 mM) in argon-saturated acetonitrile. The black bar reflects the B3LYP/6-31G(d) calculated frequency of triplet t-butyloxycarbonylnitrene (4t).

160

40 (a) triplet nitrene 4t 20 -1 1640 cm 6 -1 0 kobs = 2.3 x 10 s -20

Intensity

 -40 -6 -60x10

0 1 2 3µs Time

-6 800x10 (b)

600 oxazolidinone 5 -1 1762 cm 400 7 -1 kobs1 = 2 x 10 s 200 k = 2.6 x 106 s-1 Intensity obs2 Fast : Slow 9 : 1  0

0 1 2 3µs Time

-6 200x10 (c)

100

0 isocyanate 12 Intensity -1 2180 cm  7 -1 -100 kobs > 2 x 10 s

0 10 20 30 40µs Time

Figure 6.10. Kinetic traces observed following 266 nm laser photolysis of sulfilimine 7 in argon-saturated acetonitrile at (a) 1640 cm-1 from -0.4 to 3.6 μs, (b) 1762 cm-1 from - 0.4 to 3.6 μs, and (c) 2180 cm-1 from -5 to 45 μs. Black curves are the calculated best fit to a single- or double-exponential function.

161

Scheme 6.5. Reactivity observed upon photolysis of 7 in acetonitrile, dichloromethane, and Freon-113. Laser photolysis of sulfilimine 7 in argon-saturated dichloromethane results in the

TRIR difference spectrum shown in Figure 6.11. Again, we observe triplet nitrene 4t and oxazolidinone 5 at 1638 cm-1 and 1752 cm-1, respectively (Figure 6.12). Rapid production of isocyanate 12 is also observed at 2175 cm-1 (Figure 6.12). Interestingly, the ratio of fast component to slow component for growth of oxazolidinone 5 in dichloromethane (1:1.4,

Figure 6.12b) is significantly different from that observed in acetonitrile (9:1, Figure

6.10b). TRIR experiments in Freon-113 reveal analogous signals (Figure 6.13). In this solvent, the ratio of fast to slow component for the growth of oxazolidinone 5 is now 1:1.3

(Figure 6.14b). The greater contribution of the fast component to the observed oxazolidinone growth kinetics in acetonitrile (dielectric constant, ε = 35.9) compared to that in either dichloromethane (ε = 8.9) or Freon-113 (ε = 2.4) suggests a solvent effect on the relative stabilities of the singlet and triplet states of nitrene 4. The singlets for both nitrenes are calculated to have higher dipole moments relative to their corresponding triplet

(Supporting Information). Given that more polar solvents are expected to stabilize the single nitrene due to its zwitterionic character, these results point to a greater contribution from singlet nitrene reactivity in acetonitrile vs. dichloromethane or Freon-113.

162

-3 0.0 - 0.2 µs 3x10 oxazolidinone 5 0.2 - 0.4 µs -1 1752 cm 0.4 - 0.6 µs 0.6 - 0.8 µs 0.8 - 1.2 µs 1.2 - 2.0 µs 2 2.0 - 2.8 µs 2.8 - 3.6 µs triplet nitrene 4t -1 1638 cm 1

Absorbance

 0

-1

1850 1800 1750 1700 1650 1600 1550 -1 Wavenumber (cm )

Figure 6.11. TRIR difference spectra averaged over the time scales indicated following 266 nm laser photolysis of sulfilimine 7 (3 mM) in argon-saturated dichloromethane. The black bar reflects the B3LYP/6-31G(d) calculated frequency of triplet t- butyloxycarbonylnitrene (4t).

163

200 (a) 100 triplet nitrene 4t -1 1638 cm 0 6 -1 kobs = 1.8 x 10 s -100

Intensity

 -200 -6 -300x10

0 1 2 3µs Time

-3 2.0x10 (b) 1.5 oxazolidinone 5 -1 1752 cm 1.0 7 -1 kobs1 = 2 x 10 s 6 -1

Intensity Intensity 0.5 kobs2 = 1.9 x 10 s  Fast : Slow = 1 : 1.4 0.0

0 1 2 3µs Time

-6 200x10 (c)

100

0 isocyanate 12 -1 2175 cm

Intensity 7 -1 k > 2 x 10 s  -100 obs

0 10 20 30 40µs Time Figure 6.12. Kinetic traces observed following 266 nm laser photolysis of sulfilimine 7 in argon-saturated dichloromethane at (a) 1638 cm-1 from -0.4 to 3.6 μs, (b) 1752 cm-1 from -0.4 to 3.6 μs, and (c) 2175 cm-1 from -5 to 45 μs. Black curves are the calculated best fit to a single- or double-exponential function.

164

-3 3x10 0.0 - 0.2 µs oxazolidinone 5 0.2 - 0.4 µs -1 1764 cm 0.4 - 0.6 µs 0.6 - 0.8 µs 0.8 - 1.2 µs 2 1.2 - 2.0 µs 2.0 - 2.8 µs 2.8 - 3.6 µs triplet nitrene 4t -1 1 1640 cm

Absorbance

 0

-1

1850 1800 1750 1700 1650 1600 1550 -1 Wavenumber (cm ) Figure 6.13. TRIR difference spectra averaged over the time scales indicated following 266 nm laser photolysis of sulfilimine 7 (3 mM) in argon-saturated Freon-113. The black bar reflects the B3LYP/6-31G(d) calculated frequency of triplet t-butyloxycarbonylnitrene (4t).

165

-6 200x10 (a) triplet nitrene 4t -1 1640 cm 5 -1 100 kobs = 9.8 x 10 s

Intensity Intensity

 -100

0 1 2 3µs Time

-3 (b) 1.5x10

oxazolidinone 5 1.0 -1 1764 cm 7 -1 kobs1 = 2 x 10 s 0.5 5 -1 kobs2 = 9.6 x 10 s

Intensity Intensity Fast : Slow 1 : 1.3  0.0

0 1 2 3µs Time

-6 150x10 (c) 100

isocyanate 11

Intensity Intensity 0 -1 2180 cm  7 -1 -50 kobs > 2.0 x 10 s

0 10 20 30 40µs Time Figure 6.14. Kinetic traces observed following 266 nm laser photolysis of sulfilimine 7 in argon-saturated Freon-113 at (a) 1640 cm-1 from -0.4 to 3.6 μs, (b) 1764 cm-1 from -0.4 to 3.6 μs, and (c) 2180 cm-1 from -5 to 45 μs. Black curves are the calculated best fit to a single- or double-exponential function.

The observation of a solvent effect on the relative contributions of the singlet and triplets states of nitrene 4 implies a very small ΔEST value (ca. 1 kcal/mol or less), which is consistent with our B3LYP/6-31(d) calculations (Figure 6.3b). This solvent effect is reminiscent of our previous TRIR studies of carbonylcarbenes where singlet carbonylcarbenes were found to be favored in polar solvents, the result of their increased dipole moment.41 The singlet and triplet carbonylcarbenes in these previous studies were

166 in equilibrium, with distinct TRIR bands displaying identical kinetics. In contrast, we find no evidence that singlet 4s and triplet 4t are in equilibrium. Indeed, we do not observe singlet 4s in our TRIR experiments and the observed growth kinetics of product 5 indicate a separate kinetic contribution from the singlet nitrene (fast) and the triplet nitrene (slow).

These observed growth kinetics may be explained by two potential mechanisms: (1)

Separate production of 5 from singlet 4s and triplet 4t (Scheme 6.6a); or (2) production of

5 from only singlet 4s, with the slow component the result of thermal repopulation of 4s from 4t (Scheme 6.6b).

More polar solvents are expected to lower the energy of singlet 4s relative to that of triplet 4t, resulting in the decrease of the EST, and therefore facilitating the potential reactivity of the triplet (Scheme 6.6a). However, increasing solvent polarity results in a decrease in contribution from the triplet nitrene, therefore, the formation of oxazolidinone

5 is likely not due to direct reaction of the triplet. Enhanced reactivity through singlet nitrene 4s could be accomplished in the event triplet nitrene 4t was thermally repopulating

42 the singlet (Scheme 6.6b), which is feasible small ΔEST values. In this case, the observed kinetics of the triplet nitrene would be representative of triplet its repopulation of the singlet. These mechanisms may also similarly account for the observed growth kinetics of ylide 8 (Figure 6.6b,c) and amide 10 (Figure 6.8b).

167

Scheme 6.6. Production of 5 from either (a) direct reaction with singlet and triplet nitrenes 4s and 4t, or (b) solely through the singlet via thermal repopulation.

6.5 Conclusions

N-substituted dibenzothiophene sulifilimine-based precursors 6 and 7 are efficient photoprecursors to ethoxycarbonylnitrene (2) and t-butyloxycarbonylnitrene (4), respectively. Computational analysis indicates that both of these nitrenes are ground state triplets, albeit by small margins. TRIR spectroscopy was used to detect triplet nitrenes 2t and 4t in several solvents. Ethoxycarbonylnitrene reacts with acetonitrile to form ylide 8

168 and with cyclohexane to form amide 10. Conversely, t-butyloxycarbonylnitrene exclusively forms the intramolecular C-H insertion product oxazolidinone 5, independent of solvent. The observed product growth kinetics for ylide 8, amide 10, and oxazolidinone

5 suggest a contribution from both the triplet and singlet nitrene. In addition, solvent effects on the observed growth kinetics for oxazolidinone 5 indicate that single nitrene 4s is stabilized relative to triplet nitrene 4t in polar solvent.

6.6 Experimental Methods

6.6.1 General Methods

Unless otherwise noted, materials were obtained from Aldrich Chemical Company,

Fisher Scientific, or Cambridge Isotope Laboratories and were used without further purification. 1H NMR and 13C NMR spectra were recorded on a Bruker Avance 400 MHz

FT-NMR operating at 400 MHz and 100 MHz, respectively. All resonances are reported

1 in parts per million, and are referenced to residual CHCl3 (7.26 ppm, for H, 77.23 ppm for

13C). High-resolution mass spectra were obtained on a VG Analytical VG-70S Magnetic

(IR) absorption spectra were obtained using a Bruker IFS 55 Fourier transform infrared spectrometer.

169

6.6.2 Procedure for the Synthesis of N-Ethoxycarbonyl Dibenzothiophene

Sulfilimine (6)

This compound was prepared by two methods. Method A is analogous to that reported by Nakayama et al.43 To a solution of trifluroacetic anhydride (0.735g, 3.5 mmol) in dichloromethane at -78 °C was added dibenzothiophene S-oxide (0.350g, 1.75 mmol) slowly in dichloromethane, and stirred for 1h. Solid urethane (0.405g, 4.55 mmol) was added and stirred for an additional 2h at -78 °C, at which point the solution was allowed to warm slowly to room temperature and quenched with ice-water. The solution was then washed with aqueous sodium bicarbonate, dried, filtered, and then evaporated to give the crude product. The crude product was purified by column chromatography with 90:10 dichloromethane to ethyl acetate to give 0.17 g of 6 as an off-white solid in 37% yield.

Method B began with the synthesis of N-p-tosyldibenzothiophene sulfilimine following a literature procedure.44 N-p-tosyldibenzothiophene sulfilimine (1.0 g, 2.8 mM) was then dissolved in 1 mL of concentrated sulfuric acid (95%) at room temperature for approximately 2 hours, and the resulting solution was poured into 100 mL of cold diethyl ether. After removal of solvent, the oil mixture was dissolved in 100 mL chloroform, washed with ammonium hydroxide (2x), followed by water (5x), dried with sodium sulfate, and the solvent was removed. The resulting white solid was then dissolved in 50 mL benzene, to which diethyl pyrocarbonate (0.45 g, 2.8 mmol) was added. The solution was allowed to stir for one hour and the benzene was then evaporated, and the residue was dissolved in 50 mL dichloromethane, washed with water (5x), dried with sodium sulfate, and the solvent was removed. The residue was chromatographed on silica gel using 10%

1 ethyl acetate/hexane as an eluent to give 0.38 g (50%) of 6. H-NMR (CDCl3) δ 8.02 (d, J

170

= 7.8 Hz, 2H), 7.90 (d, J = 7.8 Hz, 2H), 7.65 (t, J = 7.7 Hz, 2H), 7.53 (t, J = 7.7 Hz, 2H),

13 4.10 (q, J = 7.1 Hz, 2H), 1.20 (t, J = 7.1 Hz, 3 H); C-NMR (CDCl3) δ 165.76, 138.79,

138.14, 132.48, 129.88, 127.61, 122.30, 62.14, 14.72; HR-MS (FAB): m/z found =

+ + 272.07309 (MH ); calc. for C15H14NO2S: 272.07453 (MH ).

6.6.3 Procedure for the Synthesis of N-t-Butyloxycarbonyl Dibenzothiophene

Sulfilimine (7)

The compound was prepared following procedures similar to those used for the synthesis of 6, where either di-t-butyldicarbonate or t-butyl carbamate was used instead of diethyl pyrocarbonate or urethane, respectively. Chromatographic purification resulted in

1 a white solid, 50% yield. H-NMR (CDCl3) δ 8.03 (d, J = 7.9 Hz, 2H), 7.63 (d, J = 7.8 Hz,

13 2H), 7.51 (t, J = 7.8 Hz, 2H), 1.43 (s, 9 H). C-NMR (CDCl3) δ 165.39, 139.22, 138.07,

132.37, 129.83, 127.80, 122.26, 79.64, 28.42. HR-MS (FAB): m/z found = 300.10546

+ + (MH ); calc. for C17H18NO2S: 300.10583 (MH ).

6.6.4 Steady-State Photolysis of Oxycarbonylnitrene Precursors 6 and 7

Photolysis of 12 mM solutions of precursors 6 and 7 in deuterated solvent

(dichloromethane or acetonitrile) was performed in a Rayonet reactor (254 nm). After 4 h of photolysis, samples were analyzed by 1H NMR spectroscopy.

6.6.5 Time-Resolved IR Methods

TRIR experiments were conducted (with 16 cm-1 spectral resolution) following the method of Hamaguchi and co-workers,45,46 as has been described previously.47 Briefly, the

171 broadband output of a MoSi2 IR source (JASCO) is crossed with excitation pulses from a

Continuum Minilite II Nd:YAG laser (266 nm, 5 ns, 2 mJ) operating at 15 Hz. Changes in

IR intensity are monitored using an AC-coupled mercury/cadmium/tellurium (MCT) photovoltaic IR detector (Kolmar Technologies, KMPV11-J1/AC), amplified, digitized with a Tektronix TDS520A oscilloscope, and collected for data processing. The experiment is conducted in dispersive mode with a JASCO TRIR 1000 spectrometer.

