A Dissertation Entitled DNA Adducts from 5

Home , Hydroperoxyl, Sulfanyl

A Dissertation

entitled

DNA Adducts from 5'-Aldehyde Lesions and their Contributions to the Endogenous

Exposome

Shin Hae Cho, Pharm.D.

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the

Doctor of Philosophy Degree in

Medicinal Chemistry

______Amanda Bryant-Friedrich, Dr. rer. Nat., Committee Chair

______Meng-Cheh Liu Ph.D., Committee Member

______Zahoor Shah, Ph.D., Committee Member

______L.M. Viranga Tillekeratne, Ph.D., Committee Member

______Cyndee Gruden, Ph.D., Dean College of Graduate Studies

The University of Toledo

May 2019

This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An abstract of

DNA Adducts from 5'-Aldehyde Lesions and their Contributions to the Endogenous

Exposome

Shin Hae Cho

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Medicinal Chemistry

The University of Toledo

May 2019

Chemical species originating from oxidatively damaged DNA contribute to the composition of the endogenous exposome. These endogenous exposome may also form biological adducts that have implications in disease development. Identifying DNA adducts are crucial in understanding the etiology of disease states and manifestations of aging. This research seeks to determine if DNA damage contributes to the endogenous exposome through the formation of reactive intermediates. Reactive oxygen species that result from oxidative stress generate carbon-centered radicals in DNA. These radicals result in strand breaks, which liberate small molecules that can be further metabolized into unwanted compounds that maybe mutagenic or carcinogenic. Radicals generated at the 5' carbon (C5') of the 2-deoxyribose moiety result in a 5' aldehyde formation.

Consequently, a deoxynucleoside intermediate, or 3',4'-didehydro-3'-deoxy-5'- oxothymidine (ddoT) is formed when the oligonucleotide with 5' aldehyde degrades to a small molecule and a phosphorylated oligomer. ddoT is a reactive molecule proposed to form adducts with constituents of the cellular environment. In this work, properties of

iii ddoT, including its half-life and the conversion to furfural and thymine, were studied. In addition, we examined the reactivity and the viability of ddoT with low molecular weight thiols (glutathione, cysteine, N-acetylcysteine) and coenzyme A as well as developing analytical methods for detections. Glutathione, N-acetylcysteine, coenzyme A, and cysteine successfully formed adducts via Michael addition to ddoT, as evidenced by LC-

MS analysis. The study also reveals the dimerization of ddoT in the presence of cysteine.

This information will be useful in identifying ddoT adducts to confirm the formation of ddoT as a constituent of the endogenous exposome.

This dissertation is dedicated to my mother, Dr. Onekyun Choi.

Acknowledgements

Arnold Bennett once said, “The real Tragedy is the tragedy of the man who never in his life braces himself for his one supreme effort—he never stretches to his full capacity, never stands up to his full stature.” I owe a debt of gratitude to my research advisor Dean Amanda Bryant-Friedrich for her continuous care, patience, advice, and scholarly support throughout my academic career to “stand up on my full stature.” I also greatly thank Dr. Mel Bedi’s mentorship as a Pharm.D./Ph.D. in the same discipline, as well as his guidance in the research. I appreciate Dr. Yong-Wah Kim who helped me greatly with his expertise in NMR, Dr. Steve Peseckis for his motivation and knowledge in chemistry, and all my committee members, Dr. Ming-Cheh Liu, Dr. Zahoor Shah, and

Dr. Viranga Tillekeratne, for their undivided attention. Lastly, I express my sincere gratitude to my family and my labmates in Bryant-Friedrich’s lab who supported me during this journey.

Table of Contents

Acknowledgements ...... vi

Table of Contents ...... vii

List of Tables ...... xi

List of Figures ...... xii

List of Schemes ...... xx

List of Abbreviations ...... xxii

List of Symbols ...... xxv

1 Introduction and Background ...... 1

1.1 The Concept of Exposome ...... 1

1.1.1 Domains of the exposome ...... 3

1.1.1.1 Exogenous Exposome ...... 3

1.1.1.2 Endogenous Exposome ...... 4

1.1.1.3 How Does the Endogenous Exposome Play A Role in

DNA Damage? ...... 6

1.1.1.4 Types of oxidative DNA damage from the endogenous

exposome ...... 9

1.1.2 DNA base damage ...... 9

1.1.3 DNA sugar damage ...... 10

vii

1.1.3.1 DNA C1' Radicals ...... 11

1.1.3.2 DNA C2' Radicals ...... 12

1.1.3.3 DNA C3' Radicals ...... 13

1.1.3.4 DNA C4' Radicals ...... 14

1.1.3.5 DNA C5' Radicals ...... 15

1.2 DNA Adducts from damage lesions ...... 18

2 Results and Discussions ...... 21

2.1 Synthesis of ddoT (58) ...... 21

2.2 Synthesis of Michael adducts of ddoT ...... 25

2.3 ddoT adduct formation with glutathione under physiological conditions

...... 33

2.3.1 Method development for analysis of ddoT ...... 33

2.3.2 Reactivity of ddoT with glutathione ...... 37

2.4 ddoT adduct formation with physiological thiols under modified

conditions ...... 39

2.4.1 Method development of ddoT and degradation products with LC-

MS in modified conditions...... 39

2.4.2 A glutathione adduct of ddoT ...... 44

2.4.3 A coenzyme A adduct of ddoT ...... 52

2.4.4 A N-acetylcysteine adduct of ddoT ...... 54

2.4.5 A cysteine adduct of ddoT ...... 57

3 Conclusion and Future Research Directions ...... 62

3.1 Summary ...... 62

viii

3.2 Future Research Directions ...... 65

3.2.1 Biological Relevance of ddoT adduct formation ...... 65

3.2.1.1 Detection of Michael adducts from ddoT from calf

thymus DNA ...... 65

3.2.1.2 Detection of Michael adducts from ddoT from

oligonucleotides with 5' lesions ...... 67

3.3 Conclusion ...... 68

4 Experimental procedures ...... 70

4.1 Materials ...... 70

4.2 Structural Analysis ...... 71

4.2.1 1H-NMR, 13C-NMR ...... 71

4.2.2 Mass Spectrometry ...... 72

4.2.2.1 ESI-MS ...... 72

4.2.2.2 MALDI-ToF MS ...... 73

4.3 Chromatographic Methods ...... 74

4.3.1 Thin Layer Chromatography (TLC) ...... 74

4.3.2 Reverse-Phase HPLC (RP-HPLC) ...... 74

4.4 Other equipment and Devices ...... 75

4.5 Synthesis of C5'-modified thymidine ...... 75

4.5.1 3',4'-Didehydro-2',3'-dideoxy-5'-oxothymidine (ddoT) ...... 75

4.5.1.1 Synthesis of 5'-O-(tert-butyldimethylsilyl)-thymidine ... 75

4.5.1.2 Synthesis of 5'-O-(tert-butyldimethylsilyl)- 3'-O-benzoyl-

thymidine ...... 76

4.5.1.3 Synthesis of 3'-O-benzoyl-thymidine ...... 77

4.5.1.4 Synthesis of 3',4'-Didehydro-2',3'-dideoxy-5'-

oxothymidine (ddoT) with IBX71, 84...... 77

4.5.2 Synthesis of Michael adduct using thiol-containing compounds43

...... 78

4.6 Reactivity and stability of C5'-modified thymidine ...... 79

4.6.1 Stability of ddoT in PBS ...... 79

4.6.2 Stability of ddoT in BBS ...... 79

4.6.3 Calibration curve of ddoT ...... 79

4.6.4 Fragmentation of ddoT ...... 80

4.6.5 Reactivity of ddoT in GSH ...... 80

4.6.6 Sample preparation of ddoT ...... 80

4.6.7 Reactivity of ddoT in GSH ...... 80

4.6.8 Reactivity of ddoT in Coenzyme A (CoA) ...... 81

4.6.9 Reactivity of ddoT in Cysteine ...... 81

4.6.10 Reactivity of ddoT in N-Acetyl-Cysteine ...... 81

4.6.11 Fragmentation of ddoT-RSH adducts ...... 82

4.6.12 Time response reaction with RSH ...... 82

Appendix A ...... 83

References ...... 105

List of Tables

Table 1.1. “The endogenous exposome steady-state amounts of endogenous DNA

damage.” The endogenous exposome. DNA repair. 2014;19:3-13.Reprinted

with permission.6 ...... 5

Table 2.1. pKa of selected thiol-containing substrates ...... 28

Table 2.2. Validation data for the determination ddoT for monitoring (n=5) ...... 40

Table 4.1. LC-MS Condition A ...... 73

Table 4.2. HPLC Condition A ...... 75

Table A- 1. Chemical properties of physiologically available Michael acceptors ...... 93

List of Figures

Figure 1-1. “Three domains of the exposome are presented diagrammatically with non-

exhaustive examples for each of these domains.” Wild C. P., et al. The

exposome: from concept to utility. Int J Epidemiol. 2012;41:24-32. Reprinted

with permission.9 ...... 3

Figure 1-2. “Risk factors for exposures that contribute to chronic-disease mortality. The

chart was compiled from World Health Organization estimates of exposures

affecting 50 million global deaths in 2010 (Lim et al. 2012). (Because some

risk factors may be correlated, the indicated percentages are approximate.).”

Rappaport, S. et al. Environ Health Perspect. 2014;122(8)769-774. Reprinted

with permission.11 ...... 4

Figure 1-3. Primary structure and the nucleobase of DNA. The dotted line represents

hydrogen bonds between the complementary base pairs...... 6

Figure 1-4. “Sequential one-electron reduction of molecular oxygen to water.” Oxygen:

how do we stand it? Fridovich I., et al. Med Princ Pract. 2013;22(2):131-7.

Reprinted with permission.17 ...... 8

xii

Figure 2-1. MALDI-TOF mass spectrum of products from Michael addition with A) 2-

mercaptobenzothiazole B) 3-mercaptobenzoic acid C) thiophenol ...... 30

Figure 2-2. Analysis of 1 mM 18, 58, and 59 by HPLC-PDA with detection at 254 nm

after incubation under physiological conditions (pH 7.4, 37 °C) in phosphate

buffered saline. The mobile phase was composed of 5 mM ammonium

acetate:CH3CN (50:50, v:v)...... 34

Figure 2-3. HPLC-DAD UV spectrum of 58 in phosphate buffered saline...... 35

Figure 2-4. HPLC-DAD UV spectrum of 18 in phosphate buffered saline...... 36

Figure 2-5. HPLC-DAD UV spectrum of 59 in phosphate buffered saline...... 36

Figure 2-6. HPLC chromatogram of 80 μM ddoT (58) in the presence of 6 mM

glutathione over time at 254 nm under physiological conditions (pH 7.4, 37

°C) in phosphate buffered saline. The mobile phase was composed of 5 mM

ammonium acetate:CH3CN (50:50, v:v)...... 38

Figure 2-7. Degradation of 80 μM of 58 in 6 mM glutathione under physiological

conditions (pH 7.4, 37 °C) in phosphate buffered saline ...... 38

Figure 2-8. Analysis of 18, 58, and 59 by HPLC-PDA at 254 nm in 10 mM borate

buffered saline at pH 8.0 at 37 °C. The mobile phase was composed of 0.1%

acetic acid:CH3CN (v:v) ...... 40

Figure 2-9. The calibration curves for 18, 58, and 59 at pH 8.0 in 10 mM BBS buffer ... 41

Figure 2-10. The change of ddoT concentration in borate buffered saline (pH 8.0 at 40

C) ...... 42

Figure 2-11. The change of thymine, furfural concentration in borate buffered saline (pH

8.0 at 40 C) ...... 42

xiii

Figure 2-12. Expected ddoT adducts formed by Michael addition with physiological

thiol-containing biomolecules...... 43

Figure 2-13. Analysis of ddoT (0.625 mM) in the presence of glutathione (0.625 mM) in

vitro at 40 °C BBS buffer (pH 8.0) by HPLC-UV-MS ...... 45

Figure 2-14. Electrospray ionization mass spectrum of GSH adduct (MH+ 530) in

positive mode. Ion chromatograms of summed ions: m/z 530, 548 in BBS

buffer (pH 8.0) at 40 oC ...... 46

Figure 2-15. Electrospray ionization mass spectrum of GSH adduct (MH- 528) in

negative mode. Ion chromatograms of summed ions: m/z 528, 546 in BBS

buffer (pH 8.0) at 40 oC ...... 47

Figure 2-16. Total ion chromatogram of GSH-ddoT adducts (pink) and ddoT (black)

from Figure 2-13...... 48

Figure 2-17. Predicted ddoT adducts from Michael addition ...... 48

Figure 2-18. Fragmentation pattern of 85 by LC-MS/MS ...... 49

Figure 2-19. Change of formation of glutathione adduct with 85 and 86 over 24 hrs ..... 50

Figure 2-20. Formation of thymine and furfural as degradation products of ddoT in the

precence of glutathione over 24 hrs ...... 51

Figure 2-21. Analysis of ddoT (0.625 mM) in the presence of CoA (0.625 mM) at 40 °C

at BBS buffer (pH 8.0) by HPLC-UV-MS at 24 hrs ...... 52

Figure 2-22. Electrospray ionization mass spectrum of CoA adduct (MH+ 990) in positive

mode. Ion chromatograms of summed ions: m/z 496 (M+2H+), 768 (CoA),

990 ([M+H]+) in BBS buffer (pH 8.0) at 40 oC...... 53

xiv

Figure 2-23. Electrospray ionization mass spectrum of CoA adduct (MH- 988) in negative

mode. Ion chromatograms of summed ions: m/z 494, 765, 988 in BBS buffer

(pH 8.0) at 40 oC...... 54

Figure 2-24. Analysis of ddoT (0.625 mM) in the presence of N-Acetylcysteine (0.625

mM) at 40 °C at BBS buffer (pH 8.0) by HPLC-UV-MS at 24 hrs ...... 55

Figure 2-25. Electrospray ionization mass spectrum of NAC-ddoT adduct (MH+ 386) and

the sodium adduct (M+Na+ 408), NAC-ddoT hydrate adduct M+Na+ (MH+

426) in positive mode. Ion chromatograms of summed ions: m/z 386, 408,

426 in pH 8 buffer at 40 °C...... 56

Figure 2-26. Electrospray ionization mass spectrum of NAC adduct (MH- 384) and NAC-

ddoT hydrate adduct (MH- 402) in negative mode. Ion chromatograms of

summed ions: m/z 384, 402 in BBS buffer (pH 8.0) at 40 oC...... 56

Figure 2-27. Analysis of ddoT (0.625 mM) in the presence of cysteine (0.625 mM) at 40

°C at BBS buffer (pH 8.0) by HPLC-UV-MS at 24 hrs ...... 57

Figure 2-28. Electrospray ionization mass spectrum of cysteine adduct (MH+ 344) in

positive mode. Ion chromatograms of summed ions: m/z 344 in BBS buffer

(pH 8.0) at 40 °C ...... 58

Figure 2-29. Electrospray ionization mass spectrum of cysteine adduct (MH- 342) in

negative mode. Ion chromatograms of summed ions: m/z 342, 360 in BBS

buffer (pH 8.0) at 40 oC...... 58

Figure 2-30. The proposed structure of ddoT from potential dimerization ...... 59

Figure 2-31. Electrospray ionization mass spectrum of 92 in positive mode. Ion

chromatograms of summed ions: m/z 447 in BBS buffer (pH 8.0) at 40 oC . 59

Figure 2-32. Electrospray ionization mass spectrum of 92 in negative mode. Ion

chromatograms of summed ions: m/z 445 in BBS buffer (pH 8.0) at 40 oC . 60

Figure 3-1. A method for the preparation of calf thymus DNA for adduct formation ..... 66

