Mechanistic Studies on the Reactions of Vitamin B12 Complexes with the Nitroxyl (Hno) Donors Angeli's Salt and Piloty's Acid

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MECHANISTIC STUDIES ON THE REACTIONS OF

VITAMIN B12 COMPLEXES WITH THE NITROXYL (HNO) DONORS ANGELI’S SALT AND PILOTY’S ACID

A dissertation submitted

to Kent State University in partial

fulfillment of the requirements for the

degree of Doctor of Philosophy

By Harishchandra Subedi August, 2014

Dissertation written by

Harishchandra Subedi

B.Sc., Tribhuvan University, Nepal, 2002

M.Sc., Tribhuvan University, Nepal, 2004

Ph.D., Kent State University, USA, 2014

Approved by

______, Chair, Doctoral Dissertation Committee Nicola E. Brasch, Ph.D. ______, Member, Doctoral Dissertation Committee Scott D. Bunge, Ph.D. ______, Member, Doctoral Dissertation Committee Jacob T. Shelley, Ph.D. ______, Member, Doctoral Dissertation Committee Darren L. Bade, Ph.D. ______, Member, Doctoral Dissertation Committee Jennifer A. McDonough, Ph.D.

Accepted by

______, Chair, Department of Chemistry & Biochemistry Michael J. Tubergen, Ph.D. ______, Interim Dean, College of Arts and Sciences James L. Blank, Ph.D.

TABLE OF CONTENTS

LIST OF FIGURES…………………………………………………………………… XI

LIST OF SCHEMES………………………………………………………………....XIX

LIST OF TABLES………………………………………………………………...….XXI

DEDICATION…………………………………………………………………...…..XXII

ACKNOWLEDGEMENTS ……………………………………….………………XXIII

ABSTRACT……………………….………………………………………………....XXV

CHAPTER 1: INTRODUCTION AND BACKGROUND…………………….………1

1.1 Vitamin B12 (cobalamins)…………………….……………………..………..…...1

1.1.1 Discovery of vitamin B12………………………………………………….1

1.1.2 Structure of vitamin B12……………..…………………………………….2

1.1.3 Other oxidation states of cobalamins……………………………………...3

1.1.4 Biochemistry of vitamin B12………………………………………...…….5

1.1.4.1 B12–dependent enzymatic reactions……………………………….5

1.1.4.2 Cellular uptake and metabolism of cobalamins…………………...8

1.1.5 Characterization methods for cobalamins………………………………....9

1.1.5.1 X–ray crystallography……………………………………………10

1.1.5.2 UV–vis spectroscopy…………………………….………………10

1.1.5.3 1H NMR spectroscopy…………………………………………...12

1.1.5.4 High performance liquid chromatography (HPLC)……………...13

1.1.5.5 Other characterization techniques………………………………..13

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1.1.6 Non–cofactor functions of vitamin B12…………………………….…….13

1.2 Reactive oxygen / nitrogen species and oxidative stress………...... ………14

1.2.1 Nitric oxide…………………………………………...………………….14

1.2.2 Nitroxyl (HNO/NO–)…………………………………………………….16

1.2.2.1 Biological production and reactivity of nitroxyl…………………17

1.2.2.2 Nitroxyl donors…………………………………………………..18

1.2.2.3 Nitroxyl detection………………………………………………..20

1.2.2.4 Reactions of nitroxyl with transition metal complexes…………..20

1.3 Cobalamins and oxidative stress…………………………………………………23

CHAPTER 2: MECHANISTIC STUDIES ON THE REACTIONS OF REDUCED

VITAMIN B12 COMPLEXES WITH THE NITROXYL DONOR PILOTY’S

ACID……………………………………………………………………………..……...25

2.1 Introduction………………………………………………………………………25

2.2 Experimental section……………………………………………………….…….26

2.2.1 Reagents………………………………………………………………….26

2.2.2 General methods and instrumentation…………………………………...27

2.2.3 Synthesis of cob(II)alamin and cob(I)alamin………...………………….28

2.2.4 Determination of cobalamin concentrations……………………………..29

2.2.5 Determination of the rate constants for spontaneous decomposition of

Piloty’s acid…………...…………………………………………………29

2.2.6 Sample preparation for kinetic measurements on the reaction of

cob(II)alamin with Piloty’s acid………………………………..………..30

2.2.7 Sample preparation for kinetic measurements on the reaction of

cob(I)alamin with Piloty’s acid……………………………………….….30

2.2.8 Sample preparation for 1H NMR experiments for the reaction of

cob(II)alamin with Piloty’s acid…………………………………………31

2.2.9 Determination of the reaction stoichiometry…………………………….31

2.2.10 Indooxine tests to determine if hydroxylamine is formed in the reaction of

cob(II)alamin with Piloty’s acid…………………………………………32

2.2.11 Indooxine tests with commercial Piloty’s acid…………………………..32

2.2.12 Nessler’s test to determine if ammonia is a reaction product……………33

2.2.13 Griess test to determine the presence of nitrite as a reaction

product…………………………………………………………………...33

2.2.14 Control experiments………………………………………………..…….34

2.3 Results and discussion…………………………………………………………...35

2.3.1 Kinetic studies on the reaction of cob(II)alamin with Piloty’s acid……..35

2.3.2 Stoichiometry of the reaction of cob(II)alamin with Piloty’s acid………39

2.3.3 Kinetic studies on the reaction of cob(I)alamin with Piloty’s acid………42

2.4 Summary…………………………………………………………………………53

CHAPTER 3: KINETIC STUDIES ON THE REACTION BETWEEN

AQUACOBALAMIN AND THE HNO DONOR PILOTY’S ACID………………..55

3.1 Introduction………………………………………………………………………55

3.2 Experimental section……………………………………………………………..56

3.2.1 Reagents………………………………………………………………….56

3.2.2 Instrumentation…………………………………………………..………56

3.2.3 Determination of the rate constants for spontaneous decomposition of

Piloty’s acid…………………………………..……………………...…..56

3.2.4 Sample preparation for kinetic measurements…………………………...57

3.2.5 Sample preparation for 1H NMR spectroscopy studies……….…………57

3.2.6 Determination of the stoichiometry of the reaction………………..…….57

3.3 Results and discussion…………………………………………………………...58

3.3.1 Kinetic studies on the reaction of cob(III)alamin with Piloty’s acid (pH ≥

10.00)…………………………………………………………………….58

3.3.2 Kinetic studies on the reaction of cob(III)alamin with Piloty’s acid (pH <

10.00)…………………………………………………………….………66

3.4 Summary…………………………………………………………………………71

CHAPTER 4: STUDIES ON THE REACTION OF THE REDUCED VITAMIN B12

COMPLEX COB(II)ALAMIN WITH THE HNO DONOR ANGELI’S SALT…...72

4.1 Introduction………………………………………………………………………72

4.2 Experimental section……………………………………………………………..73

4.2.1 Reagents……………………………………………………….…………73

4.2.2 Instrumentation…………………………………………………………..73

4.2.3 Solution preparations………………………………………………….…74

4.2.4 Synthesis of cob(II)alamin and cob(I)alamin……………………………74

4.2.5 Determination of cobalamin concentrations……………………………..74

4.2.6 Determination of rate constants for the spontaneous decomposition of

Angeli’s salt……………………………………………………………...74

4.2.7 Sample preparation for kinetic measurements on the reaction of

cob(II)alamin with Angeli’s salt…………………………………………75

4.2.8 Sample preparation for kinetic measurements on the reaction of

cob(I)alamin with Angeli’s salt………………………………………….75

4.2.9 Determination of stoichiometry of the reaction between cob(II)alamin and

Angeli’s salt……………………………………………………………...76

4.2.10 Determination of stoichiometry of the reaction between cob(I)alamin and

Angeli’s salt……………………………………………………………...76

4.2.11 Indooxine tests to determine if hydroxylamine is formed in the reaction of

cob(II)alamin or cob(I)alamin with Angeli’s salt………………………..77

4.2.12 Nessler’s test to determine whether ammonia is formed in the reaction of

cob(II)alamin or cob(I)alamin with Angeli’s salt………………………..78

4.2.13 Reaction of cob(II)alamin with nitrous oxide……………….…………...78

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4.2.14 Griess assay to quantify the amount of nitrite produced in the reaction of

cob(II)alamin or cob(I)alamin with Angeli’s salt and in Angeli’s salt

itself………………………………………………………………..……..78

4.2.15 Experiments in the presence of cyanide to probe whether cob(III)alamin is

an intermediate of the reaction between cob(II)alamin with Angeli’s

salt………………………………………………………………………..79

4.2.16 Stoichiometry experiments in the presence of excess nitrite………….…80

4.3 Results and discussion…………………………………………………………...81

4.3.1 Kinetic studies on the reaction between cob(II)alamin and Angeli’s salt

(pH ≤ 9.00)……………………………………………………………….81

4.3.2 Determination of the stoichiometry of the reaction between cob(II)alamin

and Angeli’s salt (pH ≤ 9.00)…………………………………………….84

4.3.3 Cob(I)alamin, not cob(III)alamin, is an intermediate of the reaction

between cob(II)alamin and Angeli’s salt (pH ≤ 9.00)……………...……86

4.3.4 Identification of the HNO reduction product(s)…………………...…..…92

4.3.5 Studies on the reaction between cob(I)alamin and Angeli’s salt………...93

4.4 Summary………………………………………………………………………..102

CHAPTER 5: KINETIC AND MECHANISTIC STUDIES ON THE REACTION

OF AQUACOBALAMIN WITH THE HNO DONOR ANGELI’S SALT…..……103

5.1 Introduction……………………………………………………………………..103

5.2 Experimental section……………………………………………………………104

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5.2.1 Reagents……………………………...…………………………………104

5.2.2 Instrumentation…………………………………………………………104

5.2.3 Determination of rate constants for the spontaneous decomposition of

Angeli’s salt…………………………………………………………….105

5.2.4 Griess assay for the quantification of nitrite……………………………105

5.2.5 Sample preparation for kinetic measurements on the reaction of

aquacobalamin with Angeli’s salt...... 106

5.3 Results and discussion………………………………………………………….106

5.3.1 Studies on the reaction between aquacobalamin and Angeli’s salt (pH ≤

9.90)…………………………………………………………………….106

5.3.2 Studies on the reaction between hydroxocobalamin and Angeli’s salt

under strongly alkaline conditions……………………………………...120

5.4 Summary………………………………………………………………………..126

CHAPTER 6: MECHANISTIC STUDIES ON THE REACTION OF

NITROXYLCOBALAMIN WITH DIOXYGEN……………………………...……128

6.1 Introduction……………………………………………………………………..128

6.2 Experimental section…………………………………………………………....129

6.2.1 Reagents…………………………………………...……………………129

6.2.2 Instrumentation…………………………………………………………130

6.2.3 Solution preparations…………………………………………………...133

6.2.4 Synthesis of nitroxylcobalamin…………………………………...…….133

6.2.5 Synthesis of nitrocobalamin…………………………………………….134

6.2.6 Synthesis of peroxynitrite………………………………………………134

6.2.7 Mass spectrometry identification of unknown HPLC peak in the product

of the reaction of nitroxylcobalamin with oxygen……………………...134

6.2.8 Determination of the equilibrium constant for the reaction of

nitroxylcobalamin with oxygen………………………………………...134

6.2.9 Probing for the possible cobalamin reaction intermediate by 1H NMR

spectroscopy…………………………………………………………….135

6.3 Results and discussion………………………………………………………….135

6.3.1 Kinetic studies on the reaction of nitroxylcobalamin with oxygen…….135

6.3.2 Determination of the equilibrium constant……………………………..144

6.3.3 Probing for reaction intermediates………………………………..…….146

6.4 Summary………………………………………………………………………..166

CHAPTER 7: SUMMARY AND FUTURE DIRECTIONS………………………..168

7.1 Summary………………………………………………………………………..168

7.2 Future directions………………………………………………………………..170

APPENDIX………………………………………………………………………….…172

REFERENCES………………………………………………….……………..………176

LIST OF FIGURES

CHAPTER 1: INTRODUCTION AND BACKGROUND

Figure 1.1. The general structure of vitamin B12 derivatives…….……………………….3

Figure 1.2. Absorption, transport and cellular uptake of cobalamins in mammals………………………………………………………………………….……….9

Figure 1.3. The structures of the HNO donors Angeli's salt and Piloty's acid....………..19

CHAPTER 2: MECHANISTIC STUDIES ON THE REACTIONS OF REDUCED

VITAMIN B12 COMPLEXES WITH THE NITROXYL DONOR PILOTY’S ACID

Figure 2.1. UV–vis spectra for the reaction between Cbl(II) and excess at pH 10.00, 25.0 °C.…………………………………………………………………………………..……36

Figure 2.2. Plot of absorbance versus time for the reaction of Cbl(II) with excess PA at pH 10.00, 25.0 °C ………………………………………………………….……………36

Figure 2.3. Plot of absorbance versus time for the reaction of Cbl(II) with 1.0 mol equiv PA at pH 10.00, 25.0 °C…………………………………………………………………37

Figure 2.4. Plots of absorbance versus time for the reaction of Cbl(II) with PA at pH 9.00, 8.60, 8.00 and 12.00, 25.0 °C …..…………………………………………………38

Figure 2.5. Determination of stoichiometry of the reaction between Cbl(II) and PA at 10.00……………………………………………………………………………………...39

Figure 2.6. Plot of absorbance at 312 nm and 510 nm versus mol equiv of PA for the reaction of Cbl(II) with PA at pH 10.00, 25.0 °C………………………………………..40

Figure 2.7. Aromatic region of the 1H NMR spectrum of the products of the reaction between Cbl(II) and 2.2 mol equiv PA at pD 10.00, 24 °C……………………………...41

Figure 2.8. UV–vis spectra recorded as a function of time for the reaction between Cbl(I)– and 5.0 mol equiv PA at pH 10.00, 25.0 °C……………………………………..43

Figure 2.9. a) The first 10 spectra of the reaction described in Figure 2.8 showing the formation of a Cbl(II) intermediate and b) selected spectra at longer reaction times showing the formation of the final NOCbl product…………………………………...…44

Figure 2.10. UV–vis spectra recorded as a function of time for the reaction of Cbl(I)– with 1.0 mol equiv PA at pH 10.00, 25.0 °C…………………………………………….45

Figure 2.11. UV–vis spectra recorded as a function of time for the reaction of excess Cbl(I)– with PA at pH 10.00, 25.0 °C……………………………………………………45

Figure 2.12. Plot of absorbance versus time for the reaction of excess Cbl(I)– with PA at pH 10.00, 25.0 °C………………………………………………………………………..47

Figure 2.13. Plot of absorbance versus time for the spontaneous decomposition of PA at pH 10.00, 25.0 °C………………………………………………………………………..47

Figure 2.14. Determination of stoichiometry of the reaction between Cbl(I)– and PA at pH 10.00………………………………………………………………………………….48

Figure 2.15. UV–vis spectra of the product mixture of the reaction between Cbl(II) with – 1.0 mol equiv PA in the absence and presence of excess NO2 at pH 10.00, 25.0 °C…...51

Figure 2.16. UV–vis spectrum of the product mixture of the reaction between Cbl(I)– – with 1.0 mol equiv PA in the presence of excess NO2 at pH 10.00, 25.0 °C…………...51

Figure 2.17. UV–vis spectrum of the products of the reaction of HOCbl with 1.1 mol – equiv PA in the presence of excess NO2 at pH 10.00, 25.0 °C…………………………………………………………………………………..…….52

CHAPTER 3: KINETIC STUDIES ON THE REACTION OF AQUACOBALAMIN WITH THE HNO DONOR PILOTY’S ACID

Figure 3.1. UV–vis spectra for the reaction between HOCbl and excess PA as a function of time at pH 12.00, 25.0 °C……………………………………………………………..58

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Figure 3.2. UV–vis spectra for the reaction of Cbl(III) with excess benzenesulfinate, 25.0 °C………………………………………………………………………………………...59

Figure 3.3. UV–vis spectra for the reaction between excess Cbl(III) and PA at pH 12.00, 25.0 °C…………………………………………………………………………………...60

Figure 3.4. Plots of absorbance versus time for the reaction of excess Cbl(III) with PA at pH 12.00, 25.0 °C………………………………………………………………………..61

Figure 3.5. Plot of absorbance versus time for the spontaneous decomposition of PA at pH 12.00, 25.0 °C………………………………………………………………………..62

Figure 3.6. Plot of kobs versus [Cbl(III)] for the reaction of PA with excess Cbl(III) at pH 10.00, 25.0 °C……………………………………………………………………………63

Figure 3.7. Determination of stoichiometry of the reaction between Cbl(III) and PA at pH 12.00………………………………………………………………………………….64

Figure 3.8. Aromatic region of the 1H NMR spectrum of the products of the reaction between Cbl(III) and 1.1 mol equiv PA at pD 12.00, 24 °C……………………………..64

Figure 3.9. Aromatic region of the 1H NMR spectrum of the products of the reaction between Cbl(III) and 0.5 mol equiv PA at pD 12.00, 24 °C……………………………..65

Figure 3.10. Plot of absorbance versus time for the reaction between excess Cbl(III) and PA at pH 8.00, 25.0 °C…………………………………………………………………..67

Figure 3.11. Plot of kobs versus [Cbl(III)] for the reaction between Cbl(III) and PA at pH 8.00, 25.0 °C……………………………………………………………………………..68

Figure 3.12. Plot of kobs versus [Cbl(III)] for the reaction of PA with excess Cbl(III) at pH 6.00, 25.0 °C…………………………………………………………………………69

Figure 3.13. Plot of the apparent second–order rate constant (kapp) versus pH for the reaction between Cbl(III) and PA………………………………………………………..69

Figure 3.14. Determination of stoichiometry of the reaction between Cbl(III) and PA at pH 6.00…………………………………………………………………………………...70

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Figure 3.15. Aromatic region of the 1H NMR spectrum of the products of the reaction between HOCbl and 1.1 mol equiv PA at pD 7.00, 24 °C……………………………….70

CHAPTER 4: STUDIES ON THE REACTION OF THE REDUCED VITAMIN B12 COMPLEX COB(II)ALAMIN WITH THE HNO DONOR ANGELI’S SALT

Figure 4.1. Selected UV–vis spectra for the reaction between Cbl(II) and excess AS at pH 8.00, 25.0 °C…………………………………………………………………………82

Figure 4.2. Plot of absorbance versus time for the reaction between Cbl(II) and excess AS at pH 8.00, 25.0 °C…………………………………………………………………..82

Figure 4.3. Plot of absorbance versus time for the reaction of Cbl(II) with 1.0 mol equiv AS at pH 8.00, 25.0 °C…………………………………………………………………..83

Figure 4.4. Determination of stoichiometry of the reaction between Cbl(II) and AS at pH 8.00……………………………………………………………………………………….85

Figure 4.5. Aromatic region of the 1H NMR spectrum of the products of the reaction between Cbl(II) and 2.2 mol equiv AS at pD 8.00, 24 °C……………………………….85

Figure 4.6. UV–vis spectra as a function of time for the reaction between Cbl(II) and 2.0 mol equiv AS in the presence of 20.0 equiv cyanide at pH 8.50, 25.0 °C……………….87

Figure 4.7. UV–vis spectra as a function of time for NOCbl in the absence and presence of 20.0 mol equiv cyanide at pH 8.50……………………………………………………88

+ Figure 4.8. UV–vis spectra as a function of time for H2OCbl /HOCbl with 20.0 mol equiv cyanide in the absence and presence of 1.0 mol equiv AS at pH 8.50……………90

Figure 4.9. UV–vis spectra of Cbl(II) with 20.0 mol equiv cyanide at pH 8.50………...90

Figure 4.10. UV–vis spectra of the product of reaction between Cbl(II) and different – equiv of AS in the presence and absence of excess NO2 at pH 8.00, 25.0 °C………….91

Figure 4.11. Calibration curve for the reaction of NaNO2 with the Griess reagent……..92

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Figure 4.12. UV–vis spectra as a function of time for the reaction of Cbl(I)– with excess AS at pH 7.40, 25.0 °C…………………………………………………………………..93

Figure 4.13. UV–vis spectra as a function of time for the reaction of Cbl(I)– with 0.25 mol equiv AS at pH 7.40, 25.0 °C……………………………………………………….94

Figure 4.14. Plot of absorbance versus time for the reaction of Cbl(I)– with 0.25 equiv of AS at pH 8.00, 25.0 °C…………………………………………………………………..95

Figure 4.15. Plot of absorbance versus time for the reaction of Cbl(I)– with 0.04 mol equiv AS at pH 8.00, 25.0 °C……………………………………………………………97

Figure 4.16. Determination of stoichiometry of the reaction between Cbl(I)– and AS at pH 7.00…………………………………………………………………………………...97

Figure 4.17. Determination of stoichiometry of the reaction between Cbl(I)– and AS at pH 8.00…………………………………………………………………………………...99

Figure 4.18. UV–vis spectrum of the reaction of Cbl(I)– with 1.0 equiv AS in the – presence of excess NO2 at pH 8.00, 25.0 °C……………………………………..……101

CHAPTER 5: KINETIC AND MECHANISTIC STUDIES ON THE REACTION OF AQUACOBALAMIN WITH THE HNO DONOR ANGELI’S SALT

Figure 5.1. UV–vis spectra for the reaction between Cbl(III) and excess AS at pH 9.80, 25.0 °C………………………………………………………………………………….107

Figure 5.2. Plot of absorbance versus time for the reaction between Cbl(III) and excess AS at pH 9.80, 25.0 °C…………………………………………………………………108

Figure 5.3. Plot of kobs versus AS concentration for the reaction of Cbl(III) with excess AS at pH 9.80, 25.0 °C………...……………………………………………………….109

+ Figure 5.4. UV–vis spectra for the reaction between AS and excess H2OCbl as a function of time at pH 5.00, 25.0 °C……………………………………………………112

+ Figure 5.5. Plot of kobs versus the concentration of H2OCbl at pH 5.00, 25.0 °C…….113

+ Figure 5.6. Plot of kobs versus concentration of H2OCbl for the reaction between + H2OCbl and AS at pH 4.15, 6.00, 7.05, 8.00, 9.05, 9.40 and 9.90…………………….114

+ Figure 5.7. Plot of kapp versus pH for the reaction between H2OCbl and AS…………115

+ Figure 5.8. Determination of stoichiometry of the reaction between H2OCbl and AS at pH 6.00………………………………………………………………………………….115

Figure 5.9. Aromatic region of 1H NMR spectrum of the Cbl product for the reaction between HOCbl and 1.10 mol equiv AS at pD 9.86, 24 °C…………………………….116

+ Figure 5.10. Plot of kapp versus pH for the reaction between H2OCbl and AS………..119

Figure 5.11. Plot of absorbance versus time for the reaction of HOCbl with excess AS at pH 10.80, 25.0 °C………………………………………………………………………121

Figure 5.12. Plot of absorbance versus time for the reaction of HOCbl with 1.0 mol equiv of AS at pH 10.80, 25.0 °C……………………………………………………………..123

Figure 5.13. Absorbance versus time for the reaction between excess HOCbl and AS at pH 10.80, 25.0 °C………………………………………………………………………124

Figure 5.14. Aromatic region of the 1H NMR spectrum of the product of the reaction between HOCbl and 1.1 mol equiv AS at pD 10.82, 24 °C…………………………….125

CHAPTER 6: MECHANISTIC STUDIES ON THE REACTION OF NITROXYLCOBALAMIN WITH DIOXYGEN

Figure 6.1. UV–vis spectra for the reaction between NOCbl and excess O2 at pH 7.40, 25.0 °C………………………………………………………………………………….136

Figure 6.2. Aromatic region of the 1H NMR spectrum of a solution of NOCbl exposed to air, pD 7.40, 25.0 °C……………………………………………………………………137

Figure 6.3. HPLC chromatogram for the products of the reaction between NOCbl and O2 (air) at pH 7.40, 25.0 °C………………………………………………………………...138

Figure 6.4. HPLC chromatogram for an authentic sample of NO2Cbl………………...139

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Figure 6.5. Plot of absorbance versus time for the reaction of excess NOCbl with O2 at pH 7.40, 25.0 °C………………………………………………………………………..140

Figure 6.6. Plot of kobs versus [NOCbl] for the reaction of NOCbl with O2 at pH 7.40, 25.0 °C………...………………………………………………………………………..141

Figure 6.7. Plot of kobs versus [NOCbl] for the reaction between excess NOCbl and O2 at pH 10.40, 9.00, 8.00, 6.00, 5.55, 5.00 and 4.10………………………………………...142

Figure 6.8. Plot of kapp versus pH for the reaction between NOCbl and O2…………...143

Figure 6.9. Eyring plot for the reaction of NOCbl with O2 at pH 7.40………………...144

Figure 6.10. UV–vis spectra for equilibrated solutions of NOCbl with varying mol equiv of O2 at pH 7.40………………………………………………………………………...145

Figure 6.11. Aromatic region of the 1H NMR spectrum of the products of the reaction between NOCbl and 0.5 mol equiv O2 at pD 7.40, 24 °C……………………………...146

Figure 6.12. HPLC chromatograms of nitrate, cobalamin standards and the product mixture of NOCbl with O2……………………………………………………………...148

Figure 6.13. HPLC chromatogram of the products of the reaction between NOCbl and O2 (air) at pH 7.40 treated with excess KCN………………………………………………149

Figure 6.14. ESI–MS analysis of an authentic sample of CNCbl and HPLC fractions of the product mixture of the reaction between NOCbl and O2 (air) at pH 7.40 subsequently treated with KCN……………………………………………………………………….151

Figure 6.15. HPLC chromatograms for Cbl standards, Tyr standards, Phenol standards and for the separation of the unknown corrinoid product of the reaction of NOCbl with

O2………………………………….……………………………………………………152

Figure 6.16. HPLC chromatogram for hydroxylated and nitrated phenol standards…..153

Figure 6.17. HPLC chromatogram for the products of the reaction between NOCbl and

O2 (air) in the presence and absence of excess phenol…….…………………………...154

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Figure 6.18. HPLC chromatogram for the authentic mixture of Tyr (6.00 mM), OH–Tyr

(0.18 mM) and NO2–Tyr (0.18 mM)…………………………………………………...155

Figure 6.19. HPLC chromatogram for the products of the reaction of NOCbl with air in the presence and absence of excess Tyr………………………………………………...156

Figure 6.20. ESI–MS analysis of the unknown corrinoid compound eluted from HPLC experiments……………………………………………………………………………..158

Figure 6.21. Aromatic region of the 1H NMR spectrum of a solution of NOCbl exposed to air (pD 7.40) in the absence of light, 24 °C………………………………………….163

Figure 6.22. Plot of absorbance versus time for the spontaneous decomposition of ONOO(H) at pH 10.40, 25.0 °C………………………………………………………..164

CHAPTER 7: SUMMARY AND FUTURE DIRECTIONS

Figure 7.1. The structure of diaquacobinamide………………………………………..171

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LIST OF SCHEMES

CHAPTER 1: INTRODUCTION AND BACKGROUND

Scheme 1.1. General method for the reduction of Cbl(III) to Cbl(II) and Cbl(I)–………..4

Scheme 1.2. Base–on and base–off forms of cobalamins in aqueous solution…………...5

Scheme 1.3. The enzymatic reaction catalyzed by methylmalonyl–coenzyme A mutase..6

Scheme 1.4. Catalytic cycle of methionine synthase……………………………………..7

Scheme 1.5. Schematic representation for the mechanism of enzymatic generation of nitric oxide by nitric oxide synthases…………………………………………………….15

Scheme 1.6. Mechanism of spontaneous decomposition of Angeli’s salt………………18

Scheme 1.7. Mechanism of base–catalyzed decomposition of Piloty’s acid……………19

CHAPTER 2: MECHANISTIC STUDIES ON THE REACTIONS OF REDUCED

VITAMIN B12 COMPLEXES WITH THE NITROXYL DONOR PILOTY’S ACID

Scheme 2.1. Proposed reaction pathway for the reaction of Cbl(II) with PA…………...42

Scheme 2.2. Proposed mechanism for the reaction between Cbl(II) and 1.0 mol equiv PA – in the presence of excess NO2 ……………………………………………………...…...49

CHAPTER 3: KINETIC STUDIES ON THE REACTION OF AQUACOBALAMIN WITH THE HNO DONOR PILOTY’S ACID

Scheme 3.1. Proposed mechanism for the reaction between Cbl(III) and PA at pH ≥ 10.00………………………………………………………………………………..…….66

xix

CHAPTER 4: STUDIES ON THE REACTION OF THE REDUCED VITAMIN B12 COMPLEX COB(II)ALAMIN WITH THE HNO DONOR ANGELI’S SALT

Scheme 4.1. Proposed reaction pathway for the reaction of Cbl(II) with AS at pH ≤ 9.00...... 89

Scheme 4.2. Proposed reaction pathway for the reaction of Cbl(I)– with AS at pH ≤ 9.00……………………………………………………………………………………...100

CHAPTER 5: KINETIC AND MECHANISTIC STUDIES ON THE REACTION OF AQUACOBALAMIN WITH THE HNO DONOR ANGELI’S SALT

+ Scheme 5.1. Proposed mechanism for the reaction of H2OCbl /HOCbl with AS at pH ≤ 9.90……………………………………………………………………………………...118

+ Scheme 5.2. Possible reaction scheme for the reaction of H2OCbl /HOCbl with AS – 2– assuming that both protonated (HN2O3 ) and deprotonated (N2O3 ) form of AS react + with H2OCbl …………………………………………………………………………...121

+ Scheme 5.3. Proposed major mechanism for the reaction of H2OCbl /HOCbl with AS at pH ≥ 10.80……………………………………………………………………………...126

CHAPTER 6: MECHANISTIC STUDIES ON THE REACTION OF NITROXYLCOBALAMIN WITH DIOXYGEN

Scheme 6.1. Decomposition pathways for ONOOH and ONOO–……………………..159

Scheme 6.2. Proposed reaction pathway for formation of nitrocobalamin product from the peroxynitritocobalt(III) intermediate……………………………………………….161

Scheme 6.3. Proposed reaction pathway for the reaction between NOCbl and O2…….166

LIST OF TABLES

CHAPTER 1: INTRODUCTION AND BACKGROUND

Table 1.1. UV–vis spectroscopic data for various cobalamin derivatives………………11

Table 1.2. 1H NMR chemical shifts in the aromatic region for several cobalamin complexes………………………………………………………………………………..12

CHAPTER 2: MECHANISTIC STUDIES ON THE REACTIONS OF REDUCED

VITAMIN B12 COMPLEXES WITH NITROXYL DONOR PILOTY’S ACID

Table 2.1. Comparison of the observed rate constants for the spontaneous decomposition of PA (kL) and the reaction of Cbl(II) with 1.0 mol equiv of PA (kobs) as a function of pH……………………………………………………………………………………...... 37

CHAPTER 4: STUDIES ON THE REACTION OF THE REDUCED VITAMIN B12 COMPLEX COB(II)ALAMIN WITH THE HNO DONOR ANGELI’S SALT

Table 4.1. Comparison of the observed rate constants for the reaction between Cbl(II) and AS (kobs) and the spontaneous decomposition of AS (kL) as a function of pH...... 84

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DEDICATION

To My Family

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ACKNOWLEDGEMENTS

First and foremost, I would like to extend my sincere gratitude to my research advisor Professor Nicola E. Brasch for her continuous guidance, supervision and corroboration. Her tremendous support continuously encouraged me to deal with various challenges in the completion of all research projects. None of my work would have been possible without her kind help and feedback. Thank you very much, Dr. Brasch!

I am thankful to all the members of my PhD committee – Dr. Scott D. Bunge, Dr.

Jacob T. Shelley, Dr. Darren L. Bade and previous committee member Dr. Mietek

Jaroniec for their continuous support. I would also like to thank Dr. Jennifer A.

McDonough for being the Graduate Faculty Representative for my oral defense.

I really appreciate the help and cooperation from the current Brasch lab members

Rohan Dassanayake, Yang (Joe) Zhou, Anthony Giovengo, Sonya Adas, Vivian Hogan,

Mohammad Rahman, Mary Waddington and Matthew Brining. Special thanks to Sonya for her assistance with proofreading this dissertation. I am also thankful to previous

Brasch lab members, Dr. Riya Mukherjee, Dr. Hanaa Hassanin, Dr. Edward Suarez–

Moreira, Noah Plymale, Alexandria Lesak, Jeffrey Brown, David Walker, Andrew Hunt and Jermey Roughton.

I would like to acknowledge the Kent State University’s Department of Chemistry and Biochemistry for providing the research facility and U. S. National Science

Foundation (Grants: CHE–0848397 and CHE–1306644) for the research funding. I am grateful to all the faculties and staff members of the KSU Chemistry Department,

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especially, staff members Erin Michael, Janie Viers, Arla Dee McPherson, Catherine

Taylor and Larry Maurer for their immense help and cooperation. I would also like to thank Dr. Mahinda Gangoda (Kent State University) for his assistance with mass spectrometry experiments and Dr. Pierre Moënne–Loccoz (Oregon Health & Science

University) for helpful discussions and his assistance with Raman spectroscopy measurements. Last but not least I offer my gratitude to the friends I have made from the

Kent Nepalese community, and their families, for creating a such a positive and enjoyable environment for me and my family during our time in Kent.

I am deeply indebted to all my family members specially my parents for their unconditional love and support. I would like to thank my wife Sandhya Poudel Subedi for being my strength all the time. Finally, I thank my lovely daughter Riju Subedi whose single smile worked as the strongest motivation in my work.

Harishchandra Subedi

Friday; June 6th, 2014

Kent, Ohio, USA

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ABSTRACT

Nitric oxide (NO, ●NO), the ‘1992 Molecule of the Year’, is a gaseous messenger molecule produced in cells from L–arginine by nitric oxide synthases. The chemical and biological properties of NO have been extensively studied for well over a decade.

However, recent studies have shown that nitroxyl (nitrosyl hydride, HNO), the one– electron–reduction product of NO, may also be formed in biological systems, from nitric oxide synthase–catalyzed oxidation of L–arginine in the absence of tetrahydrobiopterin and by enzyme catalyzed reduction of NO. Much less is known about the reactivity of

HNO, including its reactivity with transition metal complexes. A fundamental understanding of this is important, given that about one third of proteins are metalloproteins.

In this dissertation we present detailed kinetic and mechanistic studies of the reactions of two well characterized HNO donors, Angeli’s salt (AS) and Piloty’s acid

(PA), with cobalamins (vitamin B12 derivatives). Cobalamins have important role as cofactors in two B12–dependent enzymatic reactions in humans. Furthermore, cobalamins have three readily accessible oxidation states. Chapter 2 of this dissertation is concerned with studies on the reactions of reduced vitamin B12 complexes, cob(II)alamin (Cbl(II)) and cob(I)alamin (Cbl(I)–), with the HNO donor PA. Cob(II)alamin is a major intracellular form of B12 and cob(I)alamin is a short–lived precursor of the two B12 coenzyme forms methylcobalamin and adenosylcobalamin. Although it is well established that HNO reduces transition metals including transition metal centers of

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porphyrins and metalloproteins, oxidation of a transition metal center by HNO has yet to be reported. Kinetic studies on the reaction of Cbl(II) with PA show that PA

– decomposition to give HNO (and C6H5SO2 ) is the rate–determining step, the reaction

– stoichiometry is 1:2 Cbl(II):PA, and nitroxylcobalamin (NOCbl), N2 and C6H5SO2 being formed. A mechanism is proposed in which reduction of Cbl(II) by HNO results in formation of cob(I)alamin (Cbl(I)–) and NO. Cob(I)alamin intermediate is subsequently oxidized back to Cbl(II) by a second HNO molecule, and Cbl(II) reacts rapidly with NO to form nitroxylcobalamin, NOCbl.

