Molybdenum Oxotransferase Active Site ... - Brown University

Molybdenum Oxotransferase Active Site Models and Their

Oxygen Atom Transfer Reactivity

By Lee Taylor Elrod

B.S. University of Vermont 2010

A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy in the Department of Chemistry at Brown University

Providence, Rhode Island, May 2018

iii

This dissertation by Lee Taylor Elrod is accepted in its present form by the Department

of Chemistry as satisfying the dissertation requirement for the degree of Doctor of

Philosophy

Recommended to the Graduate Council

Date____________ _____________________________

Eunsuk Kim, Ph.D. Advisor

Date____________ _____________________________

Jerome Robinson, Ph.D. Reader

Date____________ _____________________________

Paul Williard, Ph.D. Reader

Approved by the Graduate Council

Date____________ _____________________________

Andrew G. Campbell, Ph.D.

Dean of the Graduate School

iv

Curriculum Vitae

Lee Taylor Elrod

Education:

PhD Inorganic Chemistry with Dr. Eunsuk Kim Brown University, Providence, RI

2018

B.S. Degree in Chemistry University of Vermont, Burlington, VT

2010

High School Diploma Libertyville High School, Libertyville, IL

2006

Academic Accomplishments:

• Dissertation Fellowship, Brown University • Recipient Charles E. Braun Award, University of Vermont • Recipient Clinton D. Cook Award in Chemistry, University of Vermont • Recipient Presidential Scholarship (2006-2010), University of Vermont • Recipient Academic Excellence Scholarship (2006-2010), University of Vermont

Presentations:

Elrod, L. T.; Kim, E. “Oxygen Atom Transfer Mediated By Molybdenum Oxo Complexes and Lewis Acid”. Poster presentation at 252nd ACS National Meeting & Exposition, Philadelphia, PA, August 2016

Publications:

• Elrod, L. T.; Robinson, J. R.; Victor, E.; Kim, E. “Lewis Acid Enhanced Nitrate and Perchlorate reduction by Mo(µ-O) Dimer” Manuscript in preparation.

• Elrod, L. T.; Kim, E. “Lewis Acid Assisted Nitrate Reduction with Biomimetic Molybdenum Oxotransferase Complex” Inorg. Chem. 2018, 57, 2594 -2602.

• Cao, R.; Elrod, L. T.; Lehane, R. L.; Kim, E.; Karlin, K.D. “A Peroxynitrite Dicopper Complex: Formation via Cu–NO and Cu–O2 Intermediates and Reactivity via O–O Cleavage Chemistry” J. Am. Chem. Soc., 2016, 138, 16148-16158.

v

• Roering, A. J.; Elrod, L. T.; Pagano, J. K.; Guillot, S. L.; Chan, S. M.; Tanski, J. M.; Waterman, R. “A General Synthesis of Phosphaalkenes at Zirconium with Liberation of Phosphaformamides” Dalton Trans. 2013, 42, 1159-1167.

• Elrod, L. T.; Boxwala, H.; Haq, H.; Zhao, A. W.; Waterman, R. “As-As Bond Formation Via Reductive Elimination from a Zirconocene Bis(dimesitylarsenide) Compound” Organometallics, 2012, 31, 5204-5207.

• Roering, A. J.; Maddox, A. F.; Elrod, L. T.; Chan, S. M.; Ghebreab, M. B.; Donovan, K. L.; Davidson, J. J.; Hughes, R. P.; Shalumova, T.; MacMillan, S. N.; Tanski, J. M.; Waterman, R. "General Preparation of (N3N)ZrX (N3N = N(CH2CH2NSiMe3)3

3–) Complexes from a Hydride Surrogate" Organometallics, 2009, 28, 573-581.

Teaching Experience:

2012 to Present Brown University, Department of Chemistry Providence, RI

Fall 2015:

• Head teaching assistant advanced undergraduate inorganic lab, 1 section

Spring 2014:

• Head teaching assistant undergraduate bioinorganic lab, 2 sections

Fall 2013:

• Head teaching assistant advanced undergraduate inorganic lab, 1 section per

Spring 2013:

• Teaching assistant undergraduate bioinorganic lab, 2 sections

Fall 2012:

• Teaching assistant undergraduate general chemistry laboratory, 1 section

2011-2012 University of Vermont Department of Chemistry Burlington, VT

• Teaching assistant undergraduate general chemistry laboratory, 5 sections per semester

• Teaching assistant undergraduate inorganic chemistry, one section Spring 2012

• Oversaw and assisted undergraduate and high school researchers in lab affiliated with NOYCE scholarship/ACS Project SEED

2010 Indiana University Bloomington, IN

• Graduate teaching assistant for organic chemistry laboratory

2009 University of Vermont Department of Chemistry Burlington, VT

• Teaching assistant for general chemistry laboratory, 2 sections

vi

Acknowledgments

I would like to thank the following people for their help and contributions to this

work. First, I would like to thank Dr. Eunsuk Kim. Over the past five and half years

she has help me continue to develop as a scientist. Her continued support and insight

into my project is why I can present my work here. Her enthusiasm for bioinorganic

chemistry and research has had a profound impact on how I approach research. Her

encouragement has helped me overcome the day to day obstacles of research, and I am

thankful for her support, guidance, and patience.

I would also like to thank Dr. Jerome Robinson for his continued help with

experimental techniques, discussions about science, and serving on my thesis

committee. The X-ray crystallography and structure determination of the Mo2VO3

complex presented here was solved by Dr. Robinson and was critical to the preparation

of this thesis. Thank you to Dr. Paul Williard and Dr. Wesley Bernskoetter for serving

as my committee members. Their insights and comments have fostered helpful

discussions about my research. I would also like to thank Dr. Eric Victor for

computational work performed on the Mo2VO3 complex.

Thank you to all the current and former Kim Group members.

Last, and certainly not least. I would like to thank my parents Lee and Jackie

Elrod. Without their love and support this would not have been possible. Through the

wonderful and terrible times, they have always been there for me, and that makes me

truly blessed.

vii

Table of Contents

Signature Page iii

Curriculum Vitae iv

Acknowledgments vi

Table of Contents vii

List of Figures ix

List of Schemes xv

List of Equations xvi

Chapter 1. Introduction 1

1.1. Introduction 2

1.2. Nitrate and Perchlorate 3

1.3. Nitrate and Perchlorate Reducing Enzymes 5

1.4. Biomimetic Nitrate Reducing Molybdenum Complexes 8

1.5. Perchlorate Reducing Complexes 11

1.6. Lewis Acid Additives and Oxygen Atom Transfer 13

1.7. References 16

Chapter 2: Lewis Acid Assisted Nitrate Reduction with Biomimetic Molybdenum Oxotransferase Complex

27

2.1. Abstract 28

2.2. Introduction 29

2.3. Experimental Section 34

2.4. Results and Discussion 40

2.5. Conclusions 58

viii

2.6. References 59

Chapter 3: Structure and Oxygen Atom Transfer Reactivity of Dinuclear (µ-O)Molybdenum(V) Complex

70

3.1. Abstract 71




3.5. Conclusions 105

3.6. References 106

Chapter 4: Lewis Acid Assisted Perchlorate Reduction with Dinuclear Molybdenum(V)(µ-Oxo) Complex

112

4.1. Abstract 113




4.5. Conclusions 125

4.6. References 125

ix

List of Figures

Figure 1.1. Reduction of DMSO by DMSOR from Rhodobacter sphaeroides.

6

Figure 1.2. The global nitrogen cycle.

6

Figure 1.3. Reduced active sites of a) DMSOR from Rhodobacter sphaeroides b) Nar from Escherichia coli and PcrAB from Azopira suillum c) Nap from D. desulfuricans , where Asp = aspartate, Cys = cysteine, and Ser = serine.

7

Figure 1.4. OAT with Holm dithiolene DMSOR structural and functional model complexes.

8

Figure 1.5. Model complexes a) Mo2O3(5-SO3ssp)2(sol)2 b) Mo2O3(L-NS2)2(sol)2, where sol = DMF.

9

Figure 1.6. Proposed associative mechanism for nitrate reduction by [WIV(SC6H2-2,4,6-Pri

3)(S2C2Me2)2](Et4N).

10

Figure 1.7. Catalytic nitrate reduction with [Et4N][Mo(SPh)(PPh3)(mnt)2] and triphenylphosphine.

10

Figure 1.8. Catalytic perchlorate reduction with ReV(O)(hoz)2Cl or [ReV(O)(hoz)2(OH2)]OTf and organic sulfide.

12

Figure 1.9. Nitrate reduction by N(afaCy)3FeII(OTf)](OTf).

13

Figure 1.10. Perchlorate reduction by N(afaCy)3FeII(OTf)](OTf).

13

Figure 1.11. OAT from (TBP8Cz)Mnv(O) to aryl phosphine

14

Figure 1.12. Generation of valence tautomer from (TBP8Cz)Mnv(O) and OAT with [(TBP8Cz•+)MnIV(O)-Zn2+].

15

Figure 1.13. Proposed binding of Sc3+ to [MnV(O)(TAML)][PPh4].

16

Figure 2.1. Active site structures of the oxidized forms of periplasmic nitrate reductase (Nap) from D. desulfuricans and formate dehydrogenases (Fdhs) from E. coli (X = SeCys) or R.capsulatus (X = Cys).

30

Figure 2.2. MoVI(O)2(SN)2 (1) and MoIV(O)(SN)2 (SN=bis(4-t-butylphenyl)-2-pyridylmethanethiolate)

33

x

Figure 2.3. KBr IR of Mo(O)2(SN)2 (1) (yellow trace) and Mo(O)(SN)2 (2)

41

Figure 2.4a. Room temperature 1H NMR Mo(O)2(SN)2 (1) in CD2Cl2

42

Figure 2.4b. Room temperature 1H NMR Mo(O)2(SN)2 (1) in CD2Cl2. 43

Figure 2.5a. Room temperature 1H NMR Mo(O)(SN)2 (2) in CD2Cl2.

43

Figure 2.5b. Room temperature 1H NMR Mo(O)(SN)2 (2) in CD2Cl2.

43

Figure 2.6. Room temperature UV-vis of Mo(O)2(SN)2 (1) and Mo(O)(SN)2 (2) in DCM.

44

Figure 2.7. Room temperature UV-vis of Mo(O)(SN)2 (2) and Bu3N(NO3) (10 equiv.) after 24 hours in DCM.

45

Figure 2.8. Room temperature UV-vis of Mo(O)2(SN)2 (1) generated from Mo(O)(SN)2 (2) (1 equiv.), Sc(OTf)3 (2 equiv.) and Bu4N(NO3) (10 equiv.) in DCM.

46

Figure 2.9. KBr of Mo(O)2(SN)2 (1) generated from Mo(O)(SN)2 (2) (1 equiv.), Sc(OTf)3 (2 equiv.) and Bu4N(NO3) (10 equiv.) in DCM.

46

Figure 2.10. 1H NMR of Mo(O)2(SN)2 (1) generated from Mo(O)(SN)2 (2) (1 equiv.), Sc(OTf)3 (2 equiv.) and Bu4N(NO3) (10 equiv.) and authentic Mo(O)2(SN)2 (1).

47

Figure 2.11. Reaction of Mo(O)(SN)2 (2) (0.8 mM), [Bu4N][NO3] (8.0 mM), and Sc(OTf)3 (1.7 mM) followed by UV-Vis spectroscopy at –40 ºC in dichloromethane for 1.5 hours. The spectral changes correspond to the conversion of Mo(O)(SN)2 (2) (λmax = 328, 430 nm) to Mo(O)2(SN)2 (1) (λmax = 370 nm).

48

Figure 2.12. IR spectra (KBr) of Mo(16O)2(SN)2 (1) and Mo(18O)2(SN)2 (118/18), along with Mo(16/18O)2(SN)2 (118/16) generated from 218 with [Bu4N][NO3] in the presence of Sc(OTf)3 (red solid), and Mo(16/18O)2(SN)2 (118/16) generated from 218 with trimethylamine n-oxide.

50

Figure 2.13. Room temperature UV-vis in H2O for positive Griess reagent test for nitrite generated from Mo(O)(SN)2 (2) (1 equiv.), Sc(OTf)3 (2 equiv.) and Bu4N(NO3) (10 equiv.) and prepared nitrite calibration curve (inset).

51

Figure 2.14. Room temperature 15N NMR (in CD2Cl2) spectra of a) the reaction mixture of Mo(O)(SN)2 (2) (1equiv.), Sc(OTf)3 (2 equiv.) and Bu4N(15NO3) (10 equiv.) showing excess nitrate signal at 6.46 ppm, b)

52

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authentic [Bu4N][15NO2], and c) an equimolar mixture of [Bu4N][15NO2] and Sc(OTf)3. Figure 2.15. IR spectra (gas cell) of headspace from the reaction mixtures of Mo(O)(SN)2 (2) (1 equiv.) and Bu4N(NO3) (10 equiv.) before and after addition of Sc(OTf)3 (2 equiv.), along with the spectrum from the reaction of [Bu4N][NO2] (1 equiv.) and Sc(OTf)3 (1 equiv.) and that of authentic N2O.

54

Figure 2.16. UV-vis of pyrogallol in 50% KOH (aq) before and after head space transfer from reaction of Sc(OTf)3 (3.5 x 10-5

mol) and Bu4N(NO2) (3.5 x 10-5 mol).

54

Figure 2.17. Room temperature UV-vis in DCM of Mo(O)(SN)2 (2) before and after addition of Sc(OTf)3 .

55

Figure 2.18. 1H NMR of Mo(O)(SN)2 (2) with Sc(OTf)3 and authentic Mo(O)(SN)2 (2) in CD2Cl2.

56

Figure 2.19. 1H NMR of Mo(O)2(SN)2 (1) with Sc(OTf)3 (top) and authentic Mo(O)2(SN)2 (1) (bottom) in CD2Cl2.

56

Figure 3.1. Active site structures of the oxidized forms of (a) respiratory nitrate reductase (Nar) from Escherichia coli and (b) periplasmic nitrate reductase (Nap) from D. desulfuricans where Asp = aspartate and Cys = cysteine.

71

Figure 3.2. cis-dioxomolybdenum(VI) thiosemicarbazone (X = Me, H, I, Br, OCF3, NO2).

74

Figure 3.3. KBr IR of Mo(O)2(LBr)(MeOH) (1).

82

Figure 3.4. 1H NMR Mo(O)2(LBr)(MeOH) (1) in DMSO-d6 (400 MHz).