Figure 3-2. HPLC chromatogram of supernatant from ct-DNA - control ...... 67

Figure A- 1. Positive mode ESI-MS ionization spectrum of 58 (direct injection) ...... 83

Figure A- 2. Positive mode ESI-MS ionization spectrum of 58 on column ...... 84

Figure A- 3. Positive SIM mode ESI-MS/MS ionization spectrum of 58 ...... 84

Figure A- 4. Positive mode ESI-MS/MS product ion spectrum of 58 at CE 30eV with

(top) column; without column (bottom) ...... 85

Figure A- 5. Positive mode ESI-MS/MS product ion spectrum of 58 at CE 25eV with

(top) column; without column (bottom) ...... 86

Figure A- 6. Positive mode ESI-MS/MS ionization spectrum of 58 at various injection

volumes (from top, 0.5 uL, 1 uL, 5 uL) ...... 87

Figure A- 7. Negative mode ESI-MS/MS ionization spectrum of 58 at various injection

volumes (from top, 1 uL, 5 uL) at CE 25 eV ...... 88

Figure A- 8. Positive mode ESI-MS/MS precursor ion spectrum of 58 at CE 30eV ...... 89

Figure A- 9. Positive mode ESI-MS/MS precursor ion spectrum of 58 at CE 25eV ...... 89

Figure A- 10. Positive mode ESI-MS/MS precursor ion spectrum of 58 at CE 20eV ..... 90

Figure A- 11. Positive mode ESI-MS/MS precursor ion spectrum of 58 at CE 15eV ..... 90

Figure A- 12. Positive mode ESI-MS/MS precursor ion spectrum of 58 at CE 10eV ..... 91

Figure A-13. Calibration curve of ddoT (58) absorbance as a function of the

concentration at 37 °C ...... 91

Figure A- 14. UV spectra of 58 at various wavelengths ...... 92

xvi

Figure A- 15. UV spectra of 59 at various wavelengths ...... 92

Figure A- 16. UV spectra of 18 at various wavelengths ...... 92

Figure A- 17. Electrospray ionization mass spectrum of GSH adduct (MH+ 530) in

positive mode at tR 22 min in Figure 2-13. Ion chromatograms of summed

ions: m/z 530, 548 in BBS buffer (pH 8.0) at 40 oC...... 94

Figure A- 18. Electrospray ionization mass spectrum of GSH adduct (MH- 528) in

negative mode at tR 22 min in Figure 2-13. Ion chromatograms of summed

ions: m/z 529, 546 in BBS buffer (pH 8.0) at 40 oC...... 94

Figure A- 19. Electrospray ionization mass spectrum of GSH adduct (MH+ 530) in

positive mode at tR 23.7 min in Figure 2-13. Ion chromatograms of summed

ions: m/z 530, 548 in BBS buffer (pH 8.0) at 40 oC...... 95

Figure A- 20. Electrospray ionization mass spectrum of GSH adduct (MH- 528) in

negative mode at tR 23.7 min in Figure 2-13. Ion chromatograms of summed

ions: m/z 528, 546 in BBS buffer (pH 8.0) at 40 oC...... 95

Figure A- 21. Electrospray ionization mass spectrum of GSH adduct (MH+ 530) in

positive mode at tR 25.3 min in Figure 2-13. Ion chromatograms of summed

ions: m/z 530, 548 in BBS buffer (pH 8.0) at 40 oC...... 96

Figure A- 22. Electrospray ionization mass spectrum of GSH adduct (MH- 528) in

negative mode at tR 25.3 min in Figure 2-13. Ion chromatograms of summed

ions: m/z 528, 546 in BBS buffer (pH 8.0) at 40 oC...... 96

Figure A- 23. Electrospray ionization mass spectrum of GSH adduct (MH+ 530) in

positive mode at tR 28.8 min Figure 2-13. Ion chromatograms of summed

ions: m/z 530, 548 in BBS buffer (pH 8.0) at 40 oC...... 97

xvii

Figure A- 24. Electrospray ionization mass spectrum of GSH adduct (MH- 528) in

negative mode at tR 28 min in Figure 2-13. Ion chromatograms of summed

ions: m/z 528, 546 in BBS buffer (pH 8.0) at 40 oC...... 97

Figure A- 25. Positive SIM mode ESI-MS ionization spectrum of 85 ...... 98

Figure A- 26. Negative SIM mode ESI-MS ionization spectrum of 85 ...... 98

Figure A- 27. Negative SIM mode ESI-MS ionization spectrum of 85 in the presence of

Et3N ...... 99

Figure A- 28. UV spectra of 1 at various wavelengths in Figure 2-16 ...... 100

Figure A- 29. UV spectra of 3 at various wavelengths in Figure 2-16 ...... 100

Figure A- 30. UV spectra of 4 at various wavelengths in Figure 2-16 ...... 100

Figure A- 31. Positive SIM mode ESI-MS ionization spectrum of 87 ...... 101

Figure A- 32. Negative SIM mode ESI-MS ionization spectrum of 87 ...... 101

Figure A- 33. Total ion chromatogram in positive SIM mode ESI-MS ionization

spectrum of 87, [CoA-ddoT+H]+ = 990 m/z ...... 102

Figure A- 34. Total ion chromatogram in positive SIM mode ESI-MS ionization

spectrum of 87, [CoA-ddoT-H]- = 989 m/z ...... 102

Figure A- 35. Analysis of 18 in the presence of GSH for 24 hrs by HPLC at 254 nm and

280 nm in borate buffered saline in pH 8.0 at 37 °C. The mobile phase was

composed of 0.1% acetic acid:CH3CN (v:v) ...... 102

Figure A- 36. Analysis of 59 in the presence of GSH for 24 hrs by HPLC at 254 nm and

280 nm in borate buffered saline in pH 8.0 at 37 °C. The mobile phase was

composed of 0.1% acetic acid:CH3CN (v:v) ...... 102

xviii

Figure A- 37. Analysis of 18 in the presence of CoA for 24 hrs by HPLC at 254 nm and

280 nm in borate buffered saline in pH 8.0 at 37 °C. The mobile phase was

composed of 0.1% acetic acid:CH3CN (v:v) ...... 102

Figure A- 38. Analysis of 59 in the presence of CoA for 24 hrs by HPLC at 254 nm and

280 nm in borate buffered saline in pH 8.0 at 37 °C. The mobile phase was

composed of 0.1% acetic acid:CH3CN (v:v) ...... 103

Figure A- 39. Analysis of 18 in the presence of NAC for 24 hrs by HPLC at 254 nm and

280 nm in borate buffered saline in pH 8.0 at 37 °C. The mobile phase was

composed of 0.1% acetic acid:CH3CN (v:v) ...... 103

Figure A- 40. Analysis of 59 in the presence of NAC for 24 hrs by HPLC at 254 nm and

280 nm in borate buffered saline in pH 8.0 at 37 °C. The mobile phase was

composed of 0.1% acetic acid:CH3CN (v:v) ...... 103

Figure A- 41. Analysis of 18 in the presence of cysteine for 24 hrs by HPLC at 254 nm

and 280 nm in borate buffered saline in pH 8.0 at 37 °C. The mobile phase

was composed of 0.1% acetic acid:CH3CN (v:v) ...... 103

Figure A- 42. Analysis of 59 in the presence of Cysteine for 24 hrs by HPLC at 254 nm

and 280 nm in borate buffered saline in pH 8.0 at 37 °C. The mobile phase

was composed of 0.1% acetic acid:CH3CN (v:v) ...... 104

xix

List of Schemes

Scheme 1-1. DNA sugar radicals generated at each carbon center in DNA and their

mechanistic fate24 ...... 10

Scheme 1-2. Fate of the C1' uridinyl radical in DNA25-27 ...... 11

Scheme 1-3. Fate of the C2' radical in DNA25-27 ...... 12

Scheme 1-4. Fate of the C3' radical in DNA25-27 ...... 13

Scheme 1-5. Fate of C4' radicals in DNA25-27 ...... 14

Scheme 1-6. Fate of C5' radicals in DNA16, 25, 27, 60 ...... 16

Scheme 2-1. Synthesis of 3',4'-didehydro-2',3'-dideoxy-5'-oxothymidine25, 71, 77-78 ...... 21

Scheme 2-2. Synthesis of Dess-Martin periodinane81-82 ...... 23

Scheme 2-3. Formation of hydrate71 ...... 24

Scheme 2-4. “Possible mechanistic scenarios of strand scission in DNA containing T-al.”

Rana, A., et al. Reactivity of the major product of C5′-oxidative damage in

nucleosome core particles. Chembiochem. 2019;20(5):672-676. Reprinted

with permission. (Author’s note: This is a proposed mechanistic pathways of

intermediate formation during T-al scission by computational study of T-al

expressed in lysine-rich nucleosome core particles.)75 ...... 26

Scheme 2-5. Mechanism of Michael addition of ddoT ...... 27

Scheme 2-6. Michael addition of 58 with various thiol-containing substrates ...... 29

Scheme 2-7. Synthesis of 83 from literature43 ...... 31

Scheme 2-8. Fragmentation pattern of 18, 58, and 59 obtained by ESI-MS/MS ...... 35

Scheme 2-9. The decomposition of 1 mM 58 in phosphate buffered saline at 4 C in pH

7.4 ...... 37

Scheme 2-10. The decomposition of 58 in borate buffered saline (40 C, pH 8.0) ...... 41

Scheme 2-11. Proposed mechanism of pinacol coupling of 58 ...... 61

xxi

List of Abbreviations

1H NMR ...... Proton Nuclear Magnetic Resonance Spectroscopy 13C NMR ...... Carbon-13 Nuclear Magnetic Resonance Spectroscopy 5fC…………………..5-Formylcytosine

A ...... Adenine Ac ...... Acetate Acr…………………..Acrolein AP…………………...Apurinic/Apyrimidinic Ar ...... Argon

B…………………….Base BBS…………………Borate Buffered Saline BER…………………Base Excision Repair BME…………………-mercaptoethanol

C ...... Cytosine C1……………………Carbon 1 C2 ...... Carbon 2 C3 ...... Carbon 3 C4 ...... Carbon 4 C5 ...... Carbon 5 CE…..………………Collusion Energy CoA…………………Coenzyme A Cys…………………..Cysteine ctDNA………………Calf-Thymus DNA d...... Doublet dA ...... Deoxyadenosine DAD…………………Diode array detector dC ...... Deoxycytidine dd...... Doublet of Doublet dG ...... Deoxyguanosine DCM ...... Dichloromethane ddoT ...... 3',4'-Didehydro-2',3'-Dideoxy-5'-Oxothymidine DHB…………………2,5-Dihydroxybenzoic acid DMF ...... N,N'-Dimethylformamide

xxii

DMSO ...... Dimethyl Sulfoxide DMSO-d6 ...... Deuterated Dimethyl Sulfoxide DMP………………...Dess-Martin Periodinane DMT ...... Dimethoxytrityl DNA ...... Deoxyribonucleic Acid DPC…………………DNA-protein crosslink DOB…………………1,4-Dioxobutane dsDNA ...... Double-Stranded Deoxyribonucleic Acid dT ...... Deoxythymidine

Et……………………Ethyl EtOH ...... Ethanol ESI-MS ...... Electrospray Ionization Mass Spectrometry

G ...... Guanine GSH...... Reduced Glutathione GSSG ...... Oxidized Glutathione

HPLC ...... High-Performance Liquid Chromatography

IBX………………….2-iodoxybenzoic acid IR...... Ionizing Radiation

LC-MS ...... Liquid Chromatography–Mass Spectrometry LC-MS ...... Tandem Liquid Chromatography–Mass Spectrometry LOD…………………Limit of Detection LOQ…………………Limit of Quantification Lys…………………..Lysine

M1dG………………..3-(Pyrimido[1,2-α]purin-10(3H)-one m ...... Multiplet MALDI-TOF...... Matrix-Assisted Laser/Desorption Ionization Time-of-Flight Mass Spectrometry MBA………………3-mercaptobenzoic acid MBT…………………2-mercaptobenzothiazole MRM………………..Multiple Reaction Monitoring MS ...... Mass Spectrometer/Mass Spectrometry

NAC…………………N-Acetylcysteine NER...... Nucleotide excision repair NMR ...... Nuclear Magnetic Resonance Spectroscopy

ODN ...... Oligodeoxynucleotide

PBS………………….Phosphate buffered saline PDA…………………Photodiode array detector

xxiii

PGA…………………Phosphoglycoaldehyde Ph ...... Phenyl phen…………………Phenanthroline Pyr ...... Pyridine q...... Quadruplet

RP-HPLC ...... Reverse Phase-HPLC ROS ...... Reactive Oxygen Species RSD…………………Relative Standard Deviations s ...... Singlet sep ...... Septet s/n…………………..Signal to Noise Ratio ssDNA ...... Single-Stranded DNA

T ...... Thymine, time t ...... Triplet TBDMS-Cl ...... tert-Butyldimethylsilyl Chloride tert…………………...Tertiary TFA ...... Trifluoroacetic Acid THF ...... Tetrahydrofuran TLC ...... Thin Layer Chromatography tR…………………….Retention Time in Minutes

UV ...... Ultraviolet

xxiv

List of Symbols

' ...... Prime °C ...... Degrees Celcius

α ...... Alpha β ...... Beta δ ...... Chemical Shift λ ...... Wavelength λmax ...... Maximum Wavelength

µL ...... Microliter µm ...... Micrometer eq ...... Equivalent hr ...... Hour J ...... Coupling Constant k……………………..Rate constant L ...... Liter n……………………..Number M ...... Molar mAU ...... Milli Absorption Unit min ...... Minute mL ...... Milliliter µM ...... Micromolar mM ...... Millimolar mmol ...... Millimole M-1s-1………………..Second order rate constant unit for k m/z...... Mass-to-Charge Ratio ng/mL (ng mL-1)…….One-Thousandth ppm nmol ...... Nanomole pKa………………….Dissociation Constant rpm………………….Revolutions Per Minute s-1……………………First Order Rate Constant Unit for k T½…………………...Half-life

xxv

5′-AODN……………Oligonucleotides Ending with 5′-Aldehyde Group 8-OHdG……………..8-Hydroxy-2′-Deoxyguanosine

Ac2O ...... Acetic Anhydride BzCl…………………Benzoyl chloride CD3CN ...... Deuterated Acetonitrile CDCl3 ...... Deuterated Chloroform CH3CN………………Acetonitrile EtOAc……………….Ethyl Acetate Et3N…………………Triethylamine Fe2+………………….Ferrous Ion Fe3+…………….……Ferric Ion FeSO4……………….Ferrous Sulfate H ...... Hydrogen H+ ...... Proton H2O ...... Water H2O2 ...... Hydrogen Peroxide HOO• ...... Hydroperoxyl Radical KHSO5 ...... Potassium Peroxymonosulfate Mn…………………..Manganese NaBH4………………Sodium Borohydride Na2B4O7……………..Sodium Tetraborate NaCl ...... Sodium Chloride NaHCO3 ...... Sodium Bicarbonate NaOH ...... Sodium Hydroxide NH4OAc…………….Ammonium Acetate [O]…………………..Oxidation O2 ...... Oxygen - O2• ...... Superoxide Radical Anion •OH ...... Hydroxyl Radical -OH ...... Hydroxyl Ion Pyr…………………...Pyridine RSH…………………Substrate with Thiols T-al…………………..Oligonucleotides Ending with Thymidine-5′-Aldehyde TMPyP………………5,10,15,20-Tetrakis(1-methyl-4-pyridinio)porphyrin

xxvi

Chapter 1

1 Introduction and Background Introduction and Background

1.1 The Concept of Exposome

With an increase in life expectancy in economically developed countries over past decades, the causes of mortality have shifted from communicable diseases to non- communicable chronic diseases.1 Scientists have studied to prevent these chronic illnesses by identifying risk factors in connection to genetics and the environment. In recent years, the Human Genome Project has allowed for significant improvements in investigating and preventing diseases that are associated with genetic susceptibility.