6.6.6 Computational Methods

Calculations were performed with Spartan ’14.48 Geometries were fully optimized at the B3LYP level of theory with the 6-31G(d) basis set. Vibrational frequencies were also calculated to verify minimum energy structures (no imaginary frequencies) or transition states (one imaginary frequency) and to provide zero-point vibrational energy corrections.

172

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6.8 Supporting Information Chapter 5

6.8.1 Optimized Geometries and Energies

Table S6.1. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for the syn-singlet Ethoxycarbonylnitrene 2ssyn (Dipole moment = 4.55 debye)

B3LYP/6-31G* energy -322.402226 Hartrees Zero-point correction 0.082710276 Hartrees Thermal correction to energy 0.089593919 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z O 8 0.1849282 -0.5839627 0.0000000 O 8 -1.5846722 0.9877074 0.0000000 N 7 -2.2155665 -0.7373761 0.0000000 C 6 2.5444169 -0.1759165 0.0000000 C 6 1.1834965 0.4882771 0.0000000 C 6 -1.0634316 -0.1905919 0.0000000 H 1 1.0243431 1.1051511 -0.8891371 H 1 1.0243431 1.1051511 0.8891371 H 1 3.3244967 0.5926194 0.0000000 H 1 2.6734220 -0.8009292 -0.8887391 H 1 2.6734220 -0.8009292 0.8887391

180

Table S6.2. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities for syn-singlet Ethoxycarbonylnitrene 2ssyn.

Frequency Intensity Frequency Intensity 34 1.63 1311 0.53 118 0.21 1411 77.13 181 2.35 1413 25.43 250 0.23 1452 34.43 353 15.40 1514 6.70 448 10.67 1528 3.45 581 0.24 1546 8.25 627 29.38 1826 369.21 825 0.76 3069 9.78 854 7.11 3086 11.13 1020 5.08 3131 1.83 1100 200.99 3142 16.78 1141 3.83 3155 30.15 1188 4.55

181

Table S6.3. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for syn-triplet Ethoxycarbonylnitrene 2tsyn. (Dipole moment = 3.26 debye)

UB3LYP/6-31G* energy -322.4225538 Hartrees Zero-point correction 0.082232539 Hartrees Thermal correction to energy 0.089074246 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z O 8 0.1059323 -0.5089712 -0.0081129 O 8 -1.4622653 1.1535638 0.0069326 N 7 -2.1171029 -1.0235922 0.0016209 C 6 2.4870834 -0.2676857 0.0098588 C 6 1.1702556 0.4820998 -0.0115201 C 6 -1.1410471 -0.0259254 0.0008665 H 1 2.5818281 -0.9174423 -0.8655912 H 1 2.5717111 -0.8839294 0.9102334 H 1 3.3163549 0.4477312 0.0013852 H 1 1.0590366 1.1031920 -0.9062782 H 1 1.0437024 1.1279209 0.8631153

182

Table S6.4. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities for syn-triplet Ethoxycarbonylnitrene 2tsyn.

Frequency Intensity Frequency Intensity 58 0.88 1280 326.72 150 0.35 1305 0.80 193 0.96 1413 4.18 267 0.18 1453 14.03 361 16.10 1517 6.21 430 4.28 1525 1.82 630 5.79 1541 7.48 682 36.00 1672 201.41 820 0.42 3066 12.37 870 7.15 3076 11.14 989 6.95 3118 5.55 1062 86.94 3138 19.06 1146 5.22 3148 30.89 1188 4.46

183

Table S6.5. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for the anti-singlet Ethoxycarbonylnitrene 2santi. (Dipole moment = 4.75 debye)

UB3LYP/6-31G* energy -322.402280 Hartrees Zero-point correction 0.082765276 Hartrees Thermal correction to energy 0.089640424 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z O 8 0.1845572 -0.5205227 0.0055243 O 8 -2.1431315 -0.7176314 -0.0023593 N 7 -1.6730702 1.0481120 -0.0027249 C 6 2.5622626 -0.2057985 -0.0045926 C 6 1.2265364 0.5065796 0.0037296 C 6 -1.0399068 -0.0594773 0.0011224 H 1 1.0868542 1.1315926 -0.8844323 H 1 1.0948411 1.1262961 0.8968483 H 1 3.3705360 0.5328990 -0.0038490 H 1 2.6640873 -0.8305381 -0.8969632 H 1 2.6704131 -0.8396231 0.8805927

184

Table S6.6. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities for anti-singlet Ethoxycarbonylnitrene 2santi.

Frequency Intensity Frequency Intensity 41 1.63 1312 0.95 129 0.04 1403 83.40 169 2.75 1434 22.57 257 0.13 1458 23.55 348 11.64 1515 6.90 472 7.52 1528 3.60 562 0.84 1546 9.41 628 28.75 1817 394.75 828 0.97 3070 8.61 865 13.79 3073 12.59 1022 12.70 3118 8.25 1101 156.69 3143 15.43 1149 13.03 3152 25.03 1190 4.64

185

Table S6.7. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for anti-triplet Ethoxycarbonylnitrene 2tanti.. (Dipole moment = 3.77 debye)

UB3LYP/6-31G* energy -322.4162641 Hartrees Zero-point correction 0.081918123 Hartrees Thermal correction to energy 0.088766571 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z O 8 0.1178635 -0.5002564 -0.0053311 O 8 -2.0998488 -0.8458674 0.0017670 N 7 -1.4251950 1.2888565 0.0033517 C 6 2.4956771 -0.2715079 0.0050174 C 6 1.1843073 0.4891813 -0.0050299 C 6 -1.1504205 -0.0711407 0.0000248 H 1 2.5786505 -0.9124753 -0.8779128 H 1 2.5731221 -0.8990422 0.8980353 H 1 3.3325569 0.4348759 0.0023927 H 1 1.0891711 1.1175667 -0.8972161 H 1 1.0813633 1.1268736 0.8796785

186

Table S6.8. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities for anti-triplet Ethoxycarbonylnitrene 2tanti.

Frequency Intensity Frequency Intensity 51 0.39 1279 345.68 129 0.45 1314 0.61 197 0.24 1412 22.05 268 0.42 1450 29.49 353 8.63 1516 6.31 508 10.92 1526 4.70 551 11.17 1542 9.09 656 39.12 1634 216.30 826 0.77 3063 18.54 842 14.32 3067 10.82 984 2.82 3107 12.98 1069 56.67 3140 19.04 1139 41.41 3149 26.61 1186 4.55

187

Table S6.9. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for syn-singlet t-butoxycarbonylnitrene 4ssyn. . (Dipole moment = 4.80 debye)

UB3LYP/6-31G* energy -401.037929 Hartrees Zero-point correction 0.138609656 Hartrees Thermal correction to energy 0.147923845 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z O 8 -0.2371798 -0.8614080 0.0000000 O 8 -1.8493200 0.9000120 0.0000000 N 7 -2.6312507 -0.7387603 0.0000000 C 6 0.9832474 0.0100739 0.0000000 C 6 0.9882699 0.8533212 1.2755995 C 6 0.9882699 0.8533212 -1.2755995 C 6 2.1150022 -1.0142107 0.0000000 C 6 -1.4245733 -0.3229676 0.0000000 H 1 0.9284562 0.2104678 2.1599027 H 1 0.1527961 1.5573965 1.2967731 H 1 1.9220404 1.4233166 1.3307840 H 1 0.1527961 1.5573965 -1.2967731 H 1 0.9284562 0.2104678 -2.1599027 H 1 1.9220404 1.4233166 -1.3307840 H 1 2.0616005 -1.6502424 0.8886284 H 1 3.0796701 -0.4966156 0.0000000 H 1 2.0616005 -1.6502424 -0.8886284

188

Table S6.10. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities for syn-singlet t-butoxycarbonylnitrene 4ssyn.

Frequency Intensity Frequency Intensity 73 0.05 1287 17.48 104 0.44 1297 12.34 171 3.03 1393 78.13 212 0.06 1428 14.66 262 0.00 1432 16.61 275 0.13 1458 12.39 308 4.07 1501 0.36 343 0.45 1517 0.27 361 6.62 1521 0.02 418 3.65 1526 2.58 451 1.27 1530 4.90 459 15.63 1553 5.62 582 1.36 1822 397.13 630 30.38 3062 6.46 745 7.84 3062 13.57 833 18.99 3070 8.99 937 0.07 3131 5.02 941 0.21 3132 7.39 986 0.07 3142 32.89 1061 29.32 3144 19.63 1067 0.60 3152 4.22 1094 133.36 3155 16.50 1211 92.92

189

Table S6.11. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for syn-triplet t-butoxycarbonylnitrene 4tsyn. (Dipole moment = 3.50 debye)

UB3LYP/6-31G* energy -401.055453 Hartrees Zero-point correction 0.138014188 Hartrees Thermal correction to energy 0.147310667 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z O 8 -0.3428895 -0.7469401 0.0000000 O 8 -1.7121645 1.1060193 0.0000000 N 7 -2.5990977 -0.9772631 0.0000000 C 6 1.9708044 -1.1637659 0.0000000 C 6 0.9556666 -0.0210388 0.0000000 C 6 1.0666436 0.8209539 -1.2738815 C 6 1.0666436 0.8209539 1.2738815 C 6 -1.5081790 -0.1007254 0.0000000 H 1 2.9873225 -0.7574348 0.0000000 H 1 1.8463332 -1.7910808 -0.8881016 H 1 1.8463332 -1.7910808 0.8881016 H 1 2.0685093 1.2610015 -1.3294038 H 1 0.3327918 1.6291523 -1.2841589 H 1 0.9210249 0.1946154 -2.1603947 H 1 2.0685093 1.2610015 1.3294038 H 1 0.9210249 0.1946154 2.1603947 H 1 0.3327918 1.6291523 1.2841589

190

Table S6.12. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities for syn-triplet t-butoxycarbonylnitrene 4tsyn.

Frequency Intensity Frequency Intensity 84 0.28 1281 16.02 105 0.10 1284 163.88 184 1.53 1300 42.34 185 0.08 1433 16.37 234 0.01 1435 14.38 268 0.09 1459 9.33 314 1.60 1501 0.83 330 8.80 1517 0.13 351 0.52 1519 1.43 427 0.46 1526 12.11 456 0.76 1532 1.50 459 13.90 1552 8.36 623 1.36 1672 215.45 681 38.13 3062 18.09 758 9.72 3062 5.73 839 18.14 3070 14.22 935 0.13 3125 7.00 938 0.42 3127 12.28 988 0.14 3142 33.35 1000 41.86 3143 24.14 1069 0.50 3165 1.30 1071 4.86 3169 13.09 1207 210.37

191

Table S6.13. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for anti-singlet t-butoxycarbonylnitrene 4santi. (Dipole moment = 5.03 debye)

UB3LYP/6-31G* energy -401.038454 Hartrees Zero-point correction 0.138535308 Hartrees Thermal correction to energy 0.147842908 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z O 8 -0.2534488 -0.8054490 0.0038519 O 8 -2.5802651 -0.7059322 0.0002841 N 7 -1.9175779 0.9822184 -0.0001882 C 6 1.0025075 0.0070112 -0.0005382 C 6 1.0396760 0.8597119 1.2683846 C 6 1.0394668 0.8454868 -1.2788467 C 6 2.0921441 -1.0613689 0.0062179 C 6 -1.4007152 -0.1848331 0.0016673 H 1 0.9596714 0.2278842 2.1587200 H 1 0.2285068 1.5942245 1.2904215 H 1 1.9900416 1.4018821 1.3162592 H 1 0.2272309 1.5783537 -1.3074743 H 1 0.9595943 0.2035490 -2.1619311 H 1 1.9892788 1.3879378 -1.3328696 H 1 2.0091345 -1.6912604 0.8970262 H 1 3.0775265 -0.5847827 0.0065926 H 1 2.0132968 -1.6983150 -0.8798245

192

Table S6.14. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities for anti-singlet t-butoxycarbonylnitrene 4santi.

Frequency Intensity Frequency Intensity 50 0.05 1285 16.66 112 0.44 1298 23.26 168 3.10 1405 75.01 201 0.06 1432 17.96 255 0.03 1438 16.05 268 0.16 1461 8.01 307 3.94 1501 0.63 345 0.14 1516 0.02 359 3.08 1516 0.32 424 3.73 1525 1.77 453 0.65 1529 9.07 469 7.03 1549 5.39 574 3.36 1810 379.44 639 29.67 3060 10.05 754 7.75 3062 7.52 842 29.00 3069 5.46 936 0.07 3132 0.93 944 0.24 3134 7.01 988 0.00 3138 5.00 1056 24.75 3141 23.29 1068 1.01 3145 31.56 1095 93.59 3146 24.24 1213 90.75

193

Table S6.15. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for anti-triplet t-butoxycarbonylnitrene 4tanti. (Dipole moment = 4.16 debye)

UB3LYP/6-31G* energy -401.049860 Hartrees Zero-point correction 0.137551343 Hartrees Thermal correction to energy 0.146845346 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z O 8 -0.3325283 -0.7490762 0.0002323 O 8 -2.5680307 -0.7957221 -0.0000714 N 7 -1.6505805 1.2338505 0.0001032 C 6 1.9758952 -1.1619497 -0.0011845 C 6 0.9574946 -0.0205840 0.0000005 C 6 1.0759895 0.8211867 -1.2738821 C 6 1.0770784 0.8195362 1.2748557 C 6 -1.5260984 -0.1470822 0.0000536 H 1 1.8492832 -1.7886676 -0.8891079 H 1 1.8498704 -1.7896301 0.8861259 H 1 2.9926207 -0.7561986 -0.0013079 H 1 2.0814419 1.2512416 -1.3384585 H 1 0.3571225 1.6465716 -1.2833189 H 1 0.9117293 0.1999088 -2.1603208 H 1 2.0817384 1.2516488 1.3377632 H 1 0.9160914 0.1965777 2.1607175 H 1 0.3564817 1.6433390 1.2868393

194

Table S6.16. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities for anti-triplet t-butoxycarbonylnitrene 4tanti.

