Characterization of DNA Damaging Intermediates by EPR

Chromium Carcinogenesis: Characterization of DNA damaging Intermediates by EPR

31P NMR, HPLC, ESI-MS and Magnetic Susceptibility

A dissertation presented to

the faculty of

the College of Arts and Sciences of Ohio University

In partial fulfillment

of the requirements for the degree

Doctor of Philosophy

Roberto Marín Córdoba

March 2010 2

This dissertation titled

Chromium Carcinogenesis: Characterization of DNA damaging Intermediates by EPR

31P NMR, HPLC, ESI-MS and Magnetic Susceptibility

ROBERTO MARÍN CÓRDOBA

has been approved for

the Department of Chemistry and Biochemistry

and the College of Arts and Sciences by

Rathindra N. Bose

Professor of Chemistry and Biochemistry

Benjamin M. Ogles

Dean, College of Arts and Sciences 3

ABSTRACT

MARÍN CÓRDOBA, ROBERTO, Ph.D., March 2010, Chemistry and Biochemistry

Chromium Carcinogenesis: Characterization of DNA damaging Intermediates by EPR

31P NMR, HPLC, ESI-MS and Magnetic Susceptibility (150 pp.)

Director of Dissertation: Rathindra N. Bose

The hydrolytic cleavage and oxidative degradation mechanisms of dGDP by the oxochromate-(V) complexes bis(2-ethyl-2-hydroxybutanoato)oxochromate(V) (I) and bis(hydroxyethyl)-amino-tris((hydroxymethyl)methane)oxochromate(V) (II) in the

presence of H2O2 were investigated at neutral pH. The products of the reactions were separated and characterized by chromatographic and spectroscopic methods. The oxidative degradation is supported by the detection of free G, furfural, phosphoglycolate, pyrophosphate, guaninepropenal, 8OHdG and guanidinohydantoin. These products are formed through two parallel mechanisms that start with a common Cr-dGDP intermediate in which the oxochromate binds the α phosphate moiety followed by abstraction of H from C4’ and C5’ from the ribose. The detection of inorganic phosphate and dGMP suggests that when the oxometal binds the β phosphate it mainly promotes hydrolytic cleavage of the phosphate diester bond. By estimating the amount of each catabolite it was concluded that the oxo metal ion does not show selectivity during the hydrogen abstraction and that oxidation of the substrate is preferred over its hydrolysis. The reaction between diperoxoaquaethylendiamine chromium(IV) (III) and glutathione (GSH) at neutral pH was studied by EPR and ESI-MS. The ions of m/z ratios of 450 and 757 were identified as intermediates while the ions of m/z ratios of 484 and 775 were identified as products. Three EPR signals detected at g = 1.996, 1,986 and 1.983 were attributed to Cr(V) intermediates while a signal at g = 1.975 (peak to peak line width = 259.72 G) that appeared after the Cr(V) signals had disappeared was 4 attributed to Cr(IV). Spin trapping experiments with DMPO and DEPMPO revealed that the GS radical but not OH radical was formed during this reaction. The GS radical and the Cr(V) intermediates detected by EPR confirmed that the reaction occurs in a series of one electron transactions. Overall this reaction proceeds in cycles. The reaction produces Cr(V) intermediates followed by accumulation of a chromium(IV) second intermediate that decays slowly to a Cr(III) product. The DNA damage assay suggests that during its reaction with glutathione, III becomes a DNA damaging agent.

The mixture of intermediates of the reaction between chromate and glutathione in glycine buffer (pH 2.8) was investigated with a SQUID susceptomer. The saturation magnetic moment was calculated to be 1.39 µB while the effective magnetic moment was

2.55 µB. Based on the saturation magnetic moment, the intermediates were characterized as

30 % Cr(IV) and 69 % Cr(V). Using the effective magnetic moment the mixture was

characterized as 58 % Cr(IV) and 42 % Cr(V). The spin only formula gave a total spin

angular momentum of 0.88 which supports Cr(IV) as the predominant intermediate. As

previous studies did not detect Cr(V) EPR signals, it was assumed that only a Cr(IV)

intermediate was formed. In light of the present work and stoichiometric data from previous

work, the silent part of the mixture of intermediates was interpreted as a Cr(V) dimer in

which the Cr centers are diamagnetically coupled. With this data in consideration, the

proportion of Cr(IV) in the mixture of intermediates should be at least 65%.

Approved: ______

Rathindra N. Bose

Professor of Chemistry and Biochemistry 5

ACKNOWLEDGMENTS

The end of this journey would not be possible without the people that helped me throughout it. First to Dr. Gisell Sandi, who encouraged me to apply to the graduate school and to the Department of Chemistry at Northern Illinois University. Also the NIU chemistry graduate committee that accepted my application and awarded me a teaching assistantship throughout my entire stay. In this regard I would like to thank the tax payers of the United States of America, who made possible the research and the teaching activities involved in this process.

From the very beginning Dr. Gerardo Chacon and Dr. Jorge Alvarado awarded to me their friendship and help in many situations. For this I am thankful.

Soon after my arrival to the United States of America, the Highlands made me part of their family, in particular John, Mrs. Rose Mary Highland, Angelo, Mary, Rita and Sheila. To them I give my gratitude. Thank you John for helping me to run my errands when I did not have a car, for picking up my recyclables, for introduce me to your lovely family and for your friendship.

Also thank you Dr. Gregory Ross for allowed me to participate during the school year of 2005 in the Foreign Language Residence Program at NIU.

Special thanks to Leslie and Brad Shive, who I am sure have helped hundreds of international students personally and through the Network of Nations and other organizations and activities. And thanks also to Cliff Golden and the scouts of the Troop

33 of Dekalb Illinois for accepting me as scout leader and for sharing with me the camaraderie that makes boy scouts an international community. 6

Of great help were, and still are Dr. James Erman and Dr. Lidia Vitello. From them not only did I obtain good advice and friendship, but a model upon which to look.

A particular joyful memory was to watch my first snow fall in Dr. Erman’s office.

Dr. Morley and Mrs. Maureen Russell are two sincere friends who still follow my well being, - my heartfelt thanks.

I would like also to thank all the members of Dr. Bose and Dr. Anima Bose research groups whose company, friendship, and help I enjoyed over the last five years, and in particular to Dr. Leila Maurman, Dr. Robert Mishur, Dr. Shadi Moghadas, Raghu

Kumar and Pradeep Babbury,

This dissertation was performed in the research laboratories of Northern Illinois

University and the Konneker Research Laboratory of Ohio University. Also, the EPR studies were performed at the EPR and Imaging Center of Ohio State University. To the staff members of these institutions and their Chemistry and Biochemistry Departments, I give sincere thanks. In particular I would like to mention Dr. Jon Carnahan, graduate chair of the NIU Chemistry and Biochemistry Department at my arrival in 2004 and Dr.

Periannan Kuppusamy, head of the EPR center at Ohio State University.

During the last two years of my doctoral work I also participated in the study of a catalyst for the reaction of oxygen reduction for applications in fuel cells. I would like to thank to Dr. Anima Bose for accepting me as part of her research group and for her guidance on such an important subject.

Finally I would like to thank Dr. Rathindra Bose for allowing me to work on his research projects and for his guidance and thoughtful suggestions. Few graduate 7 students, regardless of the name of the institution, have the opportunity that was given to me, and that I vigorously took. During my dissertation I had the opportunity to work with the most relevant instrumental techniques to perform state of the art science, including NMR, EPR, HPLC, ESI-MS, magnetic susceptibility, electrochemical analysis and UV spectroscopy. And through my participation in the catalyst for fuel cells project and the synthesis and chemical analysis of pyrodach-2 and 4, I also gained knowledge in

GFAA, ICP-OES and surface science techniques that include SEM, EDX, XPS and fuel cell testing.

More importantly, working with and witnessing Dr. Bose’s performance as both administrator of science and as scientist. I also refined my soft skills, in particular courage, creativity, commitment and compassion. A very important lesson that I am taking with me comes from Dr. Bose own story as scientist. I quickly noticed that his work from twenty years or more is giving fruits today, thanks to his perseverance and courage as a deep thinker. It is of the popular domain that science is about trial and error; but what most do not know is that intelligently designed and carefully performed experiments are most likely to give more meaningful data than errors, with less trials. Dr.

Bose’s guidance led me to comprehend the depth of this statement.

Dedicated to little Robin bird and all the young little faces of our families

TABLE OF CONTENTS

Page

Abstract ...... 3

Acknowledgments...... 5

List of Tables ...... 12

List of Figures ...... 13

List of Abreviations ...... 17

Preface Objectives for Study ...... 18

Chapter 1 Introduction ...... 19

Indirect Free Radical DNA Damage ...... 20

Direct Metal-Mediated Oxidative DNA Damage ...... 21

Direct Metal-DNA Binding ...... 21

The Oxidation State of Chromium Carcinogens ...... 21

Implications of the Mechanism of the Reduction of Cr(VI) in Biological Systems ..... 32

Chapter 2 Oxidative and Hydrolytic Activity of Bis(2-Ethyl-2-Hydroxy

Butanoato)Oxochromate(V) towards Deoxyguanosinediphosphate...... 35

Experimental Part ...... 35

Reagents ...... 35

Reactions of dGDP and Cr(V) ...... 35

Test For Thiobarbituric Acid (TBA) Reactive Species ...... 36

Nuclear Magnetic Resonance Measurements ...... 37

High Performance Liquid Chromatography Measurements ...... 37 10

Results ...... 38

Discussion ...... 46

Chapter 3 ESI-MS and EPR Characterization of the Intermediates and Products of the

Reaction between Diperoxoaquaethylendiaminechromium (IV) and Glutathione ...... 56

Experimental Part ...... 56

Reagents ...... 56

ESI-MS Measurements ...... 56

Nuclear Magnetic Resonance Measurements ...... 57

Electron Paramagnetic Resonance Measurements ...... 57

Reactions between III and GSH ...... 58

31P NMR studies of the reaction of III and DNA ...... 58

Results ...... 59

ESI-MS Studies ...... 59

EPR Characterization of the Reaction Intermediates ...... 73

Reactions of III and GSH with DNA ...... 97

Discussion ...... 98

Chapter 4 Magnetic Characterization of the Intermediates of the reaction between

Chromate and Glutathione in Glycine buffer (pH 2.8) ...... 109

Experimental Part ...... 109

Reagents ...... 109

UV-VIS Spectrophotometric Measurements ...... 110

Magnetic Susceptibility Measurements ...... 110 11

Kinetic Profiles ...... 115

Results ...... 116

Magnetic Measurements ...... 119

Discussion ...... 134

Chapter 5 Conclusions ...... 140

References ...... 142

LIST OF TABLES

Page

Table 1. Comparison of the chromatograms of the reaction between oxochromate (V) and dGDP in the presence of H2O2 at pH 7.0 and the control...... 54

Table 2. Assignment of the major peaks of the mass spectrum shown in Figure 10. .... 62

Table 3. Distribution of chromium in the reaction between Cr(VI) and glutathione in 100 mM glycine at the time when [Intermediate] is at maximum concentration (tmax)...... 119

LIST OF FIGURES

Page

Figure 1: Chromium (IV) ester reported by Shi59, 60 et al...... 26

Figure 2: Structures of chromium (IV) and (V) complexes employed to study the reduction of chromium by glutathione (GSH) and as model carcinogens...... 34

Figure 4. 31P NMR of reactions between dGDP and EHBA-Cr(V) in Bis Tris at pH 7 and under various concentrations of H2O2...... 40

Figure 5. Visible spectrum of the crude of the reaction between thiobarbituric acid and the mixture obtained by reacting dGDP and EHBA-Cr(V) in the presence of H2O2 at pH 7...... 41

Figure 6. HPLC of (A) the reaction of EHBA-Cr(V) (4.0mM) and dGDP (4.0 mM) in the presence of H2O2 at pH; 7 and (B) the same mixture after treatment with alkaline phosphatase...... 44

Figure 7. UV spectrum of the compound eluted at 5.93 min (peak h) during the chromatographic separation of the reaction mixture between dGDP (4.0 mM), EHBA- Cr(V) (4.0 mM), H2O2 (200 mM) at pH 7.0...... 45

Figure 8. Proposed mechanism for the reaction of compounds I or II and dGDP in the presence of H2O2 at pH 7.0...... 51

Figure 9: Structures of 8-hydroxyguanosine (8OHdG) and guanidinohydantoin (Gh). . 52

Figure 10. ESI-MS in the positive ion mode of a 20 µM solution of III in water...... 60

Figure 11. Average ESI-MS of the reaction between III and GSH in Bis Tris buffer (pH 6.5)...... 61

Figure 12. (+)-MS3 of m/z 484...... 65

Figure 13. (+)-MS2 of m/z 757...... 66

Figure 14. (+)-MS2 of m/z 775...... 67

Figure 15 ESI-MS of the reaction mixture between III and GSH shown the region of between 438 and 454 m/z units...... 68 14

Figure 16. Observed (circles) and simulated (solid line) time course SIM-ESI-MS of m/z 450...... 69

Figure 17. Observed (circles) and simulated (solid line) time course SIM-ESI-MS of m/z 484...... 70

Figure 18. Observed (circles) and simulated (solid line) time course SIM-ESI-MS of m/z 757...... 71

Figure 19. Observed (circles) and simulated (solid line) time course SIM-ESI-MS of m/z 775...... 72

Figure 20. EPR control spectra of 1.0 mM III in water (A) and 1.0 mM III in 100 mM phosphate (B) buffer pH 7.0...... 75

Figure 21. EPR spectra of the reaction mixture of 1.0 mM III, 100 mM phosphate pH 7.0 and (A) 5.00 mM GSH, (B) 10.0 mM GSH, (C) 20.0 mM GSH, (D) 40.0 mM GSH and (F) 60.0 mM GSH...... 76

Figure 22. EPR spectra of the reaction mixture of 2.0 mM III, 100 mM phosphate pH 7.0 and 20.0 mM GSH...... 77

Figure 29. Observed (circles) and simulated (solid line) EPR time course of the intermediate with g = 1.986 detected in the reaction mixture of 1 mM III, 60.0 mM GSH and 100 mM phosphate buffer (pH 7.0)...... 84

Figure 30. Observed (circles) and simulated (solid line) EPR time course of the intermediate with g = 1.996 detected in the reaction mixture of 1 mM III, 60.0 mM GSH and 100 mM phosphate buffer (pH 7.0)...... 85

Figure 31. Structures of spin trap adducts of the OH radical...... 87

Figure 32. EPR spectrum of the reaction mixture of 1.0 mM III, 20.0 mM GSH, 100 mM phosphate (pH 7.0) and 10 mM DMPO...... 88

Figure 34. Spin trap experiments with DEPMPO (10 mM in all experiments), 100 mM in phosphate (pH 7.0): (A) 1.0 mM EHBA-Cr(V) (I), 100 mM H2O2; (B) 1.0 mM EHBA-Cr(V) (I), 20.0 mM GSH; (C) 1.0 mM III, 100 mM H2O2; (D) 1 mM III, 20.0 mM GSH...... 91

Figure 35. Overlay of the EPRs of the reaction mixture of 1.0 III, 20.0 mM GSH and 10 mM DEPMPO in 100 mM phosphate (pH 7.0) (red curve) and the mixture of 1.0 mM EHBA-Cr(V) (I), 100 mM H2O2 and 10 mM DEPMPO in 100 mM phosphate (pH 7.0) (black curve)...... 94

Figure 36. Overlay of the EPRs of the reaction mixture of 1.0 III, 20.0 mM GSH and 10 mM DEPMPO in 100 mM phosphate (pH 7.0) (red curve) and the mixture of 1.0 mM EHBA-Cr(V) (I), 20 mM GSH and 10 mM DEPMPO in 100 mM phosphate (pH 7.0) (black curve)...... 95

Figure 37. Overlay of the EPRs of the reaction mixture of 1.0 III, 20.0 mM GSH and 10 mM DEPMPO in 100 mM phosphate (pH 7.0) (red curve) and the mixture of 1.0 mM EHBA-Cr(V) (I), 100 mM H2O2, 20 mM GSH and 10 mM DEPMPO in 100 mM phosphate (pH 7.0) (black curve)...... 96

Figure 38. 31P NMR spectrum acquired before (A) and after (B) the reaction of III (0.145 mM), the 28-mer oligonucleotide described in the experimental part (0.240 mM) and glutathione (0.420 mM) in the presence of Bis Tris (3 mM) pH 7.0...... 97

Figure 39. (a) Top view of the stopper, (b) Lateral view of the empty cuvette without the stopper, (c) Lateral view of the cuvette with solution inside...... 111 16

Figure 40. Magnetic susceptibility of a Palladium reference material provided by the MSPM manufacturer...... 112

Figure 41. Observed (Circles) and simulated (solid line) absorbance-time trace at 460 nm of a mixture of 1.02±0.05 mM Cr(VI), 15.0±0.8 mM glutathione in 100 mM glycine (pH 2.8)...... 117

Figure 42. Magnetization curves of several samples of CrK(SO4)2...... 121

Figure 43. Magnetization curves of the reaction mixture of 1.02±0.05 mM Cr(VI) and 15.0±0.8 mM GSH in 100 mM glycine, pH 2.8, in D2O...... 124

LIST OF ABBREVIATIONS dGDP deoxyguanosinediphosphate dGMP deoxyguanosinemonophosphate dG deoxyguanosine 8OH-dG 8-hydroxyguanosine Py pyrophosphate PG phosphoglycolic acid or phosphoglycolate EHBA bis(2-ethyl-2-hydroxy butanoic acid) EHBA-Cr(V) sodium bis(2-ethyl-2-hydroxy butanoato)oxochromate(V) Gh guanidinohydantoin MD malondialdehyde BP base propenals TBA thiobarbituric acid BT bis-tris GSH glutathione GSSG oxidized glutathione GS glutathione thyil radical GS- glutathionate or un protonated glutathione EPR electron paramagnetic resonance ESI-MS electro spray ionization mass spectrometry OH hydroxyl radical DMPO 5,5-dimethyl-1-pyrroline N-oxide DEPMPO 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide G guanine NMR nuclear magnetic resonance spectroscopy HPLC high performance liquid chromatography

PREFACE

Objectives for Study

Hypervalent chromium species, Cr(IV) and Cr(V), are considered to be the putative carcinogens that damage and mutate DNA in chromium(VI) mediated carcinogenesis. The dissertation topic is devoted to gain insight into the existence of either or both hypervalent oxidation states in cellular milleu. In particular, the study is focused to detect and characterize Cr(IV) and Cr(V) intermediates with kinetic data in conjunction with magnetic data obtained from SQUID measurements of the reaction between Cr(VI) and glutathione in glycine buffer (pH 2.8) when the intermediates of this reaction are expected to be in their maximum concentration. Also, the reaction between a chromium(IV) complex (diperoxoaquaethylenediaminechromium(IV), III) and glutathione was characterized by ESI-MS and EPR experiments. Structures of the intermediates and products of this reaction were determined using the SIM (Selected Ion

Monitoring) and CID (Collision Induced Dissociation) techniques. Chromium intermediate species can degrade the molecule of DNA by two pathways: hydrolysis and oxidation. The study is also directed to address the ability of bis[2-ethyl-2- hydroybutanoato(2-)]oxochromate(V) (EHBA-Cr(V), I) to oxidize and to hydrolyze nucleotides by studying a model reaction of this complex with 2’- deoxyguanosinediphosphate (dGDP).