However, mounting evidence suggests that multiple illnesses such as cancer, diseases originally believed to be emanating from genomic traits, have a strong causal relationship with environmental contributors. According to new findings, genetic factors only contribute to a fraction of the underlying causes of disease development.2 This shows a discrepancy in the current trends of research that heavily focus on genomics to find disease associations. It also highlights the fact that the effects of environmental factors are still not fully accounted for.2-3

In 2005, the concept of the exposome, defined as the totality of exogenous exposures and physiological responses from conception to death,4 was introduced to elucidate how environmental factors in combination with genomics can cause chronic illnesses. The pathways in the development of these chronic diseases originate from the intricate interactions of the exposome with the genome, proteasome, transcriptome, and metabolome. Consequently, secondary disease phenotypes, or comorbidities, can develop.5 The concept of the exposome has brought about a new way of approaching disease etiologies and pathways. Nonetheless, measuring the impact of the exposome on disease etiology is still underdeveloped. There are no well-established controls, no standard methods for quantification, and no predictable population-dependent variables.4,

The scientific challenges centers around the development of methodologies that can quantitatively or qualitatively measure the exposome. Biomarkers4 have been effective tools in measuring the exposome to study disease susceptibility. In the study of biomarkers, the knowledge of metabolism, DNA adduct formation, molecular structures of biomarkers, and the mechanism of biomarker formation provide the scientific rationale to identify exposure-associated biomarkers to promote early diagnosis of diseases. For example, exposure to aflatoxin, a carcinogen found from agricultural crops, can be determined by the detection of adducts such as aflatoxin-N7-guanine in urine. This adduct is formed after depurination of the aflatoxin DNA conjugate, generating apurinic sites.7 This information is useful in assessing risks associated with hepatocellular carcinomas.8 Here we focus on developing analytical methods to detect a potential biomarker resulting from adduct formation from oxidatively damaged DNA.

1.1.1 Domains of the exposome

Before moving forward, the exposome can be further categorized for better understanding of the origins of DNA damage and how it is related. The exposome can be broadly differentiated into two categories based on the source of exposure: internal exposome (endogenous) and external (exogenous) exposome (Figure 1-1).9 The initial definition of the exposome that mainly described the exogenous exposome was later redefined to include the endogenous exposome.10

Figure 1-1. “Three domains of the exposome are presented diagrammatically with non- exhaustive examples for each of these domains.” Wild C. P., et al. The exposome: from concept to utility. Int J Epidemiol. 2012;41:24-32. Reprinted with permission.9

1.1.1.1 Exogenous Exposome

The exogenous exposome includes general external and specific external constituents as illustrated in Figure 1-1Figure 1-2Error! Reference source not found..

They involve environmental exposures such as ionizing radiation (IR) i.e. UV radiation, climate, medical diagnostic procedures such as X-rays, smoking, urban versus rural environments, diet, and pollution.9 Recent studies11-12 have found that 51% of the risk factors associated with mortality caused by chronic diseases have been identified to be environmental factors, such as airborne particles or dietary intake (Figure 1-2). The other

49% are yet to be identified, but the answers to the unknown contributing factors may be found with the ongoing investigation to understand the endogenous exposome.

Figure 1-2. “Risk factors for exposures that contribute to chronic-disease mortality. The chart was compiled from World Health Organization estimates of exposures affecting 50 million global deaths in 2010 (Lim et al. 2012). (Because some risk factors may be correlated, the indicated percentages are approximate).” Rappaport, S. et al. Environ Health Perspect. 2014;122(8)769-774. Reprinted with permission.11

1.1.1.2 Endogenous Exposome

As the name implies, the endogenous exposome is composed of ubiquitous cellular components as well as products resulting from alterations of the cellular

4 environment. The alteration can occur via exogenous sources, cellular metabolism, aging, or diseases known to be associated with oxidative stress. Swenberg and colleagues introduced the biological implications with respect to DNA damage within the domains of the endogenous exposome.6 As a result of these DNA damage, base or sugar modifications, and single or double strand breaks may occur, which ultimately induce unwanted biological responses endogenously.13 Therefore, the composition of chemicals in physiological compartments as a result of DNA damage also account for a part of the endogenous exposome.

Table 1.1. “The endogenous exposome steady-state amounts of endogenous DNA damage.” The endogenous exposome. DNA repair. 2014;19:3-13.Reprinted with permission.6

Endogenous DNA lesions Number per cell AP sites 30,000 OHEtG 3,000 7-(2-Oxoethyl)G 3,000 8-OxodG 2,400 Formaldehyde 1,000-4,000 Acetaldehyde 1,000-5,000 7-methylguanine 2,300 AcrdG 120 M1dG 60 N2,3-Ethenoguanine 36 1N2-Etheno dG 30 1N2-Etheno dA 12 O6-Methyl dG 2 Total 40,000+

Shown in Table 1.1 are the steady state levels of constituents of the endogenous exposome from DNA damage.6 According to this table, the majority of the endogenous exposome in cell is apurinic/apyrimidinic (AP). Guanine is most frequently damaged due to its high oxidation potential. The primary focus of this research involves elucidation of

5 constituents of the endogenous exposome, especially from oxidative damage of the 2- deoxyribose of DNA.

1.1.1.3 How Does the Endogenous Exposome Play A Role in DNA Damage?

Figure 1-3. Primary structure and the nucleobase of DNA. The dotted line represents hydrogen bonds between the complementary base pairs.

DNA contains genetic information and is essential for replication, transcription, and translation according to the central dogma of molecular biology.14 Knowing the structure of DNA helps in understanding how DNA contribute to the endogenous exposome. DNA is composed of deoxyribose, or sugar moieties, that are linked through

N-glycosidic bonds to nitrogenous pyrimidine and purine bases. The nitrogenous bases are shown in Figure 1-3. Adenine and guanine are purines; cytosine and thymine are

6 pyrimidines. The phosphates at the 3' and 5' position form phosphodiester linkages to connect one DNA monomer to another. Watson-Crick base pairing through hydrogen bonding (A-T and G-C) facilitates double helix formation, most commonly in the B form.

B form DNA is the most common types of DNA that is right-handed and has 10 nucleotides per turn. B form DNA has C2' endo confirmation with the nucleobases in an anti position.

Accumulated DNA damage in our body encompasses a long-term deleterious effect that contributes to the development of cancer and other chronic illnesses. DNA damage may originate from the exogenous exposome such as ionizing radiation (IR), or accumulate from insults caused by normal cellular metabolism. In the case of IR, energy can be directly deposited into DNA; this direct effect of IR accounts for 40% of all IR damage. However, the most common effect of IR involves the indirect effect; when secondary low energy electrons, generated through the radiolysis of water, and interact with the cellular medium. This accounts for 60% of IR damage. Our body generates highly reactive oxygen species (ROS) by our own cellular metabolism, such as the reduction of oxygen during oxidative phosphorylation in mitochondria (Scheme 1-4).

Through interactions of ROS with molecules in the cell, one of which is DNA, severe damage can occur. Among ROS,15 the hydroxyl radical (•OH) is one of the most common to damage DNA. Hydrogen atoms at the 5 carbon centers of the sugar or 2-deoxyribose moiety can be abstracted by a hydroxyl radical (•OH), although base damage may occur as well.16

Figure 1-4. “Sequential one-electron reduction of molecular oxygen to water.” Oxygen: how do we stand it? Fridovich I., et al. Med Princ Pract. 2013;22(2):131-7. Reprinted with permission.17

During cellular homeostasis, the effect of ROS is counteracted by antioxidants.

When this fine balance is distorted, it causes oxidative stress, altering the cellular environment, and ultimately leading to DNA damage. This causes the formation of small electrophilic fragments that further degrade into products that can be carcinogenic.

Fortunately, we have number of repair mechanisms that can fix errors such as base excision repair (BER) or nucleotide excision repair (NER). However, if not fully repaired, the generated species contribute to the endogenous exposome. When damaged

DNA is not repaired properly, it can be detrimental causing mutations, senescence or apoptosis.18 Damaged DNA may have the ability to form adducts with nearby biomolecules, or alter the mechanism of normal healthy cells. Damaged DNA can also crosslink with proteins and other nucleophilic substituents during repair processes.19

These all play roles in disease development. Recent studies revealed that base and sugar damage products from DNA may form adducts that increase with oxidative stress.20-21,22-

23 If these damaged products can be identified and measured, the endogenous exposome can be understood in detail with respect to diseases associated with oxidative stress.

1.1.1.4 Types of oxidative DNA damage from the endogenous exposome

Our body has several systems to repair damaged DNA, but some damage is not easily repairable especially if the environment is altered due to disease or stress. It is therefore important to address damage to nucleic acids from oxidative stress. Oxidative

DNA damage may cause strand breaks, although not all damage leads to such consequences.20

1.1.2 DNA base damage

Electrophilic hydroxyl radicals are generated most commonly, and cause damage at the base of nucleotides. This event can generate apurinic, apyrimidinic (AP) sites or modified bases within the DNA, distorting the integrity of the double helix. One of the most common products of base damage is oxidized guanosine, 8-oxo-dG (8-oxo-dG), because of guanine’s high oxidation potential. In fact, the majority of constituents of the endogenous exposome shown in Table 1.1 are the result of base damage. Base damage is most commonly repaired by a repair mechanism called base excision repair (BER). BER replaces damaged bases with the correct ones without distorting the double helix unlike nucleotide excision repair (NER), which is used for severe types of damage.

1.1.3 DNA sugar damage

The sugar moiety of DNA is often oxidatively damaged in cells.20 Evidence suggest that deoxyribose damage is linked to oxidative stress and inflammation.20 The deoxyribose moiety has five carbon centers from which hydroxyl radicals can abstract any of the seven hydrogens. The abstraction of hydrogen atoms from 2-deoxyribose results in radical formation. The reactivity of the hydroxyl radical at each carbon center depends on the solvent accessibility of each hydrogen atom to the solvent or surrounding water. 16 The most common damage occurs at the hydrogens at the C5' position, then 4', followed by 3' and 1' due to similar rates of hydrogen atom abstraction.

Scheme 1-1. DNA sugar radicals generated at each carbon center in DNA and their mechanistic fate24

1.1.3.1 DNA C1' Radicals

The C1' position is unique in DNA as it is the only carbon directly attached to the base through an N-glycosidic bond. Therefore, the abstraction of the hydrogens at this position forms a N-glycosyl radical.

Scheme 1-2. Fate of the C1' uridinyl radical in DNA25-27

In earlier studies, the C1' radical was generated as the 2'-deoxyuridin-1'-yl radical

(6).28-29 The anaerobic reduction of 6 with -mercaptoethanol (BME) yields 2 anomeric products (11 and 12). The  (11) and  (12) anomers are distinguished by the orientation of the N-glycosidic bond at the 1' position.  anomer formation is favored in nature, and it is effective in trapping the radicals. The  anomer is less effective at trapping radicals, less favored in nature, and pre-mutagenic.12 Under aerobic conditions, peroxyl radicals are formed at the 1' position (14) in the presence of oxygen. Peroxyl radicals can quickly release superoxide anion to form an unstable carbocation intermediate. The cation intermediate (15) reacts with water, and a ribonolactone (13) is formed. This ribonolactone may also form in small amounts through anaerobic reduction.

Ribonolactone 13 is further degraded to an -unsaturated butanolide species (16). This species was found to form DNA crosslinks with lysine residues of polymerase  and

11 endonuclease III by amide formation instead of normal Schiff base formation.30-31

Butanolide 16 decomposes further ,-conjugated 5-methylene-2-furanone (17). Product

16 has the ability to undergo Michael addition with nearby biomolecules.32-33

1.1.3.2 DNA C2' Radicals

Unlike the at C1' position, the C2' carbon center has two hydrogen atoms that can be abstracted. A C2' radical (7) was generated by -irradiation as 5-uridinyl radical34 and the radical was trapped by oxygen to form a peroxyl radical.35 The peroxyl radical (20) degraded to an erythrose abasic site (21) as the major product in B form DNA with a demonstrated half-life of 3 hours in 0.1 M NaOH at 37 °C.36 The product 13 may also form as a result of 1,2-hydride shift of a carbocation generated at C1' after releasing the superoxide anion.37

Scheme 1-3. Fate of the C2' radical in DNA25-27

1.1.3.3 DNA C3' Radicals

A 3' radical has been generated by the Norrish Type I photocleavage reaction25, 38 and in the presence of Rh-phenanthrenequinone diamine complexes.39-40 Under aerobic conditions, the -phosphatoxyalkyl radical (8) can form isomeric peroxyl radicals41 (23,

24) which are reduced to the hydrogen peroxide (25) in the presence of thiols. The stable peroxide (25) then undergoes Criegee-like rearrangement to generate intermediate 26, a

6-membered ring with a positive charge dwelling at C3'. The 6-membered ring undergoes intramolecular rearrangement to release base propenoate (30) and 3'- phosphoglycoaldehyde (3'-PGA, 29). Compound 29 was studied using 2- phosphoglycoaldehyde (2-PGA) as a model, and it was found that 2-PGA is further degraded into glyoxal (31) under physiological conditions which forms diastereomeric

1,N2-gloyxal adducts with dG.42

Scheme 1-4. Fate of the C3' radical in DNA25-27

The C3' radical can also be oxidized under anaerobic conditions and undergo solvolysis to form a carbocation intermediate (32) followed by the formation of 33. The elimination of phosphate from 33 gives rise to 34 which undergoes further elimination to form a small sugar fragment (36) containing two ,-unsaturated systems. Compound 36 forms Michael adducts with thiols demonstrated using 2-mercaptobenzoic acid.43 The presence of thiols under anaerobic conditions reduces 8 in ssDNA to two anomers (37 and 38).40 In addition to the aforementioned damage products, another 3' radical degradation pathway involves the formation of damage products that may also arise from the 4' or 5' radical degradation pathways, which will be introduced later.

1.1.3.4 DNA C4' Radicals

The C4' radical degradation pathway is one of the most studied of all the carbon centers, and first to be studied. It has been observed to form from ROS generated by

44 45 46-48 enediynes antibiotics, Cu(phen)2, and iron bleomycin. Independent generation of

C4' was also studied.49-50

Scheme 1-5. Fate of C4' radicals in DNA25-27

C4' radicals undergo either anaerobic or aerobic reactions to form different sugar fragments.51 Under anaerobic conditions, the radical at C4' is trapped by glutathione or other reducing agents, resulting in two anomers (43 and 44).52 In the absence of glutathione, Under anerobic conditions heterolytic cleavage generates 39 in ss- and dsDNA.52 The radical cation can be trapped by water to form 40.52 Reduction allows the formation of 41, whereas pseudo-repaired 42 is formed under otherwise anaerobic conditions.52 Under aerobic conditions, peroxyl radicals form reversibly, or the reduced hydrogen peroxide (46) undergoes Criegee-like rearrangement to form base propenal (50) and 3'-phosphoglycolate (3'-PG, 51). Product 51 may also arise in the degradation of the

21, C4' radical. The base propenal (50) can form a base-modified adduct with dG to M1dG.

1.1.3.5 DNA C5' Radicals

Radical formation by the abstraction of a hydrogen atom at C5', which is located in the minor groove of DNA, is the result of attack by ROS in the cellular environment.16,

54-55 Although damage at the C5' position was among the first to be studied,44 the research has focused less on C5' damage affiliated with mutagenesis and polymerase bypass due to the lack of abasic sites until recently.20 The C5' radical was studied in Fenton chemistry,

45 56-57 gamma radiolysis, Cu(phen)2 derivatives, or Mn(TMPyP)/KHSO5, and enediyne derivatives.44 Depending on the environment in which the C5' radical is generated, different mechanisms such as radical trapping, cyclization, or oxidation occur (Scheme

1-6). One of the unique characteristics of radical generation at the C5' position under anaerobic conditions is the formation of sugar-base cyclization product 49. These damage products are found to be associated with an increase in oxidative stress in mammalian

15 cells.58-59 The rate of reaction at which stereoselective anaerobic damage products 5',8-

26-27 cyclopurines (B=G, A, in 49) are formed is faster than that aerobic oxidation with O2.