Frequency Intensity Frequency Intensity 76 0.05 1268 60.80 92 0.02 1281 16.32 186 0.15 1299 120.53 186 0.06 1436 16.16 233 0.04 1436 11.68 269 0.00 1462 11.01 311 2.79 1502 0.80 351 0.53 1510 0.03 360 2.21 1515 0.41 402 3.48 1529 7.12 457 2.31 1530 1.42 515 15.99 1545 5.22 562 5.87 1616 240.38 659 37.40 3057 11.21 757 8.46 3060 7.44 837 15.30 3069 7.28 933 0.10 3127 0.25 936 1.03 3130 17.56 984 0.00 3134 7.09 1006 40.34 3136 29.06 1064 0.17 3144 33.78 1067 1.18 3146 24.63 1202 228.38

195

Table S6.17. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for 1 the syn-singlet hydroxycarbonylnitrene HOC(O)Nsyn. (Dipole moment = 3.46 debye)

B3LYP/6-31G* energy -243.773552 Hartrees Zero-point correction 0.025526570 Hartrees Thermal correction to energy 0.030307679 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z O 8 1.3445373 -0.1534703 0.0000000 O 8 -0.7592090 0.9264737 0.0000000 N 7 -0.9357988 -0.9174864 0.0000000 C 6 0.0284869 -0.0862276 0.0000000 H 1 1.6970436 0.7557431 0.0000000

Table S6.18. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities 1 for syn-singlet hydroxycarbonylnitrene HOC(O)Nsyn.

Frequency Intensity 422 11.39 433 137.39 490 24.40 622 33.58 1027 47.30 1181 94.13 1485 142.75 1845 227.83 3699 100.97

196

Table S6.19. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for 3 the syn-triplet hydroxycarbonylnitrene HOC(O)Nsyn. (Dipole moment = 2.29 debye)

B3LYP/6-31G* energy -243.794368 Hartrees Zero-point correction 0.025213944 Hartrees Thermal correction to energy 0.029846852 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z O 8 1.2043522 -0.3416958 0.0000000 O 8 -0.4979783 1.1764682 0.0000000 N 7 -0.9798527 -1.0446951 0.0000000 C 6 -0.0882084 0.0263958 0.0000000 H 1 1.7372279 0.4763113 0.0000000

Table S6.20. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities 3 for syn-triplet hydroxycarbonylnitrene HOC(O)Nsyn.

Frequency Intensity 428 3.56 534 54.35 562 44.28 701 104.83 923 1.37 1169 257.92 1394 29.57 1678 208.65 3679 76.53

197

Table S6.21. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for 1 the anti-singlet hydroxycarbonylnitrene HOC(O)Nanti. (Dipole moment = 3.76 debye)

B3LYP/6-31G* energy -243.772572 Hartrees Zero-point correction 0.025567362 Hartrees Thermal correction to energy 0.030325771 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z O 8 1.3422017 0.1459845 0.0000000 O 8 -0.8792331 0.8454853 0.0000000 N 7 -0.8183908 -0.9907199 0.0000000 C 6 0.0375852 -0.0470615 0.0000000 H 1 1.7994753 -0.7143500 0.0000000

Table S6.22. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities 1 for anti-singlet hydroxycarbonylnitrene HOC(O)Nanti.

Frequency Intensity 434 120.71 438 21.36 498 7.83 625 42.12 1022 34.80 1179 138.76 1501 39.67 1818 287.42 3706 90.39

198

Table S6.23. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for 3 the anti-triplet hydroxycarbonylnitrene HOC(O)Nanti. (Dipole moment = 2.91 debye)

B3LYP/6-31G* energy -243.790114 Hartrees Zero-point correction 0.024870581 Hartrees Thermal correction to energy 0.029566144 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z O 8 1.1850682 0.4237265 0.0000000 O 8 -1.0366096 0.7940118 0.0000000 N 7 -0.3378040 -1.3540614 0.0000000 C 6 -0.0995243 0.0148493 0.0000000 H 1 1.7741055 -0.3525722 0.0000000

Table S6.24. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities 3 for syn-triplet hydroxycarbonylnitrene HOC(O)Nsyn.

Frequency Intensity 410 25.08 492 154.17 571 5.96 658 17.53 921 23.38 1160 1.02 1335 358.49 1674 111.34 3697 70.58

199

Table S6.25. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for 1 the singlet formylnitrene HC(O)N. (Dipole moment = 3.06 debye)

B3LYP/6-31G* energy -168.538743 Hartrees Zero-point correction 0.020070961 Hartrees Thermal correction to energy 0.024151556 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z O 8 0.8817724 0.2823347 0.0000000 N 7 -0.9003836 0.4049873 0.0000000 C 6 -0.1052069 -0.5721507 0.0000000 H 1 -0.1202531 -1.6606852 0.0000000

Table S6.26. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities for singlet formylnitrene 1HC(O)N.

Frequency Intensity 507 11.21 936 2.08 1080 42.63 1350 14.90 1719 13.82 3217 5.01

200

Table S6.27. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for 3 the triplet formylnitrene HC(O)N. (Dipole moment = 1.81 debye)

B3LYP/6-31G* energy -168.551707 Hartrees Zero-point correction 0.018805068 Hartrees Thermal correction to energy 0.022925351 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z O 8 1.0607928 0.2492620 0.0000000 N 7 -1.2046797 0.2580159 0.0000000 C 6 -0.0029283 -0.3848547 0.0000000 H 1 -0.0360151 -1.4910790 0.0000000

Table S6.28. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities for triplet formylnitrene 3HC(O)N.

Frequency Intensity 474 34.46 900 5.48 1034 9.38 1380 2.84 1474 33.12 2992 49.76

201

Table S6.29. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for 1 the singlet Ethoxycarbonylnitrene rotation transition state 2TS.

B3LYP/6-31G* energy -322.391856 Hartrees Zero-point correction 0.082009077 Hartrees Thermal correction to energy 0.088946423 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z O 8 -0.2022552 0.0254922 0.5407753 O 8 1.8880580 0.8949285 -0.1414016 N 7 1.9374485 -0.9439754 -0.0370750 C 6 -2.5630977 0.0670042 0.1159209 C 6 -1.2027368 -0.1222912 -0.5194385 C 6 1.0604425 -0.0465543 0.1541921 H 1 -1.0015728 0.6325078 -1.2870332 H 1 -1.0903275 -1.1182631 -0.9604677 H 1 -3.3401540 -0.0355668 -0.6490800 H 1 -2.6461325 1.0610433 0.5651179 H 1 -2.7380235 -0.6842104 0.8919511

202

Table S6.30. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities 1 for singlet Ethoxycarbonylnitrene rotation transition state 2TS.

Frequency Intensity Frequency Intensity -127 1.15 1307 2.12 29 0.72 1375 115.17 172 4.58 1415 13.59 262 0.14 1447 29.74 335 14.39 1512 7.16 438 4.46 1529 2.99 507 6.72 1545 6.49 687 4.42 1804 313.78 824 1.28 3067 20.77 845 24.93 3067 8.45 1014 6.81 3114 10.16 1087 237.53 3140 17.18 1141 7.51 3151 24.79 1183 4.42

203

Table S6.31. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for 3 the triplet Ethoxycarbonylnitrene rotation transition state 2TS.

B3LYP/6-31G* energy -322.406497 Hartrees Zero-point correction 0.081190566 Hartrees Thermal correction to energy 0.088100374 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z O 8 -0.1365246 -0.0311651 0.5132095 O 8 1.8242503 -1.0294335 -0.0860061 N 7 1.7171471 1.2320259 -0.0495643 C 6 -2.5041736 -0.0228614 0.1464102 C 6 -1.1481864 0.0205148 -0.5286150 C 6 1.1585924 -0.0213163 0.0985252 H 1 -2.6340254 0.8375033 0.8099689 H 1 -2.6103459 -0.9382758 0.7361532 H 1 -3.2949606 -0.0012761 -0.6112882 H 1 -1.0184863 0.9428507 -1.1085146 H 1 -1.0014113 -0.8382181 -1.1949186

204

Table S6.32. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities 3 for triplet Ethoxycarbonylnitrene rotation transition state 2TS.

Frequency Intensity Frequency Intensity -122 2.47 1200 309.77 63 0.26 1312 1.25 187 2.60 1417 19.62 246 0.28 1450 32.76 335 8.94 1512 6.43 436 1.65 1530 2.42 579 15.80 1548 8.55 693 13.28 1610 143.90 832 2.56 3046 26.63 839 20.77 3067 11.01 975 18.16 3092 18.84 1061 116.29 3139 19.88 1138 26.15 3149 23.25 1185 8.75

205

Table S6.33. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for 1 the singlet t-butoxycarbonylnitrene rotation transition state 4TS.

B3LYP/6-31G* energy -401.029715 Hartrees Zero-point correction 0.137859437 Hartrees Thermal correction to energy 0.147244089 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z O 8 0.2061566 -0.0025986 0.8602736 O 8 2.2221037 0.8748164 -0.0167456 N 7 2.2390164 -0.9566874 -0.0362335 C 6 -0.9927710 0.0012238 -0.0429245 C 6 -1.0650218 -1.3538514 -0.7452097 C 6 -0.8640247 1.1630065 -1.0277721 C 6 -2.1470461 0.2030776 0.9324694 C 6 1.4029820 -0.0677014 0.3162345 H 1 -1.1397269 -2.1647983 -0.0139120 H 1 -0.1810307 -1.5255437 -1.3677193 H 1 -1.9479560 -1.3887423 -1.3925644 H 1 -0.0127745 1.0295064 -1.7028397 H 1 -0.7363814 2.1096966 -0.4943243 H 1 -1.7721562 1.2275101 -1.6368100 H 1 -2.1738032 -0.6063582 1.6679653 H 1 -3.0957684 0.2087446 0.3860131 H 1 -2.0443103 1.1545234 1.4628163

206

Table S6.34. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities 1 for singlet t-butoxycarbonylnitrene rotation transition state 4TS.

Frequency Intensity Frequency Intensity -74 0.09 1284 13.95 52 0.08 1299 23.24 158 5.00 1363 87.86 206 0.04 1435 14.92 260 0.04 1437 13.95 270 0.08 1461 9.23 301 6.39 1503 0.28 322 0.58 1509 0.41 376 6.02 1513 0.08 422 3.09 1528 2.71 427 3.88 1532 6.24 463 0.47 1542 6.67 528 10.14 1799 311.22 637 7.85 3059 9.21 767 16.15 3062 8.61 836 47.61 3069 6.62 933 0.03 3130 0.32 939 0.58 3134 7.89 985 0.01 3139 10.30 1049 103.11 3141 22.88 1066 0.85 3145 25.02 1078 69.95 3147 24.16 1207 96.44

207

Table S6.35. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for 3 the triplet t-butoxycarbonylnitrene rotation transition state 4TS.

B3LYP/6-31G* energy -401.042161 Hartrees Zero-point correction 0.136692230 Hartrees Thermal correction to energy 0.146075625 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z O 8 0.2779540 -0.0905718 0.7973229 O 8 2.2010301 -1.0026846 -0.0118119 N 7 1.9970753 1.2450350 -0.0052340 C 6 -0.9517879 -1.1819694 -1.0150273 C 6 -0.9493680 -0.0093622 -0.0316406 C 6 -2.0692200 -0.1382267 0.9988137 C 6 -1.0026430 1.3419064 -0.7485921 C 6 1.5037401 -0.0248348 0.2267034 H 1 -1.8911628 -1.1916496 -1.5788074 H 1 -0.8536890 -2.1317352 -0.4816151 H 1 -0.1289065 -1.1064467 -1.7334256 H 1 -3.0434602 -0.1056514 0.4998173 H 1 -2.0221535 0.6803986 1.7237292 H 1 -1.9857141 -1.0855953 1.5400783 H 1 -1.9497468 1.4354780 -1.2911735 H 1 -0.1906521 1.4464267 -1.4766552 H 1 -0.9302420 2.1645007 -0.0309412

208

Table S6.36. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities 3 for triplet t-butoxycarbonylnitrene rotation transition state 4TS.

Frequency Intensity Frequency Intensity -66 1.27 1234 26.58 37 0.29 1278 16.22 165 1.86 1295 20.05 198 0.04 1424 15.80 254 0.01 1431 13.09 264 0.06 1455 7.99 296 2.74 1500 0.21 318 1.58 1507 0.47 380 2.45 1513 0.07 404 0.56 1523 3.93 440 5.70 1528 4.50 476 0.99 1542 4.76 570 16.58 1601 165.48 667 7.62 3056 8.38 756 23.09 3059 15.03 834 30.34 3066 4.75 927 0.12 3122 4.86 935 3.02 3128 28.68 978 2.28 3135 18.22 983 87.39 3140 3.48 1055 1.25 3142 27.47 1066 2.01 3146 27.15 1176 346.59

209

Table S6. 37. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for the syn-ethoxycarbonylnitrene-acetonitrile ylide 9.

B3LYP/6-31G* energy -455.230639 Hartrees Zero-point correction 0.133213464 Hartrees Thermal correction to energy 0.142894936 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z C 6 0.3523481 -0.0898696 0.0000000 O 8 1.5868383 0.4585713 0.0000000 C 6 2.6726596 -0.4866861 0.0000000 H 1 2.5890821 -1.1301190 0.8826815 H 1 2.5890821 -1.1301190 -0.8826815 C 6 3.9644716 0.3098778 0.0000000 H 1 4.0289178 0.9483600 0.8870024 H 1 4.0289178 0.9483600 -0.8870024 H 1 4.8230558 -0.3708331 0.0000000 O 8 0.1296893 -1.2899111 0.0000000 N 7 -0.5819369 0.9453118 0.0000000 N 7 -1.7793356 0.4945702 0.0000000 C 6 -2.8975220 0.1811652 0.0000000 C 6 -4.3015387 -0.2053898 0.0000000 H 1 -4.8143333 0.1747462 0.8908718 H 1 -4.3762147 -1.2981820 0.0000000 H 1 -4.8143333 0.1747462 -0.8908718

210

Table S6.38. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities for the syn-ethoxycarbonylnitrene-acetonitrile ylide 9.

Frequency Intensity Frequency Intensity 25 2.04 1193 4.17 31 0.20 1288 1151.09 74 7.45 1301 0.28 78 6.13 1335 600.69 116 0.09 1416 51.98 168 0.36 1444 19.77 188 3.27 1452 7.87 269 1.53 1493 10.56 274 0.41 1502 9.63 363 13.08 1517 4.62 449 2.46 1527 1.71 491 3.86 1547 4.81 491 1.15 1768 255.51 755 24.17 2441 359.24 763 0.56 3050 15.11 818 0.03 3059 20.59 825 16.67 3064 22.19 920 7.11 3101 13.49 1004 11.92 3116 2.69 1060 2.12 3128 2.70 1060 11.19 3130 29.91 1092 96.15 3139 40.28 1150 8.31

211

Table S6. 39. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for the anti-ethoxycarbonylnitrene-acetonitrile ylide 9.

