Copper Adduct Formation, Properties, and Reactivity

Heme Axial Base Effects on Heme-Peroxo- Copper Adduct Formation, Properties, and Reactivity

by Patrick J. Rogler

Dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy

Baltimore, MD October, 2018

Abstract

Over the course of the next century, a fundamental understanding of the critical factors that control the efficiency, and selectivity of the four proton, four electron reduction of O2 to H2O will likely continue to grow in importance for inorganic and materials chemists. This may be driven in large part by the necessity and promise of sustainable and economically feasible alternative energy technologies which will undoubtedly rely on fundamental chemical insights into the discrete metal-oxy species formed in the course of reductive O—O bond activation and cleavage in both homogeneous and heterogeneous media. Bio-inorganic chemists seek to address these important problems by looking to natural systems, wherein lie excellent examples of efficient, selective O2 reduction provided by Heme-Copper Oxidases (HCOs). This superfamily of integral membrane proteins serve as the terminal electron acceptors of the mitochondrial electron transport chain where they bind and reduce O2 to H2O at a hetero-binuclear active site consisting of a tris-histidyl ligated CuB ion, and a heme iron cofactor. The discrete steps of this reductive

O—O bond cleavage reaction are coupled to transmembrane proton pumping and oxidative phosphorylation.

A long standing interest of our group has been to utilize heme-peroxo-copper adducts as model systems to understand in detail the factors which result in O2 reduction to water by drawing upon structure function relationships in the binuclear active site. The

III 2- II metal bridging peroxide formulation (heme)-Fe -(O2 )-Cu has never been observed during turnover, however it has been explored computationally and is an informative starting point as a model.

ii In the chapters below we present a design approach wherein the heme axial base moiety attached to the porphyrin periphery is changed from a covalently attached pyridyl base, to an imidazole base, and we describe the effects of these axial base functionalities on heme-peroxo-copper adduct formation, and stability. Finally, we report the synthesis, and characterization of a small library of mononuclear heme-ferric peroxides which serve to sharpen relevant questions related to heme-peroxo-copper adduct reactivity towards exogenous reductant.

Advisor: Prof. Kenneth D. Karlin Thesis Committee: Dr. David P. Goldberg Dr. John P. Toscano

iii Acknowledgements

I would first and foremost like to thank my thesis advisor, Prof. Kenneth D. Karlin for allowing me the opportunity to conduct my thesis research in hi laboratory. His vast knowledge and experience within not only our field, but also on a broad range of topics in chemistry and biology has helped me to grow great deal in the course of my graduate career, and I most certainly would not have been able to complete this work without his help and guidance. I would also like to thank the members of my thesis committee, Prof.

David P. Goldberg, and Prof. John P. Toscano for their insights, as well as their patience and kindness.

During my time as PhD candidate, especially early on, senior group members have helped me to grow a great deal. In particular I would like to thank Dr. Savita K. Sharma,

Dr. Isaac Garcia-Bosch, Dr. Ryan L. Peterson, and Dr. Sunghee Kim all of whom have helped me either through their superb teaching skills, or their mentorship. I would not have grown as much as a person, or a scientist during my time in graduate school were it not for their influence. Several lab group members have also been particularly impactful as well.

Daniel, Mayukh Jeff, Hyun, Haley, Austin, and Diego are all wonderful people, and I have been privileged to be in their company both in a scientific, and personal sense, and I will look back on our time together in lab with great fondness.

I would also like to thank the NIH for funding, and the Chemistry Department for financial support. Also, several staff members including Boris, Phil, Cathy, Joel, Rosalie

Dennis and Lauren have been very helpful throughout my time here.

Lastly, and most importantly of all, I would like to thank my parents, Charles and

Leslie Rogler, my brother Chris, my sister Kimberley and Valerie. Without their love and support I would never have made it through, and I will never forget how important they are to me. In addition to my family, many of my friends have been highly supportive, and great to talk to, and I have very many fond memories of my time with Zaid, Darius, Ivan,

Jordan, James, and Eddie.

Sincerely,

Patrick J. Rogler

Table of Contents

CHAPTER 1. UNDERSTANDING BIOLOGICAL O2 REDUCTION BY HEME-COPPER OXIDASES: SMALL MOLECULE SCALE HEME-CU MODELS CAN PROVIDE ENZYME INSIGHTS ...... 1 1.1. DIOXYGEN REDUCTION AND ACTIVATION: GENERAL CONSIDERATIONS ...... 2 1.2. DIOXYGEN ACTIVATION AND REDUCTION BY METALLOENZYMES ...... 4 1.3. EFFICIENT, SELECTIVE O2 REDUCTION BY HEME-COPPER-OXIDASES ...... 6 1.4. KEY STRUCTURAL FEATURES OF THE HCO SUPERFAMILY, AND ESSENTIAL REDOX COFACTORS ... 9 1.5. CATALYTIC PHASES OF O2 REDUCTION BY BOVINE HEART CYTOCHROME C OXIDASE ...... 10 1.5.1. Catalytic mechanism of cytochrome c oxidase ...... 11 1.6. UNDERSTANDING O2 REDUCTION BY HCOS USING A SYNTHETIC MODELING APPROACH ...... 14 1.7. DIOXYGEN DERIVED HEME-PEROXO-COPPER ASSEMBLIES AS HCO SYNTHETIC MODELS: GENERATION, STRUCTURES AND SPECTROSCOPIC PROPERTIES ...... 16 REFERENCES: ...... 28 CHAPTER 2. ISOCYANIDE OR NITROSYL COMPLEXATION TO HEMES WITH VARYING TETHERED AXIAL BASE LIGAND DONORS: SYNTHESIS AND CHARACTERIZATION ...... 39 2.1. ABSTRACT ...... 40 2.2. INTRODUCTION ...... 41 2.3. EXPERIMENTAL ...... 44 2.3.1. Materials and Methods ...... 44 2.3.2. Synthesis of DIMPI, and NO bound ferrous heme porphyrinates ...... 46 2.3.3. X-Ray structure determination ...... 48 2.4. RESULTS AND DISCUSSION ...... 50 2.4.1. Stable heme–isocyanide complex formation: ...... 50 2.4.2. Crystal structure of isocyanide complex, [(PIm)FeII-(DIMPI)] (3)-DIMPI ...... 53 2.4.3. Stable heme–Fe–Nitrosyl formation ...... 61 2.5. CONCLUSIONS ...... 65 REFERENCES: ...... 67 APPENDIX A ...... 72 CHAPTER 3. REACTIONS OF A HEME-SUPEROXO COMPLEX TOWARD A CUPROUS CHELATE AND •NO(G): CCO AND NOD CHEMISTRY ...... 76 3.1. ABSTRACT ...... 77 3.2. INTRODUCTION ...... 77 3.3. EXPERIMENTAL ...... 79 3.3.1. Materials and Methods ...... 79 3.3.2. Synthesis ...... 80 3.3.3. Preparation of 2H NMR and EPR samples ...... 81 3.3.4. Procedure for Nitrate/Nitrite test ...... 82 3.3.5. Nitration of the 2,4-Di-tert-butylphenol (DTBP) ...... 82 3.4. RESULTS AND DISCUSSION ...... 83 I 3.4.1. Reactivity of the iron(III)-superoxo complex (2) towards [Cu (AN)][B(C6F5)4] ...... 83 3.4.2. Reactivity of iron(III)-superoxo complex (2) towards •NO(g) ...... 86 3.5. CONCLUSIONS ...... 90 REFERENCES: ...... 91 CHAPTER 4. MONONUCLEAR LOW-SPIN FERRIC HEME PEROXIDE AND (HYDRO)- PEROXIDE COMPLEXES DISPLAYING AN END-ON BINDING MOTIF TO FERRIC HEME PORPHYRINATE ...... 96

vi 4.1. ABSTRACT: ...... 97 4.2. INTRODUCTION ...... 97 4.2.1. Heme/dioxygen interactions: from biology to model systems ...... 97 4.2.2. Heme ferric peroxide and hydroperoxide intermediates in cytochrome P450 enzymes, and synthetic models complexes...... 104 4.3. EXPERIMENTAL ...... 114 4.3.1. General methods ...... 114 4.3.2. UV-Vis experiments ...... 115 4.3.3. EPR experiments ...... 116 4.3.4. Resonance Raman experiments ...... 116 4.3.5. H2O2 quantification by horseradish peroxidase (HRP) test: ...... 117 4.4. RESULTS AND DISCUSSION ...... 118 III 2- - 4.4.1. Synthesis and characterization of an iron-peroxide porphyrin complex, [(F8)Fe -(O2 )] , III and its corresponding hydroperoxide species, [(L)(F8)Fe (O2H)] ...... 118 4.4.2. Synthesis and characterization of an end-on heme ferric peroxide complex, III Im III 2- Im III [Co Cp2][(P )Fe (O2 )] (3), and its hydroperoxide analogue, [(L)(P )Fe (O2H)] (4) ...... 124 4.4.3. Heme axial base effects on heme ferric peroxide structure, and spectroscopic properties . 129 4.4.4. Generation and characterization of a low-spin heme-peroxo-copper complex featuring a covalently attached axial imidazole ligand, and its reactivity towards an exogenous organometallic reductant (Me10Fc) ...... 132 4.5. CONCLUSIONS ...... 139 REFERENCES: ...... 141 APPENDIX B ...... 151 Curriculum Vitae…………………………………………………………………....154

vii List of Figures and Tables:

Chapter 1

Figure 1.1. (Top) Stepwise reduction of dioxygen to water. The reduction potentials are given in - + volts vs. NHE at 25°C, 1.0 atm O2, pH = 7. Overall Reaction: O2 + 4e + 4H  2H2O ΔEº = 0.815 V (pH=7, 1 atm)3 (Bottom) Dioxygen binding, and reductive O—O bond cleavage in Cytochrome c Oxidase (CcO), and the overall stoichiometry of O2 reduction by CcO. Note: Pumped protons are designated by the subscript ‘out’………………………….…………………2

Figure 1.2. (Left) Schematic diagram depicting the role of cytochrome oxidases in mitochondrial electron transport chain, CcO is represented by Complex IV. (Right) Overview of proton pumping channels, electron transfer pathways, substrate/product channels, and critical redox cofactors in Cytochrome c Oxidase.29 Copyright 2010 National Academy of Sciences…………..8

Figure 1.3. Active site of bovine heart CcO in its fully reduced (FeII/CuI) form. ………………...9

Figure 1.4. Overview of all of the redox active centers of bovine CcO (Left). View of the 36 binuclear active site comprising hemea3, and CuB (Right). (PDB file 1OCR.)…………………10

Figure 1.5. Catalytic mechanism of O2 reduction by bovine heart Cytochrome c Oxidase. Steps in the reductive phase coupled to proton pumping are indicated by white arrows. …………….…..14

III 2- II + Figure 1.6. Generation of heme-peroxo-copper adducts [(F8)Fe -(O2 )Cu (TMPA)] (top A), III 2- II + 95 and [(F8)Fe -(O2 )Cu (AN)] (bottom B). Adapted from ref. Copyright 2010 American Chemical Society…………………………… …………………………………………………...18

Figure 1.7. Heme-peroxo-copper adducts (A-C) synthesized, and spectroscopically characterized III 2- by the Naruta group. Complex (C) includes the crystal structure for [(TMP)Fe -(O2 )- II 98 (5MeTPA)Cu ](BPh4). Part (C) created using data taken from ref. ……………..……………..19

Figure 1.9. High-spin heme iron(III)-peroxo-copper(II) complexes, utilizing either the tetradentate ligand TMPA (A), or tridentate chelate AN (B). Structural details are derived from DFT calculations. Adapted from ref.95 Copyright 2010 American Chemical Society. .…………23

Figure 1.10. Formation of end-on μ-1,2 low-spin heme-peroxo-copper adducts (A) III 2- II + III 2- II + [(DCHIm)(F8)Fe -(O2 )-Cu (TMPA)] and (B) [(DCHIm)(F8)Fe -(O2 )-Cu (AN)] from their high spin precursors. Adapted from references.103,104 Copyright 2015 and 2011 American Chemical Society. ………………………………………………………………………………..25

Figure 1.11. Distinct heme-peroxo-copper binding motifs characterized by Karlin, and Naruta.106 See text for details. ……………………………………………………………………………….26

Table 1.1. Selected resonance Raman spectroscopic data: Heme-peroxo-copper complex core vibrations. ………………………………………………………………………………………...26

viii

Chapter 2

Figure 2.1. Generation of HCO model compound adducts from ferrous porphyrinate complexes, dioxygen and cuprous chelates. ………………………………………………………………….43

Figure 2.2. Ferric superoxide species characterized by our group. …………………………….....44

Table 2.1. Crystallographic data for complex [(PIm)FeII-(DIMPI)]. …………...…………………49

Figure 2.3. Generation of bis-isocyanide ferrous heme complex, (1)-DIMPI at room temperature. …………………………………………………………………………………………………….51

Figure 2.4. Generation of six-coordinate ferrous heme isocyanide complexes; (2), and (3)- DIMPI. …………………………………………………………………………………………...53

Figure 2.5. UV-Vis spectroscopic data for the ferrous DIMPI and NO complexes of (1), (2), and (3) in THF at room temperature. Black-reduced Fe(II) species; Red-Fe(II)-DIMPI and Blue- Fe(II)-NO complexes. ……………………………………………………………………………53

Figure 2.6. Displacement ellipsoid plot (50% probability level) of [(PIm)FeII-(DIMPI)] ((3)- DIMPI) showing the imidazolyl and 2,6-dimethylphenyl isocyanide ligands bound to Fe(II) center. Lattice solvent molecules and H atoms have been omitted for the sake of clarity. Selected bond lengths and bond angles are reported in Table 2.2. ………………………………………...54

Table 2.2. Selected bond lengths (Å) and bond angles () for (3)-DIMPI. Proposed H-bonds are also listed. ………………………………………………………………………………………..55

Figure 2.7. Crystal structures showing weak intramolecular CH…F interaction identified from the green lines shown. (Left) (3)-DIMPI and (Right) (4)-DIMPI. See text for further discussion. ……57

Figure 2.8. Solid state FT-IR spectra for Fe(II)-DIMPI complexes of, 2,6- dimethylphenylisocyanide (DIMPI), (1)-DIMPI, (2)-DIMPI, and (3)-DIMPI. …………………..58

Table 2.3. Properties of ferrous heme-DIMPI and ferrous heme-NO model complexes. ………...59

Figure 2.9. 19F-NMR of (1)-DIMPI (top), and (3)-DIMPI (middle) complexes in THF at room temperature. ...……………………………………………………………………………………59

Figure 2.10. Six coordinate ferrous heme mono nitrosyl complexes of (PPy)FeII, and (PIm)FeII…61

II Figure 2.11. X-band EPR at 4K in THF for ferrous heme-NO complexes. [(F8)Fe -NO] (1)-NO) (Red, 5C species), while (2), and (3), form 6C species. [(PPy)FeII-NO] (2)-NO) (Orange, 6C), and [(PIm)FeII-NO] (3)-NO) (Green, 6C). These spectra were analyzed further using an EPR simulation computer program, and the results of those fits, giving g-values and hyperfine coupling constants, are given in Appendix A (Figure S3). ………………………………………63

Figure 2.12. 19F-NMR of (1)-NO (top), and (3)-NO (bottom) complexes in THF at room temperature. ……………………………………………………………………………………...65

Appendix A

Figure S1. Binding isotherm at 430 nm resulting from the reaction of (PPy)FeII (12 μM in 2.5 mL Py II 7 -1 Tetrahydrofuran, THF, black) and DIMPI (red, (P )Fe -DIMPI). Ka = 2.29 x 10 M See main text for detailed discussion. ………………………………………………………………………74

Figure S2. Binding isotherm at 430 nm resulting from the reaction of (PIm)FeII (12 μM in 2.5 mL Im II 7 -1 Tetrahydrofuran, THF, black) and DIMPI (red, (P )Fe -DIMPI). Ka = 1.19 x 10 M See main text for detailed discussion. ………………………………………………………………………74

Figure S3. X-band EPR spectroscopy of ferrous heme-NO complexes recorded at 8K in frozen THF (red) and fit of the spectrum using the program Easy Spin [1] (blue). Fit parameters:

NO NO (a) (1)–NO (g1 = 2.0918, g2 = 2.0074, g3 = 2.0052; N hyperfine: A1 = 48.78, A2 = 67.63, NO A3 = 35.68) NO NO NO (b) (2)–NO (g1 = 2.0746, g2 = 2.0081, g3 = 1.9904; N hyperfine: A1 = 4.0, A2 = 60.4, A3 = Py Py Py 35.8; A1 = 18.1, A2 = 20.1, A3 = 19.1) NO NO NO (c) (3)–NO (g1 = 2.0686, g2 = 2.002, g3 = 1.9662; N hyperfine: A1 = 44.10, A2 = 63.18, A3 Im Im Im = 47.45; A1 = 22.94, A2 = 1.29, A3 = 16.84)………………………………………………..75

Chapter 3

Py III •- Py II Figure 3.1. Formation of (P )Fe -(O2 ) (2) via oxygenation of (P )Fe (1) and subsequent reaction with [CuI(AN)]+ to give the meta-stable intermediate, the high-spin heme-peroxo-copper III 2- II + complex (3a), which decays to give the µ-oxo complex [(F8)Fe -(O )-Cu (TMPA)] (3). ……83

Figure 3.2. UV-Vis spectra of (1, black) a reduced (PPy)FeII + [CuI(AN)]+ 1:1 mixture; (3a, Py III 2- II + Py III red) high spin peroxo complex [(P )Fe -(O2 )Cu (AN)] ; (3, green) μ-oxo complex [(P )Fe - (O2-)-CuII(AN)]+.………………………………………………………………………….………85

2 Py II Py III 2•- Figure 3.3. H-NMR spectra at –80 °C in THF (1) (d8-P )Fe -THF, (2) (d8-P )Fe -O2 ; (3a) Py III 2- II + Py III 2- II + [(d8-P )Fe -(O2 )-Cu (AN)] and (3) [(d8-P )Fe -(O )-Cu (AN)] . …………………………85 Figure 3.4. Reaction sequence where •NO(g) is added to superoxo complex (2) to give complex nitrato complex (4). In the presence of a phenolic substrate, the same reaction gives (5) as a final product along with the ortho-nitrated phenol. …………………………………………………...87

Figure 3.5. UV-Vis spectra showing superoxo (2, red) formed from reduced (PPy)FeII (1, black) Py III by bubbling O2(g) at –80 °C; nitrato complex (P )Fe -ONO2 (4, grey) generated immediately after addition of •NO(g). …………………………………………………………………………..88

Figure 3.6. UV-Vis spectroscopy in THF at -80 ºC. (black) spectra is reduced (PPy)FeII (1); red is (2 + DTBP), and green is (5). ……………………………………………………………………89

Chapter 4

Figure 4.1. General paradigm of dioxygen activation and reductive O-O bond cleavage in hemoproteins. …………………………………………………………………………………...98

x Table 4.1. Dioxygen-Binding Hemoproteins and Their Main Catalytic Functionalities. ……...99

Figure 4.2. Crystal structure of Compound 0 intermediate in chloroperoxidase. Fe-O = 1.80 Å, O—O = 1.50 Å, and Fe—O—O = 131°. ……………………………………………………….103

III 2- - Figure 4.3. Synthetic approaches utilized by Naruta et. al. in order to generate [(P)Fe -O2 ] , and III (P)Fe (O2H) complexes utilizing the TMPIm ligand platform. ………………………………..108

Figure 4.4. Work carried out by Naruta and co-workers on a modified “hangman” porphyrin ligand platform (Mes = mesityl). Chemical reduction of the ferric heme superoxide yielded the low-spin end-on ferric heme peroxo complex, which can then be protonated to its corresponding ferric hydroperoxide species. ………………………………………………………………..….110

Figure 4.5. “Hangman” type porphyrins featuring an imidazole axial ligand, and an carboxylic acid (top), or ester (bottom) group attached via an anthracene linker. The heme superoxide species (2a) was observed to undergo reduction protonation with its parent porphyrin complex (1a), to give the ferric hydroperoxide (3a). Replacement of the carboxylic acid group with an ester moiety yielded the stable superoxide (2b), which could be reduced and protonated with exogenous reductants, and acid sources to the ferric hydroperoxide (3b). ……………………..111

Table 4.2. Spectroscopic data for mononuclear ferric heme peroxide and hydroperoxide complexes. ……………………………………………………………………………………...112

III III 2- Figure 4.6. Synthetic methods utilized in this work to generate [Co (Cp)2][(F8)Fe -(O2 )] (1), III and [(L)(F8)Fe (O2H)] (2), here L represents either solvent (THF), 2,6-lutidine, or DMF; See text for further discussion. …………………………………………………………………………..119

II III .- Figure 4.7. UV-Vis spectra depicting [(F8)Fe (THF)2] (black); [(THF)(F8)Fe (O2 )] (red), III III 2- formed by bubbling O2 through the starting solution; (1) [Co Cp2][(F8)Fe (O2 )] (blue), formed II on addition of ~1.3 equivalents of the reductant [Co (Cp)2] to the ferric superoxide. Each experiment was carried out in anhydrous THF at -80°C (193 K). See text for further discussion. …………………………………………………..……………………………………………… 120

III III 2- Figure 4.8. X-band EPR spectrum (10 K) taken of (1) [Co (Cp2)][(F8)Fe (O2 )] (blue) generated by adding ~1.3 equivalents of CoCp2 to the six-coordinate ferric superoxide III .- [(THF)(F8)Fe (O2 )] (red) in THF at -80°C. See text for discussion. …………………………121

III III 2- Figure 4.9. Resonance Raman spectra for (1) [Co (Cp2)][(F8)Fe (O2 )] (red); (2) III III [(L)(F8)Fe (O2H)] (green); and [(DCHIm)(F8)Fe (O2H)] (blue). See text for discussion. …...122

III III 2- Figure 4.10. UV-Vis spectra depicting [Co Cp2][(F8)Fe (O2 )] (1) (blue), and III [(L)(F8)Fe (O2H)] (2) (green) in THF at -80°C (193 K). See text for further discussion. ….....123

III III 2- III Figure 4.11. EPR spectra (10K) of [Co (Cp2)][(F8)Fe (O2 )] (1) (blue), and [(F8)Fe (O2H)] (2) (green), formed by adding ~1.0 equivalent of [(Lu)(H)][OTf] or [(H)(DMF)][OTf] to the side-on heme ferric peroxide (1) Generated at 2.0 mM concentration at -80°C in THF. …………………...124 III Im III 2- Figure 4.12. Synthetic methods utilized in this work to generate [Co (Cp)2][(P )Fe -(O2 )] (3), Im III and [(P )Fe (O2H)] (4), see text for discussion. ………………………………………………125

Im II Im III .- Figure 4.13. UV-Vis spectra depicting [(P )Fe (THF)] (black); [(P )Fe (O2 )] (red) obtained Im III 2- III by bubbling O2 through the reduced solution (black); [(P )Fe (O2 )][Co Cp2] (3) (blue) II obtained by adding ~1.3 equivalents of [Co (Cp)2] to the ferric superoxide in THF at -80°C (193 K). See text for further discussion. ……………………………………………………………..126

III Im III 2- Figure 4.14. X-band EPR spectrum (10 K) taken at of [Co (Cp2)][(P )Fe (O2 )] (3) (blue) Im III .- generated by adding 1.3 equivalents of CoCp2 to the ferric superoxide [(P )Fe (O2 )] in THF. See text for discussion. …………………………………………………………………………127

III Im III 2- Figure 4.15. Resonance Raman spectra for [Co (Cp2)][(P )Fe (O2 )] (3) (blue). See text for discussion. ………………………………………………………………………………………128

III Im III 2- Im III Figure 4.16. UV-Vis spectra depicting [Co Cp2][(P )Fe (O2 )] (3) (blue); (4) [(P )Fe (O2H)] (4) (green) generated by adding ~1.0 equivalent of [(Lu)(H)](OTf) to (3) in THF at -80°C (193 K). See text for further discussion. ……………………………………………………………..129

Im III Figure 4.17. X-band EPR spectrum (10 K) taken at of [(P )Fe (O2H)] (4) (green) generated by adding ~1.0 equivalents of [(Lu)(H)](OTf) to (3) in THF. See text for discussion. ……………129

Figure 4.18. Summary of spectroscopic parameters determined thus far for mononuclear heme ferric peroxides (1) & (3), and hydroperoxides (2) & (4). ……………………………………...131

Im III 2- II + Figure 4.19. Synthetic methodology for the generation of [(P )Fe -(O2 )-Cu (AN)] (5) at - 80°C in THF, see text for discussion. …………………………………………………………..132

Figure 4.20. UV-Vis spectrum depicting the starting equimolar mixture of [(PIm)FeII] / I F Im III 2- II + [Cu (AN)](BAr ) (black), and [(P )Fe -(O2 )-Cu (AN)] (5) (blue) obtained by bubbling O2 into the starting reduced solution at -80°C in THF. …………………………………………….134

Figure 4.21. Resonance Raman spectrum depicting the ν(Fe—O), and ν(O—O) vibrational Im III 2- II + modes detected for [(P )Fe -(O2 )-Cu (AN)] (5) (blue) see text for discussion. …………...135

Figure 4.22. Proposed 1e‒ reduction chemistry occurring on addition of excess reductant Im III 2- II + (Me10Fc) to [(P )Fe -(O2 )-Cu (AN)] (5) in THF at -90°C. ………………………………...136