Chapter 3 involves kinetic studies on the reaction of the oxidized form of

+ cobalamin (Cbl(III), aquacobalamin/hydroxocobalamin; pKa(H2OCbl = 7.8)) with PA.

Under alkaline conditions (pH ≥ 10.00), the rate–determining–step is decomposition of

3 – deprotonated PA to give NO (pKa(HNO) = 11.4). The hydroxo ligand of hydroxocobalamin is inert to substitution. It is therefore likely that 3NO– (and also possibly HNO) reduce Cbl(III) to Cbl(II), being oxidized to NO. Cbl(II) and NO rapidly combine to give the observed NOCbl product. At lower pH conditions (< pH 10), PA instead reacts directly with Cbl(III) to give NOCbl. The mechanism of this latter reaction is unclear.

Chapters 4 and 5 are focused on studies of the reaction between Angeli’s salt and

Cbl(II) and Cbl(III), respectively. The reaction of Cbl(II) with AS occurs via a Cbl(I)– intermediate with AS decomposition being the rate–determining–step. The reaction stoichiometry is 1:2 Cbl(II):AS and NOCbl, nitrite and N2 are the reaction products. As for the Cbl(II)/PA system, HNO reduces the Cbl(II) to Cbl(I)– and a second HNO

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molecule oxidize Cbl(I)– back to Cbl(II), followed by the combination of NO and Cbl(II) to give NOCbl. The reaction of AS with Cbl(III), however, undergoes via different mechanisms depending upon the pH conditions. At pH < 9.90 aquacobalamin reacts

– directly with the monoprotonated form of Angeli’s salt, HN2O3 , to form NOCbl and nitrite. At pH > 10.80 the reaction instead switches predominantly to a mechanism in which spontaneous decomposition of AS to give HNO and nitrite becomes the rate– determining step, followed by the rapid reaction between aquacobalamin and HNO/NO– to again give NOCbl. Both reactions proceed with a 1:1 stoichiometry.

NOCbl is formed as the cobalamin product in all of the above reactions.

Furthermore it has been proposed that NOCbl is formed in vivo. The final chapter of this thesis, Chapter 6, is concerned with the mechanistic studies on the reaction of extremely air–sensitive cobalamin complex, NOCbl, with dioxygen. Only base–on NOCbl reacts with O2 and the reaction proceeds via an associative mechanism involving a peroxynitritocob(III)alamin intermediate, Co(III)–N(O)OO–. The intermediate undergoes

O–O bond homolysis and ligand isomerization to ultimately yield NO2Cbl and

+ H2OCbl /HOCbl, respectively. Ligand isomerization may potentially occur independent

● ● of O–O bond homolysis. Formation of OH and NO2 intermediates from O–O bond homolysis is demonstrated using phenol and tyrosine radical traps and the characterization of small amounts of a corrinoid product with minor modifications to the corrin ring.

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CHAPTER 1

INTRODUCTION AND BACKGROUND

1.1 VITAMIN B12 (COBALAMINS)

1.1.1 DISCOVERY OF VITAMIN B12

The discovery that liver contains a compound (vitamin B12) that treats pernicious anemia was serendipitous [1]. In 1920, George Whipple, an American pathologist and biomedical researcher, was carrying out experiments on anemia–induced dogs. He discovered that a diet containing liver cured anemia [1, 2]. In 1926, George Minot and

William Murphy showed that patients with pernicious anemia experienced remarkable improvements in their health when they adopted the ‘raw liver diet’. In 1934, Whipple,

Minot and Murphy were awarded the Nobel Prize in Physiology and Medicine for the discovery of the ‘liver factor’ or the ‘anti–pernicious anemia factor’ [1, 2]. In 1948,

Folkers and his colleagues isolated a red crystalline compound from liver and named the compound ‘vitamin B12’ [3]. The chemical structure of the vitamin B12 was determined by Dorothy Crowfoot Hodgkin and her coworkers in 1956 using X–ray crystallography

[4]. Professor Crowfoot Hodgkin was awarded the Nobel Prize in Chemistry in 1964 for her outstanding contributions to the field of X–ray crystallography.

1 2

1.1.2 STRUCTURE OF VITAMIN B12

Vitamin B12 derivatives, also known as cobalamins (Cbls) or simply B12, are a class of redox active cobalt containing macrocyclic complexes. They belong to the corrinoid family and are synthesized by a number of microorganisms including bacteria and archaea [5]. B12 is the only vitamin which contains a metal and is one of eight B vitamins. Cobalamins are water–soluble and have a central cobalt atom coordinated by four equatorial pyrroline rings of the corrin ring (Figure 1.1). The corrin ring is structurally similar to other tetrapyrrolic macrocycles such as corroles and porphyrins [6].

In cob(III)alamins the fifth lower coordination site of the octahedral Co(III) center, also known as α– axial site, is occupied by a 5,6–dimethylbenzimidazole (DMB) base whereas the sixth coordination site, also known as β– or upper axial site, can be occupied by a wide range of ligands (Figure 1.1). Cyanocobalamin (‘vitamin B12’, CNCbl, X =

– CN ) is by far the most common pharmaceutical form of vitamin B12 and is widely used in vitamin supplements [7]. CNCbl is not naturally occurring, although it is found in the serum of smokers [8, 9]. Cob(III)alamins that occur in biological systems include methylcobalamin (MeCbl), adenosylcobalamin (AdoCbl) and

+ + aquacobalamin/hydroxocobalamin (H2OCbl /HOCbl; pKa(H2OCbl ) = 7.8 [10, 11]).

MeCbl and AdoCbl are the predominant Cbl forms found in foods [12].

Finally, numerous studies have been carried out on the kinetics of β–axial ligand

+ substitution of the aqua ligand of H2OCbl by a wide variety of ligands [13–16]. These substitution reactions occur via a dissociative interchange mechanism [13, 15] and are

very rapid compared with other Co(III) complexes including Co(III) porphyrins due to the cis effect of the corrin macrocycle [14, 15].

Figure 1.1. The general structure of vitamin B12 derivatives (cob(III)alamins, Cbl(III); X – – – – = CN , CH3, 5'–deoxy–5'–adenosyl (Ado), H2O, OH , NO , NO2 , etc.). The β–axial ligand X is cleaved upon reduction of Cbl(III) to penta–coordinate cob(II)alamin (Cbl(II)). The α–axial bond to the DMB is cleaved upon reduction of Cbl(II) to 4– coordinate cob(I)alamin (Cbl(I)–). Five protons labeled as B2, B4, B7, R1 and C10 resonate in the aromatic region of the 1H NMR spectrum. The figure on the right hand side shows the simplified representation of the Cbl(III).

1.1.3 OTHER OXIDATION STATES OF COBALAMINS

Cobalamins can exist in three oxidation states with respect to the central cobalt atom: cob(I)alamin (Cbl(I)–, Co+), cob(II)alamin (Cbl(II), Co2+), and cob(III)alamin

(Cbl(III), Co3+) [17–19]. All three forms of cobalamins exist in cells, Cbl(II) being the major intracellular form. Upon entering cells, Cbl(III)s are reduced to Cbl(II) by intracellular reductases [20, 21]. Cbl(III) can be chemically converted to Cbl(II) (Co(II),

 – Cbl(II) or B12r) and super–reduced cobalamin (Cbl(I) or B12s) by the addition of reducing agents under anaerobic conditions, Scheme 1.1 [22, 23]. The Cbl reduction

– process is thermodymically favorable (E°Cbl(III)/Cbl(II) = +0.23 V and E°Cbl(II)/Cbl(I) = –0.61

V versus SHE) [24–27]. Cbl(II) lacks the β–axial ligand and is therefore pentacoordinate, whereas tetracoordinate Cbl(I)– is devoid of both axial ligands. Both Cbl(II) and Cbl(I)– are very reactive and extremely air–sensitive cobalamin species.

OH2 Co(II) 1.2 equiv NaBH4 6.0 equiv NaBH4 Co(I) Co(III) N N N Aquacobalamin Cob(II)alamin Cob(I)alamin (Cbl(III)) (Cbl(II)) (Cbl(I)-)

Scheme 1.1. One of several methods for the synthesis of Cbl(II) and Cbl(I)–. Anaerobic conditions are required.

In aqueous solution, three forms of cob(III)alamins exist in equilibrium: a “base– on” form (the 5,6–dimethylbenzimidazole (DMB) imine N is bound at the –axial site) and two “base–off” forms (a solvent molecule is bound to the –axial site and the DMB imine N is either deprotonated or protonated); Scheme 1.2 [28, 29].

X X X pK base-off KCo Co Co Co + H+ + H O + 2 NH OH2 N OH2 N base-off base-off base-on DMB protonated DMB deprotonated

Scheme 1.2. Base–on and base–off forms of cobalamins in aqueous solution.

1.1.4 BIOCHEMISTRY OF VITAMIN B12

Vitamin B12 is essential for every cell type in mammals. Higher organisms including humans do not synthesize vitamin B12 naturally and therefore it must be obtained from dietary sources [30, 31] such as meat, seafood and dairy products. Normal adults should intake 2–5 µg/day [32]. Liver and kidney are the major storage sites for dietary vitamin B12 in mammals [33, 34]. Since two B12 forms, AdoCbl and MeCbl, serve as important cofactors for two B12–dependent enzymatic reactions in humans, cobalamin deficiency is associated with several conditions including pernicious anemia, cardiovascular diseases and neurological disorders including Alzheimer’s disease and Parkinson’s disease [35, 36]. It has been estimated that 10–40% of the US population over the age of 65 are B12– deficient [37–40].

1.1.4.1 B12–DEPENDENT ENZYMATIC REACTIONS

Cobalamins are important coenzymes in all organisms. Two B12–dependent enzyme reactions occur in humans which require AdoCbl or MeCbl as cofactors [41]. In mitochondria, methylmalonyl–coenzyme A mutase (MM–CoA mutase) utilizes AdoCbl as a cofactor to catalyze a carbon skeleton rearrangement resulting in the conversion of

methylmalonyl–CoA to its isomer succinyl–CoA (Scheme 1.3), an important substrate in

the extraction of energy from proteins and fats (citric acid cycle) [31]. This process is

compromised in B12–deficient patients and leads to elevated levels of methylmalonic acid

(MMA) in blood plasma [30, 42].

Cytosolic methionine synthase is a methyl transfer enzyme, and uses its MeCbl

cofactor to catalyze conversion of the amino acid homocysteine (Hcy) to the amino acid

methionine (Met) (Reaction 1, Scheme 1.4) [21]. During the catalytic process, MeCbl is

reduced to Cbl(I)– upon the removal of its Me group by Hcy. As part of the overall

- CO2 H H C C H

HO OH HO OH H C O - - CO2 CO2 H H HO OH O Ade O Ade SCoA H C H H C C H H C H H C C H O Ade Co(II) C O H C O H C H

HO OH N Co(II) SCoA SCoA H Co(III) N O Ade N H C H

Co(III) HO OH HO - OH CO2 H N O Ade O Ade H C H - H C C H CO2 H C H H C O Co(II) H C C H H

SCoA Co(II) N H C O

N SCoA

Scheme 1.3. The enzymatic reaction catalyzed by methylmalonyl–coenzyme A mutase. Ade = adenine.

– reaction, Cbl(I) then extracts the methyl group of methyltetrahydrofolate (CH3H4–folate) to form tetrahydrofolate (H4folate) and MeCbl to continue the cycle (Reaction 2, Scheme

1.4). This catalytic reaction plays a key role in the regulation of plasma Hcy levels. High

Hcy is an independent risk factor for cardiovascular disease [36, 43] and neurological disorders including Alzheimer’s disease and vascular dementia [44–46]. Under microaerophilic conditions, cob(I)alamin is oxidized and converted to inactive cob(II)alamin, deactivating the enzyme [31]. The enzyme is reactivated when Cbl(II) is reduced back to Cbl(I)– by flavodoxin. Cbl(I)– is methylated by S–adenosyl–L– methionine (SAM) to regenerate MeCbl in the catalytic cycle whereas SAM is converted to S–adenosyl–homocysteine (SAH); Reaction 3, Scheme 1.4 [31]. An adequate amount of Met is required to maintain a balanced SAM:SAH ratio because SAM is the principal

Scheme 1.4. Catalytic cycle of methionine synthase.

physiological methyl donor and plays an important role in DNA methylation [47].

1.1.4.2 CELLULAR UPTAKE AND METABOLISM OF COBALAMINS

There are three primary carrier proteins which are responsible for the absorption, transportation and cellular uptake of B12: haptocorrin (HC), intrinsic factor (IF) and transcobalamin (TC). Cobalamins are tightly bound to these proteins, forming B12– protein complexes [48, 49]. The pathway for the absorption and cellular transport is summarized in Figure 1.2 [31]. The dietary cobalamin is first bound to the HC present in saliva. HC takes the B12 to stomach and where B12 interacts with gastric HC. The combined effect of acidic pH and the proteolytic activity of pepsin causes the release of

60–70% of corrinoids in food [50, 51]. These corrinoids interact mainly with HC despite high concentration of another B12–binding protein, IF [50]. This is because the interaction of IF with cobalamins is pH–sensitive so B12 only binds in the intestine after the digesting material is neutralized [52]. Pancreatic enzymes help liberate the free dietary B12 in addition to cleaving HC [52]. The affinity of HC for B12 decreases after cleavage and the majority of B12 is transferred to IF to form a B12–IF complex [52]. This complex is recognized by cubilin receptor whose function is coupled with amnionless and the B12–IF complex is absorbed in the terminal ileum [53]. After the receptor–mediated absorption of B12–IF complex into the lysosomes of intestinal absorptive cells (enterocytes), the IF is degraded and the free Cbl binds to the third B12–binding protein, TC. The B12–TC complex is then released into blood plasma, taken up by the extracellular TC receptor by endocytosis and degraded in the lysosomes to release B12 [54]. B12 is metabolized further to give the two cofactor forms: MeCbl (cytosolic) and AdoCbl (mitochondrial) [55].

Figure 1.2. Absorption, transport and cellular uptake of cobalamins in mammals. The schematic illustrates the formation and function of the two cofactors, AdoCbl and MeCbl within the cell. The homolytic and heterolytic cleavage of the Co–C bond in AdoCbl and MeCbl enzymes, respectively are evidenced. The corresponding arrows indicate where the cleavage occurs: homolysis into the mitochondrion and heterolysis into the cytoplasm. [Randaccio et al. Molecules 2010, 15, 3228]

1.1.5 CHARACTERIZATION METHODS FOR COBALAMINS

Various techniques have been utilized to characterize cobalamins in solid state and solution phase.

1.1.5.1 X–RAY CRYSTALLOGRAPHY

The crystal structure of cyanocobalamin was first determined by Dorothy

Hodgkin and her coworkers in 1956 [4]. Her studies revealed the presence of a tetrapyrrole corrin ring in the complex macrocycle with a cyanide ion occupying the

β−axial position. Professor Hodgkin’s breakthrough opened doors for characterization of complex cobalamin derivatives in the solid state using X–ray crystallography. The elucidation of the structure of AdoCbl by Lenhert and Hodgkin in 1961 [56] using X–ray crystallography showed evidence for the existence of the first organometallic bond in a naturally–occurring compound. Later, high–resolution neutron and X–ray diffraction data were obtained for AdoCbl to elucidate the organization of water molecules associated with AdoCbl crystals (C72H100CoN18O17P•17H2O) [57]. The crystal structure of the second coenzyme form of B12, MeCbl was determined in 1985 [58]. X–ray crystal structure of reduced B12 form, Cbl(II), has also been solved [59–61] confirming the pentacoordinate geometry. Owing to the oxygen–sensitivity of Cbl(II), the crystals were grown directly in X–ray capillaries and the structure was determined at very low temperature (–82 °C) [59].

1.1.5.2 UV–VIS SPECTROSCOPY

Since the corrin ring consists of highly conjugated double bonds, cobalamins are intensely colored compounds with high extinction coefficients and absorb strongly in the

UV–vis region of the spectrum. Interestingly, the valency of the cobalt atom, nature of the substituent groups in corrin ring and change in axial ligands profoundly affect the electronic properties of the B12 derivatives thus the absorption pattern of these

compounds. UV–vis spectroscopy is, therefore, an important characterization technique for B12 derivatives. The strong absorption of cobalamin derivatives observed > 300 nm is attributed to the spin–allowed π–π* transitions within the conjugated corrin ring. The intensity of the strong band in 350–370 nm region (γ–band) depends on the nature of the

β–axial ligand, X (see Figure 1.1 and Table 1.1). The DMB moiety present in the cobalamins absorbs in the 260–300 nm region [62]. Charge transfer bands (axial ligand to cobalt center; LMCT) have been assigned in the case of cobalamins with the axial ligands

– 2– – such as phenolato–, I , S2O3 and RS [62]. Spin–allowed d–d transitions are expected due to the presence of five N atoms coordinating to cobalt center, however, no direct evidence of the position of d–d transitions has been reported [62]. Table 1.1 summarizes the UV–vis spectroscopic data for various cobalamins [10, 63–66].

Table 1.1. UV–vis spectroscopic data for various cobalamin derivatives in aqueous solutions [10, 63–66].

Cobalamin λmax/nm + H2OCbl 349 411 525 CNCbl 278 362 548

MeCbl 340 377 528 AdoCbl 315 340 375 522 NO2Cbl 354 413 532 – SO3Cbl 312 365 418 517 GSCbl 333 372 428 534 HcyCbl 333 372 428 534 NOCbl 289 315 478

Cbl(II) 312 405 475 Cbl(I)– 388 464 548

1.1.5.3 1H NMR SPECTROSCOPY

1H NMR spectroscopy is another important technique to characterize the cobalamins in solution. Despite being a large molecule, cobalamins have only 5 protons resonating in aromatic region (5–8 ppm) and chemical shifts for these protons are strongly dependent on the β–axial ligand, X (see Figure 1.1 and Table 1.2) [10, 67, 68].

The five peaks arise due to the resonances from three DMB nucleotide protons (B2, B4,

B7), one corrin ring proton (C10) and a ribose proton (R1); Figure 1.1. Cob(II)alamin, the major intracellular form of B12, is a paramagnetic species.

Table 1.2. 1H NMR chemical shifts in the aromatic region for several cobalamin + complexes in D2O (H2OCbl , MeCbl, AdoCbl and CNCbl), 0.10 M MES buffer; pD 6.0 – (NO2Cbl, SO3Cbl , GSCbl and HcyCbl) or 0.10 M phosphate buffer; pD 7.40 (NOCbl) [10, 67, 68].

1H NMR chemical shifts

(ppm) Cobalamin B7 B2 B4 R1 C10 + H2OCbl 7.18 6.54 6.47 6.26 6.30 NO2Cbl 7.20 6.74 6.42 6.28 6.20 NOCbl 7.19 7.44 6.77 6.26 6.34 – SO3Cbl 7.17 6.94 6.43 6.25 6.02 MeCbl 7.18 6.97 6.28 6.27 5.91 AdoCbl 7.16 6.95 6.24 6.26 5.93 CNCbl 7.28 7.10 6.51 6.36 6.09

GSCbl 7.19 6.95 6.39 6.28 6.09 HcyCbl 7.20 6.95 6.38 6.28 6.10

1.1.5.4 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

HPLC is an important technique for identifying and separating various cobalamins in solution. Research labs, including our own, have developed various HPLC methods to characterize different cobalamin species [23, 29, 69–73].

1.1.5.5 OTHER CHARACTERIZATION TECHNIQUES

Other B12 characterization methods include mass spectrometry [10, 74, 75],

Fourier–transform infrared spectroscopy [76] and X–ray atomic absorption spectroscopy

[10].

1.1.6 NON–COFACTOR FUNCTIONS OF VITAMIN B12

Cobalamins help maintain the balance in the folate pool by converting CH3H4– folate into H4–folate. Accumulation of CH3H4–folate occurs with B12 deficiency which prevents DNA methylation leading to abnormal DNA synthesis in B12–deficient marrow

[77, 78]. Cobalamins also have important roles in the central nervous system (CNS). B12 deficiency may result in neuropathological problems due to demyelination in the spinal cord and peripheral nerves, leading to nerve degeneration and irreversible neurological damage [79, 80]. Cobalamins also regulate the expression of various cytokines and growth factors including neurotoxic tumor necrosis factor–α (TNF–α) and neurotrophic epidermal growth factor (EGF) [81]. Cbls also suppress the production of activated nuclear factor–kappa B (NF–κB), an inducible transcription factor [82].

Administration of cobalamin supplement has shown to be beneficial for treating various chronic inflammatory diseases including sepsis, asthma, autism, Alzheimer’s disease, autoimmune diseases, multiple sclerosis and stroke [8, 83–89].

1.2 REACTIVE OXYGEN / NITROGEN SPECIES AND OXIDATIVE STRESS

Reactive oxygen and nitrogen species (collectively designated as RNOS) are reactive free radical species containing oxygen and nitrogen atoms including nitric oxide

  –   ( NO, NO), superoxide ( O2 ), hydroxyl ( OH), nitrogen dioxide ( NO2) and other strong oxidants such as nitroxyl (HNO/NO–), peroxynitrite (ONOO–) and hydrogen peroxide

(H2O2). RNOS are generated by immune cells to destroy pathogens and tumors [90]. In addition, RNOS play other important roles in biological systems including regulating cell growth, cell signaling and programmed cell death [90]. Since RNOS are such potent molecules, high levels of RNOS can irreversibly damage biomolecules including lipids, proteins and nucleic acids, causing oxidative/nitrosative stress [91]. Oxidative/nitrosative stress is a state that arises from the imbalance between the production of RNOS and the ability of a biological system to degrade these species or repair the resulting damage [91].

1.2.1 NITRIC OXIDE

Nitric oxide (NO, NO), the ‘1992 Molecule of the Year’ [92]), is produced in cells by family of enzymes called nitric oxide synthases (NOS) [93]. It is produced as a result of the five–electron–oxidation of L–arginine to citruline in the presence of NADPH and oxygen (Scheme 1.5). Other essential cofactors, viz. flavin mononucleotide (FMN),

flavin adenine dinucleotide (FAD), heme, calmodulin and tetrahydrobiopterin, are involved to facilitate electron transfer during the production of NO [94].

H N NH H N N H2N O 2 2 OH

NH NH NH NADPH NADP+ 1/2 NADPH 1/2 NADP+ + NO

O2 H2O O2 H2O

H2N COOH H2N COOH H2N COOH L-arginine N-hydroxy-L-arginine L-citruline (NOHA)

Scheme 1.5. Schematic representation for the mechanism of enzymatic generation of nitric oxide by nitric oxide synthases.

Nitric oxide is involved in many physiological and pathological processes in mammals. As a neurotransmitter, it is involved in memory and learning processes [95,

96], whereas in signaling it controls vascular tone, cell proliferation, and cell survival

[97]. It also plays a key role in the initiation of pro–inflammatory immune response [98].

Low NO bioavailability is associated with endothelial cell dysfunction and cardiovascular disease [99]. While elevated NO levels damage tissues and excessive vasodilation leads to septic shock [100], high NO concentrations are also associated with numerous inflammatory diseases including cancer, diabetes, neurodegenerative diseases and arthritis [101–103].

 NO reacts with oxygen to form highly toxic NO2 radical which is associated with lung disease [104]. NO forms another potent ROS peroxynitrite (ONOO–) when it reacts

– – with superoxide radical [105]. In addition, NO is readily oxidized to NO2 and/or NO3 in biological systems [106].

1.2.2 NITROXYL (HNO/NO–)

Nitroxyl, also known as nitrosyl hydride [107], hydrogen oxonitrate [107, 108], hydridooxidonitrogen [109] or azanone [109], is the one–electron reduced and protonated sibling of NO. The pKa of HNO was originally reported as 4.7 by pulse radiolysis [110], suggesting that the deprotonated form (NO–) would be the predominant species under physiological conditions. However, this study did not consider the spin states of HNO and NO–, and later, experimental and theoretical studies by Shafirovich and Lymer [111] suggested that HNO had a much weaker acidity than previously believed and the pKa value was revised to be ~ 11.4 [111]. Therefore, HNO exists solely at physiological pH.

The deprotonated form of HNO, NO–, is isoelectronic with dioxygen [112]. The ground state of HNO is a singlet state (1HNO) whereas that of NO– is a triplet state (3NO–) [111].

Importantly, HNO has unique chemical and biological properties compared to

NO. For example, HNO directly targets thiols whereas NO does not [113, 114].

 –   – Similarly, HNO does not react with superoxide ( O2 ) whereas NO reacts with O2 to give peroxynitrite (ONOO–) [115]. In addition, HNO serves as a positive cardiac inotrope whereas NO shows negligible or a negative inotropic effect [116]. Furthermore, unlike

NO, HNO inhibits aldehyde dehydrogenase and, as such, may be useful in treating alcoholism [117–119]. HNO and NO also have some biochemical properties in common.

For instance, both are synthesized by NOS under different cofactor conditions, both can

dilate the blood vessels (and thus act as vasorelaxants) and both are of interest in the treatment of heart diseases [116].

1.2.2.1 BIOLOGICAL PRODUCTION AND REACTIVITY OF NITROXYL

Although it has not yet been unequivocally demonstrated that HNO exists in biological systems [116, 120], HNO is believed to be generated by various biosynthetic pathways. HNO can be produced by the oxidation of L–arginine by nitric oxide synthase in the absence of tetrahydrobiopterin cofactor [116, 120–123], by the oxidation of N– hydroxy–L–arginine [116, 120–122] and by the decomposition of S–nitrosothiols [121,

124, 125]. In addition, reduction of NO by enzymes such as ferrocytochrome c [126] and superoxide dismutase [127] can also give rise to HNO. Recently, HNO production has been detected in the reaction between HSNO and H2S and in the heme–catalyzed reduction of nitrite with H2S [120].

The main biological targets of HNO are believed to be thiols, DNA and oxidized metals and metalloprotein centers [128–131]. Nucleophilic attack by thiol at the electrophilic nitrogen atom of HNO results in formation of N–hydroxysulfenamide [132]

(Equation 1.1). N–hydroxysulfenamide either rearranges to a sulfonamide [125]

(Equation 1.2) or, when thiol is present in excess, can react further to produce disulfide and hydroxylamine [132, 133] (Equation 1.3).

RSH + HNO → RS–NHOH ……………………………...………………….…….… (1.1)

RS–NHOH → RS(O)NH2 …………………………………………..…………..…… (1.2)

RS–NHOH + RSH → RSSR + NH2OH ………………………………………...…... (1.3)

HNO can act as an oxidant in reactions with NAD(P)H, ascorbate, nitrosothiols

and thiols [125, 130, 134] and as a reductant when reacting with transition metal

complexes (discussed in detail in Section 1.2.2.4). Like NO, elevated HNO levels are

associated with oxidative damage of cellular components [135].

1.2.2.2 NITROXYL DONORS

The biggest challenge in the study of HNO chemistry and biochemistry is that

HNO is a short–lived species due to its spontaneous and rapid dimerization to ultimately

6 –1 –1 form N2O and H2O (k = 8 x 10 M s , 22 °C [111]; Equation 1.4). HNO donor

molecules are, therefore, required to generate HNO in situ.

HNO + HNO → H2N2O2 → N2O + H2O ………..………..…………………………. (1.4)

The most commonly used HNO donors include sodium trioxodinitrate, or Angeli's

salt (AS, Na2N2O3), and N–hydroxybenzenesulfonamide, or Piloty’s acid (PA,

PhSO2NHOH); Figure 1.3. First synthesized by Angeli in 1896 [136], AS spontaneously

decomposes to give HNO and nitrite under physiological conditions (t1/2 = 2.3 min, 37

°C) [137]. The pKa1 and pKa2 of AS are 2.51 and 9.70, respectively [138]. The rate of

decomposition of AS is pH–independent in the pH 4–8 region (Scheme 1.6) with t1/2 = 17

min, 25 °C [139] and becomes slower with increasing pH. Therefore, AS stock solutions

are usually made and temporarily stored in alkaline solution (0.01 M NaOH). AS

becomes an NO donor at pH < 4 [114].

2- + Ka - k - N2O3 + H HN2O3 HNO + NO2

Scheme 1.6. Mechanism of spontaneous decomposition of Angeli’s salt.

O OH O O- S N

N N O H O- Angeli's salt Piloty's acid

Figure 1.3. The structures of the HNO donors Angeli's salt and Piloty's acid.

The synthesis of Piloty’s acid (PA) was also first reported in 1896 [140]. Unlike

AS, Piloty’s acid is stable at neutral pH but decomposes rapidly under alkaline conditions to give HNO and benzenesulfinate. The decomposition of PA at pH 7.0 is very slow (t1/2

= 5500 min) [139]. The half–life of PA decomposition (25 °C) at pH 8.0, 9.0, and 10.0 is

561, 90, and 33 min, respectively [139]. Piloty’s acid is a weak acid with pKa = 9.29

[141]. The mechanism of the base–catalyzed decomposition of PA under alkaline conditions is shown in Scheme 1.7.

Scheme 1.7. Mechanism of base–catalyzed decomposition of Piloty’s acid.

Derivatives of PA have been synthesized and also serve as HNO donors [142]. In addition, toluene sulfohydroxamic acid (TSHA) and methanesulfonylhydroxylamine

(MSHA) are also widely used as HNO donors. In recent years, various HNO donors that

release HNO thermally [143], photochemically [144, 145], hydrolytically [146], enzymatically [118] or by oxidation [147] have been designed.

1.2.2.3 NITROXYL DETECTION

Various methods have been reported to detect HNO. Gas chromatography–mass spectrometry (GC–MS) is one of the most common methods to detect HNO [148]. Since

HNO rapidly dimerizes and subsequently decomposes to N2O and H2O (Equation 1.4), detection of N2O is an indirect method of detecting and quantifying HNO. A wide variety of other HNO detection techniques including membrane inlet mass spectrometry of HNO

[149], HPLC in combination with electrospray mass spectrometry [113], xerogel optical sensing [150] and fluorescence probes [151–155] have been reported. In addition, small molecules such as nitronyl nitroxides [156], organic phosphines [157] and metal porphyrin complexes (M = Mn, Fe) [158–161] have been shown to serve as ‘HNO traps’.

1.2.2.4 REACTIONS OF NITROXYL WITH TRANSITION METAL

COMPLEXES

The mechanisms of the reactions of transition metal complexes (TM complexes) with HNO donors including TM complexes of biological relevance have been investigated. HNO donors can react directly with the HNO donor with a 1:1 stoichiometry with TM complexes, Equation 1.5.

n+ n+ (n–1)+ – M + HNO donor → M –HNO / M –NO + NO2 …………...... …..………… (1.5)

Mn+ = Mn3+, Fe3+ and HNO donor = AS

This type of mechanism has been proposed for the reactions of AS with MnIII porphyrins (MnIIITEPyP) [161] and FeIII porphyrins (FeIIITEPyP) [162]. The suggested mechanism involves the formation of an AS–bound intermediate followed by rapid decomposition of the intermediate to form MnIITEPyP(NO) and FeIITEPyP(NO), respectively.

The reaction between the HNO donors and TM complexes may follow an alternative mechanism in which the rate determining decomposition of the HNO donor to give HNO is followed by the rapid reaction of HNO with the TM complex.

HNO donor → HNO ……………...... …..………………………….……………… (1.6)

HNO + Mn+ → Mn+–HNO / M(n–1)+–NO …………………………………….…….. (1.7)

Mn+ = Mn3+, Fe3+ and HNO donors = AS, PA, TSHA, MSHA

The reaction shown in Equation 1.7 competes with the dimerization of HNO

(Equation 1.4). This mechanism has been proposed for the reaction of MnIIITPPS with

AS [161], microperoxidase–11 (FeIIIMP11) with either AS at pH 7 or TSHA at pH 10

[162], and FeIII(TPPS) with AS or TSHA [160] to produce the corresponding MnII(NO) and FeII(NO) complexes (reductive nitrosylation).

The reaction of methemoglobin (metHb), metmyoglobin (metMb) and horseradish peroxidase with AS in anaerobic buffer also results in reductive nitrosylation of the ferric center to form Fe(II)–NO [158, 163, 164], the rate–determining step again being the decomposition of AS to release HNO. Bonner et al. proposed a similar mechanism for the

2– reaction of Ni(CN)4 with AS under alkaline conditions to form

2– nitrosyltricyanonickelate, NiNO(CN)3 [165].

A FeII–HNO complex has also been reported [159]. Deoxymyoglobin (Mb–Fe(II)) rapidly reacts with HNO produced from the decomposition of AS or methylsulfonylhydroxylamine (MSHA) to form Mb–FeII–HNO as the major reaction product (as evidenced by 1H NMR spectroscopy). The stoichiometry of the reaction is 1:1

(Equation 1.8).

FeII–Mb + HNO → FeII–Mb–HNO .………………………….……………...………. (1.8)

Mb–NO is also formed from a side reaction of MSHA and NO. A small amount of NO is probably produced from the decomposition of MSHA [141]. A Ru–HNO complex was synthesized by reducing the corresponding Ru–NO complex [166].

HNO released from AS can rapidly react with oxyhemoglobin (oxyHb–FeII) to form methemoglobin (metHb–FeIII) [167]. The proposed mechanism involves the co– oxidation of the Fe(II) center and HNO by the bound dioxygen to yield metHb, NO, and hydrogen peroxide (Equation 1.9).

– HNO + oxyHb → metHb + NO + HO2 …………………...……...... ……….. (1.9)

The NO formed can then react further with oxyHb to form metHb and nitrate, giving the overall reaction:

– – HNO + 2oxyHb → 2metHb + NO3 + HO2 ……...... ………...…...... …....….….. (1.10)

– In addition, it was found that 10% metHb is coordinated by nitrite (metHb–NO2 ).

Endogenous Cu+ ions efficiently catalyze the oxidation of HNO to nitric oxide

[168]. Copper does not exist in a free state under biological conditions. Nelli et al. have suggested that copper–containing enzymes (e.g. Cu/Zn–superoxide dismutase) catalyze the oxidation of HNO to NO, which can then cause vasodilation [168].

Finally, transition metal complexes incorporating O–bound AS as a ligand have been structurally characterized. AS reacts with Zn(II) and Co(II) complexes including

II II II [Zn (bipy)Cl2] and [Co (bipy)Cl2] to form air–sensitive [Zn (bipy)(H2O)(N2O3)] and

II 2– [Co (bipy)2(N2O3)], respectively, in which N2O3 is bound to the metal center in a

II bidentate fashion via the two oxygen atoms [169]. In [Zn (bipy)(H2O)(N2O3), the central zinc atom is coordinated to two chelating ligands (one bipy and one AS) and an aqua ligand. The complex has a distorted square pyramidal geometry, with the ligated atoms of the two chelate ligands (O(1), O(2), N(1), and N(2)) forming the base of the pyramid. For

II [Co (bipy)2(N2O3)], the complex is octahedral with rhombic distortion caused by the presence of three chelating ligands (two bipy and one AS). The structural data and IR stretching absorptions indicate that the N–N bond of the trioxodinitrate is elongated upon

II coordination to the metal atom by 0.016 Å in [Zn (bipy)(H2O)(N2O3)] and by 0.069 Å in

II [Co (bipy)2(N2O3)]).