B3LYP/6-31G* energy -455.226370 Hartrees Zero-point correction 0.133374386 Hartrees Thermal correction to energy 0.143041193 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z C 6 -0.3551402 -0.6890410 0.0006956 O 8 -1.7021155 -0.5839471 0.0008247 C 6 -2.3325364 0.7120337 0.0025093 H 1 -2.0172880 1.2714107 0.8898792 H 1 -2.0106817 1.2769062 -0.8788564 C 6 -3.8325710 0.4740399 -0.0030553 H 1 -4.1379832 -0.0963266 0.8799342 H 1 -4.1333520 -0.0861660 -0.8940882 H 1 -4.3641576 1.4323226 0.0010826 O 8 0.1824679 -1.7787926 -0.0001094 N 7 0.2865589 0.5588257 0.0010641 N 7 1.5591100 0.4274010 0.0002814 C 6 2.7186744 0.3819762 -0.0011667 C 6 4.1739302 0.3327050 -0.0007562 H 1 4.5840640 0.8280349 -0.8880230 H 1 4.4993995 -0.7131845 -0.0083984 H 1 4.5833559 0.8150507 0.8939689

212

Table S6.40. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities for the anti-ethoxycarbonylnitrene-acetonitrile ylide 9.

Frequency Intensity Frequency Intensity 49 2.41 1190 3.87 71 0.59 1272 1133.02 81 6.80 1301 0.11 88 2.75 1324 405.94 129 0.00 1407 33.02 169 0.29 1443 15.39 188 0.24 1448 2.67 269 1.53 1494 11.08 286 0.44 1502 9.74 335 0.84 1518 4.55 486 2.55 1529 4.92 497 3.19 1548 0.60 544 14.67 1797 368.23 658 1.82 2445 339.15 745 25.61 3052 12.61 823 0.22 3060 24.42 860 8.19 3062 19.92 895 0.19 3103 15.01 996 9.60 3119 2.31 1060 11.35 3130 2.05 1061 2.29 3131 29.00 1089 57.08 3141 38.72 1149 90.43

213

Table S6.41. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for syn-5-ethoxycarbonyliminodibenzothiophene

B3LYP/6-31G* energy -1182.779443 Hartrees Zero-point correction 0.248654539 Hartrees Thermal correction to energy 0.263385183 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z H 1 0.8073731 -2.6454561 -0.7004214 C 6 -0.2102720 -2.5018047 -0.3537613 C 6 -2.8505430 -2.0460356 0.5547557 C 6 -0.7515278 -1.2241001 -0.2972131 C 6 -1.0126904 -3.5654987 0.0706302 C 6 -2.3199920 -3.3368979 0.5142633 C 6 -2.0577178 -0.9673578 0.1525418 H 1 -0.6162677 -4.5765617 0.0523204 H 1 -2.9331401 -4.1752833 0.8328485 H 1 -3.8683512 -1.8844373 0.8991991 C 6 -2.3889964 0.4637118 0.1332233 C 6 -2.5767061 3.2583145 0.0260297 C 6 -1.3273555 1.2569649 -0.3311952 C 6 -3.5703345 1.0969473 0.5313870 C 6 -3.6556162 2.4886373 0.4747061 C 6 -1.3938895 2.6415070 -0.3901477 H 1 -4.4116543 0.5126600 0.8931832 H 1 -4.5713032 2.9805116 0.7904748 H 1 -0.5434761 3.2233263 -0.7303921 H 1 -2.6554613 4.3410816 0.0018010 S 16 0.0700215 0.2814714 -0.8829485 N 7 1.2777348 0.8356163 0.1121213 C 6 2.4164788 0.1552327 -0.2199942 O 8 2.5099072 -0.8437529 -0.9480833 O 8 3.4889718 0.7115747 0.3938449 C 6 4.7384205 0.0278791 0.1968831 H 1 4.6346302 -1.0155328 0.5147167 H 1 4.9851180 0.0210713 -0.8705651

214

C 6 5.7865480 0.7648968 1.0115466 H 1 5.5211418 0.7694088 2.0737419 H 1 5.8802041 1.8035163 0.6779616 H 1 6.7608302 0.2758866 0.8994338

Table S6.42. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities or syn-5-ethoxycarbonyliminodibenzothiophene 6.

Frequency Intensity Frequency Intensity 35 0.61 1049 1.10 55 0.47 1057 2.21 61 0.07 1074 3.55 89 1.32 1094 4.23 95 1.95 1127 88.14 119 3.92 1148 8.42 132 1.73 1151 6.90 160 5.05 1160 0.87 185 2.37 1196 2.75 199 0.86 1196 1.55 234 10.18 1198 0.64 285 1.44 1259 3.19 286 0.12 1308 56.40 302 11.47 1309 7.78 374 13.23 1321 1268.37 400 0.76 1335 49.70 416 0.58 1350 2.35 421 3.38 1384 0.27 443 5.91 1422 91.81 480 3.44 1452 19.67 497 8.67 1475 14.76 509 21.53 1493 26.92 548 12.67 1511 10.47 569 0.36 1518 4.41 629 5.33 1523 1.29 700 1.10 1528 2.22 711 0.64 1550 4.44 726 11.92 1629 2.66 740 1.87 1637 0.26 759 2.09 1645 0.50 769 88.09 1652 0.32 772 69.16 1675 201.94 777 15.70 3058 23.38 792 3.90 3062 25.99 809 29.08 3099 14.92 824 1.76 3129 31.91 882 0.03 3138 40.39 894 0.09 3190 3.99 911 4.75 3193 0.69 950 0.71 3203 4.93 968 1.41 3205 13.43

215

992 0.06 3214 21.26 1003 0.67 3215 19.72 1018 0.02 3226 8.78 1031 24.90 3237 7.00

Table S6.43. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for anti-5-ethoxycarbonyliminodibenzothiophene 6.

B3LYP/6-31G* energy -1182.776940 Hartrees Zero-point correction 0.248537685 Hartrees Thermal correction to energy 0.263284060 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z H 1 0.2843726 -2.9759812 -0.6885767 C 6 -0.6517286 -2.6058961 -0.2853915 C 6 -3.0608709 -1.5590988 0.7693662 C 6 -0.9171112 -1.2424965 -0.2840770 C 6 -1.6163534 -3.4527635 0.2696223 C 6 -2.8085394 -2.9322823 0.7849056 C 6 -2.1013946 -0.6936168 0.2364920 H 1 -1.4365621 -4.5234690 0.2980344 H 1 -3.5507694 -3.6054427 1.2048126 H 1 -3.9920424 -1.1699972 1.1716519 C 6 -2.1354485 0.7714462 0.1381629 C 6 -1.7686110 3.5335086 -0.1700769 C 6 -0.9752767 1.2996306 -0.4511379 C 6 -3.1304249 1.6569443 0.5637439 C 6 -2.9397018 3.0304297 0.4071306 C 6 -0.7703216 2.6623955 -0.6142062 H 1 -4.0415184 1.2812181 1.0207286 H 1 -3.7110688 3.7179165 0.7424031 H 1 0.1464930 3.0363122 -1.0585823 H 1 -1.6326009 4.6057047 -0.2752986 S 16 0.1485134 0.0236353 -1.0192149 N 7 1.5103730 0.3465965 -0.1163687 C 6 2.4369512 -0.5982204 -0.4700490

216

O 8 2.2390426 -1.5978089 -1.1696569 O 8 3.6777104 -0.3981752 0.0373888 C 6 3.9355996 0.7745118 0.8304317 H 1 3.7139717 1.6712180 0.2406752 H 1 3.2713044 0.7797609 1.7014835 C 6 5.3974728 0.7235619 1.2398866 H 1 6.0472179 0.7167791 0.3587960 H 1 5.6051702 -0.1778701 1.8254138 H 1 5.6477365 1.5990582 1.8498036

Table S6.44. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities for anti-5-ethoxycarbonyliminodibenzothiophene 6.

Frequency Intensity Frequency Intensity 27 0.73 1049 1.79 51 0.99 1057 1.71 60 0.35 1073 4.85 72 4.77 1094 5.49 96 1.27 1116 52.22 127 2.05 1148 73.69 151 0.48 1152 3.95 158 4.89 1160 0.62 184 7.69 1191 3.25 202 1.68 1197 0.43 229 10.07 1198 0.57 267 0.62 1259 4.61 287 0.38 1305 35.23 295 2.99 1308 62.15 364 1.12 1315 999.07 401 0.33 1335 15.75 420 1.77 1350 2.48 434 6.33 1384 0.16 461 9.07 1413 46.35 480 9.32 1445 6.22 497 2.19 1475 13.94 536 15.13 1493 28.25 564 32.55 1510 10.77 569 0.72 1515 4.34 629 4.82 1524 1.37 641 16.70 1527 4.70 701 1.42 1549 1.51 715 2.90 1628 2.74 728 12.07 1637 0.26 750 4.39 1644 0.63 773 74.43 1652 0.77 775 33.02 1692 273.54 778 13.17 3058 27.30 793 3.26 3061 15.84 822 0.18 3098 14.52 835 23.26 3128 27.69 884 0.31 3139 37.65

217

895 0.13 3191 3.48 910 3.88 3194 0.79 952 0.83 3203 5.74 970 1.60 3206 10.31 991 0.06 3215 19.86 1006 1.02 3215 20.18 1018 0.05 3225 11.42 1025 11.60 3242 7.59

Table S6.45. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for syn-5-t-butoxycarbonyliminodibenzothiophene 7.

B3LYP/6-31G* energy -1182.776940 Hartrees Zero-point correction 0.248537685 Hartrees Thermal correction to energy 0.263284060 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z H 1 0.3674791 -2.5810075 -0.7669593 C 6 -0.6513278 -2.4713672 -0.4119692 C 6 -3.2975116 -2.1061672 0.5176281 C 6 -1.2272896 -1.2103149 -0.3303495 C 6 -1.4206305 -3.5646438 -0.0018345 C 6 -2.7309968 -3.3805929 0.4530352 C 6 -2.5379356 -0.9986345 0.1299217 H 1 -0.9958755 -4.5636000 -0.0396646 H 1 -3.3183076 -4.2409720 0.7615302 H 1 -4.3176906 -1.9798094 0.8697259 C 6 -2.9102145 0.4223580 0.1352472 C 6 -3.1816682 3.2110979 0.0691315 C 6 -1.8730117 1.2534521 -0.3181732 C 6 -4.1091129 1.0139644 0.5447033 C 6 -4.2362122 2.4030710 0.5082916 C 6 -1.9814313 2.6361159 -0.3568753 H 1 -4.9321640 0.3997848 0.8989228 H 1 -5.1656997 2.8627089 0.8321959 H 1 -1.1493752 3.2479853 -0.6896967

218

H 1 -3.2932521 4.2912181 0.0598805 S 16 -0.4475838 0.3287219 -0.8863514 N 7 0.7399711 0.8944939 0.1286155 C 6 1.9014410 0.2585879 -0.2187511 O 8 2.0035974 -0.7177516 -0.9807072 O 8 2.9418760 0.8442881 0.4154573 C 6 4.2930924 0.2820122 0.3413747 C 6 5.0916881 1.2237134 1.2480915 H 1 4.6858752 1.2147146 2.2648214 H 1 5.0464011 2.2502590 0.8702271 H 1 6.1413011 0.9125541 1.2874455 C 6 4.8168175 0.3463792 -1.0979730 H 1 5.8664211 0.0303079 -1.1265143 H 1 4.7603960 1.3740957 -1.4738141 H 1 4.2324756 -0.3016398 -1.7527135 C 6 4.3091906 -1.1454899 0.9006117 H 1 5.3415380 -1.5094132 0.9596904 H 1 3.7338930 -1.8200137 0.2647073 H 1 3.8850193 -1.1617229 1.9108648

Table S6.46. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities for syn-5-t-butoxycarbonyliminodibenzothiophene 7.

Frequency Intensity Frequency Intensity Frequency Intensity 35 0.60 761 3.57 1384 2.96 50 0.21 772 75.04 1418 15.37 56 0.43 777 11.06 1423 40.71 79 2.65 785 33.26 1448 30.72 98 1.24 792 4.24 1475 14.47 119 1.74 818 35.69 1493 27.39 136 1.25 881 0.17 1501 0.50 150 1.43 894 0.04 1511 11.43 187 1.59 898 15.86 1521 0.86 190 1.23 935 0.83 1523 3.03 228 0.21 938 0.02 1524 1.02 239 8.65 950 0.85 1525 22.35 258 11.40 968 1.36 1536 0.76 267 3.19 979 0.26 1559 14.52 290 3.82 991 0.06 1629 2.59 304 0.11 1003 0.63 1637 0.61 329 14.53 1018 0.02 1645 1.66 348 2.70 1050 1.38 1652 2.36 360 1.12 1057 2.41 1657 166.53 400 0.69 1064 0.29 3054 12.68 419 1.68 1067 0.30 3055 22.41 426 4.56 1073 3.61 3063 37.42 437 8.22 1093 7.83 3116 11.02

219

451 11.49 1099 13.07 3118 22.87 461 2.16 1152 0.97 3133 53.01 480 4.17 1160 0.89 3136 30.94 498 2.95 1196 0.61 3169 5.10 518 12.18 1198 3.61 3173 12.07 547 10.03 1221 618.44 3190 3.81 569 0.38 1259 0.98 3193 0.80 629 5.30 1282 17.19 3202 5.55 699 1.04 1290 113.58 3205 12.33 709 5.93 1308 8.20 3214 22.65 726 7.89 1334 39.13 3214 19.79 738 74.09 1349 564.01 3226 8.83 742 4.85 1351 400.28 3239 6.70

Table S6. 47. B3LYP/6-31G* optimized geometries, energies, and thermal corrections for anti-5-t-butoxycarbonyliminodibenzothiophene 7.