This damage product is repaired by NER (Nucleotide Excision Repair) pathway.

Scheme 1-6. Fate of C5' radicals in DNA16, 25, 27, 60

In the presence of enediyne compounds, radical formation has been observed via two major degradation pathways: C5' aldehyde (57) formation and a minor pathway involving abasic site 3'-formyl-phosphate (63) and 5'-(2-phosphoryl-1,4-dioxobutane)

(60) formation. When oxygen is introduced, a peroxyl radical at C5' is formed (53), which is reduced to hydroperoxide 54. GSH prompts formation of 57. Through alkoxyl disproportion, 63 and 64, are formed, where 64 is hydrolyzed to 62. Opening of the ring of 63 promotes formation of highly reactive trans-1,4-dioxo-2-butene (60) upon elimination.60-61 The ability of 60 to hinder long patch base excision repair (BER) as well

16 as its half-life and ability to form adducts with Tris buffer has been demonstrated.62-63

Metalloporphyrin was also used to initiate radical formation at the C5' position.64

In the cationic porphyrin mechanism, hydroxylation occurs to deliver 56 (B=G). Upon

62 the release of H2O, the 5' aldehyde 57 is formed. Lesion 57 is removed by short and long patch BER by DNA polymerase β (Pol β) in the presence or absence of flap endonuclease 1 (FEN1).65 However, this known repair pathway is ineffective and poses a threat to the cellular environment. A 3'-phosphate 19 is released from the 5'-aldehyde

(57), forming a dideoxynucleoside 58. Compound 58 undergoes β elimination to form furfural (59) by base release (18).60, 64, 66-68 Furfural is known to be carcinogenic.69,70

Studies have been conducted in our laboratory to understand these pathways involved in

C5' oxidation,62, 71-74 but details on the fate of the 5' aldehyde, its degradation products, and the formation of adducts in a cellular environment are still under investigation. In order to have a better understanding of oxidative damage, a T-al, thymidine-5'-aldehyde, which terminates an otherwise undamaged oligonucleotide, was generated synthetically and the half-life was determined by Abdallah25 based on methods used in the Greenberg laboratory.25, 62 The 5'-AODN has also been shown to form adducts with lysine residues at nucleosome core particles (NCP).75 Compound 58 is a damaged intermediate from 5'-

AODN degradation that has been both synthesized71 and identified under biological conditions;60, 64 however, the reactivity is not fully understood.

1.2 DNA Adducts from damage lesions

Aldehydes are electrophilic in nature, and often form adducts when found in the human body. As mentioned previously, before the 5'-aldehyde (57) degrades to furfural

(59), an intermediate (58) is formed. However, due to the chemical instability of substrate

58, its fate and mechanism of degradation are yet to be fully understood.24 In addition, no findings have confirmed the formation of adducts with cellular constituents.

ddoT (58 where B=T) appears to be highly reactive due to the presence of an

-unsaturated aldehyde moiety, therefore it is not likely to be found free in cells. ddoT

(58 where B=T) was also independently synthesized in our laboratory25 and the half-life was determined under physiological conditions.25 However, adduct formation with biological molecules such as glutathione, the mechanism of adduct formation, or the susceptibility of ddoT to adduct formation was not determined. Due to its chemical structure, ddoT is expected to act as a Michael acceptor with the potential to form adducts with physiological nucleophiles. These adducts may play a role in understanding disease manifestations as constituents of endogenous exposome. Minor groove-binding nucleophiles form DNA adducts that are potentially mutagenic or carcinogenic to cells. It is directly linked to aging and the development of various chronic disease states.4 The

-unsaturated aldehyde in ddoT can react in Michael fashion with thiol-containing biomolecules such as glutathione to form covalent adducts. In earlier studies, several groups have described the formation of adducts of thiols from reactive intermediates formed from DNA oxidation. The 5-methylene-2-furanone derivative has been synthesized via Michael addition using -mercaptoethanol.32 Stubbe successfully derivatized 2-methylene-3(2H)-furanone, an -unsaturated ketone derived from C3'-

18 radical formation in DNA, using ethanethiol through Michael addition.33 Dedon’s laboratory also identified glutathione conjugates of base-propenals which result from damage at the C4ʹ position of DNA.76 The 58 can undergo the same mechanism to form thiol adduct.

In the past, studies have focused on the effect of high dose mutagens, but it is of greater importance to acknowledge the impact of biological adducts that are present at lower concentrations for risk assessment.6 This information can be ultimately utilized to test the extent of 5'-oxidation in diseased versus healthy individuals.25 The objective of this project is to identify adducts from C5' lesions as they relate to the endogenous exposome originating from oxidative nucleic acid damage. In this dissertation, we synthesized damage products believed to be derived from 5' aldehyde lesions, and investigated their reactivity and viability with thiol-containing molecules.

Chapter 2

2 Results and Discussions Results and Discussions

2.1 Synthesis of ddoT (58)

T-al (57, where B=T) undergoes -elimination to form ddoT (3’,4’-didehydro-

2’,3’-dideoxy-5’-oxothymidine, 58), which is an  -unsaturated aldehyde. Compound

58 (B=T) further degrades by releasing the nucleobase (18, B=T) to form furfural (59).

Because it is our primary interest to investigate the stability and reactivity of 58 (B=T), it was synthesized as shown in Scheme 2-1Error! Reference source not found..25, 71, 77-78

Scheme 2-1. Synthesis of 3’,4’-didehydro-2’,3’-dideoxy-5’-oxothymidine25, 71, 77-78

Silylation of commercially available thymidine (72) was performed per published

21 literature71 to deliver 5’-O-tert-butyldimethylsilylthymidine (73) in 92% yield.77

Benzoylation of 73 led to the formation of 5’-O-tert-butyldimethylsilyl-3’-benzoyl- thymidine (74) with a yield of 63%.71 Compound 74 was deprotected using

THF/TFA/H2O (5:1:1) to form 75, then different routes of oxidation was explored.

The synthesis of 58 (B=T) using Moffat oxidation was reported,71, 79-80 but the limitations of DMSO used in this approach lies in difficulty isolating compounds from the solvent by evaporating DMSO. In addition, the overall scheme is more time- consuming because it takes overnight, and more complex despite the higher yields because of the number of reagents and workup needed for this reaction.71 Previously in our laboratory, we were able to successfully synthesize compound 58 (B=T) using Dess-

Martin reagent.25 Dess-Martin oxidation followed by base-catalyzed elimination provided

58 in 21% yield. With this knowledge we chose to try IBX in this reaction. Using IBX delivered 58 (B=T) in a slightly higher 29% yield. Compared to IBX, the oxidation of 75 with Dess-Martin reagent requires overnight stirring for the completion, while IBX takes

2-3 hours.

The idea to substitute IBX for the Dess-Martin reagent stems from the structural similarity of the two and the synthetic steps of Dess-Martin. The synthesis of Dess-

Martin reagent81-82 Error! Reference source not found.is a two-step process starting from

2-iodobenzoic acid which is oxidized to 75 using oxone. IBX (77) is then acetylated to form Dess-Martin reagent (78).

Scheme 2-2. Synthesis of Dess-Martin periodinane81-82

Dess-Martin reagent (78) is moderately soluble in solvents such as CH3CN or DCM, and slightly soluble in ether or hexane.83 On the other hand, IBX is virtually insoluble in most solvents but sufficiently soluble in EtOAc, CH3CN, and acetone to convert alcohols to ketones or aldehydes at an elevated temperature.83-84 Due to the similar chemical properties between IBX and Dess-Martin reagent and the distinct solubility differences, it makes IBX a good reagent for oxidation in our case. It was also reported that IBX can be regenerated after oxidation by reoxidizing o-iodobenzoic acid, the byproduct of IBX oxidation.84 Therefore, the oxidation of 75 using IBX is a more sustainable alternative in addition to the shorter reaction time required even if percent yield of the final product is similar to that of the Dess-Martin reaction.

IBX oxidation at 80 °C followed by organic workup with anhydrous ether followed by treatment with NEt3 led to the formation of the oxidized substrate as well as the final product 58. Although the exact mechanism is unknown, the spontaneous elimination by the abstraction of the proton  to the 5' aldehyde to form a conjugated system is expected.

The aldehyde at the C5' position is in equilibrium with the corresponding hydrate under aqueous conditions delivering a 80:20 mixture of 79 and 80.71

Scheme 2-3. Formation of hydrate71

Thus, the oxidation product was not purified to prevent the formation of the hydrate.

Compound 79 (B=T) was then treated with triethylamine (Et3N) to afford final product

25 58. The literature used 3 molar equivalents of Et3N to obtain full conversion, but a total of 1 molar eq. of Et3N was sufficient to obtain the desired product 58. It may due to the partial formation of 58 during oxidation that less Et3N was needed to fully convert to be

58. Purity and identity were confirmed by NMR and ESI-MS, respectively. Synthesized

58 was also used as an analytical standard.

2.2 Synthesis of Michael adducts of ddoT

To classify DNA damage products as constituents of the endogenous exposome,6 their involvement in disease susceptibility and etiology must be assessed. For example, 8- oxo-dG, a base-modified nucleoside, is used in risk assessment for cancer85,86 because the level of 8-oxo-dG is associated with oxidative damage in DNA, which is part of cancer etiology. It is known that DNA damage leads to the formation of adducts, which can be used as markers to detect oxidative stress.16, 24, 27, 87-88 Therefore, investigating the chemical properties of 58 and identifying any adducts that can form with it is important to understand if it is a link between the endogenous exposome and diseases associated with oxidative stress.

Greenberg et al proposed two different pathways, one of which leads to the formation of 58.75 He proposed that pathway B is more probable due to the low energy barriers associated with the rate limiting phosphate elimination step. This assertion stems from the observation of the DNA-protein crosslink of 5-formylcytosine (5fC) in the nucleosome core particle through reaction with primary amines in histones.89 From this study, they found that the imine formed through lysine reacting with the 5fC through the

Schiff-base formation is reversible. Therefore, although it requires lower energy to form imine, it is possible for the end product of path B to revert back to the end product of path

A with an aldehyde due to its high energy state (Scheme 2-4). Thus, it would be useful if one can develop ways to trap intermediate 58. Pratviel trapped 58 by reducing the aldehyde to the corresponding alcohol,64 and our approach uses thiol-containing molecules to form adducts.

Scheme 2-4. “Possible mechanistic scenarios of strand scission in DNA containing T-al.” Rana, A., et al. Reactivity of the major product of C5′-oxidative damage in nucleosome core particles. Chembiochem. 2019;20(5):672-676. Reprinted with permission. (Author’s note: This is a proposed mechanistic pathways of intermediate formation during T-al scission by computational study of T-al expressed in lysine-rich nucleosome core particles.)75

Thiols such as glutathione have been shown to conjugate to DNA damage products.25, 76 Glutathione is ubiquitous in nature and recruited into the nucleus during cell division because cells require reduced environment during replication.90 Tumor cells exposed to IR tend to have higher concentrations of glutathione within a cellular region as well.91 Therefore, glutathione would be a good candidate in studying conjugate formation. If 58 can be trapped with glutathione and metabolized before degrading to furfural which is carcinogenic, cytotoxicity may decrease.

Due to the electrophilic nature of the -unsaturated aldehyde in 58, it is expected that this substrate would react in a 1,4-fashion with thiols (Scheme 2-5). The

α,β-unsaturated carbonyl in the sugar moiety makes 58 a good Michael acceptor,

26 especially at the β position (C3') of the carbon which is partially positive. This site- specific addition at the β position is due to hard-soft acid base theory. In this α,β- unsaturated system as shown in Scheme 2-5, the carbonyl carbon in 58 is resonance stabilized, pushing reactions to 1,4-additions over 1,2-addition. The carbon at the β position has a lower charge density with dominant orbital interactions, so it is considered a “soft” electrophile. This carbon participates in 1,4-addition with soft nucleophiles. The sulfur anion is a soft nucleophile that will attack primarily at the β position.

Scheme 2-5. Mechanism of Michael addition of ddoT

Similar studies have been done in the past where thiol derivatives were used to form a substrate of interest by 1,4-Michael addition.43,71 In order to further secure the hypothesis that 58 forms Michael adducts, various thiol-containing substrates were used in test reactions. Shown in Table 2.1 is the list of substrates and their pKa values. All of the selected molecules contain a conjugated system, so that adduct formation can be monitored by HPLC with UV detection. Because the pKa of the selected substrates is near or below 7, Et3N was added to facilitate thiolate formation.

Table 2.1. pKa of selected thiol-containing substrates

Substrate (RSH) pKa

7.0353

2-mercaptobenzothiazole (MBT)

4.14 (COO-H)92

a 92 6.20 -6.62 (S-H) 3-mercaptobenzoic acid (MBA)

6.6252

Thiophenol aMarvin Sketch®

Thiophenol was utilized because of previous study.71 3-mercaptobenzoic acid43 was also previously used, and 2-mercaptobenzothiazole is another substrate that is similar in properties and structure. Michael addition was conducted by adding RSH (85, 86, or

87) to 58 in 1:1 CH3CN: DCM under anhydrous conditions, followed by addition of 3 molar equivalents of Et3N.

Scheme 2-6. Michael addition of 58 with various thiol-containing substrates

The reaction took place overnight and the final product as a crude was isolated and analyzed by NMR and MALDI-TOF. Reaction progressed over 24 hours, 48 hours, and 72 hours, and there were no significant changes found. NMR analysis indicated that no 58 was found, which could have possibly degraded to furfural and thymine, but no identifiable product formation was observed. MALDI (Figure 2-1) was used to analyze the mixture for small quantities of conjugates that may have formed in the crude mixture.

Resulting compounds from reaction with mercaptobenzothioazole (82) and 3- mercaptobenzoic acid (83) have a base peak of m/z 269 m/z in positive ion mode, which is not a molecular weight of the expected product. On the other hand, the final product through the interaction with thiophenol (84) had a base peak of m/z 225, which indicates that thiophenol exhibits different reaction conditions compared to other two compounds.

Although not included, benzyl mercaptan was also used in the same reaction condition which also did not show expected adduct formation with 58.

shc020_2_rerun after clearsum 0:A2 MS Raw Intens. [a.u.] Intens. 1500

1000

269.468

500

185.220 154.539 347.074

0 x104 269.589 shc020_3 0:A3 MS Raw

Intens. [a.u.] Intens. 1.0

0.8

0.6

0.4

0.2 218.071 332.254 347.210 0.0 225.750 shc020_1_after clear sum 0:A1 MS Raw

800 Intens. [a.u.] Intens.

600

400 115.972

125.872 152.503 200 185.162 243.722 292.255 269.207

0 100 125 150 175 200 225 250 275 300 325 350 m/z

Figure 2-1. MALDI-TOF mass spectrum of products from Michael addition with A) 2- mercaptobenzothiazole B) 3-mercaptobenzoic acid C) thiophenol

Overall, these reactions did not deliver the conjugates as expected. However, the desired products may not be obtainable under the utilized reaction conditions. Following the procedure of Begley et al,43 chloroform was used to dissolve 83. However, 58 did not dissolve completely in this solvent which could have hindered reaction progression.