1.3 COBALAMINS AND OXIDATIVE STRESS

A current central hypothesis in our lab is that cobalamins scavenge excess RNOS in biological systems. This process plays important role in modulating immune response and therefore B12 is important for treating chronic inflammation. It is well known from

  the literature that major intracellular form of B12, Cbl(II) , reacts with NO to form

NOCbl at a rate close to the rate of diffusion (k = 7.4 x 108 M–1 s–1, 25 °C [64, 170]). This process is irreversible with a very high binding constant (K ~ 1 x 108 M–1, 25 °C) [64].

These data support the hypothesis that Cbl scavenges excess NO in vivo. In addition, Cbls are capable of inhibiting some of the NO–mediated events in biological systems. For

example, Cbls suppress the NO–induced smooth muscle relaxation [86, 171, 172], NO– induced vasodilation [173] and NO–mediated cell proliferation [174]. Furthermore, NO inhibits both B12–dependent enzymatic reactions [175–177] and Cbl reverses NO– induced neural tube defects [178]. Recent studies from our lab indicate that Cbl(II) can

 – 8 –1 –1  – efficiently scavenge O2 (k ~ 7 × 10 M s ) [11] and cobalamins prevent O2 –induced oxidative stress in mammalian cells. We have also shown that cobalamins prevent hydrogen peroxide and homocysteine–induced oxidative stress [179]. To the best of our knowledge, no studies report the reactivity of cobalamins with HNO donors. The chemistry of the reactions with Angeli’s salt and Piloty’s acid are explored in this dissertation. Upon reacting with AS or PA, nitroxylcobalamin (NOCbl) is formed. The mechanism for decomposition in the presence of O2 has also been investigated.

CHAPTER 2

MECHANISTIC STUDIES ON THE REACTIONS OF REDUCED VITAMIN B12

COMPLEXES WITH THE NITROXYL DONOR PILOTY’S ACID

2.1 INTRODUCTION

The importance of the reactive nitrogen species nitrosyl hydride (HNO, nitroxyl,

– pKa(HNO/NO ) ~ 11.4 [128, 132]) in biological systems is increasingly being realized.

Although the existence of HNO in living cells has not yet been unequivocally demonstrated [109, 116, 120, 180], HNO is believed to be generated from L–arginine by nitric oxide synthases in the absence of the tetrahydrobiopterin cofactor [116, 122], by the oxidation of the N–hydroxy–L–arginine [116, 122], the reaction of thiols with S– nitrosothiols [121, 124], or by enzyme catalyzed reduction of NO [121, 124, 181].

Recent studies also demonstrate that HNO has biological activity and chemical reactivity distinct from NO [116, 182–185]. However, direct observation and use of

HNO is limited by its rapid dimerization and subsequent decomposition to N2O and H2O

(k = 8 x 106 M–1 s–1, 22 °C [111]), requiring the use of HNO donor molecules in chemical and biological studies. HNO donors show considerable promise in treating alcoholism

[119, 132], cardiovascular disease, ischemia/reperfusion injury and congestive heart failure [116, 128]. Elevated intracellular levels of HNO lead to oxidative stress [116, 186,

187].

25 26

Piloty’s acid (PA) is one of the most common HNO donors and has been known since the nineteenth century [140]. Base–catalyzed decomposition of PA gives HNO and benzenesulfinate (Scheme 1.7). PA is stable at neutral pH. The rate of decomposition PA

– to give HNO (and C6H5SO2 ) increases with increasing pH and becomes almost constant at pH > 10 [139].

Cobalamins are essential cofactors that play an important role in two B12– dependent enzymatic reactions [41]. Cob(III)alamins are reduced to cob(II)alamin by reductases upon cellular uptake [21]. Protein–bound Cbl(I)– is a short–lived precursor of the two coenzyme forms of vitamin B12, methylcobalamin and adenosylcobalamin [31].

Both B12–dependent enzymes are inactivated under oxidative stress conditions [175,

176].

In this chapter, we present kinetic and mechanistic studies on the reaction of the reduced cobalamin complexes, Cbl(II) and Cbl(I)–, with the HNO donor Piloty’s acid.

Importantly, results from kinetic measurements suggest that HNO can oxidize Co(I) center of Cbl(I)–. Oxidation of a transition metal center by HNO is, to our knowledge, unprecedented.

2.2 EXPERIMENTAL SECTION

2.2.1 REAGENTS

Hydroxocobalamin hydrochloride (HOCbl•HCl, 98% stated purity by the manufacturer) was purchased from Fluka. The percentage of water in HOCbl•HCl

(•nH2O) (10–15% water, batch dependent) was determined by converting HOCbl•HCl to

– –1 –1 dicyanocobalamin, (CN)2Cbl (0.10 M KCN, pH 11.0, ε368 nm = 30.4 mM cm ) [188].

Piloty’s acid (PA, 98%) was purchased from Cayman Chemical Company and used without further purification. NaBH4 (≥ 98%), NaNO2 (99.6%), NH2OH•HCl (≥ 97%),

KCN (99%), 8–hydroxyquinoline (≥ 99%), diethylenetriaminepentaacetic acid (DTPA; ≥

98%), D2O (99.8 atom % D), CH3OH–d4 (99.8 atom % D), acetone, triflic acid (99%),

NaOH, biological buffers (MES, TES, TAPS, CHES and CAPS) and inorganic buffers

(KH2PO4, K2HPO4, NaHCO3 and Na2CO3) were obtained from either Fisher Scientific or

Acros Organics. The Griess reagent, TSP (3–(trimethylsilyl)propionic 2,2,3,3–d4 acid, sodium salt), benzenesulfinic acid, sodium salt (98%) and methanol were obtained from

Sigma Aldrich. Nessler’s reagent was obtained from Spectrum Chemical. Water was purified using a Barnstead Nanopure Diamond water purification system.

2.2.2 GENERAL METHODS AND INSTRUMENTATION

Solutions Preparations. All solutions were prepared using standard biological buffers and inorganic buffers (5.0 mM–0.10 M) and a constant ionic strength was maintained using sodium triflate (NaCF3SO3; I = 1.0 M). All pH measurements were carried out at room temperature using an Orion Model 710A pH meter equipped with

Mettler–Toledo Inlab 423 or 421 electrodes. The electrode was filled with 3 M

KCl/saturated AgCl solution (pH 7). The electrodes were calibrated with standard buffer solutions at pH 7.00, 10.00 and 12.45. The pH of the solutions was adjusted using H3PO4 or NaOH solutions as necessary.

Air–free Chemistry. Anaerobic solvent and buffer solutions were prepared by bubbling argon through the solution for ~ 24 h. Stock solutions were stored in a

MBRAUN Labmaster 130 (1250/78) glove box filled with argon, equipped with O2 and

H2O sensors and a freezer at –24 °C. Temperature–sensitive solutions were stored in the freezer.

Air–free UV–vis spectrometric measurements were carried out in Schlenk cuvettes (cuvettes fitted with a J–Young or an equivalent stopcock) on a Cary 5000 spectrophotometer equipped with a thermostated (25.0 ± 0.1 °C) cell changer operating with WinUV Bio software (version 3.00). Freshly prepared solutions were used for kinetic measurements. For 1H NMR experiments under anaerobic conditions, air–tight J–

Young NMR tubes (Wilmad, 535–JY–7) were used. 1H NMR spectra were recorded on a

Bruker 400 MHz spectrometer equipped with a 5 mm probe at 23 ± 1 °C. TSP was used as an internal reference. UV–vis and 1H NMR data were fitted using the program

Microcal Origin version 8.0.

2.2.3 SYNTHESIS OF COB(II)ALAMIN AND COB(I)ALAMIN

Cbl(II) was prepared by reducing HOCbl•HCl with NaBH4 (1.2 equiv) under anaerobic conditions using a procedure reported in the literature [22, 23]. Cbl(I)– was prepared by reducing HOCbl•HCl with excess NaBH4 (6.0 mol equiv) under anaerobic conditions also using a literature procedure [22]. After confirming the formation of the desired cobalamins using UV–vis spectroscopy, a small amount of chilled acetone

(anaerobic) was added to destroy excess NaBH4.

2.2.4 DETERMINATION OF COBALAMIN CONCENTRATIONS

Cbl concentrations (solutions prepared in water) were determined using their

3 –1 –1 molar extinction coefficients (εCbl(III) at 475 nm = 6.99 x 10 M cm , εCbl(II) at 475 nm = 1.05 x

–4 –1 –1 – 3 –1 –1 10 M cm and εCbl(I) at 420 nm = 5.81 x 10 M cm ) [23]. Alternatively, Cbls were

– converted to dicyanocobalamin, (CN)2Cbl by reacting with KCN (0.10 M, pH 11.50).

The concentration of the final product was determined using UV–vis spectrometry

4 –1 –1 (ε368 nm = 3.0 x 10 M cm [188, 189]).

2.2.5 DETERMINATION OF THE RATE CONSTANTS FOR SPONTANEOUS

DECOMPOSITION OF PILOTY’S ACID

Stock solutions were prepared by dissolving PA (solid) in anaerobic CH3OH and further dilutions were made with anaerobic water. The amount of CH3OH in the final reaction solution was < 3% (v/v). The reaction was initiated by adding an aliquot (0.200 mL, 1.50 mM) of a stock solution of PA in water to an air–free cuvette containing buffer solution (2.80 mL) which had been thermostated in the cell holder of the Cary 5000 spectrophotometer. The decomposition of PA (1.00 x 10–4 M) was monitored at 250 nm and the absorbance versus time data fitted to a first–order rate equation. Biological buffers were found to alter the rate of the decomposition of PA; hence phosphate and carbonate buffers (I = 1.0 M, NaCF3SO3) were used in all kinetic experiments.

2.2.6 SAMPLE PREPARATION FOR KINETIC MEASUREMENTS ON THE

REACTION OF COB(II)ALAMIN WITH PILOTY’S ACID

All samples were prepared under strictly anaerobic conditions inside the glove box. Stock Cbl(II) solutions were prepared by dissolving solid Cbl(II) in the appropriate buffer. Stock PA solutions were prepared and stored following the procedure discussed in

Section 2.2.5. The major reaction product in the reaction of Cbl(II) with PA under different pH conditions is NOCbl. NOCbl is extremely air–sensitive (can undergo oxidation in the presence of air in seconds) [29]. Therefore, strictly anaerobic conditions were required for all the kinetic and stoichiometry measurements.

2.2.7 SAMPLE PREPARATION FOR KINETIC MEASUREMENTS ON THE

REACTION OF COB(I)ALAMIN WITH PILOTY’S ACID

All samples were prepared under strictly anaerobic conditions inside the glove box. Stock Cbl(I)– solutions (mM concentrations) were prepared in water since Cbl(I)– undergoes slow oxidation to Cbl(II) in presence of buffers, and were stored under strictly anaerobic conditions at –24 °C. Cbl(I)– solutions were used within a week of preparation.

Since Cbl(I)– is gradually oxidized to Cbl(II) at lower concentrations even in the absence of oxygen, high concentrations (100–200 µM) were used in all kinetic experiments. Low buffer concentrations (5–10 mM) were used to control the pH. Stock PA solutions were prepared and handled as described in Section 2.2.5.

2.2.8 SAMPLE PREPARATION FOR 1H NMR EXPERIMENTS FOR THE

REACTION OF COB(II)ALAMIN WITH PILOTY’S ACID

For 1H NMR experiments, stock PA solutions were prepared in anaerobic deuterated methanol and added to the Cbl(II) solution in anaerobic buffer (pD 10.00, 0.10

M carbonate buffer). The amount of CH3OH–d4 in the final reaction solution was < 3%

(v/v). The reaction was allowed to proceed to completion inside the glove box and subsequently transferred to an air–free NMR tube before recording the 1H NMR spectra.

TSP was added as the internal reference.

2.2.9 DETERMINATION OF THE REACTION STOICHIOMETRY

Inside the glove box, a series of vials were prepared containing Cbl(II) (50.0 µM) and varying aliquots of a stock PA solution (0, 0.25–3.5 mol equiv PA) added. The total volume of the final solution in each vial was 3.00 mL. After the addition of PA, the vials were quickly capped and wrapped with Parafilm. The reaction was allowed to proceed to completion for at least 5 half–lives for the slowest reaction (Cbl(II) + 0.25 equiv PA).

The product solutions were equilibrated at 25.0 °C for at least for 15 min prior to UV–vis measurements in air–free cuvettes.

For the stoichiometry of the reaction between Cbl(I)– and PA, Cbl(I)– (2.00 x 10–4

M) was reacted with varying mol equiv of PA (0, 1.0–5.0 equiv). All other procedures were similar to that for the Cbl(II)/PA system.

2.2.10 INDOOXINE TESTS TO DETERMINE IF HYDROXYLAMINE IS

FORMED IN THE REACTION OF COB(II)ALAMIN WITH PILOTY’S ACID

A modified literature procedure was used [190, 191]. The reaction of Cbl(II) (1.00 x 10–3 M) and 2.2 mol equiv PA at pH 10.00 (0.10 M carbonate buffer) was allowed to proceed to completion under anaerobic conditions inside the glove box for ~ 3.5 h. Then aerobic 8–hydroxyquinoline solution (1.00 mL, 4.0% w/v in ethanol) and aqueous aerobic Na2CO3 solution (1.00 mL, 1.00 M) were added to the product solution (1.00 mL) outside the glove box. After standing for 45 min at room temperature, the UV–vis spectrum was recorded. The product of the reaction between Cbl(II) and 2.2 mol equiv

PA at pH 10.00 also produced ~ 6% NH2OH which was found to originate from the commercially available PA itself (see Section 2.2.11).

Our lab and others have shown that at acidic pH conditions, NH2OH can undergo disproportionation [66, 192, 193].

2.2.11 INDOOXINE TESTS WITH COMMERCIAL PILOTY’S ACID

Indooxine tests for commercially available PA were carried out using the procedure described in Section 2.2.10, at pH 10. Commercially available PA contains

~ 6% NH2OH as an impurity. In addition, the reaction of NH2OH with PA at pH 10 under anaerobic conditions shows that HNO from PA decomposition does not react with

NH2OH.

2.2.12 NESSLER’S TEST TO DETERMINE IF AMMONIA IS A REACTION

PRODUCT

The reaction between Cbl(II) (1.00 x 10–3 M) and 2.2 mol equiv PA at pH 10.00

(0.10 M carbonate buffer) was allowed to proceed to completion in a vial for ~ 3.5 h inside the glove box. The product solution (1.00 mL) was taken outside the glove box and

8–10 drops of Nessler’s reagent added. A positive result for NH3 is indicated by a yellow or brown (at high concentrations) coloring in the reaction solution [194]. In this case, no brown or yellow coloring was observed above the pink color of the HOCbl species. A similar procedure showed that there was no formation of ammonia from the reaction between Cbl(I)– and 3.0 mol equiv of PA at pH 10.00.

2.2.13 GRIESS TEST TO DETERMINE THE PRESENCE OF NITRITE AS A

REACTION PRODUCT

Standard procedures for the Griess assay [195] were used to determine if nitrite is formed in the reaction of Cbl(II) with 2.0 mol equiv PA and that of Cbl(I)– with 3.0 mol equiv PA at pH 10.00 under anaerobic conditions.

A calibration plot of absorbance at 586 nm versus nitrite concentration was generated. Note that the calibration plots were found to be pH independent. Typically, an aliquot of Griess reagent (1.50 mL) was added to an equal volume of buffer (1.50 mL,

0.30 M TAPS buffer, pH 8.00) containing varying concentrations of nitrite (0, 20.0, 40.0,

60.0, 80.0 and 100.0 µM) and the absorbance at 586 nm was determined. Aliquots of the product of the reaction of Cbl(II) (1.00 x 10–4 M) with 2.0 mol equiv PA and that of

Cbl(I)– (2.00 x 10–4 M) with 3.0 mol equiv PA at pH 10.00 were subjected to the same

procedure. Positive Griess test is indicated by the formation of dark–pink colored solution with strong absorbance at 586 nm. However, no significant increase in absorbance at 586 nm was observed in both cases indicating the absence of nitrite as a reaction product.

2.2.14 CONTROL EXPERIMENTS

Reaction of Cbl(I)–, Cbl(II) and NOCbl with Benzenesulfinate. Cbl(II)

(50.0 µM) and Cbl(I)– (2.00 x 10–4 M) were independently reacted with excess sodium benzenesulfinate (20.0 mol equiv) overnight or for 2 h, respectively, under anaerobic conditions. No spectral changes were observed by UV–vis spectroscopy. Similarly, no reaction was observed between NOCbl and sodium benzenesulfinate.

Reaction of Cbl(II) with N2O. Since N2O is the product of HNO dimerization, the reactivity of N2O with Cbl(II) was investigated. Excess N2O saturated buffer (20.0 mM; concentration of N2O in N2O–saturated buffer = 28.8 mM [196]) was injected to a septum–capped flask containing an anaerobic solution of Cbl(II) (2.00 x 10–4 M) and the mixture left to react overnight inside the glove box. After 12 h, the UV–vis spectrum of the product solution was recorded and was identical to Cbl(II); hence there is no reaction between Cbl(II) and N2O.

2.3 RESULTS AND DISCUSSION

2.3.1 KINETIC STUDIES ON THE REACTION OF COB(II)ALAMIN WITH

PILOTY’S ACID

Kinetic studies on the reaction between Cbl(II) and PA at different pH conditions were carried out using UV–vis spectroscopy. PA is stable under neutral pH conditions and the rate of decomposition of PA increases in the pH range pH 8–10 and is essentially pH independent at pH > 10 [139]. The pKa of PA is 9.29 [139] and decomposition of PA requires deprotonation of PA. Experiments were therefore first carried out at pH 10.00.

Upon the addition of excess PA (1.00 x 10–3 M) to Cbl(II) (5.00 x 10–5 M) in buffer (pH 10.00) under strictly anaerobic conditions, Cbl(II) is cleanly converted to

– nitroxylcobalamin (NOCbl, NO –Cbl(III); λmax = 289, 315 and 478 nm [29, 64, 65, 170]), with sharp isosbestic points occurring at 330, 377, 479 and 541 nm, Figure 2.1. These values are in agreement with literature values for the Cbl(II)/NOCbl conversion [64,

170]. The corresponding plot of absorbance at 510 nm versus time is given in Figure 2.2.

The linear trend (0–5 min) indicates that the rate determining step is independent of the

Cbl(II) concentration. This suggests that the rate–determining step is decomposition of

– PA to give HNO (and C6H5SO2 ), followed by a rapid reaction between HNO and

Cbl(II). This was confirmed by obtaining kinetic data for the reaction of Cbl(II) (1.00 x

10–4 M) with 1.0 mol equiv PA at pH 10.00, which fitted very well to a first–order reaction, Figure 2.3.

2.0

1.5

1.0 Absorbance 0.5

0.0 300 400 500 600 700 Wavelength (nm) Figure 2.1. UV–vis spectra for the reaction between Cbl(II) (5.00 x 10–5 M) and excess PA (1.00 x 10–3 M) at pH 10.00 ± 0.02 (25.0 °C, 0.30 M CAPS buffer, I = 1.0 M

(NaCF3SO3)) under strictly anaerobic conditions.

0.36

0.33

0.30 Abs at510 nm 0.27

0 5 10 15 20 Time (min)

Figure 2.2. Plot of absorbance at 510 nm versus time for the reaction of Cbl(II) (5.00 x 10–5 M) with excess PA (1.00 x 10–3 M) at pH 10.00 ± 0.02 (25.0 °C, 0.30 M CAPS buffer, I = 1.0 M (NaCF3SO3)) under strictly anaerobic conditions.

The observed rate constant (kobs) is similar to the rate constant for PA

– decomposition to HNO and C6H5SO2 (kL) under the same pH conditions (kobs = (1.92 ±

–2 –1 –2 –1 0.01) x 10 min and kL = (2.19 ± 0.01) x 10 min [139], respectively; see Table 2.1.

Table 2.1. Observed rate constants for the spontaneous decomposition of PA (kL) and the reaction of Cbl(II) with 1.0 mol equiv of PA (kobs) as a function of pH under strictly anaerobic conditions (25.0 °C, 0.10 M phosphate buffer, I = 1.0 M (NaCF3SO3)).

2 pH Literature value for Experimental value 10 x kobs 2 –1 2 –1 –1 (± 0.02) 10 x kL (min ) for 10 x kL (min ) (min ) 12.00 2.53 2.39 ± 0.01 2.36 ± 0.01 10.00 2.08 2.19 ± 0.01 1.92 ± 0.01 9.00 0.77 0.94 ± 0.01 0.90 ± 0.01 8.60 0.41 0.30 ± 0.01 0.35 ± 0.01 8.00 0.12 0.08 ± 0.01 0.09 ± 0.01

0.68

0.64

0.60

Abs at510Abs nm 0.56

0.52 0 50 100 150 200 Time (min) Figure 2.3. Plot of absorbance at 510 nm versus time for the reaction of Cbl(II) (1.00 x 10–4 M) with 1.0 mol equiv of PA at pH 10.00 ± 0.02 (25.0 °C, 0.10 M phosphate buffer,

I = 1.0 M (NaCF3SO3)). The first order fit of the data gives observed rate constant (kobs) = (1.92 ± 0.01) x 10–2 min–1.

0.44 a) 0.56 b)

0.40 0.52

0.48 0.36 0.44 0.32 Abs at 510 nm Abs nmat 510 0.40 0.28 0 5 10 15 20 25 30 35 40 0 100 200 300 400 Time (min) Time (min)

c) 0.42 0.60 d) 0.39 0.56 0.36 0.52 0.33 Abs510 nmat Abs atAbs 510 nm 0.48 0.30

0 200 400 600 800 1000 0 50 100 150 200 250 Time (min) Time (min)

2.10 e) 0.45 f) g) 2.03 0.60 1.96 0.40 1.89 1.82 0.55

Abs at at Abs 510 nm 0.35

1.75 Abs510 nmat Absatnm255 1.68 0.50 0 1000 2000 3000 0 5 10 15 20 25 30 0 2500 5000 7500 10000 Time (min) Time (min) Time (s)

Figure 2.4. Plot of absorbance versus time for the reaction of a) Cbl(II) (5.00 x 10–5 M) with excess PA (1.00 x 10–3 M) at pH 9.00 ± 0.02 b) Cbl(II) (1.00 x 10–4 M) with 1.0 mol equiv PA at pH 9.00 ± 0.02 c) Cbl(II) (1.00 x 10–4 M) with 1.0 mol equiv PA at pH 8.60 ± 0.02 d) Cbl(II) (5.00 x 10–5 M) with excess PA (6.00 x 10–3 M) at pH 8.00 ± 0.02 e) Cbl(II) (1.00 x 10–4 M) with 1.0 mol equiv PA at pH 8.00 ± 0.02 f) Cbl(II) (5.00 x 10–5 M) with excess PA (1.00 x 10–3 M) at pH 12.00 ± 0.02 and g) Cbl(II) (1.00 x 10–4 M) with 1.0 mol equiv PA at pH 12.00 ± 0.02. All experiments were carried out at 25.0 °C, 0.10 M phosphate buffer, I = 1.0 M (NaCF3SO3)). For each pH condition, a linear trend is observed for the reaction of Cbl(II) with excess PA, whereas kobs becomes almost identical to kL for the reaction of Cbl(II) with 1.0 mol equiv of PA (Table 2.1).

Similar experiments at other pH values (pH 8.00–12.00) showed that NOCbl is also formed upon reacting Cbl(II) with PA at these pH conditions and that PA decomposition remains the rate–determining step for the reaction (see Table 2.1 and

Figure 2.4). The values of kL agree well with values reported in the literature [139]. At pH  7 decomposition of PA to HNO becomes very slow (t1/2 ~ 4 days) [139].

2.3.2 STOICHIOMETRY OF THE REACTION OF COB(II)ALAMIN WITH

PILOTY’S ACID

In order to probe the mechanism of the reaction, the stoichiometry of the reaction between Cbl(II) and PA at pH 10.00 was determined. From the UV–vis spectra of equilibrated solutions of Cbl(II) (50.0 µM) with PA (0–3.5 mol equiv), a plot of absorbance at 355 nm versus mol equiv of PA was generated, Figure 2.5. The absorbance

1.6 a) 0.75 b)

1.2 0.70 0.65 0.8 0.60 0.55 Absorbance 0.4 0.50 Abs 355 at nm 0.45 0.0 0.40 300 400 500 600 700 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Wavelength (nm) Mole Equiv of PA Figure 2.5. a) UV–vis spectra for equilibrated solutions of Cbl(II) (5.00 x 10–5 M) with PA (0, 0.5–3.5 mol equiv) at pH 10.00 under anaerobic conditions (25.0 °C, 0.10 M phosphate buffer). b) Plot of absorbance at 355 nm versus mol equiv of PA for the same reaction. The product solutions were equilibrated for at least 15 min at 25.0 °C prior to UV–vis measurements.

at 355 nm linearly increases with the addition of PA up to 2.0 mol equiv of PA and remains unchanged upon further addition of PA. This clearly shows that the stoichiometry of the reaction of Cbl(II) with PA is 1:2 Cbl(II):PA. A similar conclusion was reached by plotting absorbance data at 312 or 510 nm versus mol equiv PA, Figure

2.6.

NOCbl and benzenesulfinate were confirmed by 1H NMR spectroscopy as the products of the reaction of anaerobic Cbl(II) with 2.2 mol equiv PA at pD 10.00 (Figure

2.7). The 1H NMR chemical shifts for NOCbl match well with literature values [68].

1.6 0.40 a) b)

0.36 1.4 0.32

1.2 0.28 Abs at 510 nm 0.24 Abs Abs at 312 nm 1.0 0.20 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Mol Equiv of PA Mol Equiv of PA

Figure 2.6. Plot of absorbance at a) 312 nm and b) 510 nm versus mol equiv of PA for the reaction of Cbl(II) (5.00 x 10–5 M) with PA (0, 0.5–3.5 equiv) at pH 10.00 under anaerobic conditions (25.0 °C, 0.10 M phosphate buffer).

Since Cbls can exist in three oxidation states (Co+, Co2+ and Co3+), the possible

Cbl reaction intermediates are either Cbl(III) (= aquacobalamin/hydroxocobalamin, X =

– + – H2O/OH , Figure 1.1; pKa(H2OCbl ) = 7.8 [10, 11]) or cob(I)alamin, Cbl(I) .

– Importantly, control experiments showed that the C6H5SO2 byproduct from PA decomposition does not react with Cbl(I)–, Cbl(II) or NOCbl (see Section 2.2.14).

8.4 8.2 8.0 7.8 7.6 7.4 7.2 7.0 6.8 6.6 Chemical shift (ppm)

7.8 7.6 7.4 7.2 7.0 6.8 6.6 6.4 6.2 Chemical shift (), ppm

Figure 2.7. Aromatic region of the 1H NMR spectrum of the products of the reaction between Cbl(II) (6.04 x 10–3 M) and 2.2 mol equiv PA at pD 10.00 (0.10 M carbonate buffer). The peaks at 7.42, 7.20, 6.79, 6.35 and 6.25 ppm correspond to NOCbl and those at 7.66, 7.65, 7.55 and 7.53 ppm correspond to benzenesulfinate. Inset: Aromatic region of the 1H NMR spectrum for authentic sodium benzenesulfinate (δ = 7.67, 7.65, 7.55 and 7.53 ppm).

It is well established that HNO and HNO donors coordinate to metal centers and/or reduce Mn+ to M(n–1)+ (HNO is oxidized to NO (NO) and reductive nitrosylation often occurs for porphyrins) [158–165, 168]. The redox potentials (E°, NHE) for the

NO/HNO (pH 10) and Cbl(II)/Cbl(I)– redox couples are –0.768 V [197] and –0.61 V [26,

66], respectively; hence reduction of Cbl(II) by HNO to Cbl(I)– and NO is thermodynamically feasible. A control experiment showed that Cbl(II) does not react with the product of HNO dimerization, N2O (see Section 2.2.14). Although NO cannot react with Cbl(I)– to give the observed NOCbl product (Cbl(I)– + NO + H+ → Cbl(II) +

– ½N2O + ½H2O [198]), Cbl(I) could conceivably be oxidized back to Cbl(II) by a second

HNO molecule, consistent with the observed 1:2 Cbl(II):PA stoichiometry, Scheme 2.1.

Cbl(II) may then subsequently rapidly react with NO to form NOCbl (k = 7.4 ×

8 −1 −1 8 −1 10 M s , K(NOCbl) ≈ 1 × 10 M , 25 °C [64, 170]). Note that Scheme 2.1 assumes N2 is formed, which will be shown later to be the likely product of HNO oxidation by Cbl(I)–.

2C6H5SO2NHOH

+ - 2H pKa 9.29 k 2C H SO NHO- L - 6 5 2 R.D.S. 2HNO + 2C6H5SO2 fast Cbl(II) + HNO Cbl(I)- + NO + H+ - + fast Cbl(I) + HNO + H Cbl(II) + 1/2N2 + H2O fast Cbl(II) + NO NOCbl

- - Cbl(II) + 2C6H5SO2NHO NOCbl + 2C6H5SO2 + 1/2N2 + H2O

Scheme 2.1. Proposed reaction pathway for the reaction of Cbl(II) with PA.

2.3.3 KINETIC STUDIES ON THE REACTION OF COB(I)ALAMIN WITH

PILOTY’S ACID

In order to determine whether Cbl(I)– is a viable intermediate of the reaction, the reaction between Cbl(I)– and PA was directly investigated. UV–vis spectra for the reaction between Cbl(I)– and excess PA at pH 10.0 under strictly anaerobic conditions once again showed that NOCbl is ultimately formed. Figure 2.8 shows that the reaction proceeds via a Cbl(II) intermediate (the isosbestic points at 347, 417 and 542 nm agree with values for conversion of Cbl(I)– to Cbl(II) [66, 199] and Cbl(II) subsequently reacts further to give NOCbl (the isosbestic points at 377, 488 and 546 nm are consistent with

2 Absorbance 1

0 360 405 450 495 540 585 Wavelength (nm)

Figure 2.8. UV–vis spectra recorded as a function of time (spectra recorded every 0.5 min) for the reaction between Cbl(I)– (2.00 x 10–4 M) and 5.0 mol equiv PA at pH 10.00 under anaerobic conditions (25.0 °C, 0.01 M carbonate buffer, I = 1.0 M (NaCF3SO3)). The reaction occurs via a Cbl(II) intermediate ultimately leading to the formation of the final product, NOCbl. Isosbestic points occurring at 347, 417 and 542 nm are consistent with Cbl(I)– to Cbl(II) conversion whereas those occurring at 377, 488 and 546 nm are expected for Cbl(II) reacting further to give NOCbl.

Cbl(II)/NOCbl conversion [64, 170]). The selected spectra for these two consecutive reactions are shown in Figure 2.9. Further confirmation that the reaction proceeds via a

Cbl(II) intermediate was obtained by recording UV–vis spectra as a function of time for the reaction of Cbl(I)– with 1.0 mol equiv PA at pH 10.00. In this case there is insufficient PA for the second reaction to occur and Cbl(I)– is cleanly converted to Cbl(II)

(λmax = 405 and 475 nm); Figure 2.10 [66, 199]. However, with close observation of the spectra, another set of isosbestic points (374 and 545 nm) are evident corresponding to very slight formation of NOCbl.

To determine the rate constant for the oxidation of Cbl(I)– to Cbl(II), a large excess of Cbl(I)– compared with PA was used, to ensure no contribution to the observed rate constant from the subsequent reaction of Cbl(II) with HNO. Spectra given in Figure

2.11 for the reaction of excess Cbl(I)– (2.00 x 10–4 M) with PA (2.00 x 10–5 M) show that clean isosbestic points are observed, consistent with Cbl(I)–/Cbl(II) conversion. Figure

2.12 shows a plot of absorbance at 475 nm versus time for the reaction of Cbl(I)– (2.00 x

–4 –5 10 M) with PA (2.00 x 10 M). The first–order fit of the data gives kobs = (1.43 ± 0.01) x 10–2 min–1, which is very similar to the rate constant for the anaerobic decomposition of

PA at pH 10.00 ((1.96 ± 0.01) x 10–2 min–1, Figure 2.13); hence the rate determining step for the reaction between Cbl(I)– and PA at pH 10.00 is once again the decomposition of

– PA to HNO and C6H5SO2 .

5 a) 3.5 b)

4 2.8

3 2.1

2 1.4 Absorbance Absorbance 1 0.7

0 0.0 360 405 450 495 540 585 360 405 450 495 540 585 Wavelength (nm) Wavelength (nm) Figure 2.9. UV–vis spectra obtained as a function of time for the reaction between Cbl(I)– (2.00 x 10–4 M) and 5.0 mol equiv PA at pH 10.00 under anaerobic conditions (25.0 °C,

0.01 M carbonate buffer, I = 1.0 M (NaCF3SO3)). a) The first 10 spectra (spectra recorded every 0.5 min) show the formation of a Cbl(II) intermediate. b) Selected spectra at longer reaction times (10–70 min) showing the formation of the final NOCbl product.

Absorbance 1

0 350 400 450 500 550 600 Wavelength (nm) Figure 2.10. UV–vis spectra recorded as a function of time (spectra recorded every 0.5 min) for the reaction of Cbl(I)– (2.00 x 10–4 M) with 1.0 mol equiv PA at pH 10.00 (25.0

°C, 0.01 M carbonate buffer, I = 1.0 M NaCF3SO3)) under strictly anaerobic conditions. Isosbestic points at 418 and 543 nm indicate the formation of Cbl(II) from Cbl(I)–. 5

2 Absorbance 1

0 350 400 450 500 550 600 Wavelength (nm) Figure 2.11. UV–vis spectra recorded as a function of time (spectra recorded every 1 min) for the reaction of excess Cbl(I)– (2.00 x 10–4 M) with PA (2.00 x 10–5 M) at pH

10.00 ± 0.02 (25.0 °C, 0.01 M carbonate buffer, I = 1.0 M (NaCF3SO3)) under strictly anaerobic conditions. The clean isosbestic points occurring at 344, 416 and 541 nm indicate the direct conversion of Cbl(I)– to Cbl(II).

If indeed Cbl(I)– is first oxidized by 1.0 mol equiv HNO to Cbl(II), and Cbl(II)

subsequently reacts further with HNO (1:2 Cbl(II):PA) to ultimately form NOCbl, one would expect a 1:3 Cbl(I)–:PA stoichiometry for the reaction of Cbl(I)– with PA. This was confirmed experimentally, Figure 2.14.

1.04

0.96

0.88

0.80

Abs atAbs 475 nm 0.72

0.64

0 50 100 150 200 Time (min)

Figure 2.12. Plot of absorbance at 475 nm versus time for the reaction of excess Cbl(I)– (2.00 x 10–4 M) with PA (2.00 x 10–5 M) at pH 10.00 ± 0.02 (25.0 °C, 0.01 M carbonate buffer, I = 1.0 M (NaCF3SO3)) under strictly anaerobic conditions. The data fit well to a –2 –1 first order rate equation giving kobs = (1.43 ± 0.01) x 10 min .

0.36

0.34

0.32

0.30

0.28 Abs at250 nm 0.26

0.24

0 50 100 150 200 Time (min)

Figure 2.13. Plot of absorbance at 250 nm versus time for the spontaneous decomposition of PA (1.00 x 10–4 M) at pH 10.00 ± 0.02 (25.0 °C, 0.01 M carbonate buffer, I = 1.0 M (NaCF3SO3)) under strictly anaerobic conditions. The first–order fit of –2 –1 the data gives kobs = (1.96 ± 0.01) x 10 min .