82

Figure 3.5. Room temperature UV-vis of Mo(O)2(LBr)(MeOH) (1) in THF.

83

Figure 3.6. Room temperature 1H NMR (in acetone-d6, 400MHz) spectra of a) of Mo(O)2(LBr)(MeOH) (1) and b) Mo2O3(LBr)2(THF)2·2THF (2).

84

Figure 3.7. Thermal ellipsoid plot of Mo2O3(LBr)2(THF)2·2THF (2) projected at the 50% probability level.

86

Figure 3.8. Room temperature 1H NMR (in THF-d8, 400MHz) spectra of a) of Mo(O)2(LBr)(MeOH) (1) and b) Mo2O3(LBr)2(THF)2·2THF (2).

86

xii

Figure 3.9. Room temperature UV-vis of Mo2O3(LBr)2(THF)2·2THF (2) in THF.

87

Figure 3.10. KBr IR of Mo2O3(LBr)2(THF)2·2THF (2).

87

Figure 3.11. Overlay of 1H NMR Mo(O)2(LBr)(MeOH) (1) in DMSO-d6 (bottom) and 1H NMR Mo2O3(LBr)2(THF)2·2THF (2) in DMSO-d6 (top) (400 MHz) (5-0 ppm).

89

Figure 3,12. Overlay of 1H NMR Mo(O)2(LBr)(MeOH) (1) in DMSO-d6 (bottom) and 1H NMR Mo2O3(LBr)2(THF)2·2THF (2) in DMSO-d6 (top) (400 MHz) (10-5 ppm).

89

Figure 3.13 Room temperature UV-vis of Mo2O3(LBr)2(THF)2·2THF (2) before (blue trace) and after the addition of 100 equivalents DMSO (orange trace) in THF.

90

Figure 3.14. Room temperature UV-vis of Mo2O3(LBr)2(THF)2·2THF (2) (1 equivalent) with [Bu4N][NO3] (1 equivalent) in THF.

91

Figure 3.15. Overlay of a) Mo2O3(LBr)2(THF)2·2THF (2) with [Bu4N][NO3] (0.5 equiv.) b) Mo2O3(LBr)2(THF)2·2THF (2) with [Bu4N][NO3] (1 equiv.) c) Mo2O3(LBr)2(THF)2·2THF (2) with [Bu4N][NO2] (1 equiv.) d) Mo(O)2(LBr)(MeOH) (1) in acetone-d6 (400 MHz).

92

Figure 3.16. KBr IR of reaction mixture of Mo2O3(LBr)2(THF)2·2THF (2) with [Bu4N][NO3] (1 equivalent).

92

Figure 3.17. Mo2O3(LBr)2(THF)2·2THF (2) (0.15 mM) and [Bu4N][NO3] (0.15 mM) followed by UV-Vis spectroscopy at room temperature in THF for 2 hours. The spectral changes correspond to the conversion of Mo2O3(LBr)2(THF)2·2THF (2) to Mo(O)2(LBr)(THF) (3).

93


94

Figure 3.19. Room temperature UV-vis in H2O for positive Griess reagent test for nitrite generated from Mo2O3(LBr)2(THF)2·2THF (2) (1 equiv.) [Bu4N][NO3] (1 equiv.) Prepared nitrite calibration curve (inset).

95

Figure 3.20. Room temperature 15N NMR (in acetone-d6) spectra of a) [Bu4N][15NO3] showing nitrate signal at 6.46 ppm, b) reaction of Mo2O3(LBr)2(THF)2·2THF (2) and [Bu4N][15NO3].

96

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Figure 3.21. Room temperature 15N NMR (in acetone-d6) spectra of a) Bu4N[15NO2] showing nitrate signal at 243.67 ppm, b) reaction of Mo(O)2(LBr)(MeOH) (1) and [Bu4N][15NO2 ] (2 equivalents).

97

Figure 3.22. Room temperature UV-vis of Mo(O)2(LBr)(MeOH) (1) (1 equivalent) with [Bu4N][NO2] (2 equivalent) in THF.

97

Figure 3.23. Overlay of a) Mo(O)2(LBr)(MeOH) (1) b) Mo(O)2(LBr)(MeOH) (1) (1 equivalent) with [Bu4N][NO2] (2 equivalents) in acetone-d6 (400 MHz).

98

Figure 3.24. IR spectra (gas cell) of headspace from the Mo(O)2(LBr)(MeOH)

(1) (1 equivalent) with [Bu4N][NO2] (2 equivalents) (orange solid) and that of authentic N2O (blue dotted).

99

Figure 3.25. UV-vis of pyrogallol in 50% KOH (aq) before (blue trace) and after head space transfer (orange trace) from the reaction of Mo(O)2(LBr)(MeOH) (1) (1 equivalent) with [Bu4N][NO2] (2 equivalents).

100

Figure 3.26. Mo2O3(LBr)2(THF)2·2THF (2) (0.15 mM), Sc(OTf)3 (0.15 mM), and [Bu4N][NO3] (0.15 mM) followed by UV-Vis spectroscopy at room temperature in THF for 2 hours.

100


101

Figure 3.28. Overlay of a) Mo2O3(LBr)2(THF)2·2THF (2) with [Bu4N][NO2] (1 equiv.) and b) Mo(O)2(LBr)(MeOH) (1) (1 equivalent) with [Bu4N][NO2] (2 equivalents) in acetone-d6 (400 MHz).

102

Figure 3.29. Room temperature UV-vis of Mo2O3(LBr)2(THF)2·2THF (2) (1 equivalent) with [Bu4N][NO3] (0.5 equivalent) in THF.

103

Figure 3.30. Mo2O3(LBr)2(THF)2·2THF (2) with [Bu4N][NO3] (0.5 equivalents) in acetone-d6 (400 MHz).

103

Figure 3.31. IR spectra (gas cell) of headspace from the Mo2O3(LBr)2(THF)2·2THF (2) (1 equivalent) with [Bu4N][NO3] (0.5 equivalents) (orange solid) and that of authentic N2O (blue dotted).

104

Figure 4.1. Room temperature UV-vis of Mo2O3(LBr)2(THF)2·2THF (2) (1 equivalent) and [Bu4N][ClO4] (10 equivalents) in THF.

120

Figure 4.2. 1H NMR overlay of a) Mo2O3(LBr)2(THF)2·2THF (2) (1 equivalent), Sc(OTf)3 (1 equivalent) and [Bu4N][ClO4] (0.25

120

xiv

equivalents) b) Mo2O3(LBr)2(THF)2·2THF (2) (1 equivalent) and [Bu4N][ClO4] (10 equivalents) c) Mo(O)2(LBr)(MeOH) (1) in acetone-d6 Figure 4.3. 35Cl NMR overlay of a) Mo2O3(LBr)2(THF)2·2THF (2) (1 equivalent) and [Bu4N][ClO4] (10 equivalents) b) Mo2O3(LBr)2(THF)2·2THF (2) (1 equivalent), Sc(OTf)3 (1 equivalent) and [Bu4N][ClO4] (0.25 equivalents) in acetone-d6.

121

Figure 4.4. Room temperature UV-vis of Mo2O3(LBr)2(THF)2·2THF (2) (1 equivalent), Sc(OTf)3 (1 equivalent), and [Bu4N][ClO4] (1 equivalent) in THF.

122

Figure 4.5. Mo2O3(LBr)2(THF)2·2THF (2) (0.13 mM), Sc(OTf)3 (0.13 mM), and [Bu4N][NO3] (1.3mM) followed by UV-Vis spectroscopy at room temperature in THF for 5 minutes.

122

Figure 4.5. Room temperature UV-vis of Mo2O3(LBr)2(THF)2·2THF (2) (1 equivalent), Sc(OTf)3 (1 equivalent), and [Bu4N][ClO4] (1/4 equivalent) in THF.

124

xv

List of Schemes

Scheme 2.1. Nitrate reduction by R. capsulatus Fdh

31

Scheme 2.2 Reactivity of Mo(O)(SN)2 (2) and [Bu4N][NO3] in absence (top) and presence (bottom) of Sc(OTf)3 (SN = bis(4-t-butylphenyl)-2-pyridylmethanethiolate).

44

Scheme 2.3. Mo(18/16O)2(SN)2 (118/16) preparation from Mo(18O)(SN)2 (218) via Sc(OTf)3 assisted nitrate reduction (I) or trimethylamine n-oxide (II).

49

Scheme 2.4. Lewis acid assisted nitrate reduction by Mo(O)(SN)2 (2) to form Mo(O)2(SN)2 (1) by oxygen atom transfer.

58

Scheme 3.1. OAT with dimer disproportionation.

73

Scheme 3.2 OAT without dimer disproportionation.

73

Scheme 3.3. Proposed Mo2O3(LBr)2(THF)2·2THF (2) reactivity with 1.0 and 0.5 equivalents NO3−.

104

Scheme 4.1. Perchlorate reduction by A. suilum PcrAB.

114

Scheme 4.2. Reduction of perchlorate (ClO4−) to chloride (Cl−) and molecular oxygen by perchlorate reductase (PcrAB) and chlorite dismutase (Cld).

115

Scheme 4.3. Catalytic reduction of perchlorate (ClO4−) to chloride (Cl−) by ReV-oxo complexes.

116

xvi

List of Equations

Equation 1.1

6

Equation 1.2

6

Equation 1.3

6

Equation 1.4

6

Equation 1.5

6

Equation 1.6

6

Equation 1.7

11

Equation 2.1

29

Equation 3.1

71

Equation 3.2

72

Equation 3.3

72

Equation 3.4

72

1

Chapter 1: Introduction

2

1.1. Introduction

Remediation of ground water contamination associated with industrialization will

continue to be a challenge in the 21st century. As the world’s population grows, it’s use of

nitrate (NO3−) and perchlorate (ClO4−) in agricultural and industrial processes will

continue to grow with demand. With the increase in use there will be an inevitable

increase in ground water contamination associated with run off from agricultural and

industrial use. While the negative health effects and environmental impact of nitrate

contamination is well known, the long-term effects of perchlorate contamination are still

largely unknown. Both oxyanions are pervasive ground water contaminants owning to

their high solubility and mobility in water. Owing to the difficulties associated with

remediation of perchlorate and nitrate, the likely negative impacts to the environment and

health, and the likely increase in ground water contamination with population growth, the

development of new systems that can remediate perchlorate and nitrate contamination is

an important ongoing process.

Presented here are efforts to further develop bioinspired systems that can reduce

nitrate and perchlorate by oxygen atom transfer (OAT) using high valent Mo(IV/VI)

metal complexes. I will briefly discuss the environmental/health impacts and difficulties

in remediation associated with perchlorate and nitrate contamination. The enzymes that

utilize perchlorate and nitrate for metabolic processes will be presented and their active

site structures discussed. Complexes that have been successfully utilized for the reduction

of nitrate and perchlorate through OAT are presented. The effect of Lewis acid additives

on OAT will be briefly discussed.

3

1.2. Nitrate and Perchlorate

The environmental impact of nitrate is well known. The use of nitrate containing

fertilizers makes agricultural run off a common source for nitrate. Introduction of nitrate

into water sources leads to eutrophication that can result in harmful algal blooms and fish

kills in aquatic environments.1-2 While modern water chemistry quickly identified nitrate

as the one of the sources of eutrophication and lead to the development of remediation

technologies, with the increase of the global population the problem of eutrophication

will remain an issue. Disastrous environmental consequences could arise from the

combined increased waste production associated with population growth, the growing

dependence on nitrate containing fertilizers, and higher water temperatures associated

with global warming.3 The high solubility of nitrates in water complicates its removal

from ground and surface water.4

Nitrate and related nitrite (NO2−) found in the human body are from both external

and internal sources. The largest sources of nitrate are from external exposure through

consumption of contaminated water and food. Vegetables grown in nitrate contaminated

soils and water have increased nitrate levels. Nitrate and nitrite are also commonly used

as food additives commonly found in cured meats and cheeses.5 The negative health

effects of nitrate consumption are attributed related to its conversion to nitrite by bacterial

nitrite reductases found in the gastrointestinal tract and mouths.6 Consumption of nitrates

is associated with the potentially fatal blood disorder methemoglobinemia, also known as

the “blue baby syndrome”, that arises from nitrite oxidation of hemoglobin from Fe(II) to

Fe(III) producing methemoglobin. The reduced ability of the blood to carry oxygen

4

results in hypoxia.5-6 Intake of high levels of nitrate is also associated with spontaneous

abortion, premature birth, birth defects, and hypertension.4-5 While the direct link

between nitrate/nitrite and cancer has not been established, the formation of reactive N-

nitroso compounds associated with cancer have been linked to consumption of nitrate.7

Like nitrate, perchlorate (ClO4−) is a pervasive ground and surface water

contaminant owning to its high solubility in water. However, the long term environmental

effects of perchlorate are not yet known. Prior to the late 1990s, perchlorate could not be

detected in ground water below 100 µg/L. Only with better methods of detection was the

extent of perchlorate contamination in the United States realized.8-9 Like nitrate,

perchlorate contamination of ground water is linked to its use in fertilizers. The largest

source of perchlorate contamination is from the use of perchlorate salts in a wide variety

of industrial processes, including manufacture of industrial lubes, dyes, rubbers,

fertilizers, paints, in electroplating processes, and battery manufacturing. Ammonium

perchlorate is widely used in solid rocket fuels and in the production of pyrotechnics and

munitions owing to its high oxygen content.8, 10 The long term effects of perchlorate,

chlorate (ClO3−) and related chlorite (ClO2−) consumption are largely unknown. Chlorate

and chlorite can cause hemolytic anemia. Thyroid function is impacted by perchlorate

consumption, with potassium perchlorate is used to treat hyperthyroidism.8 Perchlorate

competitively inhibits the uptake of iodide by the thyroid gland, altering the production of

hormones in the body, and is a potential source of hypothyroidism.9, 11

Perchlorates physical properties make remediation difficult. As a solid perchlorate

salts are reactive and potentially explosive, and a commonly used solid oxidant for jet

propulsion systems and explosives. While perchlorate is reasonably strong oxidant

5

thermodynamically (equation 1), in solution rates of reaction are slow due to high kinetic

barriers.10 These kinetic barriers prevent reaction with reducing agents commonly used in

remediation technologies, such as sulfite and zero valent iron. Perchlorate is generally a

non-complexing anion, a poor nucleophile, and kinetically inert to reduction. These

properties make perchlorate ideal for use in the laboratory as an electrolyte in

electrochemical studies or as a non-coordinating anion in crystallizations, but makes

remediation from ground water difficult.10, 12

1.3. Nitrate and Perchlorate Reducing Enzymes

Molybdenum enzymes containing pyranopterin cofactors are utilized for a wide of

biological functions. Many eukaryotic and prokaryotic organisms utilize mononuclear

oxotransferase enzymes, capable of two-electron oxidation-reduction reactions, for

metabolic functions.13-14 The dimethylsulfoxide reductase (DMSOR) family of enzymes

utilize high valent Mo(VI-VI) metal centers bound to two pyranopterin cofactors to

catalyze the two-electron oxygen atom transfer to and from substrates (Figure 1.1).15-16

The DMSOR family of enzymes is one of the largest and most diverse family of

molybdenum containing enzymes. In addition to reduction of DMSO to dimethyl sulfide

(equation 4) performed by DMSOR, the DMSOR family contains enzymes that reduce

perchlorate and chlorate (equations 1 and 2, potentials w/respect to standard hydrogen

electrode at pH ~7), nitrate (equation 3) and trimethylamine oxide (equation 5). Transfer

of the oxygen atom to from substrate to the molybdenum center results in the formation

of a two-electron oxidized metal center. The reduced molybdenum center is reformed by

use of a redox partner, such as NADH (equation 6) and the oxygen atom transferred is

then released as water after the addition of protons.