B3LYP/6-31G* energy -1261.406954 Hartrees Zero-point correction 0.304068068 Hartrees Thermal correction to energy 0.321273556 Hartrees

Atom Atomic Cartesian Coordinates (Angstroms) Number X Y Z H 1 -0.4047087 -3.0644583 -0.7825513 C 6 -1.2854578 -2.6187402 -0.3340611 C 6 -3.5451799 -1.3805848 0.8363096 C 6 -1.4370463 -1.2379560 -0.3233485 C 6 -2.2876449 -3.3846115 0.2696428 C 6 -3.4061495 -2.7696924 0.8427405 C 6 -2.5456927 -0.5956142 0.2544348 H 1 -2.1949317 -4.4665355 0.2905645 H 1 -4.1789788 -3.3804436 1.3010799 H 1 -4.4202699 -0.9169207 1.2831714 C 6 -2.4682785 0.8673847 0.1510573 C 6 -1.9109962 3.5908501 -0.2009598 C 6 -1.3017319 1.3012237 -0.4996275

220

C 6 -3.3700496 1.8277882 0.6197624 C 6 -3.0850045 3.1819846 0.4412224 C 6 -1.0052010 2.6441701 -0.6867689 H 1 -4.2816071 1.5237996 1.1261456 H 1 -3.7834807 3.9282265 0.8091055 H 1 -0.0891603 2.9470524 -1.1837433 H 1 -1.7004879 4.6490180 -0.3245976 S 16 -0.3113583 -0.0612930 -1.1156498 N 7 1.1150792 0.1581968 -0.2749591 C 6 1.9368053 -0.8660443 -0.6674834 O 8 1.5904126 -1.8321678 -1.3606099 O 8 3.2199537 -0.8327823 -0.2400378 C 6 3.8385215 0.2297984 0.5586220 C 6 5.2805992 -0.2729633 0.6941721 H 1 5.7404463 -0.3855601 -0.2928264 H 1 5.2995138 -1.2457685 1.1956777 H 1 5.8774339 0.4344474 1.2799819 C 6 3.1728848 0.3257458 1.9357531 H 1 3.1847917 -0.6528836 2.4281072 H 1 2.1392862 0.6648503 1.8471585 H 1 3.7263119 1.0316577 2.5661547 C 6 3.8098980 1.5638946 -0.1952539 H 1 4.2401250 1.4426020 -1.1956300 H 1 4.4083562 2.3060732 0.3460077 H 1 2.7889521 1.9379531 -0.2907987

Table S6.48. B3LYP/6-31G* calculated IR frequencies (cm-1, unscaled) and intensities for anti-5-t-butoxycarbonyliminodibenzothiophene 7.

Frequency Intensity Frequency Intensity Frequency Intensity 22 1.37 773 69.44 1384 0.17 41 0.05 776 31.47 1422 12.87 50 0.72 778 15.69 1422 8.85 80 2.05 783 1.76 1451 44.45 99 1.08 793 3.46 1474 14.81 111 1.99 836 30.70 1493 27.92 136 1.85 883 1.91 1497 0.70 149 1.85 891 12.94 1509 11.14 179 7.02 895 0.26 1516 0.98 185 3.14 931 0.12 1518 0.70 210 0.98 932 1.90 1522 17.84 227 10.06 952 1.02 1523 1.99 245 1.02 970 1.81 1532 0.43 266 0.92 976 0.08 1556 7.90 267 0.12 992 0.05 1628 2.79 289 1.16 1006 1.41 1637 0.50

221

345 2.36 1018 0.01 1644 1.86 351 0.69 1049 2.39 1651 0.52 365 4.65 1057 2.02 1671 271.7 401 0.37 1061 0.52 3053 22.30 405 1.41 1063 6.52 3054 9.36 419 2.47 1072 6.07 3063 38.37 435 9.44 1087 32.26 3116 10.03 461 3.09 1094 1.69 3118 15.61 466 18.25 1151 0.94 3132 45.50 479 7.68 1159 1.45 3135 29.27 497 1.44 1196 1.11 3163 1.09 540 9.29 1197 5.07 3166 26.05 562 20.61 1214 460.09 3192 3.22 569 0.72 1259 0.65 3195 0.85 625 35.50 1277 15.03 3204 6.28 629 5.52 1287 2.68 3206 9.24 700 1.32 1307 10.05 3215 19.18 713 2.97 1331 528.45 3215 22.83 727 12.77 1336 241.63 3224 11.54 749 4.95 1350 3.25 3244 7.71

222

6.8.2 Calculated Rotation Barriers

1 1 1 2anti 2syn 2anti

Figure S6.1. Energy profile for the rotation of C-O bond of singlet ethoxycarbonylnitrene 12.

223

3 2anti

3 3 2syn 2syn

Figure S6.2. Energy profile for the rotation of C-O bond of triplet ethoxycarbonylnitrene 32.

224

1 1 4anti 1 4anti 4syn

Figure S6.3. Energy profile for the rotation of C-O bond of singlet t-butoxycarbonylnitrene 14.

225

3 4anti

3 3 4syn 4syn

Figure S6.4. Energy profile for the rotation of C-O bond of triplet t-butoxycarbonylnitrene 34.

Figure S6.5. Kinetic traces observed at 1640 cm–1 following 266 nm laser photolysis of of 6 (3 mM) in argon-saturated acetonitrile (a) without or (b) with triethylsilane (TES) in presence. The dotted curves are experimental data; the solid curves are best fits to a single-exponential function.

226

6.8.3 Compound Characterization: 1H and 13C NMR Spectra of Final

Compounds

N-ethoxycarbonyl dibenzothiophene sulfilimine_6.esp

1.21 1.0

0.9

0.40 N-ETHOXYCARBONYL DIBENZOTHIOPHENE SULFILIMINE_1H.ESP

0.8 0.35

0.30

0.7 0.25 0.20

Normalized Intensity Normalized 0.15

0.6 4.08 0.10

1.19

4.06

0.05 0.5 1.23 8.10 8.05 8.00 7.95 7.90 7.85 7.80 7.75 7.70 7.65 7.60 7.55 7.50 7.45 7.40 7.35 Chemical Shift (ppm)

0.4

Normalized Intensity Normalized

7.61

7.49

7.85

7.97

0.3 7.99

7.63

4.10

7.47 0.2 4.04

7.25

0.1

1.76

1.24

0 2.00 2.032.05 2.02 2.04 3.14

8 7 6 5 4 3 2 1 0 Chemical Shift (ppm) 1 Figure S6.6. H NMR (CDCl3) of oxycarbonylnitrene precursor 6.

227

N-ethoxycarbonyl dibenzothiophene sulfilimine_6_13C.esp

127.62 1.0

0.9

129.88 0.8

0.7

122.30

0.6 132.48

77.15

77.47 0.5

138.14

76.84

14.72

Normalized Intensity Normalized 0.4

138.79

62.14 0.3

0.2 165.77

0.1

220 200 180 160 140 120 100 80 60 40 20 0 -20 Chemical Shift (ppm) 13 Figure S6.7. C NMR (CDCl3) of oxycarbonylnitrene precursor 6.

N-t-butyloxycarbonyl dibenzothiophene sulfilimine_1H.esp

1.42

0.30

0.25

0.20

0.15

Normalized Intensity Normalized

0.10

7.62

7.50

7.49

7.86

8.01

7.25

8.03

7.87 7.51 0.05 8.03

1.64

0 2.00 2.012.05 2.07 9.11

8 7 6 5 4 3 2 1 0 Chemical Shift (ppm) 1 Figure S6.8. H NMR (CDCl3) of oxycarbonylnitrene precursor 7.

228

N-t-butyloxycarbonyl dibenzothiophene sulfilimine_13C.esp

28.42 1.0

0.9

129.83 0.8 122.27

132.37 0.7

76.83

77.46 0.6

0.5

127.80 Normalized Intensity Normalized 0.4

0.3 79.64

139.22

0.2

0.1 165.40

220 200 180 160 140 120 100 80 60 40 20 0 -20 Chemical Shift (ppm) 13 Figure S6.9. C NMR (CDCl3) of oxycarbonylnitrene precursor 7.

N-t-butyloxycarbonyl dibenzothiophene sulfilimine_CD3CN.esp

1.27

0.8

2.14

0.7

0.6

0.5

1.94

0.4

1.95

1.93

Normalized Intensity Normalized 0.3

1.95 0.2 1.93

7.95

8.06

7.72 7.59

7.97 0.1 8.08

7.60

7.70

7.74

0 2.00 2.00 2.09 2.06 9.23

8 7 6 5 4 3 2 1 Chemical Shift (ppm) 1 Figure S6.10. H NMR (CD3CN) of oxycarbonylnitrene precursor 7.

229

N-t-butyloxycarbonyl dibenzothiophene sulfilimine_CD2Cl2.esp

1.33 1.0

0.9

0.8

0.7

0.6

0.5

0.4

Normalized Intensity Normalized

0.3

5.32

0.2

7.94

7.97

7.67

7.55

7.99

7.96

7.57 7.56

0.1 7.66

7.65

1.55

0 4.002.04 2.05 9.17

8 7 6 5 4 3 2 1 0 Chemical Shift (ppm) 1 Figure S6.11. H NMR (CD2Cl2) of oxycarbonylnitrene precursor 7.

230

N-T-BUTYLOXYCARBONYL DIBENZOTHIOPHENE SULFILIMINE_CD3CN_4HR PHOTOLYSIS.ESP

2.17

0.30

0.25

1.39

0.20

0.15

Normalized Intensity Normalized

1.94

0.10

1.93

1.95

3.26

7.52

1.93

1.95

7.50

7.52 0.05 7.51

1.27

8.27 8.26

7.93

8.28

7.94

7.96

8.27

7.95

1.17

0 2.08 1.94 4.00 1.10 1.85 5.80

8 7 6 5 4 3 2 1 Chemical Shift (ppm) 1 Figure S6.12. H NMR (CD3CN) of oxycarbonylnitrene precursor 7 after 4 hour photolysis at 254nm in argon-purged CD3CN.

231

N-t-butyloxycarbonyl dibenzothiophene sulfilimine_CD2Cl2_4hr photolysis.esp

1.45

0.7

0.6

0.5

0.4

0.3

3.31

Normalized Intensity Normalized

7.49

5.32 0.2 7.47

7.48 7.48

8.20

8.18

7.88

8.20 0.1 7.87

7.88

1.57

1.43

7.46

0 2.06 1.86 4.00 0.97 1.95 5.72

8 7 6 5 4 3 2 1 0 Chemical Shift (ppm) 1 Figure S6.13. H NMR (CD2Cl2) of oxycarbonylnitrene precursor 7 after 4 hour photolysis at 254nm in argon-purged CD2Cl2.

232

N-ethoxycarbonyl dibenzothiophene sulfilimine_CD3CN_4hr photolysis.esp

2.17

0.20

0.15

0.10

1.40

Normalized Intensity Normalized

1.94

7.52

7.50

7.51

1.94

7.51

1.95

1.42

1.38

4.44 0.05 4.45

8.25

8.28

8.26

8.27 7.93

7.94

7.95

7.94

0 2.09 1.84 4.00 1.63 2.75

8 7 6 5 4 3 2 1 0 Chemical Shift (ppm) 1 Figure S6.14. H NMR (CD3CN) of oxycarbonylnitrene precursor 6 after 4 hour photolysis at 254nm in argon-purged CD3CN.

233

N-ethoxycarbonyl dibenzothiophene sulfilimine_CH3CN_4hr photolysis.esp

2.13 1.0

0.9

0.8

0.7 2.30

0.6

0.5

1.94 0.4

1.38

Normalized Intensity Normalized

7.50

7.47

7.48

7.49 0.3 1.92

1.91

1.40

1.36

4.41 0.2 4.43

1.82

7.91

7.90

8.23

8.25 8.24

7.93

8.23

4.39

4.45 0.1 7.46

7.89

0 2.01 1.98 4.00 1.42 2.13 2.15

8 7 6 5 4 3 2 1 Chemical Shift (ppm) 1 Figure S6.15. H NMR (CD3CN) of oxycarbonylnitrene precursor 6 after 4 hour photolysis at 254nm in argon-purged CH3CN.

234

Chapter 7: The Singlet-Triplet Splittings of Benzoylnitrene, Acetylnitrene, and Trifluoroacetylnitrene

7.1 Introduction

Nitrenes are reactive intermediates containing neutral, monovalent nitrogen atoms.1

Alkyl- and arylnitrenes typically favor triplet ground states by a wide margin.2 The ground state multiplicity of nitrenes is often determined experimentally by stereospecific trapping reactions with alkenes. Rapid rearrangement reactions of the singlet state and slow intersystem crossing rates, however, have made direct detection of the triplet nitrene difficult. Nonetheless, the direct observation of triplet nitrenes by low temperature EPR, matrix-isolation, and time-resolved spectroscopy methods has been reported.3–13

Nitrenes are most commonly generated from the corresponding azides. However, other precursors, including phenanthrene- and sulfilimine-based compounds have been shown to generate nitrenes efficiently.9,14,15 These compounds have the added advantage of being more stable for storage and handling compared to the corresponding azide precursor. Thus, we have chosen to use N-substituted dibenzothiophene sulfilimine photoprecursors 1a-c for these studies (Scheme 7.1).

235

Scheme 7.1. Dibenzothiophene (DBT) Sulfilimine-based precursors to carbonylnitrenes. In contrast to alkyl- and arylnitrenes, recent experimental and computational studies have indicated that carbonylnitrenes (RC(O)N) have singlet ground states.9,10,16,17

The carbonylnitrene singlet state is stabilized by the interaction between the in-plane carbonyl oxygen lone pair and the empty in-plane orbital on the nitrene nitrogen. This bonding interaction leads to an oxazirine-like structure for the singlet carbonylnitrene that is very different from that of the corresponding triplet carbonylnitrene (Figure 7.1).

Figure 7.1. Singlet carbonylnitrene and its oxazirine-like resonance contributor.

Despite the extensive study of nitrenes and the fundamental importance of their ground state multiplicity and singlet-triplet splitting (ΔEST = ES – ET) on reactivity, only three ΔEST values have been determined experimentally. Negative ion photoelectron

18 spectroscopy has been used to determine ΔEST for imidogen (NH), methylnitrene

19 20,21 (CH3N), and phenylnitrene (PhN). Herein, we are pleased to report the generation of the anions of benzoylnitrene, acetylnitrene, and trifluoroacetylnitrene from their N- substituted dibenzothiophene sulfilimine precursors (Scheme 1) and present for the first time anion photoelectron spectra of these carbonylnitrenes. In addition, we report

236 calculations, which are validated by the spectra, and are used to derive a ΔEST value for each of the three carbonylnitrenes.