Substrate 58 is most soluble in acetonitrile, and mixing or substituting with other solvents was either not compatible with 58 or RSHs (82-84). Steric hinderance also exists in 58

30 that it contains a nitrogenous base (thymine) that is bulkier than the amount of a small abasic sugar fragment that was used in the reference.43

Scheme 2-7. Synthesis of 83 from literature43

In addition, the Et3N used in the reference was 0.143 mmol compared to the

Michael acceptor (0.25 mmol), which is approximately a 0.6 to 1 ratio. In this experiment, a total of 3 molar eq. of Et3N was added. Adding a ½ molar equivalent of

Et3N as found in procedure did not cause the reaction to progress. Adding varying molar concentrations of triethylamine was conducted, but when it reached 3 molar equivalents,

58 likely undergoes base elimination. This phenomenon has been reported before, and the

71 longer the substrate was incubated with Et3N, the more degradation occurred. In some studies,76 thiol adduct formation was facilitated by enzymes. Although the thiol adduct was not confirmed in this experiment, it does not prove that Michael adduct does not occur with 58. According to the literature, non-physiological molecules have a rate of reaction that is significantly longer than that of physiological molecules.26 Therefore, using non-physiological molecules may not be appropriate to be a sole determining factor

31 if Michael addition occurs with 58. As a result, the substrates were changed to physiologically relevant molecules that contain thiols to facilitate the reaction.

2.3 ddoT adduct formation with glutathione under physiological conditions

In previous studies, the ddoT did not undergo 1,4-addition of thiols, but it could be due to the substrate and the reaction condition. Using physiological conditions with physiological thiols may still form ddoT adduct through Michael addition. ddoT was reported to degrade into furfural and thymine. Therefore, developing methods for HPLC conditions for known degradation products are important. Then, the ddoT can be incubated under physiological conditions and analyze using LC-MS to identify adducts.

2.3.1 Method development for analysis of ddoT

Given the information that 58 degrades into 18 and 59, the retention times for 58 and 18, and 59 were established, and a calibration curve obtained. Using LC-MS, the molecular weight of degradation products were determined. The analytical method was set up in an isocratic mode initially to observe separation with known degradation products (HPLC Condition A). The retention time for 80 was 6.2 minutes, 18 was 5.3 minutes, and 59 was 8.1 minutes.

Figure 2-2. Analysis of 1 mM 18, 58, and 59 by HPLC-PDA with detection at 254 nm after incubation under physiological conditions (pH 7.4, 37 °C) in phosphate buffered saline. The mobile phase was composed of 5 mM ammonium acetate:CH3CN (50:50, v:v).

In general, nucleobase is one of the first to be eluted due to its polarity.

Interestingly, using this method, 58 elutes faster than 59, indicating that 58 is more polar than 59. The dipole moment of 59 is around 3 D93 and it is of intermediate polarity.94

Compound 59 seems to be more polar than 58 according to the structure, but eluted later than 58. Substrate 58 has a λmax of 265 nm, 18 a λmax of 262 nm, and 59 a λmax of 276 nm, which are expected for their chemical structures. Although the λmax of thymine and

95-96 furfural has been determined previously, λmax of ddoT (58) was never determined, but correlates with that of thymidine at 265 nm. Using an isocratic system with a total runtime of 10 minutes is efficient because it provides separation among three crucial substrates without the need of gradients. Because the three compounds exhibited good separation, this HPLC method was used for the analysis. A calibration curve for 58 was obtained at 4°C using HPLC condition A in 10 mM BBS buffer. The calibration curves

34 for 59 and 18 were also established in the same manner. This curve allowed the quantitation of the substrate at varying concentrations with the linear equation obtained.

In addition, the fragmentation of each compound was determined in LC-MS/MS. The fragmentation patterns of 58 have never been determined, and this information will provide a foundation of future analytical studies involving 58.

Scheme 2-8. Fragmentation pattern of 18, 58, and 59 obtained by ESI-MS/MS

Figure 2-3. HPLC-DAD UV spectrum of 58 in phosphate buffered saline.

Figure 2-4. HPLC-DAD UV spectrum of 18 in phosphate buffered saline.

Figure 2-5. HPLC-DAD UV spectrum of 59 in phosphate buffered saline.

The stability of 58 was determined in 100 mM phosphate buffer at 37 C and pH 7.4 and a half-life of 13 hours under physiological conditions was determined.25 Compound 58 was dissolved in 5% CH3CN in PBS to determine the appropriate conditions for storage.

The storage conditions of 58 were determined in phosphate buffered saline (PBS) at 4 C

36 at pH 7.4 after purification. The decomposition of 18 to 58 and 59 was monitored by LC-

MS for 10 days, and a very small amount of 18 and 59 was formed over time. The degradation pattern was in parallel with previous findings.25

Scheme 2-9. The decomposition of 1 mM 58 in phosphate buffered saline at 4 C in pH 7.4

2.3.2 Reactivity of ddoT with glutathione

Thiolates from physiological thiol-containing compounds may form Michael adducts within the human body. Glutathione is a Michael donor which reacts with

Michael acceptors such as 58. In order to determine the reactivity of ddoT under physiological conditions, 80 μM of 58 was incubated with excess glutathione (6 mM) in

PBS buffer, pH 7.4, 37 °C and analyzed using HPLC Condition A. From this experiment, the rapid disappearance of 58 with a half-life of 30 minutes was observed with chromatographic separation, and new peaks were detected by UV detection.

Figure 2-6. HPLC chromatogram of 80 μM ddoT (58) in the presence of 6 mM glutathione over time at 254 nm under physiological conditions (pH 7.4, 37 °C) in phosphate buffered saline. The mobile phase was composed of 5 mM ammonium acetate:CH3CN (50:50, v:v).

Figure 2-7. Degradation of 80 μM of 58 in 6 mM glutathione under physiological conditions (pH 7.4, 37 °C) in phosphate buffered saline

However, the mass analyses of new peaks in LC-MS were not accomplished because the signals were significantly suppressed by the high concentration of glutathione used in the experiment. Previously in our laboratory, it was found by HPLC-

UV that various peaks formed when ddoT (58) was incubated with glutathione, but the peaks were not identified, and masses obtained by MALDI-TOF didn’t correlate with the expected glutathione adducts.25 Therefore, the concentration of GSH was modified to attempt to detect the adduct we were expecting. Also, overlap between new substrates formed and 58 made it difficult to analyze the mixture, thus the analytical conditions were modified.

2.4 ddoT adduct formation with physiological thiols under modified conditions

The -unsaturated aldehyde in ddoT makes it a good Michael acceptor. The 1,4 addition has been demonstrated in nitro and thiol adducts by Michael addition,97 and the physiological relevance of thiol adducts from DNA damage has been demonstrated by

Dedon’s work in studying the metabolites of adducts.76

2.4.1 Method development of ddoT and degradation products with LC-MS in modified

conditions

Due to inconclusive results from previous studies, the reaction conditions were modified such that the temperature was increased to 40 °C and the pH was increased to

8.0 using 10 mM borate buffered saline. Therefore, a new calibration curve and HPLC methods (LC-MS Condition A) for these new conditions were developed. Thymine,

39 furfural, and ddoT eluted at 5.1 minutes, 10.2 minutes, and 21.5 minutes, respectively.

The resolution of furfural and thymine was 3.41, and that of furfural and ddoT was 5.43.

The capacity factor was 1.19. Therefore, the separation of each standard provides the resolution needed for future studies of adduct formation. The diode array detector was used for detection using various wavelengths and comparing the absorbance of the compound at a given tR (Appendix A).

1 Detector A Channel 1 254nm

700 2 Detector A Channel 2 280nm 5.171

600 5.174 500

400

300

200

21.484

37.451 36.933

100 10.120 6.049 0 36.825

0 5 10 15 20 25 30 35 min

Figure 2-8. Analysis of 18, 58, and 59 by HPLC-PDA at 254 nm in 10 mM borate buffered saline at pH 8.0 at 37 °C. The mobile phase was composed of 0.1% acetic acid:CH3CN (v:v)

Table 2.2. Validation data for the determination ddoT for monitoring (n=5)

1) 2) 3) 4) tR Regression equation R LOD LOQ RSD (%) (min) (ng mL-1) (ng mL- 5) 1) Thymine 5.2 y = 6000000 x-64103 0.9997 0.98 ddoT 21.8 y = 7000000 x-28080 0.9999 37.5 75 0.98 Furfural 10.1 y = 5000000 x-1069.6 0.9999 1.00 1)Retention time in min, 2) Regression coefficient, 3) Limit of detection (3= s/n), 4) Limit of quantitation (10=s/n), 5) Relative Standard Deviations.

Figure 2-9. The calibration curves for 18, 58, and 59 at pH 8.0 in 10 mM BBS buffer

When the stability of ddoT (58) was determined in 10 mM borate buffered saline

(BBS) at pH 8.0 and 40 C, the half-life increased to 85.5 hours based on the amount of

58 recovered as shown in Figure 2-10Error! Reference source not found..

Scheme 2-10. The decomposition of 58 in borate buffered saline (40 C, pH 8.0)

The concentration of thymine (18) and furfural (59) increased as 58 degraded.

The total concentration of furfural, thymine, and ddoT at the end of the experiment summed up to be the same molar concentration of ddoT started. There was no other adduct formation or interaction occurred with borate buffered saline, and the decomposition pattern is the same as that of physiological conditions.

Figure 2-10. The change of ddoT concentration in borate buffered saline (pH 8.0 at 40 C)

Figure 2-11. The change of thymine, furfural concentration in borate buffered saline (pH 8.0 at 40 C)

During the stability studies of 58, the half-life of 58 at 37 °C and pH 7.4 was compared to the half-life at 40 °C and pH 8.0. ddoT is fairly stable at 4 °C and pH 7.4, but the increase in temperature promotes decomposition, as observed from the half-life

42 study at 37 °C.25 On the other hand, the half-life of ddoT at 40 °C and pH 8.0 is significantly longer at 85.5 hours. The half-life of ddoT is 13 hours under physiological conditions cannot be directly compared, because the incubation conditions vary. If any, the use of borate buffered saline and buffered ion may play a role in stabilizing ddoT (58) therefore increasing the half-life. Due to the long half-life of 58, the latter condition was used for the reactivity of 58 with thiol-containing molecules.

58 was shown to have a shorter half-life in the presence of GSH, but it was unclear if GSH caused the degradation of ddoT or caused reaction of ddoT to form adducts.25 Due to the electrophilic nature of the -unsaturated aldehyde in 58, it is expected that this substrate would react in a Michael fashion with thiols. To determine if adduct formation is a driving force in the disappearance of 58, experiments were performed in the presence of thiol-containing biological molecules: GSH, Coenzyme A

(CoA), cysteine (Cys), and N-acetylcysteine (NAC). Figure 2-12 shows the adducts expected when ddoT and thiol-containing biomolecules were incubated at various timepoints at pH 8.0 and 40 °C.

Figure 2-12. Expected ddoT adducts formed by Michael addition with physiological thiol-containing biomolecules.

2.4.2 A glutathione adduct of ddoT

Glutathione is an important biomolecules that is involved in reduction of accumulated oxidants in body and conjugation of xenobiotics for metabolism. GSH is also involved in the mechanism of different types of antioxidant such as vitamin C.98

GSH is a well-known Michael donor with a thiol group possessing a pKa of 9.2. As discussed previously, ddoT was shown to have a substantially decreased half-life in the presence of GSH. The GSH adduct was selected for further investigation because GSH is ubiquitous in nature.

A total of 0.625 mM of 58 was incubated in the presence of 0.625 mM GSH in 10 mM BBS buffer for 24 hours at pH 8.0 and 40 C. As seen previously, excess GSH afforded complete consumption of 58 rapidly. Although the concentration of GSH is likely in excess in biological systems compared to that of 58, the 1:1 ratio was used to monitor the reactivity of ddoT and the formation of the ddoT adduct. This was done so that the reaction process can be monitored slowly, and to determine the reactivity of ddoT in a 1:1 ratio. Upon the addition of ddoT to GSH in a 1:1 ratio, ddoT was converted to a

GSH-ddoT adduct. Under these conditions, ddoT was not fully consumed. During chromatographic separation, GSH and GSSG eluted earlier than thymine. Thymine (18) and furfural (59), degradation products of 58 (tR 22.5 min), eluted at 5.1 minutes and 10.5 minutes, respectively (Figure 2-13). The formation of two GSH adducts, or the product of hydrate formation and after Michael addition, eluted with retention times of 23.7 minutes,

25.4 minutes, and 28.8 minutes.

Detector A Channel 1 254nm 22.501

500

250

25.360

5.154

28.847

10.452 23.739 0

0 10 20 30 40 50 min

Figure 2-13. Analysis of ddoT (0.625 mM) in the presence of glutathione (0.625 mM) in vitro at 40 °C BBS buffer (pH 8.0) by HPLC-UV-MS

MS analysis was done in positive and negative ion mode to confirm the mass of

58 and the expected GSH adducts. Analysis with LC-MS showed unreacted 58 and GSH as well as expected degradation products 59 and 18 in the ion chromatogram. ddoT-GSH adducts form without nucleobase elimination show an MH+ m/z 530, or the corresponding hydrate with an MH+ m/z 548.

The formation of the hydrate was expected due to the nature of aldehyde and the conditions of the experiment. However, hydrate formation causes loss of the reactive α,β- unsaturated system required for Michael addition to occur. Therefore, the formation of the hydrate most likely occurs after the sufhydryl group adds in a Michael fashion. The abundance of the ddoT-GSH adducts in mass spectra varied depending whether the analysis was performed in positive or negative ion mode. Masses corresponding to both

GSH adducts and hydrates appeared at the same retention time. It is likely that the hydrate form of the GSH-ddoT adducts exist in equilibrium with the aldehyde, which

45 explains why GSH adducts and GSH hydrate adducts co-elute in mass spectra. The most probable order of elution is ddoT-GSH hydrate adduct followed by GSH adduct. This elution order is expected because the additional hydroxyl group at the C5' makes the compound more polar. The hydrate formation in ddoT standard was not found in LC-MS analysis.

Figure 2-14. Electrospray ionization mass spectrum of GSH adduct (MH+ 530) in positive mode. Ion chromatograms of summed ions: m/z 530, 548 in BBS buffer (pH 8.0) at 40 oC

Figure 2-15. Electrospray ionization mass spectrum of GSH adduct (MH- 528) in negative mode. Ion chromatograms of summed ions: m/z 528, 546 in BBS buffer (pH 8.0) at 40 oC

As mentioned, multiple peaks were found to correlate with the expected GSH adducts in equilibrium with hydrate formation. The most abundant adduct is 85 which elutes later at tR = 28.8 min, followed by 86, tR = 25.4 min. Through evaluation of the UV absorbance of each adduct, it was determined that all exhibited the same λmax = 268 nm

(Appendix A). During Michael addition with 58, 1,4 additions occur by either syn or anti addition of GSH relative to sugar, and it was demonstrated by glutathione conjugation to form (–)-anti- and (+)-syn-stereoisomers.99 Because of this, there are eight different possible stereoisomers. The GSH addition is not stereospecific and would deliver two isomers  at the C-3′ position. This would be converted to two isomers of the hydrate.

3:223.00(+) 100000 3:530.00(+) 3 4 ddoT (58) 75000

50000 GSH adducts 1 25000 2

0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 min

Figure 2-16. Total ion chromatogram of GSH-ddoT adducts (pink) and ddoT (black) from Figure 2-13.

In the total ion chromatogram showed in Figure 2-16, four isomers were also detected. As shown in Figure 2-17, considering that each GSH adduct is found in equilibrium with the corresponding hydrate, the formation of four isomers was expected.

The specificity in the types of stereoisomers formed and the order of elution can be further determined by either synthesis of stereospecific molecules or chiral resolution.

Figure 2-17. Predicted ddoT adducts from Michael addition

Figure 2-18 shows the observed fragmentation pattern of 85 using LC-MS/MS.