CHAPTER 1

Introduction

Chromium is a known carcinogen in several work settings1-5 where poisoning

occurs through breathing.6 Also, the carcinogenic risk associated with oral exposure to

hexavalent chromium in drinking water has been reviewed in light of recent analysis that

revealed that 38% of municipal sources of drinking water in California have detectable

levels of hexavalent chromium.7 More interestingly, human population exposed to hexavalent chromium via drinking water showed a statistically significant increase in stomach cancer.7 These remarkable health hazard facts make the topic of chromium

carcinogenesis a field of remarkable societal importance.

-3 Chromate or dichromate, being iso structural and iso electronic with PO4 and

-2 SO4 penetrate the cell membrane through the anion channels. Once in the cytoplasm or in the nucleus, Cr is reduced to Cr(IV) and Cr(V) and then ultimately to Cr(III). 8-11

Neither the hexavalent nor the trivalent chromium damages the DNA molecule. Since the participation of hypervalent chromium species in damaging DNA was established, one way to obtain information about these species is by studying the products of the reaction of chromate or chromium model compounds with DNA, oligos or individual nucleotides. In any given case, in vitro studies with both Cr(V) and Cr(IV) model compounds have shown that both types of compounds are able to produce DNA strand breaks and DNA oxidation yielding catabolites like, 5-methylen-furanone, furfural, base propenals and 8-OH-Guanosine.12-18 20

There is plenty of evidence that Cr(V) intermediates are DNA damaging agents; but the reports regarding existence of Cr(IV) are rather scarce. It is currently accepted that these intermediates are the carcinogenic form of chromium and responsible for the observed DNA lesions which include double and single strand breaks, Cr-DNA adducts,

DNA-DNA cross links, DNA-protein cross links, oxidative damage and a-basic sites.17, 19,

Although the mechanisms of the reaction of several model chromium compounds

with DNA are understood,2, 12, 13, 21-23 two major unknowns remain to be answered. It is still unclear what oxidation state of chromium is predominantly responsible for most

DNA lesions.1, 2 Moreover, the ligation environment of such species has not been well

resolved.1, 2

Three general mechanisms have been proposed to explain the DNA damage

effects of Cr(VI); these include indirect free radical DNA damage, direct metal-mediated

oxidative DNA damage, and direct metal-DNA binding.

Indirect Free Radical DNA Damage

Participation of free radicals like reactive oxygen species was based on the

observed products which include oxidized DNA bases, abasic sites, DNA strand breaks, and DNA-DNA and DNA-protein crosslinks following hexavalent chromium treatment

in vitro and in vivo, and observations that hexavalent chromium toxicity is reduced in the

presence of free radical scavengers.7

Direct Metal-Mediated Oxidative DNA Damage

Treatment of cells with both chromium (V) model compounds and chromium (VI) causes the activation of genes that respond to oxidative stress; but not of genes that are activated upon exposure to oxygen radicals. In addition, it has been observed that the genes activated by exposure of cells to chromium (VI) are different from those activated

by the exposure of cells to hydrogen peroxide, dioxygen or X-rays.7 Also, the production

of DNA damage markers like 8-OH-dG can be the result of oxidation of the base by the

metal,24 while the concomitant oxidation of the sugar moiety by hydrogen abstraction is

consistent with the direct observation of Cr(V)-DNA intermediates in vitro using EPR

spectroscopy12, 25 and the indirectly detection of Cr(III)-DNA adducts after treatment of

intact cells with Cr(VI).26-29

Direct Metal-DNA Binding

Chromium(III)-DNA adducts have been detected after treatment of whole cells

with chromate or dichromate.26-29 It has been suggested that DNA-Cr(III)-DNA links are

the most damaging among Cr(VI) induced DNA lesions, leading to DNA polymerase

arrest and S-phase blockage of the cell cycle.29, 30 As chromate treated intact cells have

also rendered DNA-protein cross links,31, 32 it has been speculated that they are likely

responsible for the disruption of DNA interactions with transcription factors.33

The Oxidation State of Chromium Carcinogens

In vivo it is well established that Cr(V) exists as intermediates in the reduction

pathway of chromate to Cr(III). These intermediates have been detected by EPR in 22 whole mice and other specimens.34-38 In studies performed in vitro the intermediates that

result upon the reaction of Cr(VI) with cellular low molecular weight reductants like

glutathione, ascorbate or cysteine and cellular extracts like mitochondria, kidney and liver homogenates and microsomes have been described as species of Cr(IV) and

Cr(V).22, 38-41 Those species that contain Cr(V) can be detected by EPR; however those

that contain Cr(IV) are typically EPR silent and are identified only indirectly. Recently

the mixture of relevant intermediates in the reaction of Cr(VI) and glutathione has been

described as mostly a Cr(VI) glutathione complex.42

The mechanism of the reduction of chromium (VI) is strongly dependant on the

concentration of the reducing agent as has been illustrated by extensive EPR experiments,

in vivo and in vitro. In the in vitro reduction of chromate by glutathione at neutral pH,

the X band EPR has shown several Cr(V) intermediates whose signal intensities are

modulated by the concentration of the reducing agent.43 Aiyar44 et al detected two Cr(V)

intermediates (g = 1.986 and g = 1.996) in the reduction of 2.7 mM Cr(VI) with 8.1-27 mM glutathione at pH 8.0. In this study the intensity of the species with g = 1.986

increased almost linearly with the increase in GSH concentration, while the g = 1.996

was seen only at the highest glutathione concentration. The species with g = 1.996 that

predominates in excess glutathione was also seen by O’Brien45, 46 et al. These workers

precipitated the intermediate by cooling quickly and adding methanol and their analysis

43 was consistent with a molecular formula of Na4(GSH)4Cr.8H2O. Goodgame and Joy

also studied the reduction of chromate and glutathione and using 1:1 mole ratio of the

reactants observed five EPR signals (g = 1.995, 1.985, 1.997, 1.972 and 1.970), the 23 strongest one being at g = 1.985. In this study, an increase in the GSH/Cr(VI) was accompanied by a decrease in intensity of the species with g = 1.985, while the species with g = 1.995 grew, becoming the dominant feature beyond 5:1 glutathione/Cr(VI).

Similar results were obtained by other groups.22 Based on the dependencies of these EPR

signals with the glutathione concentration it is apparent that they correspond to at least

mono and bis-(glutathionato)Cr(V) complexes;22, 43 however, according to Lay47 et al

only a glutathionatoaquaoxochromate(VI) is formed in the reaction of Cr(VI) and

glutathione.

A generalization regarding the reduction of chromium(VI) at neutral pH can be

made. Under low concentration of the reductant, a series of one electron reductions may

occur, producing mainly a mixture of Cr(V) and Cr(IV), while when the intracellular

levels of the reductant are present in large excess, the initial steps involves a two electron

reduction leading to Cr(IV) followed by one electron reduction to Cr(III). In the process,

free radical species generated during the reaction contribute to produce secondary

toxicological effects.48

In order to fully understand the chromium (VI) reduction mechanism, an

assessment of the relevance of chromium (IV) in the chromium (VI) reduction pathway is

required.

Bose22, 39 et al have used indirect methods to detect the presence of Cr(IV) in the

reaction of Cr(VI) and glutathione. The same group has measured the formal potential of

the couple Cr(IV/III) showing that Cr(IV) is a more powerful oxidizing agent than Cr(V), because the corresponding Cr(V/IV) couple has a higher reduction potential.49 In 24 particular, the reduction of Cr(VI) with glutathione (GSH) in an excess of the oxidized form of glutathione (GSSG) proceeds through long lived intermediates in the pH range

1.8-3.5. These intermediates were identified as Cr(V) and Cr(IV) by EPR and time course magnetic susceptibility measurements respectively.39 However; in the presence of

glycine the reduction of Cr(VI) with GSH proceeds almost exclusively through the

Cr(IV) intermediate.22

As in studies with ESI-MS and XAS spectroscopy of the reaction of Cr(VI) and

glutathione in which the presence of Cr(IV)-glutathione complexes was not observed, the

evidence obtained by dynamic magnetic susceptibility methods22 of Cr(IV) as an

intermediate in this reaction has been disputed.47 However, the participation of Cr(IV) in

the reduction pathway from Cr(VI) or Cr(V) to Cr(III) as a result of its reaction with

either cellular or inorganic reductants has been inferred chemically and instrumentally.

For example Ce(III) is capable of disproportionating Cr(V), Ti(III) reduces Cr(V) with

the production of Cr(IV) as the limiting step and the reaction of Cr(V) with Fe(II), V(IV)

and U(IV) produces a long-lived Cr(IV) intermediate.50-52 In addition, reactions of Cr(V)

with some non-metal reductants exhibit a “clock reaction” which is auto catalyzed by

Cr(IV) and the reaction of Cr(VI) and glutathione and other cellular reducing agents

afford long-lived Cr(V) and Cr(IV) intermediates.53, 54

Few reports have appeared in the literature concerning the involvement of

chromium (IV) in the reduction of chromium(VI) with the most likely physiological low

molecular weight reductants like glutathione, cysteine or ascorbic acid. 25

Wetterhanh55 et al detected the presence of Cr(IV) indirectly by reaction with

Mn(II) and a subsequent decrease in the Mn(II) EPR signal. This approach relies on the assumption that Mn(II) does not react with Cr(V) but studies of the reaction between

EHBA-Cr(V) (I) and a EDTA-Mn(II) complex in the pH range 2.7-3.7 demonstrated that these complexes of Cr(V) and Mn(II) do react slowly in a four step process that in excess

Mn (II) produces Cr (III) and Mn (III).56 Gould57 et al also have shown that Mn(II) catalyzes the disproportionation of Cr(IV)-EHBA complexes. The results that led to conclusions based on this assay might be incorrect; however, as pointed out by

Wetterhanhn,58 reliable results can be obtained if control experiments are undertaken, accompanied with spectroscopic data and interpreted with care taking into consideration the possible increase of Cr(V) concentration due to reaction with Mn(II).

Chromium (IV) model compounds have also been employed to gain insights into the carcinogenic power of this oxidation state. Shi and Dalal59, 60 et al isolated the complex IV from the reaction of CrO3 with 2,4-dimethyl-pentane-2,4-diol. The proposed structure is the result of x-ray diffraction, magnetic susceptibility and EPR experiments.

It is particularly interesting that the EPR spectrum of the powder and its solution in CCl4 contains a broad signal (peak to peak width of about 700 Gauss at g = 1.957) which is considered typical of Cr (IV) complexes. The large line broadening is considered typical of species with two or more unpaired electrons due to rapid spin relaxation induced by the electronic dipole-dipole interaction. According to these authors,59, 60 this complex alone caused DNA strand breaks and the addition of hydrogen peroxide increased the damage via hydroxyl generation by a Cr mediated Fenton-like reaction. Also, the 26 addition of Mn(II) reduced the level of damage proving that the damaging species was a

Cr(IV) species. The same workers61 also reported the synthesis of a Cr(IV)-GSH complex which resulted from mixing 100 mM GSH and 25 mM Na2Cr2O7 in water at

room temperature. The corresponding reference shows the EPR spectrum of an aqueous

solution of this ester in which a very broad line (peak to peak width of 480 gauss) was

centered at g = 1.9629.

O O Cr HO OH IV

Figure 1: Chromium (IV) ester reported by Shi59, 60 et al.

These workers ruled out Cr(V) based on the line width and used magnetic

susceptibility measurements of the powder to rule out the possibility of a Cr(III) species

and to confirm the assignment as Cr(IV). Because the complex was able to produce OH

radical in the presence of molecular oxygen in aqueous medium, and the production of

OH radical was enhanced by the addition of H2O2 and inhibited by catalase, they proposed it as a model chromium (IV) complex for studies in chromium carcinogenesis.

Unfortunately, no follow up work was found in the literature regarding the ability of this complex to produce DNA strand breaks or other lesions. Lay47 and co-workers have

disputed the occurrence of chromium (IV) intermediates or products under the 27 experimental conditions used by Shi and Dalal et al61; however, Lay and co-workers did

not perform the exact experimental work to characterize the precipitate produced upon

mixing the reagents. Instead they infused the filtrate into the ESI-MS and failed to detect

any evidence of the formation of stable Cr(IV) intermediates suggesting that the solid

was probably a mixture of Cr(III) products and traces of Cr(VI).47 Clearly, if it is a

mixture of Cr(III) and Cr(VI), the magnetic susceptibility measurements would have

rendered a magnetic moment very close to that of a Cr(III) complex, being the Cr(VI)

state, as Lay and co-workers pointed out,47 present only as a trace. In the reaction system

61 employed by Shi et al, Na2Cr2O7 (25Mm) was reacted with GSH (100mM) and the

precipitate forms by itself according to the authors. In this case Shi61 et al proposed that the precipitate is a bis(glutathionato)chromium(IV) complex. Interestingly, Lay42 et al recognized that there must be an important variation in the solubility between the low and high glutathione substituted complexes or a shift in the equilibrium among the bis and tetra glutathionato complexes when the mixture is subject to dilution. This could be the reason why this group did not find the bis(glutathionato)Cr(IV) complex in the ESI-MS of the filtrate using the conditions of Shi61 et al. The use of ESI-MS to study the

oxidation state of any metal is not a reliable method for this purpose because it has been

demonstrated62 that in this technique the ionization method is not exclusively chemical, and some electrochemistry can modify the oxidation state of the substrate, especially

metallic complexes or pure metals. However, as will be demonstrated in the body of this

work, it is possible to obtain information regarding intermediates and products and their 28 oxidation state in the reaction of chromium complexes and glutathione under certain conditions using electro spray ionization mass spectrometry.

There is evidence that suggests that the production of larger amounts of Cr(IV) over Cr(V) as intermediates in the reduction of Cr(VI) is tunable. The mechanism of the reduction of Cr(VI) with model thiols like L-cystein and and glutathione in neutral media has been investigated by various authors.63-66 McAuley and Olatunji66 seem to be the

first to have performed kinetic studies of this reaction using DL-Penicillamine, glutathione and β-mercaptoethylamine. According to the more detailed studies of

Pennington63 et al, the thioester-Cr(VI) intermediate reacts with a second molecule of the

thiol and experiences a two electron transfer to Cr(IV):

k1 - - HCrO4 + L-CysSH L-CysSCrO3 +H2O (1) k-1

k - 2 - (2) L-CysSCrO3 + L-CysSH L-Cystine + HCrO3

k L-CysSCrO - 3 - 3 L-CysS + CrO3 (3)

Similar conclusions were obtained from studies with other thiols including glutathione.63

As no Cr(V) was observed with EPR under the conditions of this study, and because I-

had an small effect on the rate values, Cr(V) was considered at best a minor

intermediate.63 Also, efforts to intercept Cr(IV) were partially successful since the addition of Mn(II) increased the rate of the Cr(VI)-L-cystein reaction.63 The products of

- 63 these reactions were identified as Cr(L-Cysteinato-N,O,S)2 and L-Cystine and their

spectroscopic data coincided with that of authentic samples prepared as reported by 29

Hodgson and Freeman67 et al and by O’Brien68 et al. These results support the idea of a

Cr(IV) intermediate being implicated because according to the ligand capture

experiments in reductions of Cr(VI) of Cooper69 et al, initial two-electron reduction

leads to Cr(III) entrapment of excess substrate. As stated by O’Brien70 et al, the

formation of cystine or oxidize glutathione for that matter occurs in an associative way.

The two thiol molecules are likely to be proximate to the chromium center when the

electron transfer occurs.70 For this reason, the second rate constant might reflect a second

pre-equilibrium with the second thiol molecule which is followed by an uni-molecular

intramolecular reduction.70

Bose39 et al performed kinetic studies of the reduction of Cr(VI) and glutathione

under acidic conditions and proposed a similar mechanism; the only difference being that

the rate of the reduction of the Cr(IV) was slower. Interestingly, although an EPR signal

was observed in this work (g = 1.997), the estimated amount of Cr(V) was 3%. Dynamic magnetic susceptibility measurements of the reaction mixture gave a spin only moment of

2.8-3.0 µB for the intermediates and 3.8-4.1µB for the products. Based on these facts the

authors concluded that the long-lived intermediate is predominantly a Cr(IV) species.39

The kinetic experiments of the reduction of Cr(VI) with several thiols performed by

Wetterhahn64 et al also agreed with this finding because the rate law better

accommodates better to an expression in which the fate of the thioester intermediate via

reduction to a Cr(V) species is negligible. However, more recent studies of the reduction

of Cr(VI) with Cys performed by Lay65 et al point to a very fast reduction of the

thioester-Cr(VI) intermediate and negligible accumulation of the Cr(V) and Cr(IV). 30

According to Lay65 et al, the Cr(III) products are formed in two sequential fast one electron transfers that are about two orders of magnitude faster than the formation of the

Cr(VI) thioester, and the formation and decay of the intermediate with λmax ∼ 430nm

was assigned by Lay65 et al to the Cr(VI) thioester with no reference to other hypervalent

Cr species.

The electronic spectra of the species involved in the reduction of Cr(VI) with

thiols has been described by several authors. O’Brien71 et al and Wetterhanh64 et al

characterized the spectrum of the Cr(V) intermediate (λmax 640 nm, sh 900 nm) and

distinguished it from the features of the Cr(VI)-thioester (λmax 420-440 nm) and from the

39 Cr(III) products (λmax 550-550 nm). According to the aforementioned studies of Bose et al, because the long-lived intermediate is a Cr(IV) species, the absorbance at 460 nm must correspond primarily to this species. Indeed, a Cr(IV)-EHBA49, 72, 73 complex can

be generated in situ by reaction of the parent compound Cr(V)-EHBA with As(III) in a solution of the ligand (EHBA) and has two forms with absorption bands at λmax = 512 nm

for the protonated one and λmax = 464 nm for the unprotonated one.