7.2 Experimental and Computational Analysis of Singlet-Triplet

Splittings

To verify the accuracy of the theoretical methods in calculating the singlet-triplet splitting for the three carbonylnitrenes, the same methods were used to calculate the VDEs and the adiabatic energy difference between the singlet and triplet states (i.e. the energy difference between the relaxed ground state singlet and relaxed ground state triplet) for the

20 19 previously studied nitrenes, phenylnitrene (PhN) and methylnitrene (CH3N) . In the previous experimental and theoretical work on phenylnitrene anion, the relaxed ground states of the singlet and triplet were both observed in the PES and the singlet-triplet splitting was determined from the spectrum.20 Our theoretical methods accurately reproduce the observed transitions from the photoelectron spectrum of phenylnitrene and found the relaxed ground state triplet is 0.57 eV lower in energy than the relaxed ground state singlet

(Table 7.1). Our calculations are in good agreement with both the previous experimental and theoretical results. For this case we find two nearly-degenerate closed-shell singlets at

2.47 eV and an open-shell singlet at 2.1 eV, which is in good agreement with the experimentally measured PES peak at 2.1 eV by Ellison et al.20 We find the lowest triplet state to be at 1.52 eV, which agrees with the experimentally measured peak by Ellison et al. at 1.45 eV.20

237

To corroborate our theoretical method further, another nitrene system previously explored by PES was examined. The experimentally observed transitions of the relaxed ground states of the singlet and triplet of CH3N were calculated. We find that the triplet neutral energy is essentially degenerate with the doublet anion. PBE-GGA predicts that the neutral triplet is 0.046 eV more stable than the doublet anion in contrast to Ellison’s result, where the neutral triplet is 0.023 eV less stable than the doublet anion.19 With the inclusion of zero-point energy correction, however, the neutral triplet lies 0.0027 eV above the anion doublet of CH3N, which is consistent with Ellison’s experiment. For the singlet neutral, we find that the lowest energy is 1.33 eV above the doublet anion, which is also in good agreement with Ellison’s value. Our theoretical methods accurately reproduce the observed transitions from the photoelectron spectrum of methylnitrene and we find the adiabatic singlet-triplet energy difference to be 1.45 eV (Table 7.1). The calculated structure of

CH3N corresponds to the fractionally occupied “metallic” case (discussed in

Computational Methods) which is also in good agreement with Ellison’s description of this state in terms of two real determinants. Based on an analysis of the Kohn-Sham eigenvalues, the next electronic levels should be approximately 3.93 eV above the doublet anion, which is also in good agreement with Ellison’s measurements of 3.95 eV.19

We relied on theory to assign the observed transitions in the PES of each carbonylnitrene studied in this work. The spectra of the anions of benzoylnitrene, acetylnitrene, and trifluoroacetylnitrene are presented in Figure 7.2. Two distinct transitions are present in the benzoylnitrene and acetylnitrene spectra (Figure 7.2 (a) and

(b), respectively), while in the spectrum of the trifluroacetylnitrene (Figure 7.2 (c)), a broad

238 low intensity peak between 2.0 and 3.0 eV along with three more intense transitions at higher EBE are observed.

Figure 7.2. Anion photoelectron spectra of (a) benzoylnitrene, (b) acetylnitrene, and (c) trifluoroacetylnitrene anions. The vertical sticks represent the calculated vertical detachment transitions.

The experimental and calculated values are reported in Table 7.1. From the theoretical calculations, the most intense observed transitions in the photoelectron spectra of all three nitrene anions are assigned as vertical detachment transitions (VDE) from the relaxed ground state anion to either the singlet or triplet neutral states, in the geometry of

- the anion. The broad low intensity peak in the CF3CON spectrum at ~2.4 eV could be a

239

- transition from the anion of a structural isomer, isocyanate (CF3NCO ). Photoisomerization of the anion from a photon (hυ) followed by the photodetachment of the structural isomer from another photon is plausible. Our calculations reveal a transition at 2.36 eV from the ground state doublet isocyanate anion to the neutral singlet isocyanate. A large difference in cohesive energies between the doublet nitrene and isocyanate structures (which we find to be 0.95 eV within PBE-GGA) is indicative of broadening in the observed peak.

Table 7.1. Experimental Observed Transitions and Calculated Values of benzoyl-, acetyl- , trifluroacetyl-, phenyl-, and methylnitrene. All ΔEST values include zero-point energy corrections.

20 19 PhC(O)N CH3C(O)N CF3C(O)N PhN CH3N Experimentally 3.15 2.85 3.75 1.45 0.022 Observed 3.68 3.30 4.25, 4.45 2.10 1.35 Transitions 3.06 2.80 3.86 1.52 0.0027a Calculated (triplet) (triplet) (triplet) (triplet) (triplet) Vertical 3.4, 3.7 3.42 4.54, 4.58 2.1, 2.47 1.33 Transitions (singlet) (singlet) (singlet) (singlet) (singlet) Calculated ΔEST eV -0.124 -0.186 -0.0123 0.568 1.447 (kcal/mol) (-2.86) (-4.29) (-0.284) (13.09) (33.37) a: zero-point energy corrected value

The origin containing transitions, i.e., the ν′= 0 ← ν′′ = 0 transition (electron affinity, EA), for both the neutral singlet and triplet states in benzoylnitrene, acetylnitrene, and trifluoroacetylnitrene are not observed due to significant geometry changes between the relaxed ground state doublet anion and the relaxed ground state neutral singlet and triplet (Figures 7.3 and 7.4). This was not the case for the previously studied phenyl- and methylnitrene, which do not show significant differences in geometries between the relaxed neutrals and the doublet anions (Figure 7.4a). Thus, the adiabatic energy difference

240 between the singlet and the triplet states of benzoylnitrene, acetylnitrene, and trifluoroacetylnitrene cannot be directly measured from the PES as shown schematically in

Figure 7.4b.

117.7° 132.1° 117.7° O 1.284 1.323 O 1.251 O 2.314 2.211 1.764 126.4° 114.6° Ph 1.532 Ph 1.448 Ph 1.483 85.6° 1.308 1.273 1.375 N N N 22a 12a 32a

124.5° 117.3° O 131.2° O 1.284 O 1.248 2.328 1.319 2.214 1.778 115.3° 128.1° H C 1.504 H3C 1.555 H3C 1.473 3 86.8° 1.306 1.268 1.371 N N N 22b 12b 32b

115.8° O 130.9° 121.7° 1.271 1.304 O 1.238 O 2.341 2.246 1.813 130.9° 119.4° F3C 1.573 F3C 1.517 F3C 1.559 89.7° 1.302 1.265 1.362 N N N 2 1 3 2c 2c 2c

Figure 7.3. NRMOL calculated bond lengths (Å) and angles for the doublet anion and the neutral singlet and triplet spin states of benzoyl- (2a), acetyl- (2b), and trifluroacetylnitrene (2c).

241

Figure 7.4. Schematic representation of vertical detachment energy transitions (VDE) and singlet-triplet energy splittings (EST) for two general cases where (a) the geometry of the doublet anion (black) is similar to the neutral spin states (blue) and (b) the geometry of the doublet anion is significantly different from one of the neutral species. Given that the theoretical methods in this work accurately reproduce the observed transitions in the photoelectron spectrum of five experimentally studied nitrenes, we are confident our calculations can correctly determine the singlet-triplet splitting (ΔEST) in the carbonylnitrenes. Thus, we find the singlet state to be lower in energy than the triplet state for benzoylnitrene, acetylnitrene, and trifluoroacetylnitrene where the ΔEST values are -

0.146 eV, -0.220 eV, - 0.0324 eV, respectively. When zero point vibrational effects are included the the ΔEST values for benzoylnitrene and acetylnitrene are -0.124 (-2.86 kcal/mol) and -0.186 eV (-4.29 kcal/mol), respectively, and for trifluoroacetynitrene, the singlet and triplet states are nearly degenerate with a splitting of -0.0123 eV (-0.284 kcal/mol).

242

7.3 Comparison with Solution Reactivity

The geometries and singlet-triplet energy splitting of the carbonylnitrenes reported here have been previously investigated computationally.10,15–17,22–24 Carbonylnitrene geometries have been shown to be relatively insensitive to the computational method employed.10,17 Indeed, the NRMOL calculated geometries of benzoyl-, acetyl-, and trifluroacetylnitrene correlate well with previously reported work (Figure 7.3).9,10,15,23 As expected, the singlet geometries show structural similarity to an oxazirine, with the O-C-

N fragment having significant three-membered ring character, i.e., much smaller O-C-N bond angles and smaller O-N distances compared to the triplet species (Figure 7.3), the result of a stabilizing interaction between the carbonyl oxygen lone pair electrons and the empty p-type orbital on the nitrene nitrogen.

Sherman and Jenks recently reported a thorough computational investigation of the effect of fluorination on the ground state multiplicity of acylnitrenes.23 They found that the inductive electron withdrawing effect of fluorine substitution reduces the stabilization of the singlet configuration, via the oxazirine-like resonance contributor (Figure 7.1). For example, similar to the work of Jenks and co-workers our calculated O-C-N bond angles of both singlet and triplet trifluroacetylnitrene are larger relative to benzoyl- and acetylnitrene (Figure 7.3).

In contrast to calculated geometries, calculated energies of carbonylnitrenes, and

10,23 therefore EST values, vary considerably with computational method. However,

Sherman and Jenks found that the EST value between acetyl- and trifluroacetylnitrene was approximately 4 kcal/mol, independent of computational method.23 This matches well

243 with our calculated EST values. Previous time-resolved infrared (TRIR) spectroscopy studies and product analysis suggested a very small (ca. -1 kcal/mol) EST value for acetylnitrene, and indicated that tifluroacetylnitrene also has a small singlet-triplet splitting, but is likely a ground state triplet.15

In the anion photoelectron spectra of benzoylnitrene, acetylnitrene, and trifluoroacetylnitrene, the peaks observed are vertical detachment transitions. The adiabatic energy transitions are not observed due to significant geometry differences between the anions and neutral nitrenes (Figure 7.3). This results in an observed splitting that is not an accurate reflection of the EST value (Figure 7.4). Thus, the adiabatic energy difference between the singlet and triplet cannot be directly determined from the photoelectron spectra. Benzoyl-, and acetylnitrene are calculated by the NRLMOL method to be ground state singlets with EST values of -2.86 and -4.29 kcal/mol, respectively. The singlet and triplet states for trifluroacetylnitrene are nearly degenerate (EST = -0.284 kcal/mol). By comparison, high-level CBS-QB3 calculations estimate a EST value of -4 kcal/mol for

22 acetylnitrene, which is in close agreement to our calculated EST value.

For trifluoroacetylnitrene, in the neutral geometry, PBE-GGA, with NRLMOL, finds that the singlet state is 0.75 kcal/mole (0.0324 eV) more stable (lower energy) than the triplet state (without zero-point energy correction). However, because the triplet has slightly softer vibrational frequencies, when zero-point corrections are included the energies are essentially degenerate such that EST is -0.284 kcal/mol (-0.0123 eV). Several vibrational calculations show that the triplet zero-point energy is 0.7 kcal/mole smaller than the singlet zero-point energy. Singlet-triplet splittings, or more generally high-spin/low- spin splittings, are known to be sensitive to the self-interaction error in density-functional

244 theory. Trifluoroacetynitrene, which experimentally has been suggested to have a triplet ground state, will be an excellent test case for the new unitarily-invariant self-interaction- corrected density-functional approach.25

7.4 Conclusions

We have shown that N-substituted dibenzothiophene sulfilimine based precursors

1a-c are efficient precursors to carbonylnitrenes 2a-b. Unlike previously studied alkyl- and arylnitrenes, the observed transitions in the PES spectra do not reflect the energy difference between the relaxed singlet and triplet nitrenes (EST) due to significant geometry differences between the doublet anions and neutral species. Computational analysis, which is validated by the spectra, indicates that benzoyl- and acetylnitrene are ground state singlets, albeit by small margins, and the energies of singlet and triplet trifluroacetylnitrene are nearly degenerate (EST = -0.284 kcal/mol, -0.0123 eV).

7.5 Experimental

7.5.1 Synthesis and Characterization

Dibenzothiophene oxide was prepared using a modified literature procedure.26 A solution of 3-chloroperbenzoic acid (m-CPBA) in CHCl3 was added slowly to a solution

of dibenzothiophene (1g, 5.4 mmol) in CHCl3 at -30-35 °C. The reaction was stirred at -

30-35 °C for 1 hour, then allowed to return to room temperature and stirred for an additional

1 hour. The reaction mixture was then filtered through celite and neutralized with saturated

NaHCO3. The organic layer was washed with NaHCO3 and brine, dried with MgSO4,

245 filtered, and evaporated. The crude material was purified by flash chromatography (80:20 dichloromethane:hexanes) to give a white solid final product (870 mg, 80%). 1H NMR

(400 MHz, CDCl3) δ 7.48-7.52 (dt, 2H), 7.58-7.62 (dt, 2H), 7.81 (dd, 2H), 7.99 (dd, 2H);

13 C (CDCl3) δ 122.0, 127.7, 129.7, 132.7, 137.2, 145.2.

N-benzoyl dibenzothiophene sulfilimine,9 1a. A solution of dibenzothiophene oxide

(243 mg, 1.21 mmol) in dichloromethane was added slowly to a solution of trifluoroacetic anhydride (509 mg, 2.42 mmol) in dichlormethane at -78 °C. The reaction was stirred at -

78 °C for 30 min at which point solid benzamide (294 mg, 2.42 mmol) was added and allowed to stir for an additional 90 min at -78 °C. After 90 min the reaction was warmed to room temperature. The organic layer was washed with saturated NaHCO3 and brine, dried over MgSO4, filtered, evaporated. The crude material was purified by flash column chromatography (gradient, 70:30 dichloromethane:hexanes to 100% dichloromethane) to

1 give a white solid (80 mg, 22%). H NMR (400 Mhz, CDCl3) δ 7.36-7.45 (m, 3H), 7.56 (t,

13 2H), 7.68 (t, 2H), 7.95 (d, 2H), 8.13 (d, 2H), 8.25 (d, 2H); C (CDCl3) δ 122.4, 127.9,

128.8, 128.9, 129.9, 131.1, 132.5, 135.9, 138.2, 138.4, 178.6.

N-acetyl dibenzothiophene sulfilimine,15 1b. This compound was prepared as was

1a with substitution of acetamide for benzamide. The crude material was purified by flash column chromatography (gradient, dichloromethane 100% – ethylacetate 100%) to give a

1 white solid: 18% yield; H NMR (400 MHz, CDCl3) δ 2.14 (s, 3H), 7.50 (t, 2H), 7.62 (t,

13 2H), 7.87 (d, 2H), 8.11 (d, 2H); C (CDCl3) δ 24.1, 122.4, 128.8, 130.0, 132.5, 137.9,

138.4, 183.9.

N-trifluoroacetyl dibenzothiophene sulfilimine,15 1c. This compound was prepared as was 1a with substitution of trifluoroacetamide for benzamide. The crude material was

246 purified by flash column chromatography (gradient, dichloromethane 100% – ethylacetate

1 100%) to give a white solid: 34% yield; H NMR (400 MHz, CDCl3) δ 7.59 (t, 2H), 7.73

13 (t, 2H), 7.94 (d, 2H), 8.17 (d, 2H); C (CDCl3) δ 117.21 (q, J = 287.8 Hz), 122.8, 129.1,

130.5, 133.5, 135.8, 138.7, 169.0 (q, J = 35.4 Hz).