+ Figure 4.23. UV-Vis spectra showing the production of ~1 eq of Me10Fc (424, 531, 784, 803 nm Im III 2- II + (purple)), on addition of 10 equivalents of Fc* to [(P )Fe -(O2 )-Cu (AN)] (5) at -80°C in THF. …………………………………………………………………………………………….136

Figure 4.24. Resonance Raman spectrum of the product mixture (6), displaying a ν(Fe-O) stretch -1 16 18 at 578 cm . The top two spectra are the rRaman spectra of the O2 (blue), and O2 isotopes, the bottom is their difference, revealing the isotope sensitive features in the product mixture (6) substituted versions o see text for discussion. ………………………………………………….138

III Im III 2- Figure 4.25. Summary of the spectroscopic properties observed for [Co (Cp)2][[(P )Fe (O2 )] (3), and the product mixture formed on reduction of (5) with excess decamethylferrocene (but it is Im III 2- - I + a one-electron reduction), proposed here to be [(P )Fe (O2 )] ⋅⋅⋅⋅[Cu (AN)] (6). See text. …138

xii Appendix B

Figure S1. UV-Vis spectrum depicting the oxidation of AzBTS-(NH4)2 with H2O2. Calibration performed at room temperature. See experimental section for details. ………………………...152

Figure S2. Calibration curves for the UV-Vis features formed at 416, and 734 nm in the HRP test. See experimental section for details. ………………………………………………………152

Figure S3. UV-Vis spectra depicting decamethylferrocenium BArF, features at 783, and 803 nm are diagnostic of concentration ranging from 0.2 mM to 1.0 mM. Spectra were taken at -80C in THF. …………………………………………………………………………………………….153

Figure S4. Calibration Curve depicting decamethylferrocenium BArF, features at 785, and 803 nm are diagnostic of concentration ranging from 0.2 mM to 1.0 mM. Spectra were taken at -80C in THF. ………………………………………………………………………………………….153

xiii

xiv

Chapter 1. Understanding Biological O2 Reduction by Heme-Copper Oxidases: Small Molecule Scale Heme-Cu Models Can Provide Enzyme Insights

1 1.1. Dioxygen reduction and activation: general considerations

Some of the most pressing, and intriguing problems pivotal to the development of sustainable energy technologies and chemical feedstock production are small molecule transformations such as O2 reduction, H2O oxidation, H2 evolution and oxidation, and

N2/CO/CO2 reduction/fixation. One of these diatoms, dioxygen (O2), not only spans all areas of chemistry, but serves as a cornerstone of aerobic life. In spite of the immense oxidizing power stored in its double bond, O2 is kinetically inert under ambient conditions due to its triplet ground state which necessitates intersystem crossing to allow for reaction with typical organic molecules in a singlet ground state. In order to access and utilize (i.e. for chemical transformations) this highly potent oxidant, the oxygen double bond must first be reductively activated both in biological, and non-biological contexts. The complete reduction of O2 to water requires 4 protons, and 4 electrons, and is well understood in aqueous media under alkaline, acidic, and neutral conditions (See Figure 1.1, below).1,2

This small molecule activation reaction is not only critical to the bioenergetics of aerobic life, where it is utilized to propel oxidative phosphorylation, but also to the implementation and development of sustainable fuel cell technologies wherein it can serve as the cathodic half reaction of a fuel cell.4–10 An in-depth understanding, and control over this reductive O—O bond cleavage reaction is essential in natural systems because leakage of partially reduced oxygen species (PROS) can lead to protein and membrane damage, and yield decreased efficiencies in fuel cells. Indeed, the implementation of oxygen reduction catalysts with low overpotential and high selectivity for the 4-electron reduction product, H2O, has been a substantial barrier to the implementation of sustainable fuel cell technology at industrial scale. Given these pivotal concerns, a long-standing goal in inorganic chemistry has been to conceive and implement chemical systems that reduce O2 to water completely and selectively. To achieve this goal, a fundamental understanding of all the discrete stepwise proton transfer, electron transfer, and proton coupled electron

11–15 transfer steps involved in O2 reduction is expedient. In the ‘natural systems’ introduced above, (see Figure 1.1, bottom) the dioxygen reduction reaction is catalyzed by a diverse superfamily of integral membrane proteins known as Heme-Copper Oxidases, which couple transmembrane proton movements and internal electron transfer reactions within cellular mitochondria to the selective, and efficient reductive activation, and O—O bond cleavage of O2 to water at a binuclear active site containing a tris-histidyl ligated copper ion, and a heme cofactor.

3 1.2. Dioxygen activation and reduction by metalloenzymes

In aerobic biological systems, stepwise, selective, and efficient activation of dioxygen, and reductive O—O bond cleavage to yield to water is carried out by metalloproteins containing redox active first row transition metal ions such as Iron, Copper,

Zinc, and Manganese. Another level of complexity in these systems arises from the diversity of primary coordination/ligand environments, as well as secondary coordination sphere interactions that include important effects such as hydrogen bonding, Lewis acid/base interactions, and in many important cases, the redox activity of amino acid residues. These variables make metalloproteins amongst the most efficient, and selective known catalysts for reductive dioxygen cleavage, a distinction made all the more exceptional by their usage of earth abundant transition metals.

Despite O2 reduction in aqueous media being well understood, very little is known about the interactions of PROS with metal ions, particularly how the redox and protonation chemistry of oxy species vary when O2 is bound to various metal ions, with differing coordination modes, and secondary coordination sphere interactions. Undoubtedly, the

0 thermodynamic reduction potential (E ) of PROS derived from O2 is different when the species is bound to a metal fragment, as opposed to the free fragment in aqueous media.

Additionally, the basicity (pKa) of the metal bound anionic species (for example, the hydroperoxide anion, –OOH) cannot be expected to have the same value when the PROS is bound to a positively charged metal ion. Not only should factors such as metal ion identity, and ligand coordination environment affect the fundamental properties of metal- oxy species, but they assuredly change the conditions, and discrete steps by which O2 is activated, and used for substrate oxidation, oxygenation, or is reductively cleaved to water.

4 Indeed, this variation is evidenced by the great structural variety of metal-oxygen adducts observed in metalloenzymes, as well as their unique functionalities, which vary depending on interactions with the protein active site, and amino acid residues.

A now classic example of these principles is illustrated by cytochrome P-450 monooxygenases, a diverse class of metalloproteins which activate dioxygen using a heme cofactor for highly selective, C-H bond functionalization such as epoxidations and oxygenations. During substrate oxidation by cytochrome P-450’s, the basicity of an

III iron(III)-hydroperoxide intermediate ((heme)Fe (O2H) is controlled such that subsequent protonation of the distal oxygen atom yields heterolytic O—O bond cleavage, to generate the high valent active oxidant in the catalytic cycle, Compound I (Cmpd I, formulated as an FeIV=O π-cation radical), which oxidizes the substrate. Additionally, a ‘push’ effect generating from an axial cysteinate residue bound to the heme, a ‘pull’ effect is facilitated by polar amino acid residues in the distal pocket of the enzyme to protonate Cmpd 0 (a low spin 6 coordinate heme ferric hydroperoxide, see Chapter 4) at the distal oxygen atom, triggering heterolysis to generate the active oxidant, Cmpd I. The focus of this project is to develop a series of small molecule inorganic model systems inspired by the heme-copper binuclear center utilized by the heme-copper oxidase superfamily to effect efficient and

16–20 selective O2 reduction.

Despite the huge number of heme, or copper containing enzymes involved in partial

O2 reduction, be it for purposes related to biosynthesis(substrate oxygenation, oxidation, etc.) or PROS mitigation/regulation, the best examples to date that allow us to study complete O2 reduction to water are heme-copper oxidases. In aerobic natural systems (both prokaryotic, and eukaryotic), an elaborate electron transport chain has been evolved where

5 many complicated enzymes, including terminal heme-copper oxidases work in concert to efficiently reduce O2 to water, while converting the thermodynamic potential energy stored in the O=O double bond to chemical energy in order to drive transmembrane proton pumping.21

A great deal of research effort has been devoted to understanding the critical biochemistry and bioenergetics of heme-copper oxidase (HCO) function and mechanism.

These studies are inevitably complicated by the fact that heme-copper oxidases are highly complex integral membrane proteins, making direct spectroscopic interrogation of intermediates formed during turnover highly complicated. This is what makes small molecule model studies of cytochrome c oxidases very attractive as a surrogate for complicated enzymatic systems, as these discrete heme-peroxo-copper adducts, by emulating the key active site features can serve to answer fundamental questions related to heme-peroxo-copper adduct properties, their reactivity towards acidic phenols, and reductants as proxies for redox active amino acids in the active site, and overall enzyme valency, respectively. They are also highly amenable to spectroscopic investigation given their relative simplicity as comparted to the enzyme structure.

1.3. Efficient, selective O2 reduction by heme-copper-oxidases

As mentioned earlier, the four proton (H+) / four electron (e-) reduction of dioxygen to water is one of the most enabling chemical transformations in aerobic life. This overall thermodynamically favorable process is utilized in Archaea, Bacteria, and Eukaryotes via terminal oxidase enzymes to generate the proton electrochemical gradients that are essential to cellular respiration, in addition to motility amongst some Bacteria and Archaea.

6 They provide a central example of the coupling of O2 reduction to the generation of transmembrane electrochemical gradients. In one critical capacity these integral membrane proteins serve as the terminal electron acceptor of the mitochondrial electron transport chain. Here, their role is to catalytically reduce dioxygen to water, while simultaneously transporting protons across the mitochondrial inner membrane via proton pumping channels utilizing a Grothuss type mechanism.22–24 (Figure 1.2, right).

+ + 4subred + 8Hin + O2  2H2O + 4subox + 4H out

In this equation, the subscripts denote 4 protons pumped per dioxygen molecule reduced to two equivalents of water. Heme Copper Oxidases can be further classified according to their source of electrons, which also has implications for the proton pumping mechanism. Cytochrome Oxidases obtain their electrons via cytochrome c, an electron transfer hemoprotein, which acts as a one electron reductant. Quinol Oxidases however obtain their electrons from ubiquinol, or menaquinol, which are oxidized in a double proton coupled electron transfer reaction to their corresponding quinones, with the protons pumped to the P-side of the membrane. Another important distinction between Quinol and

25–27 cytochrome oxidases is that quinol oxidases lack the CuA redox cofactor (see below).

Cytochrome Oxidases are one critical component for maintaining this electrochemical potential gradient, which is utilized downstream by ATP synthase to generate adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and inorganic phosphate (Pi), which is the main source of ATP for the cell.24,28

Broadly speaking, HCO’s are a large superfamily of integral membrane proteins with diversity in heme type, electron donor, and number of subunits.30–33 They have a high degree of evolutionary similarity to Nitric Oxide Reductases, both in terms of their structure, and their functionality.34,35 However, as will be explained below, there are several structural features that distinguish Cytochrome c Oxidases. Namely a redox active tyrosine residue at the binuclear center is critical to CcO’s function and is not present in nitric oxide reductases. Nitric Oxide Reductases also contain a non-heme iron cofactor in the binuclear center, as opposed to copper in Cytochrome Oxidases, and they also do not have proton pumping channels. The most well studied example of the Cytochrome c

Oxidase family, in terms of structure, mechanism, and theoretical modeling is the bovine heart (Bos taurus) cytochrome c oxidase, and in subsequent discussions the amino acid sequence from this enzyme is used as reference. (Figure 1.3, below, PDB: 5B1B(1.6A))

Figure 1.3. Active site of bovine heart CcO in its fully reduced (FeII/CuI) form.

1.4. Key structural features of the HCO superfamily, and essential redox cofactors

The elucidation of the structure of Cytochrome c Oxidase was critically important to establishing a structural motif that could be used for modeling studies of the active site.36

In Cytochrome c Oxidases, there are 4 redox active metal centers (Figure 1.2, right). The first of these are a part of the dicopper center (CuA) that serves as an electron storage and transfer site. This binuclear center is reduced by ferrocytochrome c, and serves as the entry

– point for electrons (e ) into the enzyme. The CuA electron storage site is formally a mixed- valent, fully delocalized di-Cu1.5 site, and when reduced by cytochrome c, it is reduced to a CuI—CuI oxidation state, and is ready to provide reducing equivalents to the next electron storage site in the enzyme. This is a low spin hemea cofactor which is located ~ 19.5 Å below the binuclear CuA site. This cofactor also stores reducing equivalents for use by the binuclear center, to which it is axially connected to by a His-Phe-His moiety. The binuclear

9 center consists of the high spin heme a3 cofactor, and the CuB ion, located ~5.1 Å away from the heme a3 cofactor, Figure 1.4, right. The CuB cofactor is ligated by three histidine residues (His-290, His-291, His-240), one of which is cross-linked to a nearby tyrosine residue (Tyr-244). This His-Tyr cross-link is critical to maintaining the oxidase function

37–42 of CcO. Not only does this C-N crosslink alter the basicity (pKa), and redox potential

(E0) of the tyrosine residue, which is important to its redox activity during turnover (vide infra),37,43,44 but it also facilitates hydrogen bonding interactions with water molecules in the active site, that link the tyrosine O—H to a heme a3 bound superoxide (see below) preceding O—O bond cleavage.45–48 The tyrosine residue also hydrogen bonds to a heme farnesyl sidechain, linking it to substrate and proton gating during turnover.49,50 Despite the critical nature of this cross-link to cytochrome oxidase function, its biogenesis is not well understood, and remains an important question in the sub-field of biological cofactor biogenesis.41,51,52

Figure 1.4. Overview of all of the redox active centers of bovine CcO (Left). View of the binuclear 36 active site comprising hemea3, and CuB (Right). (PDB file 1OCR.)

1.5. Catalytic phases of O2 reduction by bovine heart cytochrome c oxidase

The mechanism of reductive O—O bond cleavage by Cytochrome c Oxidase can be divided into two phases. The first is the oxidative phase, during which dioxygen binds

10 to the binuclear center of CcO, and is reductively cleaved. This is followed by the reductive phase, where the active site is ‘regenerated’ by successive electron and proton transfers from the electron storage sites (Cua and heme a), and proton storage sites within the binuclear heme-copper center (BNC). Stepwise proton coupled electron transfers restoring

CuB and heme a3 to their copper(I), and iron (II) oxidation states during the reductive phase are coupled to proton pumping across the inner mitochondrial membrane by CcO. As mentioned earlier, the membrane electrochemical gradient formed during the reductive phase of CcO turnover drives ATP synthesis, and consequently is of great interest to biophysical chemists, and biochemists.53 Extensive molecular dynamics simulations, and mutational studies have provided insights into exactly how protons are shuttled across the membrane, and transferred to the BNC, with no back-leakage.29,54–57 Briefly, proton loading sites within two proton pumping channels, D and K are linked to redox changes at the binuclear center. Conformational changes caused by redox events, can lead to proton uptake at loading sites that increase the electron affinity at the BNC.20,58–61 Coupling proton pumping to O2 reduction implies that the active site proton storage moieties, including a glutamic acid residue (E-242), and a Mg-water cluster adjacent to heme a3 gate proton pumping pathways, and cause allosteric changes in the protein that allow protons to be delivered to the active site, or pumped across the membrane.

1.5.1. Catalytic mechanism of cytochrome c oxidase

Oxidative Phase –

Numerous spectroscopic techniques including time resolved infrared, resonance

Raman (rR) spectroscopy, X-Ray techniques, and flash photolysis of heme-CO bound CcO have revealed that on entering the binuclear center O2 binds CuB transiently, which

11 increases the O2 affinity of heme a3 by conformational changes related to water channel

62,63 gating. Binding of O2 to (R), the fully reduced state of CcO, begins the oxidative phase of the catalytic cycle, by forming the ferric-superoxo species (A).64 To aid in overcoming the energetic barrier for O—O bond cleavage, an additional proton in the active site increases the redox potential of the binuclear center.46,65 This proton may be stored at a nearby Mg-water cluster, which functions as a proton loading site for the binuclear center.60

The intermediate immediately preceding O—O bond cleavage, (IP) has not been spectroscopically characterized in the active form of cytochrome c oxidase because of its rapid decay via reductive O—O bond cleavage to yield (PM). Despite this, it has not been ruled out spectroscopically, and given the close proximity of the iron center in heme a3, and CuB, this configuration is not unrealistic. The next observed intermediate is (PM), which was originally mistaken to be a peroxide species due to its Fe-O stretching frequency at 804 cm-1. However, mixed isotope labeling studies later showed that the O—O bond is cleaved in this intermediate, making it a formally CuII-OH⋅⋅⋅FeIV=O species. This iron(IV)- oxo species can also be generated via a “peroxide shunt” pathway by adding H2O2 to the fully oxidized state of CcO.66–69 At this point, three electrons have been provided by the metal centers, with the fourth reducing equivalent plus proton provided as a hydrogen atom from the tyrosine residue cross-linked to His-240 in a proton-electron transfer step mediated by hydrogen bonded water molecules (IP to PM, see Figure 1.5).

Reductive Phase –

Prior to the reductive cleavage of the O—O bond during the oxidative phase of the catalytic cycle, all of the reducing equivalents needed are stored in the redox active metal ion cofactors embedded in subunit I (CuA, Heme a, CuB, Heme a3). The regeneration of

12 these cofactors is of great interest to biophysicists because these proton and/or electron transfer events occur in concert with proton pumping by CcO.70,71 Further reduction of

(PM) yields the (F) state, wherein the tyrosyl radical has been reduced to tyrosinate, and one proton provided to the CuII-OH to yield water; this conclusion is supported by infrared spectroscopic data (IR). The close overlap of (F)’s features with those of the (P) intermediates suggest that electrons stored in CuA, or heme a go toward a proton coupled

72 reduction (PM)  (F), rather than just reduction of (PM) (PR). This step is coupled to the uptake of a proton by the D channel, and its pumping across the membrane as well as

71 internal electron transfer in subunit I from CuA to heme a. Next, proton, and electron

III transfer to the binuclear center results in the formation of (OH), formulated as an Fe -

OH…CuII species, although this could also be a bridging hydroxy species.39,50,73,74 A great deal of work has been devoted to understanding the precise mechanism by which (F) is converted to (OH), because the precise stepwise electron and proton transfer, and how they

50,74–78 are coupled to proton pumping is not fully understood. The intermediate (EH) is next, formed by an additional, rapid proton coupled electron transfer that is observed only by electron injection experiments.79 Finally, the reduced state of the enzyme is obtained by delivery of a proton and electron, both of these steps (OH)  (EH), and (EH)  (R) occur very rapidly and are coupled to proton translocation.80–82 The most important stage of this catalytic cycle for our work is the conversion of the (A/oxy) intermediate to (PM). In our

HCO adduct approach, we seek to understand the bonding, structure, and reactivity of intermediates (i.e. the hyrdoperoxo, or peroxo species, (IP)) involved in this step, and their relevance towards broader heme-peroxo-copper systems. For our model complexes of cytochrome c oxidase, the binuclear active site structure of bovine (aa3) cytochrome c

13 oxidase provides a direct biological context. The “resting” state structure (above) of this particular cytochrome c oxidase has been extensively studied, and serves as the specific structural inspiration for our model complexes.20,83

Figure 1.5. Catalytic mechanism of O2 reduction by bovine heart Cytochrome c Oxidase. Steps in the reductive phase coupled to proton pumping are indicated by white arrows.

1.6. Understanding O2 reduction by HCOs using a synthetic modeling approach

Certain aspects of the catalytic mechanism and active site structure function relationships within highly complex biological systems, such as heme-copper oxidases, can be best answered using approximations developed via synthetic model chemistry.51,84

Cooperative effects including allosteric interaction, and proton conducting channels are critical to moving proton, electrons, water, and substrates/products over (relative) long

14 distances during HCO turnover, and are thereby critical to O2 reduction in biological systems. When considering how to transfer insights from complex biological systems to more industrially relevant applications, such as the design of functional catalytic systems for fuel cell applications, it’s necessary to simplify the problem to several key questions and parameters. These may include, but are not limited to (i) the discrete structure, i.e. binding motif, of the metal-oxy species and its partially reduced forms (superoxo, peroxo, or oxo), (ii) the degree of protonation of different intermediate species formed in the course of the O2 reduction reaction, (iii) interactions that may favor, or disfavor reductive O—O bond cleavage including acid-base interactions and/or hydrogen bonding interactions, (iv) the relative rates and order of proton-transfers (PT), electron transfers (ET), and (v) the role of the solvent environment, including the coordinating ability of the solvent and its capacity to solvate charged species (dielectric). Details such as these can be probed using models that incorporate the critical features of the active site. There have been many investigations with both simple (small molecule), and elaborate (engineered proteins) HCO model compounds to systematically address the factors noted above.85–87

Synthetic inorganic models enable chemists to use conditions that are seemingly irrelevant to the enzyme, such as organic solvents, low temperatures, and unnatural pH values to eliminate side reactions, solubilize metal-ligand coordination complexes, stabilize low-temperature stable intermediates, and modulate hydrogen bonding interactions. By controlling these conditions, fundamental questions related to intermediate

0 properties, and reactivity as a function of substrate properties (i.e. pKa, and E ), can be addressed in a simplified, systematic manner. Another important aspect of these model chemistries are theoretical investigations, which have made extremely valuable

15 contributions that supplement experimental data from model systems, and help to relate these experimental data to our understanding of HCOs, both in terms of spectroscopically detectable intermediates, as well as bioenergetics. Model chemistry leading to the rigorous physical and electronic structural characterization of HCO models, and their reactivates with O2, coupled to theoretical investigations have provided great insights into structure- function relationships in O2 activation and reductive O—O bond cleavage chemistry by

HCOs. Interplay between enzyme studies and synthetic models has expanded our understanding and inspires the design of new systems.

1.7. Dioxygen derived heme-peroxo-copper assemblies as HCO synthetic models: generation, structures and spectroscopic properties

Clearly, the study of O2-reaction with heme and copper complexes, or pre- organized synthetic reduced heme-Cu assemblies, is critical in the modeling of HCOs and this strategy has been extensively employed by the Karlin, Naruta, Collman88–92 and a few other research groups. In these model compound systems, the Fe…Cu distance is determined by the bonding interactions of the Fe and Cu to the bridging ligand, which derives from dioxygen reactivity with reduced iron and copper ions. Many of the studies, must be carried out at low temperatures (~ –80 °C), under dry conditions due to the thermal instability, susceptibility of peroxo groups to protonate, releasing (usually) hydrogen peroxide, and/or their tendency to form bridging oxo species (i.e., by disproportionation, vide infra), and hydroxides (i.e., via further reaction with water). This methodology has enabled the systematic variation of the coordination environment around the Cu as well as the Fe ion, and characterization of interesting biorelevant intermediates. Additionally, these adducts can and have been studied by spectroscopic techniques which help correlate

16 aspects of heme/Cu O2-adduct molecular and electronic structure to their reactivity towards substrates (e.g., H+, e–, phenols).

Before going in depth into heme-peroxo-copper model system reported in the literature, it is instructive to cover a few pivotal aspects of this chemistry, utilizing seminal examples from by Karlin and Naruta groups, in order to give better context for other systems described, especially the more recent ones. To this end, in this section we detail the synthesis, typical geometric and electronic properties, and key spectroscopic signatures of high-spin and low-spin heme-peroxo-copper model systems, and their relationship with similar/relevant intermediates of CcO. Our discussion includes peroxo complexes supported by tethered (i.e., potential copper ion chelates covalently appended to the synthetic porphyrinate) ligand architectures, as well as those bearing appended phenol moieties as a mimic for the His-Tyr crosslink at CcO active site.

III 2- II + III 2- II + The study of [(F8)Fe -(O2 )-Cu (TMPA)] (A) and [(F8)Fe -(O2 )-Cu (AN)]

(B) (Note: both as perchlorate and/or BArF salts, BarF = tetrakis(pentafluorophenyl)borate)

(Figure 1.6) have helped to illustrate the influence of copper chelate denticity, and axial base binding on heme-peroxo-copper bonding interactions. Complex (A) was generated

I + II via the addition of O2 to a 1:1 mixture of [(TMPA)Cu (CH3CN)] , and (F8)Fe in THF at

93,94 III 2- –80 °C. Its classification as a heme peroxo copper complex [(F8)Fe -(O2 )-

II + 18/16 Cu (TMPA)] (A) was based on rR spectroscopy, which showed an O2 isotope

18 18 sensitive O–O and Fe–O stretching frequencies at 808 (Δ O2 –46) and 533 (Δ O2 –22) cm–1, respectively. The oxygenated adduct was also characterized by MALDI-TOF-MS, which showed a parent peak at m/z = 1239 corresponding to [A – ClO4 + CH3CN], and

18 showed an increase by 4 mass units upon oxygenation with O2. Dioxygen uptake

17 II I measurements confirmed a 1:1:1 stoichiometry for Fe :Cu :O2. The paramagnetically

1 19 2 8 shifted H, F, and H (via the pyrrole deuterated derivative d -F8) NMR spectra along with Evans method experiments confirmed an overall S = 2 spin state. Mössbauer spectroscopy established the iron as a high spin ferric ion, bound to an electron rich peroxide ligand.93,94 This data can be reconciled by considering that the d9 CuII is antiferromagnetically coupled to the unpaired electrons on the high-spin (d5) FeIII ion through the peroxidic bridging ligand. In order to better understand the basic coordination chemistry of this bridging peroxo species, extended X-ray absorption fine structure

(EXAFS) spectroscopy was used in conjunction with DFT calculations. Iron and copper

K-edge EXAFS data conformed to the proposed structure (but see just below) and were fit with a Cu—Fe distance of ~3.72 Å.