6

Figure 1.1. Reduction of DMSO by DMSOR from Rhodobacter sphaeroides.15-16

Figure 1.2. The global nitrogen cycle. ANAMMOX = anaerobic ammonium oxidation.

Nitrate reductase enzymes utilize nitrogen in its highest oxidation state, and play a

critical role in denitrification, assimilatory and dissimilatory nitrate reduction steps of the

global nitrogen cycle (Figure 1.2). Nitrate reductase enzymes are further classified by

7

their role. Assimilatory nitrate reductase (Nas) incorporates nitrogen from nitrate into

biomass and is utilized for growth, while dissimilatory nitrate reductase (Nap) does not

incorporate nitrogen from nitrate into biomass. Nitrate reductase (Nar) generates

metabolic energy by using nitrate as the terminal electron acceptor in nitrate respiration

and is utilized denitrification.17 Respiratory nitrate reductase (Nar) from Escherichia

coli18 and (b) periplasmic nitrate reductase (Nap) from D. desulfuricans19 are two well

studied nitrate reductase enzymes (Figure 1.3). 19-22

Figure 1.3. Oxidized active sites of a) DMSOR from Rhodobacter sphaeroides16 b) Nar from Escherichia coli18 and PcrAB from Azopira suillum11 c) Nap from D. desulfuricans19 , where Asp = aspartate, Cys = cysteine, and Ser = serine.

Perchlorate reductase (PcrAB) has an identical active site as Nar, yet has different

amino acid residues in the secondary coordination environment, and likely evolved from

a common ancestor11 Perchlorate reductase utilize perchlorate and chlorate (equations 1

and 2) as terminal electron acceptors during anaerobic respiration.23 Perchlorate reducing

organisms often contain chlorite dismutase, a heme enzyme that further converts the

chlorite (ClO2−) generated from perchlorate reduction to molecular oxygen and

chloride.23-24

8

1.4. Biomimetic Nitrate Reducing Molybdenum Complexes

Interest in the molybdenum mediated oxygen atom transfer exhibited by the

mononuclear oxotransferase enzymes, including nitrate reductase, led to the development

of numerous biomimetic molybdenum systems capable of oxygen atom transfer.25-27

Early biomimetic systems consisted of thiocarbamate (S2CNR2)28-32 and Schiff base

ligand systems, that were complicated by MoV2O3 (µ-O) dimers.33-35 While not desirable

for mononuclear enzyme models, these systems were capable of oxygen atom transfer.

Later generation biomimetic systems utilized bulky ligand systems36-37 or charged

complexes38 to prevent dimerization. Numerous structural and functional models of

DMSOR enzymes, including nitrate reductase, trimethylamine oxide reductase, and

selenate reductase, utilizing dithiolene ligand systems with molybdenum and tungsten

were reported by Holm in the late 1990s and early 2000s (Figure 1.4).39-43 In addition to

developing numerous biomimetic Mo and W complexes, Holm and coworkers

extensively studied the kinetics and thermodynamics of the OAT reactions.44-46

Development of Mo systems that are capable of OAT reactivity continues to be an active

area of research, with systems capable of light induced OAT47 and polymer supported

systems48 demonstrating varied new directions the field may take.

Figure 1.4. OAT with Holm dithiolene DMSOR structural and functional model

complexes.

9

While many molybdenum systems demonstrate stochiometric and even catalytic

oxidation of phosphines and sulfides utilizing dimethylsulfoxide, few have demonstrated

nitrate reduction capability. Binuclear Mo2O3(5-SO3ssp)2(sol)2 (ssp = 2-

(saliclideneamino)benzenethiolato(2-), sol = DMF) from Holm35 and Mo2O3(L-

NS2)2(sol)2 ( L-NS2 = 2,6-bis(2,2-diphenyl-2-thioethyl)pyridinate(2-), sol = DMF)

(Figure 1.5), originally reported as a MoIV mono-oxo complex by Holm35, 49 and later

shown to be a dimer by Young50 both demonstrate nitrate reduction. Nitrate reduction by

W(IV) and Mo(IV) bis(dithiolene) complexes, containing sterically encumbered axial

sulfido ligands were reported by Holm and coworkers, with the tungsten complex product

being isolated, due to instability of the molybdenum complex.42 The reaction was found

to obey a second-rate law and proposed to take place by an associative reaction

mechanism, suggesting nitrate reduction occurs through direct oxo transfer to the metal

center (Figure 1.6) .

Figure 1.5. Model complexes a) Mo2O3(5-SO3ssp)2(sol)2 b) Mo2O3(L-NS2)2(sol)2, where sol = DMF.

10

Figure 1.6. Proposed associative mechanism for nitrate reduction by [WIV(SC6H2-2,4,6-Pri

3)(S2C2Me2)2](Et4N).

Catalytic nitrate reduction utilizing [Et4N][Mo(SPh)(PPh3)(mnt)2] (mnt = 1,2-

dicyanoethylenedithiolate(2-)) (Figure 1.7) was reported by Sarkar and coworkers.51 The

active catalyst was proposed to be formed through loss of triphenylphosphine, resulting

in the pentacoordinate MoIV thiolate complex. After nitrate reduction occurs

triphenylphosphine reduces the MoVIO species generating triphenylphosphine oxide and

reforming the MoIV species that can further undergo reactivity with nitrate or reform the

catalytically inactive [Et4N][Mo(SPh)(PPh3)(mnt)2].

Figure 1.7. Catalytic nitrate reduction with [Et4N][Mo(SPh)(PPh3)(mnt)2] and triphenylphosphine.

11

1.5. Perchlorate Reducing Complexes

Complexes that reduce perchlorate to chloride under mild conditions are rare.

Reduction of perchlorate to chloride through oxygen atom transfer was reported by

Espenson and coworkers. Methylrhenium dioxide, (MDO = Ch3ReO2) generated in situ

from methylrhenium trioxide (MTO = Ch3ReO3) and the powerful reducing agent

hypophosphorus acid (H3PO2) at pH = 0 with 1.0 M trifluoromethanesulfonic acid

(HOTf) reacts with perchlorate to regenerate MTO (equation 7).52-53

Catalytic reduction of perchlorate to chloride under mild conditions utilizing

organic sulfides with the air and water stable ReV(O)(hoz)2Cl and

[ReV(O)(hoz)2(OH2)]OTf (hoz = [2-(2’-hydroxyphenyl)-2-oxazoline], OTf=

trifluoromethanesulfonate) (Figure 1.8) was reported by Abu-Omar in 2000.54

ReV(O)(hoz)2Cl and [ReV(O)(hoz)2(OH2)]OTf show little decomposition after hundreds

of turnovers, react cleanly to produce sulfoxide and chloride, are readily prepared from

inexpensive and commercially available starting materials. and have been utilized in the

preparation of hybrid heterogenous catalyst systems.55 Re(V) complexes containing

tetradentate iminophenolate ligands have also been utilized in the successful reduction of

perchlorate to chloride with organic sulfides.56

12

Figure 1.8. Catalytic perchlorate reduction with ReV(O)(hoz)2Cl or [ReV(O)(hoz)2(OH2)]OTf (above) with organic sulfide.

A Fe(II) azafulvene-amine ([N(afaCy)3Fe(OTf)](OTf)) reported by Fout and

coworkers is an example of a synthetic system that can reduce both nitrate and

perchlorate.57 Utilizing ligands that incorporate non-covalent interactions, inspired by

Borovik’s non-heme Fe(III)-oxo complexes58, [N(afaCy)3FeII(OTf)](OTf) was shown to

react with nitrite to form [N(afaCy)3FeIII(O)](OTf) and [N(afaCy)3FeII(NO)](OTf)2.59

Nitrate reduction was achieved with 3 equivalents of [N(afaCy)3FeII(OTf)](OTf) and 1

equivalent of [Bu4N][NO3] in the presence of triethylamine, yielding

[N(afaCy)3FeIII(O)](OTf) and [N(afaCy)3FeII(NO)](OTf)2 in a 2:1 ratio, indicating that the

nitrite generated by reduction is further reduced to NO (Figure 1.9). Perchlorate reduction

to chloride was also achieved with 5 equivalents of [N(afaCy)3FeII(OTf)](OTf) and 1

equivalent of [Bu4N][ClO4]s to form [N(afaCy)3FeIII(O)](OTf) and

[N(afaCy)3FeII(Cl)](OTf) in a 4:1 ratio (Figure1.10). Catalytic reduction of both nitrate

and perchlorate was achieved through the use of the diphenylhydrazine, a 2H+/2e− source,

and decamethylferrocenium triflate as a sacrificial oxidant.57

13

Figure 1.9. Nitrate reduction by N(afaCy)3FeII(OTf)](OTf).

Figure 1.10. Perchlorate reduction by N(afaCy)3FeII(OTf)](OTf).

1.6. Lewis Acid Additives and Oxygen Atom Transfer

The ability to alter the redox reactivity of metal oxo complexes with redox-

inactive Lewis acid metal cations has been well established. Redox-inactive Lewis acid

additives have been shown to alter redox potentials60-63, electron transfer rates60, 64-66, and

reactivity62, 67-72 of metal oxo complexes. The ability to alter oxygen atom transfer

reactivity with Lewis acids has also been demonstrated.

The MnV-oxo corrolazine complex (TBP8Cz)Mnv(O) developed by Goldberg and

coworkers are capable of OAT (Figure 1.11) to aryl phosphines to generate the

(TBP8Cz)MnIII.62, 73 The electron transfer and hydrogen atom transfer activity of

Goldberg’s high valent MnV-oxo species were enhanced with the addition of the Zn2+.

The addition of the Lewis acid binds to the diamagnetic corrolazine complex, possibly

14

through the terminal oxo, with a high affinity and induces valence tautomerization

resulting in a paramagnetic MnIV species [(TBP8Cz•+)MnIV(O)-Zn2+] (Figure 1.12).

Addition of 1,10 phenanthroline regenerates the MnV-oxo species demonstrating that the

tautomerization is reversible.69 Both (TBP8Cz)Mnv(O) and the MnIV valence tautomer are

capable of OAT. However, addition of the Lewis acid results in dramatically slower rates

of oxygen atom transfer and is attributed to a less electrophilic terminal oxo of the MnIV

valence tautomer, demonstrating the ability to tune OAT reactivity through the addition

of a Lewis acid metal cation.62

Figure 1.11. OAT from (TBP8Cz)Mnv(O) to aryl phosphine.

15

Figure 1.12. Generation of valence tautomer from (TBP8Cz)Mnv(O) and OAT with [(TBP8Cz•+)MnIV(O)-Zn2+].

A [MnV(O)(TAML)][PPh4] complex reported by Nam and coworkers

demonstrated enhanced OAT reactivity with the addition of Sc(OTf)3. The addition of

Sc3+ results in a 0.7 V increase in the one-electron reduction potential compared to the

Lewis acid free complex, and enhances the oxidizing power of the complex. Interestingly

the binding site of the Sc3+ was proposed to take place on the macrocyclic TAML ligand

and not the terminal Mn-oxo (Figure 1.13). This was attributed to the low basicity of the

oxo group compared to the amide carbonyl of the ligand. This work demonstrated the

ability to alter the oxidizing power a metal oxo system by altering the secondary

coordination sphere.

16

Figure 1.13. Proposed binding of Sc3+ to [MnV(O)(TAML)][PPh4].

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Scandium Ion. J. Am. Chem. Soc. 2016, 138 (27), 8523-8532.

25

64. Fukuzumi, S.; Ohkubo, K., Metal ion-coupled and decoupled electron transfer.

Coord. Chem. Rev. 2010, 254 (3), 372-385.

65. Fukuzumi, S.; Ohkubo, K.; Lee, Y.-M.; Nam, W., Lewis Acid Coupled Electron

Transfer of Metal–Oxygen Intermediates. Chemistry – A European Journal 2015, 21

(49), 17548-17559.

66. Lee, Y.-M.; Bang, S.; Kim, Y. M.; Cho, J.; Hong, S.; Nomura, T.; Ogura, T.;

Troeppner, O.; Ivanovic-Burmazovic, I.; Sarangi, R.; Fukuzumi, S.; Nam, W., A

mononuclear nonheme iron(iii)-peroxo complex binding redox-inactive metal ions.

Chemical Science 2013, 4 (10), 3917-3923.

67. Park, J.; Morimoto, Y.; Lee, Y.-M.; You, Y.; Nam, W.; Fukuzumi, S., Scandium

Ion-Enhanced Oxidative Dimerization and N-Demethylation of N,N-Dimethylanilines by

a Non-Heme Iron(IV)-Oxo Complex. Inorg. Chem. 2011, 50 (22), 11612-11622.

68. dPark, Y. J.; Ziller, J. W.; Borovik, A. S., The Effects of Redox-Inactive Metal

Ions on the Activation of Dioxygen: Isolation and Characterization of a Heterobimetallic

Complex Containing a MnIII–(μ-OH)–CaII Core. J. Am. Chem. Soc. 2011, 133 (24),

9258-9261.

69. Leeladee, P.; Baglia, R. A.; Prokop, K. A.; Latifi, R.; de Visser, S. P.; Goldberg,

D. P., Valence Tautomerism in a High-Valent Manganese–Oxo Porphyrinoid Complex

Induced by a Lewis Acid. J. Am. Chem. Soc. 2012, 134 (25), 10397-10400.