O’Brien71 et al obtained a precipitate upon cooling and adding MeOH to the

reaction mixture of Cr(VI) and glutathione, (5.0 mL of 1.0M GSH, pH 7.0 mixed with

5.0 mL of 0.1M Cr(VI)). The molecular formula given for this precipitate was

Na4(GSH)4Cr.8H2O. Using diluted mixtures (GSH (16.7 mM), GSSG (3.3 mM) and

74 NaCrO4 (1-20 mM) Gaggelli et al found a homo dinuclear chromium(V) complex as a

product in their reaction system. They based their assignment on the fact that the UV-

VIS bands of the reacting species (345 nm) and the intermediates (435 nm) disappear 31 after 15 minutes leaving a mixture of Cr(V) or Cr(IV) that they later identified as Cr(V) based on EPR (g =1.93) and NMR measurements. The authors identified the intermediates as Cr(VI) thioesters based on their absorbance at 435 nm. In the kinetic studies of Wetterhahn64 et al on the reaction between Cr(VI) (0.37 mM) and several

thiols (25 mM) or carboxylic acids, no Cr(V) was implicated as part of the intermediates

because little or no EPR signal of Cr(V) was observable. According to Wetterhahn64 et al the rate limiting step is the formation of the thioester followed by fast electron transfers that lead to the Cr(III) products. Lay42 et al have also questioned the results by

Gaggelli74 et al and their argument is that under Gaggelli’s conditions Cr(V) is formed as

a minor intermediate (no more than 5% of total Cr) and the results of paramagnetic NMR

spectroscopy describe the structures of the Cr(III) products. They also pointed out that a

dimeric species of Cr(V) would be EPR silent so that the g = 1.993 corresponds to only

traces of the Cr(V) formed at the beginning of the reaction. Interestingly, in the

aforementioned kinetic experiments of Bose39 et al, a dinuclear Cr(III) complex

containing two GSH molecules and one GSSG molecule was likely to be among the

products, according to HPLC and ion-exchange experiments. A dinuclear chromium

species produced in the reduction of excess Cr(VI) with ascorbic acid (AA) at low pH

(3.8 and 5.8) was seen also by Stearns and Wetterhahn.58 According to the interpretation

of the spectroscopic data, a two electron reduction of the Cr(VI) occurs first producing mainly Cr(IV). This chromium (IV) disproportionates producing Cr(V) and Cr(III).

When there is a sufficient amount of Cr(V), two Cr(V) molecules dimerize through an oxo bridge that produces an EPR silent intermediate. Finally, the extra equivalents of AA 32 reduce the mixture to Cr(III) products. These studies58 were accompanied with plasmid

DNA damage assays that show a correlation with the kinetic studies without DNA. In

fact, the number of DNA strand breaks was diminished in the presence of Mn(II) as

expected if the damaging species were a Cr(IV) one. Also, they concluded that the major

damaging species may not be the Cr(V) because maximum strand breaks did not occur

under conditions producing maximum Cr(V).

Implications of the Mechanism of the Reduction of Cr(VI) in Biological Systems

Glutathione is an ubiquitous tripeptide as it occurs in animal cells, plants and bacteria.75 Its diverse functions include the reduction of disulfide linkages in proteins and

other molecules, the synthesis of deoxyribonucleotides, the protection of cells against the

effects of free radicals and of reactive oxygen intermediates formed in metabolic

processes. Because of this, an increment in cellular GSH concentrations may be

beneficial under certain conditions.75 Indeed, it has been observed that in response to

increased oxygen tension the lung GSH levels also increased.75

Both reduced (GSH) and oxidized (GSSG) glutathione can react with reactive

oxygen species (ROS) including hydroxyl (HO) and hydrogen peroxide as has been

demonstrated in vitro in studies with classical polarography and pulse voltammetry.76

According to Wetterhahn64 et al, if the rate of the reduction of the Cr(VI) glutathionyl

ester is smaller than its formation, then the Cr(V/IV) intermediates thus formed would

react with cellular components that otherwise are unreactive towards Cr(VI) and Cr(III).

Glutathione is not only found in relatively high concentrations in the cellular milieu (0.8-8.0mM)8, its concentration is 10-1000 times higher than other biological 33 thiols that react faster with Cr(VI). Although ascorbic acid reacts at a similar rate with

Cr(VI), the ascorbic acid concentration is only in the micromolar range.8 Also, another

biological molecule with a thiol group might react faster with the Cr(VI)-glutathione ester than with chromate.64 The transformation of chromate into Cr(VI)-glutathione would be

an effective way to extend the life of Cr(VI) in the cellular environment increasing the

likelihood of interaction with other biomolecules like DNA.64

An alternative scenario is proposed by Lay65 et al based on kinetic studies using a

large [Cys]/[Cr(VI)]0 (∼200). At neutral pH, the intermediate is regarded as a five

- coordinated species with the formula [CrO3(SR)(OH2)] which is in rapid equilibrium

2- with [CrO4] . In this proposal, due to the fast transformation of any Cr(V) or Cr(IV) into

Cr(III), such intermediates would be unlikely to participate in the Cr(VI) associated carcinogenesis.

OONH2 HO

HO HN

O S S O

O NH OH

HN Glutathione, GSH HO O H2N O

- O -2 O O O O O O O NH NH O O BT Cr 2 2 Cr Et Et O O O Cr O O O O N Et Et OH2

EHBA-Cr(V), I BT-Cr(V), II DPO, III Figure 2: Structures of chromium (IV) and (V) complexes employed to study the reduction of chromium by glutathione (GSH) and as model carcinogens.

CHAPTER 2

Oxidative and Hydrolytic Activity of Bis(2-Ethyl-2-Hydroxy

Butanoato)Oxochromate(V) towards Deoxyguanosinediphosphate

Experimental Part

Reagents

Sodium bis(2-ethyl-2-hydroxy butanoato)oxochromate(V) was prepared

following the method of Krumpolc and Roček.77 The Cr(V)-Bis-Tris (II) complex was

generated in situ by direct ligand exchange of Bis Tris with EHBA-Cr(V).78 2-ethyl-2-

hydroxy-butanoic acid (EHBA), deoxyguanosine (dG), deoxyguanosine diphosphate

sodium salt (dGDP), deoxyguanosine monophosphate sodium salt (dGMP), 8-hydroxy-

2’-deoxyguanosine (8OHdG), thiobarbituric acid, sodium pyrophosphate, and

phosphoglycolic acid were purchased from Sigma. Unless otherwise stated all other

common reagents were of highest purity (Sigma or Fisher Scientific).

Reactions of dGDP and Cr(V)

Reactions with II

In a 15 mL test tube, dGDP (5.0 mg, 10.6 μmol), D2O (500 μL), 1 M Bis Tris pH

7.5 (50 μL), 1 M H2O2 (500 μL) and distilled water (1272 μL) were added; the mixture

was stirred and then fresh 56 mM EHBA-Cr(V) (178 μL) was added with immediate stirring. Then the mixture was transferred to a 10 mm NMR tube, which was then wrapped with aluminum foil and placed in a water bath at 37ºC for 2 hours. Then EDTA

(500 mM, pH 8, 50 μL) was added and the tube was incubated overnight. Final concentrations of reagents were: dGDP (4.2 mM), H2O2 (200 mM) and EHBA-Cr(V)

(4.0 mM). 36

Reactions with I

In a 15 mL test tube, D2O (500 μL), 10.0 mM I in 25 mM pH 7.0 EHBA (1.00

mL), 1 M H2O2 (500 μL) and distilled water (500 μL) were mixed. To this solution

dGDP (5.0 mg,10.6 μmol) was added, the tube was stirred vigorously and the solution

was then transferred to a 10 mm NMR tube, which was wrapped with aluminum foil and

placed in a water bath at 37ºC for 2 hours. Then 500 mM pH 8 EDTA (50 μL) was

added and another 4 hours of incubation were given. Final concentrations of reagents being: dGDP (4.2 mM), H2O2 (200 mM), EHBA-Cr(V) (4.0 mM).

Hydrolysis with Alkaline Phosphatase

To a portion of the reaction mixture after EDTA treatment (500 μL), was added an aliquot of alkaline phosphatase (2.0 μL) and the sample was placed in a water bath at

37ºC for two hours; then another aliquot of alkaline phosphatase (2.0 μL) was added to the sample and another two hours of incubation were given.

Test For Thiobarbituric Acid (TBA) Reactive Species

The presence of thiobarbituric acid (TBA) reactive species was done following known procedures.17, 79, 80 The TBA reagent was prepared by dissolving 2-tiobarbituric

acid (262 mg, 1.82 mmol) in distilled water (50 mL) with a final pH of 2.0, due to the

addition of either HCl or NaOH. Then, the sample (500 uL) was diluted to 1.0 mL with distilled water and to the diluted sample (500 uL), the thiobarbituric acid reagent (500 uL) was added. The pH was verified to be 2.0 and then the sample was put in a water bath at 65 C for 45 minutes. After that, the UV spectrum was acquired using the other half of the diluted sample as the reference. Absorption at 532 nm is considered indicative of malondihaldehyde type substances present in the mixture.17 37

Nuclear Magnetic Resonance Measurements

NMR experiments were performed on a Bruker 500 MHz (DRX 500) instrument.

Proton decoupled 31P resonances were recorded at 202.45 MHz and reported with respect

to 85% external phosphoric acid at 0.0 ppm. For 31P a 8.20 μs pulse with a repetition time of 0.2 s was used. Typically 64 k data points were collected within a frequency window of 8090 Hz. A line-broadening factor of 1.0 Hz was introduced before Fourier transformation.

High Performance Liquid Chromatography Measurements

HPLC measurements were performed on a Waters 515 pump which was connected to a Waters 996 photodiode array detector. Separations were accomplished on

a reversed phase Symmetry C18 column (Waters, 5 μm,3.9 x 150 mm) using a one step

gradient elution. Mobil phase A was 50.0 mM ammonium formate at pH 4.0 and mobil

phase B was methanol. The gradient starts with 100% solvent A and end with 100% B in

30 minutes.

Results

Figure 3 shows the 31P NMR taken minutes after the reaction between dGDP and

EHBA-Cr(V) in the presence of H2O2 was quenched with EDTA. The identity of the

peaks in these NMRs was done by adding authentic compounds to the reaction mixture

and noticing the enhancement of the peak height. Besides the production of the expected

products for the hydrolysis of the first phosphate group, dGMP and inorganic phosphate,

pyrophosphate and phosphoglycolic acid (PG) were also found.

The reaction was run in two modalities. In the first one, the pH was controlled by

Bis-Tris, which is known to react with EHBA-Cr(V) generating Cr(V)-BT in situ.78 In the second approach, dGDP and H2O2 were dissolved in a solution of the ligand that was previously adjusted to pH 7. Another portion of the same solution was used to dissolve

the metallic complex (EHBA-Cr(V)); then both solutions were mixed to start the

reaction. When the EHBA ligand was used to buffer the solution, a final adjustment of

the pH with diluted HCl was necessary. Both, the 31P NMR and HPLC of each of the two approaches gave the same results. Neither hydrogen peroxide nor EHBA-Cr(V)

separately reacted with dGDP to an appreciable extend.

The effect of the concentration of H2O2 was also monitored. Figure 4 shows the

31 P NMR of several reactions in which the concentration of H2O2 was varied. The most

relevant feature on these spectra was the absence of PG when the concentration was 50

mM or less. Meanwhile pyrophosphate appears as a small shoulder on the high field side

of the β phosphate signal.

Figure 3. 31P NMR spectrum acquired after the reaction between EHBA-Cr(V) (4.0 mM) and dGDP (4.0 mM) in the presence of H2O2 (200 mM) either in Bis Tris (20 mM) or excess EHBA ligand (25 mM) and at pH 7. A is the spectrum minutes after the reaction was quenched with EDTA, B is the same sample after addition of authentic pyrophosphate and C is the reaction mixture after addition of authentic phosphoglycolic acid (PG). 40

Figure 4. 31P NMR of reactions between dGDP and EHBA-Cr(V) in Bis Tris at pH 7 and under various concentrations of H2O2. 41

Further characterization of the products of this reaction was done by HPLC and by determining the presence of TBA reactive species.

The presence of base propenals (BP) or related malondialdehyde (MDA) compounds due to the oxidation of the sugar moiety of dGDP was examined using the

TBA test.17, 79 The crude of the reaction between dGDP and EHBA-Cr(V) in the presence of H2O2 at pH 7 was reacted with TBA at pH 2 which produced the visible

spectrum shown in Figure 5. This spectrum is identical to that produced when TBA

reacts with malondialdehyde and is characterized by an absorption maximum at 532

nm.17

1.000

(532, 0.832) 0.750

0.500 Absorvance

0.250

0.000 400.0 450.0 500.0 550.0 600.0 nm

Figure 5. Visible spectrum of the crude of the reaction between thiobarbituric acid and the mixture obtained by reacting dGDP and EHBA-Cr(V) in the presence of H2O2 at pH 7. 42

HPLC characterization of the catabolites produced by reaction of EHBA-Cr(V) with dGDP is exhibited in Figure 6. The assignment of the peaks was done by comparison of the retention times of authentic species and by matching the UV spectra.

In Figure 6, the chromatograms of the reaction (A) and the same reaction mixture after treatment with alkaline phosphatase (B) are shown. Enzymatic digestion of the reaction mixture with alkaline phosphatases was done in order to identify dG. This assignment was also verified by an independent injection of the authentic compound. It is apparent from the figure that the reaction does not produce dG because peaks g (dGDP) and i

(dGMP) were transformed into peak p (dG) as the result of the enzymatic hydrolysis of the former two compounds. Peak a is the solvent front. Peaks b and b’ are from Cr bound species. Peaks c and d are not present in the control, but their identities were not determined. Peak e is present in both the control and the reaction. Peak f is guanine and peak g is dGDP. Peak h was tentatively assigned to guanine propenal based on the fact that malondialdehyde type catabolites were detected in our assays with TBA through its retention time and UV spectrum (see Figure 7) discussed earlier. Hetch81 et al detected

guanine propenal in a synthetic oligonucleotide digested with bleomycin by the use of

reverse phase HPLC with a linear gradient of 0-100% methanol at a flow rate of 1.0 mL/min (similar to our conditions). In such studies guanine propenal eluted at 9.5 min

(our retention time being 6.0 min) and its UV spectrum is characterized by a strong band at 266 nm, a weaker band at 240 nm and a very weak band at 327 nm. UV spectrum of peak h has the strongest band at 266 nm and a weaker band at 310 nm (Figure 7). Peak i is for dGMP as shown on Figure 6B. Peaks j, k and l are present in both the reaction and

the control. Peak m is due to furfural and was determined by comparing the retention 43 time and the UV spectrum with an authentic sample of the compound. Peaks n and o have a similar UV spectrum as 8OHdG and peak n has the same retention time as

8OHdG. We conclude that n is 8OHdG and o is a related species, guanidinohydantoin, which is known to be produced upon further oxidation of 8OHdG.82-84

Guanidinohydantoin was detected at the same retention time in previous work from this

laboratory under the same chromatographic conditions.14

Figure 6. HPLC of (A) the reaction of EHBA-Cr(V) (4.0mM) and dGDP (4.0 mM) in the presence of H2O2 at pH; 7 and (B) the same mixture after treatment with alkaline phosphatase. A Symmetry C18 5.0 µm 3.9 mm x 150 mm column (Waters) with a gradient elution in two steps both at 1 mL/min was used as described in the experimental part. Peak assignments: a, solvent front; b, Cr bound species; c, d, e, j, k and l are unknowns being e, j, k, l present in the control; f, guanine; g, dGDP; h, guanine propenal; i, dGMP; and m (furfural), n (8OHG); o, guanidinohydantoine. Treatment of the reaction mixture with alkaline phosphatase transformed peaks g (dGDP) and i (dGMP) into peak p (dG). 45

Figure 7. UV spectrum of the compound eluted at 5.93 min (peak h) during the chromatographic separation of the reaction mixture between dGDP (4.0 mM), EHBA-Cr(V) (4.0 mM), H2O2 (200 mM) at pH 7.0.

Discussion

Bose and co workers using EPR spectroscopy observed that compounds I and II bind ss-DNA through the phosphate moiety.12 Oxo chromate complexes I and II cleaved

ss-DNA and oligonucleotides through an oxidative process as evidenced by the presence

of products like 5-methylene-furanone and furfural in such studies.12 The oxidation of

both the sugar and the base was caused by the oxometal and not by oxygen radicals

because the indicated catabolites and products of the oxidation of the nitrogen base like

8-oxo-dG occur in the presence of excess Bis Tris buffer which is known to be a radical

scanvenger.12

Using EPR spectroscopy, Bose14 and co workers observed peroxo-chromium

complexes in the reaction of compounds I and II with hydrogen peroxide. Complex I did

form hydroxyl radicals when reacted with hydrogen peroxide; however, complex II in the

presence of excess BT did not form hydroxyl radicals.14 A mono-peroxo chromium(V)

complex with a g value of 1.982 was also detected with EPR spectroscopy by Lay85 and coworkers in a direct reaction between Cr(VI) and H2O2, which is very similar to the g

value of 1.981 obtained by Bose14 et al. Hydrogen peroxide reacts with chromate

producing a blue intermediate identified as oxodiperoxochromium (VI). Tanaka86 et al generated this species in reactions of Cr(IV) and Cr(VI) with H2O2 using Cr

concentrations as low as 0.08 mM and H2O2 concentrations of 7.8 mM. It is known that

such intermediate can be generated over a wide range of pH. In addition, Ramasami87 et al detected the oxodiperoxochromium (VI) by reacting diperoxoaqua(ethylendiamine)chromium(IV) and diperoxo(diethylenetriamine)chromium(IV) at concentrations of 0.2 to 0.3 mM with

H2O2 at 10 to 50 mM concentrations. 47

Some considerations are necessary to dissect the hydrolytic products from those originated from the redox reactions of complexes like I and II in the presence of hydrogen peroxide. The oxo chromate complex can bind dGDP either by the α or by the

β phosphate group or both in a bi-dentate fashion as depicted in Scheme 1. Under such circumstances, the oxometal could exhibit its hydrolytic activity similar to phosphatase activity by acting as a catalyst for the hydrolysis of the diphosphate. However for most true wild or artificial phosphatases, the cleavage process would need the assistance of another species acting as a nucleophile. Such nucleophile could be just water or an oxo- hydroxo chromate generated by the reaction of the oxo chromate complex and hydrogen peroxide as indicated in equation (4). Such pathway would release inorganic phosphate, dGMP, pyrophosphate and dG. If the metal binds preferentially the β phosphate, the hydrolysis of dGDP would produce inorganic phosphate and dGMP which could be further hydrolyzed into more inorganic phosphate and dG or be oxidized to furfural, guanine propenal and phosphoglycolate. Finally, although these reactions would generate some chromium bound products, such products would not be EPR active as they would be products of the oxidation of the sugar ring, either Cr(III) or Cr(IV), the latter being EPR silent. However when the reaction proceeds exclusively through hydrolysis, the intermediates and the products should be EPR active chromium (V) species.

