INVESTIGATION OF REACTIVE INTERMEDIATES: NITROXYL (HNO ...

INVESTIGATION OF REACTIVE INTERMEDIATES:

NITROXYL (HNO) AND CARBONYLNITRENES

by

Tyler A. Chavez

A dissertation submitted to the Johns Hopkins University in conformity with the

requirements for the degree of Doctor of Philosophy

Baltimore, Maryland

February 2016

© 2016 Tyler A. Chavez

All rights reserved

ii

Abstract

Membrane inlet mass spectrometry (MIMS) is a well-established method used to

detect gases dissolved in solution through the use of a semipermeable hydrophobic

membrane that allows the dissolved gases, but not the liquid phase, to enter a mass

spectrometer. Interest in the unique biological activity of azanone (nitroxyl, HNO) has

highlighted the need for new sensitive and direct detection methods. Recently, MIMS has

been shown to be a viable method for HNO detection with nanomolar sensitivity under

physiologically relevant conditions (Chapter 2). In addition, this technique has been used

to explore potential biological pathways to HNO production (Chapter 3).

Nitrenes are reactive intermediates containing neutral, monovalent nitrogen atoms.

In contrast to alky- and arylnitrenes, carbonylnitrenes are typically ground state singlets.

In joint synthesis, anion photoelectron spectroscopic, and computational work we studied

the three nitrenes, benzoylnitrene, acetylnitrene, and trifluoroacetylnitrene, with the

purpose of determining the singlet-triplet splitting (ΔEST = ES – ET) in each case (Chapter

7). Further, triplet ethoxycarbonylnitrene and triplet t-butyloxycarbonylnitrene have been

observed following photolysis of sulfilimine precursors by time-resolved infrared (TRIR)

spectroscopy (Chapter 6). The observed growth kinetics of nitrene products suggest a

contribution from both the triplet and singlet nitrene, with the contribution from the singlet

becoming more prevalent in polar solvents.

Advisor: Professor John P. Toscano

Readers: Professor Kenneth D. Karlin

Professor Christopher Falzone

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Dedication In honor of all those who loved and supported me through this journey

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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.

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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

vi

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

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3.4 Conclusions ............................................................................................................. 62


3.5.1 General Methods............................................................................................... 63

3.5.2 Gas Chromatographic (GC) Headspace Analysis of N2O ................................ 63


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


4.7.1 General Methods............................................................................................... 93


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

ix

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

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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

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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,

(c) or with phosphines to form the phosphine oxide and aza-ylide products. .................. 23

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

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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

xiv

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

xv

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

xvi

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

xvii

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

xviii

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

xix

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

xx


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


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


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


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-



in argon-saturated dichloromethane at (a) 1638 cm-1 from -0.4 to 3.6 μs, (b) 1752 cm-1

xxi

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


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-



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

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.

......................................................................................................................................... 242

xxii

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


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

xxiii

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

xxiv

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

xxv

presence. The dotted curves are experimental data; the solid curves are best fits to a

single-exponential function. ............................................................................................ 226

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

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

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

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

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

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

Figure S6.12. 1H NMR (CD3CN) of oxycarbonylnitrene precursor 7 after 4 hour

photolysis at 254nm in argon-purged CD3CN. ............................................................... 231

Figure S6.13. 1H NMR (CD2Cl2) of oxycarbonylnitrene precursor 7 after 4 hour

photolysis at 254nm in argon-purged CD2Cl2. ............................................................... 232


photolysis at 254nm in argon-purged CD3CN. ............................................................... 233


photolysis at 254nm in argon-purged CH3CN. ............................................................... 234

xxvi

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


[SN(H)OH]+ SN-OH2 TS. ........................................................................................ 103


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


SN-OH2........................................................................................................................... 104


intensities for SN-OH2. ................................................................................................... 105


SN-OH2 [HOSN-H]+ TS. ........................................................................................... 105


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


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


intensities for [HOSN-H]+. ............................................................................................. 107


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

xxvii


intensities for [HON(H)S-W]+. ...................................................................................... 108


[HON(H)S-W]+ SN-2W TS. ..................................................................................... 108


intensities for [HON(H)S-W]+ SN-2W TS. .............................................................. 109


SN-2W. ........................................................................................................................... 109


intensities for SN-2W. .................................................................................................... 110


SN-2W [HOSN(H)-W]+ TS. ..................................................................................... 110


intensities for SN-2W [HOSN(H)-W]+ TS. .............................................................. 111


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


intensities for [HOSN(H)-W]+. ...................................................................................... 112


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


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

xxviii


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


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


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


intensities for SN-2W (H2O dielectric). ........................................................................ 115


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


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


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


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


HOSN-W. ....................................................................................................................... 117


intensities for HOSN-W. ................................................................................................ 118


HOSN-W HNSO-W TS. .......................................................................................... 119

xxix


intensities for HOSN-W HNSO-W TS. ................................................................... 120


HNSO-W ........................................................................................................................ 121


intensities for HNSO-W. ................................................................................................ 122


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


intensities for HOSN-W (H2O dielectric). .................................................................... 124


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


intensities for HOSN-W HNSO-W TS (H2O dielectric). ....................................... 126


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


intensities for HNSO-W(H2O dielectric). ..................................................................... 128


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

xxx


syn-triplet Ethoxycarbonylnitrene 2tsyn. (Dipole moment = 3.26 debye) ........................ 182


for syn-triplet Ethoxycarbonylnitrene 2tsyn. .................................................................... 183


the anti-singlet Ethoxycarbonylnitrene 2santi. (Dipole moment = 4.75 debye) ............... 184


for anti-singlet Ethoxycarbonylnitrene 2santi. ................................................................. 185


anti-triplet Ethoxycarbonylnitrene 2tanti.. (Dipole moment = 3.77 debye) ...................... 186


for anti-triplet Ethoxycarbonylnitrene 2tanti. .................................................................. 187


syn-singlet t-butoxycarbonylnitrene 4ssyn. . (Dipole moment = 4.80 debye) ................... 188


for syn-singlet t-butoxycarbonylnitrene 4ssyn. ................................................................. 189


syn-triplet t-butoxycarbonylnitrene 4tsyn. (Dipole moment = 3.50 debye) ..................... 190


for syn-triplet t-butoxycarbonylnitrene 4tsyn. .................................................................. 191


anti-singlet t-butoxycarbonylnitrene 4santi. (Dipole moment = 5.03 debye) ................... 192

xxxi


for anti-singlet t-butoxycarbonylnitrene 4santi. ............................................................... 193


anti-triplet t-butoxycarbonylnitrene 4tanti. (Dipole moment = 4.16 debye) .................... 194


for anti-triplet t-butoxycarbonylnitrene 4tanti. ................................................................ 195