5 1.5 a) b)

4 1.4

3 1.3

1.2 2 Abs at Abs 510 nm Absorbance 1.1 1 1.0 0 300 350 400 450 500 550 600 650 1 2 3 4 5 Wavelength (nm) Mol Equiv of PA

Figure 2.14. a) UV–vis spectra of equilibrated solutions of Cbl(I)– (2.00 x 10–4 M) with PA (0, 1.0–5.0 mol equiv) at pH 10.00 (0.01 M carbonate buffer) under strictly anaerobic conditions. b) Corresponding plot of absorbance at 510 nm versus mol equiv of PA for the same reaction. Note that Cbl(II) (blue trace) is formed with the addition of 1.0 mol equiv PA to the Cbl(I)– (red trace).

Additional experiments confirmed that Cbl(I)– is an intermediate of the reaction between Cbl(II) and PA. Specifically, previous studies from our lab have shown that

– Cbl(I) is rapidly oxidized by excess nitrite at pH 10.0 to Cbl(II) (+ NH2OH; HNO2 is the

– oxidant [66]; pKa (HNO2) ~ 3.2 [200]), and that Cbl(II) does not react with NO2 [170]. If indeed Cbl(I)– is the intermediate and Scheme 2.1 is correct, the complete conversion of

Cbl(II) to NOCbl in the presence of excess nitrite should require only 1.0 mol equiv

HNO. In other words, 1:2 Cbl(II):PA stoichiometry should be changed to 1:1 Cbl(II):PA

– – in the presence of excess nitrite since NO2 can replace HNO as the oxidant of the Cbl(I) intermediate, Scheme 2.2.

Scheme 2.2. Proposed mechanism for the reaction between Cbl(II) and 1.0 mol equiv PA – in the presence of excess NO2 .

This was confirmed experimentally (50.0 mol equiv nitrite, Figure 2.15).

Furthermore, the stoichiometry of the reaction between Cbl(I)– and PA should also be 1:1 in the presence of 50.0 mol equiv nitrite, and this was also observed experimentally,

Figure 2.16. Finally, experiments showed that Cbl(III) does not react with 1.1 mol equiv

PA to form NOCbl in the presence of 50.0 mol equiv nitrite, Figure 2.17. Hence all three experiments in the presence of excess nitrite support Cbl(I)–, not Cbl(III), being the Cbl reaction intermediate.

Experiments were carried out to identify the nitrogen products from HNO reduction. If Cbl(I)– is oxidized by HNO to Cbl(II), then possible HNO reduction

+ products are NH2OH, NH4 , and/or N2. Although others have shown that NH2OH can disproportionate [192, 193] or react directly with HNO to form N2 and H2O [130], control experiments using the indooxine test to determine the NH2OH concentration under our alkaline experimental conditions showed that NH2OH is stable at pH 10.00 (10 mM carbonate buffer), and that NH2OH does not react with HNO produced from the decomposition of PA (Section 2.2.10). The indooxine test of the products of the reaction between Cbl(II) and 2.2 mol equiv PA unexpectedly showed that 0.06 mol equiv of

NH2OH was formed, which was subsequently found to be originated from PA itself (see

Section 2.2.11; the standard method to synthesize PA is by reacting benzenesulfonyl chloride with NH2OH [139]).

– Both the Nessler’s test for NH3 (see Section 2.2.12) and Griess test for NO2 (see

Section 2.2.13) were negative for the reaction of Cbl(II) with 2.2 mol equiv of PA at pH

10.00. Hence N2 is the likely product of HNO reduction. The Nessler’s test for NH3 and

– the Griess test for NO2 (see Sections 2.2.12 and 2.2.13) were also both negative for the

– reaction of Cbl(I) with 3.0 mol equiv PA at pH 10.00 suggesting again that N2 is the only possible nitrogen product from the reaction of Cbl(I)– with PA. Attempts to detect nitrogen in the product mixture by mass spectrometry headspace gas analysis (at Kent

State University) or Raman spectroscopy (at Oregon Health and Science University) were unsuccessful, and subsequent control experiments showed that the low concentrations of

N2 formed using the highest concentrations of reagents possible were unfortunately insufficient for detection by the available instrumentation. Note that since NH2OH was shown to be stable in the presence of HNO under our experimental conditions, and that the indooxine test for NH2OH of the product mixture was negative, it seems unlikely that

NH2OH is a reaction intermediate.

Finally, in Scheme 2.1 we have assumed that Cbl(I)– is oxidized by HNO.

However others have reported that the product of the rapid dimerization of HNO, N2O,

– 2 –1 –1 also rapidly oxidizes Cbl(I) to Cbl(II) (2HNO  N2O + H2O; k = 1.6 x 10 M s ; pH

8) [201].

1.2

1.6 0.9

0.6 1.2

Absorbance 0.3 0.8 0.0 300 400 500 600 700 Wavelength (nm) Absorbance 0.4

0.0 300 400 500 600 700 Wavelength (nm) Figure 2.15. UV–vis spectra of the product solution for the reaction between Cbl(II) (50.0 µM) and 1.0 mol equiv PA in the absence (red dotted trace) and presence (blue – solid trace) of 50.0 mol equiv NO2 at pH 10.00 (25.0 °C, 0.10 M carbonate buffer). Inset: The spectrum for authentic NOCbl. 1:1 Cbl(II):PA reaction gives incomplete – formation of NOCbl whereas 1:50:1 Cbl(II):NO2 :PA reaction gives the complete formation of NOCbl which matches well with the spectrum of authentic NOCbl (inset). A – small bump appears at ~ 350 nm because NO2 strongly absorbs in 300–400 nm region.

3.6 0.75

2.7 0.50

0.25

1.8 Absorbance

Absorbance 0.00 0.9 400 500 600 700 Wavelength (nm)

0.0 400 500 600 700 Wavelength (nm)

Figure 2.16. UV–vis spectrum of the product solution for the reaction between Cbl(I)– –4 – (2.00 x 10 M) and 1.0 mol equiv PA in the presence of 50.0 equiv NO2 at pH 10.00 (25.0 °C, 0.10 M carbonate buffer). The final spectrum of the product matches well with the spectrum of NOCbl (inset).

1.2

1.2 0.9

0.6 0.9

Absorbance 0.3

0.0 0.6 300 400 500 600 700 Wavelength (nm)

Absorbance 0.3

0.0 300 400 500 600 700 Wavelength (nm)

Figure 2.17. UV–vis spectrum of the products of the reaction of Cbl(III) (HOCbl, 5.00 x –5 – 10 M) with 1.1 mol equiv PA in the presence of 50.0 mol equiv NO2 at pH 10.00 (25.0 °C, 0.10 M carbonate buffer). The product spectrum does not match with the spectrum of NOCbl (inset), but instead resembles the reactant HOCbl.

As discussed earlier, given that N2O does not react with Cbl(II), HNO, not N2O, must reduce Cbl(II) to Cbl(I)–. Therefore the observed rate of HNO dimerization is  5 times slower than the reduction of Cbl(II) by HNO, since both of these HNO–consuming reactions compete with each other. Complete conversion of Cbl(II) to NOCbl requires the addition of 2.0 mol equiv PA (= 2HNO), whereas upon the addition of 1.0 mol equiv PA, the product mixture consists of a 1:1 mixture of unreacted Cbl(II) and NOCbl. Given that the Cbl(I)– is never observed experimentally, all reactions subsequent to the reduction of

Cbl(II) by HNO must also therefore be significantly faster than the reduction of Cbl(II)

– by HNO. Hence oxidation of Cbl(I) by N2O is unlikely, since formation of N2O first

requires (slow) HNO dimerization, and would result in observation of the Cbl(I)– intermediate upon the addition of 1.0 mol equiv PA, which is clearly not experimentally observed. Furthermore, upon the direct addition of 1.0 mol equiv PA (= HNO) to Cbl(I)–, complete conversion to a Cbl(II) intermediate is observed, which subsequently reacts to ultimately yield NOCbl upon the addition of more PA. This means that the oxidation of

– Cbl(I) by the nitrogen oxide species (N2O or HNO) is at least 5 times faster than the reduction of Cbl(II) by HNO, which is again consistent with HNO, not N2O oxidizing

– Cbl(I) , since dimerization of HNO to give N2O is slow relative to the latter reduction reaction.

2.4 SUMMARY

Kinetic and mechanistic studies have been carried out on the reaction of Cbl(II) with the HNO donor Piloty’s acid, to form NOCbl. A stoichiometry of 1:2 Cbl(II):PA was observed. The rate–determining step involves PA decomposition to give HNO and benzenesulfinate. HNO then reduces Cbl(II) to Cbl(I)– and is itself oxidized to NO. The

Cbl(I)– intermediate is, in turn, oxidized back to Cbl(II) by a second molecule of HNO and the Cbl(II) and NO radicals subsequently rapidly combine to form NOCbl. The reaction between Cbl(I)– and PA involves an additional step in which Cbl(I)– is first oxidized by HNO to Cbl(II), which reacts further with HNO. Experiments in the presence of nitrite and kinetic and stoichiometric data for the reaction of Cbl(I)– with PA (1:3

Cbl(I)–:PA) confirm the involvement of a Cbl(I)– intermediate. The oxidation of a transition metal center by HNO which may occur for this system is, to our knowledge, unprecedented. Given that about one third of proteins are metalloproteins, our results

have important implications in regards to elucidating the potential roles and toxicity of

HNO in biological systems.

CHAPTER 3

KINETIC STUDIES ON THE REACTION BETWEEN AQUACOBALAMIN AND

THE HNO DONOR PILOTY’S ACID

3.1 INTRODUCTION

Piloty’s acid (PA), also known as N–hydroxybenzenesulfonamide or benzenesulfohydroxamic acid [202], belongs to the family of N–hydroxysulfonamides.

First synthesized by Piloty in 1896 [140], PA is a commonly used HNO donor in alkaline solution [139]. HNO is the one–electron–reduction product of the gasotransmitter NO and has biological and pharmacological properties distinct from those of NO [116, 182–

185]. Therefore, HNO donor compounds may have a unique therapeutic potential different from NO. However, the use of PA as an HNO donor under physiological conditions is limited by its very slow decomposition rate (t1/2 = 5500 min at pH 7) [139].

The pKa of PA is 9.26 [139] and decomposition of PA requires its deprotonation, Scheme

1.7. The rate of PA decomposition increases with increasing pH from 8 to 10 and becomes pH independent at higher pH values [139]. Recently, syntheses of novel derivatives of PA have been reported and their HNO releasing properties under physiological conditions evaluated [149, 203].

In this chapter, we present kinetic studies on the reaction of cob(III)alamin

(Cbl(III) = aquacobalamin/hydroxocobalamin, H2OCbl/HOCbl; pKa = 7.8 [10, 11] with the HNO donor PA.

55 56

3.2 EXPERIMENTAL SECTION

3.2.1 REAGENTS

Hydroxocobalamin hydrochloride (HOCbl•HCl, 98%) was purchased from Fluka.

The percentage of water in HOCbl•HCl (•nH2O) was determined following the procedure described in Section 2.2.1. Piloty’s acid (PA, 98%) was purchased from Cayman

Chemical Company and used without further purification. All other chemicals were purchased from various suppliers as described in Section 2.2.1.

3.2.2 INSTRUMENTATION

pH measurements were carried out as described in Section 2.2.2. The electrodes were standardized with standard buffer solutions at pH 7.00, 10.00 and 12.45. Solution pH was adjusted using H3PO4 or NaOH solutions as necessary.

1H NMR spectroscopic measurements were performed as described in Section

2.2.2. Air–tight J–Young NMR tubes (Wilmad, 535–JY–7) were used for 1H NMR measurements under anaerobic conditions. Reaction mixtures were left to equilibrate for

15 min prior to 1H NMR measurements.

UV–vis spectroscopic measurements were performed as described in Section

2.2.2. Anaerobic measurements were carried out in Schlenk cuvettes. All the anaerobic reactant solutions were prepared and stored inside the glove box.

3.2.3 DETERMINATION OF THE RATE CONSTANTS FOR SPONTANEOUS

DECOMPOSITION OF PILOTY’S ACID

Rate constants for the decomposition of PA under anaerobic conditions at various pH values were determined as described in Section 2.2.5.

3.2.4 SAMPLE PREPARATION FOR KINETIC MEASUREMENTS

All samples were prepared under strictly anaerobic conditions inside the glove box. Stock Cbl(III) solutions were prepared by dissolving solid HOCbl•HCl in the appropriate buffer. Stock PA solutions were prepared by dissolving PA in anaerobic

CH3OH and further dilutions were made in anaerobic water. Stock solutions were stored in the freezer (–24 °C) inside the glove box and used within 24 h.

3.2.5 SAMPLE PREPARATION FOR 1H NMR SPECTROSCOPY STUDIES

For 1H NMR experiments, stock PA solutions were prepared in anaerobic deuterated methanol and added to the Cbl(III) solution in anaerobic buffer (pD 12.00, or

7.00, 0.10 M phosphate buffer). The reaction was allowed to proceed to completion inside the glove box and subsequently transferred to an air–free NMR tube before recording the 1H NMR spectra. TSP was added as the internal reference.

3.2.6 DETERMINATION OF THE STOICHIOMETRY OF THE REACTION

A series of vials were prepared in a glove box containing Cbl(III) (50.0 µM) and varying aliquots of a stock PA solution (0, 0.25–2.5 mol equiv PA) added. A high concentration of Cbl(III) (6.00 x 10–3 M) was used to make the reaction proceed more rapidly to completion (cobalamins undergo self–reduction under alkaline conditions when left in solution for several hours [205, 206]). After the addition of PA, the vials were quickly capped and wrapped with parafilm. The reaction was allowed to proceed to completion for 3 h. The product solutions were diluted to 1.00 x 10–4 M, transferred to

air–free cuvettes, equilibrated at 25.0 °C for at least for 15 min and UV–vis spectra recorded.

3.3 RESULTS AND DISCUSSION

3.3.1 KINETIC STUDIES ON THE REACTION OF COB(III)ALAMIN WITH

PILOTY’S ACID (pH ≥ 10.00)

Initial experiments were carried out at pH 12.00. With the addition of PA to solution of Cbl(III) (HOCbl) at pH 12.00 under strictly anaerobic conditions, formation of NOCbl product was indicated by the change in the color of the reaction solution from red to orange. Figure 3.1 shows typical UV–vis spectra as a function of time for the reaction between HOCbl (5.00 x 10–5 M) and excess PA (1.50 x 10–3 M) under anaerobic

1.04 1.2 1.00 0.96 0.92 0.88 0.9 0.84 0.80 Abs at Abs 356 nm 0.76 0.72 0.6 0 100 200 300 400 500 600 Time (min)

Absorbance 0.3

0.0 300 350 400 450 500 550 600 Wavelength (nm) Figure 3.1. UV–vis spectra for the reaction between HOCbl (5.00 x 10–5 M) and excess PA (1.50 x 10–3 M) as a function of time under anaerobic conditions at pH 12.00 (25.0

°C, 0.10 M phosphate buffer, I = 1.0 M, NaCF3SO3). Inset: Plot of absorbance at 356 nm versus time for the same reaction. Spectra were recorded every 1 min.

conditions at pH 12.00 (25.0 °C, 0.10 M phosphate buffer, I = 1.0 M, NaCF3SO3). The conversion of HOCbl (λmax = 357, 420 and 435 nm) to NOCbl (λmax = 320 and 479 nm) is confirmed by the isosbestic points occurring at 338, 372 and 498 nm. These values are in close agreement with literature values for Cbl(III) to NOCbl conversion [64]. However, from the plot of absorbance at 356 nm versus time (inset to Figure 3.1) it is clear that more than one reaction is occurring. This is not surprising because the PA decomposition

– product, C6H5SO2 , also reacts with Cbl(III) to give benzenesulfinatocobalamin, Figure

3.2.

– In order to minimize the interference from the C6H5SO2 in the reaction of

Cbl(III) with PA, kinetic studies of the reaction between Cbl(III) and PA were instead carried out with at least 7 times excess Cbl(III) compared to the concentration of PA,

3.0

2.5

2.0

1.5

1.0 Absorbance

0.5

0.0 300 350 400 450 500 550 600 Wavelength (nm) Figure 3.2. UV–vis spectra obtained from the reaction of Cbl(III) (1.00 x 10–4 M) with –3 excess sodium benzenesulfinate (1.00 x 10 M) in water. Cbl(III) (λmax = 350, 410 and

525 nm) is converted to benzenesulfinatocobalamin (λmax = 333, 367, 424 and 528 nm) with clean isosbestics occurring at 343, 358 439 and 535 nm.

rather than with PA in excess. Under these conditions, the maximum concentration of benzenesulfinate produced is the same as the PA concentration. We utilized a similar approach to minimize the interference of the byproduct of HNO donor decomposition,

– NO2 , in the reaction between Cbl(III) and Angeli’s salt (Chapter 5) [65].

Figure 3.3 shows UV–vis spectra as a function of time for the reaction of PA

(1.40 x 10–5 M) with excess Cbl(III) (1.00 x 10–4 M) at pH 12.00 (25.0 °C, 0.10 M phosphate buffer, I = 1.0 M, NaCF3SO3). The isosbestic points were same as those observed for the reaction of Cbl(III) with excess PA at pH 12.00 [64]. Note that a small absorbance change was observed due to the reaction being incomplete. The fit of the absorbance data at 435 nm versus time to a first–order rate equation gives an observed

–4 –1 rate constant (kobs) = (2.52 x 0.01) x 10 s , Figure 3.4a. Interestingly, the rate of

2.1

1.8

1.5

1.2

0.9

Absorbance 0.6

0.3

0.0 300 350 400 450 500 550 600 Wavelength (nm) Figure 3.3. UV–vis spectra for the reaction between PA (1.40 x 10–5 M) and excess Cbl(III) (1.00 x 10–4 M) at pH 12.00 ± 0.03 under anaerobic conditions (25.0 °C, 0.10 M phosphate buffer, I = 1.0 M (NaCF3SO3)). Isosbestic points occur at 342, 372 and 498 nm.

reaction remains almost constant with the five–fold increase in concentration of Cbl(III),

–4 –1 kobs = (2.99 x 0.01) x 10 s , Figure 3.4b. This indicates that the reaction of Cbl(III) with

PA at pH 12.00 is zero–order to the concentration of Cbl(III) – that is, the reaction is not dependent on the Cbl(III) concentration.

The rate of PA decomposition at pH 12.00 ((2.95 x 0.01) x 10–4 s–1, Figure 3.5) is similar to that of the reaction of Cbl(III) with PA at pH 12.00. Therefore, the rate

– determining step is the rate of decomposition of PA to give HNO and C6H5SO2 .

Similar kinetic measurements were carried out for the reaction of Cbl(III) with PA at pH 10.00. Once again, the rate of the reaction is not dependent on the concentration of

Cbl(III), Figure 3.6. The average observed reaction rate (kobs) was found to be (9.05 ±

1.36) x 10–4 s–1. This reaction rate is more than 2 times faster than the rate of

–4 –1 decomposition of PA (kPA = 3.47 x 10 s ) at pH 10.00 [139]. A possible explanation for

0.43 a) 1.85 b) 1.80 0.42 1.75 0.41 1.70

0.40 1.65 Abs at 435 nm Abs Abs at 435 nm 0.39 1.60 1.55 0 2000 4000 6000 8000 0 2000 4000 6000 8000 Time (s) Time (s) Figure 3.4. Plot of absorbance at 435 nm versus time for the reaction of a) 1.4 x 10–5 M PA with 1.00 x 10–4 M Cbl(III) and b) 7.1 x 10–5 M PA with 5.00 x 10–4 M Cbl(III) at pH

12.00 ± 0.03 (25.0 °C, 0.10 M phosphate buffer, I = 1.0 M, NaCF3SO3), giving kobs = (2.52 x 0.01) x 10–4 s–1 for reaction a) and (2.99 x 0.01) x 10–4 s–1 for reaction b), respectively.

0.70

0.65

0.60

0.55

0.50

0.45 Abs at 250 nm 0.40

0.35 0 50 100 150 200 Time (min) Figure 3.5. Plot of absorbance at 250 nm versus time for the spontaneous decomposition of PA (2.00 x 10–4 M) at pH 12.00 ± 0.03 (25.0 °C, 0.10 M phosphate buffer, I = 1.0 M

(NaCF3SO3)) under strictly anaerobic conditions. The first–order fit of the data gives kobs = (2.95 ± 0.01) x 10–4 s–1. this is that a direct reaction of PA with Cbl(III) also occurs at this pH condition in addition to HNO reacting with HOCbl (see Section 3.3.2). We also observed this type of behavior in the reaction of Cbl(III) with Angeli’s salt (Chapter 5) [65].

The stoichiometry of the reaction between Cbl(III) and PA at pH 12.00 was also determined. Figure 3.7a gives UV–vis spectra of equilibrated anaerobic solutions of

Cbl(III) with PA (0–2.5 mol equiv) at pH 12.00. Cbl(III) was cleanly converted to NOCbl with isosbestic points at ~ 344, 371 and 502 nm, in agreement with literature values [64].

Figure 3.7b gives the corresponding plot of absorbance at 440 nm versus the mol equiv of

PA for the data shown in Figure 3.7a. This plot shows that the stoichiometry of the reaction between Cbl(III) and PA is 1:1 Cbl(III):PA.

0.006

0.005 )

-1 0.004 s (

obs 0.003 k

0.002

0.001

0.0002 0.0004 0.0006 0.0008 [Cbl(III)] (M)

Figure 3.6. Plot of kobs versus [Cbl(III)] for the reaction of PA with excess Cbl(III) (100 – 700 µM) at pH 10.00 ± 0.03 (25.0 °C, 0.10 M carbonate buffer, I = 1.0 M (NaCF3SO3)) under anaerobic conditions. The plot shows that the reaction rate is independent of the –4 –1 concentration of Cbl(III) and gives kobs = (9.05 ± 1.36) x 10 s .

The 1:1 stoichiometry and the formation of NOCbl and benzenesulfinate were confirmed by 1H NMR spectroscopy, Figure 3.8. The chemical shifts values for NOCbl match well with literature values [68] whereas those for benzenesulfinate are identical to the chemical shift values for authentic sodium benzenesulfinate. The 1H NMR spectrum of the products of the reaction between Cbl(III) (6.00 x 10–3 M) and 0.5 mol equiv PA at pD 12.00 under anaerobic conditions is shown in Figure 3.9. The NOCbl product and unreacted HOCbl were observed.

0.8 2.5 a) b)

2.0 0.7

1.5 0.6

1.0 0.5 Absorbance

0.5 atAbs440 nm 0.4

0.0 0.3 300 400 500 600 0.0 0.5 1.0 1.5 2.0 2.5 Wavelength (nm) Mol Equiv of PA

Figure 3.7. a) UV–vis spectra for equilibrated anaerobic solutions of Cbl(III) with 0, 0.25–2.50 mol equiv PA at pH 12.00 (25.0 °C, 0.10 M phosphate buffer). The concentration of Cbl(III) was 6.00 x 10–3 M and the product solutions were diluted to 1.00 x 10–4 M Cbl(III) inside the glove box before recording the UV–vis spectra. b) Plot of absorbance at 440 nm versus mol equiv PA for the same reaction.

8.4 8.2 8.0 7.8 7.6 7.4 7.2 7.0 6.8 6.6 Chemical shift (ppm)

7.8 7.6 7.4 7.2 7.0 6.8 6.6 6.4 6.2 6.0 Chemical shift (); ppm Figure 3.8. Aromatic region of the 1H NMR spectrum of the products of the reaction between HOCbl (5.00 x 10–3 M) and 1.1 mol equiv PA at pD 12.00 (0.10 M phosphate buffer) under anaerobic conditions. The peaks at 7.43, 7.20, 6.79, 6.35 and 6.25 ppm correspond to NOCbl and those at 7.66, 7.65, 7.56 and 7.53 ppm correspond to benzenesulfinate. Inset: 1H NMR spectrum (aromatic region) for authentic sodium benzenesulfinate (δ = 7.67, 7.65, 7.55 and 7.53 ppm).

7.8 7.6 7.4 7.2 7.0 6.8 6.6 6.4 6.2 6.0 Chemical shift (); ppm Figure 3.9. Aromatic region of the 1H NMR spectrum of the products of the reaction between HOCbl (6.00 x 10–3 M) and 0.5 mol equiv PA at pD 12.00 (0.10 M phosphate buffer) under anaerobic conditions. The peaks at 7.66, 7.65, 7.55 and 7.53 ppm correspond to benzenesulfinate, those at 7.40, 7.21, 6.78, 6.34 and 6.25 ppm correspond to NOCbl and those at 7.17, 6.73, 6.50, 6.25 and 6.06 ppm correspond to unreacted HOCbl.

The proposed mechanism for the reaction between Cbl(III) and PA at pH ≥ 10 is shown in Scheme 3.1. The rate–determining step is decomposition of deprotonated PA to

3 – give benzenesulfinate and NO . The pKa of HNO is estimated to be 11.4 [111]; hence

3NO– exists in solution in equilibrium with 1HNO in the pH 10–12 range. HNO is a strong reducing agent, and has been shown to reduce Cbl(II) to Cbl(I)– (see Chapter 2). Given that Cbl(III) exists as HOCbl and the hydroxo group of HOCbl is substitution inert [207,

208], it is likely that 3NO– (and potentially also 1HNO; 3NO– is expected to be the stronger reducing agent of the two species) reduces HOCbl to Cbl(II) and is oxidized to

NO. Cbl(II) and NO then react rapidly to form NOCbl.

Scheme 3.1. Proposed mechanism for the reaction between Cbl(III) and PA (pH ≥ 10.00). 1HNO may also potentially reduce HOCbl to give Cbl(II)● and ●NO.

3.3.2 KINETIC STUDIES ON THE REACTION OF COB(III)ALAMIN WITH

PILOTY’S ACID (pH < 10.00)

The reaction between Cbl(III) and PA at lower pH conditions (pH < 10.00) showed different kinetic behaviour. Once again, excess Cbl(III), instead of excess PA was used to minimize the interference from benzenesulfinate. The reaction of PA with excess Cbl(III) is dependent on the Cbl(III) concentration in the pH 5.10–8.00 region. In order to check that benzenesulfinate has little effect on the observed rate constant (kobs),

–4 –5 kobs for the reaction between the reaction of Cbl(III) (3.00 x 10 M) with PA (4.20 x 10

M) was determined in the presence and absence of additional benzenesulfinate (4.20 x

–5 –3 –1 –3 –1 10 M). The kobs values were similar (3.24 x 10 s versus 3.56 x 10 s , respectively in the presence and absence of benzenesulfinate); hence the effect of the benzenesulfinate byproduct on the observed rate constant is minimal. Figure 3.10 shows a typical plot of absorbance at 435 nm versus time for the reaction between excess Cbl(III) (4.00 x 10–4

M) and PA (5.70 x 10–5 M) at pH 8.00 ± 0.03 (25.0 °C, 0.20 M phosphate buffer, I = 1.0

2.0

1.8

1.6 Absat 435 nm

1.4

0 150 300 450 600 750 Time (s) Figure 3.10. Plot of absorbance at 435 nm versus time for the reaction between excess Cbl(III) (4.00 x 10–4 M) and PA (5.70 x 10–5 M) at pH 8.00 ± 0.03 (25.0 °C, 0.20 M phosphate buffer, I = 1.0 M (NaCF3SO3)). The first–order fit of the data gives kobs = (5.29 ± 0.01) x 10–3 s–1.

–3 –1 M (NaCF3SO3)). The first–order fit of the data gives kobs = (5.29 ± 0.02) x 10 s .

Similar experiments were carried out for a range of Cbl(III) concentrations at pH 8.00 and the data are summarized in Figure 3.11. The slope of the plot of kobs versus Cbl(III) concentration (Figure 3.11) gives an apparent second–order rate constant (kapp) = 13.2 ±

0.1 M–1 s–1. The reaction of PA with at least 7 times excess Cbl(III) was also studied at pH 6.00. Figure 3.12 shows the plot of kobs versus Cbl(III) concentration, and the slope of

–1 –1 plot gives kapp = 10.7 ± 0.2 M s . This value is similar to that obtained for the reaction

–1 –1 at pH 8.00. Surprisingly, the value of kapp drops to 1.42 M s pH 5.10 and no reaction of Cbl(III) with PA was observed at pH 4.00. These data are summarized in Figure 3.13.

The stoichiometry of the reaction of Cbl(III) with PA was determined at pH 6.00 under strictly anaerobic conditions. From the spectra of the equilibrated solutions of the

0.006

0.004 ) -1 s (

obs k 0.002

0.000 0.0000 0.0001 0.0002 0.0003 0.0004 + [H2OCbl ]

Figure 3.11. Plot of kobs versus [Cbl(III)] for the reaction between Cbl(III) and PA at pH 8.00 ± 0.03 (25.0 °C, 0.20 M phosphate buffer, I = 1.0 M (NaCF3SO3)). The data have been fitted to a straight line passing through the origin with a slope (= kapp) of 13.2 ± 0.1 M–1 s–1. products of the reaction between Cbl(III) and different mol equiv of PA, a plot of absorbance at 440 nm versus mol equiv of PA was generated, Figure 3.14. The stoichiometry was 1:1 Cbl(III):PA. Formation of NOCbl and a 1:1 Cbl(III):PA stoichiometry were confirmed using 1H NMR spectroscopy at pD 7.00 (Figure 3.15).

It is not clear to us why the apparent second–order rate constant decreases with

+ decreasing pH, given that neither PA nor H2OCbl alter their ionization state

(protonation/deprotonation) in this pH region. Under the conditions of the direct reaction

+ for the Cbl(III)/AS system, the pH dependence of kapp can be explained by H2OCbl , not

– HOCbl, reacting with AS (HN2O3 ) (see Chapter 5). However, this is clearly not the case for Cbl(III)/PA system, since kapp decreases with decreasing pH.

0.008

0.006 ) -1

s 0.004 (

obs k 0.002

0.000 0.0000 0.0002 0.0004 0.0006 0.0008 + [H2OCbl ] (M)

Figure 3.12. Plot of kobs versus [Cbl(III)] for the reaction of PA with excess Cbl(III) ((1.00 – 7.00) x 10–4 M) at pH 6.00 ± 0.03 (25.0 °C, 0.30 M phosphate buffer, I = 1.0 M

(NaCF3SO3)) under anaerobic conditions. The data have been fitted to a straight line –1 –1 passing through the origin with a slope (= kapp) of 10.7 ± 0.2 M s .

12 )

-1 9 s -1 M ( 6 app k 3

0 4 5 6 7 8 9 pH

Figure 3.13. Plot of the apparent second–order rate constant (kapp) versus pH for the reaction between Cbl(III) and PA.

0.750

0.625

0.500

Abs Abs at 440 nm 0.375

0.250 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Mol Equiv of PA Figure 3.14. Plot of absorbance at 440 nm versus mol equiv of PA for the reaction of Cbl(III) with PA at pH 6.00 (25.0 °C, 0.20 M phosphate buffer) under anaerobic conditions. Concentration of Cbl(III) was 6.00 x 10–3 M and the product solutions were diluted to 1.00 x 10–4 M after 3 h reaction inside the glove box before recording UV–vis spectra.

7.6 7.4 7.2 7.0 6.8 6.6 6.4 6.2 Chemical shift (); ppm Figure 3.15. Aromatic region of the 1H NMR spectrum of the products of the reaction between HOCbl (6.00 x 10–3 M) and 1.1 mol equiv PA at pD 7.00 (0.10 M phosphate buffer) under anaerobic conditions. The peaks at 7.66, 7.64, 7.54 and 7.53 correspond to benzenesulfinate, those at 7.44, 7.20, 6.78, 6.34 and 6.26 ppm correspond to NOCbl.

3.4 SUMMARY

To summarize kinetic experiments show that the reaction of Cbl(III) with PA proceeds via two different mechanisms depending upon the pH conditions used. At pH ≥ – 10.00, decomposition of PA to give HNO (and C6H2SO2 ) is the rate–determining step and NO– (and perhaps also HNO) reduces HOCbl to form Cbl(II) and NO, which rapidly combine to form NOCbl, Scheme 3.1. For pH < 10.00, however, PA directly reacts with – Cbl(III) to form the final products NOCbl and C6H2SO2 and kapp decreases with decreasing pH. In the latter case it was not possible to propose a mechanism based on the experimental data.

CHAPTER 4

STUDIES ON THE REACTION OF THE REDUCED VITAMIN B12 COMPLEX

COB(II)ALAMIN WITH THE HNO DONOR ANGELI’S SALT

4.1 INTRODUCTION

Reduced cobalamins, viz. cob(II)alamin and cob(I)alamin have important biological significance as all cobalamins are reduced by reductases once they enter the cells [20]. Cbl(II) takes part in the isomerization of methylmalonyl–CoA to succinyl–

CoA, an important substrate in citric acid cycle [31]. Cbl(II) is formed by homolysis of the Co–C bond of AdoCbl, a cofactor in this enzymatic transformation. Similarly,

– protein–bound Cbl(I) , the super–reduced form of B12, is a short–lived precursor of the two cofactor forms of B12, MeCbl and AdoCbl.

Nitroxyl (HNO) is a one–electron–reduction product of signaling molecule, NO.

Reactions of HNO with transition metal complexes have attracted considerable attention in literature. HNO has shown to reduce the transition metal centers (M = Fe3+, Mn3+), itself being oxidized to NO (reductive nitrosylation) [158–165, 168]. In Chapter 2, we have presented a detailed study on the reaction between cob(II)alamin and HNO donor

Piloty’s acid. HNO was found to oxidize Cbl(I)– to Cbl(II). To our knowledge, the oxidation of a TM center by HNO has not previously been reported. In this chapter kinetic studies have been carried out on the reaction of Cbl(II) with another HNO donor,

72 73

Angeli’s salt. Data is presented showing that cob(I)alamin is an reaction intermediate and once again, the Cbl(I)– intermediate is oxidized by HNO back to Cbl(II).

4.2 EXPERIMENTAL SECTION

4.2.1 REAGENTS

Hydroxocobalamin hydrochloride (HOCbl•HCl, 98%) was purchased from Fluka.

Percentage of water in HOCbl•HCl (•nH2O) (10–15% water, batch dependent) was determined using the procedure described in Section 2.2.1. Angeli’s salt (AS) was either purchased from Cayman Chemical (and used without further purification) or synthesized using a published procedure [139]. The purity of AS synthesized in our lab was checked

–3 –1 –1 by UV–vis spectroscopy (ε248 nm = 8.30 x 10 M cm in 0.01 M NaOH [139]) and was and found to be ≥ 99%. All other chemicals were purchased from Fisher Scientific, Acros

Organics, Sigma Aldrich or RICCA Chemical as described in Section 2.2.1.

4.2.2 INSTRUMENTATION

pH measurements were carried out as described in Section 2.2.2. The electrodes were standardized with standard buffer solutions at pH 4.00, 7.00, 10.00 and 12.45.

Solution pH was adjusted using H3PO4 or NaOH solutions as necessary.

1H NMR spectroscopic measurements were performed as described in Section

2.2.2. Air–tight J–Young NMR tubes (Wilmad, 535–JY–7) were used for 1H NMR measurements under anaerobic conditions. Reaction mixtures were left to equilibrate for

15 min prior to 1H NMR measurements.