The fragmentation of glutathione100 as well as glutathione adducts with various substrates are well-documented in the literature.101-103 The masses associated with the fragmentation of the unreacted glutathione accounts for m/z 76.05, 162.15, 179.05, 233.15, 215.40.100,

104 The fragmentation at carboxylic acid (m/z 512) and amide (m/z 401) is indicative of the GSH adduct which is also corresponding to the literature.101 The base loss is also commonly seen in MS/MS analysis of nucleosides.105

Figure 2-18. Fragmentation pattern of 85 by LC-MS/MS

As mentioned above, GSH-ddoT and GSH-ddoT hydrates were two types of adducts found. Shown in Figure 2-19 and Figure 2-20 are the time response curves for the formation of two major ddoT adducts at tR = 25 min and 28 min by measuring AUC over time, and the degradation products thymine (18), furfural (59) were monitored at various time points over 24 hours.

Figure 2-19. Change of formation of glutathione adduct with 85 and 86 over 24 hrs

The amount of GSH-ddoT adducts generated plateaued after 10 hours, whereas thymine and furfural increase in concentration continuously. This indicates that the unreacted ddoT is continuously degrading under the given reaction conditions. In the study of ddoT stability, the furfural (59) and thymine (18) were increased over time, but the furfural formation was decreased over time indicating possible degradation. However, in this reaction, the furfural formation was continuously increasing.

Figure 2-20. Formation of thymine and furfural as degradation products of ddoT in the precence of glutathione over 24 hrs

Therefore, ddoT degradation was facilitated by the presence of GSH by converting into

GSH-ddoT adducts. Compound 18 and 59 were also incubated in the presence of GSH to determine if any other adduct formation occurred (Appendix A). No other adduct were detected. Thus, it is expected because thymine and furfural are not likely to react with glutathione. Since it was confirmed by LC-MS that Michael addition occurs with GSH and ddoT, ddoT was incubated with CoA, N-Acetylcysteine, and cysteine.

2.4.3 A coenzyme A adduct of ddoT

Coenzyme A is an important coenzyme in TCA cycles and fatty acid transport in mitochondria during cell starvation. CoA is present in mitochondria where DNA is freely dispersed in the matrix as well as in cytosol of the cells. DNA in the mitochondria is in close proximity to the many ROS produced as a result of oxidative phosphorylation. CoA has also been used as a Michael donor to form Michael adducts.106 Therefore, it was expected to be a good substrate to investigate the formation of ddoT adducts with biological molecules. Upon addition of ddoT to CoA in a 1:1 ratio, ddoT was not consumed completely. The formation of CoA adducts was detected by HPLC-UV with a retention time of 30.1 minutes; however, no hydrate adduct was detected nor stereoisomers. Thymine (18) and furfural (59) were formed as degradation products of

58.

Detector A Channel 1 254nm

1500 30.148

1000

500

23.281 5.163

0 10.510

0 10 20 30 40 50 min

Figure 2-21. Analysis of ddoT (0.625 mM) in the presence of CoA (0.625 mM) at 40 °C at BBS buffer (pH 8.0) by HPLC-UV-MS at 24 hrs

The LC-MS analysis in positive ion mode showed 496 m/zError! Reference source not

2+ found. as a major product as MH2 . The negative ion mode provided the mass

2- correlating with MH2 . The CoA adduct was more easily detected in a negative ion mode. The pKa of the thiol of CoA is 10.07. Thus, the thiol is still protonated in these experiments. As indicated in our experiments, a significant portion of CoA undergoes

Michael addition to yield CoA-ddoT adduct (87). No hydrate formation was found because the active site of CoA is less accessible to water in the environment, because the structure is hydrophobic. Thymine and furfural were also incubated in the presence of

CoA and no other adduct formation was detected.

Figure 2-22. Electrospray ionization mass spectrum of CoA adduct (MH+ 990) in positive mode. Ion chromatograms of summed ions: m/z 496 (M+2H+), 768 (CoA), 990 ([M+H]+) in BBS buffer (pH 8.0) at 40 oC.

Figure 2-23. Electrospray ionization mass spectrum of CoA adduct (MH- 988) in negative mode. Ion chromatograms of summed ions: m/z 494, 765, 988 in BBS buffer (pH 8.0) at 40 oC.

2.4.4 A N-acetylcysteine adduct of ddoT

The pKa of the thiol in NAC is 9.2, therefore it is expected to exhibit a similar product distribution as GSH with ddoT (58). Upon addition of ddoT to NAC in a 1:1 ratio, ddoT was not consumed completely, and the formation of NAC-ddoT adducts were found at tR = 23.9, 25.4, 28.2, and 30.3 minutes as isomeric NAC-ddoT adducts. Thymine

(18) and furfural (59) were detected as degradation products of ddoT. This finding was similar to the reactivity of ddoT with GSH.

Detector A Channel 1 254nm

750 21.423

500

250

30.304

5.117 5.117

32.393

28.219

10.369 25.379 0 23.891

0 10 20 30 40 50 min

Figure 2-24. Analysis of ddoT (0.625 mM) in the presence of N-Acetylcysteine (0.625 mM) at 40 °C at BBS buffer (pH 8.0) by HPLC-UV-MS at 24 hrs

In positive ion mode, m/z 386 [NAC-ddoT+H]+ was predominant, whereas m/z

402 [NAC-ddoT hydrate-H]- was predominant in negative ion mode, indicating that

NAC-ddoT adducts are in equilibrium. Other peaks include m/z 408 [M+Na]+, m/z 426

+ [M(hydrate)+Na] . The oxidized NAC eluted at tR = 10 min which overlaps with 59, and was 325 m/z in positive ion mode and 324 m/z in negative ion mode. Therefore, ddoT reacted with NAC in a similar manner to GSH to form Michael addition products via the same mechanism of action. Thymine and furfural did not react with cysteine in the presence of N-acetylecysteine.

Figure 2-25. Electrospray ionization mass spectrum of NAC-ddoT adduct (MH+ 386) and the sodium adduct (M+Na+ 408), NAC-ddoT hydrate adduct M+Na+ (MH+ 426) in positive mode. Ion chromatograms of summed ions: m/z 386, 408, 426 in pH 8 buffer at 40 °C.

Figure 2-26. Electrospray ionization mass spectrum of NAC adduct (MH- 384) and NAC-ddoT hydrate adduct (MH- 402) in negative mode. Ion chromatograms of summed ions: m/z 384, 402 in BBS buffer (pH 8.0) at 40 oC.

2.4.5 A cysteine adduct of ddoT

Cysteine is a non-essential amino acid, and is involved in the synthesis of glutathione. The structure of cysteine is slightly different from the previous low molecular weight thiols or CoA, because it tautomerizes to cysteine zwitterion, where it

- + bears charges at COO and NH3 . Upon the addition of ddoT to Cys in a 1:1 ratio, ddoT was not consumed completely. Furfural (59) and thymine (100) eluted as usual with other adducts and unconsumed ddoT (58). Unlike in other cases where adducts show after ddoT has eluted, the Cys-ddoT adduct eluted at TR 6.3, and 11.3 minutes.

Detector A Channel 1 254nm

250 31.078

200 23.353

150

100

5.167

29.218

32.587 14.863

50 21.830

6.338

10.511

11.303

33.658 1.570 1.570 0

0 10 20 30 40 50 min

Figure 2-27. Analysis of ddoT (0.625 mM) in the presence of cysteine (0.625 mM) at 40 °C at BBS buffer (pH 8.0) by HPLC-UV-MS at 24 hrs

At 6.3 minutes, Cys-ddoT hydrate adduct (m/z MH+ 362, m/z MH- 360) was prevalent in positive and negative ion mode, and Cys-ddoT adduct (m/z MH+ 344, m/z

MH- 342) was more prevalent at the later timepoint. Early elution of the cysteine adduct

57 is expected due to zwitterions present in cysteine, which makes the overall compound more polar.

Figure 2-28. Electrospray ionization mass spectrum of cysteine adduct (MH+ 344) in positive mode. Ion chromatograms of summed ions: m/z 344 in BBS buffer (pH 8.0) at 40 °C

Figure 2-29. Electrospray ionization mass spectrum of cysteine adduct (MH- 342) in negative mode. Ion chromatograms of summed ions: m/z 342, 360 in BBS buffer (pH 8.0) at 40 oC.

At the tR = 14.9 and 21.8 minutes, m/z 447 was present in positive ion mode, and m/z 445 in negative ion mode, indicating that there is a compound with a molecular weight of 446 g/mol.

Figure 2-30. The proposed structure of ddoT from potential dimerization

Figure 2-31. Electrospray ionization mass spectrum of 92 in positive mode. Ion chromatograms of summed ions: m/z 447 in BBS buffer (pH 8.0) at 40 oC

Figure 2-32. Electrospray ionization mass spectrum of 92 in negative mode. Ion chromatograms of summed ions: m/z 445 in BBS buffer (pH 8.0) at 40 oC

ddoT dimerization was only found upon the addition of cysteine. This type of reaction mimics the pinacol coupling in which a 1,2-diol is formed when the identical compound containing a carbonyl group reacts.107 In this case, the cysteine could replace commonly used magnesium to take place such experiment as shown in Scheme 2-11. It has been found that cysteine is used as a reducing agent in general,108 but the role of cysteine specifically in pinacol reaction has not been reported. However, further investigation is needed to confirm the formation of 92. Thymine and furfural did not react with cysteine in the presence of cysteine.

Scheme 2-11. Proposed mechanism of pinacol coupling of 58

Chapter 3

3 Conclusion and Future Research Directions Conclusion and Future Research Directions

3.1 Summary

Oxidatively damaged DNA may result in the formation of adducts, which can be used as markers to detect oxidative stress.16, 24, 27, 87-88 Such products would be constituents of the endogenous exposome if they are found to be involved in disease susceptibility, development or progression. DNA damage products arising from the 5' aldehyde lesions in DNA containing an ,-unsaturated moiety can form adducts with thiol-containing cellular nucleophiles. These adducts may be useful in understanding disease manifestations or aging. Our studies to understand the damage products from 5' aldehyde lesions will help correlate elements of the endogenous exposome with diseases related to oxidative stress.

Before the 5'-aldehyde (52) degrades to furfural (59), an intermediate (58) is believed to form. However, due to the chemical instability of 58, its fate and mechanism of degradation are yet to be fully understood.24 In addition, no findings have confirmed the formation of adducts with cellular constituents. ddoT (58) has an -unsaturated

62 aldehydes that is very reactive, therefore it is not likely to be found in cells without conjugation. The -unsaturated aldehydes in ddoT can react in a 1,4-fashion with thiol- containing biomolecules such as glutathione to form covalent adducts. These adducts may be found after excretion and may play a role in building our understanding of disease manifestations as constituents of the endogenous exposome.

In order to investigate the reactivity of ddoT in the presence of thiols, the synthesis of ddoT (58) was accomplished employing organic chemistry, and the half-life in the presence of borate buffered saline at 40 °C in pH 8.0 was determined to be 85.5 hours, which is much more longer than that determined under physiological conditions.25

The oxidation step to convert a primary alcohol in 79 to an aldehyde was optimized in the synthetic approach by substituting the Dess-Martin reagent with IBX, which resulted in a slightly higher yield. The synthesis of ddoT adducts with thiols were attempted, and

MBT, MBA, and thiophenol but the expected adduct was not detected. Although the expected thiol adducts were not confirmed in this experiment, it does not conclude that

Michael adduct does not occur with 58. According to the literature, non-physiological molecules have a rate of reaction that is significantly longer than that of physiological molecules.26 Therefore, using non-physiological molecules may not be appropriate to be a sole determining factor for Michael addition with 58. As a result, the substrates were changed to physiologically relevant molecules that contain thiols to facilitate the reaction.

In earlier studies, several groups described the formation of adducts of thiols from reactive intermediates formed from DNA oxidation. Stubbe successfully derivatized 2- methylene-3(2H)-furanone, an -unsaturated ketone derived from C3'-radical formation in DNA, using ethyl thiol through Michael addition.33 Dedon’s laboratory also

63 identified glutathione conjugates of base-propenals which result from damage at the C4ʹ position of DNA.76

Analytical methods were developed to detect ddoT and its adducts. The fragmentation patterns and maximum UV absorbance of each compound was also determined. Upon incubating ddoT with glutathione under physiological conditions, ddoT disappearance accelerated, but the formation of glutathione adducts was not confirmed, therefore the incubation conditions were modified. Calibration curves were constructed with the detection limit and quantification limit determined. Under modified conditions in borate buffered saline at 37 °C and pH 8.0, ddoT has a half life of 85.5 hours.

The reactivity of ddoT in the presence of glutathione under modified conditions was studied and the formation of the desired Michael adduct was determined by HPLC-

UV-MS, and the adduct was further analyzed by fragmentation using LC-MS/MS.

According to HPLC, it was found that the ddoT-GSH adducts (85) are found in equilibrium with the corresponding hydrate (86) and formed isomers of each other, which is believed to be a result of syn/anti addition during adduct formation. The formation of

85 and 86 was monitored over time, and after 10 hours there was no significant increase in formation. Because ddoT was not fully consumed in the presence of glutathione over

24 hours, thymine and furfural were found to be continuously increasing as degradation products of unreacted ddoT.

The reactivity of ddoT over 24 hours was higher compared to that of glutathione, because the CoA adduct is more hydrophobic than other thiols. Due to this feature, once the ddoT adduct is formed with CoA, it has less contact with water molecules which

64 results in no formation of hydrate adduct. The reactivity of N-acetylcysteine is similar to that of glutathione, and ddoT was largely unreacted, although the ddoT-NAC adduct was detected. The reactivity of ddoT with cysteine was also similar, but the ddoT-cysteine adduct eluted prior to ddoT, because it tautomerizes to cysteine zwitterion. In addition, what is believed to be the dimer of ddoT through pinacol coupling was also detected.

3.2 Future Research Directions

3.2.1 Biological Relevance of ddoT adduct formation

3.2.1.1 Detection of Michael adducts from ddoT from calf thymus DNA

In the previous chapters, it was confirmed that Michael addition takes place with ddoT to form thiol adducts when incubated with thiol-containing biomolecules. Another way to confirm the biological relevance of ddoT-adduct formation is using calf thymus

DNA. Ct-DNA has been utilized after the digestion of DNA to observe damage occurring with the nucleobases.109-112 Using the analytical methods developed, calf thymus DNA will be exposed to hydroxyl radicals in water, which could ultimately lead to DNA damage including 5' aldehyde lesions. The exposure of GSH to damaged DNA fragments could facilitate the formation of GSH adducts which could be detected by LC-MS.

Calf thymus DNA will be added in a solution of 2.5 mM FeSO4 in 10 mM

phosphate buffer at pH 7.4 for a total volume of 250 µL. Then, H2O2 will be added and incubated for 2 hours. This will be followed by the addition of 7 mM GSH and incubation for 1 hr. EtOH will be added for precipitation and the DNA will be frozen at -

20 C. The supernatants will be collected and analyzed by LC-MS.

Figure 3-1. A method for the preparation of calf thymus DNA for adduct formation

Iron (II) and H2O2 acts together as part of Fenton reaction as shown in the equation below:113

2+ 3+ - Fe + H2O2 → Fe + •OH + OH

3+ 2+ + Fe + H2O2 → Fe + •OOH + H

Iron (II) is oxidized to iron (III) and hydroxyl radical and hydroxide ion is released. Iron

(III) then reacts with another molecule of peroxide to reduce to iron (II) followed by the release of a peroxyl radical. These two reactions lead to the formation of water and two types of ROS that actively participate in DNA damage.

Shown below is an analysis of ct-DNA without any treatment. Because the DNA was not digested and only the supernatant was collected for analysis after evaporation, what is shown is only the baseline deoxyribose and bases present in the DNA. The identification of each peak was determined by comparing with analytical standards.