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


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


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


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


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

......................................................................................................................................... 198


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


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


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

xxxii


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


for singlet formylnitrene 1HC(O)N. ............................................................................... 200


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


for triplet formylnitrene 3HC(O)N. ................................................................................ 201


the singlet Ethoxycarbonylnitrene rotation transition state 12TS. .................................... 202


for singlet Ethoxycarbonylnitrene rotation transition state 12TS. ..................................... 203


the triplet Ethoxycarbonylnitrene rotation transition state 32TS. ..................................... 204


for triplet Ethoxycarbonylnitrene rotation transition state 32TS. ...................................... 205


the singlet t-butoxycarbonylnitrene rotation transition state 14TS. .................................. 206


for singlet t-butoxycarbonylnitrene rotation transition state 14TS. ................................... 207


the triplet t-butoxycarbonylnitrene rotation transition state 34TS. ................................... 208

xxxiii


for triplet t-butoxycarbonylnitrene rotation transition state 34TS. .................................... 209

Table S6. 37. B3LYP/6-31G* optimized geometries, energies, and thermal corrections

for the syn-ethoxycarbonylnitrene-acetonitrile ylide 9. .................................................. 210


for the syn-ethoxycarbonylnitrene-acetonitrile ylide 9. .................................................. 211


for the anti-ethoxycarbonylnitrene-acetonitrile ylide 9. ................................................. 212


for the anti-ethoxycarbonylnitrene-acetonitrile ylide 9. ................................................. 213


syn-5-ethoxycarbonyliminodibenzothiophene ................................................................ 214


or syn-5-ethoxycarbonyliminodibenzothiophene 6. ....................................................... 215


anti-5-ethoxycarbonyliminodibenzothiophene 6. ........................................................... 216


for anti-5-ethoxycarbonyliminodibenzothiophene 6. ...................................................... 217


syn-5-t-butoxycarbonyliminodibenzothiophene 7. ......................................................... 218


for syn-5-t-butoxycarbonyliminodibenzothiophene 7. ................................................... 219

xxxiv


for anti-5-t-butoxycarbonyliminodibenzothiophene 7. ................................................... 220


for anti-5-t-butoxycarbonyliminodibenzothiophene 7. ................................................... 221

1

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

2

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

dehydrates to produce nitrous oxide (N2O) and water (k = 8 x 106 M-1 s-1, Scheme 1.2).12,13

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

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

the pKa of HNO was reported to be 4.7.15 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

HNO and nitrite (NO2-, Scheme 1.3a).17 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

3

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.

4

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).

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

5

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

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

6

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

ultimately N2O and nitrite.12,54

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

are possible.8 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.

7

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

8

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

1.5a).68,78 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.

9

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.

10

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.

Nitroxyl Improves Cellular Heart Function by Directly Enhancing Cardiac

Sarcoplasmic Reticulum Ca2+ Cycling. Circ. Res. 2007, 100, 96–104.

11

(7) Paolocci, N.; Jackson, M. I.; Lopez, B. E.; Miranda, K.; Tocchetti, C. G.; Wink, D.

A.; Hobbs, A. J.; Fukuto, J. M. The Pharmacology of Nitroxyl (HNO) and Its

Therapeutic Potential: Not Just the Janus Face of NO. Pharmacol. Ther. 2007,

113, 442–458.

(8) Fukuto, J. M.; Bartberger, M. D.; Dutton, A. S.; Paolocci, N.; Wink, D. a; Houk,

K. N. The Physiological Chemistry and Biological Activity of Nitroxyl (HNO):

The Neglected, Misunderstood, and Enigmatic Nitrogen Oxide. Chem. Res.

Toxicol. 2005, 18, 790–801.

(9) Froehlich, J. P.; Mahaney, J. E.; Keceli, G.; M, C.; Goldstein, R.; Redwood, A. J.;

Sumbilla, C.; Lee, D. I.; Tocchetti, C. G.; Kass, D. a; Paolocci, N.; Toscano, J. P.

Phospholamban Thiols Play a Central Role in Activation of the Cardiac Muscle

Sarcoplasmic Reticulum Calcium Pump by Nitroxyl. Biochemistry 2008, 13150–

13152.

(10) Fukuto, J. M.; Bianco, C. L.; Chavez, T. a. Nitroxyl (HNO) Signaling. Free Radic.

Biol. Med. 2009, 47 (9), 1318–1324.

(11) Gao, W. D.; Murray, C. I.; Tian, Y.; Zhong, X.; DuMond, J. F.; Shen, X.; Stanley,

B. A.; Foster, D. B.; Wink, D. A.; King, S. B.; Van Eyk, J. E.; Paolocci, N.

Nitroxyl-Mediated Disulfide Bond Formation Between Cardiac Myofilament

Cysteines Enhances Contractile Function. Circ. Res. 2012, 111, 1002–1011.

(12) Shafirovich, V.; Lymar, S. V. Nitroxyl and Its Anion in Aqueous Solutions: Spin

States, Protic Equilibria, and Reactivities toward Oxygen and Nitric Oxide. Proc.

Natl. Acad. Sci. 2002, 99, 7340–7345.

12

(13) Fehling, C.; Friedrichs, G. Dimerization of HNO in Aqueous Solution: An

Interplay of Solvation Effects, Fast Acid-Base Equilibria, and Intramolecular

Hydrogen Bonding? J. Am. Chem. Soc. 2011, 133 (44), 17912–17922.

(14) Bartberger, M. D.; Fukuto, J. M.; Houk, K. N. On the Acidity and Reactivity of

HNO in Aqueous Solution and Biological Systems. PNAS 2001, 98 (5), 2194–

2198.

(15) Grätzel, M.; Henglein, A.; Taniguchi, S. Pulse Radiolytic Studies on Nitrate Ion

Reduction, and on Formation and Decomposition of Pernitrous Acid in Aqueous

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Trapping by Phosphines: Kinetics and New Aqueous Ligations for HNO Detection

and Quantitation. J. Am. Chem. Soc. 2011, 133, 11675–11685.

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22

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:

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.

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

23

(N2O) (Scheme 2.1a).10 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

24

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.

15 minutes, kd = 7.7 x 10-4 s-1) 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-

25

hydroxybenzenesulfonamide (Piloty’s acid, PA), and 2-bromo-N-

hydroxybenzenesulfonamide (2-BrPA) (Figure 2.2). Typical MIMS traces observed at m/z

30 (NO+), 31 (HNO+/15NO+), 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.

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

1.2

0.8

0.4

0.0

Io

n C

urr

en

t (p

A)

12840

Time (min)

m/z 31

500

400

300

200

100

0

Io

n C

urr

en

t (p

A)

20151050 Time (min)

m/z 44 m/z 30

26

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.