III 2- II + Figure 1.6. Generation of heme-peroxo-copper adducts [(F8)Fe -(O2 )Cu (TMPA)] (top A), and III 2- II + 95 [(F8)Fe -(O2 )Cu (AN)] (bottom B). Adapted from ref. Copyright 2010 American Chemical Society.

18 III 2- Initially, the structure/coordination around the peroxo ligand in [(F8)Fe -(O2 )-

CuII(TMPA)]+ (A) was not known, but the critically important synthetic and structural work of Naruta and coworkers (see Figure 1.7, A-C) allowed the formulation of the 2:1- peroxo ligation in (A),94,96 see Figure 1.7. Naruta’s contributions consisted of in fact very similar ligand donors to those used by Karlin and coworkers.97,98 Naruta however utilized derivatives of tetraphenylporphyrinate (TPP) or tetramesitylporphyrinate (TMP) where a

TMPA or its pyridyl group methylated derivative is covalently tethered to the porphyrinate periphery. Complexes A-C (Figure 1.7) are all FeIII-peroxo-CuII complexes, and the X-ray structure shown was obtained for compound (C). (Figure 1.7, right) Extensive physical measurements and reactivity studies were carried out for this crystalline material, (C),

III 2- II + – [(TMP)Fe -(O2 )-(5MeTPA)Cu ] (as a BPh4 salt). This heme-peroxo-copper complex,

(C), displayed similar spectroscopic properties to analogous compounds created by Karlin and coworkers. Specifically, (C) is an S = 2 species with antiferromagnetic coupling

III II between the Fe and Cu , with a corresponding magnetic moment of 4.65 µB, EPR silent

18 –1 (in perpendicular mode), and has a peroxidic rR feature, (O–O) = 790 (Δ O2 –44) cm .

Some structural parameters of interest are that d(O–O) = 1.460(6) Å and d(Fe…Cu) = 3.916

To expand the scope of chelates used in heme-peroxo-copper complexes, the Karlin group utilized several tridentate copper chelates, the justification and interest in this being that (i) the HCO binuclear active site contains a tridentate copper ion (CuB) coordination and (ii) the redox properties and O2-chemistry vastly differs for tridentate vs tetradentate copper(I) chelates, as detailed above in Section 3.3. In one such example, oxygenation of

I F II a solution containing [(AN)Cu ](BAr ) and (F8)Fe results in the generation of a new high-

III 2- II + 93 spin heme peroxo copper adduct, [(F8)Fe -(O2 )-Cu (AN)] (Figure 1.6, B). The

2 III previously established UV-Vis, H-NMR, and rR spectroscopic properties of [(F8)Fe -

2- II + (O2 )-Cu (TMPA)] (Figure 1.6, A) provided precedence for the classification of

III 2- II + [(F8)Fe -(O2 )-Cu (AN)] (Figure 1.6, B) as a high spin heme-peroxo-copper adduct, which is supported by its paramagnetic pyrrole 2H chemical shift centered at 96 ppm (–80

III 2- II + °C). The rR spectrum for [(F8)Fe -(O2 )-Cu (AN)] also displays an isotope sensitive

18 –1 99 peroxide O–O stretching vibration at 756 (Δ O2 = –48) cm . This O–O bond stretch is

2 2 II 2– 2+ similar to that found for a complex with µ-η :η -peroxo binding, [{(AN)Cu }2-(O2 )] , at

18 –1 721 (Δ O2 = –38) cm , which forms as a mixture with a bis-µ-oxo complex

III 2– 2+ [{(AN)Cu }2(O )2] ) in THF. The formation of these copper-only complexes in the heterobimetallic reactions was ruled out by the presence of a single Soret band (indicating a single chemically equivalent heme Fe environment) and the pyrrole chemical shift found

III 2- II + 99 for [(F8)Fe -(O2 )-Cu (AN)] .

Invested readers will note that there is a substantial difference between the value of

2– the rR O–O bond stretching frequencies observed for heme-O2 -copper adducts having

–1 –1 tetradentate (ν(O–O) = 788-808 cm ) as opposed to tridentate (ν(O–O) = 747-767 cm ) copper coordination (Table 1.1). It is well established in dicopper dioxygen chemistry that

20 the nature of the copper ligand, and minor changes in chelate denticity can greatly affect the properties of the resultant copper-oxygen adduct; i.e., tetradentate copper chelates induce formation of end-on µ-1,2-peroxo dicopper(II) structures having high (≥ 800 cm–1)

O–O bond stretching frequencies, whereas tridentate ligands generate side-on µ-η2:η2- peroxo dicopper(II) species with lower O–O bond stretching frequencies (< 760 cm–1)

(Figure 1.8).19,100,101 The distinctly different ν(O–O) stretch values have been associated with back-bonding interactions from the copper d orbitals into the antibonding σ* orbital of the bridging peroxide moiety, resulting in a measurably weaker O–O bond.102

In order to try to understand the origin of these differences among heme-peroxo- copper complexes featuring tri- vs. tetradentate copper chelates, detailed spectroscopic analysis have been carried out along with complementary DFT calculations(Figure

1.9).95,103 As mentioned earlier, the crystal structure and physical properties of [(TMP)FeIII-

2- II + (O2 )-(5MeTPA)Cu ] (Figure 1.7, C), obtained by Naruta and coworkers were important

21 III 2- precursors to this work given the structural and spectroscopic similarities to [(F8)Fe -(O2

II + III 2- )-Cu (TMPA)] (Figure 1.6, A). When comparing the Fe–Cu distances for [(F8)Fe -(O2

II + III 2- II + )-Cu (TMPA)] (Figure 1.9, A), and [(F8)Fe -(O2 )-Cu (AN)] (Figure 1.9, B), it is noteworthy that (A) has a ~0.4 Å greater Fe…Cu distance than (B), which can be rationalized by the different binding modes. Additionally, similar Fe pre-edge XAS intensities for both (A), and (B) suggest that the binding to Fe is similar in both complexes, that is, the Fe is pulled out of the heme plane due to peroxide binding and in the high-spin state. Further rR spectroscopic studies comparing (A), and (B), showed that the spin state marker bands for both complexes are similar, ~1362 cm–1, and distinct from an

–1 authentically generated end-on O2-bound heme (1370 cm ), an observation that is consistent with the assignment of the peroxide as a (side-on bound) bidentate ligand to Fe in both cases. The optimized structures of both (A) and (B) calculated using density functional theory (DFT) are in good agreement with the EXAFS data discussed above. An updated DFT model of (A) essentially reproduced previously reported core properties, and provided additional data on the ruffling of the porphyrin macrocycles. The optimized DFT structure of (B) shows that the peroxide binding mode is η2 to the copper ion, whereas that of (A) is instead η1 (Figure 1.9).

Electronic structure calculations on (A), and (B) provide a rationalization of the distinct rR spectral results while accounting for their identical spin states which is facilitated by antiferromagnetic coupling. In both cases, the d9 CuII, and high spin d5 FeIII ions are strongly antiferromagnetically coupled through the peroxide bridge, via the σ- bonding framework (Figure 1.9, right). However, the nature of the copper ligand (tridentate vs. tetradentate) results in different singly unoccupied net bonding orbitals on the copper ion. In the case of (A), the TMPA ligand favors a five-coordinate, trigonal bipyramidal

2 geometry for Cu, with the singly occupied orbital on copper having 3dz character, which favors end-on binding by the peroxide to copper. In contrast, for (B), the more flexible AN ligand allows for a square pyramidal binding geometry, with a bidentate peroxide ligand, where the singly unoccupied orbital on Cu is of 3dx2–y2 character. The antiferromagnetic

* coupling in both cases is mediated though the πσ orbital of the peroxide. The iron

23 porphyrin fragments in both (A), and (B) were found to be essentially identical with σ

* interactions occurring between the Fe 3dxz orbital and the peroxide πσ , and  bonding

* interactions occurring between the Fe 3dxy and the peroxo πv orbital. These Fe–peroxide bonding interactions are effectively the same in both (A), and (B), and therefore these σ- bonding interactions do not account for the ~50 cm–1 shift in the O–O bond stretching

–1 –1 frequency observed between (A) (ν(O–O) = 804 cm ) and (B) (ν(O–O) = 747 cm ).

Analysis of the occupied valence orbitals of (B) revealed that they embody much greater

σ* character than those of (A), indicating that in (B), the peroxo σ* orbital acts as a π- acceptor from the 3d manifold of the CuII. Due to the highly antibonding nature of the peroxo σ* orbital, its contribution to any bonding orbitals would greatly impact the observed O–O stretch.95

For these high-spin complexes, the iron spin state of the peroxo species can be changed upon the addition of an exogenous axially ligating (to heme) base moiety (1,5- dicyclohexylimidazole, (DCHIm)) to form the corresponding low-spin heme-peroxo-

III 2- II + III 2- copper species, [(DCHIm)(F8)Fe -(O2 )-Cu (TMPA)] and [(DCHIm)(F8)Fe -(O2 )-

CuII(AN)]+, as shown in Figure 1.10, labeled (A) and (B), respectively.104,105 In order to better understand the structures and bonding and confirm the peroxidic nature of (A) and

(B), rR spectroscopy, XAS (for B) and DFT studies were used to establish optimized structures. A higher peroxidic stretching frequency was observed for (B) (Table 1.1), consistent with an end-on, end-on (i.e., -η1:η1-peroxo) coordination, a result of decreased back-donation from the CuII d-orbitals to the peroxo * orbtital (which was present in the high-spin cases) and hence a stronger O–O bond.103 At the same time, the heme FeIII recedes into the plane of the porphyrin, decreasing back bonding contribution from the FeIII

24 d-orbitals. This coordination mode was also reproduced from EXAFS spectroscopy, which is consistent with the expected optimal geometry produced by DFT calculations. The fact that this low-spin peroxo is end-on to both the CuII and FeIII is significant because this was the first confirmed example of this type of binding mode for heme-copper models.

Electronic structure calculations provided additional insight into the HS to LS conversion

III III 2- II + III of the Fe spin state while [(F8)Fe -(O2 )-Cu (AN)] converted to [(DCHIm)(F8)Fe -

2- II + (O2 )-Cu (AN)] (B), upon the addition of axially ligating base, 1,5- dicyclohexylimidazole. This causes the FeIII to recede into the plane of the porphyrin. The strong ligand field contributed by the porphyrinate thus helps account for the HS to LS conversion. The antiferromagnetically coupled ground state of the LS adduct is also favored by its acute Fe-O-O-Cu dihedral angle.103 This change from high-spin to low-spin may have implications for O–O bond cleavage in CcO, as a precursor to further proton

IV coupled electron transfer, producing the Fe =O, and Cu-OH/OH2 containing PM species

(See Section 1.3.3).

With the precedents set concerning structure and spectroscopies for heme-peroxo- copper species, generated primarily by the Naruta and Karlin groups, the binding motif of the bridging peroxo species can be inferred based on its O–O bond stretching frequency, and the denticity of the copper ligand structure. Thus, three general modes have been observed in these systems, as schematically depicted in Figure 1.11; O–O bond stretching frequencies for these and other heme-peroxo-copper adducts are given in Table 1.1.

Figure 1.11. Distinct heme-peroxo-copper binding motifs characterized by Karlin, and Naruta.106 See text for details.

Table 1.1. Selected resonance Raman spectroscopic data: Heme-peroxo-copper complex core vibrations  (∆)a  (∆)a [Ref.] System O-O Fe-O Cu-O (∆)a Tetradentate Cu Ligands III 2- 107 [(DCHIm)(TACNAcr)Fe -(O2 )- 758 (–18) n/r n/r CuII]+ III 2- II + 108 [(TPP)Fe -(O2 )-Cu (tpa)] 803 (–44) n/r n/r III 2- II + 97 [(TPP)Fe -(O2 )-Cu (5-Metpa)] 793 (–42) n/r n/r III 2- II + 97 [(TMP)Fe -(O2 )-Cu (5-Metpa)] 790 (–44) n/r n/r III 2- II OH + 109,110 [(TMP)Fe -(O2 )-Cu (L )] 799 (–47) n/r n/r III 2- II OMOM + 110 [(TMP)Fe -(O2 )-Cu (L )] 801 (–46) n/r n/r III 2- II N4-OH + 111 [(TMPIm)Fe -(O2 )-Cu (L )] 803/787 (–52) n/r n/r

III 2- II N4-OMOM + 110 [(TMPIm)Fe -(O2) -Cu (L )] 804/752 (–52) n/r n/r 6 III 2- II + 112 [( L)Fe -(O2 )-Cu ] 787 (–43) 533 n/r 5 III 2- II + 112 [( L)Fe -(O2 )-Cu ] 809 (–53) n/r n/r III 2- II + 113 [(F8)Fe -(O2 )-Cu (TMPA)] 808 (–46) 538 (–22) 516 (–24)

26 III 2- 104 [(DCHIm)(F8)Fe -(O2 )- 812 (–50) 623 (–27) 533 (–24) CuII(TMPA)]+ III 2- II N4 + 114 [(F8)Fe -(O2 )-Cu (L OH)] 813 (–44) 529 (–21) n/r III 2- II N4 + 114 [(F8)Fe -(O2 )-Cu (L OMe)] 815 (–46) 528 (–22) n/r III 2- 104 [(DCHIm)(F8)Fe -(O2 )- 876,863 (–56) 591, 585 (– n/r II + Cu (DCHIm)4] 27) Tridentate Copper Ligands III .- I + b 89 [(NMePr)Fe (O2 )Cu ] n/r 570 (–26) n/r 2 III 2- II + 115 [( L)Fe -(O2) -Cu( )] 747 (–49) n/r n/r III 2- II N3-OH + 110 [(TMP)Fe -(O2 )-Cu (L )] 810 (–46) 576 (–26) n/r III 2– II N3-ONa + 110 [(TMP)Fe -(O2 )-Cu (L )] 812 (–50) 577 (–24) 491(–28) III 2- II + 99 [(F8)Fe -(O2 )-Cu (AN)] 746 (–48) n/r n/r III 2- II + 95 [(DCHIm)(F8)Fe -(O2 )-Cu (AN)] 796 (–42) 586 (–22) n/r III 2- II Me2N + 116 [(F8)Fe -(O2 )-Cu (L )] 752 (–43) n/r n/r 767 (–40) III 2- 104 [(DCHIm)(F8)Fe -(O2 )- 876 (–55) 594 (–28) n/r II + Cu (DCHIm)3] III 2- II + 104 [(F8)Fe -(O2 )-Cu (MeTHF)3] 737 (–41) 555 (–27) n/r a –1 18 Values given in cm ; values in parenthesis are  O2 shifts. b Characterized as a heme-FeIII-superoxide...CuI complex, see Section 2.2.2 n/r = not reported.

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36 Cytochrome c Oxidase. Angew. Chemie Int. Ed. 2012, 51, 168–172.

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Chapter 2. Isocyanide or Nitrosyl Complexation to Hemes with Varying Tethered Axial Base Ligand Donors: Synthesis and Characterization

This work was co-authored with the following authors and is published under the following citation:

Savita K. Sharma,† Hyun Kim,† Patrick J. Rogler,† Maxime A. Siegler,† and Kenneth D. Karlin†

†Johns Hopkins University, Baltimore, Maryland 21218, United States

39 2.1. Abstract

A series of ferrous heme 2,6-dimethylphenyl isocyanide (DIMPI) and ferrous heme mononitrosyl complexes have been synthesized and characterized. The heme portion of the complexes studied is varied with respect to the nature of the axial ligand, including complexes where it is covalently tethered to the porphyrinate periphery. Reduced heme

II Py II Im II complexes, [(F8)Fe ] (1), [(P )Fe ] (2) and [(P )Fe ] (3), where F8 = tetrakis(2,6- difluorophenyl)-porphyrinate and PPy, and PIm are partially fluorinated tetraaryl porphyrinates with covalently appended axial base pyridyl or imidazolyl moieties, were employed. Room temperature addition of DIMPI to these iron(II) complexes affords the

II II bis-isocyanide species [(F8)Fe -(DIMPI)2] in the case of [(F8)Fe ], while for the other hemes, mono-DIMPI compounds are obtained, [(PPy)FeII-(DIMPI)] ((2)-DIMPI), and

[(PIm)FeII-(DIMPI)] ((3)-DIMPI). The structure of complex (3)-DIMPI has been determined by single crystal X-ray crystallography, where interesting H…F(porphryinate aryl group) interactions are observed. 19F-NMR spectra determined for this complex suggest that H…F(porphyrinate aryl groups) interactions also persist in solution, the H- atom coming either from the DIMPI methyl groups or from a porphyinate axial base imidazole or porphyrinate pyrrole. Similarly, nitric oxide was used to generate ferrous-

II nitrosyl complexes, a five-coordinate species for F8, [(F8)Fe -(NO)] ((1)-NO), or low-spin six-coordinate compounds, [(PPy)FeII-(NO)] ((2)-NO) and [(PIm)FeII-(NO)] ((3)-NO).

These DIMPI and mononitrosyl complexes have also been characterized using UV-Vis,

IR, 1H-NMR and EPR spectroscopies.

40 2.2. Introduction

Heme containing proteins participate in critical and diverse biological functions which include electron-transfer, catalysis and signaling. For the latter two subjects, small

1 molecule diatomic gases are often involved, such as O2, NO and CO. There exist classes of proteins which serve to discriminate between these molecules for purposes including detection, signaling and/or function.2,3 For molecular oxygen, roles include storage-

4 transfer, or activation of O2 for substrate oxidation or oxygenation chemistries. Nitric oxide (nitrogen monoxide) is a signaling molecule5 such as in its interaction with the heme center in guanylate cyclase, wherein binding leads to a signaling cascade resulting in smooth muscle relaxation.6–12 Carbon monoxide is also a diatomic gas which is

13–15 biosynthesized through heme O2-activation chemistry (i.e., in heme oxygenases ); CO can also act in biological signaling via heme protein binding.16

In the history of the study of O2 interactions with hemoproteins, the investigation of the binding of diatomic surrogate ligands, mainly CO and NO, has received considerable attention. These have been utilized as structural models, but also are useful in the study of ligand binding dynamics and electronic structure of the ligated reduced hemes.17–20 For example, CO bound hemes are amenable to vibrational spectroscopic analyses, along with

CO photo-ejection and CO re-binding studies.19,21 Additionally, reduced hemes with NO bound contain unpaired electrons and can be interrogated using EPR spectroscopy. The replacement of CO with isocyanide (RNC:) ligands has also been found to be a useful probe to investigate vibrational spectroscopy and binding kinetics or heme-ligand photodissociation and time resolved rebinding. The strong isocyanide N-C triple-bond stretching vibration can be monitored, whereas variation in the size or nature of the

41 isocyanide R-group, e.g., R = aryl vs –Me or –tBu, provides insights concerning steric effects or issues of small ligand binding to iron relative to the size or shape of a protein active-site pocket.22–26

One of our research group’s major foci has been and continues to be the study of dioxygen binding and reduction at heme-copper heterobinuclear metal ion centers.27 We seek to determine how neighboring copper-ligand moieties influence the binding of O2 to hemes, and in a complementary manner see how hemes affect O2 binding to copper ion in varying ligand environments. Then, as such synthetic heme-O2-Cu assemblies can be compared and related to the active-site chemistry of heme-copper oxidases which bind and reduce O2 to two water molecules (while also translocating protons through a mitochondrial membrane which downstream facilitates ATP biosynthesis), we are interested to elucidate detailed insights into the O–O reductive cleavage process, as a function of the exact nature (structure and electronics/bonding behavior) of the heme, the copper-ligand and the source of electrons (E° value) and protons (pKa). Factors include heme or copper-ligand electron-donating ability (and thus the FeIII/FeII and/or CuII/CuI E° value), nature of porphyrinate peripheral groups and/or copper-ligand denticity and their possible steric influences or affects upon the entire heme-O2-Cu(ligand) structure, for

27–31 example the Fe…Cu distance in the heme-O2-Cu assembly.

Figure 2.1. Generation of HCO model compound adducts from ferrous porphyrinate complexes, dioxygen and cuprous chelates.

A specific example of such a synthetic construct is shown in Figure 2.1, where also an added heme axial ‘base’, dicyclohexylimidazole (DCHIm), is present.31,32 As anticipated, the structural, spectroscopic properties and reactivity of this and other such assemblies significantly depend on the detailed nature of the heme, the copper ligand, the axial base, etc., as mentioned above.27,32

As such, it is critical that complementary investigations be carried out on surrogate ligand binding to the various components of our assemblies. One such aspect is the investigation of O2, CO, NO and/or RNC binding to varying designed porphyrinoids, wherein the axial base ligand is varied between a weak O-donor (as solvent) such as tetrahydrofuran (THF), DCHIm (Scheme 1) or a covalently linked imidazolyl or pyridyl ligand. To better our understanding of the chemistry of full heme-O2-Cu assemblies, it is very useful to understand the structural aspects, physical properties and reactivity patterns of just these heme-containing moieties with varying axial base ligands, with O2, CO, NO and RNC’s. In fact, some of this information has been obtained and previously published,

43 III in particular for heme-O2 (Fe -superoxide) complexes, for four of the five species shown in Figure 2.2.33,34 Also, some of the related (heme)FeII-CO and (heme)FeII-NO compounds have also been described.33 Here, we report on advances made from the study of new

II Im Py (heme)Fe derived adducts with DIMPI along with nitric oxide, using the F8, P , and P porpyrinate frameworks, see Figure 2.2 and the equations below. New insights have been obtained based on the X-ray structures and physical properties which are described and also compared with corresponding adducts using F8, which does not incorporate a tethered axial base ligand.

Figure 2.2. Ferric superoxide species characterized by our group.

2.3. Experimental

2.3.1. Materials and Methods

All chemicals and solvents were purchased as commercially available analytical grade unless otherwise specified. Tetrahydrofuran (THF, inhibitor free) was dried over sodium/benzophenone ketyl, and purified by distillation under argon. Pentane was dried by distillation over calcium hydride. Toluene was used after passing through a 60 cm long column of activated alumina (Innovative Technologies), under argon. 2,6-Dimethylphenyl

44 isocyanide (DIMPI) was purchased from Sigma Aldrich. •NO gas was obtained from

Matheson Gases and purified following methods previously described in the literature.35 A three-way syringe was used for the addition of •NO gas to all metal complex solutions.

Preparation and handling of air-sensitive compounds were performed under an argon atmosphere using standard Schlenk techniques or in an MBraun Labmaster 130 inert atmosphere (less than 1 ppm O2, less than 1 ppm H2O) drybox filled with nitrogen.

Deoxygenation of all solvents was accomplished by either repeated freeze/pump/thaw cycles or bubbling with argon for 45–60 min.

Instrumentation: Benchtop UV-Vis measurements were carried out by using a Hewlett

Packard 8453 diode array spectrophotometer equipped with HP Chemstation software and a Unisoku thermostated cell holder for low temperature experiments. A 10 mm path length quartz cell cuvette modified with an extended glass neck with a female 14/19 joint, and stopcock was used to perform all UV-Vis experiments. ESI-MS were acquired using a

Finnigan LCQ Duo ion-trap mass spectrometer equipped with an electrospray ionization source (Thermo Finnigan, San Jose, CA). The heated capillary temperature was 250 ⁰C and the spray voltage was 5 keV. Spectra were recorded continuously after injection.

Infrared (IR) spectra were obtained on solid samples using a Thermo Scientific Nicolet

Nexus 670 Fourier transform IR (FT-IR) spectrophotometer with ATR attachment. 1H-

NMR and 19F-NMR spectra were acquired using a Bruker 300-MHz NMR spectrometer.

Chemical shifts were reported as δ (ppm) values relative to an internal standard

(tetramethylsilane) and the residual solvent proton peak. Electron paramagnetic resonance

(EPR) spectra were recorded with a Bruker EMX spectrometer equipped with a Bruker ER

041 X G microwave bridge and a continuous-flow liquid helium cryostat (ESR900)

45 coupled to an Oxford Instruments TC503 temperature controller. Spectra were obtained at

8 K under non-saturating microwave power conditions (ν = 9.4108 GHz, microwave power

= 0.201 mW, modulation amplitude = 10 G, microwave frequency = 100 kHz, receiver gain = 5.02 × 103). EPR spectra were simulated by using the Easy Spin (see Appendix A).

II 36 Py II 33,34 Im II 33,37 The compounds (F8)Fe (1), (P )Fe (2), and (P )Fe (3) were synthesized as previously described.

2.3.2. Synthesis of DIMPI, and NO bound ferrous heme porphyrinates

II II [(F8)Fe -(DIMPI)2], (1)-DIMPI: In the drybox, to a solution of (F8)Fe (1) (10.0 mg, 0.024 mmol) in THF (5 mL) was added 2,6-dimethylphenyl isocyanide (6.3 mg, 0.048 mmol).