Datafile Name:ct 1-1_11162018_7.lcd Sample Name:ct 1-1 Sample ID:ddoT t=11 mAU 70 254nm,4nm

60 dT

dC 17 3 50

40 10

C2 30 dG 20 T 14

9 6 10

dA15

7 18 0 8 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 min Figure 3-2. HPLC chromatogram of supernatant from ct-DNA - control

In the proposed experiments, the DNA will not be digested because our molecule of interest is DNA sugar fragment that is no longer a part of DNA helices. Fenton reagent used in this experiment is one of the common reagents used to generate radicals in DNA, and GSH would be added after the supernatant is obtained.

3.2.1.2 Detection of Michael adducts from ddoT from oligonucleotides with 5' lesions

Another way of confirming biological relevance in this context is to study known

DNA damage products in oligonucleotides. The formation of 52 was studied in various studies using anti-tumor or DNA-damaging agents and it is a well-known mechanism.44,

60 Previously in our lab, a 5' aldehyde was synthesized and incubated under physiological conditions to observe the degradation. It was confirmed that the phosphorylated oligomer resulting from elimination from the 3' position of 52 was detected with a half-life of 96 hours, although ddoT was not detected. However, the most logical explanation of this detection was the formation of ddoT. If ddoT formation can be confirmed from T-al through reaction with low molecular weight thiols to form Michael adducts, this would be a first step in validating the relevance of this damage product.

A 5' aldehyde was synthesized using Greenberg’s method62 to confirm the formation of ddoT from oligonucleotides. The ddoT formed from oligonucleotides will be used to determine the ability to react with ddoT under biological conditions.

3.3 Conclusion

In conclusion, stability and reactivity of ddoT and degradation of ddoT was explored in the presence of thiol-containing biomolecules. All expected adducts from different Michael acceptors (GSH, coenzyme A, N-acetylcysteine (NAC), and coenzyme

A) was found successfully using LC-MS. Unknown adducts from cysteine will be investigated by synthesizing the expected dimers via pinacol reduction. It was shown that ddoT can be trapped by thiol-containing molecules. Previously, the formation of furfural from ddoT was prevented by reducing the aldehyde at the 5' carbon to the corresponding

64 alcohol. It was chemically done by NaBH4 as well as under physiological conditions by a reductase. Therefore, different trapping methods can be applied. ddoT can also be explored with different bases, such as ddoA with adenine, ddoC with cytosine, and ddoG with guanine, although the latter two might need base protection due to their high oxidation potentials to prevent further base modification during adduct formation. T-al can also be incubated with glutathione to determine if the glutathione adduct is formed from single-stranded or double-stranded DNA. Comparing to the data presented by

Greenberg, a competing reaction with glutathione and lysine could give interesting results in determining the fate of ddoT. The development of this method is a foundational in the future work as it will provide a means to detect the compound(s) of interest.

It is crucial to identify DNA adducts because it can help us explain that small

DNA fragments from DNA damage are potential constituents of the endogenous

68 exposome. Additionally, finding such compounds will also contribute to the development of diagnostic assays and our understanding of the pathophysiology of disease states.

Although strand cleavage is not desirable in normal, healthy DNA, it is utilized as a mechanism of action of cancer medications such as bleomycin.10 Some cancer drugs are detrimental to humans, and could possibly damage the normal, healthy cell.114 It is critical to understand the subsequent mechanisms involved in cleavage artificially initiated by drugs because not all cancer medications have a known mechanism of action and subsequent processes can impact efficacy and toxicity. In addition, identifying constituents of the endogenous exposome can be applied to pharmacogenomics to determine the probability of developing certain disease states in one’s lifetime. For example, if we can identify an adduct that participates in a specific pathway, it can be used as a biomarker to monitor the progress of a disease or its etiology.

Chapter 4

4 Experimental procedures

Experimental Procedures

Unless otherwise stated, all reactions were performed using standard laboratory procedures under inert conditions (under Argon) with magnetic stirring in oven-dried glassware. All flasks were tightly sealed with rubber septa during experiments.

4.1 Materials

All reagents, chemicals, and solvents were purchased from commercial suppliers and used without purification: Acros Organics, Sigma-Aldrich, Fisher Scientific, Chem-

Impex International Inc., and Pharmaco-Aaper. Deuterated solvents were purchased from

Cambridge Isotope Laboratories for NMR analysis. HPLC- and MS- grade solvents were purchased from Fisher Scientific and Sigma Aldrich, and used for chromatographic separations and LC-MS analysis. IBX was synthesized using published literature.115 The

Dess Martin Reagent was synthesized using published literature.82 Buffers were obtained from Burdick & Jackson (Honeywell). Deionized water was purified using a Milli-Q®

Reference Water Purification System from Millipore SAS. Coenzyme A was obtained

70 from Cayman Chemical Company. Glutathione, cysteine, and N-acetylcysteine were purchased from Sigma Aldrich. Ammonium acetate was purchased from Fluka analytical.

4.2 Structural Analysis

All products synthesized were characterized by NMR (Nuclear Magnetic Resonance) spectroscopy and electrospray (ESI) mass spectroscopy.

4.2.1 1H-NMR, 13C-NMR

All 1H-NMR spectra were obtained using either a Varian VXR-400, Varian Unity Inova

600, or Bruker Avance III 600 NMR spectrometers in CDCl3, CD3CN, or d-DMSO. All

13C-NMR, COSY, and HSQC spectra were obtained on a Bruker Avance 600. Chemical shifts are reported in parts per million (ppm) relative to the residual peak of a deuterated solvent of choice as internal standard. 31P-NMR was obtained on a Varian VXR 400. For

1H-NMR, coupling constants (J) are reported in Hertz (Hz). Multiplicity is reported as follows: s = singlet; d = doublet; t = triplet; q = quartet; dd = doublet of doublet; ddd = doublet of doublet of doublet; m = multiplet. The protons of the carbons of the furanose ring and the nucleobase thymine are designated as shown in Figure 6-1:

Figure 6-1. Proton Assignments of deoxyribose sugar and numbering of the thymine residue

4.2.2 Mass Spectrometry

4.2.2.1 ESI-MS

Mass spectra were obtained using an Esquire electrospray ionization (ESI) mass spectrometer (Bruker) operated in positive ion mode. Samples were dissolved in methanol unless otherwise stated, and masses are reported as [M+H]+ or [M+Na]+ ions of each analyte.

A Shimadzu LC-MS 2020 (ESI-LC/MS), or Shimadzu LC-MS 8050 (ESI-LC-MS/MS) were also used. Samples were dissolved in acetonitrile, water, methanol, or in combination of any of these depending on the sample to a concentration of 1 nmol/L.

The instruments were operated either in positive or negative mode, and the masses are reported as [M+H]+ or [M+Na]+ ions of each analyte.

Table 4.1. LC-MS Condition A

Instrument Shimadzu 2020 LC-MS Column Thermo Hypersil Gold aQ C-18 3µm, 150 mm X 2.1 mm Column Temperature Ambient (RT) Mobile Phases A: 0.1% Acetic Acid B: Acetonitrile Gradient Time (min) % B 0.01 0 25 4 35 98 45 98 50 0 60 0

Flow Rate 0.3 mL/min Injection volume 20 µL Drying gas flow (N2) 15 L/min Heat block temperature 400 DL temperature 300 Nebulizing gas flow 1.5 L/min UV Wavelength 254, 280 nm Detector voltage 1.25 kV Ionization mode ESI - Positive mode Negative Mode Mass scan 50-1500 Run Time 60 min

4.2.2.2 MALDI-ToF MS

An UltrafleXtreme (Bruker) was used operating in positive ion reflectron mode. The matrix solution used for small molecules was 20mg/mL of 2,5-dihydroxybenzoic acid

(DHB) or matrix free in 30% acetonitrile in 0.1% TFA in H2O. The matrix (1 µL) was spotted on a 100-well stainless steel MALDI plate and evaporated to dryness. A sample

(1 µL, ≥100 pmole) was spotted and dried. [M+H]+ or [M+Na]+ ions of each analyte was obtained.

4.3 Chromatographic Methods

4.3.1 Thin Layer Chromatography (TLC)

Analytical thin layer chromatography was used to monitor the progress of all organic reactions and the elution of compounds from column chromatography using aluminum, plastic, and glass backed silica gel plates. TLC spots were visualized by UV light at 254 nm and burned after being stained with p-anisaldehyde dip composed of the following: 180 mL of absolute ethanol; 10 mL concentrated sulfuric acid; 2 mL glacial acetic acid and a few drops of p-anisaldehyde.

4.3.2 Reverse-Phase HPLC (RP-HPLC)

All High-Performance Liquid Chromatography (HPLC) was performed on a

Shimadzu Prominence Liquid Chromatograph (LC-20A) with a PDA detector (SPD-

M20A), or a dual wavelength UV detector. Solvent systems were as follows: solvent A: 5 mM ammonium acetate buffer pH = 7.0; solvent B: 100% acetonitrile, or solvent A: 0.1% acetic acid in water; solvent B: 100% acetonitrile. Samples were dissolved in borate buffered saline (pH = 8.0, Burdick & Jackson, Honeywell) containing 4NaCl∙Na2B4O7. pH 4 citrate buffer solution (Burdick & Jackson, Honeywell) was also used for preserving reagents that are sensitive to pH.

Table 4.2. HPLC Condition A

Instrument Shimadzu Prominence PDA-HPLC Column YMC-pack 5µm, C-18 250 X 4.6 mm Column Temperature 40 °C Mobile Phases A: 5 mM ammonium acetate (pH 7.0) B: acetonitrile Gradient Isocratic, 50:50 = A:B Flow rate 0.5 mL/min Injection volume 5 µL Detection wavelength 254 nm (UV) Scan wavelength 190-400 nm (PDA) Total run time 10 min

4.4 Other equipment and Devices

High vacuum pump – Edwards RV3 pH meter – Fisher Scientific Accumet Basic AB15

Rotary Evaporator – Heidolph Collegiate Brinkmann rotary evaporator

Thermomixer – Eppendorf

4.5 Synthesis of C5'-modified thymidine

4.5.1 3',4'-Didehydro-2',3'-dideoxy-5'-oxothymidine (ddoT)

4.5.1.1 Synthesis of 5'-O-(tert-butyldimethylsilyl)-thymidine

To a stirred cold solution (0 °C) of thymidine (20 g) and imidazole (13.6 g) in anhydrous DMF was added tert-butyldimethylsilyl chloride (13.6 g) in one portion.

Stirring continued overnight at 0 °C until the reaction mixture turned from off-white to clear. The reaction was quenched by the addition of deionized water (1 L), when the reaction temperature was at 10 °C). The resulting precipitate was filtered using vacuum filtration and air dried. The crude product was refluxed in hexane (500 mL) overnight.

The precipitate was filtered and co-evaporated with toluene to afford the compound as a white powder. The course of the reaction was monitored by TLC using 6% methanol in

DCM as eluent. Yield: 52.93 g (92%). The analytical data obtained is compatible to that found in the reference.77

4.5.1.2 Synthesis of 5'-O-(tert-butyldimethylsilyl)- 3'-O-benzoyl-thymidine

To a solution of 5'-O-(tert-butyldimethylsilyl)-thymidine (3.67 g) in anhydrous pyridine (10.5 mL) was added benzoyl chloride (2.2 mL) dropwise and stirred for 1 hour.

Upon completion of the reaction (monitored by TLC with 6% methanol in DCM as eluent), the solvent was evaporated in vacuo and the crude product was triturated with water (50 mL) followed by sat. NaHCO3 (50 mL), then filtered and dried. The resulting product was azeotropically dried through co-evaporation with acetonitrile and recrystallized with acetonitrile to afford 62 as white crystals. Yield: 3.01 g (63.5%).116

4.5.1.3 Synthesis of 3'-O-benzoyl-thymidine

To a clear solution of 62 (3.0 g) in THF (60 mL) was added a TFA/H2O mixture

(1:1, 14.3 mL) at 0 °C over 20-30 minutes, then stirred at room temperature overnight.

Upon completion of the reaction, a cloudy suspension was formed. The progress was monitored using 6% methanol in DCM as eluent, sat. NaHCO3 (40 mL) and ethyl acetate

(40 mL) were added sequentially and filtered. The product was isolated as a white powder.117

4.5.1.4 Synthesis of 3',4'-Didehydro-2',3'-dideoxy-5'-oxothymidine (ddoT) with IBX71, 84

3'-O-benzoyl-thymidine (0.74 g, 1 mmol) was co-evaporated with toluene twice and dissolved in anhydrous acetonitrile (7 mL, 0.14 M) followed by the addition of IBX (0.28 g, 3 mmol). The suspension was immersed in an oil bath at 80 °C and stirred vigorously under reflux. After 1 hr (TLC monitoring with 80% ethyl acetate in hexane as eluent), the reaction was cooled to room temperature, filtered via gravity filtration, and washed with acetonitrile. The filtrate was evaporated in vacuo to yield the product as a yellowish waxy solid. Acetonitrile was added (13 mL) and Et3N (0.1 mL) was added dropwise. Upon completion of reaction (TLC monitoring in 6% methanol in DCM and 80% ethyl acetate

77 in hexane), toluene (10 mL) was added and the solvent was evaporated in vacuo. The crude mixture was purified by column chromatography with ethyl acetate in hexane to afford a white powder. Yield: 0.12 g (29.4%). δH (600 MHz, CD3CN) δ 9.43 (1H, s, H5'),

8.96 (1H, br s, NH), 7.80-7.40 (m, benzoic acid impurities), 7.08 (1H, dd, J = 2.45, 1.25

Hz, 6-H), 6.75 (1H, dd, J = 10.4, 5.2 Hz, 1H'), 6.30 (1H, t, J = 3.18 Hz, H3'), 3.35 (1H, ddd, J = 20.41, 10.40, 2.80 Hz, H2'), 2.94 (1H, ddd, J = 20.46, 5.20, 3.20 Hz), 1.81 (3H, d, J = 1.31, C6-Me). δc (600 MHz, CD3CN) δ 12.34 (CH3), 36.60 (CH2), 86.70 (CH),

112.18m 112.56, 121.18 (CH), 129.47 (impurities), 130.91 (impurities), 134.08

(impurities), 136.29 (CH), 151.05, 156.08, 164.46, 167.88, 182.15. ESI [M + Na+]: calculated for 245.054, found 245. In 0.1% acetic acid/CH3CN using LC-MS Method B,

[M + H]: 223. [M + Na]: 245.

4.5.2 Synthesis of Michael adduct using thiol-containing compounds43

To compound 58 in a mixture of 1:1 CH3CN:DCM was added thiol-containing compounds (RSH) in solid under anhydrous conditions. A total of 3 molar equivalents of

Et3N was added dropwise and the reaction was monitored for 3 days. Upon no changes detected by TLC, the resulting mixture was evaporated in vacuo and analyzed by NMR and MALDI. No changes in the reaction was observed by TLC. NMR analysis showed that peaks representing 72 (R=)was not found. MALDI was performed matrix free according to conditions provided previously to obtain the molecular weight of any products detectable in the crude mixture.

4.6 Reactivity and stability of C5'-modified thymidine

4.6.1 Stability of ddoT in PBS

ddoT (58, 30 mg) was dissolved in 5% acetonitrile in PBS buffer (total 1000 µL) pH = 7.0 at 4°C and 37 °C, respectively. A stability was determined at 4°C, and a half- life was determined at 37 °C. The expected degradation products were observed. The experiment was repeated in triplicate.

4.6.2 Stability of ddoT in BBS ddoT (58, 0.3 mg, 0.675mM) was dissolved in 5% acetonitrile in BBS buffer pH 8.0

(total 1000 µL) at 37 °C. A half-life was determined, and expected degradation products were observed. The experiment was repeated in triplicate.