Scheme 1 Nu dGMP-Cr(V) O O HnPO4-Cr(V) L2CrO, H2O2 dGDP HO Pβ O Pα OdG Cr(V)-HnP2O7 O O CrV dG*

O - Nu = H2O, CrOO

V V V VI - (4) L2Cr O + H2O2 L2Cr -O OH L2Cr -O L2Cr -O

V V V +2 L CrVII=O (5) L2Cr O + H2O2 L2Cr -O OH L2Cr -O 2

In the absence of redox activity the oxo metal complex can act as nucleophile to promote the hydrolysis of the phosphate diesters as occur in most metal activated phosphatases, nucleases88, 89 and synthetic molecular mimics.89, 90In such cases, the metallic or bimetallic site transforms a water molecule into an effective nucleophile by increasing its acidity. The metallic hydroxide then can function as nucleophile cleaving phosphates and amides.90 A behavior like this would render a true artificial nuclease/phosphatase, however metals like chromium and iron tend to have redox rather

than hydrolytic activity. Although the formation of an oxohydroxochromate as indicated

in equation (4) (Scheme 1) is possible, it would behave more as an oxidant rather than as

a base or nucleophile if a proximal reducing center is accessible. For example using ESI-

MS Sam91 and co workers showed that activated Bleomycin is a iron-hydroperoxo

species capable to abstract a hydrogen atom from deoxyribose. Wink92 et al by studying

the Fe(II)/H2O2 reaction with a wide variety of substrates concluded that instead of

hydroxyl radical this reaction generates two intermediates of which the first one is a

Fe(II)-OOH species that cleaves heterolytically to produce a second intermediate Fe=O+2, which is capable of performing one electron oxidations as in the abstraction of H from the ribosyl unit of nucleotides. If the reaction of Fe(II) and H2O2 does not generate

hydroxyl radical but instead the indicated intermediate FeO+2, a formulation like the one

indicated in equation (5) (Scheme 1) for the reaction of Cr(V) and H2O2 would be

erroneous; but if the O-O bond cleaves heterolytically, a Cr(VII) species would be

necessary as indicated in equation (5) (Scheme 1).

Although it is believed that the oxochromate or oxodiperoxo chromate behave

more as an oxidant rather than as a base or nucleophile, the proposed mechanisms by 50 which such complexes oxidize the ribose moiety of nucleotides requires the participation of a strong base in an anti elimination of hydrogen cation to produce double bonds in the skeleton of the ribose. As indicated previously, the only species capable of such chemistry would be the oxodiperoxo chromate.

Oxo chromate can have true nuclease activity. In the present study, the strongest base must come from the conjugated bases of water or hydrogen peroxide93 because the formation constant for the protonation of the oxoperoxochromate intermediate was estimated to be 0.15 M-1 86 which places the oxodiperoxo chromate as a weaker base than

94, 95 water, hydrogen peroxide and peroxide. The estimated pKa of cyclopentane is 58, then none of the bases present in the reaction mixture would be able to produce a β elimination without activation of the substrate. Possibly when the chromium binds the phosphate moiety it also increases the acidity of the hydrogen atoms of the sugar skeleton by inductive effect, reducing the thermodynamic barrier of the β eliminations.

Our HPLC did not show the presence of dG and our 31P NMR studies showed the presence of pyrophosphate. It is plausible that when the Cr(V) complex binds dGDP by the α phosphate instead of promoting hydrolysis of the phosphate ester, it mainly attacks the sugar ring, and the hydrolysis of pyrophosphate from oxidized products is a later event. The detection of phosphoglycolate in our 31P NMR experiments makes this route possible.

Phosphoglycolate would be produced by the hydrolysis of a pyrophosphoglycolate intermediate. In this case, the oxo chromate complex would bind the α phosphate and then react with the sugar ring (route a in Figure 8) producing a chromium bound pyrophosphoglycolate, which ultimately hydrolyses producing a mixture of inorganic phosphate, pyrophosphate and phosphoglycolate, as is indicated in Figure 8. 51

III P O HO3PO2P OCr OH III OH O HO PO P OCrIIIOH OCrIIIOH OCr OH 3 2 O P O O O HO PO P O HO3PO2P O O b b1 3 2 a1 a G G HO CH - G Cr(V) G [Cr(IV)] + H O O B HO-O CH [Cr(IV)] H CH CH 2 CH2 2 O O O O O H

O HO OH OH OH OH OH b2 OCrIIIOH a2

HO3PO2P O O B G O CH H2OCrO4P2O G O CH2 O CH b + PY-Cr(III) B a3 O 3 O O H O

OH H OH OH H a4 b4 B

O O O CH a5 O H OCrO P O OH 2 4 2 + EDTA PGA - G + O O- + O pyrophosphate Furfural

Figure 8. Proposed mechanism for the reaction of compounds I or II and dGDP in the presence of H2O2 at pH 7.0.

In this reaction 8-OHdG and guanidinohydantoin were also detected. Since in our reaction system radicals are scavenged by either Bis Tris or excess 2-ethyl-2- hydroxybutanoate, the production of 8OH-dG and guanidinohydantoin (Gh) cannot be attributed to hydroxyl radicals. Guanidinohydantoin is the product of the 4e- oxidation of

guanine via 8OHdG (Figure 9). 8OHdG undergoes further oxidation due to its lower reduction potential (0.74V96) compared to dG (1.29V96). It is believed that the presence

of dG domains within DNA is a protective mechanism against oxidation due to its facile

oxidation97 and its high repair efficiency.98 However Essigman99 et al have shown in

constructed circular viral genomes containing site specific Gh that although Gh is

efficiently bypassed by the DNA polymerase it is highly mutagenic causing almost exclusive G→ C transversions.

N NH O NH [O] NH OH O

N NH N NH2 NH NH2 R R 8OHdG Gh

Figure 9: structures of 8-hydroxyguanosine (8OHdG) and guanidinohydantoin (Gh).

The proposed mechanisms by which catabolites like furfural, glycolic acid and base propenals are produced upon oxidation of nucleotides or DNA require the generation of an intermediate carbocation on the sugar ring.12, 100, 101 The energetics of

the H abstraction for positions 1’, 3’ and 4’ was found to be similar presumably due to 53 the stabilization of the radical intermediate by partial π bond formation with the lone pair of electrons on the adjacent oxygen atom.100 However both crystal structures and

quantum mechanical calculations show that the 5’ and the 4’ positions of a double

stranded helix DNA are more likely to be abstracted because they are significantly more

exposed to solvent than the other hydrogen atoms.100 The generation of the

aforementioned catabolites is evidence that supports the theoretical considerations just

cited.

The production of furfural, base propenals and phosphoglycolate are indicative of reaction mechanisms that implicate the abstraction of hydrogens 4’ and 5’ from the sugar ring as is indicated in Figure 8. In such mechanisms the bifunctionality of an oxoperoxo chromate is indicated since in the generation of the carbocation on either carbons 4’ and

5’, a hydro peroxide group is transferred (step a1 in Figure 8) from the metal to the

substrate. Initial hydrogen abstraction and electron transfer from carbon 4’ followed by hydro peroxide addition to the resultant carbocation generates the hydro peroxo ribose intermediate, which upon rearrangement and two subsequent eliminations will generate guanine propenal and a chromium bound pyrophosphoglycolate. And as the chromium is bound to the α phosphate, the hydrolysis of such species will render a mixture of inorganic phosphate, phosphoglycolate, pyrophosphate and glycolate.

Hydrogen abstraction from carbon 5’, for instance, will generate a carbocation upon subsequent electron transfer to the metal center. This carbocation intermediate can react with a water molecule generating an intermediate that upon two consecutive eliminations will produce furfural and the free base (Figure 8 route b) as was detected in our assays. 54

From the HPLC experiments is possible to determine which pathway, abstraction of hydrogen 4’ or 5’, predominates. Also the HPLC data provides information that allows the determination of the extent of the oxidative and the hydrolytic activity.

If the metal is coordinated only through the α phosphate it would produce only oxidation products and pyrophosphate, while if the metal is coordinated only through the

β phosphate it would produce inorganic phosphate and dGMP. A PO4-CrO(V) would be

less reactive towards dGMP due to the electrostatic repulsion between the two phosphate

groups. By comparing the areas under each of the peaks of the chromatograms of both

the control and the reaction (Table 1) it was observed that the ratio of guanine to guanine

propenal turned out to be 1, which indicates that there is no selectivity towards the hydrogen abstraction step; the oxometal abstracts both hydrogens (4’ and 5’) with equal probability.

Table 1. Comparison of the chromatograms of the reaction between oxochromate (V) and dGDP in the presence of H2O2 at pH 7.0 and the control. Reaction Control

Peak Rt/min Area % Rt/min Area % Assignment

f 3.82 1.1 3.80 0.19 guanine

g 4.30 62 4.30 81 dGDP

h 5.93 0.85 guanine propenal

i 6.73 8.0 6.57 5.4 dGMP

m 11.65 0.43 furfural

n 14.43 0.09 8OHdG

o 18.25 0.21 Gunidinohydantoin

Area% DGMP/Area% all oxidized products = 1.00; Area % Guanine/Area % Guanine propenal = 1.06. 55

It was noticed that the change in the proportion of dGMP towards all the oxidation products turn out to be 1 as well (Table 1), which suggests that the oxochromate binds the α phosphate and the β phosphate with equal probability.

These results indicate that if the oxometal is close to an oxidizable center, like in the α phosphate binding mode, the oxidation of the ribose will be preferred over the hydrolysis of the phosphate ester. On the other hand if the oxo metal is far from the reducing center, it will instead facilitate hydrolysis.

Although the oxochromate(V) was able to oxidize the base producing 8OHdG and

Gh, the oxidation of the sugar moiety was at least 8 times higher. This clearly places the formation of the Cr-PO4 intermediate as an essential step for the damage caused by the

metallate. Consistent with this mechanistic aspect the oxidation of the sugar ring is in our

system, a more important type of damage than the oxidation of the base. The fact that the

amount of Gh is larger than double the amount of 8OHdG corroborates the more facile

oxidation of 8OHdG in comparison with dG14.

CHAPTER 3

ESI-MS and EPR Characterization of the Intermediates and Products of the

Reaction between Diperoxoaquaethylendiaminechromium (IV) and Glutathione

Experimental Part

Reagents

Diperoxoaquaethylendiaminechromium(IV) (III) was prepared according to the

method of House and Garner.102 Reduced (GSH), oxidized (SGGS) glutathione, single

and double stranded calf thymus DNA were purchased from Sigma. The oligonucleotide

5’-GATCTAGTAGGAGGACAAATAGTGTTTG-3’ (oligo I) was synthesized by Sigma

Genosys. 5,5-dimethyl-l-pyrroline-N-oxide (DMPO) and 5-(diethoxyphosphory1)-5-

methyl-l-pyrrolin-N-oxide (DEPMPO) were synthesized for the Ohio State University

EPR Research Center by Radical Vision, Faculté de St. Jérôme, Marseille, France.

Unless otherwise stated all other common reagents including solvents for HPLC were of

highest purity (Sigma or Fisher Scientific).

ESI-MS Measurements

For the MS experiments a ThermoFinnegan LCQ Advantage Mass spectrometer

was used. The Mass spec was set to positive polarity, the capillary temperature of 200

°C, source voltage of 4.0 kV, capillary voltage of 42 V, and a tube lens offset of 50 V.

The mass to charge ratios were generally collected from 200 to 2000. When the reaction

between III and GSH was studied by ESI-MS, the reaction was run in Bis Tris at pH 7.0

or in just water in the case of the direct injections of solutions of III. Data was processed

using Qual Browser version 1.3.103

Nuclear Magnetic Resonance Measurements

NMR experiments were performed on a Bruker DMX-300 MHz spectrometer.

1D Proton-decoupled 31P spectra were recorded at 121.49 MHz and reported with respect

to 85% phosphoric acid at 0.0 ppm. For 31P a 8.0 µs pulse with a repetition time of 1s

was used. Typically 32 k data points were collected within a frequency window of

14000Hz. A line-broadening factor of 4.0 Hz was introduced before Fourier transformation.

Electron Paramagnetic Resonance Measurements

The EPR measurements were performed using a Bruker EMX spectrometer at

The Ohio State University Center for Biomedical EPR Spectroscopy and Imaging. The g values and hyperfine splitting were measured directly from the magnetic field separation using DPPH as a reference. Data analysis was made using the EPR 2000 2.0 software, which was provided by the Ohio State University EPR center. Typical data acquisition parameters are as follows: frequency window, 3540±100 G; acquisition time, 100s; modulation frequency, 100 kHz; modulation amplitude, 1.000 G; and attenuation, 10 dB.

The growth and decay of the Cr(V) intermediates were monitored by following the change in signal intensities over time and the corresponding kinetic profiles were computer fitted using Sigma Stat software for Windows Version 3.5. The spin traps

DMPO and DEPMPO were used to differentiate between OH and glutathione thiyl radicals. In these experiments a frequency window of 3540±200 G with an acquisition time of 84s was used, all other parameters remain the same. 58

Reactions between III and GSH

Reactions between III and GSH were prepared by mixing aliquots of concentrated fresh solutions of III and GSH with the buffer, either phosphate or Bis Tris.

Considerations were taken when manipulating the diperoxo chromium (IV) complex (III) due to its explosive and potentially carcinogenic nature. Complex III was manipulated with plastic spatulas. To prepare fresh solutions of III, the compound was weighed first and kept dry in a vial in the refrigerator. Having a known mass of III in the vial, a fresh solution of III was ready to be prepared by simply adding an aliquot of water and stirring. Then an aliquot of the freshly made stock solution of III was pipette and mixed with the solution of pre mixed other reagents including the buffer. In all cases, these operations were performed as quickly as possible. Complex III solubility in water is low, to assure a homogeneous mixture 10 mg or less of the solid should be dissolved in 5 mL or more of water.

31P NMR studies of the reaction of III and DNA

The 28 mer oligo nucleotide (5’-GATCTAGTAGGAGGACAAATAGTGTTTG-

3’) (143.9 nmol, 0.240 mM) was dissolved in dionized water (600 µL total volume, 10%

D2O) treated with Bis Tris (3 mM, pH 7.0), then the sample was transferred to a 5mm

NMR tube and a 31P NMR was acquired. Then to 550 µL of this solution, freshly made

46mM GSH (5.0 µL, 0.420 mM) and 20mM III (4.0 µL, 0.145 mM) were added in that

order. The pH right after mixing was 7.0. Then the tube was put in a water bath at 37ºC

in the dark for 4 hours. Next, 250 mM EDTA (20 µL) was added and 4 hours of

incubation were allowed before acquiring another NMR spectrum.

Results

ESI-MS Studies

The reaction between III and GSH in Bis Tris buffer at pH 7.0 was studied y ESI-

MS. The use of Bis Tris buffer instead of phosphate buffer is required by the fact that

non volatile buffers can diminish and almost completely suppress the signal in ESI-MS

experiments.104 Initially a fresh solution of 20 µM of III in water was injected to observe

the behavior of III under the conditions of the electro spray ion source as this kind of

ionizer is known to behave as an electrochemical cell.62 As shown in Figure 10, III

forms four main ions of m/z of 192, 193, 194 and 195. Based on the number of electrons

around the metal, the m/z values could be interpreted as species in which the metal has

been oxidized due to the potential applied to provoke the ionization of the sample. m/z

194 corresponds to the parent ion in which the metal center must be a Cr(V) while the m/z 195 could be a M+H in which case the metal would be a Cr(IV). m/z 192 and 193 could be seen as two species of Cr(VI). The chromium V and VI species are in agreement with the possibility of oxidation of the metal taking place in the capillary as a result of the applied potential to favor the formation of positive ions.105

Figure 11 shows the average spectrum acquired when a reaction mixture of 0.250

mM III and 3.75 mM GSH in 7 mM Bis Tris (pH 6.5) was injected. No significant ion

counts of those ions other than the buffer (m/z 210), reduced glutathione (GSH) and

oxidized glutathione (GSSG) were observed when lower concentrations of III and GSH were employed.

Figure 10. ESI-MS in the positive ion mode of a 20 µM solution of III in water.

Figure 11. Average ESI-MS of the reaction between III and GSH in Bis Tris buffer (pH 6.5). Peaks 342 and 529-530 are impurities.

Table 2. Assignment of mechanistically relevant peaks of the mass spectrum shown in Figure 11.

m/z Assignment m/z Assignment

308 [GSH-H]+ 824 [GS-GSH-BT-H]+

+ + 338 [CrL’2L”BT] 920 [(GSSG)(GSH)H]

+ + 418 [CrL”GS] 922 [(GSH)3H)]

+ + 450 [CrL’L”SG] 984 [Cr(O)(SG)3]

+ + 484 [CrO(OH)3L”-SG] 1029 [Cr(GS)3L”]

+ + 481 [Cr(O)4L”-SG] 1064 [CrL”(OH2)(O)(SG)3]

+ + 517 [BT:GSH] 1082 [CrL”(OH2)(OH)2(SG)3]

+ + 613 [GSSG-H] 1129 [CrL”O3(OH)3(SG)3]

+ + 615 [GSH:GSH(H)] 1227 [(GSSG)(GSH)2H]

+ + 724 [CrL”(GS)2] 1229 [(GSH)4H]

+ + 757 [CrL’L”(GS)2(H)] 1292 [Cr(GS)4(O) ]

+ + 775 [Cr(OH)3L”(SG)2] 1384 [CrL”O3(SG)4]

= L’= O2 , L” = H2N(CH2)2NH2, GS = unprotonated glutathione, GSH = reduced glutathione, GSSG = oxidized glutathione, BT = Bis Tris.

Table 2 shows the assignment of those peaks found relevant according to

mechanistic considerations. Of the peaks attributed to chromium compounds, some

clearly showed intensity profiles of either intermediates or products and were isolated and

fragmented for further characterization.