27

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

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0.0

Io

n C

urr

en

t (p

A)

121086420-2 Time (min)

m/z 31

2000

1500

1000

500

0

Io

n C

urr

en

t (p

A)

121086420-2 Time (min)

m/z 44 m/z 30

350

300

250

200

150

100

50

0

Io

n C

urr

en

t (p

A)

121086420-2 Time (min)

m/z 30

2.0

1.5

1.0

0.5

0.0 I

on

Cu

rre

nt

(pA

)

121086420-2 Time (min)

m/z 31

(a) (b)

(c) (d)

28

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

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

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

NO from those due to HNO and/or N2O is the use of liquid nitrogen cold trap.25 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

2-BrPA) and N2O gas effectively, but not NO (produced from DEA/NO).25 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

29

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

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

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

30

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

of hydrogen peroxide (H2O2).34 Generation of HNO is observed from a variety of FeIII

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

31

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

results of hydroxylamine, oxidation of hydroxamic acids by H2O2/FeIII 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

32

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

33

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

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

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

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

34

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

35

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.

Further, H2S and NO have been shown to react to ultimately generate HNO.63 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

36

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

°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)

37

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.

38

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Babes, A.; Lampert, A.; Lennerz, J. K.; Jacobi, J.; Martí, M. A.; Doctorovich, F.;

Högestätt, E. D.; Zygmunt, P. M.; Ivanovic-Burmazovic, I.; Messlinger, K.; Reeh,

P.; Filipovic, M. R. H2S and NO Cooperatively Regulate Vascular Tone by

Activating a Neuroendocrine HNO-TRPA1-CGRP Signalling Pathway. Nat.

Commun. 2014, 5, 4381.

(64) Nelson, D. P.; Kiesow, L. A. Enthalpy of Decomposition of Hydrogen Peroxide by

Catalase at 25 °C (with Molar Extinction Coefficients of H2O2 Solutions in the UV).

Anal. Biochem. 1972, 49, 474–478.

48

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

dehydrates to form nitrous oxide (N2O).7 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

49

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

corresponding disulfide,23,24 ferric-heme peroxidation of hydroxylamine (NH2OH),25 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

50

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

51

(NH2OH),25 N-hydroxyguanadines,22,27,40 and hydroxamic acid scaffolds22 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

52

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

determined by GC headspace analysis of N2O (SEM ± 5%; n ≥ 3). aSubstrates (500 µM)

were incubated under the same conditions in the presence of H2O2 (2.5 mM) only. b

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

15NO+ that has fragmented from 15N2O. 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

53

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

concentrations (500 µM substrate with 100 µM H2O2 and 5 µM heme).25 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).

54

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

35

30

25

20

15

10

5

0

Ion C

urr

ent

(pA

)

20151050 Time (min)

m/z 30

m/z 30 (LN2)

0.25

0.20

0.15

0.10

0.05

0.00

Ion C

urr

ent

(pA

)

20151050 Time (min)

m/z 31

150

100

50

0

Io

n C

urr

en

t (p

A)

20151050 Time (min)

m/z 44

m/z 31

m/z 30

(a)

(b)

(c)

55

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

(NH3) in glutamine synthetase.41–43 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

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.

14

12

10

8

6

4

2

0

-2

Ion C

urr

ent

(pA

)

1086420-2

Time (min)

m/z 30 (100 µM H2O2)

m/z 30 (2.5 mM H2O2)

56

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).

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.

2.0

1.5

1.0

0.5

0.0

Ion C

urr

ent

(pA

)

151050

Time (min)

m/z 30

m/z 30 (LN2)

57

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.

58

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

25

20

15

10

5

0

Ion C

urr

ent

(pA

)

151050

Time (min)

m/z 30 (NH2OH)

m/z 30 (NHG)

m/z 30 (NHA)

59

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

60

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

NH2OH and HNO to produce ultimately N2 and H2O (Scheme 3.4a).47–49 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.

61

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).

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

62

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

63

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.





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

64

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.












65

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(33) Curi, R. Glutamine, Gene Expression, and Cell Function. Front. Biosci. 2007, 12,

344.

(34) Newsholme, P.; Procopio, J.; Lima, M. M. R.; Pithon-Curi, T. C.; Curi, R.

Glutamine and Glutamate--Their Central Role in Cell Metabolism and Function.

Cell Biochem. Funct. 2003, 21, 1–9.

(35) Khogali, S. E.; Harper, A. A.; Lyall, J. A.; Rennie, M. J. Effects of L-Glutamine on

Post-Ischaemic Cardiac Function: Protection and Rescue. J. Mol. Cell. Cardiol.

1998, 30, 819–827.

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(36) Khogali, S. E. O.; Pringle, S. D.; Weryk, B. V; Rennie, M. J. Is Glutamine Beneficial

in Ischemic Heart Disease? Nutrition 2002, 18, 123–126.

(37) Wischmeyer, P.; Vanden Hoek, T.; Li, C.; Shao, Z.; Ren, H.; Riehm, J.; Becker, L.

Glutamine Preserves Cardiomyocyte Viability and Enhances Recovery of

Contractile Function after Ischemia-Reperfusion Injury. J. Parenter. Enter. Nutr.

2003, 27, 116–122.

(38) Lauzier, B.; Vaillant, F.; Merlen, C.; Gélinas, R.; Bouchard, B.; Rivard, M.-E.;

Labarthe, F.; Dolinsky, V. W.; Dyck, J. R. B.; Allen, B. G.; Chatham, J. C.; Des

Rosiers, C. Metabolic Effects of Glutamine on the Heart: Anaplerosis versus the

Hexosamine Biosynthetic Pathway. J. Mol. Cell. Cardiol. 2013, 55, 92–100.

(39) DuMond, J. F.; King, S. B. The Chemistry of Nitroxyl-Releasing Compounds.


(40) Fukuto, J. M.; Stuehr, D. J.; Feldman, P. L.; Bova, M. P.; Wong, P. Peracid

Oxidation of an N-Hydroxyguanidine Compound: A Chemical Model for the

Oxidation of N-Hydroxyl-L-Arginine by Nitric Oxide Synthase. J. Med. Chem.

1993, 36, 2666–2670.

(41) Shapiro, B. M.; Stadtman, E. R. Glutamine Synthetase (Escherichia Coli). In

Methods in Enzymology; 1970; pp 910–922.

(42) Boehlein, S. K.; Schuster, S. M.; Richards, N. G. Glutamic Acid Gamma-

Monohydroxamate and Hydroxylamine Are Alternate Substrates for Escherichia

Coli Asparagine Synthetase B. Biochemistry 1996, 35, 3031–3037.