70. Hong, S.; Pfaff, F. F.; Kwon, E.; Wang, Y.; Seo, M.-S.; Bill, E.; Ray, K.; Nam,

W., Spectroscopic Capture and Reactivity of a Low-Spin Cobalt(IV)-Oxo Complex

Stabilized by Binding Redox-Inactive Metal Ions. Angew. Chem. Int. Ed. 2014, 53 (39),

10403-10407.

26

71. Park, J.; Morimoto, Y.; Lee, Y.-M.; Nam, W.; Fukuzumi, S., Metal Ion Effect on

the Switch of Mechanism from Direct Oxygen Transfer to Metal Ion-Coupled Electron

Transfer in the Sulfoxidation of Thioanisoles by a Non-Heme Iron(IV)−Oxo Complex. J.

Am. Chem. Soc. 2011, 133 (14), 5236-5239.

72. Bang, S.; Lee, Y.-M.; Hong, S.; Cho, K.-B.; Nishida, Y.; Seo, M. S.; Sarangi, R.;

Fukuzumi, S.; Nam, W., Redox-inactive metal ions modulate the reactivity and oxygen

release of mononuclear non-haem iron(III)–peroxo complexes. Nat Chem 2014, 6 (10),

934-940.

73. Prokop, K. A.; Neu, H. M.; de Visser, S. P.; Goldberg, D. P., A Manganese(V)–

Oxo π-Cation Radical Complex: Influence of One-Electron Oxidation on Oxygen-Atom

Transfer. J. Am. Chem. Soc. 2011, 133 (40), 15874-15877.

27

Chapter 2: Lewis Acid Assisted Nitrate Reduction with

Biomimetic Molybdenum Oxotransferase Complex

28

2.1 Abstract

The reduction of nitrate (NO3−) to nitrite (NO2−) is of significant biological and

environmental importance. While MoIV(O) and MoVI(O)2 complexes that mimic the

active site structure of nitrate reducing enzymes are prevalent, few of these model

complexes can reduce nitrate to nitrite through oxygen atom transfer (OAT) chemistry.

We present a novel strategy to induce nitrate reduction chemistry of a previously known

catalyst MoIV(O)(SN)2 (2), where SN = bis(4-tert-butylphenyl)-2-pyridylmethanethiolate,

that is otherwise incapable of proceeding OAT with nitrate. Addition of nitrate together

with the Lewis acid Sc(OTf)3 (OTf = trifluoromethanesulfonate) to 2 results in an

immediate and clean conversion of 2 to MoVI(O)2(SN)2 (1). The Lewis acid additive

further reacts with the OAT product, nitrite, to form N2O and O2. This work highlights

the ability of Sc3+ additives to expand the reactivity scope of an existing MoIV(O)

complex together with which Sc3+ can convert nitrate to stable gaseous molecules.

29

2.2. Introduction. Nitrate reduction to nitrite is of significant biological and

environmental importance. Due to widespread use of nitrate in agricultural applications

and high solubility in water, it is a pervasive contaminant in ground water and can lead to

eutrophication.1 Nitrate consumed can be reduced to nitrite by nitrate reducing bacteria in

the mouth and gut, and further reduced to NO in the body, and is linked to negative

health effects including the formation of reactive N-nitroso compounds associated with

cancer and the blood disorder methemoglobinemia.2-3 Nitrate reductases found in

prokaryotic and eukaryotic microorganisms catalyze the reduction of nitrate to nitrite for

metabolic processes, and play an important role in denitrification and nitrate assimilation

steps within the global nitrogen cycle.2, 4

One of the well-studied nitrate reductases is periplasmic nitrate reductase (Nap)

from Desulfovibrio desulfuricans that belongs to the dimethylsulfoxide reductase

(DMSOR) family of enzymes. The active site of Nap for the reduction of nitrate to nitrite

contains a MoIV/VI metal center bound by two pyranopterin cofactors (Figure 2.1).5 The

two-electron reduction of nitrate (NO3–) to nitrite (NO2

–) is coupled with oxygen atom

transfer (OAT) from NO3– to MoIV to form the MoVI-oxo species which subsequently

releases water upon the addition of two external electrons and protons (equation 1)6-8.

Formate dehydrogenase (Fdh), another DMSOR family enzyme which catalyzes the

oxidation of formate to CO2 and has a very similar active site structure as Nap (Figure 1),

has also been shown to be capable of reducing nitrate to nitrite.9

NO3– + 2 H+ + 2 e– → NO2

– + H2O (1)

30

Figure 2.1. Active site structures of the oxidized forms of periplasmic nitrate reductase (Nap) from D. desulfuricans5 and formate dehydrogenases (Fdhs) from E. coli (X = SeCys)10 or R.capsulatus (X = Cys).11

Artificial systems that remediate nitrate contamination are desirable to combat

against ground water contamination associated with increased use of fertilizers and global

industrialization. Bioinspired catalysts could play an important role in the reduction of

nitrate to nitrite in the remediation processes. There are a number of biomimetic model

complexes that replicate the active site structure and reactivity of DMSOR, utilizing a

variety of ligand scaffolds including dithiolenes12-13, tris(pyrazolyl)borates14-15, salan16,

pyridylmethanethiolate17-18, and Schiff base derivatives19. However, few are known to

reduce nitrate to nitrite.20-24 Binuclear (µ-oxo)molybdenum(V) and complexes reported

by Holm and Young have demonstrated nitrate reduction capability.20-22, 25 Catalytic

nitrate reduction to nitrite coupled to triphenyl phosphine oxidation using a MoIV

dithiolene complex was reported by Sarkar.24 Nitrate reduction by a W(IV)

bis(dithiolene) complex containing a sterically encumbered axial sulfido ligand was

reported by Holm and coworkers.23

31

One of the reasons very few complexes out of many structural biomimetic models

can replicate the activity of nitrate reductases might be the absence of secondary

coordination environment found in the metalloenzyme. Recent studies by Moura and

Cerqueira emphasize that the small differences in the amino acid residues in the

secondary coordination spheres dictate the type of catalytic reactions between Fdh and

Nap who share remarkably similar structures.26-27 The latest study by Leimkühler showed

that the highly conserved, positively charged arginine residue in the secondary

coordination environment of Fdh and Nap is important in nitrate reduction by facilitating

proper binding and stabilization of the substrate during the catalytic cycle (Scheme 2.1).9

Scheme 2.1. Nitrate reduction by R. capsulatus Fdh (adapted from Ref. 9)

The importance of the secondary coordination environment in enzyme catalysis is

not limited to Nap and Fdh. Noncovalent interactions in the secondary coordination

sphere of metalloenzymes, such as H-bonding networks present in cytochrome P45028,

32

horseradish peroxidase29, and hemoglobin30, play a critical role in the chemical

transformations they take part in. Strategies to incorporate non-covalent interactions into

synthetic model complexes through ligand design have been developed in recognition of

the importance of the secondary coordination sphere.31-35 Seminal examples include

Collman’s picket fence porphyrins36-37 and Borovik’s non-heme iron scaffold bearing an

amide microenvironment.38 More recently, the research group of Fout applied this

strategy to successfully achieve nitrate reduction with a bioinspired iron catalyst.39

Addition of redox inactive Lewis acid metal cations can offer an alternative

approach to influence reaction environment without directly tailoring the ligand

backbone. Nature employs redox-inactive metals in conjunction with high valent metal-

oxo species in metalloenzymes, such as the Mn4CaO4 cluster found in the oxygen

evolving complex (OEC) found in photosystem II (PSII).40 While the role of Ca2+ in the

oxidation of water is not completely consensus its presence is essential to the observed

reactivity of OEC.41-42 Interest in PSII and the role of Ca2+ in OEC has led to the

investigation of the effects of redox-inactive Lewis acid metal cations on reactivity of

metal-oxo species.43-44 Redox-inactive Lewis acid additives have been shown to alter

redox potentials45-48, electron transfer rates45, 49-51, and reactivity47, 52-57 of metal oxo

complexes. Oxygen atom transfer (OAT) reactivity can be influenced by the addition of

Lewis acids. The rate of OAT reactivity of Goldberg’s MnV(O) porphyrinoid complexes

is dramatically decreased with the addition of Zn2+.47 Another MnV(O) system reported

by Nam demonstrates the ability of Sc3+ to enhance OAT activity by increasing the

oxidizing power of MnV(O) through binding to the ligand.48

33

A biomimetic complex MoIV(O)(SN)2 (2), where SN = bis(4-tert-butylphenyl)-2-

pyridylmethanethiolate), reported by the research group of Holm17 is a very well

documented system that facilitates efficient OAT from an organic/inorganic substrate to

yield MoVI(O)2(SN)2 (1) (Figure 2.2). Complex 2 mediates OAT chemistry with a wide

variety of substrates including various N- and S-oxides.17 However, the original report

indicated that nitrate reduction to nitrite by 2 was unsuccessful. Inspired by recent reports

demonstrating the ability of redox-inactive metal cations to alter the OAT reactivity of

MnV(O) systems47-48, we investigated the effect of Lewis acid additives on the OAT

activity of 2. Herein, we report a strategy to induce novel nitrate reduction with 2 through

addition of Sc(OTf)3 (OTf = trifluoromethanesulfonate), demonstrating the ability of

Lewis acids additives to expand the reactivity scope of previously developed metal-oxo

systems.

Figure 2.2. MoVI(O)2(SN)2 (1) and MoIV(O)(SN)2 (SN=bis(4-t-butylphenyl)-2-pyridylmethanethiolate)

34

2.3. Experimental Section

General Considerations. Unless otherwise specified all reactions and manipulations

were carried out under an inert nitrogen atmosphere using a MBraun Labmaster SP

gloveblox or under argon using standard Schlenck line techniques. 3Å molecular sieves

were dried under vacuum for 24 hours at 250 °C prior to use. THF, pentane, and

acetonitrile were degassed then dried using an MBraun solvent purification systems

under an Ar atmosphere, and stored over activated 3 Å molecular sieves. Anhydrous

dichloromethane and methanol were purchased from Aldrich, degassed by sparging with

Ar for 30 minutes, and stored over activated 3 Å molecular sieves for 48 hours prior to

use. Ultrapure, deionized water was obtained from a Millipore Direct-Q 3 UV Water

Purification System. Tetrabutylammonium nitrate, triphenylphosphine, trimethylamine n-

oxide, 99% nitrous oxide, Amberlite IRA-400 chloride form resin, potassium hydroxide,

and pyrogallol were purchased from Sigma-Aldrich and used as received. Scandium

triflate was purchased from Strem and used as received. Tetrabutylammonium nitrite was

purchased from Sigma-Aldrich and recrystallized from THF at –35 °C prior to use. H218O

(98%), Na15NO3, and Na15NO2 were purchased from Cambridge Isotopes and used as

received. CD2Cl2 was purchased from Cambridge Isotopes, degassed via freeze-pump-

thaw cycle (5x) and dried over 3 Å molecular sieves for 48 hours prior to use. Griess

Reagent Kit for Nitrite Determination (G-7921) was purchased from Molecular Probes.

Samples were lyophilized using a Labcono FreeZone 4.5 freeze dry system. Mo(O)2(SN)2

(1), and Mo(18/18O)2(SN)2 (118/18) were prepared according to the published procedure.17

Physical Methods. All samples for spectroscopic analysis were prepared inside a

nitrogen glovebox unless otherwise noted. Infrared spectra were recorded on a Bruker

35

Tensor 27 FTIR spectrometer. Headspace detection of N2O was performed with a Bruker

A131 IR gas cell equipped with quartz windows. Room temperature UV-vis spectra were

recorded on a Varian Cary 50 Bio spectrometer. Low temperature UV-Vis spectra were

recorded on a Varian Cary 50 Bio spectrometer equipped with a 2 mm Hellma All-Quartz

Immersion probe fitted in a 24/40 Schlenk tube with a 14/20 female joint sealed with a

rubber septum. 1H and 15N NMR were recorded with a Bruker 400MHz Avance III

ultrashield spectrometer. 1H NMR was referenced to CD2Cl2 residual solvent signal (δ

5.32). 15N NMR was externally referenced to nitromethane in CD2Cl2. Elemental analyses

were carried out by Intertek Pharmaceutical Services (Whitehouse, NJ).

Synthesis of Mo(O)2(SN)2 (1). Mo(O)2(SN)2 was synthesized following the published

procedure.17 An additional recrystallization from THF/pentane (1:9) at -35 °C is was

needed to obtain pure compound, resulting in a lower yield than previously reported

(85%). UV-vis (dichloromethane): λmax (εM) 370 nm (7399). IR (KBr): 901, 936 cm-. 1H

NMR (CD2Cl2, 400 MHz): δ 9.42 (d, 1 H, J = 4.7 Hz, py), 7.57 (t, 1 H, J = 8.0 Hz, py),

7.28-7.24 (m, 4H, ph), 7.17 (d, 2H, J = 8.7 Hz, ph), 7.00 (d, 2H, J = 8.7 Hz, ph), 6.96 (t, 1

H, J = 5.7 Hz, py), 6.90 (d, 1H, J = 8.2 Hz, py), 1.32 (s, 9H, t-Bu), 1.28 (s, 9H, t-Bu).

Anal. Calcd for C52H60MoN2O2S2 • 0.6 CH2Cl2: C, 66.08; H, 6.45; N, 2.83. Found: C,

65.61; H, 6.53; N, 2.86.

Synthesis of Mo(O)(SN)2 (2). Mo(O)2(SN)2 (1) (500.0 mg, 0.552 mmol) and

triphenylphosphine (290.0 mg, 1.10 mmol) were dissolved in 20 mL of THF. The yellow

solution was refluxed under N2 for 5 h at 70 °C. The resulting dark brown solution was

dried to a brown oil and washed with 20 mL of acetonitrile (3x) resulting in the

precipitation of a dark brown solid. Recrystallization from dichloromethane/pentane

36

(1:15) at -35 °C yielded 314 mg (64%) of 2 as a semi-crystalline dark brown solid.