After stirring the reaction mixture for 30 min, the solvent was removed under vacuum to yield a red solid. The crude solid obtained was further dissolved in THF and layered with pentane to obtain a very fine crystalline material. UV-Vis spectrum [λmax, nm] in THF: 430,

1 527. H-NMR (THF-d8, 300 MHz; δ, ppm): 10.45 (pyrrole-H), 2.43 (s, -CH3 (DIMPI)), 7.2

19 (m, ArH (DIMPI)); F-NMR (THF-d8, 282 MHz; δ, ppm): -109 (d). FT-IR spectrum

-1 (solid): νCN = 2124 cm .

[(PPy)FeII-(DIMPI)], (2)-DIMPI: In the dry box, to the THF solution of (PPy)FeII, (2) (10.0 mg, 0.011 mmol) in a 10 mL Schlenk flask, we added one equivalent of DIMPI (1.5 mg,

0.012 mmol) and reaction mixture stirred for half an hour. The solvent was removed under vacuum to yield a deep red colored solid, which was further recrystallized by dissolving in a minimal amount of THF and layering it with pentane to obtain the fine crystalline

46 material. UV-Vis spectrum [λmax, nm] in THF: 430, 534. FT-IR spectrum (solid): νCN =

2104 cm-1.

[(PIm)FeII-(DIMPI)], (3)-DIMPI: This complex was synthesized in a similar manner to complex (2)-DIMPI. X-ray quality crystals were obtained from the solution of

1 MeTHF/pentane. UV-Vis spectrum [λmax, nm] in THF: 430, 532. H-NMR (THF-d8, 300

19 MHz; δ, ppm): 9.1 (pyrrole-H), 7.2 (m, ArH (DIMPI)), 2.43 (s, -CH3 (DIMPI)); F-NMR

(THF-d8, 282 MHz; δ, ppm): -110.6 (d), -110.8 (d), -111.0 (d), -111.7 (d). FT-IR spectrum

-1 (solid): νCN = 2098 cm .

II [(F8)Fe -NO], (1)-NO:38 The ferrous mono-nitrosyl complex (1)-NO was generated by

II bubbling excess NO gas through the THF solution of (F8)Fe (1) (2 mM) under argon atmosphere at room temperature. After the reaction mixture stirred for 2 hours, the solvent was removed under vacuum to obtain a dark red solid. A highly pure material was obtained by dissolving red solid in the minimum amount of THF and layering it with pentane inside

1 the dry box. UV-Vis spectrum [λmax, nm] in THF: 408, 547. H-NMR (THF-d8, 300 MHz;

19 δ, ppm): 6.9 (br, pyrrole-H); F-NMR (THF-d8, 282 MHz; δ, ppm): -106 (br). FT-IR

-1 spectrum (solid): νNO = 1688 cm . EPR spectra (X-band spectrometer, ν = 9.428 GHz): g

= 2.09, 2.02, 1.99 (hyperfine) in THF at 7 K.

[(PPy)FeII-NO], (2)-NO: A method similar to that used to synthesize complex (1)-NO was used to make complex (2)-NO. Excess of NO gas was bubbled through the 2 mM THF

Py II 1 solution of (P )Fe (2). UV-Vis spectrum [λmax, nm] in THF: 417, 543. H-NMR (THF-

47 -1 d8, 300 MHz; δ, ppm): 8.0 (pyrrole-H). FT-IR spectrum (solid): νNO = 1648 cm . EPR spectra (X-band spectrometer, ν = 9.428 GHz): g = 2.07, 2.01 (br-hyperfine), 1.98 in THF at 7 K.

[(PIm)FeII-NO], (3)-NO: This complex was prepared in the same manner as complexes (1)-

1 NO and (2)-NO. UV-Vis spectrum [λmax, nm] in THF: 423, 542. H-NMR (THF-d8, 300

19 MHz; δ, ppm): 8.8 (pyrrole-H); F-NMR (THF-d8, 282 MHz; δ, ppm): -106.2 (br), -107.9

-1 (br), -110.9 (br). FT-IR spectrum (solid): νNO = 1650 cm . EPR spectra (X-band spectrometer, ν = 9.428 GHz): g = 2.07, 2.00 (hyperfine), 1.97 in THF at 7 K.

2.3.3. X-Ray structure determination

X-ray structure determination of (3)-DIMPI was performed at the X-ray diffraction facility at Johns Hopkins University. CIF files have been deposited with the Cambridge

Crystallographic Data Centre (CCDC). CCDC 1455862 contains the supplementary crystallographic data for (3)-DIMPI. These data can be obtained free of charge from the

CCDC via http://www.ccdc.cam.ac.uk/data_request/cif. All reflection intensities were measured at 110(2) K using a SuperNova diffractometer (equipped with Atlas detector) with Cu Kα radiation (λ = 1.54178 Å) under the program CrysAlisPro (Version 1.171.36.32

Agilent Technologies, 2013). The program CrysAlisPro (Version 1.171.36.32 Agilent

Technologies, 2013) was used to refine the cell dimensions and for data reduction. The structures were solved with the program SHELXS-2013 (Sheldrick, 2013) and was refined on F2 with SHELXL-2013 (Sheldrick, 2013). Analytical numeric absorption correction based on a multifaceted crystal model was applied using CrysAlisPro (Version 1.171.36.32

48 Agilent Technologies, 2013). The temperature of the data collection was controlled using the system Cryojet (manufactured by Oxford Instruments). The H atoms were placed at calculated positions using the instructions AFIX 23, AFIX 43 or AFIX 137 with isotropic displacement parameters having values 1.2 or 1.5 times Ueq of the attached C or N atoms.

Crystals of (3)-DIMPI were obtained from a MeTHF solution of complex and layered with pentane. The structure of (3)-DIMPI is partly disordered. Some unresolved electron density  i.e., a very disordered lattice methyl THF solvent molecule  has been taken out in the final refinement (SQUEEZE details are provided in the CIF file, Spek,

2009).39 In addition, the imidazole / amide arm may be slightly disordered, but the disorder is not significant enough to model it in the final refinement.

Table 2.1. Crystallographic data for complex [(PIm)FeII-(DIMPI)]. Compounds (3)-DIMPI Formula Weight 1106.89 (g/mol) T (K) 110(2) Crystal shape small dark red plate (0.11  0.05  0.02 mm3) Space group triclinic, P-1 (no. 2) a (Å) 12.3852(6) b (Å) 12.5816(6) c (Å) 19.3657(12) α (⁰) 104.424(5) β (⁰) 95.293(4) γ (⁰) 111.823(5) V (Å3) 2655.1(3) Z 2 −3 Dx (g cm ) 1.385 μ (mm-1) 2.897 Absorption 0.7970.947 correction range −1 (sin /)max (Å ) 0.60 Total, Unique, 24276, 9448, 6441 and Observed Reflections Rint 0.0467 GOF 1.041

49 R1/wR2 [I > 0.0703/0.1902 2(I)] R1/wR2 0.1038/0.2151 max, min, rms 1.126, -0.478, 0.083

2.4. Results and discussion

2.4.1. Stable heme–isocyanide complex formation:

DIMPI reacts immediately with the reduced synthetic ferrous-heme complexes,

II Py II Im II [(F8)Fe ], [(P )Fe ], and [(P )Fe ] to yield six-coordinate low-spin ferrous heme isonitrile species, as shown in Figures 2.3 and 2.4.

II Generation of bis-isocyanide-porphyrin complex [(F8)Fe -(DIMPI)2]

II When one equivalent DIMPI is added to a THF solution of [(F8)Fe ] at room temperature, a new UV-Vis peak at 430 nm is observed, but the absorption at 422 nm characteristic of the starting complex still remains. However, addition of another equivalent of DIMPI leads to the full formation of the 430 nm peak in the Soret region (Figures 2.3 and 2.5). Additional DIMPI added to the solution does not change the UV-Vis spectral features. Based on these observations, we postulate that two DIMPI molecules are bound to the iron(II) center, as also confirmed by integration of peaks in the 1H-NMR spectrum

II of [(F8)Fe -(DIMPI)2]. Similar UV-Vis spectral features were observed for a structurally characterized bis-isocyanide iron(II) complex with tetraphenylporphyrin (TPP),

II 40 [(TPP)Fe -(tBuNC)2]. The reactivity of DIMPI with reduced synthetic hemes is similar to that in general and previously observed for carbon monoxide (CO) as the heme ligand.33,41 Complex (1)-DIMPI was further characterized by FT-IR spectroscopy where a

-1 single ν(C≡N) stretch is observed at 2124 cm (vide infra, Figure 2.8, Table 2.3), as would be expected for this highly symmetric compound, even with the presence of two DIMPI

50 ligands per molecule. Also, the 19F-NMR spectrum of (1)-DIMPI (see Figure 2.12) shows one sharp absorbance at -109.0 ppm for the o-difluoro substituted phenyl rings of the F8 porphyrin.42

Figure 2.3. Generation of bis-isocyanide ferrous heme complex, (1)-DIMPI at room temperature.

Generation of six-coordinate (PPy/PIm) iron(II)-DIMPI complexes

[(PPy)FeII] and [(PIm)FeII] in THF solution at RT exhibit different structures in this solvent, as previously deduced by observation of the positions of NMR spectroscopic pyrrole resonances. [(PPy)FeII] and [(PIm)FeII] are both five-coordinate high-spin, the former at all temperatures between RT and -90 °C. However, at low temperature [(PIm)FeII] is six-coordinate low-spin (S = 0), with the pyrrole resonances appearing in the diamagnetic region, indicating that both the imidazolyl group and a THF solvent molecule act as axial ligands

Addition of one equivalent of DIMPI solution to each of the reduced iron(II) porphyrinates ([(PPy)FeII], and [(PIm)FeII]) in THF at room temperature leads to a substantial change in the UV-Vis Soret region, with the formation of a band at 430 nm in all three cases. Additional equivalents of DIMPI do not yield any change in the UV-Vis spectra. (Figures 2.4, 2.5) This may indicate that only one DIMPI molecule is bound to the

51 iron(II) center axially while the covalently linked axial base imidazole/pyridine is coordinated to the iron(II) center giving an overall six-coordinate low-spin ferrous-DIMPI complex. These conclusions are borne out by the X-ray structures determined for complex

(3)-DIMPI (see below, Figure 2.6).

We have also carried out experiments to determine binding constants of DIMPI with the ferrous hemes with covalently attached axial ligand bases. Titrations with DIMPI were performed using (PPy)FeII (2), and (PIm)FeII (3), and isosbestic behavior was seen for all the titrations (Figure. S1–2, in Appendix A). A plot of the absorbance at 430 nm versus

[DIMPI] (Figure. S1–2, in Appendix A) reaches a maximum at ∼1 equiv of DIMPI, and no further spectral changes are observed with the addition of more DIMPI. Assuming that

DIMPI reversibly binds to the ferrous heme complexes (PPy)FeII (2), and (PIm)FeII (3) under equilibrium conditions, a good fit of the data can be obtained with a model for a one-to-

7 −1 one binding isotherm. This fit gives association constants (Ka) of 2.29 × 10 M , and 1.19

× 107M-1 for (2), and (3)-DIMPI, respectively. These values are comparable to those

8 - measured for DIMPI binding to hemoglobin (Hb) and myoglobin (Mb) [Ka = 1.0 × 10 M

1].43 The binding constant for complex (2)–DIMPI is 2-fold greater than (3)–DIMPI, which is consistent with a less strong binding of the pyridine axial base in (2)–DIMPI when compared to the imidazole complex.

Figure 2.4. Generation of six-coordinate ferrous heme isocyanide complexes; (2), and (3)- DIMPI..

2.4.2. Crystal structure of isocyanide complex, [(PIm)FeII-(DIMPI)] (3)-DIMPI

In order to better understand the similarities and differences in the coordination geometry of these iron(II)-DIMPI complexes, crystal structure of (3)-DIMPI was

53 determined. The structure of (3)-DIMPI is depicted in figure 2.6 and important structural parameters are listed in Tables 2.1 and 2.2. The geometry around the iron is octahedral, where the 5th ligand is the covalently linked imidazole, while the 6th ligand is the isocyanide

(DIMPI). The metal center lies in the porphyrinate plane. The observed bond distances and angles for (3)-DIMPI indicate very little strain within the linker arm. A slight perturbation from the expected linear Fe–C–N bond angle is seen for complex (3)-DIMPI, with a Fe–

C–N value of 173.8(4). This difference could arise due to crystal packing effects.44 The

Fe–C(DIMPI) bond distance is ca. 1.82 Å (3)-DIMPI, and Fe–N(imidazole) bond distances are ca. 2.02 Å, while the average Fe–N(porphyrin) bond distances are 1.99 Å, similar to

II 45 our previously reported [(F8)Fe ]•2THF complex. The structure of (3)-DIMPI is well ordered, see Materials and Methods. Lehnert and co-workers have previously published on a crystal structure of a zinc(II) analogue, a five-coordinate complex with the PIm ligand.46

Figure 2.6. Displacement ellipsoid plot (50% probability level) of [(PIm)FeII-(DIMPI)] ((3)-DIMPI) showing the imidazolyl and 2,6-dimethylphenyl isocyanide ligands bound to Fe(II) center. Lattice solvent molecules and H atoms have been omitted for the sake of clarity. Selected bond lengths and bond angles are reported in Table 2.2.

54 Table 2.2. Selected bond lengths (Å) and bond angles () for (3)-DIMPI. Proposed H-bonds are also listed.

Compound (3)-DIMPI Bond length (Å) Fe – N1 1.998 (4) Fe – N2 2.000 (3) Fe – N3 1.990 (4) Fe – N4 2.001 (3) Fe – N5 2.034 (4) Fe – C56 1.824 (4) N8 – C56 1.163 (6)

Bond angle (⁰) N1 – Fe – N5 91.38 (15) N2 – Fe – N5 89.77 (15) N3 – Fe – N5 88.85 (15) N4 – Fe – N5 88.58 (14) N5 – Fe – C56 176.03 (16) Fe – C56 – N8 173.8 (4) Weak C-H---F [Bond Length interaction: (Å)/Bond Angles (⁰)] F1…H64methyl (C64) 2.731/ (133.93) F2…H7pyrrole (C7) 3.490 / (95.04) F3…H8pyrrole (C8) 2.966 / (95.57) F4…H39imid (C39) 3.117 / F5…H2pyrrole (C2) (153.44) 2.926 / (100.23) F6…H41imid (C41) 2.991/ (166.53)

Other potentially important (from a structural perspective) observations obtained from the crystal structures of both complexes are that weak but noticeable intramolecular

CH…F interactions occur, as shown in Figure 2.7 and listed in Table 2.2. The observed range of C–H…F interactions for our new structure lie between 2.73–3.08 Å, which is greater than the sum of reported Van der Waals radii for hydrogen and fluorine

(approximately 2.3–2.5 Å).47 Based on reported structural observations and DFT calculations,48,49 the very strong H-bonding ability of fluorine gives rise to such C–H…F

55 interactions which vary between ~ 2.7 to 3.1 Å , similar to what is observed for (3)-DIMPI, and (4)-DIMPI. Such literature examples of organic compounds with longer distance H⋅⋅⋅F interactions occur even where the CH⋅⋅⋅F angle lies in the range between 130 and 145 and even in some cases it is close to 100.48 Further comparisons may be made to examples of non-bonded CH…O contacts made between an O-atom in an iron(IV)-oxo complex with surrounding ligand methyl group H-atoms; there, short CH…O distances are observed (2.3 to 2.7 Å) while very acute CH…O angles are present (~ 100 – 109°).50

With these precedents in mind, we postulate that in the solid state structure of (3)-

DIMPI, a CH⋅⋅⋅F interaction occurs between the methyl group on the DIMPI ligand (H64C) and F1 from the proximate o-F aryl porphyrinate substituent, see the green line in the middle top of the (3)-DIMPI structure in Figure 2.7; here the CH⋅⋅⋅F distance and angle are

2.73 Å and 134°, respectively (Table 2.2). A far closer to linear interaction occurs between

F6 and H41 from the ligated imidazolyl group with a bond distance and angle of 2.99 Å and 167°, respectively (Table 2.2 and Figure 2.7, bottom right). Notice that F6 and F5, on the same porphyrinate aryl group both H-bond, the latter (‘back right’ in Figure 2.7 left) to a pyrrole H2 hydrogen atom, a F5⋅⋅⋅H2C interaction, which likely is a synergistic interaction. Envisioning the iron atom as a kind-of pseudo center of symmetry, the F3 and

F4 atoms on the left hand o-F2-phenyl ring forms two H-bonds, between F3⋅⋅⋅H8pyrrole (C8) and F4⋅⋅⋅H39imid (C39) (Table 2.2). The overall result is that the H-bonding interactions formed by the four F-atoms just described fixes the orientation of the imidazolyl axial ligand, which is already close to perpendicular to the porphyrinate plane, to be nearly coplanar with both of the aryl rings containing these F3, F4, F5 and F6 atoms {Notice how in viewing the structure of (3)-DIMPI (Figure 2.7) that these two aryl rings and the

56 imidazolyl ring seem to lie in the plane of the sheet}. This imidazole orientation could also

19 lead to a close interaction of F2⋅⋅⋅H7pyrrole (C7) in the solution state (see F-NMR spectroscopy, below), whereas in solid state structure this distance seems too long for this interaction to occur (Table 2.2).

Figure 2.7. Crystal structures showing weak intramolecular CH…F interaction identified from the green lines shown. (Left) (3)-DIMPI and (Right) (4)-DIMPI. See text for further discussion.

Complexes (2)-DIMPI, and (3)-DIMPI were also characterized by FT-IR spectroscopy (Figure 2.8, Table 2.3). The IR spectrum for (1)-DIMPI shows a single sharp

-1 ν(C≡N) band (2124 cm ), corresponding to the absorption for both isonitrile ligands in

II -1 [(F8)Fe -(DIMPI)2], which is shifted 4 cm higher in energy relative to the stretching frequency of the uncomplexed ligand. The shift to higher energy is relatively small and

2 could be attributed to a ligand (σ NC) to metal (dσ) interaction, consistent with the expected

57 behavior for an isonitrile ligand acting as a σ-donor to a metal center. [(TPP)FeII-

40 (tBuNC)2], as mentioned above, was structurally characterized but no IR data were given.

Figure 2.8. Solid state FT-IR spectra for Fe(II)-DIMPI complexes of, 2,6- dimethylphenylisocyanide (DIMPI), (1)-DIMPI, (2)-DIMPI, and (3)-DIMPI.

On the other hand, for all three porphyrin Fe(II)-DIMPI complexes with covalently attached axial bases (pyridine/imidazole/histamine moieties), we observe a shift in the C≡N bond stretch of the isonitrile ligand to lower energy with respect to the uncomplexed ligand.

-1 -1 The ν(C≡N) band for (2)-DIMPI is 2104 cm , 16 cm lower in energy compared to that of free DIMPI, and also 20 cm-1 lower in energy with respect to (1)-DIMPI. For (3)-DIMPI,

-1 -1 the value of ν(C≡N) is 2098 cm , which is 23 cm lower in energy with free ligand. (As suggested by a reviewer, the room-temperature molecular structure for (3)-DIMPI may be dynamic with respect to Fe-C-N bending; the IR band observed for these complexes do seem to be asymmetric, and composed of two bands, possibly two conformers.) The shift to lower energy in all these cases could be due to the presence of a more strongly donating axial base trans to the isonitrile ligand. This will most likely decrease the σ-donation to the

58 iron(II) from the isocyanide ligand, while increasing backbonding to the ligand C≡N * orbitals from the iron(II) d orbitals.

Table 2.3. Properties of ferrous heme-DIMPI and ferrous heme-NO model complexes. Compound UV-Vis IR (cm-1) EPR (nm) ν(C≡N) / ν(N‒O) (g values) II [(F8)Fe -(DIMPI)2] 430 2124 Silent

[(PPy)FeII-(DIMPI)] 430 2104 Silent

[(PIm)FeII-(DIMPI)] 430 2098 Silent II [(F8)Fe -NO] 408 1688 2.09/2.01/1.99 [(PPy)FeII-NO] 418 1648 2.08/2.00/1.97 [(PIm)FeII-NO] 423 1650 2.07/2.00/1.97

Figure 2.9. 19F-NMR of (1)-DIMPI (top), and (3)-DIMPI (middle) complexes in THF at room temperature.

Diamagnetic 1H-NMR spectra were observed for all iron(II)-DIMPI (S = 0, d6) complexes. The pyrrole hydrogens of the iron(II)-DIMPI porphyrinates resonate between

8.5-9.5 ppm compared with the starting reduced high-spin five-coordinated paramagnetic

6 33 iron(II) (d ) species ranging in pyrole = 12 to 58 ppm. We also observed a singlet δ 2.65 ppm for the DIMPI o-methyl protons and a multiplet for the aromatic protons of bound

DIMPI at δ 7.2-7.5 ppm; both sets of peaks are slightly shifted downfield from what is observed for free DIMPI.

59 19 II In the F-NMR spectra for bis-isocyanide complex, [(F8)Fe -(DIMPI)2] / (1)-

DIMPI, we observe one sharp singlet peak at -109.0 ppm, as shown in Figure 2.9. So, unlike what we have suggested (and described above) for what is observed in the X-ray crystal structures for the supers-structured heme (3)-DIMPI there are no observed F-atom interactions or coupling to porphyrin or isocyanide H atoms.

On the other hand, CH⋅⋅⋅F couplings for, (3)-DIMPI (Figure 2.9) with peaks for the ortho F-atom resonances on the porphryinate aryl groups occurring between -110 to -112 ppm.42 In the case of (3)-DIMPI, doublet peaks, proposed to be due to 19F resonances coupled to a S = ½ H-atom, are observed at -111.7 and -111.0 ppm and both of these appear to integrate to two F-atoms. Our suggested assignments are as follows: (a) One of the two upfield (more negative delta value) doublets corresponds to F4 and F6 coupling to imidazole H-atoms H39 and H41, which spatially line up rather well, see Table 2.2, Figure

2.7. (b) The other upfield doublet represents 2 o-fluorine H-bonds with pyrrole H-atoms, possibly F3 and F5 with pyrrole H-atoms H8 and H2 (see Figure 2.7), and these also seem to be in very closely matching chemical environments. (c) One of the two absorptions, -

110.8 or -110.6 corresponds to a single o-fluorine atom (F2) also coupling to a pyrrole H7 atom, but by symmetry this is in a different chemical environment then for the interactions discussed just above for F5 and F3. (d) Then, the other upfield absorption is a unique interaction of F1 with the DIMPI methyl group H-atoms (CH64⋅⋅⋅F1).

As mentioned, for (1)-DIMPI, we do not observe any DIMPI ligand H-atom coupling to F-atoms of the F8-heme. Also, we do not observe any such couplings for the

II compound (THF)(F8)Fe -CO (unpublished observation). We suggest that when there is a tethered axial ligand such as in PIm or PImH, there are constraints in the movement or rotation

60 of the axial ligand, which thereby allow for these weaker imidazole-H⋅⋅⋅F interactions to be observed. Further studies may be warranted.

2.4.3. Stable heme–Fe–Nitrosyl formation

II Py II In this study, we have also investigated the reactivity of [(F8)Fe ] (1), [(P )Fe ]

(2), and [(PIm)FeII] (3), towards nitric oxide (NO). All iron(II) complexes form NO-adducts at room temperature following bubbling NO(g) through the solution of each of the reduced iron complexes (Figure 2.10). We have systematically characterized iron(II)–NO complexes by using UV-Vis, FT-IR, 1H-NMR, 19F-NMR spectroscopy and low temperature EPR spectroscopy, to access the binding properties of our covalently tethered

N-donor ligand containing heme complexes.

Figure 2.10. Six coordinate ferrous heme mono nitrosyl complexes of (PPy)FeII, and (PIm)FeII.

In-depth studies have been carried out by Lehnert and coworkers,12,51 using synthetic heme porphyrins with tethered N-donor ligands which indicate a direct correlation between the coordination geometry of the iron center, and the observed spectroscopic properties obtained from UV-Vis, IR, and EPR. For five-coordinate (5C) heme nitrosyls the Soret band (UV-Vis) is typically about 405 nm whereas in six- coordinate (6C) porphyrinoids, where the proximal ligand (N-donor) is bound to the Fe

61 center, the Soret max shifts to ~ 426 nm. Similarly, in IR spectroscopy 5C and 6C ferrous heme mononitrosyl species have distinctive N-O stretching modes. For 5C complexes the

N-O stretch typically lies between 1675-1700 cm-1, whereas for 6C complexes the N-O stretch occurs at ~1630 cm-1. Low temperature EPR spectroscopy studies conducted by several authors, also reveal interesting differences between the 5C and 6C iron(II) porphyrin NO adducts.52,53 Hyperfine lines resulting from the bound nitrogen of NO are observed at the lowest g value (g min) in 5C ferrous heme nitrosyls. The coordination of the proximal nitrogen atom in 6C ferrous heme nitrosyls causes a broadening in the EPR spectrum at g-mid resulting from the hyperfine lines of the bound NO, and the trans-N

II donor ligand. Based on spectroscopic data available from the literature, our [(F8)Fe -NO]

((1)-NO) complex is a typical 5C ferrous heme mononitrosyl complex with a Soret band

max at 408 nm in the UV-vis region at room temperature (Figure 2.5), along with a characteristic N–O stretching band ν(N–O) at 1680 cm-1 in its IR spectrum.38,53 Additional evidence for the 5C nitrosyl complex can be seen in its EPR spectrum, which displays g values at 2.09, 2.02 and 1.99, with three hyperfine splittings at g(min) (Figure 2.11).38 In a

19F-NMR spectrum, a broad o-phenyl fluorine signal is observed at -106.0 ppm.