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(43) Geisseler, D.; Doane, T. A.; Horwath, W. R. Determining Potential Glutamine

Synthetase and Glutamate Dehydrogenase Activity in Soil. Soil Biol. Biochem.

2009, 41, 1741–1749.

(44) Minet, R.; Villie, F.; Marcollet, M.; Meynial-Denis, D.; Cynober, L. Measurement

of Glutamine Synthetase Activity in Rat Muscle by a Colorimetric Assay. Clin.

Chim. acta 1997, 268, 121–132.

(45) Knott, M. E.; Dorfman, D.; Chianelli, M. S.; Sáenz, D. A. Effect of Angeli’s Salt on

the Glutamate/glutamine Cycle Activity and on Glutamate Excitotoxicity in the

Hamster Retina. Neurochem. Int. 2012, 61, 7–15.

(46) Evans, A. S.; Cohen, A. D.; Gurard-Levin, Z. a.; Kebede, N.; Celius, T. C.; Miceli,

A. P.; Toscano, J. P. Photogeneration and Reactivity of Acyl Nitroso Compounds.

Can. J. Chem. 2011, 89, 130–138.

(47) Bonner, F. T.; Dzelzkalns, L. S.; Bonucci, J. A. Properties of Nitroxyl as

Intermediate in the Nitric Oxide-Hydroxylamine Reaction and in Trioxodinitrate

Decomposition. Inorg. Chem. 1978, 17, 2487–2494.

(48) Bonner, F. T.; Wang, N.-Y. Reduction of Nitric Oxide by Hydroxylamine. 1.

Kinetics and Mechanism. Inorg. Chem. 1986, 25, 1858–1862.

(49) Doctorovich, F.; Bikiel, D. E.; Pellegrino, J.; Suárez, S. A.; Martí, M. A. How to

Find an HNO Needle in a (Bio)-Chemical Haystack. In Progress in Inorganic

Chemistry; Karlin, K. D., Ed.; John Wiley & Sons, Inc., 2014; Vol. 58, pp 145–184.

(50) Palmer, L.; Frazier, C.; Read de Alaniz, J. Developments in Nitrosocarbonyl

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Discoveries. Synthesis. 2013, 46, 269–280.

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Anal. Biochem. 1972, 49, 474–478.

73

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

physiological roles.4–6 In mammals, H2S is generated by three enzymes, cystathionine-β-

synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase

(3-MST).7 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

sensitivity and selectivity,12–15 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,

74

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

excess H2S.17 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.

75

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

H2S.22,23 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.

Based on the pKa of H2S (pKa = 6.98 at 20 °C),24 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.

76

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.

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

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77

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.

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.

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78

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.

79

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

contain polysulfide contaminants (HS-(n+1), n ≥ 1).28 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;

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

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.

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80

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

81

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.

82

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

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83

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

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

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

84

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.

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.

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

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

85

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).

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.

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86

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.

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

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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

88

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),

which has a pKa of 3.5.33 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

89

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.

90

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.

91

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).

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

92

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.

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).

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

93

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.





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-

94

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.












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

95

(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

96

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.

97

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102

4.9 Supporting Information Chapter 4

4.9.1 Optimized Geometries and Energies


[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

103


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


[SN(H)OH]+ SN-OH2 TS.





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

104


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


SN-OH2.





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

105


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


SN-OH2 [HOSN-H]+ TS.





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

106


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


[HOSN-H]+.





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

107


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


[HON(H)S-W]+.





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

108


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


[HON(H)S-W]+ SN-2W TS.





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

109


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


-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


SN-2W.





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

110


intensities for SN-2W.


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


SN-2W [HOSN(H)-W]+ TS.





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

111


intensities for SN-2W [HOSN(H)-W]+ TS.


-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


[HOSN(H)-W]+.





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

112


intensities for [HOSN(H)-W]+.


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


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





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

113


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


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


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





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

114


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


-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


SN-2W (H2O dielectric).





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

115


intensities for SN-2W (H2O dielectric).


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


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





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

116


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


-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


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





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

117


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


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


HOSN-W.





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

118


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

119


HOSN-W HNSO-W TS.





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

120


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

121


HNSO-W





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

122


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

123


HOSN-W (H2O dielectric).





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

124


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

125


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





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

126


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

127


HNSO-W (H2O dielectric).





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

128


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

129

4.9.2 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.

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.

2500

2000

1500

1000

500

0

Ion C

urr

ent

(pA

)

86420-2

Time (min)

m/z 30

130

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.

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.

131

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.

132

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.

133

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.

134

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

state triplet, by a smaller margin of 18.5 kcal/mol.16 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

135

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.

136

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

calculations), singlet-triplet energy gaps (ΔEST = ES – ET) are overestimated.10,22,24 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

137

their ground state multiplicity and singlet-triplet splitting on reactivity, only three ΔEST

values have been determined experimentally. Negative ion photoelectron spectroscopy has

been used to determine ΔEST for imidogen (NH),26 methylnitrene (CH3N),27 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).

138

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.

139

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

140

States: Photochemistry of Acetyl-Substituted Aroyl and Aryloxycarbonyl Azides.

J. Am. Chem. Soc. 1988, 110, 4297–4305.



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.

141

(16) Travers, M. J.; Cowles, D. C.; Clifford, E. P.; Ellison, G. B. Photoelectron

Spectroscopy of the Phenylnitrene Anion. J. Am. Chem. Soc. 1992, 114, 8699–

8701.

(17) Platz, M. S. Comparison of Phenylcarbene and Phenylnitrenel. Acc. Chem. Res.

1995, 28, 487–492.

(18) Platz, M. S. Nitrenes. In Reactive Intermediate Chemistry; Moss, Robert A., Platz,

Matthew S., Jones, M., Ed.; John Wiley & Sons, Inc., 2004; pp 501–559.



J. Am. Chem. Soc. 1988, 110, 4297–4305.

(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

State? Mendeleev Commun. 2001, 11, 94–95.

(22) Liu, J.; Mandel, S.; Hadad, C. M.; Platz, M. S. A Comparison of Acetyl- and

Methoxycarbonylnitrenes by Computational Methods and a Laser Flash Photolysis

Study of Benzoylnitrene. J. Org. Chem. 2004, 69, 8583–8593.



J. Org. Chem. 2007, 72, 6848–6859.

142

(24) Pritchina, E. a.; Gritsan, N. P.; Bally, T. Ground State Multiplicity of Acylnitrenes:

Computational and Experimental Studies. Russ. Chem. Bull. 2005, 54, 525–532.

(25) Sherman, M. P.; Jenks, W. S. Computational Rationalization for the Observed

Ground-State Multiplicities of Fluorinated Acylnitrenes. J. Org. Chem. 2014, 79,

8977–8983.