UV–vis spectroscopic measurements were performed as described in Section

2.2.2. Anaerobic measurements were carried out in Schlenk cuvettes. All the anaerobic reactant solutions were prepared and stored inside the glove box.

4.2.3 SOLUTION PREPARATIONS

All solutions were prepared using standard biological buffers and phosphate buffers (0.05–0.30 M) and a constant ionic strength was maintained using sodium triflate

(NaCF3SO3; I = 1.0 M).

4.2.4 SYNTHESIS OF COB(II)ALAMIN AND COB(I)ALAMIN

These complexes were synthesized as described in Section 2.2.3.

4.2.5 DETERMINATION OF COBALAMIN CONCENTRATIONS

Cobalamin concentrations were determined either by using their extinction coefficients or by converting them to dicyanocobalamin as described in Section 2.2.4.

4.2.6 DETERMINATION OF RATE CONSTANTS FOR THE SPONTANEOUS

DECOMPOSITION OF ANGELI’S SALT

Rate constants for the spontaneous decomposition of Angeli’s salt as a function of pH under anaerobic conditions were measured using UV–vis spectrophotometry by following the decay in the Angeli’s salt absorbance at 245 nm. The reaction was initiated by adding an aliquot (0.050 mL, 6.00 mM) of a stock solution of AS in 10.0 mM NaOH to a cuvette containing buffer (2.95 mL) which had been thermostated in the cell holder of the Cary 5000 spectrophotometer. CAPS, CHES and TAPS buffers (0.30 M) were

used, and the total ionic strength was maintained at 1.0 M (NaCF3SO3). The absorbance at 245 nm versus time data were fitted to a first–order rate equation.

4.2.7 SAMPLE PREPARATION FOR KINETIC MEASUREMENTS ON THE

REACTION OF COB(II)ALAMIN WITH ANGELI’S SALT

All samples were prepared under strictly anaerobic conditions inside the glove box. Stock Cbl(II) solutions were prepared by dissolving solid Cbl(II) in the appropriate buffer. Stock AS solutions were prepared by dissolving solid AS in NaOH (0.01 M).

Stock solutions were stored in the freezer (–24 °C) inside the glove box and used within

24 h.

4.2.8 SAMPLE PREPARATION FOR KINETIC MEASUREMENTS ON THE

REACTION OF COB(I)ALAMIN WITH ANGELI’S SALT

All samples were prepared under strictly anaerobic conditions inside the glove

– – box. Stock Cbl(I) solutions were prepared in H2O (Cbl(I) undergoes slow oxidation to

Cbl(II) in presence of buffers), stored under strictly anaerobic conditions at –24 °C and used within 1 week of preparation. Since Cbl(I)– is extremely air–sensitive and undergoes self–oxidation to Cbl(II) at lower concentrations even in the absence of oxygen, high concentrations (≥ 2.00 x 10–4 M) were used in all kinetic experiments. Low buffer concentrations (5–10 mM) were used to control the pH. Stock AS solutions were prepared and handled as described in Section 4.2.6.

4.2.9 DETERMINATION OF STOICHIOMETRY OF THE REACTION

BETWEEN COB(II)ALAMIN AND ANGELI’S SALT

In the glove box, a series of vials were prepared containing a fixed volume of

Cbl(II) solution (5.00 x 10–5 M) and an aliquot of a stock AS solution (prepared in 0.01

M NaOH) was added to achieve various mol equiv AS (0, 0.25–3.5 mol equiv; pH 8.00).

The total volume of final solution in each vial was 3.00 mL. After the addition of AS, the vials were capped and wrapped with parafilm to minimize evaporation. The reactions were allowed to proceed to completion for at least 5 half–lives for the slowest reaction

(Cbl(II) + 0.25 mol equiv AS). The product solutions were systematically transferred to an air–free cuvette and the cuvette equilibrated at 25.0 °C in the cell compartment of the spectrometer for at least for 15 min prior to UV–vis measurements.

4.2.10 DETERMINATION OF STOICHIOMETRY OF THE REACTION

BETWEEN COB(I)ALAMIN AND ANGELI’S SALT

High concentrations of Cbl(I)– (2.00 x 10–4 M) were used to avoid the spontaneous self–oxidation of Cbl(I)– to Cbl(II). Varying aliquots of AS stock solutions

(0.0–5.0 mol equiv) were added to the vial containing Cbl(II) (2.00 x 10–4 M) in buffer, and the vials capped and wrapped with parafilm. The reaction was allowed to go to completion for ~ 4 h. The product solutions were equilibrated in an air–free cuvettes at

25.0 °C for at least 15 min prior to UV–vis measurements. A control experiment showed that Cbl(I)– is stable in solution for 4 h under the conditions of these experiments.

4.2.11 INDOOXINE TESTS TO DETERMINE IF HYDROXYLAMINE IS

FORMED IN THE REACTION OF COB(II)ALAMIN OR COB(I)ALAMIN WITH

ANGELI’S SALT

A modified literature procedure was used [190, 191]. The reaction of Cbl(II) (1.00 x 10–3 M) with 2.0 mol equiv AS at pH 8.00 (0.30 M CHES buffer) was allowed to proceed to completion under anaerobic conditions inside the glove box for ~ 1 h. Then aerobic 8–hydroxyquinoline solution (1.00 mL, 4.0% w/v in ethanol) and aqueous aerobic Na2CO3 solution (1.00 mL, 1.00 M) were added to the product solution (1.00 mL) outside the glove box. After standing for 45 min at room temperature, the UV–vis spectrum was recorded.

The stability of authentic NH2OH in pH 8.00 buffer was also checked using the same procedure. Our lab and others have shown that at lower pH conditions, NH2OH can undergo disproportionation [66, 192, 193]. In a typical experiment, NH2OH (50.0 µM) was prepared in TAPS buffer (0.30 M, pH 8.00). After 30 min, 8–hydroxyquinoline

(4.0% w/v in ethanol) and Na2CO3 (1.00 M) were added, the reaction allowed to proceed for 40 min and UV–vis spectra recorded (carried out in duplicate). From the absorbance at 710 nm the concentration of NH2OH was calculated to be > 49.0 µM (> 98% recovery); hence NH2OH is stable at pH 8.00. Finally, the indooxine test for the product solution of the reaction between NH2OH (50.0 µM) and 1.0 mol equiv AS at pH 8.00 shows that AS reacts with NH2OH. Only ~ 40.0 µM of NH2OH was recovered after 3 h of reaction.

4.2.12 NESSLER’S TEST TO DETERMINE WHETHER AMMONIA IS

FORMED IN THE REACTION OF COB(II)ALAMIN OR COB(I)ALAMIN WITH

ANGELI’S SALT

The reaction between Cbl(II) (5.50 x 10–3 M) and 2.2 mol equiv AS at pH 7.00

(5.00 x 10–3 M phosphate buffer) was allowed to proceed to completion in a vial for ~ 2.5 h inside the glove box. The product solution (1.00 mL) was taken outside the glove box and 8–10 drops Nessler’s reagent added under aerobic conditions. Formation of a brown colored precipitate indicates the presence of ammonia in the product solution [194].

However, a red precipitate formed at the bottom of the reaction vial. From similar experiments for Cbl(II) in the absence (red precipitate) or presence of ammonia (brown precipitate) it was clear that significant amounts of ammonia are not formed. Similar procedures were used to test for ammonia in the products of the reaction between Cbl(I)– and AS at pH 8.00. Once again, no evidence was found for ammonia production.

4.2.13 REACTION OF COB(II)ALAMIN WITH NITROUS OXIDE

Reaction of Cbl(II) with N2O was carried out as described in Section 2.2.14.

4.2.14 GRIESS ASSAY TO QUANTIFY THE AMOUNT OF NITRITE

PRODUCED IN THE REACTION OF COB(II)ALAMIN OR COB(I)ALAMIN

WITH ANGELI’S SALT AND IN ANGELI’S SALT ITSELF

Experiments were carried out under strictly anaerobic conditions following standard literature protocols [195]. A calibration plot of absorbance at 586 nm versus nitrite concentration was generated (see Figure 4.11). Note that the calibration plots were

found to be pH independent. Typically, an aliquot of Griess reagent (1.50 mL) was added to an equal volume of buffer (1.50 mL, 0.30 M TAPS buffer, pH 8.00) containing varying concentrations of nitrite (0, 20.0, 40.0, 60.0, 80.0 and 100.0 µM) and NOCbl (40.0 µM) and the absorbance at 586 nm was determined. An aliquot of the product of the reaction between Cbl(II) (25.0 µM) and 2.0 mol equiv AS at pH 8.00 was subjected to the same procedure. The resulting absorbance of 0.653 ± 0.003 at 586 nm corresponded to 48.5

– µM (1.94 equiv) NO2 produced. Similarly, an aliquot of the product of the reaction between Cbl(I)– (25.0 µM) and 2.25 mol equiv AS at pH 8.00 was subjected to the Griess

– assay procedure, and in this case 49.5 µM (~ 2.0 mol equiv) NO2 was recovered.

The Griess assay was also carried out to test the presence of nitrite in the commercially available AS and in AS synthesized in our lab. Both AS samples were found to contain ~ 6% nitrite.

4.2.15 EXPERIMENTS IN THE PRESENCE OF CYANIDE TO PROBE

WHETHER COB(III)ALAMIN IS AN INTERMEDIATE OF THE REACTION

BETWEEN COB(II)ALAMIN WITH ANGELI’S SALT

Experiments were carried out to trap the potential cob(III)alamin intermediate of the reaction between Cbl(II) and AS. All experiments were carried out under strictly anaerobic conditions. UV–vis spectra were recorded as a function of time for the reaction of Cbl(II) (5.00 x 10–5 M) with 2.0 mol equiv AS in the presence of 20.0 mol equiv cyanide at pH 8.50 (25.0 °C, 0.10 M phosphate buffer). Spectral changes and the

isosbestic points of the spectra remained the same as those obtained from the reaction of

Cbl(II) with 2.0 equiv AS in the absence of cyanide, consistent with NOCbl formation.

Separate experiments showed that neither Cbl(II) nor NOCbl react with 20.0 mol

+ –5 equiv cyanide. Reacting H2OCbl /HOCbl (5.00 x 10 M) with cyanide (20.0 mol equiv) in the presence or absence of 1.0 mol equiv AS at pH 8.50 under anaerobic conditions results in CNCbl formation.

4.2.16 STOICHIOMETRY EXPERIMENTS IN THE PRESENCE OF EXCESS

NITRITE

To confirm the presence of Cbl(I)– as an intermediate in the reaction of Cbl(II) with AS, experiments were carried out in the presence of excess nitrite. UV–vis spectra were recorded for the product solution of the reaction between Cbl(II) (5.00 x 10–5 M)

– with 1.0 mol equiv AS in the presence of 5.0 mol equiv NO2 at pH 8.00 (5.0 mM TAPS buffer) under anaerobic conditions. The product spectrum is identical to that of NOCbl.

Similarly, the UV–vis spectrum of the product solution of the reaction between Cbl(I)–

–4 – (2.00 x 10 M) and 1.0 mol equiv AS in the presence of 20.0 mol equiv NO2 at pH 8.00

(5.0 mM TAPS buffer) under strictly anaerobic conditions is also identical with that for authentic NOCbl.

4.3 RESULTS AND DISCUSSION

4.3.1 KINETIC STUDIES ON THE REACTION BETWEEN COB(II)ALAMIN

AND ANGELI’S SALT (pH ≤ 9.00)

Kinetic studies on the reaction between Cbl(II) and AS at different pH conditions were carried out using UV–vis spectroscopy. The rate of AS decomposition to give HNO

– and NO2 is pH independent in the pH 4–8 range (t½ = 17 min at 25 °C [139]), and becomes increasing slower for pH > 8 [139]. Upon the addition of excess AS (4.00 x 10–4

M) to Cbl(II) (4.00 x 10–5 M) in buffer (pH 8.00) under strictly anaerobic conditions,

– Cbl(II) is cleanly converted to nitroxylcobalamin (NOCbl, NO –Cbl(III) [64, 170]; λmax =

256, 280 (shoulder), 289, 315 and 478 nm [64, 170]). The isosbestic points at 330, 387,

484 and 542 nm (Figure 4.1) are in agreement with literature values for the

Cbl(II)/NOCbl conversion [64, 170]. The corresponding plot of absorbance at 308 nm versus time is given in Figure 4.2. The linear trend (0–250 s) indicates that the rate of reaction is independent of the Cbl(II) concentration and suggests that the rate–

– determining step is decomposition of AS to give HNO (and NO2 ), followed by a rapid reaction between HNO and Cbl(II).

1.4

1.2

1.0

0.8

0.6

Absorbance 0.4

0.2

300 400 500 600 Wavelength (nm) Figure 4.1. Selected UV–vis spectra for the reaction between Cbl(II) (4.00 x 10–5 M) and excess AS (4.00 x 10–4 M) at pH 8.00 ± 0.03 (25.0 °C, 0.30 M TAPS buffer, I = 1.0 M

(NaCF3SO3)) under anaerobic conditions. Spectra are shown every 0.5 min.

1.14

1.08

1.02

Abs Abs at 308 nm 0.96

0.90 0 100 200 300 400 500 Time (s)

Figure 4.2. Plot of absorbance at 308 nm versus time for the reaction between Cbl(II) and excess AS at pH 8.00 as described in Figure 4.1.

In order to test this further, kinetic data were collected for the reaction of Cbl(II)

(4.00 x 10–5 M) with 1.0 mol equiv AS at pH 8.00. The absorbance at 308 nm versus time

data were fitted to a first–order rate equation giving an observed rate constant (kobs) =

(3.58 ± 0.01) x 10–2 min–1 (Figure 4.3). This rate constant is similar to the rate of

–2 –1 spontaneous decomposition of AS at pH 8.00 (kL = (3.06 ± 0.01) x 10 min ; L = ligand,

Table 4.1); hence decomposition of AS is the rate–determining step of the reaction between Cbl(II) and AS.

1.15

1.10

1.05 308 nm 1.00 Abs atAbs

0.95

0 20 40 60 80 100 Time (min)

Figure 4.3. Plot of absorbance at 308 nm versus time for the reaction of Cbl(II) (4.00 x 10–5 M) with 1.0 mol equiv AS at pH 8.00 (25.0 °C, 0.30 M TAPS buffer, I = 1.0 M

(NaCF3SO3)) under strictly anaerobic conditions. The data were fitted to a first–order rate –2 –1 equation giving the observed rate constant (kobs) = (3.58 ± 0.01) x 10 min .

Control experiments showed that the rate constant is unchanged upon addition of

–5 –2 the free metal scavenger DTPA (5.00 x 10 M) to the reaction mixture (kobs = 3.52 x 10 min–1 and 3.69 x 10–2 min–1 in the presence and absence of DTPA, respectively (1.0 mol equiv AS, pH 7.40, 0.10 M phosphate buffer, 25.0 °C, I = 1.0 M (NaCF3SO3)); hence free metals are not involved in the reaction. Studies were also carried out on the reaction

between Cbl(II) and AS at pH 7.00 and 9.00. NOCbl is formed under all conditions and the rate of the reaction was again found to be the same as that for AS salt decomposition at each pH condition, Table 4.1.

Table 4.1. Comparison of the observed rate constants for the reaction between Cbl(II)

and AS (kobs) and the spontaneous decomposition of AS (kL) as a function of pH under anaerobic conditions (25.0 °C, I = 1.0 M (NaCF3SO3)).

2 –1 a 2 –1 pH (± 0.03) kobs x 10 (min ) kL x 10 (min )

9.00 2.43 ± 0.01 2.40 ± 0.01 8.00 3.58 ± 0.01 3.06 ± 0.01

7.00 3.58 ± 0.02 3.25 ± 0.01

aDetermined at [Cbl(II)] = [AS] = 4.00 x 10–5 M

4.3.2 DETERMINATION OF THE STOICHIOMETRY OF THE REACTION

BETWEEN COB(II)ALAMIN AND ANGELI’S SALT (pH ≤ 9.00)

In order to probe the mechanism of the reaction, the stoichiometry of the reaction between Cbl(II) and AS at pH 8.00 was determined. From UV–vis spectra of equilibrated solutions of Cbl(II) (50.0 µM) with AS (0, 0.25–3.5 mol equiv), a plot of absorbance at

316 nm versus mol equiv of AS was generated, Figure 4.4. The absorbance at 316 nm decreases linearly up to 2.0 mol equiv of AS and is unchanged upon the further addition of AS. The stoichiometry of the reaction of Cbl(II) with AS at pH 8.00 is therefore 1:2

Cbl(II):AS.

2.0 1.7 a) b) 1.6 1.6 1.5 1.2 1.4

0.8 1.3 Absorbance 0.4 atAbs nm316 1.2

1.1 0.0 300 400 500 600 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Wavelength (nm) Mol Equiv of AS

Figure 4.4. a) UV–vis spectra for anaerobic equilibrated solutions of Cbl(II) (50.0 µM) with AS (0, 0.25–3.5 equiv) at pH 8.00 (TAPS buffer). b) Corresponding plot of absorbance at 316 nm versus mol equiv of AS.

The formation of NOCbl as cobalamin product was also confirmed by 1H NMR spectroscopy, Figure 4.5. The aromatic region of the 1H NMR spectrum of the products of the reaction between Cbl(II) and 2.2 mol equiv AS at pD 8.00 gives the chemical shift values, δ, = 7.41, 7.20, 6.79, 6.34, 6.27 ppm, Figure 4.5. These δ values match well with the literature values for NOCbl [68].

7.6 7.4 7.2 7.0 6.8 6.6 6.4 6.2 6.0 Chemical shift (), ppm Figure 4.5. Aromatic region of the 1H NMR spectrum of the products of the reaction between Cbl(II) (7.0 x 10–4 M) and 2.2 mol equiv AS at pD 8.00 (0.10 M TAPS buffer) under anaerobic conditions.

The unusual 1:2 Cbl(II):AS stoichiometry suggests that the reaction undergoes multiple steps involving intermediate species. The potential cobalamin reaction intermediates are Cbl(III) or Cbl(I)–. The redox potentials (E°) for the NO/HNO,

+ – 2H /NH2OH, Cbl(III)/Cbl(II) and Cbl(II)/Cbl(I) redox couples are –0.68 V (pH 7,

NHE), +0.30 V (pH 7, NHE), +0.19 V (pH 7.4, NHE) and –0.61 V (pH 7.2, NHE) respectively [25–27, 186, 197]. Hence, the oxidation of Cbl(II) by HNO to Cbl(III) (=

+ + H2OCbl /HOCbl; pKa(H2OCbl = 7.8 [10, 11]) and the reduction of Cbl(II) by HNO to

Cbl(I)– are both thermodynamically feasible processes.

4.3.3 COB(I)ALAMIN, NOT COB(III)ALAMIN, IS AN INTERMEDIATE OF

THE REACTION BETWEEN COB(II)ALAMIN AND ANGELI’S SALT (pH ≤

9.00)

Given that NOCbl has a single NO– ligand, a 1:1 Cbl(II):AS stoichiometry was anticipated. However, the reaction between Cbl(II) and AS is complete only after the addition of 2.0 mol equiv AS. Importantly, the same stoichiometry was observed for the

Cbl(II) + Piloty’s acid (PA) system and for this system it was found that the reaction proceeds via a cob(I)alamin intermediate (see Chapter 2). However studies on the Cbl(II)

+ PA system were carried out under alkaline solution since decomposition of PA to release HNO is very slow at pH < 8 [139]. Given that a substantial fraction of

+ cob(III)alamin (Cbl(III)) exists as aquacobalamin (H2OCbl /HOCbl; pKa 7.8 [10, 11]) rather than substitution–inert hydroxocobalamin under the pH conditions of this present

+ study, and that independent experiments have shown that H2OCbl reacts rapidly with AS to give NOCbl (see Chapter 5) [65], it is possible that the Cbl(II) + AS reaction could

proceed instead via a Cbl(III) intermediate. In order to probe this further, experiments

+ were carried out in the presence of an efficient H2OCbl trap, cyanide, which reacts

+ –1 rapidly and essentially irreversibly with H2OCbl to form cyanocobalamin (kf = 250 M

–1 12 –1 + s and K(CNCbl) ≥ 10 M [2, 209, 210]). If H2OCbl is produced as an intermediate of the reaction between Cbl(II) and AS in the presence of excess cyanide, CNCbl, not

NOCbl, would be the anticipated cobalamin product.

Figure 4.6 shows spectral changes which occur upon reacting Cbl(II) (5.00 x 10–5

M) with 2.0 mol equiv AS in the presence of 20.0 mol equiv cyanide (pH 8.50, 0.10 M phosphate buffer) under anaerobic conditions. The spectral changes and isosbestic points

(330, 385 and 484 nm) are identical to those observed in the absence of cyanide, and

NOCbl is formed. Importantly, control experiments showed that the presence of 20.0 mol

1.6 1.4 1.2 1.0 0.8 0.6

Absorbance 0.4 0.2 0.0 300 350 400 450 500 550 600 Wavelength (nm) Figure 4.6. UV–vis spectra as a function of time for the reaction between Cbl(II) (5.00 x 10–5 M) and 2.0 mol equiv AS in the presence of 20.0 equiv cyanide at pH 8.50 ± 0.02 (25.0 °C, 0.10 M phosphate buffer). Spectra are shown every 2 min.

equiv cyanide does not affect the rate of decomposition of AS (pH 8.00; kL = (4.76 ±

0.01) x 10–4 s–1 and (5.10 ± 0.01) x 10–4 s–1, in the presence and absence of cyanide, respectively), and that NOCbl does not react significantly with cyanide (Figure 4.7).

Furthermore, the reaction of Cbl(III) (5.00 x 10–5 M) with 20.0 mol equiv cyanide in the presence or absence of 1.0 mol equiv AS results in instantaneous formation of CNCbl

(λmax = 362 and 548 nm [63]), Figure 4.8. Finally, note that Cbl(II) does not react significantly with cyanide, Figure 4.9. The results of these multiple experiments do not support a Cbl(III) intermediate for the reaction of Cbl(III) with AS, despite the fact that

Cbl(III) reacts rapidly with AS with a 1:1 stoichiometry to form NOCbl (Chapter 5) [65].

+ Since experiments in the presence of the efficient H2OCbl trap cyanide suggest that Cbl(I)– is formed and consumed during the reaction between Cbl(II) and HNO, this suggests that Cbl(II) is reduced to Cbl(I)– by HNO, with concurrent oxidation of HNO to

1.0 1.0

0.8 0.8

0.6 0.6

0.4 0.4 Absorbance Absorbance 0.2 0.2

0.0 0.0 300 350 400 450 500 550 600 300 350 400 450 500 550 600 Wavelength (nm) Wavelength (nm)

Figure 4.7. UV–vis spectra as a function of time for NOCbl (5.00 x 10–5 M) in the a) absence and b) presence of 20.0 mol equiv cyanide at pH 8.50 (0.10 M phosphate buffer). Data were collected for 3 h under strictly anaerobic conditions. A small bump occurs at ~ 362 nm when excess cyanide is added to NOCbl.

NO. Cbl(I)– could subsequently react with the second HNO molecule to regenerate

Cbl(II), which rapidly reacts with NO to form NOCbl, Scheme 4.1. This scheme assumes

– that N2 is the product of HNO oxidation of Cbl(I) , which is shown later to likely be the case. The reaction between Cbl(II) and NO is very rapid and thermodynamically

8 −1 −1 8 −1 favorable (k = 7.4 × 10 M s , K(NOCbl) ≈ 1 × 10 M , 25 °C [64, 170].

To probe whether Cbl(I)– is indeed a reaction intermediate, the products of the reaction between Cbl(II) and 1.0 mol equiv AS in the presence of excess nitrite were determined. Previous experiments from our lab have established that Cbl(I)– is rapidly oxidized to Cbl(II) by nitrite [66]; hence it was anticipated that nitrite could replace the second HNO molecule as the Cbl(I)– oxidant, Scheme 4.1. This would mean that only 1.0

k 4HN O - L - 2 3 R.D.S. 4HNO + 4NO2 fast 2Cbl(II) + 2HNO 2Cbl(I)- + 2NO + 2H+ - + fast 2Cbl(I) + HNO + 2H 2Cbl(II) + NH2OH fast NH2OH + HNO N2 + 2H2O fast 2Cbl(II) + 2NO 2NOCbl - - 2Cbl(II) + 4HN2O3 2NOCbl + 4NO2 + N2 + 2H2O - - Or, Cbl(II) + 2HN2O3 NOCbl + 2NO2 + 0.5N2 + H2O Scheme 4.1. Proposed reaction pathway for the reaction of Cbl(II) with AS (pH ≤ 9.00). mol equiv AS would be required for full conversion of Cbl(II) to NOCbl by HNO in the presence of excess nitrite. This was observed experimentally, Figure 4.10, providing further support for a Cbl(I)– intermediate. Importantly, Cbl(I)– was also shown to be the intermediate of the reaction between Cbl(II) and another HNO donor, PA (a nitrite–free

HNO donor). The stoichiometry of 1:2 Cbl(II):PA was also changed to 1:1 Cbl(II):PA in

– the presence of excess NO2 (Chapter 2).

1.4 1.4 a) b) 1.2 1.2

1.0 1.0

0.8 0.8

0.6 0.6 Absorbance Absorbance 0.4 0.4

0.2 0.2

0.0 0.0 300 350 400 450 500 550 600 650 300 350 400 450 500 550 600 650 Wavelength (nm) Wavelength (nm) + –5 Figure 4.8. UV–vis spectra as a function of time for H2OCbl /HOCbl (5.00 x 10 M) with 20.0 mol equiv cyanide in the a) absence and b) presence of 1.0 mol equiv AS at pH 8.50 (0.10 M phosphate buffer) under strictly anaerobic conditions. Spectra were collected every 1 min for 60 min.

2.20

1.65

1.10

Absorbance 0.55

0.00 250 300 350 400 450 500 550 600 Wavelength (nm) Figure 4.9. UV–vis spectra of Cbl(II) (7.00 x 10–5 M) with 20.0 mol equiv cyanide at pH 8.50 (0.10 M phosphate buffer) under anaerobic conditions. Spectra were recorded for 2 h. The λmax characteristic of Cbl(II) (312, 405 and 475 nm) remain unchanged even after 2 h indicating that cyanide does not react appreciably with Cbl(II).

1.8 Cbl(II) only 1:2.1 Cbl(II):AS 1.5 1:5:1 Cbl(II):nitrite:AS 1.2

0.9

0.6 Absorbance 0.3

0.0 300 400 500 600 700 Wavelength (nm) Figure 4.10. UV–vis spectra of the product solution of reaction between Cbl(II) (5.00 x –5 – 10 M) and varying equiv of AS in the presence and absence of excess NO2 at pH 8.00 (25.0 °C, 0.30 M TAPS buffer). Cbl(II) (black dotted trace) is converted to NOCbl with 2.1 equiv of AS (red dashed trace) or with 1.0 mol equiv of AS in the presence of 5.0 – – equiv NO2 (blue solid trace). Note that NO2 absorbs strongly at 300–400 nm region.

The mechanism shown in Scheme 4.1 assumes that the nitrite released from AS decomposition is not of a sufficiently high concentration to oxidize Cbl(I)– to Cbl(II).

– Note that reaction of Cbl(II) with NO2 is negligible under the experimental conditions

[170]. The Griess assay is a well–established method to quantify nitrite [195]. A calibration plot of absorbance at 586 nm versus nitrite concentration (0–100 µM) was generated, Figure 4.11. The Griess assay was initially carried out to determine the amount of nitrite released upon decomposition of AS in the absence of Cbl(II). Upon

– decomposition of 50 µM AS, 51 ± 1 µM NO2 is recovered (two experiments). If 2.0 mol equiv AS is allowed to react with Cbl(II) (25 µM Cbl(II), 50 µM AS), 49 ± 1 µM nitrite

1.4

1.2

1.0

0.8

0.6

Abs at 586nm 0.4

0.2

0.0 0 20 40 60 80 100 [NaNO ]; M 2

Figure 4.11. Calibration plot for the reaction of NaNO2 (0–100 µM) with the Griess reagent in the presence of NOCbl (25.0 µM). NOCbl was added to the solutions; however it was subsequently shown that NOCbl does not absorb appreciably at 586 nm. is recovered (two experiments). Hence nitrite is not consumed in the reaction between

Cbl(II) and AS, Scheme 4.1.

4.3.4 IDENTIFICATION OF THE HNO REDUCTION PRODUCT(S)

Since the reaction pathway in Scheme 4.1 involves oxidation of Cbl(I)– by HNO,

HNO is reduced. Possible HNO reduction products from the reaction between Cbl(II) and

+ AS are NH2OH, NH4 and/or N2.

The indooxine test and Nessler’s test were independently performed to check the presence of NH2OH or NH3 respectively, in the product solution of the reaction between

Cbl(II) and 2.0 mol equiv AS at pH 8.00. Both tests were negative (see Sections 4.2.11 and 4.2.12). Importantly, a control experiment showed that NH2OH is stable at pH 8.00

(at lower pH conditions NH2OH can undergo disproportionation [66, 192, 193]). Results from these multiple experiments suggest that N2 is the HNO reduction product.

Experiments to detect nitrogen using Raman spectroscopy (Oregon Health and Science

University) and headspace gas analysis of product mixture (Kent State University) were unfortunately unsuccessful. Control experiments suggested that the small amounts of N2 in the product mixture were not sufficient for detection using this instrumentation. Note, however, that bubbles were clearly visible upon the addition of AS to Cbl(II), consistent with N2 formation.

Finally, it was of interest to see if N2O (the product of HNO dimerization) reacts with Cbl(II). The UV–vis spectrum of a solution of Cbl(II) exposed to excess N2O (20.0 mM) was identical to that for Cbl(II) (λmax = 405 and 475 nm [66, 199]). There is, therefore, no reaction between Cbl(II) and N2O, as expected based on work of others who

– – propose that Cbl(II) and N2O are the products of oxidation of Cbl(I) by NO (Cbl(I) +

+ NO + H → Cbl(II) + ½N2O + ½H2O) [198].

4.3.5 STUDIES ON THE REACTION BETWEEN COB(I)ALAMIN AND

ANGELI’S SALT

Given that Cbl(I)– is likely to be a reaction intermediate as observed for the

2 Absorbance 1

0 350 400 450 500 550 Wavelength (nm) Figure 4.12. UV–vis spectra as a function of time for the reaction of Cbl(I)– (2.00 x 10–4 M) with excess AS (4.5 mol equiv) at pH 7.40 (25.0 °C, 5.00 x 10–3 M phosphate buffer, I = 1.0 NaCF3SO3).

Cbl(II)/PA system, kinetic studies on the reaction between Cbl(I)– and AS were independently carried out. Figure 4.12 shows UV–vis spectra for the reaction of Cbl(I)–

(2.00 x 10–4 M) with excess AS (4.5 mol equiv) under anaerobic conditions at pH 7.40

–3 (25.0 °C, 5.00 x 10 M phosphate buffer, I = 1.0 M NaCF3SO3). High cobalamin concentrations were used to ensure that Cbl(I)– was stable in solution. All the product solutions were diluted prior to determining the nitrite concentration by UV–vis spectroscopy. The reaction is rapid, and follows two steps to give the final product,

– NOCbl. Upon addition of AS to the Cbl(I) solution (λmax = 388, 464 and 547 nm),

Cbl(II) is formed (λmax = 405 and 475 nm), which is subsequently converted to NOCbl

(λmax = 478 nm).

To confirm that Cbl(I)– is first oxidized to Cbl(II) which can react further with

HNO generated by AS decomposition, spectral scans were obtained for the reaction of

Cbl(I)– (2.00 x 10–4 M) with 0.25 mol equiv AS (pH 7.40, 25.0 °C, 5.00 x 10–3 M phosphate buffer), Figure 4.13. Since Cbl(I)– is in excess, one would expect that Cbl(I)– is

3.6

3.0

2.4

1.8

Absorbance 1.2

0.6

350 400 450 500 550 Wavelength (nm) Figure 4.13. UV–vis spectra as a function of time for the reaction of Cbl(I)– (2.00 x 10–4 M) with 0.25 mol equiv AS at pH 7.40 (25.0 °C, 5.00 x 10–3 M phosphate buffer, I = 1.0

NaCF3SO3).

oxidized cleanly to Cbl(II) and that the amount of AS is insufficient for NOCbl formation. Figure 4.13 shows the clean conversion of Cbl(I)– to Cbl(II) with isosbestic points occurring at 417 and 542 nm, which are in good agreement with literature values for Cbl(I)–/Cbl(II) conversion [66, 199]. The absorbance at 475 nm versus time data for the reaction of Cbl(I)– (2.00 x 10–4 M) with 0.25 equiv of AS at pH 8.00 (25.0 °C, 5.00 x

–3 10 M TAPS buffer, I = 1.0 M (NaCF3SO3)) fit well to a first–order rate equation giving

–2 –1 kobs = (4.33 ± 0.04) x 10 min , Figure 4.14. This kobs value is slightly higher than that

–2 –1 for AS decomposition at pH 8.00 (kAS = (3.25 ± 0.01) x 10 min , Table 4.1). This is not

– – surprising because Cbl(I) reacts rapidly with the NO2 produced from the AS decomposition. To confirm this, the amount of AS was reduced to 0.04 mol equiv (7 times less than stoichiometric amount). As expected, the reaction becomes slower (kobs =

2.0 1.8 1.6 1.4 1.2 1.0

Absat 475 nm 0.8 0.6 0.4 0 20 40 60 80 100 120 140 Time (min) Figure 4.14. Plot of absorbance at 475 nm versus time for the reaction of Cbl(I)– (2.00 x 10–4 M) with 0.25 equiv of AS at pH 8.00 (25.0 °C, 5.00 x 10–3 M TAPS buffer, I = 1.0

M (NaCF3SO3)) under strictly anaerobic conditions. The data were fitted to a first–order –2 –1 rate equation giving the observed rate constant (kobs) = (4.33 ± 0.04) x 10 min .

3.71 ± 0.01) x 10–2 min–1), Figure 4.15. These results indicate that the rate–determining step of the reaction between Cbl(I)– and AS is decomposition of AS to give HNO and nitrite.

1.0

0.9

0.8

0.7 Abs at475Abs nm

0.6

0 20 40 60 80 100 120 140 Time (min) Figure 4.15. Plot of absorbance at 475 nm versus time for the reaction of Cbl(I)– (2.00 x 10–4 M) with 0.04 mol equiv AS at pH 8.00 (25.0 °C, 5.00 x 10–3 M TAPS buffer, I = 1.0

M (NaCF3SO3)) under strictly anaerobic conditions. The data were fitted to a first–order –2 –1 rate equation giving the observed rate constant (kobs) = (3.71 ± 0.01) x 10 min .