In the case of [(PPy)FeII-NO] ((2)-NO), the Soret band absorption is observed at 417 nm (Figure 2.5), which lies in between that known for 5C and 6C iron-nitrosyl complexes.

This indicates that in solution at room temperature, the proximal pyridine is weakly bound to the iron center to give 6C species. Further evidence comes from the low temperature

EPR spectrum of [(PPy)FeII-NO], shown in Figure 2.11, and Table 2.3, which clearly resembles the spectra of other 6C low-spin heme-Fe(II)-nitrosyl complexes;51 lowering the temperature allows for stronger binding of the pyridyl group, as would be expected. The spectrum shows small, unresolved hyperfine splittings at g(mid) due to the presence of the proximal pyridine ligand (g = 2.07, 2.01, 1.98). The lack of UV-Vis spectral features at

63 400, and 470 nm, as well as EPR data for [(PPy)FeII-NO] ((2)-NO) confirms that in solution it forms a 6C species, but where the pyridyl group is weakly bound to the iron center at room temperature.

On the other hand complex [(PIm)FeII-NO] ((3)-NO) is a very stable 6C iron(II) nitrosyl species. In the UV-vis region, the observed Soret bands shift to 423 nm for (3)-

NO, which match very well with reported 6C iron(II) nitrosyl complexes.51 To further investigate the strength of the proximal (imidazole) ligand binding to the iron center in (3)-

NO, an EPR spectrum was recorded. The observed g values are 2.07, 2.00, and 1.97 for

(3)-NO (Figure. 2.11, Table 2.3), the hyperfine pattern is on g(mid) and the hyperfine lines, are not well resolved. Also, the stretching frequency, ν(N–O) for complexes (3)-NO is 1650 cm-1, and this lowered stretching frequency is due to the binding of the N-donor ligand

(Imidazole) trans to the NO, which weakens the Fe–NO σ-bond.54 Interestingly, this frequency is higher in energy compared to a similar ferrous heme nitrosyl with a free axial

-1 base, [Fe(To-F2PP)(MI)(NO)] (MI = methylimidazole; ν(N–O) = 1624 cm ). The trend observed is in line with a previous study by Scheidt,55 further suggesting that the tethered axial ligand bases bind to the heme more weakly that would or does a freely added (or present) base. The IR data matches closely with reported work by Lehnert and co-workers51 and indicates that the benzyl-imidazole linker impedes the binding of the proximal N-donor ligand when compared to the free base, but still allows for the formation of very stable 6C complexes at room temperature.

Figure 2.12. 19F-NMR of (1)-NO (top), and (3)-NO (bottom) complexes in THF at room temperature.

In 1H-NMR, the pyrrole hydrogen atoms resonate at 8.8 ppm for (3)-NO, while in

19F-NMR, the o-fluorine atoms resonate at -106, -107, -110 ppm for (3)-NO as seen in

Figure 2.12, above. Here peaks are very broad compared with all the Fe(II)-DIMPI complexes, where peaks were very sharp, and displayed visible H-F coupling interactions.

2.5. Conclusions

Using a small family of derivatized hemes featuring covalently tethered axial ligands (pyridine/imidazole) synthesized in our lab, we explored their reactivity towards

2,6-dimethlyphenyl isocyanide (DIMPI), and nitric oxide (NO). Towards this aim, we generated and characterized the six coordinate ferrous heme complexes;

[(PPy)FeII(DIMPI)], and [(PIm)FeII(DIMPI)] which have UV-Vis, IR, and EPR properties

II that are clearly distinguishable from those of [(F8)Fe (DIMPI)2]. The X-ray structures reveal a significant contribution from H-bonding between porphryinate meso-phenyl ortho-fluorine atoms, and these have been described. These are emphasized in large part because 19F-NMR spectroscopy clearly indicates that most if not all of these interactions are maintained in solution. We have also characterized several ferrous heme mononitrosyl complexes by multinuclear NMR, UV-Vis, EPR and solid state FT-IR spectroscopies. At

65 room temperature, [(PIm)FeII] forms very stable six-coordinate ferrous iron-NO complex.

II Py II [(F8)Fe ] forms a five coordinate ferrous heme nitrosyl complex, while [(P )Fe ] appears to be somewhere between 5 coordinate, and 6 coordinate due to its weakly binding axial pyridyl ligand.

As mentioned, the impetus for synthesizing ferrous heme porphyrinates using the

(PPy), and (PIm) ligand systems is to utilize these porphyrins in our ongoing research into modeling the active site chemistry of cytochrome c oxidase. An understanding of the nature of reactions and structures, i.e., coordination numbers, ligation preferences (e.g., pyridyl vs imidazolyl vs solvent THF), and other bonding or structural aspects can help to better understand the types of structures obtained in heme-O2-copper chemistry, and also inform on the design of hemes utilized for such studies.

Acknowledgement This work was supported by the National Institutes of Health (R01 GM 060353 to K.D.K).

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APPENDIX A

SUPPORTING INFORMATION FOR CHAPTER 2

UV-vis Spectral Titrations for (P)FeII + DIMPI. To a solution of (P)FeII (12 μM, THF;

P = PPy, PIm, PImH) was added 0.1 – 2.5 equiv of DIMPI in 0.1 equiv increments from a stock solution in THF. UV-vis spectrum was taken after each addition of DIMPI, showing isosbestic conversion of (P)FeII to (P)FeII-DIMPI. The reaction mixture was allowed to equilibrate fully until no further spectral change was observed prior to the next equivalent of DIMPI. A plot of the change in absorbance at 430 nm vs DIMPI resulted in the binding curve shown in Fig. S9 – S11 and could be well fit by a 1:1 binding model, eq 1–4. Using

Py Im ImH 7 - this equation, the best fit of the plot for P , P and P system gives Ka = 2.29 x 10 M

1, 1.19 x 107 M-1 and 1.29 x 107 M-1.

Figure S3. X-band EPR spectroscopy of ferrous heme-NO complexes recorded at 8K in frozen THF (red) and fit of the spectrum using the program Easy Spin [1] (blue). Fit parameters:

NO NO NO (a) (1)–NO (g1 = 2.0918, g2 = 2.0074, g3 = 2.0052; N hyperfine: A1 = 48.78, A2 = 67.63, A3 = 35.68) NO NO NO (b) (2)–NO (g1 = 2.0746, g2 = 2.0081, g3 = 1.9904; N hyperfine: A1 = 4.0, A2 = 60.4, A3 = Py Py Py 35.8; A1 = 18.1, A2 = 20.1, A3 = 19.1) NO NO NO (c) (3)–NO (g1 = 2.0686, g2 = 2.002, g3 = 1.9662; N hyperfine: A1 = 44.10, A2 = 63.18, A3 Im Im Im = 47.45; A1 = 22.94, A2 = 1.29, A3 = 16.84)

References:

1. S. Stoll and A. Schweiger (2006) Journal of Magnetic Resonance 178:42-55; http://easyspin.org/

Chapter 3. Reactions of a Heme-Superoxo Complex Toward a Cuprous Chelate and •NO(g): CcO and NOD Chemistry

This work was co-authored with the following authors and is published under the following citation:

Savita K. Sharma,† Patrick J. Rogler,† and Kenneth D. Karlin†

†Johns Hopkins University, Baltimore, Maryland 21218, United States

J. Porphyrins Pthalocyanines 2015; 19: 352-360.

76 3.1. Abstract

Following up on the characterization of a new (heme)iron(III)-superoxide species formed from the cryogenic oxgyenation of a ferrous-heme (PPy)FeII (1) (PPy = a tetraarylporphryinate with a covalently tethered pyridine group as potential axial base),

Py III •- 1 giving (P )Fe -O2 (2), we report here on (i) its use in forming a cytochrome c oxidase

(CcO) model compound, and (ii) in a reaction with nitrogen monoxide (•NO; nitric oxide) to mimic nitric oxide dioxygenase (NOD) chemistry. Reaction of (2) with the cuprous

I chelate [Cu (AN)][B(C6F5)4] (AN = bis[3-(dimethylamino)propyl]amine]) gives a meta-

Py III 2- II stable product [(P )Fe -(O2 )-Cu (AN)][B(C6F5)4] (3a), possessing a high-spin iron(III) and copper(II) side-on bridged peroxo moiety with a μ-η2:η2- binding motif. This complex

Py III 2- II thermally decays to a corresponding μ-oxo complex [(P )Fe -(O )-Cu (AN)][B(C6F5)4]

Py III 2- II (3). Both (3) and [(P )Fe -(O2 )-Cu (AN)][B(C6F5)4] (3a) have been characterized by

2 UV-Vis, H-NMR and EPR spectroscopies. When (2) is exposed to •NO(g), a ferric heme nitrato compound forms; if 2,4-di-tertbutylphenol is added prior to •NO(g) exposure, phenol ortho-nitration occurs with the iron product being the ferric hydroxide complex

(PPy)FeIII(OH) (5). The latter reactions mimic the action of NOD’s.

3.2. Introduction

Metalloenzymes form an essential component of the various biological and physiological functions that are essential for life.2 Hemoprotiens are perhaps the best- known class of metalloenzymes, and their reactions with dioxygen are foundational to aerobic life. These proteins are critical to dioxygen storage and transport (myoglobin and hemoglobin),3 substrate oxygenation (cytochrome P-450 family), as well as dioxygenation4 and peroxidation. Proteins with a heme-copper active site are critical to cellular respiration

77 (e.g., in cytochrome c oxidase). Nitric oxide is biosynthesized and interacts with hemoproteins, as part for this molecule’s involvement in inflammatory responses, cellular signaling, and vasodilation. Nitric oxide dioxygenase (NOD)5 and nitric oxide reductase

(NOR)6 enable cellular NO regulation/removal when it is present in excess.

It has been shown that CcO functionality is inhibited in the presence of NO7,8 via competition with O2 for binding at the binuclear center. Under certain physiological conditions the NO concentration in vivo can reach levels that significantly affect the reaction rate of CcO.9 To compensate, myoglobin (Mb) and/ or hemoglobin (Hb) play a major role in scavenging •NO, helping to keep respiratory homeostasis. This maintains the proton gradient over the mitochondrial inner membrane, driving ATP synthesis. When •NO is overproduced in vivo as a component of inflammatory response, reactive nitrogen species (RNS) can be formed by the reaction of NO with reactive oxygen species (ROS) such as superoxide, to generate a peroxynitrite (O=NOO-). Peroxynitrite10,11 is a strong oxidant and nitrating agent, and reacts with a number of biological substrates such as

12 13 14–16 17 thiols, tyrosine residues, lipids, CO2 , DNA, and metalloprotiens. Hence, •NO and peroxynitrite scavenging by Hb/Mb, is critical not only to respiration, but also for the mitigation of oxidative damage via NOD activity.18,19

Our own research group is particularly interested in providing basic coordination chemistry insights into the possible reactive intermediates formed during CcO turnover by

20–24 using synthetic functional models. CcO is responsible for the reduction of O2 to water as a terminal step of the respiratory chain of mitochondria and many aerobic bacteria.25,26

A ferric superoxo species is the most well studied dioxygen intermediate generated upon

II I 27 initial O2-reaction with the fully reduced active-site heme Fe –Cu center. This species

78 forms prior to O–O bond cleavage and as such has attracted considerable interest.1,28,29

Here, we describe the reactivity of an iron(III)-superoxo species (2) towards (i) a cuprous-

I chelated complex [Cu (AN)][B(C6F5)4] and (ii) •NO, these reactions representing synthetic functional models for CcO and NOD, respectively.

3.3. Experimental

3.3.1. Materials and Methods

All reagents and solvents used were of commercially available analytical quality except as noted. Dioxygen was dried by passing through a short column of supported P4O10

(Aquasorb, Mallinkrodt). Nitrogen monoxide (•NO) gas was obtained from Matheson

Gases (High Purity Grade, Full cylinder ~500 psig @ 70 ºF) and purified as follows: it was first passed through multiple columns containing Ascarite II (Thomas Scientific) to remove higher nitrogen oxide impurities. Further purification by distillation was completed by warming frozen •NO (as crystalline N2O2) from 78 K in a liquid N2 cooled vacuum trap to

193 K through use of an acetone/dry–ice (– 78 ºC) bath, and collection in a second liquid

N2 cooled evacuated vacuum trap. This secondary flask was again warmed to 193 K and the purified •NO was collected in an evacuated Schlenk flask (typically 50 mL) closed with a rubber septum secured tightly by copper wire. The •NO in the Schlenk flask is collected and kept at higher pressures (> 1 atm). Addition of •NO and O2 to the metal complex solutions was accomplished using a three-way long needle syringe connected to a Schlenk line.

THF and Pentane were distilled over Na/benzophenone ketyl and calcium hydride, respectively. 2,4–di–tert–butylphenol (DTBP) was purchased from Sigma–Aldrich and

79 purified by multiple recrystallizations in toluene under Ar. All other reagents were used as received. Preparation and handling of air–sensitive compounds were performed under an argon atmosphere using standard Schlenk techniques or in an MBraun Labmaster 130 inert atmosphere (<1 ppm O2, <1 ppm H2O) drybox filled with nitrogen gas. Solvents were purged with Ar prior to use. All UV-Vis measurements were carried out by using a Hewlett

Packard 8453 diode array spectrophotometer with a 10 mm path quartz cell. The spectrometer was equipped with HP Chemstation software and a Unisoku thermostated cell holder for low temperature experiments. All NMR spectra were recorded in 7 inch, 5 mm o.d. NMR tubes. Low–temperature 2H NMR (Bruker 300 MHz spectrometer equipped with a tunable deuterium probe to enhance deuterium detection) measurements were performed

2 at – 80 °C under a N2 atmosphere. The H chemical shifts are calibrated to natural abundance deuterium solvent peaks. EPR measurements of the frozen solutions were carried out at 14K on an X-Band Bruker EMX CW EPR spectrometer controlled with a

Bruker ER 041 XG microwave bridge operating at the X-band (~9 GHz). Gas chromatography (GC) was performed on an Agilent 6890 gas chromatograph fitted with a

HP-5 (5%-phenyl)-methylpolysiloxane capillary column (30 m * 0.32 mm* 0.25 mm) and equipped with a flame-ionization detector.

3.3.2. Synthesis

Py II Py II I F (P )Fe /(d8-P )Fe (1) and [Cu (AN)]BAr were synthesized as previously described.1,30

Synthesis of (PPy)FeIII-OH (5)

(PPy)FeIII-(OH) (5) was prepared using a modified procedure for the synthesis of its previously published1 chloride analogue, (PPy)FeIII-(Cl). In this case (PPy)FeIII-(Cl) was

80 dissolved in ~250 ml DCM, and this DCM layer was then stirred vigorously with ~250 ml of 3.0 M NaOH in a 1000 ml round-bottom flask for three hours. Separation of the organic layer, followed by drying with magnesium sulfate, and solvent removal yielded the

(PPy)FeIII-(OH) (5) Yield 600 mg (57.8 %). This compound was then characterized by UV-

1 Vis, and H-NMR spectroscopies as well as ESI-MS. UV-Vis (THF): λmax, nm 412, 563.

1H-NMR (300 MHz; CD2Cl2): δpyrrole 80.63 ppm. MS (ESI) m/z 924.

3.3.3. Preparation of 2H NMR and EPR samples

In-situ generation of complexes (3a) and (3)

Py II In a typical experiment, 0.57 mL of a complex (d8-P )Fe (5 mM) solution in THF were placed in a 5 mm rubber septum capped NMR tube. After cooling down the NMR tube to –80 ⁰C (acetone/N2(liq) bath), dioxygen was bubbled through the solution mixture

Py III ·- to form the (d8-P )Fe (O2 ) complex (2). The NMR tube was transferred rapidly into the

NMR instrument which was precooled at -85 ºC. Similar to our UV-Vis experiments, complex (3a) was prepared by removing any excess of dioxygen by Vacuum/Ar cycles from (2) and careful addition of 1 equiv. of [CuI(AN)]BArF complex. Finally, Complex

(3a) was warmed to RT to obtain decomposed product (3). EPR samples were prepared in a similar way by using 9 mm EPR tube.

In-situ generation of complex (4)

In a typical experiment, 0.650 mL of (PPy)FeII (1 mM) in THF was placed in a 9 mm rubber septum capped EPR tube. After cooling down the EPR tube to –80 ⁰C

(acetone/N2(liq) bath), 3 mL dioxygen was bubbled through the solution mixture to form the

Py III ·- (P )Fe (O2 ) complex (2). Similar to our UV-Vis experiments, complex (4) was prepared by removing excess of dioxygen by Vacuum/Ar cycles from (2) and careful addition of 2

81 mL •NO g via 3-way gas tight syringe. Excess gas was removed by vacuum/Ar cyles. After generation of all complexes, the tubes were frozen in N2(liq) and brought to the spectrometer for measurement.

3.3.4. Procedure for Nitrate/Nitrite test

In a 25 mL-Schlenk flask compound (1) was prepared in 10 mL THF (0.1 mM solution, inside dry box). In a typical bench top reaction, the flask was cooled to -80 ⁰C

(Acetone/dryice bath) and dioxygen was bubbled through complex 1, resulting in the formation of superoxo species, (2). Excess O2(g) was removed by several cycles of Ar bubble followed by vacuum. Addition of •NO(g) to (2) resulted in formation of (4), immediately. Reaction stirred for 10 minutes at -80 ⁰C. After 10 mins, solvent was removed and solid product was isolated in 5 mL of DCM and extracted with 10 mL of aqueous NaCl solution (6 mM). The presence of a significant amount of nitrate ion in the aqueous layer was confirmed by semiquantitative QUANTOFIX nitrate/nitrite test strips.

3.3.5. Nitration of the 2,4-Di-tert-butylphenol (DTBP)

The formation of the superoxo complex (2) in THF at - 80 °C was carried out as described above for (1) (0.1 mM) in a schlenk cuvette, and the reaction was monitored by

UV-Vis. Excess O2(g) was removed by bubbling the solution with Ar and vacuum purge cycles as before. Two equivalents of 2,4-di-tert butylphenol (DTBP) (0.1 mmol) were added. Upon addition of the DTBP no change in the UV-vis spectrum was observed. •NO(g) was added by using a three-way gas tight syringe, leading to the formation of (5). The resulting solution was concentrated in vacuo and pentane was added to precipitate the Fe product. The pentane solution was collected by decanting. The Fe product was washed several times with pentane, and the pentane solution was removed and collected by

82 Py III decanting after each wash. The solid Fe product (P )Fe -(S) (S = solvent, H2O) was dried in vacuo, redissolved in THF and its UV-vis spectrum was recorded [λmax = 410, 563 nm,]; these spectral parameters matched those of authentically synthesized (PPy)FeIII(OH) (see above). The pentane solution containing the phenolic products was filtered to remove any trace of Fe product and the solvent was removed in vacuo. The resulting solid was redissolved in MeOH and dodecane used as internal standard and injected into a GC. This showed 2,4-di-t-butyl-6-nitrophenol (NO2-DTBP) (82% yield) and unreacted DTBP as the only products of the reaction. These were identified by comparison to the spectra obtained from commercial 2,4-di-t-butyl-6-nitrophenol, and 2,4-di-t-butylphenol respectively.

3.4. Results and Discussion

3.4.1. Reactivity of the iron(III)-superoxo complex (2) towards I [Cu (AN)][B(C6F5)4]

83 Earlier work in our group1 described the synthesis and characterization of (PPy)FeII

Py (1) [(P ) = pyridyl tailed porphyrinate (2-)] (λmax = 416, 525, 554 (sh) nm) which reacts

Py III •- reversibly with dioxygen to give a diamagnetic iron(III)-superoxo species (P )Fe -(O2 )

(2) (UV-vis, λmax = 419, 535 nm; EPR, silent). This is stable in solution below -30 °C in coordinating solvents such as tetrahydrofuran (THF), acetone, or acetonitrile as well as in non-coordinating solvents like dichloromethane (DCM). The use of copper ion complexes with tridentate alkylamino ligand AN has previously been useful23,30–32 and as such this

I copper chelate was employed here. Addition of one equivalent of [Cu (AN)][B(C6F5)4]

(Figure 3.1) to the superoxo compound (2) in THF at -100 ⁰C, monitored by UV-Vis spectroscopy, leads to the immediate formation of a heme-copper-O2 adduct (3a) [λmax =

420, 530, 555 (sh) nm], see Figure 3.2. The UV-vis spectrum of (3a) is very similar to our

III 2– II + previously described high-spin [(heme)Fe -(O2 )-Cu (L)] species where L is a tri- or tetradentate alkylamino or pyridylalkylamino ligand; these possess a side-on binding of peroxide to both metal ions, as depicted in Figure 3.1.33–35 Thermal decomposition of (3a) leads to the formation of complex (3) with a red shifted Soret band at 443 nm and Q band at 556 nm (Figure 3.2). These are characteristic features for μ-oxo FeIII-O-CuII like species,

III such as the previously structurally and spectroscopically characterized complex [(F8)Fe -

(O2-)-CuII(TMPA)]+ (TMPA = tris(2-pyridylmethyl)amine).36 Complex (3) can also be obtained by direct bubbling of dioxygen to a 1:1 mixture of (PPy)FeII (1) and

I Py III 2- II + [Cu (AN)][B(C6F5)4] at -80 ⁰C in THF. The μ-oxo complex [(P )Fe -(O )-Cu (AN)]

(3) (or rather a protonated μ-hydroxo conjugate acid form), are of interest since such species derive from dioxygen reactivity, thus perhaps related to CcO reaction chemistry.37,38

Figure 3.2. UV-Vis spectra of (1, black) a reduced (PPy)FeII + [CuI(AN)]+ 1:1 mixture; (3a, red) Py III 2- II + Py III 2- high spin peroxo complex [(P )Fe -(O2 )Cu (AN)] ; (3, green) μ-oxo complex [(P )Fe -(O )- CuII(AN)]+.

Further characterization of μ-oxo complex (3) was provided by low temperature

2 Py II Py II H-NMR spectroscopy of the pyrrole deuterated analogue of (P )Fe (1), (d8-P )Fe .

Figure 3.3, shows a 2H-NMR spectra of the solution derived from the oxygenation reaction of a PPyFeII-THF (1) and it exhibits a pyrrole resonance at δ 10 ppm, indicative of a low- spin (S = 0) six-coordinate ferrous heme at low temperature, but we also observe pyrrole resonances at δ 98 and 86 ppm, which are characteristic of a penta-coordinated high spin

85 heme. We interpret this observed NMR spectroscopic data as indicating that complex (1) is a mixture of 6-coordinate (pyridyl + THF) low-spin iron(II) and 5 or 6-coordinate high- spin iron(II) (e.g., pyridyl arm off, THF bound, or vice versa, and also possibly a bis-THF ligated ferrous heme). Direct bubbling of dioxygen to (1) gives superoxo complex (2) where a pyrrole resonance occurs in the diamagnetic region, at δ 9.0 ppm. Subsequent addition of one equiv. of [CuI(AN)]+ to the cold solution of superoxo complex (2) in an

NMR tube leads to the formation of [(PPy)FeIII-(O2-)-CuII(AN)]+ (3), with a downfield shifting of the pyrrole resonance to δ 102 ppm (Figure 3.3), indicative of a high-spin ferric heme (also, see below). Complex (3) is stable at room temperature. We have previously reported this characteristic pattern of downfield shifted pyrrole resonance for (P)FeIII-X-

II 2- 2- 35,39–41 Cu (X = O2 or O ) systems having overall S = 2 ground spin states, which arise from the antiferromagnetic coupling of the S = 5/2 high-spin heme-iron(III) center to an S

= ½ copper(II) moiety, through the bridging X ligand in (3) or (3a). When monitoring peroxo complex (3a) by 2H-NMR spectroscopy, a clean thermal transformation to (3) is observed. EPR spectroscopic interrogation of (3) and (3a) revealed that both are EPR inactive, consistent with their formulations.

3.4.2. Reactivity of iron(III)-superoxo complex (2) towards •NO(g)

Figure 3.4. Reaction sequence where •NO(g) is added to superoxo complex (2) to give complex nitrato complex (4). In the presence of a phenolic substrate, the same reaction gives (5) as a final product along with the ortho-nitrated phenol.

Using a gas-tight three way syringe, addition of •NO(g) to superoxo species

Py III •- (P )Fe -(O2 ) (2) at -80 ºC in THF, as monitored by UV-Vis spectroscopy, led to the

Py III immediate formation of a five-coordinate nitrato compound (P )Fe -(ONO2) (4) [UV-vis,

λmax = 397, 410 (sh), 505, 573, 650 nm; EPR. g = 6, 14 K, high-spin iron(III)], as indicated in Figure 3.4 and with spectra shown in Figure 3.5. Product (4) yields a positive test for

- - nitrate ion, as determined using semiquantative QUANTOFIX nitrate (NO3 )/nitrite (NO2

– ) test paper; no NO2 ion was detected and the yield of nitrate ion was estimated to be > 75

% (see Experimental). These results are very similar to what we observed with in a previous

II study of (F8)Fe (F8 = tetrakis(2,6-difuorophenyl)porphyrinate(2-)) where addition of

III •- •NO(g) to the superoxo complex (F8)Fe -(O2 ), yields a five-coordinate heme nitrato

42 complex. While no transient species were detected following addition of •NO(g) to (2) and isolation of (4), the formation of a nitrite complex supports the intermediacy of a peroxynitrite –OON=O species (4a) which formed during the reaction (Figure 3.4), indicating an NOD type of reaction mechanism (see below).