(26) Engelking, P. C. Laser Photoelectron Spectrometry of NH−: Electron Affinity and

Intercombination Energy Difference in NH. J. Chem. Phys. 1976, 65, 4323.


Photoelectron Spectroscopy of the CH3N- Ion. J. Chem. Phys. 1999, 111, 23–25.

(28) Wijeratne, N. R.; Fonte, M. Da; Ronemus, A.; Wyss, P. J.; Tahmassebi, D.;

Wenthold, P. G. Photoelectron Spectroscopy of Chloro-Substituted Phenylnitrene

Anions. J. Phys. Chem. A 2009, 113, 9467–9473.

143

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-


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

144

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.

Scheme 6.2. Photolysis of t-butyloxycarbonylazide (3) to produce t-


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

145

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

146

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

calculations), singlet-triplet energy gaps (ΔEST = ES – ET) are overestimated.18,27,28 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).

147

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

carboxynitrene (HOC(O)N) by 7 kcal/mol,28 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)

correction,28 the ΔEST values of the syn- and anti-rotamers of t-butoxycarbonylnitrene are

148

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

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

149

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.

150

Previous TRIR work on other carbonylnitrenes has shown that they can be detected in

solution at frequencies similar to those calculated.29,33,38

151

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.

152

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),

upon photolysis of carboethoxyazide (1), and did not observe reactivity with O2.11 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).

153

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

-1.5x10-3

-1.0

-0.5

0.0

0.5

1.0

1.5

A

bso

rba

nce

1800 1750 1700 1650

Wavenumber (cm-1

)

1300 1250 1200 1150

ylide 8

1690 cm-1

triplet nitrene 2t

1640 cm-1

ylide 8

1285 cm-1

ylide 8

1160 cm-1

0.0 - 0.2 µs 0.2 - 0.4 µs 0.4 - 0.6 µs 0.6 - 0.8 µs 0.8 - 1.2 µs 1.2 - 2.0 µs 2.0 - 2.8 µs 2.8 - 3.6 µs

154

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).

155


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.

150x10-6

100

50

0

-50

Inte

nsity

8µs6420Time

ylide 8

1160 cm-1

kobs1 = 2.0 x 107 s

-1

kobs2 = 5.5 x 105 s

-1

Fast : Slow = 2 : 1

300x10-6

200

100

0Inte

nsity

800µs6004002000

Time

ylide 8

1690 cm-1

kobs = 3.5 x 103 s

-1

300x10-6

200

100

0

-100

In

ten

sity

8µs6420

Time

ylide 8

1690 cm-1

kobs1 = 2.0 x 107 s

-1

kobs2 = 4.7 x 105 s

-1

Fast : Slow = 1.1 : 1

-40x10-6

-20

0

20

40

In

ten

sity

8µs6420

Time

triplet nitrene 2t

1640 cm-1

kobs = 5.5 x 105 s

-1

(c)

(a)

(d)

(b)

156

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).

157


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).

1.0x10-3

0.5

0.0

-0.5

A

bsorb

ance

1800 1750 1700 1650 1600 1550

Wavenumber (cm-1

)


amide 10

1728 cm-1

triplet nitrene 2t

1640 cm-1

158


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.

600x10-6

400

200

0Inte

nsity

8µs6420

Time

amide 10

1728 cm-1

kobs1 = 2 x 107s

-1

kobs2 = 6.8 x 105s

-1

Fast : Slow = 2 : 1

-100x10-6

-50

0

50

In

tensity

8µs6420

Time

triplet nitrene 2t

1640 cm-1

kobs = 7.1 x 105s

-1

100x10-6

50

0

-50Inte

nsity

40µs3020100

Time

isocyanate 11

2180 cm-1

kobs > 2 x 107s

-1

(a)

(b)

(c)

159

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).

160


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).

3x10-3

2

1

0

-1

A

bsorb

ance

1850 1800 1750 1700 1650 1600

Wavenumber (cm-1

)

0.0 - 0.2 s

0.2 - 0.4 s

0.4 - 0.6 s

0.6 - 0.8 s

0.8 - 1.2 s

1.2 - 2.0 s

2.0 - 2.8 s

2.8 - 3.6 s

oxazolidinone 5

1762 cm-1

triplet nitrene 4t

1640 cm-1

161


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.

-60x10-6

-40

-20

0

20

40

Inte

nsity

3µs210

Time

triplet nitrene 4t

1640 cm-1

kobs = 2.3 x 106 s

-1

800x10-6

600

400

200

0Inte

nsity

3µs210

Time

oxazolidinone 5

1762 cm-1

kobs1 = 2 x 107 s

-1

kobs2 = 2.6 x 106 s

-1

Fast : Slow 9 : 1

200x10-6

100

0

-100

In

tensity

40µs3020100

Time

isocyanate 12

2180 cm-1

kobs > 2 x 107s

-1

(a)

(b)

(c)

162

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.

163


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).

3x10-3

2

1

0

-1

A

bso

rba

nce

1850 1800 1750 1700 1650 1600 1550

Wavenumber (cm-1

)


oxazolidinone 5

1752 cm-1

triplet nitrene 4t

1638 cm-1

164


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.

200x10-6

100

0

-100In

ten

sity

40µs3020100

Time

isocyanate 12

2175 cm-1

kobs > 2 x 107s

-1

-300x10-6

-200

-100

0

100

200

In

tensity

3µs210

Time

triplet nitrene 4t

1638 cm-1

kobs = 1.8 x 106s

-1

2.0x10-3

1.5

1.0

0.5

0.0

Inte

nsity

3µs210

Time

oxazolidinone 5

1752 cm-1

kobs1 = 2 x 107s

-1

kobs2 = 1.9 x 106s

-1

Fast : Slow = 1 : 1.4

(a)

(b)

(c)

165


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).

3x10-3

2

1

0

-1

A

bsorb

ance

1850 1800 1750 1700 1650 1600 1550

Wavenumber (cm-1

)


oxazolidinone 5

1764 cm-1

triplet nitrene 4t

1640 cm-1

166


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

1.5x10-3

1.0

0.5

0.0Inte

nsity

3µs210Time

oxazolidinone 5

1764 cm-1

kobs1 = 2 x 107s

-1

kobs2 = 9.6 x 105s

-1

Fast : Slow 1 : 1.3

150x10-6

100

50

0

-50

Inte

nsity

40µs3020100

Time

isocyanate 11

2180 cm-1

kobs > 2.0 x 107s

-1

200x10-6

100

0

-100Inte

nsity

3µs210

Time

triplet nitrene 4t

1640 cm-1

kobs = 9.8 x 105s

-1

(a)

(b)

(c)

167

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

the singlet (Scheme 6.6b), which is feasible small ΔEST values.42 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).