Figures 16 through 19 show the time course ion count for the ions of m/z 450

(Figure 16), 484 (Figure 17), 757 (Figure 18) and 775 (Figure 19). These four peaks were found to be the only ones with a significant change over time with the exception of

the peaks regarding glutathione (m/z 308) and oxidized glutathione (m/z 613). Clearly the

indicated ions are either products or intermediates in the reaction between III and GSH. The 63

tendency shown in the time course ESI-MS of ions m/z 484 (Figure 17) and m/z 775 (Figure

19) suggests that these ions are probably products, while ions m/z 450 (Figure 15) and m/z

757 (Figure 17) are intermediates. In particular, the ion count of m/z 775 (Figure 18) continually increases until about 1600 minutes and then stabilizes, while the other ions acquired a steady ion count after approximately one hundred and fifty minutes which is in agreement with the approximate time at which the peak of the reaction’s profile is reached.

The fragmentation of m/z 484 was achieved by CID and it is shown in Figure 12.

In agreement with it being a product, it was drawn with four OH groups, one

ethylenediamine and one glutathione coordinated from the axial positions. The

fragmentation pattern fully agrees with the proposed structure. CID of m/z 484 resulted

in the ion of m/z 355 from the loss of glutamate, the ion of m/z 337 from the loss of water

and m/z 211 from the loss of a fragment that includes glycine and part of the Cysteine residue.

In Figure 13, the MS2 of the peak of m/z 757 is shown. This peak is an

intermediate that resulted after the first oxidation and reduction round by the one peroxo

group and two glutathiones. The peaks of m/z 450 and 418 are generated by the loss of

one glutathione (GSH) and further loss of the peroxo group, respectively. In order to

justify the formation of the peaks of m/z 665 and 679 a transient m/z 697 was proposed;

although it was not seen in the spectrum.

CID of peak m/z 775 generated the spectrum shown in Figure 14. MS2 of m/z

775 produced the ions of m/z 715 (loss of ethylenediamine), 745 (loss of methyleneamine), 757 (loss of water). The ion m/z 757 is postulated to be a transient species that breaks apart producing the ions of m/z 697, 670, 727 and m/z 484. That is probably why m/z 757 has low intensity in the fragmentation spectrum of m/z 775. 64

Figure 15 shows the MS in the region of m/z units from 438 to 454. It includes the ion of m/z 450 and its proposed structure. It is also indicated in brackets the calculated isotopic distribution. The purpose of this figure is to call the attention to the fact that in all cases the calculated abundance of the M+1 peak agrees with the observed one but the observed abundance of the M+2 peak differs from the calculated one, even though the fragmentation studies support the structural assignments. This is because as was noticed in the ms spectrum of III (Figure 10), the same ion could be ionized either by a redox reaction or by protonation and that alters the isotopic distribution of the protonated and the oxidized species because both groups of peaks overlap.

An attempt to find out the possible relationship between the ions of m/z 450, 484, 757

and 775 was made by monitoring their ion intensity over the most part of the reaction. The

ion count profiles for these ions are shown in Figures 16 through 19. Interestingly, the profiles of m/z 450 and 757 clearly correspond to an exponential decay with estimated rate constants of 2.95 x 10-4 s-1 and 3.74 x 10-4 s-1 respectively. Unfortunately the first minutes of data, which would belong to their growth, were too erratic and inappropriate for any computer fitting. The ions of m/z 484 and 775 (Figures 17 and 19) show a typical profile of the products of a consecutive reaction in which their growth depends on how fast their precursors accumulate. The estimated rate constants for these two species are 2.94 x 10-4 s-1 for m/z 484 and 1.88 x 10-5 s-1 for m/z 775.

According to stoichiometric studies,106 7 to 9 moles of GSH are consumed in this

reaction. That suggests that 2 to 4 moles of GSH are coordinated to the metal in the final

mixture of Cr(III) products. In agreement with this finding, the ESI-MS of the reaction of III

+ and 15 equivalents of GSH showed that peaks of m/z of 724 ([CrL”(GS)2] ), 984

+ + + ([Cr(O)(SG)3] ), 1029 ([Cr(GS)3L”] ), 1064 ([CrL”(OH2)(O)(SG)3] ), 1082

+ + + ([CrL”(OH2)(OH)2(SG)3] ), 1129 ([CrL”O3(OH)3(SG)3] ), 1292 ([Cr(GS)4(O) ] ) and

+ 1384 ([CrL”O3(SG)4] ) appear in the spectrum with time courses that correspond to the

raising of a product; however, in general all these peaks were small. 65

Figure 12. (+)-MS3 of m/z 484. Brackets to indicate the ionic nature of the molecules have been omitted for clarity.

Figure 13. (+)-MS2 of m/z 757. 1Very unstable, not seen in the spectrum. 67

Figure 14. (+)-MS2 of m/z 775. Brackets to indicate the ionic nature of the molecules have been omitted for clarity.

Figure 15 ESI-MS of the reaction mixture between III and GSH shown the region of between 438 and 454 m/z units. The abundance of the M+1 peak agrees with the calculated one but not the abundance of the M+2 peak. 69

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Figure 16. Observed (circles) and simulated (solid line) time course SIM-ESI-MS of m/z 450. The simulation is based on the equation y = a+ b e-kt, a = 151885, b = 742863 and k = 2.95 x 10-4 s-1. 70

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140000 0 5000 10000 15000 20000 25000 30000 Time (s) Figure 17. Observed (circles) and simulated (solid line) time course SIM-ESI-MS of m/z 484. The simulation is based on the equation y = a+ b e-kt, a = 232282, b = -143212, k = 2.94 x 10-4 s-1. 71

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Figure 18. Observed (circles) and simulated (solid line) time course SIM-ESI-MS of m/z 757. The simulation is based on the equation y = a+ b e-kt, a = 275229, b = 1973478, k = 3.74 x 10-4 s-1. 72

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Figure 19. Observed (circles) and simulated (solid line) time course SIM-ESI-MS of m/z 775. The simulation is based on the equation y = a+ b e-kt, a = 1486159, b = -1433807, k = 1.88 x 10-5 s-1. 73

EPR Characterization of the Reaction Intermediates

The reaction between III and GSH at pH 7.0 (phosphate buffer 100mM) was studied by X band EPR. Reactions in which the ratio of GSH to III were 10, 20, 40 and

60 were investigated. In all reactions the reagents were fresh and final reactions were made in 1.5 mL plastic PCR capsules from which 50µL were taken with a capillary to perform the EPR measurements. Mixing of reagents, filling up the capillary and instrument set up, including electromagnet tuning, was made in less than 60 seconds.

Controls were made in which no glutathione was added. In this case the chromium complex was dissolved in either water or 100 mM phosphate buffer pH 7.0 and the magnetic field was scanned at different times well beyond the time at which the intermediate was expected. In both cases no intermediates or other signals were detected as Figure 20 shows.

In the reaction of III with GSH three intermediates were detected with the EPR spectrometer. These intermediates have g values of 1.996, 1.986 and 1.983 and their occurrence was dependant on the relative concentration of GSH. For convenience all reactions monitored by EPR spectroscopy were run using 1.0 mM of III in 100 mM phosphate buffer at pH 7.0. At low GSH concentration the dominant intermediate was the one with g = 1.986. While at larger than 20 mM GSH the intensity of the signals at g

= 1.996 and g = 1.983 increased at the expense of the former one as Figure 21 shows.

However, the intensity of the intermediate with g = 1.983 did not significantly increase with the increment in the GSH concentration. Instead, the intermediate with g = 1.996 increases its intensity, and as seen in Figure 21D and Figure 21E it became the dominant 74 species at GSH/III larger than 40. Interestingly, after these intermediates have decayed (t

> 4000 s), another species with g = 1.975 was detected using a modulation amplitude of 5

G. The peak to peak band width of this signal was 259.72G and its spectrum is shown in

Figure 22.

The experimental and the simulated kinetic profiles of the reaction of 1.0 mM III and 5-

60 mM GHS are presented in Figures 23 – 30. The simulated kinetic profiles were constructed by least square computer fitting of the equation (6).

-k t -k t Intensity = a + b e 1 +d e 2 (6)

Figure 20. EPR control spectra of 1.0 mM III in water (A) and 1.0 mM III in 100 mM phosphate (B) buffer pH 7.0.

Figure 21. EPR spectra of the reaction mixture of 1.0 mM III, 100 mM phosphate pH 7.0 and (A) 5.00 mM GSH, (B) 10.0 mM GSH, (C) 20.0 mM GSH, (D) 40.0 mM GSH and (F) 60.0 mM GSH. Each spectrum approximately corresponds to the peak of the kinetic profile of each reaction. Signals: g = 1.996, peak to peak line width = 4.84 G, g = 1.986, peak to peak line width = 1.24 G and g = 1.983, peak to peak line width = 2.20 G.

Figure 22. EPR spectra of the reaction mixture of 2.0 mM III, 100 mM phosphate pH 7.0 and 20.0 mM GSH. Signal: g = 1.975, peak to peak line width = 259.72 G. Center field: 3570G, sweep width = 1000 G, modulation amplitude = 5G.

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Figure 23. Observed (circles) and simulated (solid line) EPR time course of the intermediate with g = 1.986 detected in the reaction mixture of 1mM III, 5.00 mM GSH and 100 mM phosphate buffer (pH 7.0). The calculated profile was generated -3 -1 -4 -1 using the rate constants k1 = 5.3 x 10 s , k2 = 5.5 x 10 s . 79

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Figure 24. Observed (circles) and simulated (solid line) EPR time course of the intermediate with g = 1.986 detected in the reaction mixture of 1mM III, 10.0 mM GSH and 100 mM phosphate buffer (pH 7.0). The calculated profile was generated -3 -1 -4 -1 using the rate constants k1 = 4.5 x 10 s , k2 = 3.8 x 10 s . 80

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Figure 25. Observed (circles) and simulated (solid line) EPR time course of the intermediate with g = 1.986 detected in the reaction mixture of 1 mM III, 20.0 mM GSH and 100 mM phosphate buffer (pH 7.0). The calculated profile was generated -3 -1 -3 -1 using the rate constants k1 = 2.2 x 10 s , k2 = 1.9 x 10 s . 81

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Intensity 8000 6000 4000 2000 0 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 Time (s)

Figure 26. Observed (circles) and simulated (solid line) EPR time course of the intermediate with g = 1.996 detected in the reaction mixture of 1 mM III, 20.0mM GSH and 100 mM phosphate buffer (pH 7.0). The calculated profile was generated -3 -1 -3 -1 using the rate constants k1 = 2.4 x 10 s , k2 = 2.1 x 10 s .

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Figure 27. Observed (circles) and simulated (solid line) EPR time course of the intermediate with g = 1.986 detected in the reaction mixture of 1 mM III, 40.0 mM GSH and 100 mM phosphate buffer (pH 7.0). The calculated profile was generated -3 -1 -3 -1 using the rate constants k1 = 4.1 x 10 s , k2 = 3.7 x 10 s . 83

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Figure 28. Observed (circles) and simulated (solid line) EPR time course of the intermediate with g = 1.996 detected in the reaction mixture of 1mM III, 40.0 mM GSH and 100 mM phosphate buffer (pH 7.0). The calculated profile was generated -3 -1 -3 -1 using the rate constants k1 = 4.2 x 10 s , k2 = 3.8 x 10 s . 84

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These kinetic profiles were computer fitted in order to evaluate the rate constants.

As mentioned before, in the reactions with less than 20 equivalents of glutathione the

intermediate with g = 1.996 was not detected and no rate constants were calculated. For

all other cases, Table 3 contains the rate constants for both intermediates at each ratio of

III to GSH. For the reaction of 1.0 mM III and 20.0 mM GSH additional replicates of

the rate constants are given in parenthesis because each experimental set of data was

fitted to a truncated version of equation 1 with only two exponentials - one for the raise

and one for the decay of the intermediate. In the case of the GSH/III = 60, only the decay

of the intermediates was followed and only the corresponding rate constant for the decay

of such intermediate is provided. The signal of the intermediate with g = 1.983 that

appeared in the EPR of the reactions with GSH/III >20 was always too weak and no rate

constants were evaluated.

Table 3. Rate constants for the raise and decay of the intermediates followed by X-band EPR of the reaction between III and GSH in 100 mM phosphate buffer (pH 7.0).

Initial Concentrations g =1.986 g = 1.996 (mM) -3 -3 -3 -3 III GSH k1 (x10 ) k2 (x10 ) k1 (x10 ) k2 (x10 ) 1.0 5.00 5.3 0.55 ND ND 1.0 10.0 4.5 3.8 ND ND 1.0 20.0 5.6(2.2)* 1.1 (1.9)* 4.2 (2.4)* 1.9 (2.1)* 1.0 40.0 4.1 3.7 4.2 3.8 1.0 60.0 ND 4.2 ND 3.0 *Results of a second experiment given in parenthesis.

Complex III is expected to produce either OH or glutathione thiyl radicals or both

during its oxidation by the peroxo ligands and reduction by glutathione. In order to 87 determine if there is production of any of these radical species, experiments with the spin trap DMPO were carried out.

H H OEt N R R N P OEt O O O

DMPO-OH DEPMPO-OH

Figure 31. Structures of spin trap adducts of the OH radical.

As seen in Figure 32, in the presence of DMPO, the X band EPR spectrum of a mixture of 1.0 mM III and 20.0 mM GSH in phosphate buffer pH 7.0, a four line signal with an average coupling constant of 15.6 G was observed. This four line signal reaches its maximum intensity before the first EPR was taken and only its decay was observed. The four line signal was centered at g = 2.006 and presented a hyperfine coupling constant of

15.8 G. This four line signal was accompanied most of the time by a signal at g = 1.991 which decayed faster than the DMPO radical adduct. Typically, DMPO adducts present a quartet with an intensity of 1:2:2:1. Clearly in this case, the fourth line at higher field overlaps with the signal of g = 1.991 which changes the intensity pattern. 88

g=1.991

Figure 32. EPR spectrum of the reaction mixture of 1.0 mM III, 20.0 mM GSH, 100 mM phosphate (pH 7.0) and 10 mM DMPO. The main signal is centered with a g = 2.006 with a hyperfine coupling constant of 15.8 G. A second signal with a g = 1.991 was also detected but it decays faster than the main signal.

In order to determine if this quartet was due to DMPO-OH or DMPO-GS, control experiments were performed. In the first experiment, OH radical were generated via a

Fenton like reaction using the reaction between bis(2-ethyl-2-hydroxybutanoato)oxochromate(V) (I) and H2O2:

- Cr(V) + H2O2 → Cr(VI) + OH + OH (7)

In a second control experiment, glutathione thyl radicals were produced by

reacting glutathione and bis(2-ethyl-2-hydroxy-butanoato)oxochromate(V) (I).

The generation of OH radical in the reaction of Cr(V) and H2O2 is well

documented and the assignment of the DMPO-OH spectrum is typically made based on

the hyperfine coupling constant between the β H and 15N of the DMPO adduct rather than 89

on its g value. For example for the reaction of 0.22 M DMPO, 2.5 mM Na2CrO4, and 25

107 mM H2O2 in 100 mM Tris-HC1 at pH 8.0 Kawanishi et al measured an aN= aβH of

44 14.83G. Aiyar et al also detected OH radical in the reduction of Cr(VI) with H2O2 in

25mM Tris-HCl, pH 8.0 by EPR. The hyperfine coupling constant of the DMPO-OH adduct was 14.9 G and the g value of the adduct 2.0044. Chiu108 et al in the reduction of

K2Cr2O7 with ascorbic acid in the presence of hydrogen peroxide and DMPO also

detected the DMPO-OH adduct and reported a hyperfine coupling constant of 14.9G.

The g value, splitting pattern and hyperfine coupling constant of DMPO-

glutathione thiyl radical have also been investigated. For example Bose et al22,

39determined a g value of 2.006 with a hyperfine coupling constant of 15.6 G for the

DMPO-GS adduct in the reaction of 5.0 mM Cr(VI), 50 mM GSH and 53 mM DMPO at

pH 7.1. This system was also investigated by Aiyar et al44, and in this case the

parameters of the DMPO-GS adduct were g = 2.0047, aN = 15.2 G and aH = 16.4 G.

The radical trapped with DMPO in the reaction of 1.0 mM III and 20.0 mM GSH

in 100 mM phosphate (pH 7.0) had a g value of 2.006 and a hyperfine coupling constant

of 15.8G (Figure 33). The hyperfine coupling constant of the DMPO-GS adduct ranges

in the literature109 from 14.9 to 16.4G and as seen the hyperfine coupling constants of the

DMPO-OH adduct fall in between and a similar situation occurs with the g value of these

radicals; both being very close. For this reason spin trapping with only DMPO is not sufficient to differentiate between DMPO-GS and DMPO-OH in a reaction system that might generate both radicals. This motivated a study using the spin trap DEPMPO

110 (Figure 31). In DEPMPO-OH, the aH = aN = 13-14G giving a quartet pattern, which is

110 further split by the phosphorus coupling (aP = 47.4G). 90

Figure 33. EPR spectrum of the reaction mixture of (A) 1.0 mM I (EHBA-Cr(V)), 100 mM phosphate (pH 7.0), (B) 1.0 mM I (EHBA-Cr(V)), 100 mM H2O2, 10 mM DMPO and 100 mM phosphate (pH 7.0) and (C) 1.0 mM I (EHBA-Cr(V)), 20 mM GSH, 10 mM DMPO and 100 mM phosphate (pH 7.0). From (B) the DMPO-OH spin adduct parameters are: g = 2.006, aN = aβH = 14.83G, while from (C) the DMPO-GS spin parameters are: g = 2.007, aN = aβH = 15.38G. The disappearance of the Cr(V) EPR signal (spectrum A) and the appearance of the quartet with intensity pattern 1:2:2:1 is indicative of the reduction of the Cr(V) by either H2O2 or GSH and the formation of OH and GS radicals which react with the spin trap. 91

Figure 34. Spin trap experiments with 10mM DEPMPO and 100 mM in phosphate (pH 7.0) (all experiments): (A) 1.0 mM EHBA-Cr(V) (I), 100 mM H2O2; (B) 1.0 mM EHBA-Cr(V) (I), 20.0 mM GSH; (C) 1.0 mM I, 100 mM H2O2, 20.0mM GSH; (D) 1 mM III, 100 mM H2O2; (E) 1 mM III,. 20.0 mM GSH. *Hyperfine bands associated to complex I due to coupling with 53Cr (I = 3/2, 9.8%). In (A) the numbers are used to identify the peaks of the DEPMPO radical adduct and the separation between peaks 4 and 5, also indicated with arrows, is used as a parameter to characterize the DEPMPO adducts. 92

In a control experiment (Figure 34A), a mixture of 1.0mM EHBA-Cr(V) (I),

100mM H2O2 and 10mM DEPMPO in 100mM phosphate (pH 7.0) was employed to

form the DEPMPO-OH adduct. In order to characterize the different adducts and to

compare the spectra, the separation between peaks 4 and 5 (Δ4,5) (see Figure 34A) was

taken as parameter. The Δ4,5 of DEPMPO-OH from Figure 31A was 6.0 G while the

value from the literature was 5.5 G.