Additional recrystallization from dichloromethane/pentane (1:15) at -35 °C afforded

brown/black crystalline material. UV-vis (dichloromethane): λmax (εM) 328 (5926), 430

(4676), 518 (892), 700 (546). IR (KBr): 947 cm-. 1H NMR (CD2Cl2, 400 MHz): δ 9.37 (d,

1 H, J = 5.6 Hz, py), 7.86 (t, 1 H, J = 7.8 Hz, py), 7.47 (d, 1H, J = 8.1 Hz, py), 7.37 (t,

1H, J = 6.5 Hz, py), 7.32 (d, 2H, J = 7.8 Hz, ph), 7.24 (d, 2H, J = 7.6 Hz, ph), 7.16 (q,

4H, J = 6.2 Hz, ph), 1.32 (s, 9H, t-Bu), 1.26 (s, 9H, t-Bu). Anal. Calcd for

C52H60MoN2OS2 • 0.5 CH2Cl2: C, 67.69; H, 6.60; N, 3.01. Found: C, 68.04; H, 6.64; N,

2.98.

Reaction of Mo(O)(SN)2 (2), Sc(OTf)3 and [Bu4N][NO3]. Sc(OTf)3 (11.1 mg, 22.5

μmol) dissolved in 2.0 mL dichloromethane was added to a stirring solution of

Mo(O)(SN)2 (2) (10.0 mg, -11.2 μmol), and [Bu4N][NO3] (34.2 mg, 112 μmol) in 3.0 mL

of dichloromethane resulting in the rapid formation of a bright yellow solution. The

solution was dried to a bright yellow oil after an additional 30 min of stirring. MeOH (10

mL x 2) was added to remove excess Sc(OTf)3 and [Bu4N][NO3] and resulted in the

precipitation of a yellow solid. Recrystallization of the yellow solid dissolved in minimal

dichloromethane and layered pentane at –35 °C resulted in 8.2 mg (80%) of Mo(O)2(SN)2

(1) as a yellow microcrystalline solid whose spectroscopic characterizations match those

from independently synthesized 1.17

Detection and Quantification of Nitrite. The methanol washes of above were dried and

extracted with 1.00 mL of ultrapure, deionized H2O and filtered through celite to remove

any insoluble material. Detection of nitrite with Griess reagent was performed according

37

to manufacturer’s instructions. Nitrite concentration was determined using a NaNO2

calibration curve prepared in ultrapure, deionized H2O.

Low temperature UV-Vis monitoring of the reaction of Mo(O)(SN)2 (1), Sc(OTf)3

and [Bu4N][NO3] at –40 °C. After blanking spectrometer with 4.0 mL dichloromethane

at –40°C in a dry ice/acetonitrile cold bath, Mo(O)(SN)2 (2) (5.0 mg, 5.6 μmol) in 2.0 mL

dichloromethane was injected and cooled to –40 °C, and the spectrum of 2 was recorded.

A mixture of Sc(OTf)3 (6.0 mg, 12.2 μmol) and [Bu4N][NO3] (17.1 mg, 56.2 μmol) in 1.0

mL dichloromethane was injected to the solution of 2, after which a series of spectra were

measured for 90 minutes at –40°C.

Synthesis of Mo(18O)(SN)2 (218). Compound 218 was prepared by following the same

procedure for 2 using an 18O-labled precursor, Mo(18O)2(SN)2 (118/18) (80.0 mg, 0.088

mmol) and triphenylphosphine (46.1 mg, 0.176 mmol). The reaction resulted in 43.1 mg

(55%) of 218. UV-vis (dichloromethane): λmax (εM) 328 (5926), 430 (4676), 518 (892),

700 (546). IR (KBr): 901 cm–1

Reaction of Mo(18O)(SN)2 (218), Sc(OTf)3 and [Bu4N][NO3). The reaction was followed

by the same procedure for 2/Sc(OTf)3/[Bu4N][NO3] with slightly different amounts of

reagents. A mixture of Sc(OTf)3 (10.2 mg, 0.21 μmol) and [Bu4N][NO3] (31.5 mg, 1.03

μmol) dissolved in 1 mL of dichloromethane was added to a stirring solution of 218 (9.2

mg, 0.10 μmol) in 4 mL dichloromethane resulting in the rapid formation of a bright

yellow solution. Removal of dichloromethane yielded the 5.5 mg (59%) of

Mo(18/16O)2(SN)2 (118/16) as a yellow powder. UV-vis (dichloromethane): λmax (εM) 370

nm (4389). IR (KBr): 924, 867 cm–1.

38

Synthesis of Mo(18/16O)2(SN)2 (118/16) from Mo(18O)(SN)2 (218) and trimethylamine n-

oxide (TMAO). 1.01 mL of a 13.3 mM stock solution of TMAO in DMF (0.13 μmol)

was added to a stirring solution of 218 (12.0 mg, 0.13 μmol) in dichloromethane. After 30

min the volatiles were removed in vacuum to afford a yellow oil that was washed with

MeOH (10 mL x 2) resulting in the precipitation of a yellow solid. Recrystallization of

the yellow solid dissolved in minimal dichloromethane and layered pentane at –35 °C

resulted in 9.3 mg (76%) of Mo(18/16O)2(SN)2 (118/16) as a yellow powder. UV-vis

(dichloromethane): λmax (εM) 370 nm (4389). IR (KBr): 924, 867 cm–1.

Synthesis of [Bu4N][15NO3]. 4 mL of Amberlite IRA-400 chloride form resin (5.6 mEq)

was packed into a 1 x 10 cm column and washed with 200 mL deionized H2O. A solution

of Na(15NO3) (525 mg, 6.17 mmol) dissolved in 2 mL of deionized H2O was slowly

eluted through the column. The column was then washed with additional 200 mL

deionized H2O. Subsequently, a solution of Bu4NI (750 mg, 2.03 mmol) dissolved in 20

mL of deionized H2O was slowly eluted through the column and the fractions were

saved. Removal of water under vacuum afforded a clear oil. The clear oil was then

lyophilized for 24 hours resulting in 0.5081 g (71.8 %) of [Bu4N][15NO3] as white

powder. 1H NMR(CD2Cl2, 400 MHz): δ 3.22 (t, 2 H, J = 8.6), 1.66 (m, 2H), 1.45 (m, 2H),

1.04 (t, 3H, J = 7.4). 15N NMR(CD2Cl2) δ 6.46.

Synthesis of [Bu4N][15NO2]. [Bu4N][15NO2] was prepared analogously to

[Bu4N][15NO3] using 450 mg Na(15NO2) (6.52 mmol) and 750 mg Bu4NI (2.03 mmol)

resulting in 0.4441 g (75.8 %) of white powder 1H NMR(CD2Cl2, 400 MHz): δ 3.22 (t, 2

H, J = 8.6), 1.66 (m, 2H), 1.45 (m, 2H), 1.04 (t, 3H, J = 7.4). 15N NMR(CD2Cl2) δ

243.67.

39

Detection of N2O by IR spectroscopy. In a glovebox, Mo(O)(SN)2 (2) (50.0 mg, 56.2

μmol) and [Bu4N][NO3] (171.2 mg, 0.562 mmol) were dissolved in 5.0 mL of

dichloromethane in a 10 mL Schlenk flask. Separately, a solution of Sc(OTf)3 (55.0 mg,

0.112 mmol) in 2.0 mL of dichloromethane was prepared and loaded into an air-tight

syringe inside glovebox. After recording the background of the evacuated IR gas cell, the

Schlenk flask containing 2 and [Bu4N][NO3] was connected to the IR gas cell with a

three-way stopcock and Tygon tubing. After evacuating the tubing connecting the gas

cell and flask, the system was left under static vacuum. The Sc(OTf)3 solution was

quickly injected into the flask containing 2/[Bu4N][NO3] via syringe resulting in a bright

yellow solution. After 5 min the headspace from the reaction flask was transferred by

opening the stopcocks of the flask and IR gas cell. The stopcocks were then closed, and

the IR spectrum was recorded. The same procedure was employed for the detection of

N2O from the reaction of Sc(OTf)3 and [Bu4N][NO2] using a solution of [Bu4N][NO2]

(32.2 mg, 0.112 mmol) in 5.0 mL dichloromethane and a solution of Sc(OTf)3 (55.0 mg,

0.112 mmol) in 2.0 mL dichloromethane.

Detection of O2 from the reaction of Sc(OTf)3 and [Bu4N][NO2] with alkaline

pyrogallol. In a 25 mL Schlenk flask Pyrogallol (500.0 mg) was dissolved with stirring

in 10.0 mL of deoxygenated 50% KOH (aq) solution. A Schleck cuvette (1 cm

pathlength) fitted with a 14/20 male connector was connected to the 25 mL flask containg

the alkaline pyrogallol solution under positive Ar flow. The UV-vis spectrum of the faint

yellow alkaline pyrogallol solution was recorded. The flask was then reconnected to the

Schlenck line through a three-way stopcock. In a glovebox [Bu4N][NO2] (10.0 mg, 34.7

μmol) was dissolved in 3.0 mL of dichloromethane in a 10 mL Schlenk flask. Separately,

40

a solution of Sc(OTf)3 (17.2 mg, 34.7 μmol) dissolved in 2.0 mL methane was prepared

and loaded into an air-tight syringe inside glovebox. The Schlenck flask containing

[Bu4N][NO2] was connected to the pyrogallol flask with Tygon tubing through the 3-way

stopcock. The system was left under static vacuum after evacuating the pyrogallol

containg flask and tubing connecting the two flasks. The solution of Sc(OTf)3 was

quickly injected into the nitrite containing flask via syringe. After 1 min the headspace

was transferred by opening the stopcocks on both Schlenck flasks. The light yellow

solution rapidly turned light brown. After 30 mins the stopcocks were resealed and the

UV-vis of the darkened pyrogallol solution was recorded.

2.4. Results and Discussion

Preparation of Mo(O)2(SN)2 (1) and Mo(O)(SN)2 (2). Complex Mo(O)2(SN)2 (1) was

prepared following the reported procedure17 with minor modifications. Additional

recrystallization from THF/pentane was necessary to obtain the pure yellow

microcrystalline solid in 85% yield. The IR (νMo=O = 901, 936 cm–1) (Figure 2.3) and

diamagnetic 1H NMR spectral features (Figure 2.4a-b) are in good agreement with the

previously published values.17 Complex Mo(O)(SN)2 (2) was prepared using excess

triphenylphosphine in place of previously used triethylphosphine17 and was isolated as a

dark brown solid in 64% yields. Additional recrystallizations from CH2Cl2/pentane

afforded brown/black crystalline material and were found to aid the long-term stability of

2 at –35 °C under N2. The IR spectral features including a characteristic νMo=O frequency

at 947 cm–1 (Figure 2.3) and well resolved diamagnetic 1H NMR features of 2 (Figure

2.5a-b) matched the previously published values, with minor discrepancies in the

chemical shifts probably due to different instruments employed. The UV-Vis absorption

41

spectra of 1 (λmax = 370 nm) and 2 (λmax = 328 nm) originally reported in DMF, are

nearly identical when dichloromethane is used as the solvent (Figure 2.6). Elemental

analysis was performed on 1 and 2 to ensure purity of the bulk material. 1H NMR

samples of 1 and 2 in CDCl3 indicates that 0.6 and 0.5 equivalents of dichloromethane

from recrystallization are present in crystalline samples of 1 and 2, respectively, and is

accounted for in the elemental analysis.

Figure 2.3. IR spectra (KBr) of Mo(O)2(SN)2 (1) (yellow trace) and Mo(O)(SN)2 (2) (brown trace).

42

Figure 2.4a. Room temperature 1H NMR Mo(O)2(SN)2 (1) in CD2Cl2.

Figure 2.4b. Room temperature 1H NMR Mo(O)2(SN)2 (1) in CD2Cl2.

43

Figure 2.5a. Room temperature 1H NMR Mo(O)(SN)2 (2) in CD2Cl2.

Figure 2.5b. Room temperature 1H NMR Mo(O)(SN)2 (2) in CD2Cl2.

44

Figure 2.6. Room temperature UV-vis of Mo(O)2(SN)2 (1) (yellow trace) and Mo(O)(SN)2 (2) (brown trace) in DCM.

Lewis Acid requirement for nitrate reduction. Complex Mo(O)(SN)2 (2) carries out

an efficient OAT reaction with a record number of substrates.17 However, there is a limit

in the substrate scope. Holm and coworkers reported that the reaction between 2 and

nitrate does not yield 1 without providing further experimental details and

characterization. With fresh insights on the substrate activation mechanisms for Fdh and

Nap (Section 1), we thought that altering the secondary coordination environment by

Lewis acid may induce previously unseen OAT activity with 2. The OAT activity with

nitrate can be readily examined by UV-Vis spectroscopy from pronounced spectroscopic

differences of 1 and 2 (Figure 2.6). Consistent with the previous report,17 we did not

observe the formation of 1 from the reaction of Mo(O)(SN)2 (2) and [Bu4N][NO3] at

room temperature in CH2Cl2 up to 24 hours (Figure 2.7, Scheme 2.2).

45

Scheme 2.2 Reactivity of Mo(O)(SN)2 (2) and [Bu4N][NO3] in absence (top) and presence (bottom) of Sc(OTf)3 (SN = bis(4-t-butylphenyl)-2-pyridylmethanethiolate).

Figure 2.7. Room temperature UV-vis of Mo(O)(SN)2 (2) and [Bu3N][NO3] (10 equiv.) after 24 hours in DCM.

The reactivity of 2 and nitrate is remarkably altered with the addition of Lewis acid

(Scheme 2.2). Addition of Sc(OTf)3 (2 equiv.) to a CH2Cl2 solution of 2 (1 equiv.) and

[Bu4N][NO3] (10 equiv.) results in a rapid color change from brown to bright yellow. The

UV-Vis spectrum of the of the reaction mixture contains a single absorption band at 370

nm and matches that of authentic Mo(O)2(SN)2 (1) (Figure 2.8). Complex 1 generated

from the Sc3+ assisted OAT from nitrate by 2 was isolated in good yields (80%) following

46

recrystallization from dichloromethane/pentane after the removal of byproducts with

MeOH. The formation of 1 was further confirmed by IR (Figure 2.9) and 1H NMR

spectroscopy (Figure 2.10). The generation of nitrite (NO2–), the other OAT product, was

confirmed through positive Griess reagent tests58 on the MeOH soluble material.

Figure 2.8. Room temperature UV-vis of Mo(O)2(SN)2 (1) generated from Mo(O)(SN)2

(2) (1 equiv.), Sc(OTf)3 (2 equiv.) and [Bu4N][NO3] (10 equiv.) in DCM

Figure 2.9. IR spectra (KBr) of Mo(O)2(SN)2 (1) generated from Mo(O)(SN)2 (2) (1 equiv.), Sc(OTf)3 (2 equiv.) and [Bu4N][NO3] (10 equiv.) in DCM.