168

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

169

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.





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

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.

170

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%

ethyl acetate/hexane as an eluent to give 0.38 g (50%) of 6. 1H-NMR (CDCl3) δ 8.02 (d, J

171

= 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),

4.10 (q, J = 7.1 Hz, 2H), 1.20 (t, J = 7.1 Hz, 3 H); 13C-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

a white solid, 50% yield. 1H-NMR (CDCl3) δ 8.03 (d, J = 7.9 Hz, 2H), 7.63 (d, J = 7.8 Hz,

2H), 7.51 (t, J = 7.8 Hz, 2H), 1.43 (s, 9 H). 13C-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

172

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.

173

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180

6.8 Supporting Information Chapter 5

6.8.1 Optimized Geometries and Energies


the syn-singlet Ethoxycarbonylnitrene 2ssyn (Dipole moment = 4.55 debye)




Atom Atomic

Number

Cartesian Coordinates (Angstroms)

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

181


for syn-singlet Ethoxycarbonylnitrene 2ssyn.


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

182


syn-triplet Ethoxycarbonylnitrene 2tsyn. (Dipole moment = 3.26 debye)

UB3LYP/6-31G* energy -322.4225538 Hartrees



Atom Atomic

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

183


for syn-triplet Ethoxycarbonylnitrene 2tsyn.


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

184


the anti-singlet Ethoxycarbonylnitrene 2santi. (Dipole moment = 4.75 debye)




Atom Atomic

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

185


for anti-singlet Ethoxycarbonylnitrene 2santi.


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

186


anti-triplet Ethoxycarbonylnitrene 2tanti.. (Dipole moment = 3.77 debye)




Atom Atomic

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

187


for anti-triplet Ethoxycarbonylnitrene 2tanti.


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

188


syn-singlet t-butoxycarbonylnitrene 4ssyn. . (Dipole moment = 4.80 debye)




Atom Atomic

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

189


for syn-singlet t-butoxycarbonylnitrene 4ssyn.


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

190


syn-triplet t-butoxycarbonylnitrene 4tsyn. (Dipole moment = 3.50 debye)




Atom Atomic

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

191


for syn-triplet t-butoxycarbonylnitrene 4tsyn.


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

192


anti-singlet t-butoxycarbonylnitrene 4santi. (Dipole moment = 5.03 debye)




Atom Atomic

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

193


for anti-singlet t-butoxycarbonylnitrene 4santi.


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

194


anti-triplet t-butoxycarbonylnitrene 4tanti. (Dipole moment = 4.16 debye)




Atom Atomic

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

195


for anti-triplet t-butoxycarbonylnitrene 4tanti.


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

196


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




Atom Atomic

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


for syn-singlet hydroxycarbonylnitrene 1HOC(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

197


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




Atom Atomic

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


for syn-triplet hydroxycarbonylnitrene 3HOC(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

198


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




Atom Atomic

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


for anti-singlet hydroxycarbonylnitrene 1HOC(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

199


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




Atom Atomic

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


for syn-triplet hydroxycarbonylnitrene 3HOC(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

200


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




Atom Atomic

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


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

201


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




Atom Atomic

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


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

202


the singlet Ethoxycarbonylnitrene rotation transition state 12TS.




Atom Atomic

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

203


for singlet Ethoxycarbonylnitrene rotation transition state 12TS.


-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

204


the triplet Ethoxycarbonylnitrene rotation transition state 32TS.




Atom Atomic

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

205


for triplet Ethoxycarbonylnitrene rotation transition state 32TS.


-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

206


the singlet t-butoxycarbonylnitrene rotation transition state 14TS.




Atom Atomic

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

207


for singlet t-butoxycarbonylnitrene rotation transition state 14TS.


-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

208


the triplet t-butoxycarbonylnitrene rotation transition state 34TS.




Atom Atomic

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

209


for triplet t-butoxycarbonylnitrene rotation transition state 34TS.


-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

210


for the syn-ethoxycarbonylnitrene-acetonitrile ylide 9.




Atom Atomic

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

211


for the syn-ethoxycarbonylnitrene-acetonitrile ylide 9.


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

212


for the anti-ethoxycarbonylnitrene-acetonitrile ylide 9.




Atom Atomic

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

213


for the anti-ethoxycarbonylnitrene-acetonitrile ylide 9.


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

214


syn-5-ethoxycarbonyliminodibenzothiophene




Atom Atomic

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

215

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


or syn-5-ethoxycarbonyliminodibenzothiophene 6.


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

216

992 0.06 3214 21.26

1003 0.67 3215 19.72

1018 0.02 3226 8.78

1031 24.90 3237 7.00


anti-5-ethoxycarbonyliminodibenzothiophene 6.




Atom Atomic

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

217

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


for anti-5-ethoxycarbonyliminodibenzothiophene 6.


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

218

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


syn-5-t-butoxycarbonyliminodibenzothiophene 7.




Atom Atomic

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

219

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


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

220

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


for anti-5-t-butoxycarbonyliminodibenzothiophene 7.




Atom Atomic

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

221

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


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

222

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

223

6.8.2 Calculated Rotation Barriers

Figure S6.1. Energy profile for the rotation of C-O bond of singlet ethoxycarbonylnitrene 12.

12anti 12syn 12anti

224

Figure S6.2. Energy profile for the rotation of C-O bond of triplet ethoxycarbonylnitrene 32.

32syn

32anti

32syn

225

Figure S6.3. Energy profile for the rotation of C-O bond of singlet

t-butoxycarbonylnitrene 14.

14anti 14syn 14anti

226

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.

34syn

34anti

34syn

227

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

Compounds

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

N-ethoxycarbonyl dibenzothiophene sulfilimine_6.esp

8 7 6 5 4 3 2 1 0

Chemical Shift (ppm)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Norm

aliz

ed Inte

nsity

3.142.042.022.052.032.00

1.1

91.2

11.2

31.2

4

1.7

6

4.0

44.0

64.0

84.1

0

7.2

57.4

7

7.4

97.6

17.6

1

7.6

37.8

57.9

77.9

7

7.9

9

N-ETHOXYCARBONYL DIBENZOTHIOPHENE SULFILIMINE_1H.ESP

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


0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

Norm

aliz

ed Inte

nsity

228

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

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

N-ethoxycarbonyl dibenzothiophene sulfilimine_6_13C.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Norm