5 a) 4.5 b) 4 4 4.0 3 3.5 3 3.0 2 Absnm 387 at 2.5 2 1 0.0 0.5 1.0 Absorbance 2.0 Mol Equiv of AS 1 Abs at 387nm 1.5

0 1.0 300 400 500 600 0.0 0.4 0.8 1.2 1.6 2.0 2.4 2.8 3.2 Wavelength (nm) Mol Equiv of AS

Figure 4.16. a) UV–vis spectra of equilibrated solutions of Cbl(I)– (2.00 x 10–4 M) and AS (0, 0.05, 0.10–3.0 mol equiv) at pH 7.00 (5.00 x 10–3 M phosphate buffer) under strictly anaerobic conditions. b) Plot of absorbance at 387 nm versus mol equiv of AS obtained from spectra shown in Figure 4.16a. Inset: Expanded version of the plot for 0– 1.0 mol equiv of AS. The stoichiometry of the reaction between Cbl(I)– and AS was investigated (pH

7.00). High concentrations of Cbl(I)– were used to stabilize this complex. Anaerobic solutions of Cbl(I)– (2.00 x 10–4 M) with varying mol equiv AS (0, 0.05–3.0 equiv) were equilibrated (~ 4 h; at least 5 half–lives for the slowest reaction), and UV–vis spectra recorded. From the spectra of the equilibrated product solutions, a plot of absorbance at

387 nm versus mol equiv of AS was generated, Figure 4.16. This wavelength was chosen since 387 nm is an isosbestic point for the Cbl(II)/NOCbl conversion. The absorbance at

387 nm linearly decreases up to 0.25 equiv of AS and becomes constant upon the further addition of AS, consistent with a 1:0.25 Cbl(I)–:AS stoichiometry for oxidation of Cbl(I)– to Cbl(II)– by AS or its decomposition products (HNO, nitrite). A similar plot of absorbance at 360 nm (which is not an isosbestic point for Cbl(II)/NOCbl conversion) versus mol equiv AS shows that the absorbance decreases linearly up to 0.25 equiv of

AS, then increases linearly up to 2.25 mol equiv AS, and is unchanged upon further addition of AS, Figure 4.17. This is consistent with an overall reaction stoichiometry of

1:2.25 Cbl(I)–:AS. This intriguing stoichiometry was initially difficult to rationalize and led us to carry out a detailed study on the nitrite–free Cbl(I)– + PA system, see Chapter 2.

In this latter system a 1:3 Cbl(I)–:PA stoichiometry was obtained, and HNO was found to be the Cbl(I)– oxidant. Previous studies by our lab have shown that Cbl(I)– is oxidized to

– – – Cbl(II) with a stoichiometry of 1:0.25 Cbl(I) :NO2 [66]. A 1:2.25 Cbl(I) :AS stoichiometry is therefore consistent with the reaction scheme shown in Scheme 4.2, in which nitrite carries out the initial oxidation of Cbl(I)–. If this is indeed the case, 0.25 mol

– equiv NO2 will be consumed during the reaction, which was observed experimentally using the Griess assay. Why nitrite oxidizes only one molecule of Cbl(I)– is not entirely

2.8

2.6

2.4

2.2

2.0 Abs at 360 nm 1.8

1.6 0 1 2 3 4 5 6 Mol Equiv of AS

Figure 4.17. Plot of absorbance at 360 nm versus mol equiv of AS for the reaction of Cbl(I)– (2.00 x 10–4 M) with AS (0–5.0 equiv) at pH 8.00 (5.00 x 10–3 M phosphate buffer) under strictly anaerobic conditions. clear to us. One possible explanation is that the first Cbl(I)– oxidation consumes the nitrite impurity in AS, (both commercial AS and AS synthesized in our lab contain small amounts of nitrite, see Section 4.2.14).

Since Cbl(I)– is first oxidized to Cbl(II), experiments with excess nitrite were carried out to confirm that the reaction of Cbl(I)– with AS follows similar chemistry once

Cbl(II) is formed. In the presence of excess nitrite (20.0 mol equiv), 1.0 mol equiv of AS is sufficient to convert Cbl(I)– to the final product NOCbl, Figure 4.18. That is, the 1:2.25

Cbl(I)–:AS reaction stoichiometry changes to 1:1 Cbl(I)–:AS in the presence of excess nitrite at pH 8.00.

The amount of nitrite consumed was also investigated using the Griess assay.

Upon the reaction of Cbl(I)– (25 µM) with 2.25 mol equiv AS (56.25 µM), 50 ± 1 µM nitrite is produced (two experiments). In the absence of Cbl(I)–, AS decomposes to

100

produce 58 ± 1 µM nitrite (two experiments). Hence ~ 0.25 mol equiv nitrite is consumed during the reaction, consistent with initial oxidation of Cbl(I)– to Cbl(II) by nitrite,

Scheme 4.2. The Nessler’s test for the presence of ammonia as the reaction product of the reaction of Cbl(I)– with AS was negative.

k 9HN O - L - 2 3 R.D.S. 9HNO + 9NO2 - - + fast 4Cbl(I) + NO2 + 5H 4Cbl(II) + NH2OH + H2O fast NH2OH + HNO N2 + 2H2O fast 4Cbl(II) + 8HNO 4NOCbl + 2N2 + 4H2O - - + - 4Cbl(I) + 9HN2O3 + 5H 4NOCbl + 8NO2 + 3N2 + 7H2O - - + - Or, Cbl(I) + 2.25HN2O3 + 1.25H NOCbl + 2NO2 + 0.75N2 + 1.75H2O

Scheme 4.2. Proposed reaction pathway for the reaction between Cbl(I)– and AS (pH ≤ 9.00).

Finally, indooxine tests were carried out to check the presence of hydroxylamine as a product of the reaction of Cbl(I)– (2.00 x 10–4 M) with 3.0 mol equiv AS at pH 8.00.

A small amount of NH2OH (~ 9 µM) was detected which may be formed as a result of

– – the reaction between Cbl(I) and NO2 produced from AS decomposition.

In the proposed mechanism of the reaction between Cbl(II) and AS, Scheme 4.1,

HNO reduces Cbl(II) to Cbl(I)–, being itself oxidized to NO. Others have observed 1e– reduction of transition metal centers by HNO in which HNO is oxidized to NO [158–165,

168]. Cbl(I)– is then oxidized back to Cbl(II) by a second molecule of HNO. The Cbl(II) produced from oxidation of Cbl(I)– by HNO reacts rapidly with NO to form NOCbl [64,

170]. NO could potentially oxidize Cbl(I)– back to Cbl(II) (Cbl(I)– + NO + H+ → Cbl(II)

+ ½N2O + ½H2O [198]); however in this case NO would be consumed and NOCbl would therefore not be formed; hence the oxidation of Cbl(I)– by HNO must be faster than this

101

0.9 5

0.6 4

0.3

3 Absorbance

2 0.0 400 500 600 700

Absorbance Wavelength (nm) 1

0 400 500 600 700 Wavelength (nm) Figure 4.18. UV–vis spectrum of the reaction of Cbl(I)– (2.00 x 10–4 M; dotted purple – trace) with 1.0 mol equiv AS in the presence of 20.0 equiv NO2 at pH 8.00 (25.0 °C, 5.00 x 10–3 M TAPS buffer). The final product spectrum (orange solid trace) resembles that of authentic NOCbl (blue dashed trace in the Inset). In other words, 1:2.25 Cbl(I)– – – :AS stoichiometry can be changed to 1:1 Cbl(I) :AS in the presence of excess NO2 . reaction. Although nitrite produced from AS decomposition can oxidize Cbl(I)– to Cbl(II)

[66], the observation of a 1:2 Cbl(II):AS stoichiometry and the results of the Griess assay which show that nitrite is not consumed in the reaction do not support this occurring for this system. Finally, a 1:2 Cbl(II):HNO stoichiometry was also observed for the

Cbl(II)/Piloty’s acid system (Chapter 2); providing further support for no nitrite involvement.

The stoichiometry of the reaction changed to 1:1 Cbl(I)–:AS in the presence of excess nitrite, as expected. Schemes 1 and 2 assume that NH2OH is a reaction intermediate. The reaction between HNO and NH2OH to give N2 + H2O is well known

[130] and experiments confirmed that NH2OH reacts with AS at pH 8.00 (see Section

102

4.2.11), although unequivocal evidence for the reaction proceeding via a NH2OH intermediate was not obtained.

4.4 SUMMARY

Kinetic and mechanistic studies have been carried out on the reaction of Cbl(II) with the HNO donor Angeli’s salt. A stoichiometry of 1:2 Cbl(II):AS is observed. NOCbl

– is formed as the sole cobalamin product and NO2 and N2 are the non–cobalamin products. The rate–determining step of the reaction is the decomposition of AS to give

– – HNO and NO2 . Studies on the reaction between Cbl(I) and AS and experiments in the presence of cyanide and nitrite provide support for Cbl(I)– reaction intermediate. A mechanism is proposed in which Cbl(II) is reduced by HNO, giving Cbl(I)– and NO. A second HNO molecule oxidizes Cbl(I)– back to Cbl(II), which reacts rapidly with NO to form NOCbl.

CHAPTER 5

KINETIC AND MECHANISTIC STUDIES ON THE REACTION OF

AQUACOBALAMIN WITH THE HNO DONOR ANGELI’S SALT

5.1 INTRODUCTION

Angeli’s salt (AS) is one of the most commonly used HNO donors in chemical and biochemical studies of HNO reactivity. It releases HNO in a pH–independent manner from pH 4–8. Several studies have recently been published on the chemistry of HNO donor molecules, including AS, with porphyrin complexes [109, 122, 211]. In most of the cases, these porphyrin complexes are shown to be ‘HNO traps’. However, thus far no studies have been reported on the reactivity of HNO or HNO donor molecules with the structurally related vitamin B12 complexes. We reported mechanistic studies on reactions of reduced cobalamins, Cbl(II) and Cbl(I)–, with AS in Chapter 4. In this chapter, we present kinetic and mechanistic studies on the reaction of the cob(III)alamin

+ – aquacobalamin/hydroxocobalamin (H2OCbl /HOCbl; X = H2O/OH , Figure 1.1) with

+ AS. Our studies show that H2OCbl /HOCbl reacts either directly with AS and/or with its decomposition product, HNO, depending upon the pH conditions.

103 104

5.2 EXPERIMENTAL SECTION

5.2.1 REAGENTS

Hydroxocobalamin hydrochloride (HOCbl•HCl, 98%) was purchased from Fluka.

Water content in HOCbl•HCl was determined as described in Section 2.2.1. Angeli’s salt

(AS) was obtained from Cayman Chemical and used without further purification. In addition, AS was synthesized using a published procedure [139]. All other chemicals were purchased from Fisher Scientific, Acros Organics, Sigma Aldrich or RICCA

Chemical as described in Section 2.2.1.

5.2.2 INSTRUMENTATION

pH measurements were carried out as described in Section 2.2.2. The electrodes were standardized with standard buffer solutions at pH 4.00, 7.00, 10.00 and 12.45.

Solution pH was adjusted using H3PO4 or NaOH solutions as necessary.

1H NMR spectroscopic measurements were performed as described in Section

2.2.2. Air–tight J–Young NMR tubes (Wilmad, 535–JY–7) were used for 1H NMR measurements under anaerobic conditions. Reaction mixtures were left to equilibrate for

15 min prior to 1H NMR measurements.

UV–vis spectroscopic measurements were performed as described in Section

2.2.2. Anaerobic measurements were carried out in Schlenk cuvettes.

Experiments with rapid kinetics were carried out under strictly anaerobic conditions using an Applied Photophysics SX20 stopped–flow instrument equipped with a photodiode array detector, operating with Pro–Data SX (version 2.1.4) and Pro–Data

105

Viewer (version 4.1.10) software, using either a 2 or 10 mm pathlength cell. The system was pre–treated with anaerobic sodium dithionite (for at least 1 h) to remove oxygen and subsequently thoroughly flushed with anaerobic water. The instrument was continuously purged with nitrogen gas during data collection. Hamilton gas–tight syringes filled with the anaerobic reactant solutions in the glove box were used to introduce the reactant solutions into the reservoir syringes of the stopped–flow instrument. Kinetic data were fitted using the program Microcal Origin version 8.0.

Anaerobic manipulations were carried out using a glove box and Schlenk line techniques as described in Section 2.2.2.

5.2.3 DETERMINATION OF RATE CONSTANTS FOR THE SPONTANEOUS

DECOMPOSITION OF ANGELI’S SALT

5.2.4 GRIESS ASSAY FOR THE QUANTIFICATION OF NITRITE

The amount of nitrite in the product solutions was determined using the Griess assay [195] under strictly anaerobic conditions. Detailed procedures for the Griess test is described in Section 4.2.14. The concentration of nitrite was estimated from the calibration plot (see Figure 4.11).

106

The percentage nitrite impurities in commercially available AS was determined using the Griess assay under strictly anaerobic conditions. The percentage of nitrite impurity was found to be 4.8 ± 0.1%.

5.2.5 SAMPLE PREPARATION FOR KINETIC MEASUREMENTS ON THE

REACTION OF AQUACOBALAMIN WITH ANGELI’S SALT

All samples were prepared under strictly anaerobic conditions inside the glove box. Stock Cbl(III) solutions were prepared by dissolving solid HOCbl•HCl in the appropriate buffer. Stock AS solutions were prepared by dissolving solid AS in NaOH

(10.0 mM). Stock solutions were stored in the freezer (–24 °C) inside the glove box and used within 24 h.

5.3 RESULTS AND DISCUSSION

5.3.1 STUDIES ON THE REACTION BETWEEN AQUACOBALAMIN AND

ANGELI’S SALT (pH ≤ 9.90)

Upon the addition of AS to a solution of aquacobalamin/hydroxocobalamin

+ (H2OCbl /HOCbl; pKa = 7.8 [10, 11] under strictly anaerobic conditions, significant changes in the UV–vis spectrum were observed and the reaction solution changed from red to orange. Figure 5.1 shows typical UV–vis spectra for the reaction between HOCbl

(5.00 x 10–5 M) and excess AS (2.50 x 10–3 M) as a function of time under anaerobic conditions at pH 9.80 (25.0 °C, 0.30 M CHES buffer, I = 1.0 M, NaCF3SO3). The inset to

Figure 5.1 shows a comparison between the initial and final spectrum, and it is clear that

–_ the product is nitroxylcobalamin, NO Cbl(III) (λmax = 256, 278 (shoulder), 289, 315 and

107

478 nm), with sharp isosbestic points observed at 341, 370 and 498 nm, in agreement with literature values for the HOCbl to NOCbl conversion [64].

0.8 Initial (HOCbl) Final (NOCbl) 1.0 0.6

0.8 0.4

Absorbance 0.2 0.6 0.0 300 400 500 600 Wavelength (nm) 0.4 Absorbance 0.2

0.0 300 350 400 450 500 550 600 Wavelength (nm) Figure 5.1. UV–vis spectra for the reaction between HOCbl (5.00 x 10–5 M) and excess AS (2.50 x 10–3 M) at pH 9.80 under anaerobic conditions (25.0 °C, 0.30 M CHES buffer, I = 1.0 M (NaCF3SO3)). Isosbestic points occur at 341, 370 and 498 nm. Inset: First and last spectra.

Figure 5.2 gives the best fit of the absorbance data at 356 nm versus time to a

–3 –1 first–order rate equation, giving an observed rate constant (kobs) = (2.85 ± 0.02) x 10 s

(t1/2 = 4.1 min). The rate constant for the spontaneous acid–catalyzed decomposition of

–4 –1 AS at pH 9.80 (kL = (1.34 ± 0.01) x 10 s [65]) was found to be more than one order of magnitude (~ 20 times) slower than the reaction of AS (2.50 x 10–3 M) with HOCbl (5.00

–5 –3 –1 x 10 M) at pH 9.80 (kobs = (2.85 ± 0.02) x 10 s ). Therefore, decomposition of AS is not important under these reaction conditions and a direct reaction occurs between

+ H2OCbl /HOCbl and AS. The dependence of kobs on AS concentration (2.50 – 10.0 mM)

108

at pH 9.80 was determined and the data are summarized in Figure 5.3. Fitting the data to a straight line passing through the origin gives a slope of 1.18 ± 0.01 M–1 s–1.

0.92

0.88

0.84

0.80

0.76 Abs at 356 Abs nmat 356 0.72

0.68 0 5 10 15 20 25 30 35 40 Time (min) Figure 5.2. Plot of absorbance at 356 nm versus time for the reaction between Cbl(III) and excess AS at pH 9.80 as described in Figure 5.1. The fit of the data to a first–order –3 –1 rate equation gives kobs = (2.85 ± 0.02) x 10 s .

It occurred to us that nitrite could interfere with the determination of the rate

+ constant for the reaction of interest, since H2OCbl reacts rapidly with nitrite to form nitrocobalamin, NO2Cbl [212]. There are several possible sources of nitrite. Nitrite is

+ produced in the reaction between H2OCbl /HOCbl and AS (see Schemes 5.1 and 5.3) and is also a product of AS decomposition (Scheme 1.6). Furthermore, a control experiment showed that commercial AS can contain ~ 5% nitrite impurity (see Section 5.2.4).

+ Importantly, the UV–vis spectra of H2OCbl and NO2Cbl are practically identical [10];

+ hence if H2OCbl is partially converted to NO2Cbl by reacting with nitrite as the reaction proceeds prior to being converted to NOCbl, little or no change in the isosbestic points

109

0.012

0.010

0.008 ) -1 s ( 0.006 obs

k 0.004

0.002

0.000 0.000 0.002 0.004 0.006 0.008 0.010 [Angeli's salt] (M)

Figure 5.3. Plot of kobs versus AS concentration for the reaction of Cbl(III) with excess AS at pH 9.80 under anaerobic conditions (25.0 °C, 0.30 M CHES buffer, I = 1.0 M

(NaCF3SO3)). The data have been fitted to a straight line passing through the origin, –1 –1 giving a second–order rate constant (kapp = slope) of 1.18 ± 0.01 M s .

5 –1 –1 –1 would be observed. Values of K = 2.2 x 10 M and k1 = 99.82 M s at 25 °C have

+ been reported for the formation of NO2Cbl from the reaction of H2OCbl with nitrite,

Equation 5.1 [208, 212, 213].

Control experiments conducted by a previous lab member of the Brasch lab [214]

+ showed that the rate of the reaction between H2OCbl /HOCbl and nitrite is still rapid

+ even in alkaline solution despite the reaction proceeding through H2OCbl

+ + (pKa(H2OCbl ) = 7.8 [10, 11]). Specifically, the reaction between H2OCbl /HOCbl and nitrite at pH 9.80 and 8.80 (25.0 ºC, 0.30 M CAPS or TAPS buffer, I = 1.0 M

–1 –1 (NaCF3SO3)) give the apparent rate constants (kapp) of 5.95 ± 0.04 M s and 41.5 ± 0.2

M–1 s–1, respectively [214]. Note that the hydroxo ligand of HOCbl is inert to substitution

110

[207, 208]. It therefore is possible that NO2Cbl is formed as an intermediate in the reaction. To determine whether forming NO2Cbl would have an effect on the observed rate constant, the rate of the reaction between the authentic NO2Cbl and AS was determined at pH 9.80 and 9.09 (25.0 °C, 0.30 M CHES buffer, I = 1.0 M (NaCF3SO3))

–5 –3 [214]. The reaction of HOCbl or NO2Cbl (5.00 x 10 M) with excess AS (5.00 x 10 M) at pH 9.80 (25.0 °C, 0.30 M CHES buffer, I = 1.0 M (NaCF3SO3)) gave kobs = (8.29 ±

0.01) x 10–3 s–1 and (5.79 ± 0.04) x 10–3 s–1 for the reaction of AS with HOCbl and

NO2Cbl, respectively [214]. Substituting HOCbl by NO2Cbl still results in the complete formation of NOCbl, but leads to a slight decrease in the rate of the reaction. It is likely that NO2Cbl does not itself react directly with AS, but that NO2Cbl is in equilibrium with

+ H2OCbl and the latter species reacts with AS to form NOCbl. However experiments were not carried out to probe this mechanism further.

It was, therefore, likely that nitrite interferes with the reaction of interest. Given

– + that NO2 is the non–cobalamin product of the reaction between H2OCbl /HOCbl and AS

(see Schemes 5.1 and 5.3), the maximum concentration of nitrite produced during the

– reaction is ~ [AS]. In order to minimize the amount of NO2 produced during the reaction

+ + of H2OCbl /HOCbl with AS, the kinetics of the reaction between H2OCbl and AS were

+ instead investigated with excess H2OCbl (at least 7 times compared to the concentration of AS), rather than with AS in excess. Importantly, control experiments demonstrated

– –5 that the addition of 1.0 mol equiv NO2 (5.00 x 10 M) decreases the rate constant by

–5 + –4 only ~ 10% for the reaction between AS (5.00 x 10 M) and excess H2OCbl (5.00 x 10

–2 –1 –2 –1 M) at pH 4.15 ± 0.03 (kobs = (4.91 ± 0.03) x 10 s and (4.40 ± 0.02) x 10 s in the

111

–5 absence and presence of 5.00 x 10 M NaNO2, respectively), whereas there was no

–1 –2 –1 difference observed at pH 7.20 ± 0.03 (kobs = 6.78 ± 0.03 s and 6.86 x 10 s ) in the

–5 absence and presence of 5.00 x 10 M NaNO2, respectively). Hence since only ~ 0.14

– + mol equiv NO2 (= 7 times excess H2OCbl /HOCbl) is produced in our experiments,

– NO2 production will not significantly alter the observed rate of the reaction. Control experiments also demonstrated that the rate constant is unaffected by the presence of the

–2 –1 –2 –1 metal chelator DTPA (kobs = (4.91 ± 0.03) x 10 s and (4.94 ± 0.05) x 10 s in the absence and presence of 50.0 µM DTPA, respectively; at pH 4.15 ± 0.03), showing that free metal ions are not involved in the reaction.

It was established that spontaneous decomposition of AS is not significant on the timescale of the experiments. Typical UV–vis spectra as a function of time for the

–5 + –4 reaction of AS (6.50 x 10 M) with excess H2OCbl /HOCbl (3.25 x 10 M) at pH 5.00

(25.0 ºC, 0.30 M acetate buffer, I = 1.0 M (NaCF3SO3)) under anaerobic conditions are shown in Figure 5.4. The isosbestic points were the same as those observed for the

+ conversion of H2OCbl /HOCbl to NOCbl at pH 7.40 within the limitations of the small absorbance changes due to an incomplete reaction [64].

112

1.6

1.2

0.8

Absorbance 0.4

0.0 300 400 500 600 700 Wavelength (nm) Figure 5.4. UV–vis spectra for the reaction between AS (6.50 x 10–5 M) and excess + –4 H2OCbl /HOCbl (3.25 x 10 M) as a function of time at pH 5.00 (25.0 °C, 0.30 M acetate buffer, I = 1.0 M (NaCF3SO3)). Isosbestic points occur at 339, 361 and 488 nm. Spectra were recorded every 1 s using the stopped–flow instrument.

+ Kinetic data were collected at a range of H2OCbl concentrations in order to determine the apparent second–order rate constant for the reaction at pH 5.00,

+ maintaining a H2OCbl :AS ratio of ~ 7:1. The data are summarized in Figure 5.5 which

+ shows a plot of kobs versus the concentration of H2OCbl at pH 5.00 ± 0.03. The data fit to a straight line passing through origin giving a slope (second–order rate constant; kapp) =

129.4 ± 1.4 M–1 s–1.

Values of kapp were similarly determined in the pH range 4.15–9.90 (Figure 5.6)

+ Plots of kobs versus [H2OCbl /HOCbl] at pH 9.05, 9.40 and 9.90 have a small but significant y–intercept. This arises because under these pH conditions decomposition of

AS is no longer insignificant compared to the reaction of interest. In this case, the data

113

0.14 0.12 0.10 )

-1 0.08 s (

0.06 obs k 0.04 0.02 0.00 0.00000 0.00025 0.00050 0.00075 0.00100 + [H2OCbl ] (M) + Figure 5.5. Plot of kobs versus the concentration of H2OCbl at pH 5.00 ± 0.03 (25.0 °C, 0.30 M acetate buffer, I = 1.0 M (NaCF3SO3)). The data have been fitted to a straight line –1 –1 passing through origin giving a slope (kapp) = 129.4 ± 1.4 M s . were fitted to Equation 5.2. Figure 5.7 gives the pH profile for the reaction of AS with

+ [H2OCbl /HOCbl].

+ The stoichiometry of the reaction between H2OCbl and AS was determined using

+ UV–vis spectroscopy. UV–vis spectra were taken of equilibrated solutions of H2OCbl

(5.00 x 10–5 M) and AS (0, 0.25–2.5 mol equiv) at pH 6.00. The isosbestic points shown in Figure 5.8 are in agreement with the literature values for the conversion of

+ H2OCbl /HOCbl to NOCbl at pH 7.4 [64]. The inset to Figure 5.8 shows the plot of absorbance at 351 nm versus mol equiv of AS. The absorbance decreases with the addition of up to 1.0 mol equiv AS and remains unchanged upon further addition of AS.

+ These results suggest a stoichiometry of 1:1 H2OCbl :AS at pH 6.00.

114

0.12 a) 0.12 0.05 b) c) 0.10 0.10 0.04 0.08 0.08 )

) 0.03 ) -1 -1 -1 s

0.06 s 0.06 s ( ( (

0.02 obs obs obs k k 0.04 0.04 k

0.01 0.02 0.02

0.00 0.00 0.00 0.00000 0.00025 0.00050 0.00075 0.00100 0.0000 0.0002 0.0004 0.0006 0.0008 0.0010 0.0000 0.0002 0.0004 0.0006 + + [H OCbl+] (M) [H OCbl ] (M) [H2OCbl ] (M) 2 2

0.06 0.012 d) e) 0.05 0.009 0.04 ) ) -1 -1 s 0.03 s 0.006 ( (

obs obs k 0.02 k 0.003 0.01

0.00 0.000 0.0000 0.0002 0.0004 0.0006 0.0000 0.0003 0.0006 0.0009 + [H OCbl+] (M) [H2OCbl ] (M) 2 0.0020 f) g) 0.006 0.0015 ) )

-1 0.004 -1 s ( s

0.0010 (

obs obs k k 0.002 0.0005

0.0000 0.000 0.0000 0.0003 0.0006 0.0009 0.0000 0.0025 0.0050 0.0075 + [H OCbl+] (M) [H2OCbl ] (M) 2 + Figure 5.6. Plot of observed rate constant, kobs, versus concentration of H2OCbl for the + –3 –5 reaction between a) H2OCbl ((0.10–1.00) x 10 M) and AS ((1.50–7.50) x 10 M) at + –4 –5 pH 4.15 ± 0.03 b) H2OCbl ((1.00–9.00) x 10 M) and AS ((1.50–6.50) x 10 M) at pH + –4 –5 6.00 ± 0.03 c) H2OCbl ((1.00–7.00) x 10 M) and AS ((1.50–8.00) x 10 M) at pH 7.05 + –3 –5 ± 0.03 d) H2OCbl ((1.00–7.00) x 10 M) and AS ((1.50–6.50) x 10 M) at pH 8.00 ± + –3 –5 0.03 e) H2OCbl ((0.10–1.00) x 10 M) and AS ((1.50–9.00) x 10 M) at pH 9.05 ± 0.02 + –3 –5 f) H2OCbl ((0.10–1.00) x 10 M) and AS ((1.50–10.0) x 10 M) at pH 9.40 ± 0.03 g) + –3 –4 H2OCbl ((1.5–7.5) x 10 M) and AS (1.50 x 10 M) at pH 9.90 ± 0.03 under anaerobic conditions. I = 1.0 M NaCF3SO3, 25.0 °C. Acetate, MES, TES, TAPS, TES and CAPS buffers (0.30 M) were used to control the pH. Data have been fitted to a straight line A) –1 –1 passing through origin to get a second–order rate constant, kapp = 108 ± 1 M s a), 115.7 ± 1 M–1 s–1 b), 59.1 ± 0.8 M–1 s–1 c), 65.8 ± 1.4 M–1 s–1 d) and B) fixing the –4 –1 –4 –1 –4 –1 intercept (kL) to 4.00 x 10 s , 2.87 x 10 s and 1.17 x 10 s to get kapp = 9.12 ± 0.2 –1 –1 –1 –1 –1 –1 M s , 1.35 ± 0.1 M s and 0.71 ± 0.1 M s for e), f) and g), respectively.

115

140 120 100 ) -1 80 s -1

M 60 (

app 40 k 20 0 4 5 6 7 8 9 10 pH + Figure 5.7. Plot of kapp versus pH for the reaction between H2OCbl and AS. The best fit + – the data to Equation 5.3 fixing pKa(H2OCbl ) = 7.8 and pKa(HN2O3 ) = 9.48 gives kCbl(III) = 122.6 ± 5.3 M–1 s–1.

1.50

1.35 1.6 1.20

1.2 1.05 Absat 351nm 0.90 0.8 0.0 0.5 1.0 1.5 2.0 2.5

Absorbance Mol Equiv of AS 0.4

0.0 300 400 500 600 Wavelength (nm)

+ –5 Figure 5.8. UV–vis spectra obtained for equilibrated solutions of H2OCbl (5.00 x 10 M) with AS (0, 0.25–2.5 mol equiv) at pH 6.00 under anaerobic conditions. Isosbestic points occur at 337, 365 and 490 nm. Inset: Plot of absorbance at 351 nm versus mol equiv of AS at pH 6.00 (0.30 M MES buffer).

116

The stoichiometry and the Cbl product(s) were also determined at pD 9.86 using

1H NMR spectroscopy. HOCbl is completely converted to NOCbl upon reacting HOCbl with 1.1 mol equiv AS (pD 9.86, 24.0 °C, 0.30 M CHES buffer, Figure 5.9. The chemical shift values for aromatic region of the spectrum (δ = 7.40, 7.20, 6.79, 6.33 and 6.25 ppm) match well with the literature values for NOCbl [170].

7.6 7.4 7.2 7.0 6.8 6.6 6.4 6.2 Chemical shift (); ppm

Figure 5.9. Aromatic region of 1H NMR spectrum of the Cbl product (= NOCbl) for the reaction between HOCbl and 1.10 mol equiv AS at pD 9.86 under anaerobic conditions (24 °C, 0.30 M CHES buffer).

The non–cobalamin product(s) was also identified. Nitrite was shown to be the non–cobalamin product using the Griess assay [195]. A calibration plot of absorbance versus nitrite concentration was generated (see Figure 4.11 and Section 5.2.4 for details).

– From the Griess test, it was found that 0.79 mol equiv NO2 was produced in the reaction between HOCbl (1.00 x 10–4 M) and AS (1.0 mol equiv) at pH 9.80, using a calibration plot of absorbance versus nitrite concentration. Interestingly, others have reported

– significantly less NO2 formation than expected from AS decomposition based on the stoichiometry of the reaction [149, 215, 216]. This has been attributed to N–N bond

117

 homolysis (resulting in formation of 2 NO and H2O) in addition to N–N heterolysis of

– – HN2O3 (HNO and NO2 formed) [217]. We have noticed in our studies with AS that a slight excess of AS (~ 1.1 mol equiv) is always required for reactions to proceed to completion.

+ From Figure 5.7 it is clear that the reaction of HOCbl/H2OCbl with AS becomes faster with decreasing pH, becoming pH independent at pH ≤ 6. Scheme 5.1 gives the

+ proposed reaction pathways involved for pH ≤ 9.90. The equilibrium between H2OCbl , nitrite and NO2Cbl is shown for completeness; however the effect of this equilibrium under the conditions of the kinetic experiments is insignificant. It is likely that rapid

+ substitution of the aqua ligand of H2OCbl by AS occurs prior to AS undergoing N–N bond cleavage. It is well established that β–axial ligand substitution reactions for cobalamins occur via a dissociative interchange mechanism [218]. Furthermore, only

+ H2OCbl , not HOCbl, undergoes these reactions, since HOCbl is inert to β–axial ligand

+ substitution [207, 208]; hence in Scheme 5.1 only H2OCbl (pKa = 7.8 [11]), not HOCbl, reacts with AS. The corresponding rate equation is given in Equation 5.3.

+ The best fit of the data in Figure 5.7 to Equation 5.3 with pKa(H2OCbl ) = 7.8 and

– –1 –1 pKa(HN2O3 ) = 9.48 gives kK = 123 ± 5 M s . This value is of a comparable magnitude

+ to values reported for the reactions of H2OCbl with a wide variety of ligands [13, 208].

+ By fitting the data to this equation we assume that the reaction between H2OCbl and

2– N2O3 is not important under the pH conditions of our study. Indeed, if the data is

118

+ Scheme 5.1. Proposed mechanism for the reaction of H2OCbl /HOCbl with AS (pH ≤ 9.90).

– 2– + instead fitted to a model in which both HN2O3 and N2O3 react with H2OCbl (Equation

–1 –1 5.4), kK is the same within experimental error as expected (kK = 121 ± 5 M s ; Figure

+ 5.10), since practically no Cbl remains in the reactive H2OCbl form at pH conditions

2– where the concentration of N2O3 is significant (pH > 9).

119

140 120 100 )

-1 80 s -1 60 M ( 40 app k 20 0 4 5 6 7 8 9 10 pH

+ Figure 5.10. Plot of kapp versus pH for the reaction between H2OCbl and AS. The data + have been fitted to Equation 5.4 fixing pKa(H2OCbl ) and pKa(AS) to 7.8 and 9.48, – 2– respectively, assuming that both forms of AS (HN2O3 and N2O3 ; pKa = 9.48) react with + H2OCbl (see Scheme 5.2 for the reaction scheme). The fit of the data gives k1K1 = 121 ± –1 –1 –1 –1 5 M s and k2K2 = 690 ± 430 M s (that is; the value of k2K2 is ~ 0 within experimental error).

Two mechanisms have been proposed for the reaction of AS with porphyrins and heme proteins. AS reacts directly with MnIII porphyrins (MnIIITEPyP [161]) and FeIII porphyrins (FeIIITEPyP [162]) to form an AS–bound intermediate (R.D.S.) followed by rapid decomposition of intermediate to form the final nitrosyl products, MnIITEPyP(NO) and FeIITEPyP(NO), respectively. Alternatively, the reaction may occur via a mechanism in which HNO, not AS, reacts. This mechanism has been proposed for the reaction of AS with MnIIITPPS [161], microperoxidase–11 (FeIIIMP11) [162], FeIII(TPPS) [160], methemoglobin (metHb) and metmyoglobin (metMb) [158, 163] and horseradish peroxidase [164], to form the corresponding MnII(NO) and FeII(NO) complexes. Indeed,

120

evidence for this latter mechanism occurring in our system was obtained at pH 10.80 and

11.40 (see Section 5.3.2).

5.3.2 STUDIES ON THE REACTION BETWEEN HYDROXOCOBALAMIN

AND ANGELI’S SALT UNDER STRONGLY ALKALINE CONDITIONS

Figure 5.11 gives a plot of absorbance versus time for the reaction of HOCbl

(1.00 x 10–4 M) with excess AS (1.00 x 10–2 M) at pH 10.80 (25.0 oC, 0.30 M CAPS buffer, I = 1.0 M (NaCF3SO3)). An almost linear dependence is observed, with HOCbl reacting with AS to again give NOCbl. A similar result was obtained at 5.00 x 10–4 M

HOCbl. The linear dependence suggests that the reaction is independent of HOCbl concentration (i. e., zero–order in [HOCbl]), and that the decomposition of AS is instead the rate–determining step at these pH conditions. Therefore the same experiment was repeated using 1.0 mol equiv AS at pH 10.80 (0.30 M CAPS buffer, I = 1.0 M,

NaCF3SO3). Under these conditions the absorbance at 356 nm versus time data fit well to

–5 a first–order rate equation, giving an observed rate constant (kobs) of (7.45 ± 0.01) x 10 s–1 (Figure 5.12).

121

+ Scheme 5.2. Possible reaction scheme for the reaction of H2OCbl /HOCbl with AS – 2– assuming that both protonated (HN2O3 ) and deprotonated (N2O3 ) form of AS react + with H2OCbl .