47

Figure 2.10. 1H NMR of Mo(O)2(SN)2 (1) generated from Mo(O)(SN)2 (2) (1 equiv.), Sc(OTf)3 (2 equiv.) and [Bu4N][NO3] (10 equiv.) (top) and authentic Mo(O)2(SN)2 (1) (bottom) in CD2Cl2.

No intermediates were observed for the novel OAT reactivity even at low temperatures.

Instead, monitoring the reaction at –40 °C shows a clean conversion of Mo(O)(SN)2 (2)

to Mo(O)2(SN)2 (1) (Figure 2.11). Addition of Sc(OTf)3 (2 equiv.) to the dichloromethane

solution of 2 (1 equiv.) and [Bu4N][NO3] (10 equiv.) results in the loss of the absorption

bands 328 and 430 nm and the emergence of a single absorption band at 370 nm with the

tight isosbestic points at 340 and 407 nm (Figure 2.11). The observed isosbestic points

match those reported for the OAT from Ph3AsO to 2 to form 1 at room temperature,17

which indicates that Sc3+ enables 2 to undergo OAT with nitrate in the same manner with

other known substrates.

48

Figure 2.11. Reaction of Mo(O)(SN)2 (2) (0.8 mM), [Bu4N][NO3] (8.0 mM), and Sc(OTf)3 (1.7 mM) followed by UV-Vis spectroscopy at –40 ºC in dichloromethane for 1.5 hours. The spectral changes correspond to the conversion of Mo(O)(SN)2 (2) (λmax = 328, 430 nm) to Mo(O)2(SN)2 (1) (λmax = 370 nm).

Nitrate as oxygen atom source: 18O labeling experiments. Isotopic labeling studies

were conducted to confirm nitrate as the oxygen atom source for the observed conversion

of Mo(O)(SN)2 (2) to Mo(O)2(SN)2 (1). Attempts at monitoring the reaction using N18O3–

were unsuccessful due to insufficient purity of commercially available 18O-labeled

nitrate. As an alternative approach, we have prepared 18O labeled 2 for the reaction with

NO3–. Doubly 18O labeled Mo(18O)2(SN)2 (118/18) was prepared through the treatment of 1

with H218O by adopting a known procedure.17 Upon 18O substitution, the molybdenum

oxygen stretches were shifted from 901 and 936 cm–1 to 887 and 858 cm–1 in the IR

spectra, consistent with the known report17 and are in good agreement with theoretical

values from reduced mass calculations (890 and 856 cm–1) (Figure 2.12). As reported for

the synthesis of 2 (see above), the 18O labeled 2, Mo(18O)(SN)2 (218) was prepared from

118/18 and triphenylphosphine.

49

Treatment of Mo(18O)(SN)2 (218) with [Bu4N][NO3] in the presence of Sc(OTf)3

was accompanied by the brown to yellow color change observed for the analogous

unlabeled reaction. The IR spectrum of the recrystallized reaction product shows major

molybdenum-oxo stretches at 867 and 924 cm–1 in between the values for 116/16 and 118/18,

suggesting the formation of the mixed labeled di-oxo species Mo(18/16O)2(SN)2 (118/16)

(Figure 2.12). Holm and co-workers reported 218 reacts with Ph2SO to yield the mixed

labeled bis-oxo compound 118/16 that was characterized by mass spectrometry,17 but the

IR features of 118/16 were not reported. To confirm the product generated from 218/NO3–

1/Sc(OTf)3 is the same O-atom transferred product obtained from 218 with other known

substrates, the reaction of 218 with trimethylamine n-oxide (TMAO) was carried out

(Scheme 2.3). The product obtained from 218/TMAO resulted in the identical IR spectrum

as the one from 218/NO3–1/Sc(OTf)3 (Figure 2.12), indicating that nitrate is the source of

the oxygen atom to generate Mo(18/16O)2(SN)2 (118/16) from Mo(18O)(SN)2 (218).

Scheme 2.3. Mo(18/16O)2(SN)2 (118/16) preparation from Mo(18O)(SN)2 (218) via Sc(OTf)3 assisted nitrate reduction (I) or trimethylamine n-oxide (II).

50

Figure 2.12. IR spectra (KBr) of Mo(16O)2(SN)2 (1, black dashed) and Mo(18O)2(SN)2

(118/18, black solid), along with Mo(16/18O)2(SN)2 (118/16) generated from 218 with [Bu4N][NO3] in the presence of Sc(OTf)3 (red solid), and Mo(16/18O)2(SN)2 (118/16) generated from 218 with trimethylamine n-oxide (blue dotted).

Fate of Nitrate. The Lewis acid assisted OAT from nitrate by Mo(O)(SN)2 (2) would

generate nitrite (NO2–) as a reaction product. The formation of nitrite was first probed by

the Griess reagent test,58 the most commonly used method of detection of nitrite. All the

reaction products and byproducts excluding 1 were extracted with MeOH, on which the

Griess test was conducted. Treatment of the MeOH soluble material with the Griess

reagent resulted in the formation of the azo dye with an absorbance band at 548 nm

(Figure 2.13) indicative of the presence of nitrite. However, only a trace amount (<1%) of

nitrite was obtained based off the prepared nitrite calibration curve although a higher

yield of nitrite was expected from the high conversion yield of 1 from 2/NO3–/Sc3+,

which indicates that there must be a secondary reaction for nitrite.

51

Figure 2.13. Room temperature UV-vis in H2O for positive Griess reagent test for nitrite generated from Mo(O)(SN)2 (2) (1 equiv.), Sc(OTf)3 (2 equiv.) and Bu4N(NO3) (10 equiv.) Prepared nitrite calibration curve (inset)

To further investigate the fate of nitrite, 15N NMR studies were carried out, for

which [Bu4N][15NO3] and [Bu4N][15NO2] were independently prepared (see section 2).

The formation or consumption of nitrate or nitrite can be readily identified by the 15N

chemical shifts for [Bu4N][15NO3] at 6.46 ppm and [Bu4N][15NO2] at 243.67 ppm in

CD2Cl2. There was no reaction between [Bu4N][15NO3] and Sc(OTf)3 (1:1 ratio), from

which only unreacted nitrate signal at 6.46 ppm was observed in 15N NMR spectrum

(data not shown). Likewise, when Mo(O)(SN)2 (2) was reacted with [Bu4N][15NO3] (1-10

equiv.) in the absence of Sc(OTf)3, only the unreacted starting reagents, 2 and

[Bu4N][15NO3] were observed in 15N- and 1H-NMR, IR, and UV-vis spectroscopy. When

the reaction of 2 (1 equiv.) and [Bu4N][15NO3] (10 equiv.) was carried out in the presence

of with Sc(OTf)3 (2 equiv.) in a sealed J Young NMR tube, the clean formation of the O-

52

atom abstracted metal product, Mo(O)2(SN)2 (1), was detected by 1H NMR spectroscopy,

which was further confirmed by UV-Vis and IR spectroscopy. However, the 15N NMR

spectrum of the reaction products did not show a signal for 15NO2– nor for any other 15N-

containing products beside excess unreacted substrate, 15NO3–. (Figure 2.14a).

Figure 2.14. Room temperature 15N NMR (in CD2Cl2) spectra of a) the reaction mixture of Mo(O)(SN)2 (2) (1equiv.), Sc(OTf)3 (2 equiv.) and Bu4N(15NO3) (10 equiv.) showing excess nitrate signal at 6.46 ppm, b) authentic [Bu4N][15NO2], and c) an equimolar mixture of [Bu4N][15NO2] and Sc(OTf)3.

The lack of 15N- signal for the anticipated product, nitrite, led us to consider a

secondary reaction for nitrite involving gas evolution. Detection of NO by utilizing

established chemical method59 and IR spectroscopy was negative. However, when the

headspace of the reaction mixture of Mo(O)(SN)2 (2)/Sc(OTf)3/[Bu4N][NO3] was

analyzed by IR spectroscopy, the characteristic signals60 reported for N2O was observed

53

at 2236 and 2212 cm–1 (Figure 2.15). N2O can be potentially generated from nitroxyl

(NO–) that may be produced from OAT reactivity of 2 with NO2–, but the presence of

large excess nitrate (10 equiv.) in the reaction mixture would make this scenario unlikely.

This led us to investigate the reaction between [Bu4N][NO2] and Sc(OTf)3. To our

surprise, gas evolution was observed upon mixing of Sc(OTf)3 and [Bu4N][NO2], in the

absence of molybdenum complexes. Addition of Sc(OTf)3 to [Bu4N][15NO2] (1:1 ratio) in

CD2Cl2 results in the complete loss of the nitrite signal at 243 ppm in the 15N NMR

(Figure 2.14c). The gas generated from the reaction of [Bu4N][NO2] with Sc(OTf)3 was

examined and identified through headspace analysis using an IR gas cell. Two features

present at 2236 and 2212 cm–1 in the IR spectrum from the headspace match the N-N

stretches of authentic N2O (figure 2.15). In addition to N2O, the headspace for the

reaction of [Bu4N][NO2]with Sc(OTf)3 was tested positive for O2 generation.

Transferring the headspace to an alkaline solution of pyrogallol, a known dioxygen

scavenger that does not react with N2O61-62, resulted in the rapid formation of a light

brown solution. The color change and appearance of an absorbance band at 409 nm in the

UV-vis spectrum (Figure 2.16) are in agreement with the alkaline pyrogallic O2 detection

reported by Karlin and coworkers.63-64 The presence of O2 and N2O in the head space of

the reaction of [Bu4N][NO2] with Sc(OTf)3 suggests that Sc(OTf)3 carries out the

unexpected disproportionation of nitrite to form O2 and NO– in which the latter dimerizes

to N2O. Although the conversion of nitrite to O2 and NO– by Sc3+ is an interesting

chemical transformation in its own right, the mechanism for the reaction is beyond the

scope of this manuscript.

54

Figure 2.15. IR spectra (gas cell) of headspace from the reaction mixtures of Mo(O)(SN)2 (2) (1 equiv.) and [Bu4N][NO3] (10 equiv.) before (blue solid) and after (red solid) addition of Sc(OTf)3 (2 equiv.), along with the spectrum from the reaction of [Bu4N][NO2] (1 equiv.) and Sc(OTf)3 (1 equiv.) (green solid) and that of authentic N2O (black dotted).

Figure 2.16. UV-vis of pyrogallol in 50% KOH (aq) before (blue trace) and after head space transfer (red trace) from reaction of Sc(OTf)3 (3.5 x 10-5

mol) and Bu4N(NO2) (3.5 x 10-5 mol).

409

55

Role of Sc3+ in observed nitrate reduction. The addition of the Lewis acidic Sc(OTf)3 is

crucial for the observed nitrate reduction by Mo(O)(SN)2 (2). It is also important how

Sc(OTf)3 is introduced to the reaction. The direct interaction between Sc(OTf)3 and

Mo(O)(SN)2 (2) is disruptive and needs to be avoided for nitrate reduction to occur.

Addition of Sc(OTf)3 to 2 in the absence of nitrate results in fast decomposition of 2 as

judged by UV-vis (Figure 2.17) and 1H NMR (Figure 2.18) spectroscopy. Likewise,

addition of Sc(OTf)3 to Mo(O)2(SN)2 (1) also leads to decomposition in the absence of

nitrate (Figure 2.19). When excess nitrate (5-10 equivalents) is present, the

decomposition of 1 or 2 by Sc(OTf)3 is no longer observed and a clean OAT chemistry

can be achieved.

Figure 2.17. Room temperature UV-vis in DCM of Mo(O)(SN)2 (2) (solid trace) before and after addition of Sc(OTf)3 (dashed).

56

Figure 2.18. 1H NMR of Mo(O)(SN)2 (2) with Sc(OTf)3 (top) and authentic Mo(O)(SN)2

(2) (bottom) in CD2Cl2.

Figure 2.19. 1H NMR of Mo(O)2(SN)2 (1) with Sc(OTf)3 (top) and authentic Mo(O)2(SN)2 (1) (bottom) in CD2Cl2.

57

Effects of Lewis acid on OAT have been reported in two different MnV(O)

systems. Nam and coworkers reported48 an advantageous effect of Lewis acid in OAT

chemistry of a non-heme Mn(V)-oxo complex in which the binding Sc3+ to the ligand

scaffold enhances the oxidizing power of Mn(V)-oxo and the OAT activity. Goldberg

and coworkers reported47 an inhibitory effect of Lewis acid in OAT reaction with a

Mn(V)-oxo porphyrinoid complex, in which the binding of Zn2+ to the ligand periphery

leads to a valence tautomerization to form a Mn(IV)-oxo π-radical cation which proceeds

OAT dramatically slower than the Mn(V)-oxo valence tautomer. In our system, the

acquired OAT activity of Mo(O)(SN)2 (2) with nitrate by Lewis acid is not likely due to

the binding of Sc3+ to the ligand frame of 2 because the SN ligand does not have basic

sites outside of the Mo chelating donor atoms, and the direct interaction between 2 and

Sc3+ leads to decomposition. However, the binding of Sc3+ to the substrate would likely

pull electron density from nitrate and induce the weakening of N-O bond which would

favor the OAT by 2 (Scheme 2.4). The proposed role for Sc3+ in the activation of NO3– is

similar to the role of Lewis acid for N2 activation on a recently reported Fe system by

Szymczak and coworkers, in which secondary sphere Lewis acids weaken and polarize

the N−N bond via ‘push-pull mechanism’.65 The importance of the substrate bond

strength in the OAT chemistry of DMSOR has been thoroughly discussed by Solomon

and coworkers.66-67 X-ray absorption spectroscopic and DFT calculation studies on the

DMSOR model complexes suggest that OAT requires the S-O bond elongation which

then subsequently facilitates electron transfer from MoIV to the sulfur atom.66 Although

molybdenum oxotranferase enzymes do not utilize Lewis acidic metal ions for the

58

substrate activation, our study shows that application of Sc3+ as an additive can emulate

the substrate bond activation, one of the key effects of the secondary environment of the

enzyme.

Scheme 2.4. Lewis acid assisted nitrate reduction by Mo(O)(SN)2 (2) to form

Mo(O)2(SN)2 (1) by oxygen atom transfer.