Figure 3.5. UV-Vis spectra showing superoxo (2, red) formed from reduced (PPy)FeII (1, black) by Py III bubbling O2(g) at –80 °C; nitrato complex (P )Fe -ONO2 (4, grey) generated immediately after addition of •NO(g).

Thus, we sought chemical evidence which might support our supposition involving the formation of a peroxynitrite species during the reaction of (2) with •NO(g). Here, a tyrosine analogue, (2,4-di-tert-butylphenol (DTBP), was added into the solution of

Py III •– superoxo complex (P )Fe -(O2 ) (2). When •NO(g) was subsequently added, we observed

Py III the immediate formation of (P )Fe (OH) (5) [λmax = 400, 563 nm] as indicated by the Q- band feature in the UV-Vis spectrum (Figure 3.6). We then isolated the pure (PPy)FeIII(OH)

(5) (λmax = 410, 563) from this mixture (see Experimental). An authentic sample of

Py III (P )Fe (OH) (5) (λmax = 412, 563) was prepared (see Experimental), and has nearly identical UV-Vis features as those observed for the reaction product. The UV-vis and EPR

III spectroscopic features observed here also closely match those known for (F8)Fe (OH)

42 (λmax = 408, 572 nm high-spin, EPR, g = 6.0) (Figure 3.6). Workup of the reaction solution revealed that the product (5) forms along with high yields (>85%) of 2,4-di-tert-

88 butyl-6-nitrophenol (NO2-DTBP), as confirmed via gas-chromatography. This reaction

- - mixture was also tested for the presence of any NO3 /NO2 ion and yielded a negative result for both.

Figure 3.6. UV-Vis spectroscopy in THF at -80 ºC. (black) spectra is reduced (PPy)FeII (1); red is (2 + DTBP), and green is (5).

These studies indicate the involvement of a heme-peroxynitrite like intermediate

[(PPy)FeIII-OONO] (4a), which we could not detect, but that formed when superoxo

Py III •– complex (P )Fe -(O2 ) (2) was reacted with •NO(g) (Figure 3.4). There were previous reports that suggested the detection of heme-peroxynitrite species in the reaction of oxy-

18,43 heme (i.e., ferric superoxo) with •NO(g), either via UV-Vis or EPR spectroscopy.

However these results were refuted by Moënne-Loccoz and co-workers44 using rapid freeze quench resonance Raman spectroscopy with Mb, which revealed that such intermediates were in fact iron-bound nitrate species formed prior to their decay to metMb. Still, the generally accepted mechanism of •NO dioxygenase involves direct reaction of the FeIII-

•- (O2 ) oxy complex with •NO, giving a peroxynitrite intermediate. Subsequent homolytic

O-O bond cleavage produces an oxo-ferryl (FeIV=O) species and the free radical nitrogen

89 dioxide (•NO2); the latter attacks the ferryl O-atom to produce a N-O bond, yielding nitrate.45–48 Recent work with oxy-coboglobin models49,50 exhibiting NOD-like activity have led to the detection of peroxynitrite intermediates using low temperature FTIR, this work has helped to shed light onto favorable conditions for generating peroxynitrite intermediates.51 Also, recent work by Nam and Karlin52 has shown an alternative method for mimicking NOD activity that is isoelectronic to the methods discussed above. In this case, NOD activity was exhibited using a nitrosonium ion added to a non-heme iron peroxo species. Following these literature precedents, we can hypothesize that (4a) undergoes homolysis to give a ferryl + •NO2 radical, which however can be captured by phenol present in solution. The ferryl would oxidize the phenol to a phenoxyl radical which will further react with •NO2 to give ortho-nitration of the phenol and form a very stable ferric hydroxo complex (4), as we observed (Figure 3.4).

3.5. Conclusions

From this investigation we can conclude that our present ferric superoxo complex

(2) can show cytochrome c oxidase (CcO) in the presence of a cuprous complex, generating a peroxo-bridged heme-Cu intermediate which can thermally transform to a heme-(O2-)-

Cu(L) μ-oxo product, which is derived from dioxygen reactivity. Alternatively, (2) can also function as a •NO scavenger by oxidizing it to the biologically benign nitrate ion, complex

(4).

Acknowledgements

We are grateful to the US National Institutes of Health for support of this research (GM60353).

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Chapter 4. Mononuclear Low-Spin Ferric Heme Peroxide and (Hydro)-Peroxide Complexes Displaying an End-on Binding Motif to Ferric Heme Porphyrinate

Patrick J. Rogler,a Hyun Kim,a Savita K. Sharma,a Andrew W. Schaefer,b Edward I. Solomonb and Kenneth D. Karlina

aDepartment of Chemistry, Johns Hopkins University, Baltimore, Maryland 21218, USA bDepartment of Chemistry, Stanford University, Stanford, California 94305, USA

96 4.1. Abstract:

We report herein the synthesis and characterization of a small library of mononuclear ferric heme peroxide complexes, and their hydroperoxide species formed on protonation in aprotic media under cryogenic conditions, as well as a new heme peroxo copper adduct featuring a covalently attached axial imidazole ligand, and its reactivity towards the exogenous reductant, decamethylferrocene (Me10Fc, Fc*). Addition of ~1.0 equivalents of the strong outer sphere reductant cobaltocene to the ferric superoxides

III .- Im III .- [(F8)Fe (O2 )] (λmax 415 (Soret), 535 nm), and [(P )Fe (O2 )] (λmax 423 (Soret), 535 nm) results in 1e- reduction to form the corresponding heme ferric peroxy anions

III III 2- III Im III 2- [Co (Cp)2][(F8)Fe (O2 )] (1) (λmax 433(Soret), 560 nm), and [Co (Cp)2][(P )Fe (O2

)(3) (λmax 425 Soret), 533, 567 nm) which differ in their coordination mode to the heme.

III Im III 2- Complex [Co (Cp)2][(P )Fe (O2 )] (3) represents only the second well characterized

(UV-Vis, EPR, rRaman) example of an end-on (η1) heme ferric peroxide species reported for synthetic models in homogeneous solution. In related chemistry, it is shown that

I F F – oxygenation of an equimolar solution of [Cu (AN)][(BAr ) (BAr = B(C6F5)4 ) and

[(PIm)FeII] in tetrahydrofuran at -80°C yields the new heme peroxo copper adduct

Im III 2- II + [(P )Fe -(O2 )-Cu (AN)] (λmax 423 (Soret), 533, 789, 949 nm), which reacts with the

Im III 2- - I + reductant Fc* to yield [(P )Fe (O2 )] , and [Cu (AN)] .

4.2. Introduction 4.2.1. Heme/dioxygen interactions: from biology to model systems

Hemoproteins are one of the most omnipresent types of enzymes across a huge diversity of cellular and extracellular systems in both aerobic, and anaerobic life. Their

97 roles in physiology span across critical functions including gas transportation, substrate oxidation (and oxygenation), biomolecule degradation, small molecule transformation for immune response, and PROS metabolism/regulation. To a great extent, this diversity in function is achieved in nature by modifying the proximal axial base ligand, and the properties of the distal heme binding pocket.1,2 Dioxygen binding and activation by heme enzymes involves several partially reduced metal-oxygen species, each of which are formed by stepwise protonation-reduction, or reduction protonation reactions (See Figure

4.1, below) Reductive O—O bond cleavage occurs concomitantly to the oxidation of the heme iron moiety, enabling hemoprotiens to harness the superior oxidizing power of oxygen’s double bond. As mentioned above (see chapter 1), understanding the basic

0 properties, including basicity (pKa), and redox potential (E ) of metal oxy species bound to hemes in synthetic and natural (enzymatic) systems will help inform the design of better catalyst materials for organic substrate activation, as well as the practical utilization of O2 in the cathodic half-cell reaction of sustainable fuel cells.

Figure 4.1. General paradigm of dioxygen activation and reductive O-O bond cleavage in hemoproteins.

98 Generally speaking enzymes that transport and store O2 utilize histidine ligands as the proximal axial base in the heme active site, as opposed to anionic cysteinate, and tyrosinate axial bases found in cytochrome P450’s and catalases, respectively. The presence of more highly polar amino acid side chains in the distal binding pocket also aid in stabilizing charged intermediates, such as the highly reactive [(P+)FeIV=O]+ -cation- radical, which is formed as a result of heterolytic O—O bond cleavage. In fact, the activity of an O2 carrier/storage protein can be changed by replacing the axial histidine with a cysteinate residue, making O2 activation proceed further, giving rise to oxidizing high valent intermediates.3 The combined effects of proximal axial base, and distal pocket interactions designed by evolution are known as ‘push-pull’ machinery, and they facilitate the heterolytic O—O bond cleavage step. The precise tuning of the active site is critical considering that any irregularities can lead to possible PROS leakage, or protein damage, which can manifest macroscopically in organism disease states. A huge amount of research about multi-functional heme enzymes has been reviewed elsewhere.4–8

Table 4.1. Dioxygen-Binding Hemoproteins and Their Main Catalytic Functionalities. Name proximal ligand catalytically functionality on heme active species Hemoglobin (Hb) Histidine N/A O2 binding and storage Myoglobin (Mb) Histidine N/A O2 binding and storage Cytochrome P450 Cysteine (P•+)FeIV=Oa Oxidation/oxygenation of organic substrates Aromatase Cysteine (P)FeIII-OO-a Biosynthesis of estrone from androstenedione III a Heme Oxygenase Histidine (P)Fe -OOH Heme degradation to biliverdin, CO(g), and free FeII Nitric Oxide Cysteine (P)FeIII-OO•-a Generation of NO from L-arginine Synthase (P)FeIII-OO-a •+ IV a Peroxidases Histidine (P )Fe =O H2O2 activation for substrate oxidation Chloroperoxidase Cysteine (P•+)FeIV=Oa Monooxygeation and halogenation of organic substrates •+ IV a Catalase Tyrosine (P )Fe =O H2O2 disproportionation to H2O and O2(g) a(P) = heme or porphyrinate supporting ligand.

99 Unlike dioxygen transport and storage proteins (hemoglobin and myoglobin), dioxygen activating enzymes specifically take O2 reduction a step further than ferric-heme- superoxide. Another reducing equivalent gives a ferric peroxo species, which on protonation forms a ferric heme-hydroperoxide species, known as compound 0. These hydroperoxides are critical intermediates in biology, and they sometime serve as the active oxidant, attacking substrates (as in heme oxygenase), and generally serve as precursors to much more reactive high-valent oxidants that oxidize or oxygenate unreactive hydrocarbon substrates. This final reductive cleavage step is critical, whether it occurs in a homolytic or heterolytic manner, and this is deterministic of the turnover outcome, and has been explored for decades in cytochrome P450 monooxygenase, peroxidases, and synthetic model chemistries.6,9–16

Cytochrome P450’s comprise an extremely widespread, and comprehensively studied class of metalloproteins. They catalyze a very broad breadth of chemical transformations including hydrocarbon (and aromatic) hydroxylation, olefin (and arene) epoxidation, alkyne oxygenation to carboxylic acids, N-/S-/O- dealkylation reaction, alcohol and aldehyde oxidations, and nitric oxide reduction.5,6 One notable subclass of

P450 enzymes are known as aromatases, and they catalyze several physiological interconversions critical to the production of sex hormones including progesterone to androstenedione,17,18 androstenedione to estrogen,19–22 and pregnenolone to dehydroepiandrostenedione.17,18 This subclass is of particular interest to us given the critical role that a ferric heme peroxide is expected to play in the third step of substrate, wherein an aldehyde deformylation occurs.23

100 The most extensively studied example of cytochrome P450 enzymes is P450cam which regioselectivity and stereospecifically hydroxylates camphor to 5- exohydroxycamphor in camphor oxidation. P450s also catalyze xenobiotic degradation in the liver, and epoxidize DNA4 In its resting state, cytochrome P450 contains a low spin, six coordinate ferric center with a proximal axially coordinated cysteinate residue, with a ligated water molecule at the axial distal site. Hydrogen bonding interactions with

Threonine (Thr-252), and aspartate residues (Asp-251), and several other amino acid side chains are key features tuning the iron centers redox potential. In the enzyme’s resting state, the FeIII/FeII redox potential is not amenable to reduction (between -400 to -170 mV).6) Binding of a substrate molecule leads to the displacement of water molecules from the binding pocket, including an axially coordinated water molecule. This dramatically increases the redox potential of the iron center by ~400 mV. Subsequently FeIII is now reduced to FeII, and the active site is ready to bind oxygen. This reduction mechanism has been evolved to prevent the spontaneous reduction of FeIII to FeII by cellular reducing agents such as NADPH, stopping the production of unwanted cytotoxic PROS when P450

4 24–27 is not turning over. Iron(II) then binds O2 to form the initial oxy-intermediate. The distal oxygen atom is stabilized by several hydrogen-bonding interactions mediated by two new water molecules that enter the cavity.6 Superoxide is then subsequently reduced to an

III 2- end-on bound [(P)Fe -O2 ] adduct, which has been modeled, and detected by Davydov and Hoffman.28 Synthetic models for this intermediate have been characterized by Naruta, and they are highly basic based on computational and model compound investigations.7,29,30 Protonation of this end-on peroxo yields a low spin six coordinate

III [(P)Fe -O2H] intermediate, and the enzyme is now primed for O—O bond cleavage. The

101 reductive O—O bond cleavage event is facilitated by the ‘push-pull’ effect deriving from the anionic cysteinate proximal axial base, which donates electron density to the dz2 orbital of the FeIII, and hydrogen bonding interactions from distal active site Threonine (Thr-252),

III and Aspartate (Asp-251) residues to the distal OH of the [(P)Fe -O2H] moiety. The effect of the interactions is to strengthen the Fe-O, and OH bonds while weakening the peroxidic

O—O bond,5,31 heterolytic cleavage then gives the powerful active oxidant, compound I.

Another class of heme containing oxidases comprise chloroperoxidases and catalases. Hydrogen peroxide has a critical role in cellular redox signaling in in oxidative

32,33 stress, and is also a co-substrate for peroxides (and peroxygenases). The H2O2 bond can be cleaved homolytically in the presence of reduced copper or iron ions, to yield problematic hydroxyl radicals via Fenton chemistry, or the Haber Weiss process, thereby, control over H2O2 production and mitigation in vivo is critical. Heterolytic cleavage, occurring in peroxidases and catalases, detoxifies or utilizes H2O2 for substrate oxidation.

Peroxidases produce Cmpd I to mediate substrate dehydrogenative oxidations, while catalases use it to oxidize another H2O2 molecule, detoxifiying H2O2 by disproportionation to O2 and H2O. These enzymes differ from P450 type monooxygenases in that they have a tyrosinate (catalases), or histidine (peroxidases) axial base. Note that the proximal histidine of peroxidases is hydrogen bonded to an aspartate residue (249, 250) which significantly increases its anionic character, aiding in heterolytic O-O bond cleavage, and differentiating peroxidases from globins which do not activate O2 further on binding. The effect of this proximal charge donation was first demonstrated in model systems,34,35 and confirmed in proteins by site directed mutagenesis.36 Similar types of enzymes, such as chloroperoxidases (CPO) (which are generally referred to as haloperoxidases)37–40 catalyze

102 halogenation, dehalogenation, N-demethylation, dismutation, epoxidation and oxidation reactions38,40–45 although their primary role is the chlorination of organic substrates following C-H bond activation. They are a hybrid of P450s and peroxidases, in spite of their differing structure from both of those classes. Their distal amino acid structure resembles that of peroxidases, with polar side chains that aid heterolytic O—O bond cleavage, and they contain a cysteinate proximal axial ligand. Hydrogen bonding interactions are critical to effect precise regioselectivity, and also mediate fast reactivity

46 with H2O2. Compound 0 of chloroperoxidase has been structurally characterized, and it

III 41,47,48 possesses a low spin iron center with a [(P)Fe -(O2H)] moiety.

Figure 4.2. Crystal structure of Compound 0 intermediate in chloroperoxidase. Fe-O = 1.80 Å, O—O = 1.50 Å, and Fe—O—O = 131°.

Understanding the mechanistic details involved in the O—O bond scission process in heme enzymes is of momentous importance, considering the potential that controlling and applying the reactivity of Cmpd I type species has for challenging synthetic transformations, but also due to the implications that these events have for the design of

103 new catalysts for energy applications that are based on earth abundant transition metals.49–

4.2.2. Heme ferric peroxide and hydroperoxide intermediates in cytochrome P450 enzymes, and synthetic models complexes.

Highly oxidizing intermediates such as compound I are not the only reactive intermediates utilized by cytochrome P450 type enzymes. While the mononuclear ferric heme peroxide intermediate (see Figure 4.1) in the majority of P450 enzymes is rapidly protonated, and converted to an oxoferryl species, some enzymes including aromatase, lanosterol 14-demethylase, progesterone 17-hydroxylase/17,20-lyase, and nitric oxide synthase utilize this anionic intermediate as their active oxidant. Aromatase catalyzes the conversion of human androgens to estrogens52 in three steps, each of which consumes one equivalent of dioxygen, and one equivalent of NADPH. The first and second steps involve typical P450 type hydroxylation carried out by an oxoferryl porphyrin cation radical species. the third step involves the cleavage of the C19 aldehyde to yield an aromatized ring.53 Extensive research efforts have been directed towards determining the active oxidant in this third step. Akhtar, et. al. utilized isotopic labeling studies to show that the oxygen atom incorporated into the formaldehyde produced is derived from dioxygen, and that the vinylic hydrogen of formic acid was bound to C19 in the substrate, implicating a nucleophilic attack by a peroxide intermediate.52–54 Indeed, site directed mutagenesis studies on microsomal P450 enzymes55 including P450 2B4 and cytochrome P450 2E1 which carry out hydroxylation, and deformylate aldehydes56,57 have shown evidence for peroxide and hydroperoxide intermediates when proton delivery to the active site is inhibited.58 Mutation of an active site threonine in P450 2B4 (Thr-302), believed to act as

104 a proton donor, to alanine, decreased substrate hydroxylation, but yielded increased rates of cyclohexanecarboxaldehyde deformylation.59 This suggested that mutation of the Thr-

302 active site residue favors a ferric peroxide species as the active oxidant. Similarly, in

P450 2E1, the same mutation increased epoxidation reactivity, and decreased allylic hydroxylation activity. The ratios of epoxidation and hydroxylation rates in P450 2E1 support a ferric hydroperoxide active oxidant. This work suggested that iron peroxide and hydroperoxide intermediates can have a long enough lifetime to react with a substrate as a nucleophile (peroxide) if proton delivery to the distal oxygen atom is constrained.58,60–62

Given the relevance of ferric heme peroxide and hydroperoxide species to enzymatic transformations such as deformylation, epoxidation, and the production of NO in biological systems, it is not surprising that researchers for many years have been developing synthetic small molecule heme ferric-peroxide model complexes. Here we will very briefly cover the characterization and basic reactivity properties of some ferric peroxo porphyrin complexes (See Table 4.2, below) as they relate to our own work.

The earliest synthetic heme iron(III)-peroxide complexes were reported by

Valentine and coworkers using 2,3,7,8,12,13,17,18-Octaethyl-21H,23H-porphine (OEP), and 5,10,15,20-tetraphenylporphyrin (TPP). They were synthesized by adding potassium

63 superoxide KO2 to the ferric porphyrins in DMSO or acetonitrile. Two equivalents of potassium superoxide are necessary in this reaction because the first reduces FeIII to FeII, and the second equivalent forms the peroxide. Reactivity patterns for this anionic peroxo species were highly dependent on solvent, iron concentration, moisture, and temperature.

In solvents that are non-coordinating, or strongly coordinating, no trace of heme ferric peroxo intermediates were detected, instead a stable hemochrome, and a -oxo iron

105 porphyrin dimer were observed. Although the thermal instability of these peroxo species did not allow for structural determination, they were characterized spectroscopically by

UV-Vis, EPR, IR, and Resonance Raman spectroscopy (See Table 4.2). The peroxide was proposed, based partially on its EPR spectrum, to have a side on binding motif to iron. The tetramethylammonium salt of the iron(III)-peroxo complex with the OEP porphyrin framework was isolated as a microcrystalline solid. Mossbauer, and zero-field splitting parameters calculated from temperature dependent EPR measurements were consistent with the proposed side-on binding to FeIII.64 Dolphin, James, et. al. also reported the synthesis of this heme ferric peroxo species by electrochemical reduction of

III .- 65 [(OEP)Fe (O2 )].

Metal peroxo adducts can be electrophilic,66 or nucleophilic67–69 and their reactivity is dependent on metal identity, the supporting ligand, solvent properties, and conditions.

III 2- - Valentine investigated the reactivity of [(TPP)Fe (O2 )] , and the analogous complex with the 5,10,15,20-Tetrakis(2,4,6-trimethylphenyl) (TMP) porphyrin framework. Both complexes exhibited nucleophilic reactivity towards electron deficient olefins.70 The authors also compared the reactivity of side-on ferric heme peroxo complexes with electron rich porphyrins such as OEP, TPP, and TMP to those featuring electron withdrawing porphyrin frameworks such as 5,10,15,20-(pentafluorophenyl)porphyrin (TPFPP). They observed that electron withdrawing substituents can turn off nucleophilic reactivity, while at the same time increasing peroxide stability. Electron withdrawing substituents did not

III 2- - make [(TPFPP)Fe (O2 )] into an electrophile, however they do increase the tendency to bind axial bases such as PPh3, and DMSO, which cause dramatic increases in epoxidation reaction rates (i.e. nucleophilicity).71,72 Comparison to analogous compounds possessing

106 other metals including MnIII, PtII, and TiIV revealed much greater nucleophilic character for heme ferric peroxide species. Interestingly, heme ferric peroxides bearing the natural protoporphyrin IX ligand showed even greater reactivity compared to other synthetic

72 III 2- - porphyrin systems. Side-on bound heme ferric peroxides [(TMP)Fe (O2 )] , and

III 2- - [(TMP)Fe (O2 )] are also competent for deformylation chemistry, a reaction that is critical to the third step of aromatase, as noted above.73,74

Within the research areas discussed above, a major goal of bioinorganic model

III chemistry has been to definitively characterize [(P)Fe -O2H] intermediates within model systems and to carry out detailed investigations into their O—O bond cleavage chemistry.

III This is a paramount goal because of the centrality of (P)Fe -O2H species to functionality in metalloenzymes such as P450’s, peroxidases, catalases, and heme-oxygenase. Studies concerning these intermediates are made difficult due to the highly de-stabilized nature of

III low-spin 6C (heme)Fe -O2H intermediates, especially when compared to their alkyl, or acyl peroxide derivatives.75–79 Tajima and company were able to characterize the first

III example of a (heme)Fe -O2H model compound during H2O2 activation in DMF as solvent.80 This intermediate could only be produced under basic conditions, using tripropylamine or KOH as base. Spectroscopic evidence for the formation a low spin iron center was provided by EPR spectroscopy, and the appearance an intense red color characteristic of ferric hydroperoxide species. Subsequent work by Tajima and coworkers

III characterized Fe -O2H complexes with OEP, TMP, and TPP porphyrinate frameworks using a rapid freeze/UV-Vis/EPR coupled technique.81,82 These hydroperoxide species were generated by reduction of each respective ferric-superoxide precursor complex using sodium ascorbate in basic media, and the reaction only worked in DMF/H2O at -40 °C.

107 The first fully characterized mononuclear ferric heme hydroperoxide model systems with critical vibrational spectroscopic characterization (including UV-Vis, rRaman, EPR, and Mossbauer spectroscopies) have been more recently characterized by

Naruta and coworkers.83–85 A modified version of the TMP Porphyrin, bearing a covalently

III attached axial imidazole base was used to make a low spin six coordinate [(P)Fe O2H]

III complex denoted here as [(TMPIm)Fe (O2H)]. This ferric heme hydroperoxide displayed a diverse synthetic profile. (Figure 4.3, below) Not only could this ferric heme

II hydroperoxide be made directly by adding KO2 to [(TMPIm)Fe ] in the presence of methanol, but also via dioxygen chemistry, and reduction with a strong 1e- reducing agent.

III .- Reduction of the ferric superoxide [(TMPIm)Fe (O2 )] prior to adding MeOH yielded a

III 2- side-on heme-ferric peroxide [CoCp2] [(TMPIm)Fe (O2 )] critical data including UV-

Vis, EPR, and Fe-O/O-O vibrational frequencies for these intermediates are tabulated in

Table 4.2, below.84,86

III 2- - Figure 4.3. Synthetic approaches utilized by Naruta et. al. in order to generate [(P)Fe -O2 ] , and III (P)Fe (O2H) complexes utilizing the TMPIm ligand platform.

108 Although the synthetic profile reported by Naruta and coworkers with the TMPIm porphyrinate framework is compelling from a synthetic perspective, they were not able to isolate, or detect an anionic ‘end-on’ mononuclear ferric heme peroxide complex. In pursuit of this goal the authors also investigated the effect of secondary coordination sphere interactions (i.e. steric interactions and hydrogen bonding) in a similar ‘hangman’ porphyrin system, denoted here as TMPXOEt, bearing an axial base and a bulky xanthene substituent, with the objective being to effect steric encumbrance (Figure 4.4, below).