aliz

ed Inte

nsity

14.7

2

62.1

4

76.8

477.1

577.4

7122.3

0

127.6

2129.8

8132.4

8

138.1

4138.7

9

165.7

7

N-t-butyloxycarbonyl dibenzothiophene sulfilimine_1H.esp

8 7 6 5 4 3 2 1 0


0

0.05

0.10

0.15

0.20

0.25

0.30

Norm

aliz

ed Inte

nsity

9.112.072.052.012.00

1.4

2

1.6

4

7.2

57.4

97.5

07.5

17.6

27.6

27.8

67.8

78.0

18.0

18.0

38.0

3

229

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

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

N-t-butyloxycarbonyl dibenzothiophene sulfilimine_13C.esp

220 200 180 160 140 120 100 80 60 40 20 0 -20


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Norm

aliz

ed Inte

nsity

28.4

2

76.8

3

77.4

679.6

4

122.2

7

127.8

0129.8

3132.3

7

139.2

2

165.4

0

N-t-butyloxycarbonyl dibenzothiophene sulfilimine_CD3CN.esp

8 7 6 5 4 3 2 1


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Norm

aliz

ed Inte

nsity

9.232.062.092.002.00

1.2

7

1.9

31.9

31.9

41.9

51.9

52.1

4

7.5

97.6

07.6

07.7

07.7

27.7

47.9

57.9

78.0

68.0

8

230

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

N-t-butyloxycarbonyl dibenzothiophene sulfilimine_CD2Cl2.esp

8 7 6 5 4 3 2 1 0


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Norm

aliz

ed Inte

nsity

9.172.052.044.00

1.3

3

1.5

5

5.3

2

7.5

57.5

67.5

77.6

57.6

67.6

77.9

47.9

67.9

77.9

9

231


photolysis at 254nm in argon-purged CD3CN.

N-T-BUTYLOXYCARBONYL DIBENZOTHIOPHENE SULFILIMINE_CD3CN_4HR PHOTOLYSIS.ESP

8 7 6 5 4 3 2 1


0

0.05

0.10

0.15

0.20

0.25

0.30

Norm

aliz

ed Inte

nsity

5.801.851.104.001.942.08

1.1

71.2

71.3

9

1.9

31.9

31.9

41.9

51.9

52.1

7

3.2

6

7.5

07.5

17.5

2

7.5

27.9

37.9

47.9

57.9

68.2

68.2

78.2

78.2

8

232

Figure S6.13. 1H NMR (CD2Cl2) of oxycarbonylnitrene precursor 7 after 4 hour

photolysis at 254nm in argon-purged CD2Cl2.

N-t-butyloxycarbonyl dibenzothiophene sulfilimine_CD2Cl2_4hr photolysis.esp

8 7 6 5 4 3 2 1 0


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Norm

aliz

ed Inte

nsity

5.721.950.974.001.862.06

1.4

31.4

51.5

7

3.3

1

5.3

2

7.4

6

7.4

77.4

87.4

87.4

97.8

77.8

87.8

88.1

88.2

08.2

0

233


photolysis at 254nm in argon-purged CD3CN.

N-ethoxycarbonyl dibenzothiophene sulfilimine_CD3CN_4hr photolysis.esp

8 7 6 5 4 3 2 1 0


0

0.05

0.10

0.15

0.20

Norm

aliz

ed Inte

nsity

2.751.634.001.842.09

1.3

81.4

01.4

2

1.9

41.9

41.9

52.1

7

4.4

44.4

5

7.5

07.5

17.5

17.5

17.5

27.9

37.9

47.9

47.9

58.2

58.2

68.2

78.2

8

234


photolysis at 254nm in argon-purged CH3CN.

N-ethoxycarbonyl dibenzothiophene sulfilimine_CH3CN_4hr photolysis.esp

8 7 6 5 4 3 2 1


0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Norm

aliz

ed Inte

nsity

2.152.131.424.001.982.01

1.3

61.3

81.4

01.8

21.9

11.9

21.9

4

2.1

32.3

0

4.3

94.4

14.4

34.4

5

7.4

6

7.4

77.4

87.4

97.5

0

7.8

97.9

07.9

1

7.9

38.2

38.2

38.2

48.2

5

235

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).

236

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

spectroscopy has been used to determine ΔEST for imidogen (NH),18 methylnitrene

(CH3N),19 and phenylnitrene (PhN).20,21 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

237

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

previously studied nitrenes, phenylnitrene (PhN)20 and methylnitrene (CH3N)19. 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

238

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

239

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

240

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.

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

PhC(O)N CH3C(O)N CF3C(O)N PhN 20 CH3N 19

Experimentally

Observed

Transitions

3.15

3.68

2.85

3.30

3.75

4.25, 4.45

1.45

2.10

0.022

1.35

Calculated

Vertical

Transitions

3.06

(triplet)

3.4, 3.7

(singlet)

2.80

(triplet)

3.42

(singlet)

3.86

(triplet)

4.54, 4.58

(singlet)

1.52

(triplet)

2.1, 2.47

(singlet)

0.0027a

(triplet)

1.33

(singlet)

Calculated ΔEST

eV

(kcal/mol)

-0.124

(-2.86)

-0.186

(-4.29)

-0.0123

(-0.284)

0.568

(13.09)

1.447

(33.37)

241

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.

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).

Ph 1.532

N

O1.284

1.308

126.4° Ph 1.448

132.1°

N

O1.323

1.27385.6°

1.7642.314

117.7°

Ph 1.483

N

O1.251

1.375

114.6°

2.211

117.7°

H3C 1.555

N

O1.284

1.306

128.1°

2.328

117.3°

H3C 1.473

N

O1.319

1.26886.8°

1.778

131.2°

H3C 1.504

N

O1.248

1.371

115.3°

2.214

124.5°

F3C 1.573

N

O1.271

1.302

130.9°

2.341

115.8°

F3C 1.517

N

O1.304

1.26589.7°

1.813

130.9°

F3C 1.559

N

O1.238

1.362

119.4°

2.246

121.7°

22a 12a 32a

22b 12b 32b

22c 12c 32c

242

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).

243

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

therefore EST values, vary considerably with computational method.10,23 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

244

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

acetylnitrene, which is in close agreement to our calculated EST value.22

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

245

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,

246

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);

13C (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

give a white solid (80 mg, 22%). 1H NMR (400 Mhz, CDCl3) δ 7.36-7.45 (m, 3H), 7.56 (t,

2H), 7.68 (t, 2H), 7.95 (d, 2H), 8.13 (d, 2H), 8.25 (d, 2H); 13C (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

white solid: 18% yield; 1H NMR (400 MHz, CDCl3) δ 2.14 (s, 3H), 7.50 (t, 2H), 7.62 (t,

2H), 7.87 (d, 2H), 8.11 (d, 2H); 13C (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

247

purified by flash column chromatography (gradient, dichloromethane 100% – ethylacetate

100%) to give a white solid: 34% yield; 1H NMR (400 MHz, CDCl3) δ 7.59 (t, 2H), 7.73

(t, 2H), 7.94 (d, 2H), 8.17 (d, 2H); 13C (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).