2.5. Conclusions. Nitrate is one of the most common contaminants in the groundwater

and poses a number of health problems. Reduction of nitrate (NO3–) to nitrite (NO2

–) is

biologically and environmentally important but is difficult to achieve outside of the

enzymatic systems. Complex Mo(O)(SN)2 (2)17 is one of the best known biomimetic

complexes possessing the oxygen atom transfer (OAT) reactivity with a wide variety of

substrates, yet 2 is not capable of reducing nitrate. In this work, we present a new

strategy that expands the reactivity of 2. Addition of a small amount of Sc3+ to the

mixture of 2/NO3– results in the activation of nitrate through a secondary coordination

interaction, which leads to an immediate and clean formation of Mo(O)2(SN)2 (1) and

nitrite (NO2–). In addition to activating nitrate for reduction, the Sc3+ additive can further

convert nitrite to gaseous molecules, N2O and O2, which can be easily removed from the

reaction solution. This work demonstrates the ability of Lewis acid additives to expand

59

the reactivity scope for existing metal complexes and bring forth novel reactivity without

the need to develop new ligand systems.

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Iron(IV)−Oxo Complex. J. Am. Chem. Soc. 2011, 133, 5236-5239.

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69

Chapter 3: Structure and Oxygen Atom Transfer

Reactivity of Dinuclear (µ-O)Molybdenum(V) Complex

70

3.1. Abstract

Nature utilizes mononuclear molybdenum oxotransferase enzymes for a wide

variety of oxygen atom transfer (OAT) reactions, including the biologically and

environmentally important reduction of nitrate (NO3−) to nitrite (NO2–) achieved by

nitrate reductase enzymes. While an abundance of MoIV(O) and MoVI(O)2 complexes

mimic the OAT reactivity displayed by dimethylsulfoxide reductase (DMSOR) enzymes,

few examples utilize nitrate as the oxygen atom donor, replicating the reactivity of the

nitrate reductase enzymes. The reactivity of early model systems was often complicated

by the rapid and sometimes irreversible formation of the dinuclear MoV2O3 species

through comproportionation of MoIV(O) and MoVIO2 species. While synthetic systems

that form MoV2O3 species are avoided as active site models, they are capable of

stochiometric and catalytic OAT reactivity. Presented here is the synthesis and crystal

structure of a MoV2O3 thiosemicarbazone species previously utilized in the catalytic

oxidation of triphenylphosphine by DMSO. Additionally, nitrate and nitrite reduction to

gaseous products is presented, demonstrating that the often maligned MoV2O3 complexes

can achieve difficult OAT chemistry.

71

3.2. Introduction. Nature utilizes mononuclear molybdenum oxotransferase enzymes for

a wide variety of oxygen atom transfer (OAT) reactions.1 The biologically and

environmentally relevant two-electron reduction of nitrate (NO3−) to nitrite (NO2−) is

carried out by nitrate reductase enzymes including periplasmic nitrate reductase (Nap)

from Desulfovibrio desulfuricans2 and membrane-bound respiratory nitrate reductase

(Nar) from Escherichia coli.3 Nap and Nar, both members of the dimethylsulfoxide

reductase (DMSOR) family of enzymes, utilize a high valent MoIV/VI metal center bound

by two pyranopterin cofactors (Figure 3.1). During the reduction of nitrate an oxygen

atom is transferred to the MoIV center generating a MoVI-oxo species. The MoIV center is

regenerated upon addition of externally provided protons and electrons completing the

catalytic cycle with the release of water resulting in the overall reactivity in equation 1.1, 4

Figure 3.1. Active site structures of the oxidized forms of (a) respiratory nitrate reductase (Nar) from Escherichia coli3 and (b) periplasmic nitrate reductase (Nap) from D. desulfuricans2 where Asp = aspartate and Cys = cysteine.

72

Interest in oxotransferase enzymes and molybdenum mediated OAT led to the

development of numerous synthetic systems that take advantage of the MoIV/VI redox

couple, utilizing a wide variety ligand scaffolds and oxygen atom donors, that are capable

of oxygen atom transfer to (equation 2) and from (equation 3) substrates.5-7 While the

oxygen atom transfer from dimethylsulfoxide (DMSO) to phosphine oxygen atom

acceptors is a hallmark of DMSOR model complexes, with systems capable of catalytic

OAT8-9, there are far less examples of oxotransferase model systems capable of OAT

from nitrate.10-13 The further development of bioinspired molybdenum complexes that

can reduce nitrate could be a useful tool in the remediation of nitrate, a pervasive

groundwater contaminant linked to adverse health effects.14

Unlike the mononuclear active sites of oxotransferase enzymes, the reactivity of

early model systems was often complicated by the formation of µ-oxo molybdenum (V)

dimers.15-17 The rapid and sometimes irreversible formation of the dinuclear MoV2O3

species through comproportionation of MoIV(O) and MoVIO2 species (equation 4) has

been well studied.5

Strategies to prevent dimer formation were developed, including the use of sterically

bulky ligands and charged complexes, and have been successfully implemented in later

73

generation oxotransferase model complexes. While dimer formation is not desired for

model systems of the mononuclear oxotransferase enzymes, its formation does not

necessarily prevent oxygen atom transfer from occurring. Stoichiometric and catalytic

OAT are possible if the dimer formation is reversible and equilibrates rapidly (scheme

3.1)16, 18-20. There are also examples of MoV2O3 species proposed to directly interact with

oxygen atom donors to generate MoVIO2 species21, with a catalytic example utilizing

Mo2O3(dtc)2I2(THF)2 (dtc = S2CNEt2) reported by Baird and co-workers (Scheme 3.2)22-

23. Schiff base and 2,6-bis(2,2-diphenyl-2-thioethyl)-pyridinate containing MoV2O3

complexes reported by Holm10, 21 and Young12 are also capable of nitrate reduction,

further demonstrating µ-oxo molybdenum (V) dimers OAT capabilities.

Scheme 3.1. OAT with dimer disproportionation.

Scheme 3.2 OAT without dimer disproportionation.

74

Recent work by Duhme-Klair and co-workers demonstrated the ability to tune the

oxygen atom transfer of a series of di-oxo molybdenum(VI) thiosemicarbazone

complexes with ligand modification (Figure 3.2)9. The rate of catalytic oxidation of

triphenylphosphine with DMSO increased with the strength of electron withdrawing

substituents of the thiosemicarbazone ligand. Absent from the work was information on

the reduced molybdenum species in the catalytic cycle, however the authors alluded to

formation of Mo(V) dimers as a possible source of catalyst deactivation in the oxygen

atom transfer activity. Our interest in the identity of the reduced molybdenum species of

the catalytic cycle and the possibility to expand the substrate scope of the system to

include nitrate led us to further develop the thiosemicarbazone system reported by

Duhme-Klair and co-workers. The isolation and characterization of the MoV2O3

thiosemicarbazone complex and its nitrate reduction ability is presented.

Figure 3.2. cis-dioxomolybdenum(VI) thiosemicarbazone (X = Me, H, I, Br, OCF3,

NO2).9

75




gloveblox or under argon using standard Schlenk line techniques. 3Å molecular sieves

were dried under vacuum for 24 hours at 250 °C prior to use. THF and pentane were

degassed then dried using an MBraun solvent purification systems under an Ar

atmosphere, and stored over activated 3 Å molecular sieves. Anhydrous dimethyl

sulfoxide was purchased from Aldrich, degassed by sparging with Ar for 30 minutes, and

stored over activated 3 Å molecular sieves for 72 hours prior to use. Ultrapure, deionized

water was obtained from a Millipore Direct-Q 3 UV Water Purification System.

Tetrabutylammonium nitrate, polymer-bound triphenylphosphine (100-200 mesh, ~3.0

mmol/g loading), trimethylamine n-oxide, 99% nitrous oxide, potassium hydroxide,

THF-d8 and pyrogallol were purchased from Sigma-Aldrich and used as received.

Scandium triflate was purchased from Strem and used as received. Tetrabutylammonium

nitrite was purchased from Sigma-Aldrich and recrystallized from THF at –35 °C prior to

use. Acetone-d6 was purchased from Cambridge Isotopes, degassed via freeze-pump-

thaw cycle (5x) and dried over 3 Å molecular sieves for 24 hours prior to use. DMSO-d6

and THF-d8 were purchased from Sigma-Aldrich and used as received. Griess Reagent

Kit for Nitrite Determination (G-7921) was purchased from Molecular Probes.

[Bu4N][15NO3] and [Bu4N][15NO2] were prepared according following the previously

reported procedure (Section 2.3).


nitrogen glovebox unless otherwise noted. Infrared spectra were recorded on a Bruker

76

Tensor 27 FTIR spectrometer. Headspace detection of N2O was performed with a Bruker

A131 IR gas cell equipped with calcium fluoride windows. Room temperature UV-Vis

spectra were recorded on a Varian Cary 50 Bio spectrometer with using screw cap UV-

Vis cuvettes, Schlenk cuvette, or a 2 mm Hellma All-Quartz Immersion probe fitted in a

24/40 Schlenk tube with a 14/20 female joint sealed with a rubber septum. NMR were

recorded with a Bruker 400MHz Avance III ultrashield spectrometer. 1H NMR were

referenced to acetone residual solvent signal (δ 2.05), DMSO residual signal (δ 2.50), or

THF residual solvent signal (δ 1.72). 15N NMR was externally referenced to nitromethane

in CD2Cl2. Elemental analysis was carried out by Intertek Pharmaceutical Services

(Whitehouse, NJ). X-ray crystallographic data was collected with a Bruker Smart Apex I

diffractometer. The structure was solved and refined using Bruker SHELXTL Software

Package.

Synthesis of Mo(O)2(LBr)(MeOH) (1). Mo(O)2(LBr)(MeOH) was synthesized following

the published procedure.9 UV-Vis (THF): λmax (εM) 233 nm (32000), 252 nm (35400),

320 nm (18000), 414 nm (5860). IR (KBr): 906, 940 cm-. 1H NMR (Acetone-d6, 400

MHz): δ 8.65 (s, 1 H), 7.83 (d, 1H, J= 2.5 Hz) 7.59 (dd, 1 H, J = 8.6 Hz, 2.5 Hz), 6.90 (d,

1H, J = 8.8 Hz ), 6.84 (br, 1H), 3.45 (q, 2H), 3.33 (s, 3H), 1.24 (t, 3H).

Synthesis of Mo2O3(LBr)2(THF)2·2THF (2). Polymer supported triphenylphosphine

(125 mg, ~0.375 mmol) was added to a stirring solution of Mo(O)2(LBr)(MeOH) (1)

(115.0 mg, 0.250 mmol) in 10.0 mL THF. The resulting dark brown solution was stirred

for 6 h then filtered through a pad of celite and concentrated to approximately 5 mL.

Recrystallization by vapor diffusion of pentane into the THF solution yielded 115.9 mg

(82.2 %) of 2 as blue-black crystals. UV-Vis (THF): λmax (εM) 236 nm (22200), 252 nm

77

(19700), 316 nm (13300), 454 nm (12200). IR (KBr): 970 cm-. 1H NMR (Acetone-d6, 400

MHz): δ 9.00 (s, 1 H), 7.93 (dd, 1 H, J = 5.9 Hz, 2.5 Hz) 7.83 (d, 1H, J= 2.5 Hz) 7.64

(dd, 1 H, J = 8.9 Hz, 2.4 Hz), 7.09 (br, 2H), 3.64 (m, 8H), 3.53 (m, 2H), 1.80 (m, 8H),

1.27 (q, 3H). Anal. Calcd. for C36H52Br2Mo2N6O9S2: C, 38.10; H, 4.64; N, 7.45. Found:

C, 38.21; H, 4.65; N, 7.50.

Reaction of Mo2O3(LBr)2(THF)2·2THF (2) and DMSO. Method 1:

Mo2O3(LBr)2(THF)2·2THF (2) (5.0 mg, 4.4 μmol) was dissolved in 0.5 mL of DMSO-d6

in a screw cap NMR tube. After half an hour the dark brown solution turned orange and

the 1H NMR was recorded. Method 2: In a screw cap UV-Vis cuvette, 100.0 uL of a 0.89

mM stock solution of 2 was added to 2.9 mL of THF and 50 μL of DMSO and the UV-

Vis was recorded after a half an hour (85% yield by UV-Vis).

Reaction of Mo2O3(LBr)2(THF)2·2THF (2) and trimethylamine n-oxide (TMAO).

TMAO (330 μL of 13.3 mM stock solution in THF/MeOH 95:5, 4.4 μmol) was added to

a stirring solution of 2 (5.0 mg, 4.4 μmol) dissolved in 5.0 mL THF. The resulting bright

yellow solution was stirred for an additional 5 minutes. After recording UV-Vis spectrum

the solution was dried to a yellow residue, then dissolved in acetone-d6. (97% yield by

UV-Vis)

Catalytic Oxygen atom transfer reactivity of Mo2O3(LBr)2(THF)2·2THF (2) with

DMSO and triphenylphosphine. Following the procedure from Duhme-Klair and

coworkers9 0.4 mL of a 255 mM triphenylphosphine in CD2Cl2 (25.5 equivalents) and 0.4

mL of 5mM 2 (0.5 equivalents) in DMSO-d6 were mixed in a J-Young NMR tube. 31P

NMR were recorded periodically for 70 hours. The concentration of triphenylphosphine

at time t , [PPh3]t, was determined from integrating the triphenylphosphine signal at -6

78

ppm and triphenylphosphine oxide signal at 26 ppm. The pseudo-first-order rate

constants (kobs) was determined from the slope of the equation ln([PPh3]t/[PPh3]0) = -kobst

where [PPh3]o was the starting triphenylphosphine concentration. Calculated kobs 107[s-] =

18. Reported kobs 107[s-] = 21(1).

Reaction of Mo2O3(LBr)2(THF)2·2THF (2) and [Bu4N][NO3]. 680 μL of a 13.1 mM

stock solution of [Bu4N][NO3] (8.86 μmol) in THF was added to a stirring solution of 2

(10.0 mg, 8.86 μmol) in 5.0 mL THF. The dark brown solution gradually turned orange-

brown over 2 hours. After recording a UV-Vis of an aliquot of the reaction mixture, the

sample was dried to an oily brown residue that was then dissolved in acetone-d6 and 1H

NMR was recorded. After recording the 1H NMR, the sample was dried onto KBr and the

IR recorded. (89% yield by UV-Vis)

Reaction of Mo2O3(LBr)2(THF)2·2THF (2) and 0.5 equivalents of [Bu4N][NO3]. 340

μL of a 13.1 mM stock solution of [Bu4N][NO3] (4.43 μmol) in THF was added to a

stirring solution of 2 (10.0 mg, 8.86 μmol) in 5.0 mL THF. The dark brown solution

gradually turned orange-brown over 2 hours. After recording a UV-Vis of an aliquot of

the reaction mixture, the sample was dried to an oily brown residue that was then

dissolved in acetone-d6. (93% yield by UV-Vis)

Reaction of Mo2O3(LBr)2(THF)2·2THF (2) and [Bu4N][NO2]. 640 μL of a 13.9 mM

stock solution of [Bu4N][NO2] (8.86 μmol) in THF was added to a stirring solution of 2

(10.0 mg, 8.86 μmol) in 5.0 mL THF. The dark brown reaction turned orange with 5

minutes of stirring. The reaction was dried to a yellow/orange residue after taking aliquot

for UV-Vis following an additional 30 minutes of stirring. The residue was dissolved in

acetone-d6 and 1H NMR was recorded.