XOEt III .- Chemical reduction of the ferric superoxide, [(TMPIm )Fe (O2 )], with cobaltocene yielded a ferric peroxide with electronic absorption features similar to that of peroxy- myoglobin (peroxy-Mb) generated by cryo-reduction of oxy-Mb crystals.87 The EPR

XOEt III 2- - spectrum of [(TMPIm )Fe (O2 )] is characteristic of end-on ferric peroxo heme intermediates, indicating a low spin ferric species with small dispersion of g-tensor components (g = [2.27, 2.16, 1.96]). Like the electronic absorption data, these EPR data are similar to those obtained for end-on heme ferric peroxides in proteins88 and are clearly distinct from high spin rhombic species which have a signal at g ~ 4.2.74,84,89,90 (See Table

XOEt III 2- - 4.2) Resonance Raman spectra obtained for [(TMPIm )Fe (O2 )] further confirmed its assignment as an ‘end-on’ peroxo species. The observed O—O stretch at 808 cm-1 was

-1 18 -1 16 18 isotope sensitive, shifting to 771 cm (Δ O2 = -37) cm on substitution of O2 with O2, and is similar to the previously characterized O—O stretch observed for

III 2- - -1 18 -1 [(TMPIm)Fe (O2 )] (807 cm (Δ O2 = -49) cm )). An important difference in the

XOEt III 2- - vibrational data obtained for [(TMPIm )Fe (O2 )] is the Fe—O stretch, which was 585

-1 18 -1 cm (Δ O2 = -25) cm for this new low spin peroxo complex. This value is close to the

109 Fe—O stretches observed in peroxide bridged heme-peroxo-copper complexes confirmed to have an end-on binding motif to both heme-iron(III) and copper(II).

Protonation of this heme ferric peroxide by adding an excess of methanol converted the ferric heme peroxide to its hydroperoxide isomer. Similar electronic absorption features

XO2Et III were observed for [(TMPIm )Fe (O2H)], although the EPR spectrum of the hydroperoxide displayed a small increase in the spread of the g values ([2.32, 2.19, 1.95]), and a 10 cm-1 decrease in the Fe—O stretch to 575 cm-1 when compared to the ‘naked’ anionic peroxide. This was proposed to occur as a result of hydrogen bonding interactions between the axial imidazole, distal hydroperoxide O-H, and methanol that disrupts back- donation from the axial imidazole into the hydroperoxide π* orbital. This shift in Fe-O does not correlate with the typical model wherein protonation activates the peroxide for

O—O bond cleavage by strengthening the Fe-O bond and weakening the O—O bond.

XO2Et III Nevertheless, the spectroscopic data for [(TMPIm )Fe (O2H)] are consistent with previously observed models,84 and comparable to hydroperoxide species isolated from cytochrome P450 by cryoreduction.91

110

More recently, Naruta has reported the formation of another heme ferric- hydroperoxide adduct (Figure 4.5) featuring a covalently attached axial imidazole base, and either a carboxylic acid, or ester functionality attached to the porphyrin periphery by an anthracene linker, to provide either a proton source (1a), or steric hindrance (1b) in the porphyrin periphery. In this model study, oxygenation of the high spin ferrous starting material (1a) yielded the corresponding ferric superoxide (2a), with some low spin

III Fe (O2H) impurity. This ferric hydroperoxide impurity was formed from proton coupled electron transfer to the ferric superoxide (2a) from unreacted ferrous heme (1a), and the authors were able to show this by adding one equivalent of (1a) to the superoxide, which yielded the ferric hydroperoxide and the high spin ‘naked’ FeIII complex. Incorporation of an ester linkage into the secondary coordination sphere, as in (1b) enabled the production

111 of a stable superoxide complex (2b), which could be reduced and protonated to the ferric hydroperoxide with exogenous reductant (CoCp2). This work demonstrated that secondary coordination sphere interactions can serve to enhance the reactivity of ferric superoxide towards reduction-protonation to ferric-hydroperoxide, giving mechanistic insights for future design approaches towards dioxygen reduction electrocatalysts.85

Table 4.2. Spectroscopic data for mononuclear ferric heme peroxide and hydroperoxide complexes. System UV- EPR (g) rRaman (cm-1) Binding Ref. Vis Motif (nm) Peroxides XOEt III 2- - 18 -1 83 [(TMPIm )Fe (O2 )] 430 2.27 Fe-O: 585 (Δ O2 = -25) cm End-On 18 -1 568 2.16 O-O: 808 (Δ O2 = -37) cm 610 1.96 III 2- - 18 -1 84 [(TMPIm)Fe (O2 )] 440 4.2 Fe-O: 475 (Δ O2 = -20) cm Side-On 18 -1 574 O-O: 807 (Δ O2 = -49) cm 615 III 2- - 18 -1 84 [(TMP)Fe (O2 )] 439 4.2 Fe-O: 470 (Δ O2 = -19) cm Side-On 18 -1 569 O-O: 809 (Δ O2 = -45) cm 614 III 2- - a 63 [(TPP)Fe (O2 )] 437 8 n.r. Side-On 565 4.2(main) 610 2 III 2- - 63 [(OEP)Fe (O2 )] 423 8 rRaman Side-On 18 -1 543 4.2(main) 780 (Δ O2 = -49) cm a.k.a. 573 2 IR rhombic 18 806 (Δ O2 = -47) Fe(III) III 2- - a 90 [(F8TPP)Fe (O2) ] 435 4.2 n.r. Side-On 540 561 III 2- - 18 -1 b [(F8)Fe (O2 )] 433 4.2 Fe-O: 467 (Δ O2 = -22) cm Side-On TW 18 -1 560 O-O: 806 (Δ O2 = -40) cm

Im III 2- - 18 -1 b [(P )Fe (O2 )] 425 2.25 Fe-O: 577 (Δ O2 = -26) cm End-On TW 18 -1 533 2.14 O-O: 812 (Δ O2 = -35) cm 567 1.95 Hydroperoxides III 18 -1 84 [(TMPIm)Fe (O2H)] 428 2.31 Fe-O: 566 (Δ O2 = -25) cm End-On 18 -1 532 2.19 O-O: 814 (Δ O2 = -46) cm 562 1.95 608 XO2Et III 18 -1 83 [(TMPIm )Fe (O2H)] 429 2.32 Fe-O: 575 (Δ O2 = -25) cm End-On 18 -1 566 2.19 O-O: 807 (Δ O2 = -41) cm

112 608 1.95 AOH III 18 -1 85 [(TMPIm )Fe (O2H)] 429 2.29 Fe-O: 579 (Δ O2 = -28) cm End-On 18 -1 538 2.17 O-O: 807 (Δ O2 = -40) cm 568 1.95 Fe-O-O-H “bend”: 518 cm-1 AOEt III 18 -1 85 [(TMPIm )Fe (O2H)] 430 2.27 Fe-O: 576 (Δ O2 = -27) cm End-on 18 -1 570 2.17 O-O: 811 (Δ O2 = -42) cm 610 1.96 III 18 -1 b [(L)(F8)Fe (O2H)] 416 2.23 Fe-O: 575 (Δ O2 = -23) cm End-On TW 18 -1 537 2.14 O-O: 806 (Δ O2 = -40) cm 558 1.96 Im III b [(P )Fe (O2H)] 424 2.25 Not Observed End-On TW 531 2.14 559 1.95 aN.R. – Not Reported; bThis Work

III Reactivity studies of well-defined model (P)Fe O2H intermediates are very uncommon, and are normally discussed as they relate to the production of high valent oxidants within enzymatic catalytic cycles. Experimental,92–94 and computational studies have suggested that these intermediates may be sluggish oxidants as compared to high valent compound I, and compound II species. Van Eldik and coworkers carried out in-

III depth investigations of axial ligand influences on O—O bond scission of heme-Fe (O2H) complexes in the presence of organic substrates, employing TPFPP as porphyrinate ligand.95 Similar to earlier work done by Valentine et. al. they observed enhanced stability

III of [(TPFPP)Fe (O2H)] compared to its TMP derived analogue due to the electron withdrawing nature of the porphyrin.81,82,96 They were also able to prepare both the five,

III and six coordinate versions of [(TPFPP)Fe (O2H)], the six coordinate species being more stable, and favoring heterolytic cleavage, versus homolytic cleavage for the five coordinate complex, the latter not possessing an axial ligand base. Neither the five, or the six- coordinate species in this case reacted with electron poor olefins and triphenylphosphine, results similar to those published previously by Valentine et al.71,72 The unreactive nature of this hydroperoxide is in contrast to conclusions from Newcomb, Hollenberg, Coon, and coworkers which suggested that Cytochome P450 compound 0 is an alternative

113 electrophilic oxidant in alkane hydroxylation. These differences are due to the large difference in geometric and electronic structure between the model systems of Van Eldik, and the active site of P450.

The subtleties involved in O—O bond cleavage are known to depend on several critical factors including (1) the electronic properties of the supporting porphyrinate ligand,

(2) the electron donating strength of axial ligands, (3) the reaction medium including solvent, pH, and temperature. Despite the central importance of the O—O bond cleavage

III process by Fe O2H intermediates in biological systems including P450 type monooxygenases, catalases, peroxidases, and cytochrome c oxidases, a comprehensive

III 2- III understanding of these factors is still in its infancy. Discrete Fe O2 and Fe -O2H models, such as those discussed above, and herein, may reveal critical factors relating to axial ligand, solvent, and pH effects that facilitate, or inhibit O—O bond cleavage.

4.3. Experimental 4.3.1. General methods

Unless otherwise specified, all reagents and solvents were purchased from commercial sources and were of reagent quality. All air sensitive compounds were handled under argon atmosphere by using standard Schlenk line techniques, or under nitrogen atmosphere, in a VAC Genesis inert atmosphere (< 1 ppm O2, < 1 ppm H2O) glovebox.

Un-inhibited tetrahydrofuran (THF) was purchased from EMD (Omnisolv®), distilled over sodium-benzophenone ketyl under Argon, degassed by bubbling with Argon gas for 1.5 hours, and stored over 4Å molecular sieves for at least 36 hours prior to use. Butyronitrile was obtained from Sigma Aldrich, prior to use it was stirred at 80°C over Na2CO3 and

114 KMnO4 for 3 hours, cooled, and purified by distillation. It was then degassed with five consecutive freeze-pump-thaw cycles, and stored in the glovebox over 4 Å molecular sieves for at least 24 hours. Cobaltocene was obtained from Sigma Aldrich, sublimed at

75°C, and stored under nitrogen in the glovebox freezer at -30 °C. The 2,6-Lutidinium

Triflate [(Lu)(H)](OTf) was synthesized according to previously published literature

97 II 98 Im II 99,100 procedures. Porphyrin complexes (F8)Fe (1), and (P )Fe (3) were synthesized

18 as previously described. O2 (99 atom %) was obtained from ICON istotopes (Dexter, Mi).

4.3.2. UV-Vis experiments

All UV-Vis Measurements were carried out using a Hewlett-Packard 8453 diode array spectrophotometer with a 1 cm path length quartz Schlenk cuvette cell. The spectrophotometer was equipped with a thermostated cell holder for low temperature

III III 2- III experiments. The complexes [Co (Cp)2][(F8)Fe (O2 )] (1), [(F8)Fe (O2H)] (2),

III Im III 2- Im III [Co (Cp)2][(P )Fe (O2 )] (3), and [(P )Fe (O2H)] (4) were generated at 10 μM

Im concentration. For each respective porphyrin (here P is a placeholder for the F8, and P porphyrins) a Schlenk cuvette was first charged with 3mL of a solution of [(P)FeII] in dry

THF in a glovebox under N2 atmosphere, and sealed with a rubber septum, parafilm, and electrical tape. After removal from the glovebox, the solution was inserted into the UV-Vis cryostat chamber, at -80 °C. After cooling for 5 minutes, dioxygen was bubbled through

III .- the cold solution to generate the [(P)Fe (O2 )] complex. Following addition of O2, excess dioxygen was removed from the headspace and solution by vacuum/Ar cycles, and bubbling Argon through the solution for approximately 20 seconds. Following removal of excess O2, ~1.0 equivalents of cobaltocene dissolved in butyronitrile was then added using

115 a gastight syringe, and mixing via bubbling of argon gas, this afforded

III III 2- [Co (Cp)2][(P)Fe (O2 )] After 12 minutes, 1.0 equivalents of [(Lu)(H)](OTf) in THF was

III added using a gas-tight syringe to afford the ferric hydroperoxide species [(P)Fe (O2H)], and Ar bubbling was used to mix the solution.

4.3.3. EPR experiments

Each EPR sample was prepared in a 5mM quartz EPR tube, with a total volume of

0.6 mL, at either 1.0 mM or 2.0 mM concentration. After being charged with the starting

[(P)FeII] solution in the glovebox, and sealed using a rubber tube cap, and parafilm, the samples were removed from the glovebox and placed into a -90 °C bath made with acetone and liquid nitrogen. Dioxygen to form the superoxide complex, and argon for purging and mixing, were added using a 10 mL Hamilton gas-tight three-way syringe, and each of the ferric heme peroxide, and hydroperoxide complexes were prepared as discussed above, with reagents added via a gas-tight Hamilton syringe. Spectra were recorded with an

ER073 magnet equipped with a Bruker ER041 X-Band microwave bridge, and a Bruker

EMX 081 power supply: microwave frequency = 9.41 GHz, microwave power = 0.201 mW, attenuation = 30 db, modulation amplitude 10G, modulation frequency 100 kHz, temperature 10K.

4.3.4. Resonance Raman experiments

Resonance Raman samples were prepared in similar fashion to the EPR samples, however with an NMR tube instead of a quartz tube. Isotopically labeled samples were

18 prepared using O2. Resonance RAMAN samples were collected at a variety of wavelengths, using either a Coherent I90C-K Kr+ ion laser, a Coherent 25/7 Saber Ar+ ion

116 laser, or an Ar+ pumped Coherent Ti:Saph laser while the sample was immersed in a liquid nitrogen cooled (77 K) EPR finger dewar (Wilman). Power was ~2mW at the sample for the high energy lines. Data were recorded using a Spex 1877 CP triple monochromator with either a 600, 1200, or 2400 grooves/mm holographic spectrograph grating, and detected by an Andor Newton CCD cooled to -80°C (for high energy excitation). Spectra were calibrated on the energy axis to toluene. Excitation profiles were intensity calibrated to the solvent (THF) by peak fitting in the program Origin. This work was done by Andrew

W. Schaefer of the Professor E. I. Solomon laboratory at the Department of Chemistry,

Stanford University.

4.3.5. H2O2 quantification by horseradish peroxidase (HRP) test:

Quantification of hydrogen peroxide was carried out by analyzing the intensity of the diammonium 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonate(AzBTS-(NH4)2) peaks (at different wavelengths to minimize error (see Figure S2 in Appendix B), which was oxidized by horseradish peroxidase (HRP), this procedure was adapted from previously published procedures.101 Prior to quantification three stock solutions were prepared including, a 300 mM phosphate buffer, pH 7.0 (solution A), 1 mg/mL AzBTS-

(NH4)2 (solution B), 4mg of HRP (type II salt free (Sigma)) with 6.5 mg of sodium azide in 50 ml of water (solution C). In a typical experiment, 3.0 mL of the desired ferric heme

III 2- - III peroxide, [(P)Fe (O2 )] , or hydroperoxide, [(P)Fe (O2H)] were generated at -80 °C in

THF, as previously described. The reaction was then ‘quenched’ by adding 150 uL of a

- strong acid [(DMF-H+](CF3SO3 ) (2.5 equivalents). Subsequently an aliquot of the quenched solution was removed via a gastight syringe and very quickly added to another cuvette containing 1.3 mL of water 500 uL of solution A, 100 uL of solution B, and 50 uL

117 of solution C (all chilled in an ice bath prior to use). After mixing for at least 10-20 seconds, the samples were allowed to sit at room temperature for ~2 min until full formation of the

418 nm band was observed. (See Appendix Figure S1)

4.4. Results and Discussion

4.4.1. Synthesis and characterization of an iron-peroxide porphyrin complex, III 2- - [(F8)Fe -(O2 )] , and its corresponding hydroperoxide species, III [(L)(F8)Fe (O2H)]

As mentioned above, ferric peroxo porphyrin complexes have been shown to act as strong nucleophiles, being capable of epoxidizing electron deficient olefins, and are also postulated as active oxidants in cytochrome P450 like enzymes such as aromatases.70 Some

III 2- - of the earliest characterized [(P)Fe (O2 )] complexes, as well as several recent examples discussed above are listed in Table 4.2, and their spectroscopic properties have been inspirational for this work. In cytochrome c oxidase (CcO), bridging heme peroxide and hydroperoxide species are highly likely to serve as transient species during catalytic turnover following O2 binding by the reduced form of the enzyme. Hence, an understanding of the basic structural and spectroscopic properties of mononuclear heme peroxide species can serve to better inform our studies reductive O—O bond activation and cleavage in an enzymatic context as well as for our heme-peroxo-copper adducts in that they provide a useful standard for comparison, and benchmarking of spectroscopic properties.

Herein, we expand upon previously published work from our group by Chufán and

Karlin where a mononuclear heme peroxo complex bearing the F8 (F8 = 5,10,15,20- tetrakis(2,6-difluorophenyl)porphyrin) ligand framework was characterized and utilized as a synthetic precursor to a well characterized heme peroxo copper adduct from our group

118 III 2- II + (see section 1.5), [(F8)Fe -(O2 )-Cu (AN)] . Whereas previous work had involved adding a cupric complex as a Lewis acid to activate the peroxide for reductive cleavage, here we protonate the side-on peroxide complex to induce a change in its binding mode from side- on (η2) to end-on (η1), and yield the corresponding ferric hydroperoxide species

III [(L)(F8)Fe -(O2H)] (2) (Figure 4.6).

III III 2- Figure 4.6. Synthetic methods utilized in this work to generate [Co (Cp)2][(F8)Fe -(O2 )] (1), and III [(L)(F8)Fe (O2H)] (2), here L represents either solvent (THF), 2,6-lutidine, or DMF; See text for further discussion.

II Bubbling dioxygen into a 10 μM solution of [(THF)2(F8)Fe ] (λmax 422 (Soret), 542 nm) at -80°C in tetrahydrofuran (THF) immediately resulted in the formation of the six-

III .- coordinate ferric superoxide species, [(THF)(F8)Fe -(O2 )] (λmax 415 (Soret), 535 nm), see

Figure 4.7, below. After formation of this well characterized superoxide species,98 excess

O2 was removed from the headspace by 7 consecutive vacuum purge cycles, and bubbling

Argon gas through the solution. Subsequent addition of 1.3 equivalents of the strong outer

‒ II 102 sphere 1 e reductant [Co Cp2] yielded the previously characterized mononuclear side-

III III 2- on ferric heme-peroxide complex [Co (Cp)2][(F8)Fe (O2 )] (1) (λmax 433 (Soret), 560 nm).

119

III III 2- The EPR spectrum of [Co (Cp)2][(F8)Fe (O2 )](1) displays a signal at g = 4.2,

2 103,104 III 2- - which is typical of η peroxo-iron species in nonheme systems, and [(P)Fe (O2 )] complexes of rhombic symmetry, see Figure 4.8.64,72 Although photodecomposition previously prohibited resonance Raman characterization of (1), we have been able to successfully record two isotope sensitive features comprising the Fe—O, and O—O stretches. The O—O stretch was observed at 806 cm-1, and shifted to 760 cm-1 on

16 18 substitution of O2 with O2 (see Figure 4.9, and Table 4.2). The observed Fe—O stretch

-1 18 -1 is 467 cm (Δ O2 = -20 cm ) which is low relative to those of the typical ferric heme superoxides, and peroxides. However, this matches well with other reported non-heme η2- peroxo iron(III) complexes, further confirming the formulation of (1) as a side-on peroxo- heme species.103–105

120

III III 2- Figure 4.8. X-band EPR spectrum (10 K) taken of (1) [Co (Cp2)][(F8)Fe (O2 )] (blue) generated III .- by adding ~1.3 equivalents of CoCp2 to the six-coordinate ferric superoxide [(THF)(F8)Fe (O2 )] (red) in THF at -80°C. See text for discussion.

III 2- - Protonation of (1) [(F8)Fe (O2 )] with ~1.0 equivalents of [(Lu)(H)](OTf) or

[(H)(DMF)](OTf) resulted in immediate conversion to a ferric heme hydroperoxide

III species, [(F8)Fe (O2H)] (2) (λmax 416 (Soret), 537, 556 nm), see the green spectrum Figure

4.10, below. The g value at 4.21 also disappears, and new signals corresponding to a low spin heme ferric peroxide species appear at g = 2.23, 2.19, and 1.96 (see Figure 4.11, below), and these features are consistent with many other published examples of

III [(P)Fe (O2H)] species (see Table 4.2), as well as a heme ferric hydroperoxide observed in hemoglobin.82,106 Note that there are also features corresponding to some leftover side-on peroxide at g = 4.21, as well as high spin ferric iron impurities most likely from degradation

III + to [(F8)Fe (THF)2] and H2O2.

121

III III 2- Figure 4.9. Resonance Raman spectra for (1) [Co (Cp2)][(F8)Fe (O2 )] (red); (2) III III [(L)(F8)Fe (O2H)] (green); and [(DCHIm)(F8)Fe (O2H)] (blue). See text for discussion.

Vibrational spectroscopy (rRaman) also showed a clear change in the Fe-O

-1 III III 2- vibrational mode on protonation, shifting from 467 cm in [Co (Cp2)][(F8)Fe (O2 )] (1)

-1 III to 575 cm in [(L)(F8)Fe (O2H)] (2) see Figure 4.9, green spectrum. However, the ν(O—

-1 18 O) stretch in this case did not measurably change, remaining the same at 806 cm (Δ O2

= -40 cm-1). These vibrational modes are similar to those obtained in other end-on bound

III [(P)Fe (O2H)] model systems (see Table 4.2), and by cryoreduction of the oxyferrous

107 complex of the D251N mutant of cytochrome P450cam. With these spectroscopic parameters in mind, we can envision (2) as a 6-coordinate low-spin η1 heme ferric hydroperoxide, as depicted in Figure 4.6, where the axial ligand could be 2,6-lutidine, dimethylformamide (DMF), or a THF solvent molecule.

122

III III 2- Figure 4.10. UV-Vis spectra depicting [Co Cp2][(F8)Fe (O2 )] (1) (blue), and III [(L)(F8)Fe (O2H)] (2) (green) in THF at -80°C (193 K). See text for further discussion.

The results here are distinctive from similar chemistry shown by Naruta and coworkers wherein protonation of a similar side-on heme ferric peroxide bearing a TMP

III 2- - supporting ligand, [(TMP)Fe (O2 )] did not yield a heme ferric hydroperoxide, but instead leads to decomposition of the peroxide. Therein, a covalently attached imidazole axial base was critical for protonation induced isomerization yielding a stable 6-coordinate heme ferric hydroperoxide species.84 In our system discussed above, we have been able to

III characterize a new low spin six-coordinate heme ferric hydroperoxide, [(L)(F8)Fe (O2H)]

(2), which is clearly distinguishable from its side-on mononuclear heme ferric peroxide

III III 2- precursor, [Co (Cp)2][(F8)Fe (O2 )] (1). It is possible that such a hydroperoxide intermediate was not observed earlier by Naruta et. al. with the TMP ligand framework because the authors used methanol as a proton source, and in the absence of an axial base ligand ‘push’ effect, a stronger acid is needed to protonate the stable side-on peroxide. Our

123 use of 2,6-Lutidinium Triflate, and [(DMF)(H)][OTf] provided a stronger acid, and potentially an axial base for heme in the form of 2,6-Lutidine, or DMF, which should help

III III 2- to activate the stable side-on peroxide [Co (Cp)2][(F8)Fe (O2 )] (1), forming its

III corresponding hydroperoxide [(L)(F8)Fe (O2H)] (2).

4.4.2. Synthesis and characterization of an end-on heme ferric peroxide III Im III 2- complex, [Co Cp2][(P )Fe (O2 )] (3), and its hydroperoxide analogue, Im III [(L)(P )Fe (O2H)] (4)

In order to evaluate the role of a covalently attached axial base on the formation, and structural properties of mononuclear heme ferric peroxides, the synthetic methodology discussed above was expanded to another ferrous porphyrinate, [(PIm)FeII]. We have previously characterized the O2, CO, NO, and 2,6-dimethylphenylisocyanide binding chemistry of [(PIm)FeII] (vide supra), and it is known to form stable six-coordinate species

Im III .- with small molecule diatoms. Generating the ferric superoxide, [(P )Fe (O2 )] (λmax 423

124 Im II (Soret), 535 nm) (red) from the starting ferrous heme porphyrinate [(P )Fe ] (λmax 418

99,108 (Soret), 525, 552(sh) nm) (black) by bubbling O2 at -80°C in THF (see Figure 4.13) provides an entry point into the synthetic strategy outlined in Figure 4.12, below. Following superoxide formation, excess O2, is then removed by successive vacuum/purge cycles, and bubbling the solution with an inert gas (argon).

III Im III 2- Figure 4.12. Synthetic methods utilized in this work to generate [Co (Cp)2][(P )Fe -(O2 )] (3), Im III and [(P )Fe (O2H)] (4), see text for discussion.