22 Benzoyl azide, 3a. A solution of NaN3 (105 mg, 1.62 mmol) in H2O (10 mL) was added slowly to a solution of benzoyl chloride (200 mg, 1.42 mmol) in anhydrous acetone

(12.5 mL) at 0 °C. The reaction was allowed to stir at 0 °C for 30 minutes. At this time the product was extracted with ethylacetate. The organic layer was dried over MgSO4, filtered, and evaporated. The crude material was run through a short silica plug (90:10 hexane:ethylacetate) to give the desired product (167 mg, 80%). 1H NMR (400 Mhz,

13 CDCl3) δ 7.42 (m, 2H), 7.58 (t, 1H), 8.01 (d, 2H); C (CDCl3) δ 128.8, 129.6, 130.8, 134.5,

172.7.

7.5.2 Spectroscopic Measurements

Anion photoelectron spectroscopy is conducted by crossing a mass-selected beam of negative ions with a fixed-frequency photon beam and energy-analyzing the resultant photodetached electrons. Photodetachment transitions occur between the ground state of a mass-selected negative ion and the ground and energetically accessible excited states of its neutral counterpart. This process is governed by the energy-conserving relationship hν =

EBE + EKE, where hν is the photon energy, EBE is the electron binding energy, and EKE is the electron kinetic energy. Measuring electron kinetic energies and knowing the photon energy provide electron binding (photodetachment transition) energies. Because these are vertical transitions, their relative intensities are determined by the extent of

Franck−Condon overlap between the anion and its corresponding neutral. Our apparatus

247 consists of a laser photoemission/oven anion source, a linear time-of-flight mass spectrometer for mass analysis and mass selection, a momentum decelerator, a magnetic bottle electron energy analyzer, and an Nd:YAG laser. The magnetic bottle has a resolution of ∼50 meV at an EKE of 1 eV. In these experiments, photoelectron spectra (PES) were recorded with 266 nm (4.66 eV) photons. The photoelectron spectra were calibrated against the well-known transitions of atomic Cu−.27

- - To produce the benzoylnitrene (C7H5ON ), acetyl nitrene (CH3CON ) and

- - trifluoroacetylnitrene (CF3CON ) anions, ~0.25 g of sample (N-benzoyl dibenzothiophene sulfilimine, N-acetyl dibenzothiophene sulfilimine, or N-trifluoroacetyl dibenzothiophene sulfilimine, respectively,) was placed in a small oven (40-55 °C) attached to the front of a pulsed (10 Hz) valve (General Valve Series 9), where helium (85-120 psi) was expanded over the sample in a high vacuum chamber (10−6 Torr). Just outside the orifice of the oven, low-energy electrons were produced by laser/photoemission from a pulsed Nd:YAG laser beam (10 Hz, 532 nm) striking a translating, rotating, copper rod (6.35 mm diameter).

Negatively charged anions were then pulse-extracted into the spectrometer prior to mass selection and photodetachment. Benzoylnitrene anion was also produced from benzoylazide under similar experimental conditions (Supporting Information). The PES was identical to the benzoyl nitrene anion spectrum from the N-benzoyl dibenzothiophene sulfilimine sample.

7.5.3 Computational Analysis

The electronic structure calculations discussed here used NRLMOL to perform density-functional based calculations, using the Perdew-Burke-Ernzerhof (PBE) generalized-gradient approximation (GGA). Large Gaussian-orbital all-electron basis sets,

248 which satisfy the Z(10/3) theorem,28 and are roughly of quadruple-zeta quality, were used for representing the Kohn-Sham Orbitals. Discussions on the NRLMOL methodology and its use in problems with multiple unpaired electrons, relevant to the most of the systems in this work, have been discussed previously.29–31 An early use of NRLMOL for the calculation of vertical detachment energies, on systems with pure singlet-states has been reported.32 In all cases, the geometry of the doublet anion molecule has been relaxed. Once the doublet anion geometry has been found, the vertical electron detachment energies are determined by calculating the energy of the neutral triplet (at the anion geometry) and by calculating the energies of the singlet states.

For doublet anions that are well approximated by a single reference state, but have two nearly degenerate HOMO levels, the lowest energy triplet neutral state that occurs when an electron is removed is relatively straightforward to construct in terms of a single determinantal wavefunction for the high-spin case (Ms=1), and standard methods may be used to determine the energetically degenerate Ms=0 triplet wavefunctions once the high- spin case is known. However, the representation of the lowest singlet state(s) is more complicated especially when the doublet anion paired state (referred to as |P>) and unpaired state (referred to as |U>) are energetically nearly degenerate. Several possible neutral singlet states that can be constructed from theses orbitals are explained further.

We first discuss the case where a closed-shell singlet is the lowest-energy geometry in density functional theory (DFT). For such cases, the singlet-triplet energy splitting is determined by the energy difference between the spin-unpolarized singlet and the high- spin triplet (ΔEST = ES – ET). There are several possible singlet states that are effectively considered during an SCF calculation. First, there are a continuous range closed-shell

249 singlet solutions of the form, |RR,00>= R(1)R(2) [21/2 (with |R>= cos()|P>+sin()|U>) that can be determined from the optimal combination of the two nearly-degenerate orbitals. Generally, due to Koopmans’ theorem, one expects that the lowest singlet would be closely approximated by the |PP,00> wavefunction(e.g. =0).

However, Koopmans theorem is not even exact in Hartree-Fock so violations could occur in cases where the |P> and |U> states are nearly or exactly degenerate. In such cases, the lowest closed-shell singlet could in principle be more closely represented as |UU,00> (e.g.

=/2). In the limit of exact degeneracy one must also consider the complex singlet state that is constructed by equally weighted complex admixtures of the |U> and |P> orbitals given by |C+>= (|P> + i|U>)/21/2. The wavefunction for this case can be constructed as a single determinant given by |CC,00> = [C+(1)C+(2)] [21/2which, with a bit of algebra, may be re-represented in terms of the two real determinants, discussed above, and the di-radical system, discussed below, according to

[P(1)P(2)+U(1)U(2)+i{P(1)U(2)+U(1)P(2)}] [23/2. In a density- functional-based picture this state can either be treated as a real solution with two half- occupied degenerate orbitals, for both  and spin, or as a complex solution with fully occupied orbitals. This generalization of this to closed-shell singlets, with |C>= (|P> + i|U>)/21/2 is relevant in at least one case studied here. Within DFT, there are further generalizations of this with complex Kohn-Sham orbitals of the form

|C>={cos()|P>+isin()}|U>. This orbital leads unpolarized spin densities with degenerate real orbitals showing occupations of cos2() and sin2() respectively for both  and  spins.

The first discussions on these so-called fractionally occupied or metallic singlets was by

Janak,33 and then later within the context of the carbon dimer by Pederson et al.25

250

In molecular magnets, and in dissociated molecules, it is generally the states with di-radical character that dominate in the ground-state wavefunction. But this type of open- shell singlet appears whenever the frontier orbitals are relatively localized. For such cases the broken spin-symmetry case, U(1)P(2)is much lower in energy than the closed shell singlet solutions and is exactly half way between the singlet energy and the triplet energy. In cases where this state is lowest in energy, the singlet-triplet energy splitting can be determined by doubling the energy difference between this broken symmetry state and the pure triplet state EST = 2[ES() - ET()]. Most of the results reported in this paper require this sort of analysis for the determination of the lowest singlet state.

In a multi-configurational picture, the lowest neutral singlet state would be an arbitrary linear combination of |PP,00>, |UU,00> and |UP,00> and would not necessarily coincide with any of the density-functional configurations discussed above. Further, an experiment over the entire energy range would pick up three different singlet energy peaks assuming each peak is observable from the standpoint of optical selection rules. For cases where these states are nearly degenerate a broader singlet PES is expected and this broadening would be due to electronic effects rather than vibrational effects.

Since there is no a priori reason for knowing whether the lowest-energy singlet state is indeed a di-radical state, we have also calculated energies of the spin-restricted singlet states (ΔEST = ES – ET) and the di-radical states (EST = 2[ES() - ET()]). In some cases these states are only slightly higher in energy. The energy splitting, and sign of the energy splitting, between the closed-shell singlet states and the open-shell di-radical states indicates the degree to which these system is an ideal di-radical. For cases where the splitting is small, one expects level-repulsion to push the states apart and further reduce

251 the singlet-triplet splitting. As mentioned above, pure di-radical systems are cousins of inorganic molecular magnets.30,31 However, since the spin-orbit coupling is small in first row elements and since the spin-ordering-energetics in the systems studied here are driven by coulomb rather than kinetic exchange, they do not exhibit the same type of collective magnetic signatures and the spin-splittings are much larger.

In addition, to the experimentally measurable vertical detachment transitions that are calculated at the doublet anion geometry, the adiabatic transitions from the relaxed doublet anion to the relaxed neutral singlet or triplet are calculated to determine the adiabatic singlet-triplet energy splitting.

252

7.6 References

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Chapter 8: Miscellaneous Work

8.1 Photochemical Precursors to HNO

8.1.1 Photochemistry of N, N’, N”-Trihydroxyisocyanuric acid (THICA)

Although recent generation of alkoxy- and aryloxynitrenes from photoprecursors has been reported, analogous precursors to the parent hydroxynitrene (HON) have remained elusive.1 Since HON is expected to isomerize to nitroxyl (HNO, Scheme 8.1),2 we have investigated N,N’,N’’-trihydroxyisocyanuric acid (THICA, Scheme 8.2) as a potential photochemical precursor to HON. The photochemistry of THICA has yet to be investigated, however, thermolysis of THICA has been shown to produce N- hydroxyisocyante (Scheme 8.2),3 which can be considered the carbon monoxide (CO) adduct of HON.4,5

Scheme 8.1. Isomerization of oxynitrenes (RON) to the corresponding nitroso (RNO) species.

Scheme 8.2. Thermolysis and potential photochemistry of N,N’,N’’-trihydroxyisocyanuric acid (THICA).

Photochemical experiments were performed in quartz cuvettes containing 0.1 M pH 7.4 phosphate buffered saline (PBS) with 100 µM diethylene triamine pentaacetic acid

258

(DTPA). Samples were irradiated in a Rayonet reactor, fitted with eight 254 nm bulbs.

Simultaneously, the irradiated solutions were passed through our membrane inlet mass spectrometry (MIMS) sample cell for the detection of photoproduced gasses (Figure 8.1).

Presumed decomposition of N-hydroxyisocyanate generated via THICA photolysis, has led to the detection of carbon monoxide (CO m/z 28), HNO (m/z 31), and HNO- or HON- derived N2O (m/z 44) by MIMS (Figure 8.2).

Figure 8.1. Experimental setup for MIMS detection of photochemically produced gasses.

259

Figure 8.2. MIMS signals observed at m/z 44, 31, and 28 upon photolysis of 1 mM THICA in 0.1 M pH 7.4 PBS containing 100 µM DTPA.

The m/z 44 MIMS signal may also contain contribution from CO2 (m/z 44) that results from hydrolysis of N-hydroxyisocyante (Scheme 8.3). This is evident in the increase in the m/z 44 to 30 ratio (~12:1) relative to N2O standards (~5:1). Sampling of the headspace of this reaction by GC analysis confirms CO2 production with a ca. yield of

76%. CO2 also has a fragment at m/z 28, therefore to confirm CO production we performed

MIMS experiments in the presence of a liquid nitrogen cold trap that would trap CO2 (bp

= -78.5 °C) and not CO (bp = -191.5 °C, Figure 8.3). This experiment also confirms that

NO (bp = -152 °C) is not produced during this reaction as a m/z 30 signal is not observed.

GC headspace analysis experiments also revealed a ca. 14% yield of HNO-derived N2O.

260

Scheme 8.3. Potential products formed via THICA photolysis.

1600 LN2 Cold Trap 1400

1200

1000 800 m/z 28 600 m/z 44

Ion Current (pA) 400 m/z 30 200 m/z 31 0

0 5 10 15 Time (Minutes) Figure 8.3. MIMS signals observed at m/z 44, 31, and 28 upon photolysis of 1 mM THICA in 0.1 M pH 7.4 PBS containing 100 µM DTPA in the presence of a liquid nitrogen cold trap.

Due to solubility limitations of THICA in solvents that are ideal for IR spectroscopy, we examined the benzyl derivative of THICA (Bn-THICA, Scheme 8.4) for spectroscopic analysis. Time-resolved infrared (TRIR) spectroscopy of the benzyl derivative revealed evidence for the formation of benzyl-isocyanate at 2250 cm-1, which is formed within the time resolution of our experiment (50 ns) and is stable on the microsecond time scale (Figure 8.4). Further reactivity of benzyl-isocyanate was not observed on the TRIR timescale (10’s of milliseconds). Unfortunately, we were unable to detect the same isocyanate signal by FTIR spectroscopy suggesting the lifetime is between milliseconds and seconds.

261

Scheme 8.4. Products resulting from the photogeneration of benzyloxynitrene in the presence of argon (Ar) or dioxygen (O2).

-3 1.2x10

1.0 0.0 - 50.0 s 50.0 - 100.0 s 0.8 100.0 - 150.0 s 150.0 - 200.0 s 0.6 200.0 - 300.0 s 300.0 - 500.0 s 0.4 500.0 - 700.0 s

Absorbance 700.0 - 900.0 s 0.2



0.0

2300 2250 2200 2150 2100 2050 2000 Wavenumber Figure 8.4. TRIR difference spectra averaged over the time scales indicated following 266 nm laser photolysis of Bn-THICA (5 mM) in argon-saturated dichloromethane.

If the fate of the observed benzylisocyante leads to similar products to that observed by THICA photolyis, benzyloxynitrene (BnON) should be the intermediate involved.

BnON has been observed previously following the photolysis of a phenanthrene

262 photoprecursor (Scheme 8.4). Photolysis resulted in the production of benzaldehyde oxime

(PhCHNOH), or benzylnitrate (PhCH2ONO2) and benzaldehyde (PhCHO) in argon- or oxygen-saturated solvents, respectively (Scheme 8.4).1 HPLC analysis of Bn-THICA photolysis samples revealed the primary products are benzaldehyde and benzylalcohol.

These results suggest that benzyloxynitrene is not involved. Mass spectrometry, following photolysis of solid samples of Bn-THICA, analysis suggests both CO and N2 production and therefore the intermediacy of benzyloxynitrene. Nitrene production, dimerization, and further decomposition to the observed prodcuts is possible (Scheme 8.5).