2.0

1.9

1.8

1.7 Absnm356 at

1.6

0 4 8 12 16 20 Time (min) Figure 5.11. Plot of absorbance at 356 nm versus time for the reaction of HOCbl (1.00 x 10–4 M) with excess AS (1.00 x 10–2 M) at pH 10.80 (25.0 °C, 0.30 M CAPS buffer, I =

1.0 M (NaCF3SO3)). The linear dependence suggests that the reaction is independent of HOCbl concentration.

122

A similar rate constant was observed with 0.5 mol equiv AS at the same

–5 –1 conditions ((8.00 ± 0.05) x 10 s ). Given that the reaction is stoichiometric, kobs would be expected to be independent of HOCbl for [HOCbl]:[AS]  1.0. The reaction of excess

HOCbl (1.00 x 10–4 M) with AS (1.40 x 10–5 M) at pH 10.80 (25.0 °C, 0.30 M CAPS

–5 –1 buffer, I = 1.0 M, NaCF3SO3) gives kobs = (5.32 ± 0.06) x 10 s , Figure 5.13. The observed rate constant for the spontaneous decomposition of AS at the same pH is (3.17

± 0.01) x 10–5 s–1. The observed rate constants for the reaction between HOCbl and AS are therefore up to 2.5 times larger than one would expect based solely on a pathway involving rate–determining AS decomposition. Possible explanations for this discrepancy are that there is a small contribution to the observed reaction from the direct reaction,

Scheme 5.1, or that weak association of HOCbl with AS in aqueous solution promotes

AS decomposition.

123

2.0

1.9

1.8

1.7

1.6

Abs at356 nm 1.5

1.4

0 200 400 600 800 1000 1200 1400 Time (min) Figure 5.12. Plot of absorbance at 356 nm versus time for the reaction of HOCbl (1.00 x 10–4 M) with 1.0 mol equiv of AS at pH 10.80 (25.0 °C, 0.30 M CAPS buffer, I = 1.0 M

(NaCF3SO3)). The best fit of the data to a first–order reaction gave kobs = (7.45 ± 0.01) x 10–5 s–1.

The reaction between HOCbl and AS was also studied at pH 11.40 (0.40 M phosphate buffer), using 0.50 and 1.0 mol equiv AS. The best fit of the absorbance versus

–5 –1 time data to a first–order equation gives kobs = (1.10 ± 0.01) x 10 s and (1.15 ± 0.01) x

10–5 s–1, respectively. At pH 11.40 the observed rate constant for the spontaneous decomposition of AS is (4.70 ± 0.02) x 10–6 s–1; hence once again the rate of the reaction is slightly faster than one would expect based purely on AS decomposition.

Since the reaction of AS with HOCbl at higher pH conditions occurs via a different mechanism, the stoichiometry of the reaction of HOCbl with 0.55, 1.1 and 2.2 mol equiv AS was investigated by 1H NMR spectroscopy at pD 10.86 (0.50 M CAPS). A minimum of 1.1 mol equivalents of AS are required for the reaction to proceed to completion, Figure 5.14, with NOCbl once again formed as the Cbl product. Reacting

124

2.00

1.96

1.92

1.88 Abs at 357 nm

1.84

0 200 400 600 800 1000 Time (min)

Figure 5.13. Absorbance at 357 nm versus time for the reaction between excess HOCbl (1.00 x 10–4 M) and AS (1.40 x 10–5 M) at pH 10.80 (25.0 °C, 0.30 M CAPS buffer, I =

1.0 M (NaCF3SO3)). The best fit of the data to a first–order rate equation gives kobs = (5.32 ± 0.06) x 10–5 s–1.

0.50 mol equiv AS yielded approximately ~ 50% NOCbl, and ~ 50% unreacted starting material (HOCbl). An attempt was also made to determine the stoichiometry of the reaction at pD 11.40. However since the reaction at this pH condition is very slow and

Cbls are not stable in alkaline solution [205, 206], considerable cobalamin decomposition to Co(II) corrinoid species was observed at this pH value.

– Finally, the amount of NO2 produced at pH 10.80 was determined using the

Griess assay [195] under strictly anaerobic conditions. From the calibration plot of absorbance versus nitrite concentration, the resulting absorbance corresponded to 0.81 and 0.78 mol equiv nitrite produced. Hence nitrite is the non–Cbl product.

125

7.6 7.4 7.2 7.0 6.8 6.6 6.4 6.2 6.0 Chemical shift (); ppm

Figure 5.14. Aromatic region of the 1H NMR spectrum of the product of the reaction between HOCbl (9.00 x 10–3 M) and 1.1 mol equiv AS at pD 10.82 (24 °C, 0.50 M CAPS buffer). The NOCbl product has chemical shifts, δ = 7.48, 7.24, 6.82, 6.39 and 6.27 ppm in the aromatic region. Identical results were obtained when 2.2 mol equiv AS was used. Therefore, the stoichiometry of the reaction between HOCbl and AS is 1:1 HOCbl:AS.

+ The proposed major reaction pathway for the reaction between H2OCbl /HOCbl with AS at pH ≥ 10.80 is summarized in Scheme 5.3. Decomposition of mono–

– – protonated form of AS (HN2O3 ; pKa 9.48) to give HNO/NO is the rate–limiting step of

+ – the reaction followed by fast substitution of aqua ligand of H2OCbl by HNO/NO to

– form NOCbl. Since decomposition of HN2O3 is rate–limiting, our experimental data

3 – 1 1 does not allow us to determine whether NO in addition to HNO (pKa ( HNO) ~ 11.4

1 3 – + + [111]; HNO ⇌ NO + H ) reacts with H2OCbl and/or HOCbl to form NOCbl.

Furthermore no information is obtained on the mechanism of this reaction.

126

st + fa bl C O H 2

fa H st 2 O C bl +

+ Scheme 5.3. Proposed major mechanism for the reaction of H2OCbl /HOCbl with AS at pH ≥ 10.80

5.4 SUMMARY

Kinetic and mechanistic studies have been carried out on the reaction of aquacobalamin with the HNO donor Angeli’s salt using UV–vis and 1H NMR spectroscopy. Since nitrite also reacts with aquacobalamin to form nitrocobalamin

(NO2Cbl), kinetic studies were carried out with aquacobalamin in excess to minimize the effect of nitrite in the reaction of our interest. At lower pH conditions (≤ 9.90),

– aquacobalamin reacts directly with the monoprotonated form of ASt, HN2O3 , to form

– NOCbl and NO2 , with a 1:1 stoichiometry. A direct transfer of a nitroxyl group to the

+ cobalt(III) center of H2OCbl from the ligand was also observed for the reaction of R2N–

+ NONOates with H2OCbl [219].

Under strongly alkaline conditions (pH ≥ 10.80) the rate–determining step instead

– – – involves AS decomposition to give HNO/NO and NO2 (R.D.S.), with HNO/NO

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+ subsequently rapidly reacting with H2OCbl to give NOCbl. Formation of nitrite is confirmed using Griess assay.

CHAPTER 6

MECHANISTIC STUDIES ON THE REACTION OF NITROXYLCOBALAMIN

WITH DIOXYGEN

6.1 INTRODUCTION

It has been postulated that cobalamins react rapidly with NO to form the Co3+

– NO derivative of vitamin B12, nitroxylcobalamin (NOCbl) in vivo [83, 174].

Cob(II)alamin, a major intracellular form of B12, reacts with NO at almost diffusion controlled rates to form NOCbl [64, 170]. Therefore, NOCbl has attracted considerable attention in literature. The two mammalian B12–dependent enzymes, methylcobalamin– dependent methionine synthase, and adenosylcobalamin–dependent L–methylmalonyl–

CoA mutase, are inhibited by NO in vitro and in vivo [175–177] and the inhibition is attributed to the formation of NOCbl [174]. Cbls show potential in treating pathologies associated with elevated NO levels including sepsis/septic shock [8, 83]. Cbls also inhibit

NO–induced vasodilation [173] and NO–induced smooth muscle relaxation [86, 171].

Finally, it has been proposed that NOCbl can act as a chemotherapeutic agent to treat cancer [220].

NOCbl itself is extremely air sensitive [17, 65, 68, 170]. Interestingly, others choose not to report this fact when discussing the biological relevance of the trapping of

NO by Cbl to form NOCbl, or in studies focused on therapeutic properties of NOCbl

[171, 173–176, 178, 221].

128 129

Most of the research projects discussed in this dissertation involved NOCbl as the product of the reaction between cobalamins with HNO or HNO donors. Therefore, we were interested in studying the reaction between this highly air–sensitive cobalamin derivative with dioxygen. Although UV–vis spectral studies reported by us and others suggest that NOCbl is simply oxidized to nitrocobalamin (NO2Cbl) by O2 (although this is not so simple as it initially seems, since it requires cleavage of the O=O bond) [10, 17,

– 68, 170], studies by others on the reactions of other NO –Co(III) complexes with O2 suggest that the mechanism is complex, with multiple products produced under some conditions [222].

In this chapter we present a detailed mechanistic study of the reaction between

NOCbl and O2, which provides support for formation of a peroxynitrito Co(III) intermediate.

6.2 EXPERIMENTAL SECTION

6.2.1 REAGENTS

Acetone (99.8%), glacial acetic acid (HPLC grade), ammonium hydroxide, phenol (≥ 99%), catechol (99%), 2–nitrophenol (99%), 4–nitrophenol (98%), L–tyrosine

(≥ 99%), n–octylamine (≥ 99%), tetrabutylammonium hydrogen sulfate (99%), sodium nitrate (≥ 99%), sodium dithionite (85%) and HPLC grade water and acetonitrile were purchased from either Fisher Scientific or Acros Organics. Hydroquinone (≥ 99%), 3,4– dihydroxy–L–phenylalanine (≥ 98%), 3–nitro–L–tyrosine (≥ 98%), and methanol (HPLC grade) were obtained from Sigma Aldrich. 2–(N,N–Diethylamino)–diazenolate 2–oxide

130

(DEA–NONOate, Na+ salt, (≥ 98%)) was purchased from Cayman Chemical. All other chemicals were purchased and used without further purification as described in Section

2.2.1.

6.2.2 INSTRUMENTATION

pH measurements were carried out as described in Section 2.2.2. The electrodes were standardized with standard buffer solutions at pH 4.00, 7.00, 10.00 and 12.45.

Solution pH was adjusted using H3PO4 or NaOH solutions as necessary.

1H NMR spectroscopic measurements were performed as described in Section

2.2.2. Air–tight J–Young NMR tubes (Wilmad, 535–JY–7) were used for 1H NMR measurements under anaerobic conditions. Reaction mixtures were left to equilibrate for

15 min prior to measurements.

UV–vis spectroscopic measurements were performed as described in Section

2.2.2. Anaerobic measurements were carried out in Schlenk cuvettes.

Kinetic data for rapid reactions were collected under strictly anaerobic conditions at using an Applied Photophysics SX20 stopped−flow instrument as detailed in Section

5.2.2. All other instruments were used as described in Section 5.2.2. Kinetic data were fitted using the program Microcal Origin version 8.0.

Anaerobic manipulations were carried out using a glove box and Schlenk techniques as described in Section 2.2.2.

HPLC analyses were carried out in an Agilent 1100 series HPLC system equipped with a degasser, quaternary pump, autosampler and a photodiode array detector

(resolution of 2 nm).

131

Method A: A Phenomenex Luna C18 semi–preparative column (5 μm, 100 Å, 10 mm × 250 mm) was used. Peaks were monitored at 254, 280 and 350 nm and a mobile phase consisting of acetic acid buffer (0.1% v/v in H2O, pH adjusted to 4.0 with 4.0 N

NH4OH), A, and 0.1% acetic acid (v/v) in pure CH3CN (pH not adjusted), B was used.

Gradient conditions: 0–2 min, 95:5 A:B (isocratic); 2–14 min, linear gradient to 85:15

A:B; 14–19 min, linear gradient to 82:18 A:B; 19–32 min, linear gradient to 65:35 A:B;

32–33 min, linear gradient to 40:60 A:B; 33–35 min, linear gradient to 95:5 A:B; 35–37 min, 95:5 A:B (isocratic). A flow rate of 3 mL/min was used in all experiments.

Method B: For tyrosine radical trap experiments, an Alltech Alltima (Grace) C18 semi–preparative column (5 μm, 100 Å, 10 mm × 300 mm) was used. Peaks were monitored at 220, 254, 280 and 350 nm and a mobile phase consisting of acetic acid buffer (0.1% v/v in H2O, pH adjusted to 4.0 ± 0.1 with 4.0 N NH4OH), A, and pure

CH3OH, B was used. Gradient conditions: 0–2 min, 70:30 A:B (isocratic); 2–5 min, linear gradient to 60:40 A:B; 5–27 min, linear gradient to 55:45 A:B; 27–30 min, linear gradient to 70:30 A:B and 30–32 min, 70:30 A:B (isocratic). A flow rate of 3 mL/min was used in all experiments. All the solutions for tyrosine derivatives were prepared in

0.1 M phosphate buffer; pH 7.40 and diluted as needed. The tyrosine solution was sonicated for 15 min to increase its solubility in aqueous solution.

Method C: For phenol radical trap experiments, a Phenomenex Luna C18 analytical column (5 μm, 100 Å, 4.6 mm × 250 mm) was used. Peaks were monitored at

254, 280, 315 and 350 nm and a mobile phase consisting of acetic acid buffer (0.1% v/v in H2O, pH adjusted to 4.0 with 4.0 N NH4OH), A, and 0.1% acetic acid (v/v) in pure

132

CH3CN (pH not adjusted), B was used. Gradient conditions: 0–2 min, 95:5 A:B

(isocratic); 2–14 min, linear gradient to 85:15 A:B; 14–19 min, linear gradient to 82:18

A:B; 19–45 min, linear gradient to 45:55 A:B; 45–50 min, linear gradient to 20:80 A:B;

50–52 min, linear gradient to 95:5 A:B; 52–53 min, 95:5 A:B (isocratic). A flow rate of 1 mL/min was used in all experiments. All the solutions for phenol standards (phenol, catechol, hydroquinone, 2–nitrophenol and 4–nitrophenol) were prepared in 0.1 M phosphate buffer; pH 7.40 and diluted as needed.

Method D: An isocratic method (94:6 A:B; 3 mL/min flow rate) was used. The solvents and HPLC column used were the same as in Method A.

– Method E: For NO3 detection experiments, an HPLC method reported in the literature was used with minor modifications [223, 224]. A Phenomenex Luna C18 analytical column (5 μm, 100 Å, 4.6 mm × 250 mm) was used. Peaks were monitored at

220, 280 and 350 nm and a mobile phase consisting of 0.01 M n–octylamine (pH adjusted to 6.6 with 4.0 M CH3COOH), A, and pure CH3OH, B was used. An isocratic method (80:20 A:B; 1 mL/min flow rate) was used.

For HPLC experiments, the product mixture of the reaction between NOCbl with

O2 was prepared as follows: solid NOCbl was dissolved in anaerobic phosphate buffer

(0.10 M, pH 7.40) inside the glove box and removed from the glove box to expose the sample to air. Tyrosine and phenol were added as needed prior to exposing the sample to

+ air. HPLC chromatograms of authentic samples of cobalamins (H2OCbl and NO2Cbl), phenols (phenol, catechol, hydroquinone, 2–nitrophenol and 4–nitrophenol), tyrosines

(tyrosine (Tyr), 3–hydroxytyrosine (OH–Tyr) and 3–nitrotyrosine (NO2–Tyr)) and nitrate

133

(NaNO3) were independently run to determine the exact retention times for these species.

Chromatograms for these standards are given in Figure 6.15. Duplicate HPLC experiments were carried out, giving identical results.

ESI–MS (+ve mode) measurements were carried out using an Agilent 6220 Time of Flight (TOF) instrument equipped with a multimode source (MMI) at the Central

Instrumentation Facilities in the Department of Chemistry at Colorado State University.

6.2.3 SOLUTION PREPARATIONS

All solutions were prepared using standard biological buffers and inorganic buffers (acetate and phosphate; 0.10 M) and a constant ionic strength was maintained using sodium triflate (NaCF3SO3; I = 1.0 M). Air–saturated solutions were prepared by bubbling air through the solutions for at least ~ 6 h. Solutions of varying oxygen

–3 concentration were prepared by mixing air–saturated buffer solution ([O2] = 1.22 x 10

M at 25 °C [225]) and deoxygenated buffer solution in different proportions inside the glove box.

6.2.4 SYNTHESIS OF NITROXYLCOBALAMIN

NOCbl was synthesized under anaerobic conditions inside the glove box using a published procedure [68]. The formation of NOCbl product was confirmed by 1H NMR and UV–vis spectroscopy. NOCbl concentrations in stock solutions were determined by

– converting NOCbl to (CN)2Cbl , as described in Section 2.2.1. Alternatively, concentrations were determined by UV–vis spectroscopy (extinction coefficient for

–1 –1 NOCbl at 478 nm, ε478 nm = 6.91 x 103 M cm [68]).

134

6.2.5 SYNTHESIS OF NITROCOBALAMIN

1 NO2Cbl was synthesized following a procedure reported in literature [10]. H

NMR and UV–vis spectroscopy were used to characterize NO2Cbl and check its purity.

6.2.6 SYNTHESIS OF PEROXYNITRITE

ONOO– was synthesized following a procedure published in literature [23]. Stock

– –1 –1 ONOO solutions were standardized using UV–vis spectroscopy (ε302 nm = 1670 M s

[226]) prior to use. The peroxynitrite stock solution was diluted with 0.01 M NaOH, to prevent spontaneous decomposition.

6.2.7 MASS SPECTROMETRY IDENTIFICATION OF UNKNOWN HPLC

PEAK IN THE PRODUCT OF THE REACTION OF NITROXYLCOBALAMIN

WITH OXYGEN

HPLC fractions were collected, combined and the pH of the solution (~ 40 mL) was adjusted to 7.10. The solution was taken to dryness using rotary evaporator. The pH of the solution was readjusted to 7.2 (from 6.0) when the volume of the solution was ~ 4 mL during evaporation process (cobalamins are prone to corrin ring destruction and amide hydrolysis under highly acidic conditions [2]). ESI–MS (+ve mode) measurements were carried out using instrumentation detailed in Section 6.2.2.

6.2.8 DETERMINATION OF THE EQUILIBRIUM CONSTANT FOR THE

REACTION OF NITROXYLCOBALAMIN WITH OXYGEN

Varying amounts of an air–saturated buffered solution (0.10 M phosphate buffer; pH 7.40) were injected using a gas tight syringe into an anaerobic solution of NOCbl in

135

phosphate buffer (0.10 M, pH 7.40, final concentration = 1.00 x 10–4 M) in HPLC vials (~

1.8 mL) capped with a septum screw cap. The final volume of the solution was 1.5 mL.

The reaction was allowed to proceed to completion for 2 h and each product solution subsequently diluted to 4.50 x 10–5 M Cbl with anaerobic phosphate buffer solution (0.10

M phosphate buffer; pH 7.40) inside the glove box before recording the UV–vis spectrum. Selected spectra were repeated and the data found to be reproducible to within

± 1% absorbance units.

6.2.9 PROBING FOR THE POSSIBLE COBALAMIN REACTION

INTERMEDIATE BY 1H NMR SPECTROSCOPY

An aliquot of air–saturated buffer (0.50 equiv O2, 0.10 M phosphate buffer, pD

7.40) was injected into an anaerobic NOCbl solution (final concentration = 1.00 x 10–3

M, 0.10 M phosphate buffer, pD 7.40) and the product mixture was transferred to an air– free NMR tube after 15 min to record the 1H NMR spectrum.

6.3 RESULTS AND DISCUSSION

6.3.1 KINETIC STUDIES ON THE REACTION OF NITROXYLCOBALAMIN

WITH OXYGEN

136

2.0 2.0 NOCbl 1.6 Products

1.6 1.2

0.8

1.2 Absorbance 0.4

0.0 0.8 300 350 400 450 500 550 600 650 Wavelength (nm) Absorbance 0.4

0.0 300 350 400 450 500 550 600 650 Wavelength (nm) Figure 6.1. UV–vis spectra for the reaction between NOCbl (5.00 x 10–5 M) and excess –4 O2 (6.10 x 10 M) at pH 7.40 ± 0.02 (25.0 °C, 0.10 M TES buffer, I = 1.0 M;

NaCF3SO3). Spectra were recorded every 0.25 s. Inset: Initial spectrum (black solid trace = NOCbl) and final spectrum (red dotted trace = products).

–3 Upon the addition of air–saturated buffer (O2 = 1.22 x 10 M at 25 °C [225]) to a

solution of NOCbl, UV–vis spectral scans show that NOCbl (λ = 318 and 478 nm [170])

is cleanly converted to species with spectral features characteristic of nitrocobalamin (λ =

352, 410 and 526 nm [10]); see Figure 6.1 (pH 7.40, 0.10 M TES buffer, I = 1.0 M;

NaCF3SO3). However the UV–vis spectra of aquacobalamin/hydroxocobalamin

+ + (H2OCbl /HOCbl, pKa(H2OCbl ) = 7.8 [10, 11]; λmax = 351, 412 and 526 nm at pH 7.4)

and NO2Cbl (λ = 354, 413 and 531 nm at pH 7.4) are practically indistinguishable [10].

1H NMR spectroscopy is a useful tool to distinguish between these complexes, since Cbls

137

have 5 characteristic chemical shifts in the aromatic region which are strongly dependent on the β–axial ligand [10, 67]. The 1H NMR spectrum of a NOCbl solution (1.00 x 10–3

M, 0.10 M phosphate buffer, pD 7.40) solution exposed to air show that both NO2Cbl and

+ H2OCbl /HOCbl are formed, in a ~ 37:63 ratio (± 2%; mean value of 3 experiments),

Figure 6.2.

1, 2

1 2 1 2 1 2 2

7.2 7.0 6.8 6.6 6.4 6.2 Chemical shift (; ppm

Figure 6.2. Aromatic region of the 1H NMR spectrum of a solution of NOCbl (1.00 mM) exposed to air for 30 min (pD 7.40, 0.10 M phosphate buffer). The peaks at 7.17, 6.55, + 6.47, 6.28 and 6.24 ppm correspond to H2OCbl /HOCbl (1) and those at 7.19, 6.74, 6.42, 6.27 and 6.21 ppm correspond to NO2Cbl (2). From the integration of the peaks, the ratio + of (H2OCbl /HOCbl):NO2Cbl is ~ 63:37.

138

80 + H2OCbl 70 60 50 40 30

20 NO2Cbl Unknown

Abs atAbs350 (mAU)nm 10 0 12 14 16 18 20 22 24 Retention time (Rt); min Figure 6.3. HPLC chromatogram for the products of the reaction between NOCbl (1.00 x –4 10 M) and O2 (air) at pH 7.40 (0.10 M, phosphate buffer, 25.0 °C). The peaks at 14.0 + and 22.4 min correspond to H2OCbl (~ 74%) and NO2Cbl (~ 26%), respectively. + H2OCbl and NO2Cbl amounts are estimated using peak areas and molar extinction coefficients at 350 nm. An unknown corrinoid complex elutes at 13.7 min.

The HPLC chromatogram of this same NMR solution and an aqueous solution of

NOCbl (1.00 x 10–4 M, 0.10 M phosphate buffer, pH 7.40) exposed to air also show

+ formation of NO2Cbl/(H2OCbl /HOCbl), Figure 6.3. The fraction of NO2Cbl observed by

HPLC is smaller than that observed by 1H NMR spectroscopy, due to partial decomposition of NO2Cbl on the HPLC column. This was confirmed by injecting an

1 authentic sample of NO2Cbl, Figure 6.4. Hence both H NMR spectroscopy and HPLC

+ clearly show that NOCbl is oxidized by air to form a NO2Cbl/(H2OCbl /HOCbl) mixture, rather than simple conversion to NO2Cbl. Figure 6.2 shows an additional small corrinoid peak is observed in the HPLC chromatogram at 350 nm immediately prior to elution of

+ H2OCbl , whose identity will be discussed in Section 6.3.3.

139

200

160

120

80 at 350 nm (mAU) 40 Abs

0 10 12 14 16 18 20 22 24 Retention time (Rt); min

Figure 6.4. HPLC chromatogram for an authentic sample of NO2Cbl dissolved in HPLC + grade H2O. The peaks at 14.5 and 22.3 min correspond to H2OCbl and NO2Cbl, + respectively. This indicates that NO2Cbl decomposes to give H2OCbl under the + conditions of our HPLC experiments (H2OCbl :NO2Cbl ~ 15:85, using molar extinction 3 –1 –1 3 –1 –1 + coefficients (at 350 nm) of 26.4 x 10 M cm and 21.2 x 10 M cm for H2OCbl and NO2Cbl, respectively).

Kinetic measurements were carried out on the reaction between NOCbl and O2 under pseudo–first order conditions. Figure 6.5 gives a plot of absorbance at 315 nm

–5 –5 versus time for the reaction of NOCbl (7.50 x 10 M) with O2 (1.00 x 10 M) at pH 7.40

(25.0 °C, 0.10 M TES buffer, I = 1.0 M; NaCF3SO3). The data fit well to a single first–

–2 –1 order rate equation giving an observed rate constant, kobs = (3.89 ± 0.01) x 10 s . Data were collected at other NOCbl concentrations, with at least 7.5 times excess NOCbl compared to the concentration of oxygen, to achieve essentially pseudo–first order conditions. Keeping NOCbl rather than O2 in excess improved the reproducibility of the rate constants. A second–order rate constant (kapp) was calculated from the plot of kobs versus NOCbl concentration (see Figure 6.6). A straight line passing through the origin is

140

1.08

1.06

1.04

1.02

1.00 Abs at 315 nm 0.98

0.96 0 10 20 30 40 50 60 70 80 90 Time (s) Figure 6.5. Plot of absorbance at 315 nm versus time for the reaction of NOCbl (7.50 x –5 –5 10 M) with O2 (1.00 x 10 M) at pH 7.40 ± 0.02 (25.0 °C, 0.10 M TES buffer, I = 1.0 M; NaCF3SO3). The first order fit of the data gives the observed rate constant (kobs) = (3.89 ± 0.01) x 10–2 s–1.

consistent with the rate–determining step involving the reaction of O2 with NOCbl,

–1 –1 giving an apparent second–order rate constant, kapp = 621 ± 6 M s at pH 7.40.

Similar experiments were carried out at other pH conditions and kapp values determined from plots of kobs versus [NOCbl] at each pH condition, see Figure 6.7. The results of these experiments are summarized in Figure 6.8 which shows that the reaction rate increases with increasing pH to become pH independent for pH > 7. Importantly, in acidic aqueous solution NOCbl exists in a mixture of its “base–on” (the N–bound 5,6– dimethylbenzimidazole (DMB) is coordinated at the α–axial site of the Cbl) and “base– off” form (a water molecule displaces the 5,6–DMB from the α–axial site), Scheme 1.2.

141

0.30

0.25

) 0.20 -1 s ( 0.15 obs k 0.10

0.05

0.00 0 1 2 3 4 5 10-4 [NOCbl] (M)

Figure 6.6. Plot of observed rate constant (kobs) versus [NOCbl] for the reaction of NOCbl with O2 at pH 7.40 ± 0.02 (25.0 °C, 0.10 M TES buffer, I = 1.0 M; NaCF3SO3). – The linear fit of the data gives an apparent second–order rate constant (kapp) = 621 ± 6 M 1 s–1.

142

0.35 a) 0.24 b) 0.28 0.18 ) )

-1 0.21 s -1 (

s 0.12 ( 0.14 obs k obs k 0.06 0.07

0.00 0.00 0.0000 0.0002 0.0004 0.0006 0.0000 0.0001 0.0002 0.0003 0.0004 [NOCbl] (M) [NOCbl] (M) 0.35 0.30 c) d)

0.24 0.28 )

) 0.21 0.18 -1 s -1 (

s ( 0.12 0.14 obs k obs k 0.06 0.07

0.00 0.00 0.0000 0.0001 0.0002 0.0003 0.0004 0.0000 0.0002 0.0004 0.0006 [NOCbl] (M) [NOCbl] (M)

e) g) 0.21 0.15 f) 0.020

0.12 0.016 ) ) 0.14 ) -1 -1

-1 0.09 s 0.012 ( s s

( (

0.06 obs 0.008 obs obs k

k 0.07 k 0.03 0.004

0.00 0.00 0.000 0.0000 0.0002 0.0004 0.0006 0.0000 0.0002 0.0004 0.0006 0.0000 0.0001 0.0002 0.0003 0.0004 [NOCbl] (M) [NOCbl] (M) [NOCbl] (M)

Figure 6.7. Plot of observed rate constant, kobs, versus NOCbl concentration for the –4 –5 reaction between a) NOCbl ((1.00–5.00) x 10 M) and O2 ((1.00–2.00) x 10 M) at pH –4 –5 10.40 ± 0.02 b) NOCbl ((0.75–3.50) x 10 M) and O2 ((1.00–2.00) x 10 M) at pH 9.00 –4 –5 ± 0.02 c) NOCbl ((1.00–4.00) x 10 M) and O2 ((1.00–2.00) x 10 M) at pH 8.00 ± 0.02 –4 –5 d) NOCbl ((1.00–5.00) x 10 M) and O2 ((1.00–2.00) x 10 M) at pH 6.00 ± 0.02 e) –4 –5 NOCbl ((1.00–5.00) x 10 M) and O2 ((1.00–2.00) x 10 M) at pH 5.55 ± 0.02 f) NOCbl –4 –5 ((1.00–5.00) x 10 M) and O2 ((1.00–2.00) x 10 M) at pH 5.00 ± 0.02 g) NOCbl –4 –5 ((1.00–4.00) x 10 M) and O2 ((1.00–2.00) x 10 M) at pH 4.10 ± 0.02 under anaerobic conditions. I = 1.0 M NaCF3SO3, 25.0 °C. CAPS, phosphate, TAPS, MES and acetate buffers (0.10 M) were used to control the pH in solution. Data have been fitted to a straight line passing through origin giving a slope (second–order rate constant, kapp) = 606 ± 4 M–1 s–1 a), 593 ± 18 M–1 s–1 b), 626 ± 5 M–1 s–1 c), 577 ± 7 M–1 s–1 d), 392 ± 4 M–1 s–1 e), 234 ± 2 M–1 s–1 f) and 44.8 ± 0.9 M–1 s–1 g).

143

650

520 ) -1

s 390 -1 M ( 260 app k 130

0 4 5 6 7 8 9 10 11 pH

Figure 6.8. Plot of second–order rate constant (kapp) versus pH for the reaction between NOCbl and O2. The data have been fitted to the Equation 6.1 in the text fixing pKbase– –1 –1 off(NOCbl) = 5.1 and KCo = 1.9, giving the rate constant (kNOCbl) = 926 ± 20 M s .

The data in Figure 6.8 were fitted to Equation 6.1 assuming that only base–on

NOCbl, not the two base–off complexes, reacts with O2, with the value of pKbase– off(NOCbl) fixed to 5.1 [170] and KCo fixed to 1.9 [227]. The best fit of the data in Figure

–1 –1 6.8 to Equation 6.1 gives the rate constant, kNOCbl = 926 ± 20 M s . The role of the ligand trans to the NO– with respect to the reactivity of nitroxyl cobalt complexes with

O2 is well established, with strong  donating ligands significantly increasing the rate of the reaction [222, 228].

k app x Kbase-off x KCo k = ...... (6.1) NOCbl -pH (10 + Kbase-off) (KCo + 1)

144

To determine the thermodynamic parameters ΔH‡ and ΔS‡, temperature dependence studies were carried out for the reaction of NOCbl with O2 in the 15.0–45.0

°C range at pH 7.40 ± 0.02. Under these conditions NOCbl exists in its base–on form.

Figure 6.9 gives the Eyring plot of ln (k/T) versus 1/T. The slope and intercept of the plot give ΔH‡ = 29 ± 1.4 kJ mol–1 and ΔS‡ = –94.0 ± 4.2 J K–1 mol–1, respectively.

1.65

1.10 ) / T k k ( ln 0.55

0.0031 0.0032 0.0033 0.0034 0.0035 1/T (K-1)

Figure 6.9. Plot of ln (k/T) versus 1/T (Eyring plot) for the reaction of NOCbl with O2 at pH 7.40 ± 0.02 (0.10 M TES buffer, I = 1.0 M; NaCF3SO3). The slope and the intercept of Eyring plot give ΔH‡ = 29 ± 1.4 kJ mol–1 and ΔS‡ = –94.0 ± 4.2 J K–1 mol–1, respectively.

6.3.2 DETERMINATION OF THE EQUILIBRIUM CONSTANT

–4 Equilibrated solutions of NOCbl (1.00 x 10 M) with varying equivalents of O2

(0–12.2 mol equiv) were prepared and the product solutions diluted to 4.50 x 10–5 M before recording UV–vis spectra, Figure 6.10. Note that scatter in the absorbance spectra is observed, since individual solutions were prepared for each O2 concentration and

145

transferred one by one to a cuvette to record the UV–vis spectrum. From a plot of absorbance at 352 nm versus concentration of O2 (inset to Figure 6.10), it is clear that the reaction requires excess O2 to proceed to completion. The data were fitted to Equation

6.2.

A0 and A∞ represent the absorbance in the absence of O2 and the absorbance after the complete formation of products, respectively, and Aobs is the observed absorbance at a

1.0 1.0 0.9

0.8 0.8

Absat 352nm 0.7 0.6 0.6 0.0000 0.0004 0.0008 0.0012 [O ] (M) 0.4 2 Absorbance

0.2

300 350 400 450 500 550 600 650 Wavelength (nm)

Figure 6.10. UV–vis spectra for equilibrated solutions of NOCbl (1.00 x 10–4 M) with varying mol equiv of O2 (0–12.2 mol equiv) at pH 7.40 (0.10 M phosphate buffer) at 25.0 °C. Inset: Plot of absorbance at 352 nm versus [O2]. The best fit of the data to equation 3 –1 6.2 in the text gave an equilibrium constant, Keq = (1.62 ± 0.34) x 10 M .

146

specific O2 concentration. The best fit of the data to Equation 6.2 gave an equilibrium

3 –1 constant (Keq) of (1.62 ± 0.34) x 10 M (pH 7.40).

6.3.3 PROBING FOR REACTION INTERMEDIATES

–3 0.50 mol equiv of O2 was added to an anaerobic solution of NOCbl (1.00 x 10

M) and the 1H NMR spectrum of the product mixture recorded (pD 7.40). Three sets of

Cbl peaks were observed in the product mixture corresponding to unreacted NOCbl and

+ the NO2Cbl and H2OCbl /HOCbl products, Figure 6.11. Hence significant amounts of other Cbl complexes are not observed by 1H NMR spectroscopy with the addition of less than 1 mol equiv O2.

7.6 7.4 7.2 7.0 6.8 6.6 6.4 6.2 Chemical shift (); ppm

Figure 6.11. Aromatic region of the 1H NMR spectrum of the products of the reaction –3 between NOCbl (1.00 x 10 M) and 0.5 mol equiv O2 at pD 7.40 (0.10 M phosphate buffer, 24 °C). Unreacted NOCbl gives prominent peaks at 7.45, 7.21, 6.83, 6.36, 6.26 ppm. The peaks at 7.19, 6.73, 6.42, 6.26, 6.20 ppm can be assigned to NO2Cbl and the + peaks at 7.17, 6.55, 6.45, 6.25, 6.24 ppm are those of H2OCbl /HOCbl. No reaction intermediates were observed.