A second control experiment was exercised to generate DEPMPO-GS using a mixture of 1.0 mM EHBA-Cr(V) (I), 20.0mM GSH and 10 mM DEPMPO in 100 mM phosphate (pH 7.0). The spectrum of this mixture is shown in Figure 34B and peaks 4 and 5 are identified with arrows. Δ4,5 was found to be 3.3G.

Figure 34C is the result of another control experiment, in this case 1.0 mM I was mixed with 100 mM H2O2, 20.0mM GSH and 10 mM DEPMPO in 100mM phosphate

(pH 7.0). This experiment was performed to generate a mixture of OH, GS and any

potential combination of these radicals that can be trapped with DEPMPO. In this case

the Δ4,5 was 2.75 G, closer to the value for the GS-DEPMPO (3.3 G) than to the HO-

DEPMPO value (vide infra).

Figure 34D is the spectrum of a mixture of 1.0 mM III, 100 mM H2O2 and 10 mM DEPMPO in 100 mM phosphate (pH 7.0). This experiment was performed in order to trap OH originated from the reaction between Cr(IV) and H2O2; but the Δ4,5 for this

system was 12G which agrees better with the DEPMPO-OOH radical adduct as Δ4,5 from the literature110 for this species was 13G.

The mixture of 1.0 mM III, 20.0mM GSH and 10 mM DEPMPO in 100 mM

phosphate (pH 7.0) gave the EPR spectrum of Figure 34E. This spectrum is particularly 93 weak in comparison with the previous ones. The identification of the peaks was made based on their position along the field and peaks 4 and 5 are indicated with arrows. For this system Δ4,5 turned out to be 3.3, exactly as in the mixture of EHBA-Cr(V) (I) and

GSH. However, this spectrum and those of Figure 34B, C, D and E contain more than

eight peaks. In the spectra attributed to DEPMPO-OH and DEPMPO-OOH (Figures

34B and D) a further splitting due to the γ and δ H from the OH and OOH groups

respectively can make from 12 to 16 peaks depending on the magnitude of the coupling

constants. In addition, the formation of the cis and trans stereoisomers of the spin adduct

and the decomposition of the superoxide adduct (DEPMPO-OOH) into DEPMPO-OH

can account for the extra lines in these spectra.110 In the case of Figure 34E, the complex

spectrum could be a mixture of DEPMPO-GS and DEPMPO-OH; but based on the Δ4,5

criteria, the spin adduct is mostly due to the production of glutathione thiyl rather than

hydroxyl radicals.

To further test the assignment, overlays of the EPRs of the reaction mixture and each of the controls were made. The overlays are shown in Figures 35 to 37. It is apparent that the mixture (Figure 36) of EHBA-Cr(V) (I) and GSH in phosphate buffer generated the same radicals that react with DEPMPO producing the same adducts that are produced by the reaction under investigation.

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Reactions of III and GSH with DNA

The reaction between a 28- mer oligonucleotide with III was performed at pH 7.0

using Bis Tris buffer. Due to the difficulties of working with such a short piece of DNA,

a better choice to assess the nuclease activity of this complex was the use of 31P NMR.

Figure 38 shows the 31P NMR of the reaction mixture after the addition of EDTA (B) and the control (A). EDTA binds the Cr(III) releasing it from the phosphate moieties which due to the line broadening effect of the paramagnetic atom would appear together as a broad peak. The 31P NMR of Figure 38B shows several shoulders that do not

appear in the NMR of the pure oligo as seen in Figure 38A. These shoulders are due to fragmented oligonucleotides generated upon cleavage of the main oligo.

Figure 38. 31P NMR spectrum of the mixture of (0.145 mM) and 28-mer 5’- GATCTAGTAGGAGGACAAATAGTGTTTG-3’ (0.240 mM) in Bis Tris (3 mM, pH 7.0) acquired before (A) and after (B) reaction with glutathione (0.420 mM). 98

Discussion

The characterization of the reaction between III and glutathione at neutral pH was

initiated in this laboratory by Bose and Ahuja,106 yet the data has not been published.

Kinetic, HPLC and electrochemical studies lead the workers106 to the conclusion that

depending on the conditions, 7 to 9 moles of GSH were consumed in the reaction. These

additional 2 to 4 moles of GSH per mole of Cr(IV) could be coordinated to the chromium(III) product. Therefore, the reaction can be written as

H N NH 2 2 + 2 O O + 18 GSH 2 (en)Cr(III)(GS)4 + 5GSSG + 8 H2O + 2H (8) CrIV O O

When the absorbance-time profiles were computer fitted, a triphasic reaction involving two intermediates was the model that best described the kinetics of the reaction106:

k1 k2 k3 A B C D (9)

The non linear dependence of the two rate constants on the GSH concentration

was interpreted as a rate saturation in which initially III is in a rapid equilibrium with a

GSH molecule. The formation of the intermediate reaches a limiting value at higher

GSH concentrations followed by an electron transfer:

K Cr(IV) + GSH Cr(IV)-GSH (10) k et (11) Cr(IV)-GSH Int1

Therefore, k1 can was expressed as

k1 = K ket [GSH] (12) 1 + K [GSH]

A similar scenario follows for the decomposition of the first intermediate.

However, in this case a parallel mechanism in which the first intermediate decomposes directly into the second intermediate and a second route in which the first intermediate reacts with one glutathione molecule to produce the second intermediate:

Int1 Int2 (13)

Int + GSH (14) 1 Int2

In the same way, (14) can be decomposed in to steps:

K' Int1 + GSH Int1-GSH (15) k Int1-GSH Int2 (16)

Then the rate law can be expressed as: k2 = K' k [GSH] (17) 1 + K' [GSH]

106 The first order dependence of k3 on GSH concentration was apparent.

Moreover, the cyclic voltammograms measured at several time intervals show the evolution of two distinct electron transfer processes whose oxidation potentials were similar to that of III. These data clearly support the existence of two intermediates.106

100

The ESI-MS studies under our conditions afforded prolific formation of ions in the positive ion mode even though there was sufficient Bis Tris to maintain a pH of 6.5.

On the contrary, no ions were seen in the negative mode. Two other groups have used the electrospray ionization technique to monitor the reaction between chromium and glutathione using mass spectrometry. Lay47 et al used it to study the reaction between 2.0

mM Cr(VI) from Na2Cr2O7 and 20 mM GSH in the presence of 50 mM GSSG at pH 2.7 and the reaction of 2.0 mM Cr(VI), 20 mM GSH in 100 mM glycine, pH 2.4; while

Gaggelli74 et al also recorded the ESI-MS of a Cr containing liver extract. The first

group used the negative ion mode and indicated that no ions were observed in the

positive mode. The results of the second group are confusing and debated by the first

group of researchers.

In this work no definitive assignment of the oxidation state of chromium based on

ESI-MS results will be done. The results obtained in this study support the idea of the

same precursor being affected by redox and acid based behavior producing more than one

ion with unsuspected ionization efficiencies which affects the isotopic distribution and prevents structural assignment based on this parameter. For example, the spectrum of the

species of m/z 757, regarded as an intermediate, was fully characterized by CID in

Figure 13. However, it is calculated isotopic pattern [M+1(40.4%), M+2(16.2%)] did not

fully match that of the spectrum (M+1 (38%), M+2 (21%). It was observed that in all

cases the M+1 and M+2 peaks are stronger than predicted and one explanation is that the

parent peak is formed from a species that exists in an acid-base equilibrium that produces

new species that are ionized by protonation/deprotonation while the precursor has the

101 charge in the metal. For example, m/z 757 owes its charge to the oxidation state of the chromium center, probably +4, while a fraction of it is protonated, giving a new species with one charge in the ligands but now +4 in the metal. The first ion is more stable than the second and its ion count larger; however, the second overlaps with the M+1 ion of the first and gives in this way a stronger M+1 peak for the first ion.

The results of spin trapping with DMPO and confirmed with DEMPO demonstrated that the reaction between III and GSH produces glutathione thyil radicals.

Second, the fragmentation pattern and the ESI-MS time course of the ion of m/z 757 clearly demonstrate that this ion is an intermediate in the early stages of the reaction; the key evidence being the loss of OOH to produce the ion of m/z 724. The detection with

EPR spectroscopy of Cr(V) species in the reaction mixture of III and glutathione proves that III is oxidized by the peroxo groups to higher oxidation states and then reduced by glutathione. The species of m/z 757 must be the intermediate produced after the first round of oxidation and reduction activity in which the first peroxo group has oxidized III to Cr(VI) and two equivalents of GSH have reduced it back to Cr(IV).

Based on the structural diversity found in the MS studies a more comprehensive mechanism for the reaction of III with GSH is exposed in Scheme 3.

In Scheme 3 the ethylenediamine group has been omitted in all structures. The ethylenediamine ligand is coordinated to the metal and does not add to the formal charge of the molecule. Also, the coordinated water molecule present in III has been removed because the two identified precursors with m/z of 450 and 757 are better explained if this water molecule is exchanged by a glutathione molecule.

102

The mechanism starts with the addition of two glutathiones to III (step 1) followed by a 2e- oxidation to produce V. The formation of glutathionato complexes as the initial step is required because in the absence of glutathione, the peroxo groups neither appreciably oxidize nor appreciably reduce the chromium center. Evidence of the

lack of reactivity of III is provided in Figure 20 where control experiments show that no

EPR signals of Cr(V) species were detected over one hour when III was dissolved in 100

mM phosphate buffer. In this oxidation the two hydrogen cations from the glutathione

molecules are used to protonate the first peroxo group and form water as leaving group in

one step. However in steps 9 and 10, the same type of oxidation is shown in 1e- steps.

This is because the Cr(V) intermediates detected by EPR can be produced both ways, during the reductions effected by glutathione and during the oxidations effected by the peroxo groups. The detection of glutathione thyil radicals with EPR spectroscopy supports the formation of Cr(V) intermediates during the reductions with glutathiones and the lack of detection of hydroxyl radicals supports the 2e- oxidations with a water

molecule leaving group. The oxidation of the Cr center with O= produces an

oxochromate as in VII. In order to transform this oxo group into a water molecule, VII

is protonated by reaction with 2GSH molecules. In order to protonate the oxo group, two

extra e- were rearranged from the metal to the oxygen atom, that is another oxidation that will require two more GSH to bring the metal back to Cr(IV) (steps 4 to 6).

In order to put three hydroxo groups onto the Cr product XXIII, steps 7 to 18

were necessary. XIII does not have a positive charge, however, following the rules of

valence and bonding, XXI, that is a Cr(IV), turned out to be positively charged. All in

103 agreement with Cr(IV) being the second intermediate, which accumulates before it is reduced to Cr(III) when there is an excess of glutathione. It seems that the reduction of these Cr(IV) species by glutathione is not a facile one. Glutathione activates Cr(IV) to be oxidize by the peroxo groups. This process may be pH dependent because the protonation of the peroxo group after addition of glutathione could be step limiting. The chromium(VI) and chromium(V) species instead are easier to reduce by glutathione but the reduction of the intermediates of Cr(IV) is not easily achieved. Interestingly, the chemistry saw in this work resembles the findings of Bose49 et al regarding the redox

potentials of Cr(V/IV) and Cr(IV/III) couples using 2-ethyl-2-hydroxybutanoato ligands.

Bose49 and co-workers found that although the reduction potential of the Cr(IV/III) couples are more positive than the corresponding Cr(V/IV), the process Cr(IV)→Cr(III) is slowed down by an electro-kinetic barrier that resulted from the structural changes required by the conversion of a penta-coordinated Cr(IV) species to a hexa-coordinated

Cr(III) species. We need to remember that all the structures in Scheme 3 contain the ethylenediamine ligand. This augments the coordination number in all of the structures shown in one or two units, depending on whether the ethylenediamine is binding the Cr in a mono or a bi-dentate fashion. Then, the products are at least hexacoordinated chromium complexes.

+ H O Scheme 3 IV 2 O + 2 GSH SG SG SG IV O [Ox] O [Red] O [Red] Cr O V O O CrIV SG CrVI SG Cr + SG O 1 23VII + GS III CrIV O O OH O O O O 2 V VI O O O CrVI O- + SG + SG SG GS SG HO HO HO HO SG XXIII O SG SG 17 SG [Red] 18 [Red] IV CrIII 2GSH MW 775 CrVI Cr XXI CrIV 16 - XXII 4 XX HO GS HO OH HO OH OH HO OH MW 775 H O +2 + +2 2 SG H + +2 SG O 15 SG + 20 OH SG VI VIII 22 O 21 O [Red] O Cr SG IV VI 19 SG Cr IV CrIV Cr O XXVII Cr OH2 H2O O - SG GSH O GS O O XIX SG XXVI GSSG SG 5 CrVI XXIV +2 MW 757 MW 450 XXV OH GS HO CrVI IX OH VI 8GSH Cr 4H2O O SG 14 - GS OH

H2O + XVIII SG V V [Red] X VI Cr2 Cr1 GSSG Cr 6 OH +2 HO OH CrIV OH = XI 4GSSG CrIV 2O2 O GSH 13 H2O 7 + GSH XVII SG +2 VI OH2 SG HO Cr IV CrIII Cr XII OH O OH 12 11 SG + SG +2 + GSH XV XIV XIII SG + [Red] VI IV IV - -O Cr O Cr O CrVI OH [Ox] CrV [Ox] Cr GS SG H OO SG 8 H2OO 2 OH XVI OH GSSG H2O + 10 9 SG OH OH According to the stoichiometric studies performed earlier,106 7 to 9 equivalents of glutathione are consumed in the reaction. In Scheme 3, it is shown that precisely 9

equivalents of glutathione (highlighted with bold red) are needed in order to completely reduced the chromium to chromium(III).

Overall, the mechanism is condensed in the inset in Scheme 3 where the cycling

nature of the reaction between III and glutathione is depicted. If more peroxo groups

were available, the cycle could be extended more rounds and if n is the number of moles

of O=, 4n moles of GSH would be required to complete all cycles.

The proposed mechanism based on the UV-VIS measurements is triphasic with a

-2 -1 sharp fast raise of the first intermediate with rate constant of the order of 10 s . Int1

must correspond, as indicated in the analysis of the UV-VIS spectrophotometric data, to

the mono-glutathionato complexes. This phase is fast and not seen by the EPR. The EPR

detects Int1 until it decays by reduction to a Cr(V) species (g = 1.986) that forms at a rate of the order of 0.5 x 10-2 s-1. This occurs within the time frame of the first and second

phases determined by UV-VIS. Because the ligation of glutathione and electron transfers

for the oxidation and reduction of the chromium center are fast, g = 1.986 is a collection

of the two or more possible mono-glutathionatochromium(V) complexes that form during

the two redox cycles that transform the diperoxochromiun(IV) into Cr(VI) and then back to Cr(IV). The two redox cycles produce a net accumulation of a Cr(IV) species, Int2,

whose slow decay (10-5s-1) is only slightly accelerated by larger glutathione

concentrations due to the competing effect of the electron transfer and the ligation of

more glutathione molecules to an already hindered coordination sphere. The broad signal

(peak to peak band width = 153 G) detected using a modulation amplitude of 5G several 106 minutes after the Cr(V) species disappeared might correspond to Int2 rather to Cr(III)

products because the EPR of Cr(III) species found in the literature are much more broader. For example, Tanaka86 et al detected EPR signals corresponding to the Cr(III)

products of the decomposition of oxodiperoxochromate that spread over 1000 G. A

similar spectrum of a Cr(III) species was seen by Morrow111 et al in their studies of the

oxidation of Cr(III) with H2O2. In addition, the formation of the two ions that are

regarded as products based on their time course, m/z 484 and ,m/z 775, and that

correspond to mono and bis-glutathionato complexes, have rate constants (2.95x10-4 s-1

-5 -1 -5 -1 and 1.88 x 10 s respectively) similar to k3 (∼10 s ) obtained by UV-VIS

spectrophotometry.

The three Cr(V) intermediates detected by EPR (g = 1.996, g = 1.986 and g =

1.983) raise and decay within the same time frame and peaked almost at the same time.

The formation of three EPR active chromium species with these exact g values, was

reported by O’Brien71 et al in their studies of a isolated Cr(V) glutathione complex.

When dissolved in water, the EPR of such complex showed two signals (g = 1.996 and g

= 1.986) but when the compound was dissolved in excess glutathione, the g = 1.996 predominated and another signal appeared, g = 1.983.71 This is consistent with the results

of this work as initially under “low” GSH concentrations only g = 1.986 was observed;

while with higher GSH concentrations g = 1.996 and g = 1.983 appeared. At large GSH

concentrations (>20 equivalents), the formation of poly glutathionatochromiun complexes during the 1st and 2nd phases of the reaction becomes important and new Cr(V)

species are detected with EPR. According to Lay42 et al, that repeated the experiments of

107

O’Brien71 et al, the signal with g = 1.996 corresponds to the bis-

glutathionatochromium(V), which decomposes partially by re organizing the ligand

environment, replacing some of the Cr-S bonds with Cr-N and Cr-O bonds from the

amino and carboxylato groups and water molecules. In such an analysis, g = 1.986 and g

= 1.983 are the minor intermediates produced during the decomposition of the species

with g = 1.996.42 This interpretation suggests that g = 1.986 and g = 1.983 would be

either isomers of g = 1.996 or isomers of mono-glutathionatochromium(V). But the

results presented in this work exclude this interpretation because the species with g =

1.996 and 1.983 appear with increasing glutathione concentration. A better interpretation

is that g = 1.983 and g = 1.986 are mono-glutathionatochromium(V) complexes, the latter

an isomer of the former one and that g = 1.996 is a bis-glutathionatochromium(V)

complex. Also, it is possible that because g = 1.983 appears in the spectrum only at

>20mM GSH and the fact that the ion count of ions like m/z 984, 1029, 1064, 11129 and

1384 are small in comparison with m/z 775, g = 1.983 is a tris or tetra glutathionato

chromium complex instead of an isomer of g = 1.986.