Benzoyl azide,22 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,

CDCl3) δ 7.42 (m, 2H), 7.58 (t, 1H), 8.01 (d, 2H); 13C (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

248

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,

249

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

250

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)] [21/2which, 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

251

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

252

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.

253

7.6 References

(1) W. Lwowski. Nitrene. In Nitrenes; Lwowski, W., Ed.; John Wiley & Sons, Inc.,

New York, 1920; p 1.

(2) Platz, M. S. Nitrenes. In Reactive Intermediate Chemistry; Moss, Robert A., Platz,

Matthew S., Jones, M., Ed.; John Wiley & Sons, Inc., 2004; pp 501–559.

(3) Wasserman, E.; Smolinsky, G.; Yager, W. a. Electron Spin Resonance of Alkyl

Nitrenes. J. Am. Chem. Soc. 1964, 86, 3166–3167.

(4) Ferrante, R. F. Spectroscopy of Matrix-Isolated Methylnitrene. J. Chem. Phys.

1987, 86, 25.

(5) 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.



J. Am. Chem. Soc. 1988, 110, 4297–4305.

(7) Wasylenko, W. a; Kebede, N.; Showalter, B. M.; Matsunaga, N.; Miceli, A. P.;



13142–13150.


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Org. Chem. 2008, 73, 4398–4414.



J. Org. Chem. 2007, 72, 6848–6859.




1018.


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) Wasylenko, W. a.; Kebede, N.; Showalter, B. M.; Matsunaga, N.; Miceli, A. P.;



13142–13150.


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(18) Engelking, P. C. Laser Photoelectron Spectrometry of NH−: Electron Affinity and

Intercombination Energy Difference in NH. J. Chem. Phys. 1976, 65, 4323.


Photoelectron Spectroscopy of the CH3N- Ion. J. Chem. Phys. 1999, 111, 23–25.

(20) Travers, M. J.; Cowles, D. C.; Clifford, E. P.; Ellison, G. B. Photoelectron

Spectroscopy of the Phenylnitrene Anion. J. Am. Chem. Soc. 1992, 114, 8699–

8701.

(21) Wijeratne, N. R.; Fonte, M. Da; Ronemus, A.; Wyss, P. J.; Tahmassebi, D.;

Wenthold, P. G. Photoelectron Spectroscopy of Chloro-Substituted Phenylnitrene

Anions. J. Phys. Chem. A 2009, 113, 9467–9473.






256

8977–8983.

(24) Zeng, X.; Beckers, H.; Willner, H.; Grote, D.; Sander, W. The Missing Link:

Triplet Fluorocarbonyl Nitrene FC(O)N. Chem. - A Eur. J. 2011, 17, 3977–3984.

(25) Pederson, M. R.; Ruzsinszky, A.; Perdew, J. P. Communication: Self-Interaction

Correction with Unitary Invariance in Density Functional Theory. J. Chem. Phys.

2014, 140, 121103.

(26) Nakayama, J.; Yu, T.; Sugihara, Y.; Ishii, A. Synthesis and Characterization of

Thiophene 1-Oxides Kinetically Stabilized by Bulky Substituents at the 3- and 4-

Positions. Chem. Lett. 1997, 26, 499–500.

(27) Thomas, O. C.; Zheng, W.; Bowen, K. H. Magic Numbers in Copper-Doped

Aluminum Cluster Anions. J. Chem. Phys. 2001, 114, 5514.

(28) Porezag, D.; Pederson, M. Optimization of Gaussian Basis Sets for Density-

Functional Calculations. Phys. Rev. A 1999, 60, 2840–2847.

(29) Pederson, M. R.; Porezag, D. V; Kortus, J.; Patton, D. C. Strategies for Massively

Parallel Local-Orbital-Based Electronic Structure Methods. Phys. Status Solidi

2000, 217, 197–218.

(30) Pederson, M. R. Density-Functional-Based Prediction of a Spin-Ordered Open-

Shell Singlet in an Unpassivated Graphene Nanofilm. Phys. Status Solidi Basic

Res. 2012, 249, 283–291.

(31) Nossa, J. F.; Islam, M. F.; Canali, C. M.; Pederson, M. R. First-Principles Studies

of Spin-Orbit and Dzyaloshinskii-Moriya Interactions in the {Cu3} Single-

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Molecule Magnet. Phys. Rev. B - Condens. Matter Mater. Phys. 2012, 85, 1–10.

(32) Ashman, C.; Khanna, S.; Pederson, M.; Porezag, D. Thermal Isomerization in

Cs4Cl3-. Phys. Rev. A. 1998, 58, 744–747.

(33) Janak, J. F. Proof That dE/ni = Ei in Density-Functional Theory. Phys. Rev. B.

1978, 18, 7165–7168.

258

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

259

(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.

260

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.

261

Scheme 8.3. Potential products formed via THICA photolysis.

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.

1600

1400

1200

1000

800

600

400

200

0

Ion C

urr

ent (p

A)

151050Time (Minutes)

m/z 28m/z 44m/z 30m/z 31

LN2 Cold Trap

262

Scheme 8.4. Products resulting from the photogeneration of benzyloxynitrene in the

presence of argon (Ar) or dioxygen (O2).


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

1.2x10-3

1.0

0.8

0.6

0.4

0.2

0.0

A

bso

rba

nce

2300 2250 2200 2150 2100 2050 2000

Wavenumber

0.0 - 50.0 s

50.0 - 100.0 s

100.0 - 150.0 s

150.0 - 200.0 s

200.0 - 300.0 s

300.0 - 500.0 s

500.0 - 700.0 s

700.0 - 900.0 s

263

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.

264

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

significantly (pKa ~ 1).10 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).

265

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

266

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.

267

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-

workers suggested that H2S was responsible for the modification of cysteine;15,16 however,

the species responsible for production of RSSH was found not to be H2S.17,18 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

pKa units compared with their corresponding thiols.21 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

268

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

(see reaction sequence below).27–30 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

269

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.

270

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

of these species. For example, isocyantes (νNCO ca. 2200 cm-1) are known to react with

thiols to produce the corresponding S-thiocarbamates (νCO ca. 1650 cm-1).31 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

271

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

272

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).

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).

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

273

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

274

Scheme 8.12. Modification of peptide cysteine residues with methoxycarbonylsulfenyl

chloride to generate protected persulfides that can be released upon exposure to amine

nucleophiles.

275

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282

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.


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.


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.


Ernest M. Marks Award for Teaching Excellence (2013)

Organic Chemistry Teaching Assistant (August 2011 – May 2012)


Krieger School of Arts and Sciences Teaching Assistant Award (2012)

https://www.linkedin.com/in/tylerachavez

283

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).


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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