It has been proposed that a transition metal–bound peroxynitrite intermediate is formed upon reacting nitrosyl transition metal complexes with O2 [222, 228–233]. This

–   complex may decompose to generate NO3 , OH and NO2 intermediates [229, 230, 234,

147

– 235]. HPLC experiments showed that NO3 is present in the product mixture, Figure

+ 6.12, in an amount consistent with the (H2OCbl /HOCbl):NO2Cbl product ratio of ~

63:37.

148

1000 a) 800 b) 700 NO - + 800 3 H2OCbl NO Cbl 600 2 500 600 400 400 300 200 200 Abs 220at (mAU) nm 100 Abs Abs at 220(mAU)nm 0 0 0 4 8 12 16 20 24 28 32 12 14 16 18 20 22 24 Retention time (Rt); min Retention time (Rt); min 1000 c) 2000 d) + NO - H2OCbl 800 + 3 H2OCbl 1600

600 1200

400 800 Unknown NO Cbl NO - 2 200 400 3 Abs at 220 nm (mAU) Abs 220at (mAU) nm

0 0 0 2 4 6 8 10 12 14 16 18 20 22 24 0 4 8 12 16 20 24 28 32 36 Retention time (R ); min Retention time (Rt); min t Figure 6.12. HPLC chromatograms (220 nm) of nitrate, cobalamin standards and the product mixture of NOCbl with O2 using Method E) described in Section 6.2.2. a) An authentic sample of NaNO3 (1 g/mL or 11.76 mM) elutes at 17.0 min. b) An authentic + mixture of H2OCbl (1.5 mM) and NO2Cbl (1.5 mM). The peaks at 4.4 and 31.5 min + + correspond to H2OCbl and NO2Cbl, respectively. c) An authentic mixture of H2OCbl + – and NaNO3. The peaks at 4.4 and 17.0 min correspond to H2OCbl and NO3 , –3 respectively. d) Product mixture of the reaction of NOCbl (1.00 x 10 M) with air (O2). + – The peaks at 4.4, 17.2 and 30.7 min correspond to H2OCbl (peak area 54.8%) NO3 (peak area 4.8%) and NO2Cbl (peak area 26.2%), respectively. The major unknown corrinoid peak occurs at 7.1 min and two further unidentified minor peaks are also + observed at 5.6 and 9.2 min. Molar extinction coefficients for H2OCbl , NO2Cbl and – 4 4 3 –1 –1 NO3 at 220 nm are 5.44 x 10 , 5.06 x 10 and 3.98 x 10 M cm , respectively. From – + the peak areas and extinction coefficients, [NO3 ] / ([H2OCbl ] + [NO2Cbl]) ~ 0.8. + Assuming that only H2OCbl and NO2Cbl are the corrinoid products of the reaction and  – –  – – that NO2 reacts with H2O to form NO2 and NO3 (2 NO2 + H2O → NO2 + NO3 ) [Goldstein, S. and Merenyl, G. Method Enzymol 2008, 436, 49], a value of 0.63 + + (0.37/2) = 0.82 would be expected, Scheme 6.3, since the ratio of (H2OCbl +

HOCbl):NO2Cbl ~ 63:37 (Figure 6.2).

149

Experiments were also carried out to probe for formation of other non–cobalamin

  corrinoid products, which may arise as a result of OH and/or NO2 intermediates attacking the corrin ring of the Cbl complex. The product mixture of the reaction between

–3 NOCbl (1.00 x 10 M) and O2 was treated with excess solid KCN (pH > 10) to convert all corrinoid complexes to their dicyano forms. The pH was subsequently lowered to 3

– before bubbling the solution with air to convert dicyanocobalamin ((CN)2Cbl ) to CNCbl and to remove excess cyanide (as HCN) [188]. Figure 6.13 shows a major peak (~ 96% at

280 nm) in the HPLC chromatogram of the product mixture, with a retention time and

UV–vis spectrum identical to CNCbl. This suggests that almost all of the corrinoid

100 160 140 80 120 100 60 80 60 40

40 Abs280 at (mAU) nm 20 0 20 16 18 20 22 24 26 28 30

Abs atAbs280 (mAU)nm Retention time (Rt); min

0 16 18 20 22 24 26 28 30 Retention time (Rt); min Figure 6.13. HPLC chromatogram (280 nm) of the products of the reaction between NOCbl (1.00 x 10–3 M) and air (0.10 M phosphate buffer, pH 7.40) treated with excess KCN for ~ 1 h. The pH of the product solution was lowered to 3.0 by the addition of phosphoric acid before injecting the sample into the HPLC instrument using Method B) described in Section 6.2.2. A single dominant peak occurs at 23.4 min which is assigned to CNCbl. Additional small peaks are also observed at 21.2, 25.1 and 27.5 min. The inset shows the HPLC chromatogram for an authentic CNCbl sample dissolved in H2O.

150

products are cobalamin complexes. HPLC fractions for this peak were collected and taken to dryness. ESI–MS confirmed that this peak does indeed correspond to CNCbl; that is, corrin ring modifications are not present to any significant extent in the major

+ cobalamin products ((H2OCbl /HOCbl) + NO2Cbl), Figure 6.14.

151

Figure 6.14. ESI–MS (+ve mode) analysis of a) an authentic sample of CNCbl and b)

HPLC fractions of the product mixture of the reaction between NOCbl and O2 (air) at pH 7.40 which have been subsequently treated with KCN to convert all corrinoids to their respective cyano forms (Figure 6.13). Observed m/z = 1355.58 (calculated m/z + for [CNCbl + H] , C63H89CoN14O14P = 1355.58); 1377.56 (calculated m/z for [CNCbl + + Na] , C63H88CoN14NaO14P = 1377.56).

152

140 120 a) b) 120 100 100 80 80 60 60 40 40

Abs Abs at 280 nm (mAU) 20 20 Abs at 280 nm (mAU)

0 0 10 15 20 25 30 16 18 20 22 24 26 28 30 Retention time (Rt); min Retention time (Rt); min 1600 60 c) d) 50 1200 24 40 16 30 800 8

20 350(mAU)Abs at nm 0 32.8 33.2 33.6 34.0 400 Retention time (Rt), min 10 Abs at 280 nm (mAU) Abs at 280Abs (mAU)nm 0 0 12 14 16 18 20 22 24 8 12 16 20 24 28 32 36 40 Retention time (Rt); min Retention time (Rt); min 1200 44 e) f) 1000 33 800

600 22

400 11 200 Absat nm350 (mAU) Abs at 280 (mAU) nm

0 0 12 16 20 24 28 0 5 10 15 20 25 30 35 Retention time (R ); min Retention time (Rt); min t Figure 6.15. HPLC chromatograms obtained from the experiments using various methods described in Section 6.2.2. Rt = retention time. a) Cbl standards (Method A); Rt + = 14.2 and 22.7 min for H2OCbl and NO2Cbl, respectively. b) Cbl standards (Method + B); Rt = 20.7 and 26.0 min for H2OCbl and NO2Cbl, respectively. c) Tyr standards (Method B); Rt = 14.8, 16.0 and 21.9 min for OH–Tyr, Tyr and NO2–Tyr, respectively. d) Phenol standards (Method C). Rt = 8.8, 17.3, 33.3 and 38.7 min for hydroquinone, catechol, 4–nitrophenol and 2–nitrophenol, respectively. Inset: Plot of Abs350 nm versus Rt (4–nitrophenol does not absorb at 280 nm). e) Cbl standards in the presence of phenol + (Method C); Rt = 12.6, 20.4 and 26.6 min for H2OCbl , NO2Cbl and phenol, respectively. f) The separation of the unknown corrinoid product (24.9 min) of the reaction between + NOCbl and O2 at pH 7.4 using isocratic Method D). Rt = 29.1 min for H2OCbl .

153

  To further investigate whether the free radicals OH and NO2 are formed in solution upon reacting NOCbl with O2, the products of the reaction between NOCbl and

  O2 were determined in the presence of the established OH/ NO2 trapping agent phenol.

  Phenol traps the OH and NO2 intermediates formed upon the spontaneous decomposition of ONOOH in aqueous solution (pH 6.8) to form hydroquinone (3%), catechol (0.2%), 2–nitrophenol (3%), and 4–nitrophenol (2.2%) [236, 237]. An HPLC method was developed suitable for separating the hydroxylated and nitrated phenol derivatives from phenol, Figure 6.15. A control experiment showed that formation of 1%

(relative to the Cbl concentration) hydroquinone, catechol, 4–nitrophenol and 2– nitrophenol would be detectable in the HPLC chromatogram of the product mixture,

Figure 6.16. Both 4–nitrophenol (33.3 min, < 1%; no attempt was made to quantitate the

3 2.0 1.5

2 1.0 0.5 Abs (mAU) at 350 nm 0.0 1 32.4 32.8 33.2 33.6 34.0 Retention time (Rt); min Abs atAbs280 (mAU)nm 0

8 12 16 20 24 28 32 36 40 Retention time (Rt); min Figure 6.16. HPLC chromatogram for hydroxylated and nitrated phenol standards (hydroquinone, catechol, 4–nitrophenol and 2–nitrophenol; 10 µM each i.e. 1% relative to the Cbl concentration) in H2O. The peaks at 7.5, 17.3, 33.3 and 38.7 min correspond to hydroquinone, catechol, 4–nitrophenol and 2–nitrophenol, respectively. Inset: Plot of absorbance at 350 nm versus retention time (4–nitrophenol does not absorb strongly at 280 nm).

154

amounts of the phenol products) and 2–nitrophenol (38.7 min, < 1%) were clearly observed in the HPLC chromatogram of the products of the reaction of NOCbl with air in

 the presence of excess phenol (0.10 M), Figure 6.17, indicating that free NO2 radicals are formed during the reaction. However, hydroxylated phenol derivatives (hydroquinone and catechol) proved more difficult to observe in the presence of the Cbl products. The peak for catechol (17.3 min) overlaps with cobalamin impurities and hydroquinone was also not detected.

   In order to probe further for formation of a OH intermediate, a second OH/ NO2

1000 1000 a) b)

+ + H2OCbl H2OCbl 800 800

0.50 0.50 4-NO Ph 2-NO Ph 600 2 2 600

0.25 0.25 NO Cbl 400 400 2 NO Cbl 2 atAbsnm(mAU) 350 Abs nmatAbs 350 (mAU)

Abs at Abs (mAU)350 nm 0.00 0.00

Unknown 350 (mAU)at Abs nm 32 34 36 38 40 200 32 34 36 38 40 200 Unknown Retention time (Rt); min Retention time (Rt); min

0 0 8 12 16 20 24 28 32 36 40 8 12 16 20 24 28 32 36 40 Retention time (R ); min t Retention time (Rt); min

Figure 6.17. HPLC chromatogram for the products of the reaction between NOCbl (1.00 –3 x 10 M) and O2 (air) in the a) presence and b) absence of excess phenol (0.10 M). The prominent peaks at 12.0, 12.6 and 20.5 correspond to a novel corrinoid complex, + H2OCbl and NO2Cbl, respectively. Note that the peak for phenol (26.6 min; Figure 6.12e) is not observed in Figure 6.17a since phenol does not absorb at 350 nm. The insets are the enlarged representations of the 32–40 min region, showing the presence of

4–nitrophenol (4–NO2Ph; 33.3 min) and 2–nitrophenol (2–NO2Ph; 38.7 min) in a) only. Two further minor corrinoid peaks are observed at 15.0 and 15.5 min, which decrease in relative intensity in the presence of phenol. No attempts were made to characterize these complexes.

155

  trapping agent, tyrosine (Tyr), was used. Tyr traps the OH and NO2 intermediates formed during ONOOH decomposition (pH 7.40) to give a much larger yield of the hydroxylated trapped product 3–hydroxytyrosine (OH–Tyr, 9%) in addition to 3– nitrotyrosine (NO2–Tyr, 5%) [23, 238–242].

Control experiments showed that both OH–Tyr (3%) and (NO2–Tyr) (3%) are detected in the presence of the Cbl reaction products, Figure 6.18. When NOCbl (1.00 x

10–3 M) was reacted with air in the presence of excess Tyr (6.00 x 10–3 M), a small

500

400

300

200

100 Abs atAbs280 (mAU)nm

0 10 12 14 16 18 20 22 24 Retention time (Rt); min Figure 6.18. HPLC chromatogram for the authentic sample mixture of Tyr (6.00 mM),

OH–Tyr and NO2–Tyr (0.18 mM each) at pH 7.40 (0.10 M, phosphate buffer). The peaks at 14.8, 16.0 and 21.9 min correspond to OH–Tyr, Tyr and NO2–Tyr, respectively. amount (14.8 min, < 1%) of OH–Tyr was clearly observed in the HPLC chromatogram,

Figure 6.19. Using this HPLC method NO2–Tyr was not observed, most likely due to overlap with Cbl impurities.

An additional small corrinoid peak eluting immediately prior to elution of

+ H2OCbl was observed at 350 nm in the HPLC chromatogram of the product mixture of

156

6 6 OH-Tyr

b) 4 a) Tyr 4 450 120 + H2OCbl 2 + 2 H2OCbl 0 0 Abs at 280 Abs nm (mAU) 300 Absat nm(mAU) 280 80 14.4 14.6 14.8 15.0 15.2 14.4 14.6 14.8 15.0 15.2 Retention time (Rt); min Retention time (Rt); min

NO Cbl 2 40 150 NO2Cbl Abs atAbs280 nm (mAU) Abs Abs at 280 (mAU) nm

0 0 14 16 18 20 22 24 26 28 30 14 16 18 20 22 24 26 28 30 Retention time (Rt); min Retention time (Rt); min Figure 6.19. HPLC chromatogram for the products of the reaction of NOCbl (1.00 x 10–3 M) with air in the a) presence and b) absence of excess Tyr (6.00 x 10–3 M). The peaks at + 14.8, 16.1, 20.8 and 26.0 min correspond to OH–Tyr, Tyr, H2OCbl and NO2Cbl, respectively. The insets are enlarged representations of the region where OH–Tyr elutes from the column.

the reaction between NOCbl and O2, Figures 6.3 and 6.17, with a UV–vis spectrum

+ indistinguishable from H2OCbl . HPLC fractions of the unknown complex were collected

+ using an isocratic HPLC method to achieve complete separation from H2OCbl , pooled together, and analyzed by mass spectrometry (see Section 6.2.2 and Figures 6.15f and

6.20 for details). The complex has m/z peaks assignable to a Cbl complex minus two hydrogen atoms, Cbl–2H (observed m/z = 1327.53 (calculated m/z for [(Cbl – 2H) + H]+,

C62H87CoN13O14P = 1327.56) and observed m/z = 664.27 (calculated m/z for [(Cbl – 2H)

2+ + 2H] , C62H88CoN13O14P = 664.28)). Although corrinoid ring modifications have been proposed to occur in numerous studies [2, 243, 244], typically these complexes are not fully characterized. One possibility is that abstraction of a H atom by a OH radical at the

C8 position (Figure 1.1) of the corrin occurs. In addition, data from control experiments

 show that NO2 does not react with the corrin ring. This results in intramolecular transfer

157

of an unpaired electron to the Co center, and subsequent attack of a deprotonated N– amide group of side–chain c at the C8 carbocation results in formation of the well–known

Co(II) c–lactam derivative of aquacobalamin [2]. This complex is oxidized to the corresponding Co(III) complex in the presence of air (a “dehydrovitamin B12” derivative)

[2]. c–Lactam derivatives of B12 have very similar UV–vis spectra and chromatographic behavior to the parent B12 complexes [244]. Furthermore the c–lactam of CNCbl elutes immediately prior to CNCbl on a C18 reverse phase HPLC column, consistent with the retention time of our unknown corrinoid species [245]. However an X–ray structure of the corrinoid product is clearly required to unequivocally characterize this complex, which is beyond the scope of this study.

158

Figure 6.20. ESI–MS (+ve mode) analysis of the unknown corrinoid peak eluted using isocratic Method D) in the Experimental section. Observed m/z = 1327.53 (calculated m/z + for [(Cbl – 2H) + H] , C62H87CoN13O14P = 1327.56); 664.27 (calculated m/z for [(Cbl – 2+ 2H) + 2H] , C62H88CoN13O14P = 664.28); 675.26 (calculated m/z for [(Cbl – 2H) + H + 2+ Na] , C62H87CoN13NaO14P = 675.27); 684.27 (calculated m/z for [(Cbl – 2H) + H + Na + 2+ H2O] , C62H89CoN13NaO15P = 684.28). Others have proposed the formation of a peroxynitrite intermediate upon the

oxidation of nitrosyl/nitroxyl metal complexes by oxygen [222, 228–233], Equation 6.3,

or the reaction of NO with superoxometal complexes [235, 236, 246–248], Equation 6.4.

Thus far two peroxynitrito complexes have been isolated. Koppenol et al.

3– 3– synthesized [Co(CN)5OONO] by reacting [Co(CN)5O2] with 1 equiv NO(g) [236]. This

complex was remarkably stable in aqueous solution, eventually hydrolyzing. Recently a

Cu(I) peroxynitrito complex was synthesized by reacting Cu(II)–NO with H2O2 [249]. X–

ray structures were not reported for either complex.

159

(n-1)+ n+ - n+ - n+ - M NO/ M NO + O2 M N(O)OO (or M OONO ) products ..(6.3) (n-1)+ n+ - n+ - M O2 /M O2 + NO M OONO products ...... (6.4)

While peroxynitrite (ONOO–) is relatively stable in solution [246], ONOOH

rapidly undergoes O–O bond homolysis to produce caged ONO and OH, which

– + recombine to form NO3 + H , Scheme 6.1 [250–252]. It has also been proposed that

– + direct intramolecular rearrangement of ONOOH occurs, to also yield NO3 + H [250].

Controversy currently exists concerning the extent of intramolecular rearrangement

versus O–O bond homolysis [250–252]. ONOO– decomposes much more slowly via

similar reaction pathways, Scheme 6.1.

Scheme 6.1. Decomposition pathways for ONOOH and ONOO–. ONOOH also reacts – – with ONOO to produce 2NO2 + O2 [Koppenol et al. Dalton Trans 2012, 41, 13779]

160

Given that pKa(ONOOH) is 6.8 [253] and that the pKa of a ligand lowers upon coordination of the ligand to a metal center [254], it is likely that the intermediate is

Co(III)–N(O)OO– rather than Co(III)–N(O)OOH in the pH range of our study. From kinetic studies on the reaction of NOCbl with O2 it was found that the rate–determining step is first–order in both [NOCbl] and [O2], and that only base–on NOCbl, not base–off

–1 –1 NOCbl, reacts with O2 (kNOCbl = 926 ± 20 M s , 25.0 °C, pH 7.40, 0.10 M phosphate buffer, I = 1.0 M; NaCF3SO3). Others have also observed that the presence of strong base coordinated trans to the nitrosyl group of cobalt nitrosyl complexes leads to an accelerated rate of reaction with O2 [222, 228, 255], and the same is true for other metal–

NO complexes [256]. Reaction intermediates were not observed for the NOCbl + O2 reaction even with 0.5 equiv O2, consistent with rate–determining electrophilic attack of

+ O2 on NOCbl, with subsequent rapid reactions to generate the H2OCbl /HOCbl and

3 –1 NO2Cbl products. An overall equilibrium constant of (1.62 ± 0.34) x 10 M was determined at pH 7.40 (25.0 °C). Interestingly, others have reported that the reaction between Co–NO and ½O2 was stoichiometric [228], and several additional experiments were carried out by us to confirm that this is not the case for the NOCbl + O2 reaction. On retrospect this result is expected, given that in solution the base–off, 5,6– dimethylbenzimidazole (DMB) deprotonated form of NOCbl exists equilibrium with base–on NOCbl, Scheme 1.1 [227], and since H2O is a weak trans  donor ligand, the base–off complex is unlikely to react with O2.

Others have proposed for their systems that the nitrosyl ligand initially dissociates from the metal center followed by formation of the superoxometal complex which

161

subsequently reacts with NO to form an O–bound peroxynitritometal complex [229, 230,

232, 233]. It has also been suggested that oxygen is first reduced by the metal center of a

Re–NO complex to superoxide, which then reacts with the metal complex to form an N– bound peroxynitritorhenium intermediate [231]. However these reaction pathways seem unlikely for the NOCbl/O2 system since the rate–determining step is first–order in both

‡ the NOCbl and O2 concentrations. Furthermore a large negative value of ΔS supports an associative rate–determining step for the reaction between NOCbl and O2, consistent with electrophilic attack of O2 on the N of NOCbl to form a N–bound peroxynitrito complex.

A bimolecular mechanism for formation of the corresponding nitro–Co(III) complex from the peroxynitritocobalt(III) intermediate has been proposed by others, with Co(III)–

– bound peroxynitrite reacting with Co(III)–NO to give NO2Cbl, Scheme 6.2 [228, 229]. If

Scheme 6.2. Proposed reaction pathway for formation of nitrocobalamin (NO2Cbl) product from the peroxynitritocobalt(III) intermediate.

+ the reaction proceeds via this type of reaction pathway, the NO2Cbl:(H2OCbl + HOCbl) product ratio will be dependent on the total cobalamin concentration [229], with more

NO2Cbl expected at higher cobalamin concentrations. However, HPLC experiments

+ showed that the NO2Cbl:H2OCbl product ratio is the same within experimental error for products of the reaction between NOCbl and air at 1.00 x 10–4 or 1.00 x 10–3 M Cbl

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concentrations, which does not support the involvement of a second Co(III) complex in

NO2Cbl formation. Note that this type of mechanism was initially proposed prior to more

 recent studies showing that ONOOH decomposes via O–O bond homolysis to give NO2 and OH [228].

– NO3 does not bind appreciably to the Co(III) center of cobalamins [2]; therefore

– any NO3Cbl potentially formed from isomerization of Co–ONOO will decompose to

+ – H2OCbl /HOCbl and NO3 . Note that formation of the corresponding nitrato complex has been observed for the reaction of the nitrosyl cobaloxime complex BCo(DH)2NO (B = base) with O2 and for the reaction of other metal nitrosyl complexes with O2 [222]. The

– observation of ~ 37% NO2Cbl suggests that like ONOOH and ONOO , Co(III)–bound

ONOO– decomposes by O–O homolysis. Ligand isomerization ultimately gives

+ – H2OCbl /HOCbl after dissociation of the ONO2 ligand, although it is not clear to us if this occurs via direct intramolecular rearrangement of the ligand in addition to O–O bond homolysis and recombination of the caged radical species. Others have reported that light can affect the products observed for the hydrolysis of a Co(III)–OONO– complex,

3– [Co(CN)5(OONO)] [257]. Additionally Richter–Addo et al. [256] found that the rate of the reaction of the picket fence porphyrin complex [Fe(tpivpp)(NO)] with air in CHCl3 solution is enhanced by light. A control experiment in the absence of regular laboratory light showed that light has no effect on the observed product ratio for our system, Figure

6.21.

It occurred to us that as for other cob(III)alamins with inorganic ligands bound at

– + the β–axial site, Co(III)–ONOO is in equilibrium with H2OCbl /HOCbl plus ONOO(H)

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7.2 7.0 6.8 6.6 6.4 6.2 Chemical shift (); ppm

Figure 6.21. Aromatic region of the 1H NMR spectrum of a solution of NOCbl (5.00 x 10–3 M) exposed to air for 30 min (pD 7.40, 0.10 M phosphate buffer) in the absence of + light. The peaks at 7.18, 6.56, 6.47, 6.27 and 6.25 ppm correspond to H2OCbl and those at 7.20, 6.74, 6.43, 6.28 and 6.21 ppm correspond to NO2Cbl. From the integration of the + peaks, the ratio of H2OCbl :NO2Cbl is ~ 63:37. at pH 7.4, and that decomposition of the intermediate may therefore occur via hydrolysis of Co–ONOO–. However, a comparison of the observed rate constants for the reaction

–1 between NOCbl and O2 at pH 10.40 (kobs = 0.05 – 0.35 s , Figure 6.7a) and the observed

–4 –1 rate constant for ONOO(H) decomposition at pH 10.40 (kobs = 4.68 x 10 s ; Figure

6.22) makes it clear that decomposition via this pathway is not occurring. Given that

ONOO– is so stable in aqueous solution [258], it is likely that the metal center substitutes for the proton of ONOOH, to assist in the decomposition of Co(III)–bound peroxynitrite

[246, 259].

However, note that the proton of ONOOH is located on an O atom, whereas it is likely that N–bound Co(III)–N(O)OO– is initially formed. Whether or not this latter complex isomerizes to O–bound Co(III)–OONO– prior to undergoing O–O bond

  homolysis of the ligand to give NO2 and OH is not known.

164

0.14

0.13

0.12

0.11

0.10

Abs atAbs 302 nm 0.09

0.08

0 20 40 60 80 100 120 140 Time (min) Figure 6.22. Plot of absorbance at 302 nm versus time for the spontaneous –5 decomposition of ONOO(H) (8.00 × 10 M, pH 10.40, 25.0 C, 0.10 M phosphate buffer). The data have been fitted to a first–order rate equation, giving kobs = (4.68 ± 0.01) x 10–4 s–1.

  Phenol and tyrosine are established trapping agents for NO2 and OH. Aerial oxidation of NOCbl in the presence of phenol (0.10 M) gave nitrated phenol products (<

 1%), providing evidence for a NO2 intermediate. In the presence of a Tyr trap OH–Tyr was detected, consistent with formation of an OH intermediate. HPLC and mass spectrometry also provided evidence for formation of a small amount of a corrinoid species with two less H atoms than Cbl, consistent with attack by OH on the corrin ring.

Scheme 6.3 summarizes the proposed reaction pathways for the reaction between

NOCbl and O2. In this scheme the rate–determining reaction of NOCbl with O2 to form an N–bound peroxynitritocobalamin intermediate is followed by O–O bond homolysis of the Co(III)–bound peroxynitrite ligand and ligand isomerization. It is not clear to us whether ligand isomerization occurs via direct intramolecular ligand rearrangement in

165

addition to O–O bond homolysis followed by radical recombination. In any event, ligand isomerization leads to formation of nitratocobalamin (NO3Cbl), which rapidly hydrolyzes

+   to H2OCbl [2]. O–O bond homolysis results in the formation of caged NO2 and OH.

–   – Theoretical studies show that ONOO decomposes to NO2 and O [260]; the latter is

  rapidly protonated to form OH (pKa( OH) ~ 11.5 [261]). Upon release from the cage

 – NO2 reacts rapidly with itself and H2O to produce NO2 [251, 262], which then

+ substitutes the β–axial H2O ligand of H2OCbl to form NO2Cbl. The yields of the phenol

  and Tyr – trapped NO2 and OH products (< 1%) are considerably less than those observed for spontaneous ONOOH decomposition under the neutral pH conditions of this study, suggesting that Cbl can also trap these species. In support of this, a novel corrinoid complex is observed in the HPLC chromatogram with a mass which is 2 units smaller than Cbl itself, consistent with scavenging of OH by the corrin ring. OH could potentially also react with the corrin ring of the Cbl within the caged complex.

166

6.4 SUMMARY

Kinetic and mechanistic studies on the reaction between NOCbl and O2 show that the reaction is rapid, and proceeds via an associative mechanism with the formation of a peroxynitritocob(III)alamin intermediate, Co(III)–N(O)OO–. The intermediate undergoes

O–O bond homolysis and ligand isomerization to ultimately yield NO2Cbl and

+ H2OCbl /HOCbl, respectively. Ligand isomerization may potentially occur independent

  of O–O bond homolysis. Formation of small amounts of OH and NO2 intermediates is demonstrated using phenol and tyrosine radical traps. The amounts of the hydroxylated and nitrated products in the phenol and Tyr trapping experiments were considerably

Scheme 6.3. Proposed reaction pathway for the reaction between NOCbl and O2.

167

smaller than that observed for ONOO(H) decomposition in the presence of the identical trapping agents. Observation of a small amount of a (non–Cbl) corrinoid species by

+ HPLC with a retention time close to that of H2OCbl is consistent with formation of an

 OH intermediate. A modified corrinoid complex with 2 H less than the Cbl unit was

– characterized using ESI–MS. Furthermore, the presence of NO3 in the product mixture of the reaction of NOCbl with O2 was also demonstrated using HPLC. Finally, the oxygen concentration in cells varies from 4–40 µM [263]. Assuming a concentration of

20 µM, the half–life for the reaction of NOCbl with O2 is ~ 1 min (25 °C). In air the half– life for NOCbl decomposition is ~ 5 s.

168168

CHAPTER 7

SUMMARY AND FUTURE DIRECTIONS

7.1 SUMMARY

Nitric oxide (NO) is a signaling molecule produced by nitric oxide synthases involved in several important processes including vasodilation and neurotransmission.

The chemistry and biochemistry of NO with numerous molecules, including transition metal (TM) complexes, have been extensively studied in the literature. The closely related species, HNO, is also produced by nitric oxide synthases and is formed by enzyme–catalyzed reduction of NO. Recent studies provide strong support for HNO formation in biological systems. The chemical and biological properties of HNO are much less well understood, with HNO having properties distinct from those of NO.

The chemistry of HNO with TM complexes is a relatively unexplored area. HNO can substitute ligands of TM complexes and/or reduce the TM center. However, the redox potential of HNO/NH2OH suggested to us that HNO could also oxidize TM complexes. As cobalamins are redox–active molecules with readily accessible Co(III),

Co(II) and Co(I) oxidation states, these complexes are excellent candidates to explore the redox chemistry of HNO with transition metal complexes. The cis effect of the macrocyclic corrin ring also means that ligand substitution rates are conveniently rapid for cobal(III)amins, unlike cobalt(III) complexes with monodentate ligands.

168 169

This dissertation explores the reactions of all three oxidation states of vitamin B12

(cob(III)alamin, cob(II)alamin and cob(I)alamin) with two of the most commonly used and best understood HNO donors, nitroxyl (HNO) donors Angeli’s salt (AS) and Piloty’s acid (PA). We initially studied the reactions of the Cbl(II), the major intracellular form of

B12, with these two HNO donors. We observed an unusual stoichiometry of 1:2

Cbl(II):HNO donor which suggested the involvement of cobalamin intermediate. The

+ – possible cobalamin intermediate was either Cbl(III) (H2OCbl /HOCbl) or Cbl(I) . If

Cbl(III) is the intermediate, then reactions carried out in the presence of excess cyanide for the Cbl(II)/AS system should result in CNCbl formation since the reaction between

Cbl(III) and cyanide to form CNCbl is both kinetically and thermodynamically favorable under the close to neutral pH conditions under which this reaction was observed. CNCbl formation was not experimentally observed. The Cbl(I)– intermediate was confirmed by carrying out the reactions in the presence of excess nitrite. It is well known that Cbl(I)–

– reacts rapidly with NO2 and the stoichiometry of both systems changed from 1:2

Cbl(II):HNO donor to 1:1 Cbl(II):HNO donor in the presence of excess nitrite. This was consistent with Cbl(II) being reduced to Cbl(I)– by HNO and HNO being oxidized to NO.

– – The Cbl(I) is then oxidized back to Cbl(II) by HNO (or NO2 in the presence of nitrite) and finally Cbl(II) and NO combine to form NOCbl. Experiments with Cbl(I)– in the absence and presence of nitrite were also consistent with this mechanism.

Studies of the reaction of Cbl(III) with HNO donors were complicated by the

– presence of byproducts which also react with Cbl(III). AS decomposition generates NO2 and PA decomposition generates benzenesulfinate in addition to HNO. Nitrite and

170

benzenesulfinate react with Cbl(III) to form nitrocobalamin and benzensulfinatocobalamin, respectively. We therefore used excess Cbl(III), rather than

HNO donors in the kinetic experiments to circumvent this problem.

7.2 FUTURE DIRECTIONS

Our studies highlight the major drawback of these commonly used HNO donors for mechanistic studies – namely decomposition of AS and PA to release HNO is typically the rate–determining step of the reaction and therefore no kinetic information is obtained on the actual reaction of interest (the reaction of the complexes with HNO).

However HNO donors that decompose rapidly to give purely HNO are currently not available. A current focus of research in our lab concerns the development of photocleavable HNO donor molecules that decompose rapidly (ms or faster) under physiological conditions. Studying the reactions of cobalamins with these novel HNO donors would allow us to probe the kinetics and mechanisms of the reactions between cobalamins and HNO.

Another potential future direction involves studies on the kinetics of the reactions between cobinamides and HNO donor molecules. Cobinamides (Cbis) are biosynthetic precursors of cobalamins and are chemically formed from cobalamins by hydrolysis at the phosphate ribose moiety [264]. Cbis are also model complexes of the “base–off” form of cobalamins, which are always present in aqueous cobalamin solutions in small amounts. The structure of diaquacobinamide is shown in Figure 7.1. Unlike cobalamins, cobinamides have two axial sites that are readily substituted, which can result in different

171

CONH2 CONH2 CH3 CONH2 CH3

H C CONH2 3 N H2O N H3C CoIII H H N N H2O CH3 CH H NOC 3 2 CH3 CH3

CONH2 CH3 NH O

H C OH Figure 7.1. The structure of diaquacobinamide. and interesting reactivities in comparison to cobalamins. For example, cobinamide has an increased affinity for NO compared with cobalamin by a factor of 100 [265].

Finally, although there are quite a few kinetic studies on the reactions of Fe(III) porphyrins with HNO donors, there are few reports for their Fe(II) counterparts. Water– soluble reduced porphyrins would be useful for these studies viz. Fe(II)TPPS,

Fe(II)TMPS, Fe(II)TMPyP, etc.

APPENDIX

A.1. DERIVATION OF EQUATIONS 5.3 AND 5.4

For

K k A + L A L B

rate = k [A–L]

If [L] >> [A] (pseudo–first–order conditions), then rate = kobs ([A] + [A–L])

Hence k [A–L] = kobs ([A] + [A–L])

So,

Rearranging gives,

Since plots of kobs versus [L] = [Cbl] are linear, then

172 173

1 >> K [L] and kobs = kK [L] = kapp [L]

For

The apparent rate constant (kapp) can be calculated using the formula

For

174

kapp can be calculated using the formula

+ + + [H ] [H ] [H ] Ka(AS) = x x k1K1 + x x k2K2 kapp + + + + + [H+]+K (AS) [H ]+Ka(H2OCbl ) [H ]+Ka(AS) [H ]+Ka(H2OCbl ) a

+ 2 + [H ] k K [H ] Ka(AS) k2K2 k = 1 1 + app + + + + + + ([H ]+Ka(H 2OCbl )) ([H ]+Ka(AS)) ([H ]+Ka(H 2OCbl )) ([H ]+Ka(AS))

[H +]2 k K + [H+] K (AS) k K k = 1 1 a 2 2 app + + + ([H ]+Ka(H 2OCbl )) ([H ]+Ka(AS))

A.2. DERIVATION OF EQUATION 6.2

Reaction: NOCbl + O2 → products

So, Aobs = fNOCbl . ANOCbl + fproducts . Aproducts

where fNOCbl and fproducts are mole fractions of NOCbl and the Cbl products, and ANOCbl and Aproducts are their absorbances, respectively.

Therefore,

[] [] = + [] []

Since ANOCbl = A0 and Aproducts = A∞,

[] [] = + [] [] where [Cbl]total = total cobalamin concentration

Rearranging,

175

( − )[] [] = … … … … . () ( − )

( − )[] [] = … … … . () ( − )

The equilibrium constant (Keq) for the reaction is expressed as:

[] = [][]

Substituting values from Equation 1 and 2 and rearranging,

( − )[] = ( − )[] []

Rearranging further,

( + [] ) = ( + [])

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