This has implications towards the understanding of the carcinogenicity of

chromium. Lay42 et al have pointed out that a chromium glutathione complex is more

likely to form Cr(III) products by intra molecular electron transfer and so is less likely to

react with external reductants such as DNA. The results of this work indicate that at

lower than 20 mM glutathione concentration, III and glutathione form mainly a mono

glutathionato chromium complex, obviously, the degradation of this Cr-GSH complex

108 will not happen by intra molecular reduction and because the decay of the Int2 is slow,

the Cr(IV)-GS thus formed will have the opportunity to react with reductants like DNA.

Also, the implication of this study regarding the carcinogenic power of

peroxochromates is evident. Although the concentration of hydrogen peroxide in vivo is

low,1, 7 it is well documented that chromium (IV) and (V) can react with hydrogen

peroxide, produce OH radical and damage the DNA.59, 60 Also, oxygen activation by

hypervalent chromium species can generate peroxochromate and damage the DNA by

direct attack of either peroxo or OH radical.13, 112, 113 The present study is the first in

pointing out the potential synergism between glutathione and hydrogen peroxide/activated oxygen in making of chromium a more deadly carcinogen.

Peroxochromates may react with glutathione or other reductants in vivo to activate the carcinogen and to recycle the chromium once it has been deactivated by reaction with important cellular constituents like DNA, RNA and proteins. Evidence that supports this idea comes from a preliminary test in which III was reacted with an oligonucleotide. III was unable to cut the single strand oligonucleotide. However, in the presence of glutathione, the 31P NMR of the chromium treated oligonucleotide showed shoulders that

are indicative of fragmentation of the main oligo. This suggests that during its reduction

with glutathione, III becomes a DNA damaging agent.

109

CHAPTER 4

Magnetic Characterization of the Intermediates of the reaction between Chromate

and Glutathione in Glycine buffer (pH 2.8)

Experimental Part

Reagents

Deuterated water (D2O), chromium potassium sulfate [CrK(SO4)2.12H2O],

anhydrous potassium chromate (K2CrO4), reduced glutathione (GSH) and glycine (Gly)

were purchased from Aldrich and used as received.

683mM K2CrO4

Anhydrous potassium chromate from Sigma (99.99%, 194.2g/mol, 2.731 g/mL),

0.2556 g, 1.316 mmol, was dissolved in D2O (99.9%, 1.1047 g/mL), 1.8326 mL. The

two compounds were measure by mass and then their volumes were calculated using their densities, then the true volume was calculated by adding the volumes of both reagents. The final concentration of the K2CrO4 solution in D2O was 683±9 mM. No

correction was attempted based on the mixing volume as no change in the temperature of

the system upon mixing was observed.

2.02 mM and 20.3 mM CrK(SO4)2

Chromium potassium sulfate dodecahydrate, 102.2 mg was put in a 100.00 mL

volumetric flask, then sufficient dionized water was added to reach the mark of 100.00

mL and assure the complete dissolution of the salt. This gave a concentration of Cr of

2.02±0.02 mM. Following similar steps a 20.3±0.2 mM solution of CrK(SO4)2 was

prepared.

110

1 M Glycine

Glycine (750.0 mg, 0.0100 mmol) was dissolved in enough D2O, then 5% HCl

was added until the pH was 2.8. The final volume was 10.00 mL.

200 mM Glutathione

Glutathione (614.0 mg, 2.00 mmol) was dissolved in enough D2O to make a total

volume of 10.00 mL in a plastic 14 mL centrifuge tube. The final glutathione

concentration was 200.0±0.6 mM.

UV-VIS Spectrophotometric Measurements

Kinetic profiles were measured with a Shimadzu UV160U spectrophotometer at room temperature. The absorbance vs time data was subjected to iterative non linear computer fitting using Sigma Stat for Windows Version 3.5.

Magnetic Susceptibility Measurements

The Magnetic Property Measurement System or SQUID (Quantum Design) was employed.

Cuvet for Magnetic Measurements

The cuvette was made up by cutting off the conic part of a 200µL PCR polypropylene tube and using the cap of another PCR tube as a stopper. An example is shown in Figure 39.

111

Figure 39. (a) Top view of the stopper, (b) Lateral view of the empty cuvette without the stopper, (c) Lateral view of the cuvette with solution inside.

Calibration of the Magnetometer

The Quantum Design MPMS susceptometer is calibrated by direct comparison made with measurements performed on calibrated palladium samples originally purchased from the National Bureau of Standards (NBS Standard Reference Material

765) and provided by the instrument manufacturer.

The calibration of the instrument was verified by measuring a Palladium reference material at 298 K. As shown in Figure 40, the expected magnetic susceptibility per gram of the reference was χg = 5.25x10-6 cm3/g while the measured magnetic susceptibility

was χg = 5.27x10-6 cm3/g.

0.400

0.300

0.200

0.100

emu/g 0.000

-0.100

-0.200

-0.300 Pd-995 Reference , mass 248.40 mg, measured at 298 K reference χg = 5.25x10-6 cm3/g, measured χg = 5.27x10-6 cm3/g -0.400 -80 -60 -40 -20 0 20 40 60 80 KOe

Figure 40. Magnetic susceptibility of a Palladium reference material provided by the MSPM manufacturer. The magnetic susceptibility is given by the slopee of the curve which is obtained by linear least-square fitting of the experimental data. The r2 value is 0.99989. 113

SQUID Measurements

As was indicated in the experimental part, when the analyte’s concentration is lower than approximately 3 mM the effects of most paramagnetic impurities become important. For that reason it was better to maximize the concentration of the analyte.

The evaluation of the kinetic profile of this reaction was necessary to calculate the time at which the intermediate’s concentration was the highest so that the reaction could be frozen at this time and then be subjected to the magnetization measurements in the

SQUID. These constraints imposed the need to adjust the concentration of chromium and all other reagents so that there was enough time to perform the whole operation, which included:

1. Once the reaction mixture or the control and the chromium stock solutions

were Ar purged inside the glove box, chromium is poured into the reaction

mixture which is then stirred.

2. Quickly a known volume (typically 100.0 µL) of the reaction mixture or the

control is pipette into the cuvette.

3. The cuvette is capped.

4. The cuvette is removed from the glove box and weighed.

5. The cuvette is mounted into the sample holder.

6. At the time given by the peak of the kinetic profile the sample holder is

immersed enough in liquid nitrogen to freeze the reaction.

7. After 2 minutes the sample holder is mounted into the SQUID, allocated in the

airlock and evacuated to remove air and hence oxygen, then lowered into the

superconducting magnet and centered to get the most accurate measurements.

114

The magnetic susceptibility of various Cr(VI) GSH reactions was measured with and without the presence of oxygen. For the measurements with oxygen the samples were pipette as such into the cuvette described above. When oxygen was removed, a plastic dry box was employed. Initially the dry box was purged with 99.994% Ar gas three times. Then using a slow flow of Ar, and a Pasteur pipette, the reagents were bubbled with Ar.

For the reaction of Cr(VI) and GSH in D2O and 100 mM glycine, the procedure

was as follows: 200 mM glutathione (150 µL), 1 M glycine (200µL), and D2O (1647 µL)

were mixed in a 5 ml glass vial. Then both the previous mixture and the 683 mM K2CrO4 solution were purged inside of the dry box by bubbling 99.994% Ar for 10 minutes in each case. Next, 3.0 µL of the K2CrO4 solution were mixed with the glutathione solution and the timer started. Then the vial was capped, shacken and then 100.0 µL of this reaction mixture were pipette into the cuvette, the cuvette was tightly capped and its mass

measured. Afterwards, the cuvette was put onto the SQUID sample holder and at the peak of the profile of the reaction, which was at 200 s, the sample holder was immersed for 2 minutes in liquid nitrogen to freeze the reaction. Following that, the frozen sample was transferred to the SQUID. As part of the protocol to avoid condensed oxygen on the exterior of the cuvette, the sample is lowered into the airlock and purged. Immediately after the sample holder is lowered into the SQUID to start the magnetic measurements. A reference solution containing all the reagents except for the chromium reactant was prepared, argon purged and measured to subtract it from the data of the reaction. The same cuvette used to measure the magnetic properties of the sample was used to measure the reference solution. Before it was put into the SQUID, the reference solution was

115 added carefully to the cuvette with a micro syringe until its weight matched the weight of the sample.

Kinetic Profiles

The reaction of Cr(VI) from K2CrO4 and glutathione in 100 mM glycine was

monitored at 460 nm by UV-VIS spectroscopy. In a typical experiment, a solution

containing 200 µL of 1 M glycine, 150 µL of 200 mM glutathione and 1647 µL of D2O was prepared in a quartz spectrophotometer cuvette. Then to this solution 683 mM

K2CrO4 (3.0 µL) was added; the cuvette was capped, shacken and then put into the spectrophotometer. The spectrophotometer was initiated at the same time at which the chromium solution was mixed with the glutathione solution so that no time adjustments to the data collected were needed afterwards. The final concentrations of all reagents in the reaction mixture ended up being: 100 ± 5 mM glycine, 1.02 ± 0.05 mM Cr, 15.0 ±

0.8 mM glutathione. The pH of this solution was 2.8.

116

Results

The kinetic profile of the reaction between Cr(VI) and glutathione in 100 mM

glycine was developed by performing a time course of the absorbance at 460 nm. A

solution with all reagents but chromium was prepared first. The concentrations and

quantities of all stock solutions were adjusted to add the smallest volume of the

chromium solution to the reaction mixture at time zero.

The reaction studied in this section was 1.02 mM in chromium, 100 mM in

glycine (pH 2.8) and 15 mM in glutathione, with all reagents prepared in D2O. This

reaction mixture gave the kinetic profile that is shown in Figure 41.

117

1.200

1.000

0.800

0.600 Absorbance

0.400

0.200

0.000 0 500 1000t (s) 1500 2000 2500 3000

Figure 41. Observed (Circles) and simulated (solid line) absorbance-time trace at 460 nm of a mixture of 1.02±0.05 mM Cr(VI), 15.0±0.8 mM glutathione in 100 mM glycine (pH 2.8). The calculated absorbances are based on equation (18). The -2 -1 -1 -3 -1 parameters used to plot the simulated curve are: a = 2.4 x 10 , b = -1.4, k1 = (1.0 x 10 ) s , c = 1.6, k2 = (1.5 x 1 ) s .

118

As indicated previously by Bose22, 39 et al, this reaction’s profile corresponds to a

biphasic process:

k1 k2 Cr(VI) + GSH Intermediate Products (18)

The advance of a reaction expressed as absorbance vs. time can be described by a function with two exponentials in which the first exponential corresponds to the raise of

the Intermediate’s absorbance while the second exponential corresponds to its decay:

-k t -k t A = a + be 1 +ce 2 (19)

The interest of this study is to determine the nature of the ‘Intermediate’ by

measuring its magnetic properties. For that reason it is necessary to calculate the mole

fraction of all paramagnetic chromium species at the peak of the reaction’s profile. This

information will then be used to determine the contribution of each of these species to the

total magnetic moment measured.

In order to calculate the mole fraction of each of the major chromium species, it is

necessary to determine the analytical expressions for the concentration of such species.

This can be achieved by integrating the set of differential equations that describe the

process given in equation 18 as follows:

d[Cr(VI)]/dt = -k1[Cr(VI)] (20)

d[Intermediate]/dt = k1[Cr(VI)]-k2[Intermediate] (21)

d[P]/dt = k2[Intermediate] (22)

Assuming that the initial concentration of Cr is [Cr(VI)]o and that at time zero

[intermediate]=[Products]=0, the equations can be integrated to give expressions for each

of the reactant’s concentration as a function of time:

-k t [Cr(VI)] = [Cr(VI)]o e 1 (23)

119

-k t -k t [Intermediate] = [k1/(k2-k1)][Cr(VI)]o(e 1 -e 2 ) (24)

[Products] = [Cr(VI)]o-[Cr(VI)]-[Intermediate]

-k t -k t -k t [Products] = [Cr(VI)]o{1-[k1/(k2-k1)](e 1 -e 2 )-e 1 } (25)

The time at the peak of the curve is given by

tmax=ln(k2/k1)/(k2-k1) (26)

Now, as [Cr(VI)]0 is known and from the computer fitting the rate constants are also

known, the concentrations of each of the major chromium contributors can be calculated

by substituting the known values in the above expressions. The results are given in

Table 3:

Table 3. Distribution of chromium in the reaction between Cr(VI) and glutathione in 100 mM glycine at the time when [Intermediate] is at maximum concentration (tmax). [GSH]/[Cr(VI)] 15

tmax/s 200

[Cr(VI)]o/mM 1.02±0.05 Mole Fraction

[Cr(VI)]max/mM 0.128±0.006 0.12±0.07

[Intermediate]max/mM 0.74±0.04 0.72±0.07

[Products]max/mM 0.155±0.008 0.15±0.07

Magnetic Measurements

Saturation Moment Method

The saturation magnetic moment is the magnetic moment measured at 2 K and at

the highest magnetic field. In this technique, the sample is positioned inside the

superconducting magnet under high vacuum initially at the temperature of liquid nitrogen

120

(100 K), then the temperature is lowered to 2 K which takes approximately one hour.

Next, the field strength is raised until the magnetization of the sample reaches the plateau which occurs at approximately 60 kOe. This second step takes approximately 1.5 hours.

For each sample. two measurements are necessary - the sample itself and the control. The experimental time is approximately four hours.

Before the magnetic moment of the frozen reaction was measured, it was necessary to evaluate the method’s accuracy with similar samples. To do this, CrK(SO4)2

114 whose magnetic properties are known was measured as follows: the saturation magnetic moment of a powder sample of CrK(SO4)2 and of a 20.3mM solution of

CrK(SO4)2 in water was measured as such, Figure 42. In both cases the saturation

magnetic moment agreed with the expected value of approximately 3 µB, actual values

being 3.02 and 2.94 µB. However, when a 2.02 mM solution of this same compound was

measured in a similar way, the saturation magnetic moment was 2.6 µB and the

Magnetization vs Field curve showed an erratic behavior. According to Day115 et al., this

is due to the effect of the paramagnetic contributions of oxygen that are difficult to match

in both the sample and the reference, especially when the analyte’s concentration is lower

than 3 mM. For that reason, all samples were argon purged and as Figure 42 shows,

when the same 2.02 mM solution was prepared under Ar, the saturation magnetic

moment was 3.12 µB and the saturation curve behaved as expected.

121

3.500

3.000

2.500 /Cr) B 2.000 Powder M(µ

1.500 20.3 mM

1.000 2.02 mM

0.500 2.02 mM without O2

0.000 0 10000 20000 30000 40000 50000 60000 70000 Oe

Figure 42. Magnetization curves of several samples of CrK(SO4)2.

122

Following the guidelines of Day115 et al, the samples for the study of the Cr(VI)

vs GSH reaction were prepared in D2O to reduced the paramagnetic contribution of the

proton nuclei of water whose nuclear paramagnetism expressed as concentration of spins

½ accounts for about 0.26 mM; the deuteron’s contribution is only 0.02 mM.

As indicated previously, the reaction mixture without chromium and the chromium stock solution were put in the glove box under argon and then purged by bubbling argon for 10 minutes. Then the chromium stock solution was added to the

reaction mixture, quickly stirred and poured in the cuvette, and the cuvette was tightly

capped, taken to the balance and its mass measured. After that, the cuvette was mounted

on the sample holder which was then immersed in liquid nitrogen when the reaction’s

time reached the tmax. Then the sample was taken to the SQUID and evacuated in the

airlock to avoid oxygen condensation on the cold walls of the cuvette and the sample

holder. Following this, the sample holder was lowered into the superconducting magnet,

centered and the measurements started. The same process is repeated for the control

except that no time frame is observed before the sample is taken into the SQUID.

The mass of both the sample and the control are measured to ensure that the

cuvette does not have leaks that will allow water to escape during the readings as the

measurements are taken at low temperature and under vacuum.

Figure 43 shows the magnetization vs magnetic field data for the sample for which the chemical and kinetic parameters were described in the previous paragraphs.

As the reaction of Cr(VI) in excess glutathione affords the complete reduction of the

Cr(VI), the final products should be a purple mixture of Cr(III) complexes. To be composed of true Cr(III), such mixture should show a saturation magnetic moment of

123

about 3 µB. As indicated in Figure 43, the saturation magnetic moment of the reaction

mixture between Cr(VI) and glutathione was measured at tmax ,and twelve hours later

when the reaction was complete. The saturation magnetic moment of the reacted solution

was measured as 3.04 µB, while the saturation magnetic moment of the reaction at tmax was 1.39 µB.

124

3.500

3.000

2.500

2.000 M/µB 1.500

1.000 Reacted after 12 hours at tmax 0.500

0.000 0 10000 20000 30000 40000 50000 60000 70000 H/Oe

Figure 43. Magnetization curves at 2 K of the reaction mixture of 1.02±0.05 mM Cr(VI) and 15.0±0.8 mM GSH in 100 mM glycine, pH 2.8, in D2O.

125

To determine the nature of the paramagnetic species that evolves as the reaction between Cr(VI) and GSH proceeds, the molar fractions of such species were calculated from the kinetic curve (Figure 41). As Cr(VI) is diamagnetic it does not contribute to the magnetic moment of the mixture. For the ‘Intermediate’, there is a contribution of 2 µB

per atom of Cr(IV) and 1 µB per atom of Cr(V) while ‘Products’ contribute as Cr(III), with 3 µB per chromium. In this way, the addition of the magnetic moments of each

contributor should total the measured magnetic moment at the specified time of

measurement according to the kinetic curve.

The expected saturation moment of the mixture at a given time is calculated

below using the mole fractions (fIntermediate, fProducts) of the intermediate and products that

were calculated from the data of the reaction with [Cr(VI)]/[GSH] = 15. Also the data

was broken down in two limiting cases: the hypothetical case in which Intermediate is

composed exclusively of Cr(IV) and the hypothetical case in which Intermediate is

composed exclusively of Cr(V):

[Cr(VI)]/[GSH] = 15

Intermediate is µ= fIntermediate µCr(IV) + fProducts µCr(III)

only Cr(IV) µ= 0.72±0.07* 2 + 0.15±0.07 * 3 = 1.9±0.1

Intermediate is µ= fIntermediate µCr(V) + fProducts µCr(III)

only Cr(V) µ= 0.72±0.07* 1 + 0.15±0.07 * 3 = 1.2±0.1

As Figure 43 shows, the saturation magnetization for the reaction of Cr(VI) and

excess glutathione falls in between the values for the case in which Intermediate is just

126

Cr(IV) and the case in which Intermediate is just Cr(V) which means that the intermediate must be a mixture of both chromium species.