Scheme 8.5. Potential reactivity of the presumed benzyloxynitrene intermediate resulting from photolysis of Bn-THICA.

263

On the basis of these observations we suggest that THICA may potentially be a precursor to HON through secondary photolysis of N-hydroxyisocyanate, which can be considered as a CO adduct of HON. Observation of HNO and HNO-derived N2O by both

MIMS and GC headspace analysis confirms that THICA has the potential to serve as a photochemical precursor to HNO, albeit with small yields. However, HPLC product analysis of Bn-THICA photolysis suggests that a limited amount of the nitrene intermediate is involved. Further studies will be require to confirm the fate of N-hydroxyisocyante and the source of the observed HNO.

8.1.2 Development of o-Quinonemethide-Based Photoprecursors to HNO

A handful of photochemical precursors to HNO have been explored, however, restrictions including thermal stability, solubility, and the requirement of UV irradiation for release have limited further application of these precursors.6 Recent work by Popik and co-workers has established o-naphthoquinone methide (oNQM) precursors as a viable photochemical protecting group for alcohols and amines.7–9 The oNQM scaffold has the ability to release leaving groups such as alcohols (pKa ~15) and amines (pKa ~12), therefore, its application to HNO (pKa 11.4) may be a viable option. Photolysis of these compounds produce an excited state where the acidity of the O-H proton increases

10 significantly (pKa ~ 1). Once deprotonated, formation of the quinone-methide leads to release of the leaving group (Scheme 8.6a). Compound 6 is a candidate for HNO production via the mechanism described above (Scheme 8.6b).

264

Scheme 8.6. (a) Mechanism responsible for the photorelease of X from o-naphthoquinone methide (oNQM) precursors. (b) Potential HNO precursor (6) reactivity upon photolysis in aqueous solution.

The dimethyl substitution of photoprecursor 6 is necessary to prevent the rapid tautomerization of the nitroso to the corresponding oxime. This requirement has made synthesis of photoprecursor 6 difficult. However, successful production of amine 11 or hydroxylamine 13 would allow for further oxidation to the desired nitroso in a similar fashion to what was reported by Quek et al.11 We have tested a variety of synthetic methods to reach these anticipated intermediate products without success (Scheme 8.7). However, other routes remain including the use of the in situ generated CeCl2Me to react with nitrile

10 to generate amine 11, which has worked for analogous scaffolds.12 Comparable to the work of Nikitin et al.,13 reaction of a tertiary alcohol using Ritter reaction conditions could

265 afford 11. Further synthetic methodology will be required in order to test the viability of these scaffolds as photochemical precursors to HNO.

Scheme 8.7. Proposed synthetic methods for the formation of target compound 6.

266

8.2 Investigation of the Solution Chemistry of Persulfides (RSSH)

Persulfides (RSSH) are well known in literature,14 but only recently gained attention as a proposed post-translational modification of cysteine.15,16 Snyder and co-

15,16 workers suggested that H2S was responsible for the modification of cysteine; however,

17,18 the species responsible for production of RSSH was found not to be H2S. Persulfide modifications have potentially unique effects on enzyme reactivity,16,19,20 leading to many questions regarding its fundamental chemistry in solution, which is still poorly understood.21,22 Persulfides are expected to be better nucleophiles due to the alpha effect,23 better reductants due to a resonance-stabilized radical, and more acidic by ca. 2

21 pKa units compared with their corresponding thiols. In many ways this expected reactivity is analogous that of selenocysteine and one might anticipate that persulfides can serve as analogues of selenols whose reactivity can easily be controlled by simple reduction of the persulfide to the corresponding thiol. Persulfides are also interesting in that they have the potential to react either as a nucleophile or an electrophile. Characterizing the reactivity of persulfides relative to that of thiols will be critical to understanding their biochemistry. Unfortunately, persulfides are inherently difficult to study, as they are unstable and decompose to a variety of species. Therefore, long-term storage of persulfides is an issue that necessitates their generation in situ. Several reports of small molecule persulfide generation using a variety of methods have appeared in the literature,22 however there is still an unmet need for precursors that generate persulfides efficiently without the addition of exogenous nucleophiles.

Currently, the most common method for RSSH generation involves the reaction between a disulfide and H2S or reaction of a thiol with a protected disulfide (PG-SSR in

267

Scheme 8.8a).21,24 However, production of persulfides in the presence of other thiols and disulfides can lead to a complicated mixture of species (see Scheme 8.8b). The development of photoprecursors to persulfides would allow unwanted reactions between persulfides and thiols, disulfides, or potentially persulfides to be avoided and the chemistry of persulfides to be studied without these complications. In addition, such precursors would allow for spatial and temporal control of persulfide release, unlike other currently available precursors. For reactivity comparison, we also propose to develop and analyze photoprecursors to thiols, H2S, and selenols.

Scheme 8.8. (a) Generation and (b) further reactivity of persulfides (RSSH).

A variety of scaffolds can potentially be implemented for the photochemical release of persulfides (and thiols, H2S, and selenols). We have begun to synthesize and study o- napthoquinone methide derivative 23 and p-hydroxyacetophenone derivative 24, inspired by the work of Popik and Givens, respectively, who have used these scaffolds to photorelease a variety of species including thiols (Scheme 8.9).25–29 Synthesis of proposed o-napthoquinone methide precursors will mainly follow established literature procedures

27–30 (see reaction sequence below). Synthesis of the H2S, thiol, and selenol precursors will involve simple substitution of alkylbromide 18 with the corresponding nucleophile.

However, synthesis of the persulfide precursor will be more involved. We envision

268 reaction of the initially generated thiol 19 in the presence of base with ethanesulfenyl chloride (ClSEt) to generate the mixed disulfide 20, which will serve as the persulfide photoprecursor (23, X = SSEt). The synthetic strategy for the p-hydroxyacetophenone derivatives will involve the same alkylbromide derivative 18 and analogous approaches to the synthesis of thiol, H2S, selenol, and presulfide photoprecursors 24. In preliminary studies, we have synthesized the thiol precursor utilizing both scaffolds, and have successfully shown by reaction with 5,5’-dithiobis-2-nitrobenzoic acid (DTNB) to generate the observed TNB at 412 nm, that p-hydroxyacetophenone 24 (X = SEt) releases ethanethiol following 254 nm Rayonet photolysis.

Scheme 8.9. Proposed synthesis of potential precursors 23 and 24.

269

With efficient photochemical precursors in hand we propose to probe the relative reactivity of thiols, H2S, selenols, and persulfides with TRIR spectroscopy. Using well- established electrophilic traps for thiols, we will derive relative trapping kinetics for each

-1 of these species. For example, isocyantes (νNCO ca. 2200 cm ) are known to react with

-1 31 thiols to produce the corresponding S-thiocarbamates (νCO ca. 1650 cm ). In the presence of excess trap, we hope to be able to eliminate the potential issue of the self-reactivity of persulfides discussed above. We anticipate that these fundamental reactivity studies will help to clarify the chemistry of H2S, selenols, and persulfides relative to the well- established reactivity of thiols. In doing so, we hope to shed light on the biochemical roles of these species.

Following our recent work investigating HNO modifications of thiols,32–36 we are interested in probing the reactivity of HNO with persulfides. Considering the known reactivity of thiols with HNO, we would expect persulfides to be efficient traps for HNO as well; however, the reaction between HNO and persulfides has not yet been investigated.

Recent reports suggest that persulfides could be produced in vivo at micomolar concentrations,37 making an understanding of their potential reactivity with HNO important. We have begun to investigate several outstanding questions regarding HNO reactivity with persulfides: (1) Are persulfides more efficient traps for HNO compared to their corresponding thiols? (2) What are the products of HNO reaction with persulfides?

(3) Does the local environment (e.g., in peptide or protein) affect persulfide reactivity?

Initial studies have been performed using the recently reported thermal precursor to penicillamine persulfide,38 which was based on the work of Xian and co-workers.39,40

The synthesis of this compound is a simple one-step process from commercially available

270 starting materials.38 Precursor 26 releases persulfide in neutral aqueous solutions through neutralization of the HCl-salt followed by an intramolecular S-N methoxycarbonyl transfer to produce 28 (Scheme 8.10). We propose to explore the scope of this strategy by synthesizing derivatives of 26 with various R groups to tune the persulfide release rate. For example, by modifying R1 we should be able to modulate the rate of intramolecular S-N carbonyl transfer. Further, substituting R2 with various alkyl substituents has the potential to slow the rate of reaction at the carbonyl resulting in a slower release rate of persulfide.

Generation of the persulfide in the presence of an HNO donor would allow for investigation of HNO induced modifications of persulfides. Initial GC headspace experiments measuring

HNO-derived N2O in the presence or absence of persulfide (generated from 26) or thiol, suggest that persulfides are better traps for HNO compared to their corresponding thiols.

Scheme 8.10. Persulfide precursor scaffold and release mechanism at neutral pH.

Product studies of the reaction between persulfide (e.g, derived from precursor 26) and HNO will be beneficial in predicting its reactivity in a protein environment. A variety outcomes for the reaction of HNO with persulfides are possible. Presumably, the initial reaction of the persulfide with HNO will proceed though an N-hydroxysulfinamide intermediate (RSSNHOH), similar to the reaction with thiols. Under high concentrations of persulfide, we expect further reactivity with RSSNHOH to generate polysulfide

(RSSSSR) and hydroxylamine (Scheme 8.11a). However, since there are two available

271 sulfurs in the proposed RSSNHOH intermediate, the reactivity with a second equivalent of persulfide could occur at the internal sulfur (Scheme 8.11b). This pathway would ultimately lead to the production of disulfide (RSSR) and potentially HNS. In the presence of low concentrations of persulfide rearrangement of RSSNHOH to what would resemble a persulfide analog of a sulfinamide (RSS(O)NH2, Scheme 8.11c) is possible. If generated, it is plausible that, unlike the related thiol derivative, this species would not be stable.

Heterolytic cleavage of the S-S bond would generate the corresponding thiol and HNSO.

Lastly, decomposition of RSSNHOH could lead to loss of thiol and generation of HONS

(Scheme 8.11d).

Following incubation of persulfides with HNO donors samples can by chromatographic and mass spectrometry techniques. Similar to methods reported in literature, we will attempt to separate and characterize thiols, disulfides, and polysulfides by HPLC and MS techniques. Previous work by Filipovic et al. suggested the ability of thionitrous acid (HSNO) to permeate cell membranes,41 and so should be detectable by

272

MIMS at m/z 63. Further, recent computational analysis has suggested the possibility of

HSNO isomerization in aqueous environmentsm 42 and we expect that HNSO and HONS could also be detected by MIMS at m/z 63 (Chapter 4). To distinguish HNSO from HONS, we will have to correlate the observed MIMS signal to the accompanying product (i.e.,

RSH for HNSO, and RSSR for HONS). In addition, we also expect to be able to detect

HNS (m/z 47) and N2S (m/z 60) by MIMS.

After gaining an understanding of small molecule persulfide reactivity with HNO we will to move onto small peptide systems to investigate the effects of a peptide environment on reactivity with HNO, similar to our previous investigations with thiols.33,35,43 Modification of cysteine containing peptides can be accomplished using methoxycarbonylsulfenyl chloride (ClSC(O)OMe) in a reaction analogous to that used to generate persulfide precursor 26 (Scheme 8.12). The cysteine residue is anticipated to be the best nucleophile in the peptide (e.g., VYPCLA),35 and will outcompete other nucleophiles present in the peptide for reactivity with methoxycarbonylsulfenyl chloride.

This strategy has potential for success as the thiol of penicillamine reacted exclusively over the amine and carboxylic acid functional groups in methanol in synthesis of 26. As shown below, in the case of a peptide system, we would rely on release of the persulfide by the addition of exogenous amine. Initially, we will confirm that this approach successfully releases the persulfide by applying the “Tag-Switch” technique, developed by Xian and co-workers, to detect and quantify persulfide modifications of cysteine residues.44

273

Scheme 8.12. Modification of peptide cysteine residues with methoxycarbonylsulfenyl chloride to generate protected persulfides that can be released upon exposure to amine nucleophiles.

274

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Curriculum Vitae

Tyler Alexander Chavez [email protected] linkedin.com/in/tylerachavez Education

Johns Hopkins University Baltimore, MD Ph.D., Organic Chemistry (February 2016) Dissertation Title: Investigation of Reactive Intermediates: Nitroxyl (HNO) and Carbonylnitrenes. Johns Hopkins University Baltimore, MD M.A., Chemistry (September 2013) Sonoma State University Rohnert Park, CA B.A., Chemistry (June 2011)

Experience John Hopkins Technology Ventures Baltimore, MD Commercialization Academy Intern (October 2014 – Present)  Completed a thorough analysis of 22 invention disclosures.  Developed a reputation for quality and efficient analysis of technologies.  Collaborate with intellectual property management teams and technology licensing associates to develop technology transfer paths.  Mentored junior interns in the use of software and approach to technology analysis. Johns Hopkins University Baltimore, MD Graduate Researcher (August 2011 - Present)  Perform organic chemistry research, applied to biologically relevant questions, under the advisement of Professor John P. Toscano.  Apply a wide variety of experimental and computational techniques.  Successfully communicated with collaborators in a diverse set of technical fields leading to manuscripts to be submitted in the near future.  Managed and trained three undergraduates to be independent researchers. Key accomplishments:  Primary author of an invited book chapter in In The Chemistry and Biology of Azanone (HNO) and published a peer-reviewed journal article in The Journal of Inorganic Biochemistry.  Presented research at the National Organic Chemistry Symposium and the Reaction Mechanisms Conference.  Ford Foundation Predoctoral Fellowship Honorable Mention (2013) Organic Chemistry Head Teaching Assistant (August 2012 – May 2014)  Prepared and delivered lectures on organic chemistry to more than 50 students.  Proficient in motivating and teaching others to successfully complete delegated tasks. Key accomplishments:  Ernest M. Marks Award for Teaching Excellence (2013) Organic Chemistry Teaching Assistant (August 2011 – May 2012) Key accomplishments:  Krieger School of Arts and Sciences Teaching Assistant Award (2012)

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Sonoma State University Rohnert Park, CA Research Assistant (February 2009 – May 2011)  Performed organic chemistry research under Professor Jon M. Fukuto.  Synthesized novel thermal release precursors to nitroxyl (HNO). Key accomplishments:  Published a peer-reviewed article in Free Radical Biology and Medicine.  Presented research at the ACS Undergraduate Research Symposium.  Louis Stokes Alliance for Minority Participation (2010)  McNair Scholarship Recipient (2010)

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