Im III .- After formation of the well characterized superoxide species [(P )Fe (O2 )], addition of ~1.3 equivalents of cobaltocene results in the formation of a new end-on (η1)

III Im III 2- heme ferric peroxide complex, [Co (Cp)2][(P )Fe (O2 )] (3) (λmax 425 (Soret), 533, 567 nm), see Figure 4.13. Note that this end-on peroxide has a Soret band maximum at 425 nm, which is substantially higher in energy than those of typical side-on heme ferric peroxides

III Im III 2- (see Table 4.2). The EPR spectrum of [Co (Cp)2][(P )Fe (O2 )] (3) (Figure 4.14, below) displays three distinctive signals g = 2.25, 2.14, 1.95. These low spin ferric heme signals

125 with small g dispersion are similar to other end-on low-spin ferric peroxo intermediates observed by cytoreduction methods in heme enzymes, such as myoglobin.88

Im II Im III .- Figure 4.13. UV-Vis spectra depicting [(P )Fe (THF)] (black); [(P )Fe (O2 )] (red) obtained Im III 2- III by bubbling O2 through the reduced solution (black); [(P )Fe (O2 )][Co Cp2] (3) (blue) obtained II by adding ~1.3 equivalents of [Co (Cp)2] to the ferric superoxide in THF at -80°C (193 K). See text for further discussion.

III Im III 2- Vibrational spectroscopic interrogation of [Co (Cp)2][(P )Fe (O2 )](3) using rRaman spectroscopy revealed two isotope sensitive vibrational modes assigned as Fe—

18 -1 18 -1 O, and O—O stretches at 577 (Δ O2 = -26) cm , and 812 (Δ O2 = -35) cm (see Figure

4.15, below), respectively. This reported O—O stretch is similar to the only other reported

XOEt III 2- - end-on heme ferric peroxide species in model systems, [(TMPIm )Fe (O2 )] , which as discussed above, was characterized by Naruta et. al. It is important to note that although the observed Fe—O stretch of 577 cm-1 is very similar to those of other, protonated heme hydroperoxides (See Table 4.2), the value of the Fe-O stretch varies greatly depending on not only the local solvent (and protein environment). In fact, ferric heme peroxides have

126 107 109 been characterized in both Cytochrome P450cam mutants, and lactoperoxidase with

18 -1 18 -1 ν(Fe-O) stretches of 553 (Δ O2 = -27) cm , and 570 (Δ O2 = -22) cm , respectively.

III Im III 2- The intermediate [Co (Cp)2][(P )Fe (O2 )] (3)could also reasonably be assigned as a mononuclear ferric heme hydroperoxo species, given the similarity of it’s Fe-O

III vibrational mode, and it’s EPR spectrum to that of [(L)(F8)Fe (O2H)] (2), discussed above in section 4.3.1. This could be the case given that such an anionic end-on ferric heme hydroperoxide would be highly basic, capable of deprotonating even trace amounts of water in the solvent. However, when ~1.0 equivalent of a proton source (in this case

III Im III 2- [Lu(H)](OTf))) is added to a solution of [Co (Cp)2][(P )Fe (O2 )] (3), a distinctive change is observed in the UV-Vis spectrum (Figure 4.16, below), wherein the Soret band increases in intensity and shifts to 424 nm, and there are also changes in the Q-band region, see Figure 4.16, below. The EPR spectrum of this purported new hydroperoxide species,

Im III [(P )Fe (O2H)] (4), displays very similar characteristics to the starting complex

127 III Im III 2- [Co (Cp)2][(P )Fe (O2 )], see Figure 4.17, below. If the starting compound

III Im III 2- [Co (Cp)2][(P )Fe (O2 )]were indeed already a hydroperoxide, subsequent addition of a proton should trigger heterolytic O—O bond cleavage, yielding a Cmpd I analogue, which would rapidly oxidize the solvent forming an FeIV=O species (Cmpd II), which is

EPR silent. Given that we do observe low spin heme ferric signal similar to many known heme ferric hydroperoxide species, we thus identify species (4) as a new heme ferric

Im III hydroperoxide, [(P )Fe (O2H)].

III Im III 2- Figure 4.15. Resonance Raman spectra for [Co (Cp2)][(P )Fe (O2 )] (3) (blue). See text for discussion.

128

Im III Figure 4.17. X-band EPR spectrum (10 K) taken at of [(P )Fe (O2H)] (4) (green) generated by adding ~1.0 equivalents of [(Lu)(H)](OTf) to (3) in THF. See text for discussion.

4.4.3. Heme axial base effects on heme ferric peroxide structure, and spectroscopic properties

An important distinction between the mononuclear heme ferric peroxide complexes

III III 2- III Im III 2- [Co (Cp)2][(F8)Fe (O2 )] (1), and [Co (Cp)2][(P )Fe (O2 )] (3) discussed above is

129 illustrated by the difference in their Fe—O vibrational frequencies. Reduction of the six-

III .- coordinate heme ferric superoxide species [(THF)(F8)Fe O2 ] with cobaltocene yields a side-on bound mononuclear heme ferric peroxide species displaying an Fe-O stretching

-1 18 -1 vibration at 467 cm (Δ O2 = -22 cm ). Whereas, reduction of the six-coordinate ferric

Im III .- heme superoxide [(P )Fe (O2 )], which features a covalently attached axial imidazole ligand, results in a purported mononuclear ferric heme peroxide with an Fe—O vibration

-1 -1 18 -1 that is ~110 cm higher in energy, at 577 cm (Δ O2 = -26 cm ), which is characteristic of an end-on bound iron-oxy species. Both of these mononuclear heme ferric peroxides display O—O bond stretching frequencies that are typical of metal-peroxy species, at around ~800 cm-1. Clearly in this case the incorporation of a relatively strongly coordinating axial base ligand (imidazole) into the porphyrinate framework favors O2 activation via an end-on heme ferric peroxide species, instead of a side-on bound heme ferric peroxide. This discrepancy is important given that such side-on ferric peroxy hemes are generally not activated for O—O scission.103,110 The increased Fe—O stretching

III Im III 2- frequency observed for [Co (Cp)2][(P )Fe (O2 )] (3) is likely a result of axial ligand donation by the covalently attached imidazole. This may occur as a result of a trans effect wherein the axial imidazole ligand increases the energy of the iron d-orbitals causing greater back donation into the peroxo σ* orbital, and greater back donation from the

* peroxide π v orbital into the iron d orbitals. These effects should strengthen the Fe-O bond,

III Im III 2- and favor the end-on binding mode for the heme ferric peroxide [Co (Cp)2][(P )Fe (O2

III III 2- )] (3). No such strongly donating axial ligand is present in [Co (Cp)2][(F8)Fe (O2 )] (1), favoring the side-on η2 binding mode. Interestingly, addition of an axial base, 1,5-

130 III III 2- dicyclohexylimidazole (1,5-DCHIm), to [Co (Cp)2][(F8)Fe (O2 )] (1) did not result in a change in binding mode from side-on (η2) to end-on (η1). However, after protonation to

III make the heme ferric hydroperoxide, [(L)(F8)Fe (O2H)] the imidazole does appear to bind,

-1 18 -1 -1 as indicated by a shift in the Fe—O stretch from 575 cm (Δ O2 = -23 cm ) to 592 cm

18 -1 (Δ O2 = -25 cm ), see Figure 4.9, (blue). This may indicate that the presence of a strong axial ligand donor prior to superoxide reduction to peroxide is necessary to avoid producing side-on bound heme ferric peroxy species, which are not as easily activated for

O—O bond cleavage. Alternatively, protonation with acid, or the presence of a Lewis acid110 can provide the needed ‘pull’ effect, stabilizing the end-on hydroperoxide. Thus, it can be postulated that in these model systems the ‘push’ effect of the axial ligand, and/or the ‘pull’ effect of a proton, or Lewis acid critically shapes the nature of the metal bound peroxide species observed during the stepwise reductive activation of O2 with synthetic model systems, and may be needed to avoid relatively stable side-on peroxide species such as (1).

Figure 4.18. Summary of spectroscopic parameters determined thus far for mononuclear heme ferric peroxides (1) & (3), and hydroperoxides (2) & (4).

131 4.4.4. Generation and characterization of a low-spin heme-peroxo-copper complex featuring a covalently attached axial imidazole ligand, and its reactivity towards an exogenous organometallic reductant (Me10Fc)

Recent work from our group related to small molecule heme-peroxo-copper models for peroxide, and hydroperoxide intermediates formed in the course of the four proton, four electron reduction of dioxygen to water by cytochrome c oxidase has focused on monodentate imidazole ligation of copper as a means of imparting flexibility into the copper coordination environment.111–113 Here we have taken a different approach to the design of heme-peroxo-copper complexes, by covalently attaching an axial imidazole ligand to the heme periphery, to evaluate the effect that such a covalently attached axial base may have on heme-peroxo-copper adduct structure, physical properties and reactivity.

Im III 2- II + Figure 4.19. Synthetic methodology for the generation of [(P )Fe -(O2 )-Cu (AN)] (5) at -80°C in THF, see text for discussion.

Im III 2- II + The new heme-peroxo-copper adduct [(P )Fe -(O2 )-Cu (AN)] (5) (Figure

4.19) was prepared similarly to other previously published heme peroxo copper adducts

(see Chapter 1), by bubbling dioxygen through an equimolar mixture of the ferrous heme, in this case [(PIm)FeII], and the cuprous chelate of interest, [CuI(AN)](BArF). The UV-Vis spectra (Figure 4.20, below) displayed a clear change on bubbling dioxygen through the

132 II I starting equimolar Fe /Cu mixture (λmax 417 (Soret), 526, 553 nm), yielding the heme-

Im III 2- II + peroxo-copper adduct (5) [(P )Fe -(O2 )-Cu (AN)] (5) (λmax 423 (Soret), 533, 789, 949 nm). The presence of the strong low energy bands observed at 789, and 949 nm are consistent with other previously characterized low spin end-on peroxide bridged heme- peroxo-copper adducts,110,111 and correspond to peroxo π*-to-iron d ligand to metal charge transfer (LMCT) bands. Low temperature 2H-NMR spectroscopy, and EPR spectroscopy helped to confirm that this heme-peroxo-copper adduct has an overall S=0 spin state, mediated by antiferromagnetic coupling of the unpaired electrons on the low spin d5 iron center, and the d9 copper center through the peroxide bridge. Specifically the silent X-band

Im III 2- II + EPR spectrum of [(P )Fe -(O2 )-Cu (AN)] (5) confirms that there are no unpaired electrons, and the pyrrole chemical shift at 7.98 ppm in THF at -85 °C indicates a diamagnetic S = 0 species. Intermediates such as these are of interest to biophysicists, and biochemists in particular because they provide a model for how cytochrome oxidases may overcome the kinetic barriers associated with reductive O—O bond activation using metal cofactors including heme a3, and CuB of the binuclear center (see Chapter 1).

133

Im III 2- II + The complex [(P )Fe -(O2 )-Cu (AN)] (5) has also been characterized using

Im III 2- vibrational spectroscopy, i.e. rRaman. Excitation of a 2 mM frozen solution of [(P )Fe -(O2 )-

CuII(AN)]+ (5) at 785 nm (recall that the LMCT bands at 789 and 949 are peroxo-to-iron charge transfer bands) resulted in the formation of isotope sensitive vibrational bands, corresponding to the Fe—O, and O—O vibrational modes. The Fe—O stretch was

18 -1 observed as 577 (Δ O2= -27) cm which is consistent with an end-on iron-oxy species

18 -1 (see above), and the observed O—O stretch at 796 (Δ O2 = 43) cm is similar to that of other μ-η1:η1 peroxo bridged heme-peroxo-copper adducts, see Figure 4.21, below.

134

Figure 4.21. Resonance Raman spectrum depicting the ν(Fe—O), and ν(O—O) vibrational Im III 2- II + modes detected for [(P )Fe -(O2 )-Cu (AN)] (5) (blue) see text for discussion.

In order to better understand the reactivity properties of heme-peroxo-copper adducts such as (5) towards exogenous reductants, a 10-fold excess of decamethylferrocene

(Fc*) was added to (5) at -80 °C in THF solvent (Figure 4.22). In this case, Fc* was chosen

0/+ specifically because of its highly reducing [Fe(Cp*)2] nature (E°’ = -0.59V (vs

0/+ [Fe(Cp)2] in acetonitrile), it’s high solubility in THF, and the presence of clear d-d

– III + transitions at 784, and 803 nm in its 1 e oxidized form [Fe (Cp*)2] which enables us to precisely quantify the amount of reductant consumed in the course of a reaction. (see Figure

S4, Appendix B)

135

Figure 4.22. Proposed 1e‒ reduction chemistry occurring on addition of excess reductant Im III 2- II + (Me10Fc) to [(P )Fe -(O2 )-Cu (AN)] (5) in THF at -90°C.

Immediately following addition of excess decamethylferrocene, a change in the

UV-Vis spectra was observed where new heme features at λmax = 424 and 531 nm, and d- d bands corresponding to Fc* at 784 and 804 nm rapidly appeared (Fig. 4.23), the intensity observed corresponding to 1.0 equivalents of decamethylferrocenium consumed.

Confirmation of this conclusion comes from noting that addition of a second equivalent of

Fc* resulted in a doubling of intensity of the features at 784, 804, thus confirming the 1 e– stoichiometry in this reaction.

136 Subsequent vibrational spectroscopic characterization of the product mixture using rRaman spectroscopy confirmed that a reaction had occurred on addition of the reductant.

-1 18 -1 One isotope sensitive vibrational mode was detected at 578 cm (Δ O2= -25 cm ) corresponding to a heme Fe—O stretch, however, a band assignable to an O—O stretching mode was not observed, see Figure 4.24, below. Additionally, the EPR spectrum of the product mixture is silent indicating that the unpaired electron on CuII is either still antiferromagnetically coupled, or has been reduced to CuI which is EPR silent, although no clear low spin ferric heme peroxide signals have been observed. Given the similarity of the UV-Vis features, and the Fe—O stretch detected in the product mixture, to the corresponding values for the mononuclear ferric heme peroxide complex

III Im III 2- - [Co (Cp)2][(P )Fe (O2 )] (3) discussed above, it can be postulated that a 1e reduction

Im III 2- II + Im III 2- - of [(P )Fe (O2 )-Cu (AN)] (5) yields an equimolar mixture of [(P )Fe (O2 )] , and

[CuI(AN)(L)]+ (6), the latter which may ion-pair with the mononuclear heme ferric peroxide complex, see Figure 4.25, below. It is possible that the covalently attached axial imidazole base of the PIm porphyrinate ligand which is known to favor end-on binding for

III Im III 2- - [Co (Cp)2][(P )Fe (O2 )] (3) favors this 1e reduction reaction by donating electron density to the peroxide stabilizing the mononuclear end-on heme ferric peroxide.

Ultimately further characterization of the products in this chemistry is needed to definitively determine what reaction has occurred, and this system is still currently under investigation.

137

138 4.5. Conclusions

We have prepared a small library of mononuclear ferric heme peroxides as synthetic models for Cmpd 0 type intermediates formed in the course of enzymatic turnover by cytochrome P450 monooxygenases, peroxidases, catalases, and aromatase. The stepwise conversion of the side-on mononuclear ferric heme peroxide

III III 2- III [Co (Cp)2][(F8)Fe (O2 )] (1), to its hydroperoxide analogue [(L)(F8)Fe (O2H)] (2) is compelling in that it shows the flexibility of mononuclear ferric heme iron-oxy species for reductive O-O bond activation. Recall that conversion of the side on-peroxide (1), to its end-on hydroperoxide (2) also results in a ~110 cm-1 increase in the Fe—O stretch. In enzymatic systems, reduction of the bound ferric superoxide yields an end-on ferric peroxide species which is subsequently protonated, its end-on binding geometry is likely favored by the proximal axial ligand bound to the heme cofactor. These model systems and those characterized by Naruta, present a different pathway to Cmpd 0 type species in model systems, wherein the presence of an axial base ligand is deterministic of the binding mode for the mononuclear ferric heme peroxide species. Indeed, in our synthetic model featuring

III Im III 2- a covalently attached axial imidazole ligand [Co (Cp)2][(P )Fe (O2 )] (3) produces an end-on mononuclear heme ferric peroxide complex on reduction by 1e- with the strong outer sphere reductant cobaltocene, and represents only the second reported example of such a small molecule complex in aprotic homogeneous media. We have also characterized

Im III 2- II + a new heme-peroxo-copper adduct [(P )Fe -(O2 )-Cu (AN)] (5), which features a μ-

η1:η1 bridging peroxide moiety, and its reactivity towards excess exogenous reductant.

Im III 2- II + - I + Thus, [(P )Fe -(O2 )-Cu (AN)] (5) undergoes a 1e reduction, to yield [Cu (AN)] , and

139 Im III 2- - [(P )Fe -(O2 )] based on the observed UV-Vis, EPR, and rRaman spectra of the product mixture, and their similarity to an authentically generated mononuclear end-on heme ferric

III Im III 2- peroxide, [Co (Cp)2][(P )Fe (O2 )]. However, further characterization of the products in this system is needed.

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APPENDIX B

SUPPORTING INFORMATION FOR CHAPTER 4

151

Figure S1. UV-Vis spectrum depicting the oxidation of AzBTS-(NH4)2 with H2O2. Calibration performed at room temperature. See experimental section for details.

Figure S2. Calibration curves for the UV-Vis features formed at 416, and 734 nm in the HRP test. See experimental section for details.

152

Figure S3. UV-Vis spectra depicting decamethylferrocenium BArF, features at 783, and 803 nm are diagnostic of concentration ranging from 0.2 mM to 1.0 mM. Spectra were taken at -80C in THF.

Figure S4. Calibration Curve depicting decamethylferrocenium BArF, features at 785, and 803 nm are diagnostic of concentration ranging from 0.2 mM to 1.0 mM. Spectra were taken at -80C in THF.

153 PATRICK J. ROGLER [email protected] (914)-391-5495 716 Washington Place, Baltimore, MD 21201, Apt. 1007

EDUCATION The Johns Hopkins University October 2018 PhD Candidate in Chemistry Master of Arts in Chemistry (M.A.) August 2014 Advisor: Professor Kenneth D. Karlin

Rutgers, The State University of New Jersey May 2012 Bachelor of Arts in Chemistry (B.A., ACS Certified) Advisor: Professor John G. Brennan

RESEARCH SKILLS

Leadership and Project Management  Excel in scientific writing and assisted with NIH Grant renewal preparation and writing. o (Project #: 2R01GM060353-18) awarded July 25th, 2018, renewing yearly until 2021.  Strong communication and writing skills. – English o Co-authored a major Chemical Reviews article (see below) on my thesis topic.  Excel at oral presentation of scientific results to peers and collaborators.  Trained junior graduate students in standard schlenk line, synthetic, and instrumental techniques.  Facilitated the purchase, setup, and troubleshooting of major laboratory equipment and repairs. o VAC GENESIS Inert Atmosphere Glovebox, ESI-MS troubleshooting and repair.  Superb interpersonal skills.  Rutgers Chemistry Society - Treasurer (Spring 2011-Spring 2012).

Synthesis and Mechanistic Analysis:  Development, troubleshooting, and optimization of methodologies for organic ligand syntheses, and organometallic coordination complexes.  Highly skilled in the synthesis, and characterization of air-sensitive inorganic compounds, and manipulations under inert atmospheres. (Schlenk-line and dry-box techniques)  Mechanistic analysis of organometallic and inorganic reaction mechanisms.

Experimental Physical Methods:  Electronic Absorption Spectroscopy (UV-Vis, UV-Vis-NIR), Electron Paramagnetic Resonance Spectroscopy (EPR), Fourier Transform Infrared Spectroscopy (ATR FT-IR), Multinuclear Magnetic Resonance Spectroscopy (NMR), Electrospray Ionization Mass Spectrometry (ESI-MS), Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS), Gas Chromatography-Mass Spectrometry (GC-MS).  Thin Layer Chromatography (TLC), Flash Column Chromatography, Air-Free Synthesis, Schlenk Line Technique, Inert Atmosphere Glovebox Maintenance and Usage.

HONORS, AWARDS, AND PROFESSIONAL ORGANIZATIONS  Johns Hopkins University Owen Scholars Award (Fall 2012). o Awarded to an outstanding entering graduate student for the first year of graduate studies.  Spring 2012 Chemical Resources Award for Service – Treasurer (Rutgers Chemistry Society)  Spring 2011 Chemical Resources Award for Distinction in Undergraduate Research  Rutgers University Aresty Undergraduate Research Fellowship Award

154 o $800 Awarded for Undergraduate Research to purchase reagents, and project materials.

RESEARCH EXPEREINCE & INTERESTS

Prof. Kenneth D. Karlin Laboratory, PhD Candidate, Johns Hopkins University Fall 2012-Present  Developed biomimetic small molecule model systems for peroxidic intermediates formed during dioxygen activation and O-O bond cleavage by Cytochrome c Oxidases, proteins which are critical to the bioenergetics of aerobic life.

Prof. John G. Brennan Laboratory, Rutgers University Spring 2010-Spring 2012  Synthesized novel Tin, and Lead complexes with fluorinated selenolate ligands as potential Chemical Vapor Deposition (CVD) precursors to semiconductor films and organically soluble nanocrystals.

SURP Albert Einstein College of Medicine of Yeshiva University, Das Laboratory June 2010-August 2010  Synthesized novel boron-containing 3,5-disubstituted-1,2,4-oxadiazoles as retinoic acid, and combretastatin A4 analogues for screening as transcriptional modulators in zebrafish embryos.

ABSTRACTS AND PRESENTATIONS

1. Rogler, P.J.; Sharma, S.K.; Schaefer, A.W.; Kim, H.; Solomon, E.I.; Karlin, K.D. “Heme-Copper and Heme Peroxide Complexes as Bio-mimics for Reductive O‒O Bond Cleavage in CcO, and Compound 0 of Cyt- P450”. 2018 Frontiers in Metallobiochemistry Symposium. The Pennsylvania State University. State College, PA. June 6-7, 2018. Poster Presentation.

2. Rogler, P.J.; Sharma, S.K.; Adam, S.; Karlin, K.D. “Axial Base Effects on Heme-Peroxo-Copper Adduct Reactivity: Evaluating the Role of Axial Base Tether and Type”. 254th ACS National Meeting and Exposition. “Many Colors of Copper” Symposium. Washington, DC. August 23rd, 2017. Oral Presentation.

3. Rogler, P.J.; Sharma, S.K.; Schaefer, A.W.; Adam, S.; Garcia-Bosch, I.; Solomon, E.I.; Karlin, K.D. “Varying Reactivity of Low-Spin Heme-Peroxo-Copper Species Towards a Reductant (Me10Fc) Plus a Phenolic Acid: Covalently Attached Versus Exogenous Heme Axial Bases”. 2016 Mid-Atlantic Seaboard Inorganic Symposium, Philadelphia, PA, July 20, 2016. Poster Presentation.

PUBLICATIONS

1. Adam, S.M.; Wijeratne, G.B.; Rogler, P.J.; Diaz-Romero, D.; Quist, D.; Liu, J.; Karlin, K.D. “Synthetic Fe/Cu Complexes Toward Understanding Heme-Copper Oxidase Structure and Function”. Chemical Reviews (2018). Accepted, in revision.

2. Sharma,S.K.; Kim, H.; Rogler, P.J.; Karlin, K.D. “Isocyanide or nitrosyl complexation to hemes with varying tethered axial base ligand donors: synthesis and characterization”. Journal of Biological Inorganic Chemistry. 21, 729 (2016).

3. Sharma, S.K.; Rogler, P.J.; Karlin, K.D. “Reactions of a heme-superoxo complex toward a cuprous chelate and NO(g): CcO and NOD chemistry”. Journal of Porphyrins and Pthalocyanines 19, 352-360 (2015).

4. Holligan, K.; Rogler, P.; Rehe, D.; Pamula, M.; Kornienko, A.Y.; Emge, T.J.; Krogh-Jespersen, K.; Brennan. J.G. “Copper, Indium, Tin, and Lead Complexes with Fluorinated Selenolate Ligands: Precursors to MSex”. Inorganic Chemistry. 54, 8896 (2015).

155 5. Tang, X.; Sanyal, S.; Mohapatra, S.; Rogler, P.; Nayak, S.; Das, B.C.; Evans, T. “Design and synthesis of 3,5-disubstituted-1,2,4-oxadiazole containing retinoids from a retinoic acid receptors agonist”. Tetrahedron Letters 52(19): 2433-2435 (2011).

6. Tang, X.; Rogler, P.; Das, B.C.; Evans, T. “Design and synthesis of 3,5-disubstituted boron-containing 1,2,4-oxaadiazoles as potential combretastatin A-4 (CA-4) analogues”. Tetrahedron Letters 53: 3947-3950.

TEACHING EXPERIENCE Teaching Johns Hopkins University:  Introductory Chemistry Lab & Lecture – Graduate Student Teaching Assistant Fall 2012-Spring 2013  Introductory Organic Chemistry Lab – Head Graduate Student Teaching Assistant Fall 2013  Chemical Structure and Bonding with Laboratory – Graduate Student Teaching Assistant Spring 2014

REFERENCES

Kenneth D. Karlin Ira Remsen Professor of Chemistry [email protected] 1-410-516-8027

David P. Goldberg Professor of Chemistry [email protected] 1-410-516-6658

John P. Toscano Professor of Chemistry and Vice Dean for Natural Sciences [email protected] 1-410-516-6534

Sunita Thyagarajan Senior Lecturer [email protected] 1-410-516-7864

156