In order to find the real composition of the intermediate, a set of two linear equations with two unknowns was solved. If f4 and f5 are the mole fractions of Cr(IV)

and Cr(V) at the peak of the reaction’s profile, then for the data acquired with

[Cr(VI)]/[GSH] = 15:

µ = (0.72-f4)*1 + f4*2 + 0.15*3 = 1.39 and f4 + f5 = 0.72

µ = 0.72-f4 +2f4 + 0.45 = 1.39

⇒ 0.72+f4 = 0.94 ⇒ f4 = 0.22, f5 = 0.50

Now with this information, the relative proportion of Cr(IV) and Cr(V) in the

Intermediates can be calculated as:

f4/XIntermediate = 0.22/0.72 = 0.30 or 30 % of Cr(IV). f5/XIntermediate = 0.50/0.72 = 0.69 or 69 % of Cr(V).

127

Temperature Dependence Magnetization

In the temperature dependence magnetization experiment, at constant field strength, the temperature is increased slowly from 2 K up to 250 K while the magnetization is measured. The isofield curve experiment takes approximately 6 hours.

The total time of the experiment is about 13 hours considering that each sample consists of two measurements, the sample and the control. The total experimental time for the measurement of the sample’s saturation magnetic moment and the temperature dependence magnetic moment would be approximately 20 hours.

The temperature dependence magnetization measurement is based on the Curie-

Weis equation:

χ = C/T, (27) where χ is magnetic susceptibility of the substance, C is the so called Curie constant and

T the absolute temperature.

The Curie constant can be evaluated from the slope of a plot of χ against the inverse of the temperature at constant field, this kind of plot is known as the isofield curve. In any given case the expression for C is:

2 C = NA µeff /3k, (28)

where NA is the Avogadro’s number, k is the Boltzmann constant, T is the absolute

temperature and µeff is the effective number of Bohr Magnetons (µB), which is related to

the total angular momentum by the expression:

2 2 µeff = g S(S+1), where S = Σsi, (29)

128

The former expression is known as the spin only effective magnetic moment.

Experimentally it has been observed that at least for first row transition metals it is generally valid, in particular for species with electronic configurations d1, d2 and d3. 116

By substitution of equation (27) into equation (28) and regrouping all constants an expression that relates the effective magnetic moment to the magnetic susceptibility is obtained:

1/2 µeff = 2.828 (χ T) (30)

Experimentally, the region from 100 K to 200 K is used to calculate µeff by linear-

squares computer fitting of the data.

Initially reference samples of CrK(SO4)2 were measured. Figure 41 shows the

results for various samples of CrK(SO4)2. Of these samples, the calculated value of µeff for the powder was the closest to the theoretical value for Cr(III) using the spin only

2 1/2 formula: µeff = {(1.977) (3/2) [(3/2)+1]} = 3.83; where g was assumed to be 1.977 which is a typical value for the g factor found in the literature of EPR experiments of

Cr(V) compounds.25, 39, 43However, the effective magnetic moment calculated from the

curve of the 2.0 mM CrK(SO4)2 without oxygen was closer to the theoretical value than

the 2.0 mM CrK(SO4)2 solution with oxygen present. As indicated in Figure 42, when

the reaction mixture of Cr(VI) and glutathione was studied with this technique, the

effective magnetic moment calculated for the products of the reaction was 3.87 µB, very close to the expected three unpaired electrons of the Cr(III) sample (Figure 42).

129

0.100 2.0 mM O2 Present 0.090 Powder Simulations 0.080 2.0 mM O2 Removed 0.070

0.060

0.050 (emu/mol) χ 0.040

0.030

0.020

0.010

0.000 0.0 50.0 100.0 150.0 200.0 250.0 300.0 T/K

Figure 41. Observed (markers) and simulated (solid lines) magnetic susceptibility at 10 kOe of solid and aqueous solutions of 2 -4 CrK(SO4)2. The simulation was done using the equation χ = χ0 + µeff /8(T-θ). The calculated parameters are χ0 = -3.3 x 10 -3 emu/mol, µeff = 3.881 µB and θ = 0.133 K for the powder sample, χ0= 4.701 x 10 emu/mol, µeff = 3.705 µB and θ = 7.44 K for -3 the 2.0 mM Oxygen Removed solution and χ0= 6.358 x 10 emu/mol, µeff = 3.1499 µB and θ = 30.728 K for the 2.0 mM Oxygen Present solution.

130

The reaction mixture of 1.02 mM Cr(VI) and 15 mM GSH in 100 mM glycine pH

2.8 was measured at 10 KOe within the temperature range of 2 to 300 K. This reaction was initiated in the glove box by first bubbling argon for 10 minutes to both the solution containing glutathione and the Cr stock solution, which were mixed afterwards as explained in the previous section. The temperature dependence magnetization experiment was performed right after the saturation magnetization experiment and the sample was not removed from the SQUID in between the experiments.

Figure 42 shows the temperature dependence magnetization results for the reaction of 1.02 mM Cr(VI) and 15 mM glutathione in 100 mM glycine. The effective magnetic moment obtained from that curve was 2.55 µB while the effective magnetic

moment of the products of the reaction was found to be 3.87 µB.

131

0.250

0.200 Intermediates Products

0.150 /(emu/mol) χ 0.100

0.050

0.000 0.0 50.0 100.0T/K 150.0 200.0 250.0

Figure 42. Observed (markers) and simulated (solid lines) magnetic susceptibility at 10 kOe of the reaction of 1.02 ±0.05 mM Cr(VI) and 15±0.8 mM glutathione in 100 mM glycine taken at the peak (Intermediates) of the reaction’s profile and at the end 2 (Products) of the reaction. The simulation was done using the equation χ = χ0 + µeff /8(T-θ). The calculated parameters are χ0 -3 -2 = 6.95 x 10 emu/mol, µeff = 2.545µB and θ = 27.36 K for the Intermediates and 2.075 x 10 emu/mol, µeff = 3.8679µB and θ = 12.88 K for the products.

The contribution of each of the chromium species present when the reaction was

stopped by immersing it in liquid nitrogen is calculated using the molar fractions

obtained from the kinetic studies and shown in Table 3. Two hypothetical limiting cases

can be assumed:

[Cr(VI)]/[GSH] = 15

2 2 1/2 Intermediate µeff= [fIntermediate (µeffCr(IV)) + fProducts (µeffCr(III)) ]

2 2 1/2 is only Cr(IV) µeff= [0.72 * (2.83) + 0.15 * (3.87) ] = 2.8

2 2 1/2 Intermediate µeff = [fIntermediate (µeffCr(V)) + fProducts (µeffCr(III)) ]

2 2 1/2 is only Cr(V) µeff = [0.72 * (1.73) + 0.15 * (3.87) ] = 2.1

Because the effective magnetic moment for the reaction of Cr(VI) and excess

glutathione in glycine buffer was 2.55 µB, the intermediate must be a mixture of both

chromium species.

In order to find the real composition of the intermediate, a set of two linear

equations with two unknowns was solved. If f4 and f5 are the mole fractions of Cr(IV)

and Cr(V) at the peak of the reaction profile:

2 2 2 2 2 µeff = (0.72-f4)*(1.73) + f4*(2.83) + 0.15*(3.87) = (2.55) and f4 + f5 = 0.72

2 2 2 2 ⇒ 0.72*(1.73) -f4*(1.73) + f4*(2.83) + 0.15*(3.87) = 6.5025

⇒ 2.1549- f4*2.9929 + f4*8.0089 + 2.2465 = 6.5025

⇒ 4.4014 - f4*5.016 = 6.5025

⇒ 5.016*f4 = 2.101

⇒ f4 = 0.42 ⇒ f5 = 0.30 133

Now with this information the relative proportion of Cr(IV) and Cr(V) in the intermediates can be calculated as: f4/XIntermediate = 0.42/0.72 = 0.58 or 58 % of Cr(IV). f5/XIntermediate = 0.30/0.72 = 0.42 or 42 % of Cr(V).

Total Spin Angular Momentum Of The Intermediate

The total spin angular momentum or S can be obtained using equation 26:

2 2 µeff = g S(S+1) (28)

2.552 = 1.9772 S(S+1)

⇒ S2 + S – 1.66 = 0

⇒ S = 0.88, theoretical S = 1 for a d2 system.

This calculation has been done assuming a quenching of the angular momentum (L = 0).

The value of 0.88 for the total spin angular momentum indicates that within 12%, the

spin state of the collection of intermediates in the reaction between Cr(VI) and

glutathione at low pH is a triplet and that the oxidation state of chromium is +4.

134

Discussion

Atoms or molecules with an unpaired number of electrons are paramagnetic. That

means they or the material they are in is attracted into a magnetic field; on the contrary,

materials in which there are atoms or molecules with no unpaired electrons are weakly repelled by the magnetic field are called diamagnetic.117 Therefore, the measurement of paramagnetism can be used to infer the redox state of a chemical compound.

Several techniques can be used to measure magnetization. In studies with metalloproteins Day115 et al have demonstrated that the SQUID susceptometer provides

the lowest detection limits of all.115 This group has successfully measured samples in the

order of 0.2 µmoles and 1mM of metalloprotein.118-120

A matched control has to be subtracted from the sample because the

magnetization measurement detects the contributions from the paramagnetic sample,

paramagnetic impurities in the sample or the cuvette, and the diamagnetic contributions

from the cuvette, the solvent and any reagents present.

Due to the large sensitivity of the SQUID susceptometer, the nuclear spin

magnetization of the protons in water or any material including the cuvette is detectable.

This can deter the reproducibility in magnetization studies due to the long relaxation time

(hours) of spin ½ nuclei in frozen samples. The use of deuterated water in all reagents

overcomes this limitation as the fast relaxing nuclear paramagnetism can be subtracted

using a matched control115.

135

In studies with metalloproteins, it was found that when the sample concentration drops below 7 mM, the background signal due to oxygen must be removed in both the sample and the match control.115

In this study, both the isofield magnetization curve (Curie Law) and the isotherm

magnetization (saturation magnetization) curve at the lowest temperature (2 K) was used

to determine the redox state of the intermediates of the reaction between Cr(VI) and

glutathione in glycine buffer.

The saturation magnetization is the highest induced magnetic moment achievable

at a given magnetic field, at the highest magnetic field no further increase in magnetization occurs.115 The saturation magnetization expressed in Bohr Magnetons

(µB) of a sample of pure spins ½ is 1, for a sample with 2 unpaired ½ spins the value will

be 2 and 3 for a sample with three unpaired ½ spins and so forth.

According to Day115, saturation magnetization data provides a better test of

theoretical models than susceptibility data limited to the Curie Law region.

No evidence in the literature was found regarding the use of the SQUID susceptometer to

measure the magnetic moment of a reaction mixture.

It was assumed that within the process of freezing, the reaction is effectively

stopped. However, during the temperature dependence magnetization, at a certain

temperature, it is expected that the reaction will continue. This introduces a certain

degree of error in the results derived from this method. This could be the reason why the

magnetic moment obtained by measuring the saturation magnetization was lower than the

magnetic moment obtained by the temperature dependence magnetization.

136

Several authors22, 39, 63-66, 70 have studied the reaction between Cr(VI) and glutathione or related thiols and according to the available kinetic models, the reaction can be preceeded by a series of one electron transfers or first by a two electron transfer followed by a one electron transfer to produce the Cr(III) products. According to the variety of conditions used to perform these studies, the mechanism is determined by the initial ratio of Cr and GSH, the pH and the reactivity of the buffer towards chromate.

The kinetic studies performed at low pH indicate an initial two electrons transfer that leads to the production of a Cr(IV) intermediate22, while at neutral pH, more diversity in the nature of the intermediates is expected. For example, it is possible to select conditions that produce exclusive formation of Cr(V) intermediates71 or a Cr(IV) intermediate64 at neutral pH. In the first case, a relatively large concentration of chromate

(50 mM) and a large concentration of glutathione (500 mM) were used; while in the second case the concentrations of both chromate (0.37 mM) and glutathione (25 mM) were low.

The conditions employed here, 1.02 mM Cr(VI) and 15 mM GSH in 100 mM glycine (pH 2.7), were expected to generate mainly a Cr(IV) intermediate as previously reported.22 Bose39 and co workers in the study of the reaction of Cr(VI) and GSH in excess oxidized glutathione (50 mM), pH 2.7; identified as intermediates two peaks in the

HPLC that grew and then decayed. The two peaks showed a maximum absorption at 460 nm; at this wavelength none of the reagents or products absorbed. The reaction produced only Cr(III) as evidenced by the UV spectrum of the mixture at the end of the reaction.

The main bands were centered at 572 (ε = 33 M-1 cm-1) and 408 nm (ε = 26 M-1 cm-1).

137

The EPR of the reaction mixture showed a weak peak with g value of 1.989. When compared with an authentic Cr(V) complex (bis(2-hydroxy-2- ethylbutyrato)oxochromate(V), (EHBA-Cr(V), I, g = 1998), this complex produced an intense signal even at 0.5 mM concentration in acidic solution. Using the intensity of this complex as a calibration point, the authors estimated that among the intermediates, less than 3% of the total chromium was Cr(V). When the same reaction was performed in the absence of an initial excess of oxidized glutathione and in glycine buffer, these same workers22 found only one intermediate by HPLC and the EPR experiments failed to show

evidence of Cr(V) at pH lower than 3.4. In both cases, in order to further characterize the

EPR silent intermediate, time course magnetic susceptibility measurements by NMR

were performed. In this way, the magnetic moments of both intermediates and products

were evaluated, the values being 2.6 and 4.3 µB respectively. These data led the

investigators to conclude that the intermediates were composed exclusively of Cr(IV).

The temperature dependence magnetization studies that are part of this work gave

an effective magnetic moment and a spin-only magnetic moment that is only 12 %

different from a sample of pure Cr(IV). These results fully support the previous findings

of Bose et al in which the intermediate of the Cr(VI) glutathione reaction in glycine

buffer was mainly a Cr(IV) species. However, the results of the magnetization studies at

2 K indicated that at the moment of freezing, approximately 30% of the intermediates

were Cr(IV) and the other 70% were Cr(V). Two issues need to be addressed. First, why

the magnetic moment of the mixture indicates the presence of such high amount of

Cr(V), and secondly, why the Cr(V) was not detected in the EPR experiments in previous

138 work?22, 39 It is possible that a mixed mechanism could be in operation, one in which an

initial two electron transfer leads to Cr(IV) while a second mechanism with consecutive

one electron transactions leads to Cr(V). Consistent with this idea is the fact that the

reaction between monoglutathionatochromate(VI) and another GSH molecule is faster

than the electron transfer. Considering that up to now there has been no quantitative

accounting of the glutathione thiyl radicals produced in the reaction under study, it is not

prudent to exclude the possibility of intra molecular oxidation of two glutathione

molecules in conjunction with one electron transactions within the GSH-Cr(VI) complex

on the basis of spin trapping of this radical.

Accordingly, the HPLC and the titration data regarding the kinetic studies of the

reaction in excess oxidized glutathione showed that at least one GSH and half of a GSSG

molecule were attached to a Cr(III) center.39 When the reaction was run in glycine buffer with no initial GSSG, the authors found 1.5 molecules of GSSG and one GSH molecule attached to the Cr(III) products.22 Because of the inertness of this oxidation state, it is

expected that the coordination of glutathione molecules occurred during the reduction

process. A dinuclear complex would be consistent with these stochiometries and the

mechanism proposed in such studies. The two Cr(V) centers would be diamagnetically

coupled and would not contribute to the magnetic moment of the mixture. Then, using

the saturation magnetic moment, the new fraction of Cr(IV) in the mixture of

intermediates would be:

f4’*2µB + 0.15*3 µB = 1.39 µB, ⇒ f4’ = 0.47, where f4’ is the fraction of Cr(IV) forming part of the intermediates, 0.15 is the molar fraction of products (Cr(III)) and 1.39 is the

139 measured saturation magnetic moment. Then f4’ is used to calculate the proportion of

Cr(IV) in the intermediate: f4’/XIntermediates = 0.47/0.72 = 0.65 or 65 %.

In the same way, using the effective magnetic moment, the new fraction of Cr(IV)

in the mixture of intermediates would be:

f4’*(2.83)^2 + 0.15*(3.87)^2 = (2.55)^2 ⇒ f4’ = 0.53, ⇒ f4’/XIntermediates = 0.53/0.72 =

0.74 or 74%.

140

CHAPTER 5

Conclusions

The mechanism by which chromium damages the molecule of DNA was investigated by studying the reaction between compounds I or II and dGDP. Based on the analysis of the reaction mixture it was concluded that these model compounds can degrade dGDP by hydrolysis of the phosphate moiety and oxidation of the ribose; although the oxidation will be preferred over the hydrolysis if the metal has access to the sugar skeleton. Also, during the oxidation, there was no preference between the abstraction of H5 or H4 from the sugar moiety.

The mechanism of the reaction between the chromium (IV) complex III and glutathione was also investigated by ESI-MS and EPR. The detection of Cr(V) signals with EPR supported the mechanism in which the recycling of the metal between Cr(IV) and Cr(VI) had been proposed.106 The detection of ions with intensity profiles of

intermediates and products supported the multiphasic nature of the kinetics of this

reaction. Also, the early detection of Cr(V) with EPR in relation to the late appearance of

the products of this reaction and the detection of an EPR signal that resembles a Cr(IV)

species led to the conclusion that this reaction produces a net accumulation of Cr(IV)

before the chromium species are completely reduced to Cr(III) and that the reaction

overall proceeds through one electron transfers. Moreover, the fact that III was able to

cleave an oligonucleotide in the presence of glutathione indicated that glutathione

transforms this chromium complex into a DNA damaging agent and also enhances the

importance of Cr(IV) in chromium induce carcinogenesis.

141

The investigation of the oxidation state of chromium during the reduction of chromate with glutathione in the presence of glycine buffer at low pH was performed with the SQUID magnetometer. An effective magnetic moment of 2.55 and a spin only moment of 0.88 suggested that within 12%, the chromium intermediate of this reaction is

Cr(IV). The low magnetic moment obtained with the saturation magnetization method can be explained by considering the presence of a diamagnetically coupled dinuclear

Cr(V). With that in consideration, it was calculated that Cr(IV) is at least 90 % of the reaction mixture at the moment of measurement.

142

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