4.6.3 Calibration curve of ddoT

ddoT (58, 30 mg, 1 mM) was dissolved in 5% acetonitrile in water (total 1000

µL) and serial dilution was performed for a calibration curve by using LC-MS. A total of

5uL was injected for each sample. The experiment was repeated 5 times. HPLC-MS analyses were performed on a Shimadzu LC-20A-PDA HPLC with a Shimadzu LC-MS

8050 mass detector. HPLC separations were performed on a YMC Pack ODS-A/C-18

(4.6 x 250 mm, 5 µm particle size) using an isocratic method (5 mM ammonium acetate:CH3CN (50:50, v:v)) with a 0.5 ml/min flow rate at 37 °C, detection at λ 190-

300nm using a photodiode array (PDA) detector.

4.6.4 Fragmentation of ddoT

LC-MS condition A was used. ddoT (58, 46mg) was dissolved in 1000 µL acetonitrile, and diluted 1000 times to be used for LC-MS/MS. Then, 5 µL of the sample was injected to obtain fragmentation patterns. The experiment was repeated in triplicate.

4.6.5 Reactivity of ddoT in GSH

ddoT (58, 80 µM) and GSH (60 mM) were incubated in in 5% acetonitrile in PBS buffer (total 1000 µL) pH = 7.0 at 37°C and 4°C, respectively, and half-life was determined. Each fraction was desalted with Ziptip® and analyzed with ESI-MS,

MALDI, and NMR. The experiment was repeated in triplicate.

4.6.6 Sample preparation of ddoT

ddoT (58, 1.16 mg) was dissolved in 50 µL of acetonitrile to facilitate with solubility, then added deionized water to 1045 µL (= 5 nmol/µL = 5 mM). Serial dilution was done with deionized water only to obtain lower concentrations and stored in refrigerator.

4.6.7 Reactivity of ddoT in GSH

ddoT (58, 0.625 mM) and equimolar of reduced R-SH (0.625 mM) was added into pH 8 buffer with or without 0.625 mM of Et3N. The mixture was incubated in thermomixer at 40 °C at 650 rpm overnight and analyzed the following day. A small aliquot was collected for time response reaction at discrete timepoint. Fractions were

80 collected and analyzed in LC-MS/MS using LC-MS Condition A. The experiment was repeated in triplicate.

4.6.8 Reactivity of ddoT in Coenzyme A (CoA)

ddoT (58, 0.625 mM) and equimolar of reduced GSH (0.625 mM) was added into pH 8 buffer with or without 0.625 mM of Et3N. The mixture was incubated in thermomixer at 40 °C at 650 rpm overnight and analyzed the following day using LC-MS

Condition A. A small aliquot was collected for time response reaction at discrete timepoint. Fractions were collected and analyzed in LC-MS/MS. The experiment was repeated in triplicate.

4.6.9 Reactivity of ddoT in Cysteine ddoT (58, 0.625 mM) and reduced Cysteine (0.625 mM) in degassed pH 4 NaCl∙

Na2B4O7 solution, and was added into pH 8 NaCl∙Na2B4O7 buffer. The mixture was incubated in thermomixer at 40 °C at 650 rpm overnight and analyzed the following day using LC-MS. The experiment was repeated in triplicate.

4.6.10 Reactivity of ddoT in N-Acetyl-Cysteine

ddoT (58, 0.625 mM) and reduced N-acetylcysteine (Sigma Aldrich, 0.625 mM) was added into pH 8 BBS buffer. The mixture was incubated in thermomixer at 40 °C at

650 rpm overnight and analyzed the following day using LC-MS. The experiment was repeated in triplicate.

4.6.11 Fragmentation of ddoT-RSH adducts

Fractions of ddoT-RSH adducts were collected manually using LC-MS 2020, and solvents were evaporated. The resulting compound was dissolved in 100 µL of 1:1 H2O:

CH3CN (v:v) and injected directly using LC-MS. The experiment was repeated in triplicate.

4.6.12 Time response reaction with RSH ddoT (58, 0.625 mM, or 312.5 nmol) and an equimolar amount of reduced R-SH (0.625 mM, 312.5 nmol) was added to a total of 500 µL (pH = 8.0). The mixture was incubated at 40 °C overnight and analyzed using LC-MS. A small aliquot was collected at discrete timepoints. Fractions were collected and analyzed with LC-MS/MS using Condition A.

The experiment was repeated in triplicate.

Appendix A

Supplemental information

Figure A- 1. Positive mode ESI-MS ionization spectrum of 58 (direct injection)

Figure A- 2. Positive mode ESI-MS ionization spectrum of 58 on column

Figure A- 3. Positive SIM mode ESI-MS/MS ionization spectrum of 58

Figure A- 4. Positive mode ESI-MS/MS product ion spectrum of 58 at CE 30eV with (top) column; without column (bottom)

Figure A- 5. Positive mode ESI-MS/MS product ion spectrum of 58 at CE 25eV with (top) column; without column (bottom)

Figure A- 6. Positive mode ESI-MS/MS ionization spectrum of 58 at various injection volumes (from top, 0.5 uL, 1 uL, 5 uL)

Figure A- 7. Negative mode ESI-MS/MS ionization spectrum of 58 at various injection volumes (from top, 1 uL, 5 uL) at CE 25 eV

Figure A- 8. Positive mode ESI-MS/MS precursor ion spectrum of 58 at CE 30eV

Figure A- 9. Positive mode ESI-MS/MS precursor ion spectrum of 58 at CE 25eV

Figure A- 10. Positive mode ESI-MS/MS precursor ion spectrum of 58 at CE 20eV

Figure A- 11. Positive mode ESI-MS/MS precursor ion spectrum of 58 at CE 15eV

Figure A- 12. Positive mode ESI-MS/MS precursor ion spectrum of 58 at CE 10eV

Figure A-13. Calibration curve of ddoT (58) absorbance as a function of the concentration at 37 °C

Datafile Name:cys adduct5.lcd Sample Name:cys adduct Sample ID:cys adduct mAU 229nm 234nm 239nm 750 244nm 249nm 254nm 259nm 264nm 500 269nm 274nm 279nm

250

0 15.8 15.9 16.0 16.1 16.2 16.3 16.4 16.5 16.6 16.7 16.8 min

Figure A- 14. UV spectra of 58 at various wavelengths Datafile Name:gsh add 24 hr_10302018_16.lcd Sample Name:gsh add 24 hr mAU 229nm 274nm 234nm 279nm 239nm 244nm 2.5 249nm 254nm 259nm 264nm 269nm 0.0 9.25 9.50 9.75 10.00 min

Figure A- 15. UV spectra of 59 at various wavelengths Datafile Name:gsh add 24 hr_10302018_16.lcd Sample Name:gsh add 24 hr mAU 229nm 269nm 10 234nm 274nm 239nm 279nm 244nm 5 249nm 254nm 259nm 0 264nm 4.6 4.7 4.8 4.9 min

Figure A- 16. UV spectra of 18 at various wavelengths

Table A- 1. Chemical properties of physiologically available Michael acceptors

Structure (pKa) MW Use

(g/mol)

767.54 TCA cycle

Fatty acid metabolism

Oxidative phosphrylation

Coenzyme Aa

121.16 Precursor to glutathione

Protein structure

Metal-ion binding Cysteine118

307.32 Antioxidant

Conjugation

Glutathione118

163.20 Antioxidant

Precursor to glutathione

N-Acetylcysteinea aMarvin Sketch®

Figure A- 17. Electrospray ionization mass spectrum of GSH adduct (MH+ 530) in positive mode at tR 22 min in Figure 2-13. Ion chromatograms of summed ions: m/z 530, 548 in BBS buffer (pH 8.0) at 40 oC.

Figure A- 18. Electrospray ionization mass spectrum of GSH adduct (MH- 528) in negative mode at tR 22 min in Figure 2-13. Ion chromatograms of summed ions: m/z 529, 546 in BBS buffer (pH 8.0) at 40 oC.

Figure A- 19. Electrospray ionization mass spectrum of GSH adduct (MH+ 530) in positive mode at tR 23.7 min in Figure 2-13. Ion chromatograms of summed ions: m/z 530, 548 in BBS buffer (pH 8.0) at 40 oC.

Figure A- 20. Electrospray ionization mass spectrum of GSH adduct (MH- 528) in negative mode at tR 23.7 min in Figure 2-13. Ion chromatograms of summed ions: m/z 528, 546 in BBS buffer (pH 8.0) at 40 oC.

Figure A- 21. Electrospray ionization mass spectrum of GSH adduct (MH+ 530) in positive mode at tR 25.3 min in Figure 2-13. Ion chromatograms of summed ions: m/z 530, 548 in BBS buffer (pH 8.0) at 40 oC.

Figure A- 22. Electrospray ionization mass spectrum of GSH adduct (MH- 528) in negative mode at tR 25.3 min in Figure 2-13. Ion chromatograms of summed ions: m/z 528, 546 in BBS buffer (pH 8.0) at 40 oC.

Figure A- 23. Electrospray ionization mass spectrum of GSH adduct (MH+ 530) in positive mode at tR 28.8 min Figure 2-13. Ion chromatograms of summed ions: m/z 530, 548 in BBS buffer (pH 8.0) at 40 oC.

Figure A- 24. Electrospray ionization mass spectrum of GSH adduct (MH- 528) in negative mode at tR 28 min in Figure 2-13. Ion chromatograms of summed ions: m/z 528, 546 in BBS buffer (pH 8.0) at 40 oC.

Figure A- 25. Positive SIM mode ESI-MS ionization spectrum of 85

Figure A- 26. Negative SIM mode ESI-MS ionization spectrum of 85

Figure A- 27. Negative SIM mode ESI-MS ionization spectrum of 85 in the presence of Et3N

Datafile Name:gsh add 24 hr_10302018_16.lcd Sample Name:gsh add 24 hr mAU 15.395/ 1.00 mAU 204 229nm 5 234nm 10.0 239nm 244nm 4 249nm 7.5 254nm 259nm 3 264nm 269nm 5.0 274nm 2 279nm 268

2.5 242

1 0.0 15.1 15.2 15.3 15.4 15.5 15.6 15.7 15.8 min 200.0 225.0 250.0 275.0 nm

Figure A- 28. UV spectra of 1 at various wavelengths in Figure 2-16

mAU 18.584/ 1.00 Datafile Name:gsh add 24 hr_10302018_16.lcd Sample Name:gsh add 24 hr 10

mAU 268 229nm 274nm 7.5 234nm 279nm 239nm 244nm 5 5.0 249nm 218 254nm 235 259nm 2.5 264nm 269nm 0 18.3 18.4 18.5 18.6 18.7 18.8 min 200.0 225.0 250.0 275.0 nm

Figure A- 29. UV spectra of 3 at various wavelengths in Figure 2-16

mAU 10 21.448/ 1.00 Datafile Name:gsh add 24 hr_10302018_16.lcd

Sample Name:gsh add 24 hr 268 5 mAU 218

229nm 274nm 235 5.0 234nm 279nm 239nm 0 244nm 249nm 2.5 254nm 259nm -5 264nm 269nm 0.0 21.2 21.3 21.4 21.5 21.6 min 200.0 225.0 250.0 275.0 nm

Figure A- 30. UV spectra of 4 at various wavelengths in Figure 2-16

100

Figure A- 31. Positive SIM mode ESI-MS ionization spectrum of 87

Figure A- 32. Negative SIM mode ESI-MS ionization spectrum of 87

101

3:990.00(+) 200000

100000 0 Figure A- 33. Total ion chromatogram in positive SIM mode ESI-MS ionization spectrum of 87, [CoA-ddoT+H]+ = 990 m/z

4:988.00(-) 2000000

1000000

0 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 min

Figure A- 34. Total ion chromatogram in positive SIM mode ESI-MS ionization spectrum of 87, [CoA-ddoT-H]- = 989 m/z Datafile Name:T-GSH_AK_5neg batch and shutdown_8302018_3.lcd Sample Name:T-GSH Sample ID:T-GSH mV Detector A Ch1 254nm Detector A Ch2 280nm

1000

500

0 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 min

Figure A- 35. Analysis of 18 in the presence of GSH for 24 hrs by HPLC at 254 nm and 280 nm in borate buffered saline in pH 8.0 at 37 °C. The mobile phase was composed of 0.1% acetic acid:CH3CN (v:v)

Datafile Name:F-GSH_AK_5neg batch and shutdown_8302018_7.lcd Sample Name:F-GSH Sample ID:F-GSH mV 1000 Detector A Ch1 254nm Detector A Ch2 280nm 750

500

250

0 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 min

Figure A- 36. Analysis of 59 in the presence of GSH for 24 hrs by HPLC at 254 nm and 280 nm in borate buffered saline in pH 8.0 at 37 °C. The mobile phase was

composed ofDatafile 0.1% Name:T-CoA_AK_5neg acetic acid:CH batch and shutdown_8302018_6.lcd3CN (v:v) Sample Name:T-CoA Sample ID:T-CoA mV Detector A Ch1 254nm 400 Detector A Ch2 280nm

300

200

100

0 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 min

Figure A- 37. Analysis of 18 in the presence of CoA for 24 hrs by HPLC at 254 nm and 280 nm in borate buffered saline in pH 8.0 at 37 °C. The mobile phase was composed of 0.1% acetic acid:CH3CN (v:v)

102

Datafile Name:F-coA_AK_5neg batch and shutdown_8302018_10.lcd Sample Name:F-coA Sample ID:F-coA mV 300 Detector A Ch1 254nm Detector A Ch2 280nm

200

100

0 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 min

Figure A- 38. Analysis of 59 in the presence of CoA for 24 hrs by HPLC at 254 nm and 280 nm in borate buffered saline in pH 8.0 at 37 °C. The mobile phase was composed of 0.1% acetic acid:CH3CN (v:v)

Datafile Name:T-NAC_AK_5neg batch and shutdown_8302018_4.lcd Sample Name:T-NAC Sample ID:T-NAC mV Detector A Ch1 254nm Detector A Ch2 280nm

1000

500

0 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 min Figure A- 39. Analysis of 18 in the presence of NAC for 24 hrs by HPLC at 254 nm and 280 nm in borate buffered saline in pH 8.0 at 37 °C. The mobile phase was composed of 0.1% acetic acid:CH3CN (v:v)

Datafile Name:F-NAC_AK_5neg batch and shutdown 5uL_942018_6.lcd Sample Name:F-NAC Sample ID:F-NAC mV SPD-20AV Ch1 254nm SPD-20AV Ch2 280nm 150

100

0 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 min

Figure A- 40. Analysis of 59 in the presence of NAC for 24 hrs by HPLC at 254 nm and 280 nm in borate buffered saline in pH 8.0 at 37 °C. The mobile phase was

composed ofDatafile 0.1% Name:T-Cys_AK_5neg acetic acid:CH batch and shutdown_8302018_5.lcd3CN (v:v) Sample Name:T-Cys Sample ID:T-Cys mV 1500 Detector A Ch1 254nm Detector A Ch2 280nm

1000

500

0 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 min

Figure A- 41. Analysis of 18 in the presence of cysteine for 24 hrs by HPLC at 254 nm and 280 nm in borate buffered saline in pH 8.0 at 37 °C. The mobile phase was composed of 0.1% acetic acid:CH3CN (v:v)

103

Datafile Name:F-cys_AK_5neg batch and shutdown 5uL_942018_7.lcd Sample Name:F-cys Sample ID:F-cys mV SPD-20AV Ch1 254nm SPD-20AV Ch2 280nm 150

100

0 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 min

Figure A- 42. Analysis of 59 in the presence of Cysteine for 24 hrs by HPLC at 254 nm and 280 nm in borate buffered saline in pH 8.0 at 37 °C. The mobile phase was composed of 0.1% acetic acid:CH3CN (v:v)

104

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