79

Reaction of Mo2O3(LBr)2(THF)2·2THF (2) and [Bu4N][15NO3]. In a J-Young NMR

tube 2 (18.5 mg, 34.8 μmol) and [Bu4N][NO3] (5.0 mg, 16.4 μmol) were dissolved in 0.4

mL acetone-d6. The dark brown solution gradually turned orange over 2 hours. 15N NMR

was then recorded.

Reaction of Mo(O)2(LBr)(MeOH) (1) and 2 equivalents [Bu4N][NO2]. [Bu4N][NO2]

(5.0 mg, 16.4 μmol) in 1.0 mL THF was added to a stirring solution of 1 (16.0 mg, 16.4

μmol) in 4.0 mL THF. The dark brown/orange solution rapidly turned bright orange and

generated yellow precipitate. After recording a UV-Vis of the reaction mixture, the

orange solution was filtered, dried, and dissolved in acetone-d6 and 1H NMR recorded.

Reaction of Mo(O)2(LBr)(MeOH) (1) and 2 equivalents [Bu4N][15NO2]. In a J-Young

NMR tube 1 (16.0 mg, 16.4 μmol) and [Bu4N][NO2] (5.0 mg, 16.4 μmol) were dissolved

in 0.6 mL acetone-d6. The dark brown solution rapidly turned orange, and developed

yellow precipitate over the time it took to record the 15N NMR.

Detection and Quantification of Nitrite. The above reaction mixtures were dried and

extracted with 1.00 mL of ultrapure, deionized H2O with sonication then filtered through

celite to remove any insoluble material. Detection of nitrite with Griess reagent was

performed according to manufacturer’s instructions. Nitrite concentration was determined

using a NaNO2 calibration curve prepared in ultrapure, deionized H2O.

UV-Vis monitoring of the reaction of Mo2O3(LBr)2(THF)2·2THF (2) and

[Bu4N][NO3]. After blanking spectrometer with 4.0 mL THF, 1.0 mL of a stock solution

of 2 (0.86 μmol) was injected and the spectrum of 2 was recorded. A stock solution of

80

[Bu4N][NO3] was diluted to 1.0 mL (0.86 μmol) in THF was injected into the solution of

2, after which a series of spectra were measured for 3 hours.

UV-Vis monitoring of the reaction of Mo2O3(LBr)2(THF)2·2THF (2) and

[Bu4N][NO3], synthetic scale. [Bu4N][NO3] (5.0 mg, 16.3 μmol) were dissolved in 1.0

mL of THF was added to a stirring solution of MoO3(LBr)2(THF)2·2THF (2) (18.5 mg,

16.3 μmol) in 9.0 mL THF. The reaction progress was monitored for 3 hours by

periodically recording UV-Vis spectra of 50 μL aliquots of the reaction mixture diluted to

3 mL of THF.

UV-Vis monitoring of the reaction of Mo2O3(LBr)2(THF)2·2THF (2), Sc(OTf)3 and

[Bu4N][NO3] (1:1:10). ]. After blanking spectrometer with 5.0 mL THF, 1.0 mL of a

stock solution of 2 (0.86 μmol) was injected and the spectrum of 2 was recorded. A stock

solution of [Bu4N][NO3] (8.6 μmol) was mixed with a stock solution of Sc(OTf)3 (0.86

μmol) and diluted to 2.0 mL in THF then injected into the solution of 2, after which a

series of spectra were measured for 2 hours.

Detection of N2O by IR spectroscopy. In a glovebox, of Mo(O)2(LBr)(MeOH) (1) (50.0

mg, 108 μmol) and [Bu4N][NO2] (15.7 mg, 54.0 μmol) were dissolved in 10.0 mL of

THF in a 25 mL Schlenk flask. After recording the background of the evacuated IR gas

cell, the Schlenk flask containing the reaction was connected to the IR gas cell with a

three-way stopcock and Tygon tubing. After evacuating the tubing connecting the gas

cell and flask, the system was left under static vacuum. The headspace from the reaction

flask was transferred by opening the stopcocks of the flask and IR gas cell. The stopcocks

were then closed, and the IR spectrum was recorded. The same procedure was employed

for the detection of N2O from the reaction of [Bu4N][NO3] and MoO3(LBr)2(THF)2·2THF

81

(2) and using a solution of [Bu4N][NO3] (6.7 mg, 22.0 μmol) and 2 (50.0 mg, 44.0 μmol)

in 25.0 mL of THF.

Detection of O2 from the reaction of of Mo(O)2(LBr)(MeOH) (1) and [Bu4N][NO2]

with alkaline pyrogallol. In a 25 mL Schlenk flask Pyrogallol (500.0 mg) was dissolved

with stirring in 10.0 mL of deoxygenated 50% KOH (aq) solution. A Schleck cuvette (1

cm pathlength) fitted with a 14/20 male connector was connected to the 25 mL flask

containg the alkaline pyrogallol solution under positive Ar flow. The UV-Vis spectrum of

the faint yellow alkaline pyrogallol solution was recorded. The flask was then

reconnected to the Schlenck line through a three-way stopcock. In a glovebox

[Bu4N][NO2] (10.0 mg, 34.7 μmol) and 1 (32.0 mg, 69.3 μmol) were dissolved in 10.0

mL of THF in a 25 mL Schlenk flask. Once the reaction was completed the Schlenck

flask connected to the pyrogallol flask with Tygon tubing through the 3-way stopcock.

The system was left under static vacuum after evacuating the pyrogallol containg flask

and tubing connecting the two flasks. The headspace was transferred by opening the

stopcocks on both Schlenck flasks. The light yellow solution rapidly darkened. After 30

mins the stopcocks were resealed and the UV-Vis of the darkened pyrogallol solution

was recorded.

3.4. Results Discussion

Preparation of Mo(O)2(LBr)(MeOH) (1) and Mo2O3(LBr)2(THF)2·2THF (2). Complex

Mo(O)2(LBr)(MeOH) (1) (Figure 3.2, X = Br) was prepared following the reported

procedure and isolated as an orange brown powder in 95% yield (99% reported).9 The IR

82

(νMo=O = 906, 940 cm–1) (Figure 3.3) and diamagnetic 1H NMR in DMSO-d6 spectral

features (Figure 3.4) are in good agreement with the published values.9, 24 The UV-Vis

absorption spectra of 1 was not previously reported and is presented in THF (λmax = 252

nm) (Figure 3.5). The 1H NMR of 1 is also presented in Acetone-d6 (Figure 3.6a).

Figure 3.3. KBr IR of Mo(O)2(LBr)(MeOH) (1).

Figure 3.4. 1H NMR Mo(O)2(LBr)(MeOH) (1) in DMSO-d6 (400 MHz).

83

Figure 3.5. Room temperature UV-vis of Mo(O)2(LBr)(MeOH) (1) in THF.

Initial attempts to prepare a MoIVO or MoV2O3 species from 1 utilizing a variety

of phosphines, including triphenyl-, trimethyl-, and trioctyl phosphine, were hindered by

the inability to remove the phosphine oxides generated in the reaction, due to similar

solubility of the phosphine oxides as the molybdenum containing product. The phosphine

oxide free product was obtained by stirring a solution of 1 in THF with polymer

supported triphenylphosphine. Following the reaction, the polymer bound phosphine

oxide is removed from solution by filtration. Vapor diffusion of pentane into the dark

brown THF solution over two days resulted in the formation of a blue-black diffraction-

quality crystals identified as Mo2O3(LBr)2(THF)2·2THF (2) through X-ray structure

determination (Figure 3.7).

84

Figure 3.6. Room temperature 1H NMR (in acetone-d6, 400MHz) spectra of a) of Mo(O)2(LBr)(MeOH) (1) and b) Mo2O3(LBr)2(THF)2·2THF (2).

The crystal structure of 2 contains bridging and terminal molybdenum oxygen

bonds and molybdenum oxygen angles that are in good agreement with structurally

similar Mo2VO3 dimers22, 25-26. In addition to the two THF molecules bound to the N-H of

the ligand in the solid state, two THF molecules are bound trans to the terminal

molybdenum oxygen bonds. The Mo-O(THF) bond lengths of 2 are similar in length to

the long Mo-O(THF) bond lengths of Mo2O3(dtc)2I2(THF)2 (dtc = S2CNEt2) reported by

Baird and co-workers.25 Like Mo2O3(dtc)2I2(THF)2, when 2 is dissolved in poorly

coordinating, such as acetone-d6, only unbound THF is observed in the 1H NMR (δ 3.63

85

and 1.79), suggesting that the THF is dissociated from 2. A diamagnetic 1H NMR spectra

is obtained for 2 in acetone-d6 (Figure 3.6b) indicative of antiferromagnetic coupling of

the MoV centers and appears as one species is in solution. However, when

Mo2O3(LBr)2(THF)2·2THF (2) is dissolved in THF-d8 the peaks in the 1H NMR are split

into two sets of signals with ~7:3 intensity (Figure 3.8) with unbound THF signals

located at δ 3.62 and δ 1.79. This could be attributed to disproportionation of

Mo2O3(LBr)2(THF)2·2THF (2) into MoIV(O) and MoVI(O)2 species (equation 4) that does

not occur in acetone but is possible in THF. Mo(O)2(LBr)(MeOH) (1) 1H NMR was

recorded in THF-d8 in attempts to identify peaks present in the NMR of

Mo2O3(LBr)2(THF)2·2THF (2) to a dioxo species in solution. While some of the signals

split signals of Mo2O3(LBr)2(THF)2·2THF (2) match the signals present in 1 in THF-d8,

the presence of MeOH in 1 complicates the comparison. Diffusion ordered spectroscopy

(DOSY) NMR experiments will be undertaken to approximate the molecular weight of

the species or specie in solution. The split signals could also be caused by dynamic

processes in THF. Separate signals could arise from ligand exchange of THF, resulting in

bound and unbound THF species. Exchange of bridging and terminal oxos through a low

energy intramolecular process, resulting in syn and anti complexes in equilibrium has

also been reported for MoV2O3 27. Future work will include variable temperature NMR

experiments to see if dynamic processes are occurring for 2 in THF.

The UV-Vis of 2 in THF (λmax = 252 nm) is reminiscent of previously reported

Mo2VO3 Schiff base dimers with a strong visible band near 450 nm (Figure 3.9). KBr IR

of 2 shows a single strong absorption at 970 cm-1 assigned to ν(Mo=O) (Figure 3.10).

86

Complex 2 is readily soluble in THF, acetone, dimethylformamide, has poorer solubility

in dichloromethane, toluene, and benzene, and is insoluble in pentane.

Figure 3.7. Thermal ellipsoid plot of Mo2O3(LBr)2(THF)2·2THF (2) projected at the 50% probability level. Selected bond distances (Å): Mo(1)–O(1) 1.8722(2), Mo(1)–O(2) 1.6724(18), Mo(1)–O(3) 2.0092(18), Mo(1)–O(4) 2.4262(18), Mo(1)–N(1) 2.178(2), Mo(1)–S(1) 2.4025(7), N(3)–O(5) 2.8961. Selected bond angles (°): O(2)–Mo(1)–O(1) 102.66(7), O(2)–Mo(1)–O(3) 101.58(9), O(2)–Mo(1)–S(1) 101.75(7), O(2)–Mo(1)–N(1) 96.27(9), O(2)–Mo(1)–O(4) 173.78(8). H-atoms except H(3) (H attached to N(3)) have been omitted for clarity.

Figure 3.8. Room temperature 1H NMR (in THF-d8, 400MHz) spectra of a) of Mo(O)2(LBr)(MeOH) (1) and b) Mo2O3(LBr)2(THF)2·2THF (2).

a

b

87

Figure 3.9. Room temperature UV-vis of Mo2O3(LBr)2(THF)2·2THF (2) in THF.

Figure 3.10. KBr IR of Mo2O3(LBr)2(THF)2·2THF (2).

88

Mo2O3(LBr)2(THF)2·2THF (2) with O-Atom Donors. The ability of 2 to take part in

oxygen atom transfer was investigated using several commonly used O-atom donors.

Dissolution of Mo2O3(LBr)2(THF)2·2THF (2) in DMSO-d6 resulted in the formation of an

orange solution whose 1H NMR (Figures 3.11 and 3.12) matches authentic

Mo(O)2(LBr)(MeOH) (1) without MeOH, where the vacant coordination site is occupied

by THF resulting in of Mo(O)2(LBr)(THF) (3). Di-oxo formation is further evident from

the UV-Vis spectra of 2 in THF with added DMSO (Figure 3.13), which results in the

loss of the 454 nm (εM =12200) absorbance band, and the increase and shift of the 316

nm (εM = 13300) absorbance band to 320 nm (εM = 18000), that matches the spectra of

authentic 1 in THF (85% yield by UV-Vis). Reaction of 2 with trimethylamine n-oxide

(TMAO) in THF resulted in the rapid formation of a yellow/orange solution and

formation of the molybdenum dioxo product as demonstrated in the UV-Vis in THF (97

% yield by UV-Vis) and 1H NMR in acetone-d6.

89

Figure 3.11. Overlay of 1H NMR Mo(O)2(LBr)(MeOH) (1) in DMSO-d6 (bottom) and 1H NMR Mo2O3(LBr)2(THF)2·2THF (2) in DMSO-d6 (top) (400 MHz) (5-0 ppm).

Figure 3,12. Overlay of 1H NMR Mo(O)2(LBr)(MeOH) (1) in DMSO-d6 (bottom) and 1H NMR Mo2O3(LBr)2(THF)2·2THF (2) in DMSO-d6 (top) (400 MHz) (10-5 ppm).

90

Figure 3.13 Room temperature UV-vis of Mo2O3(LBr)2(THF)2·2THF (2) before (blue trace) and after the addition of 100 equivalents DMSO (orange trace) in THF.

The possible role of 2 in the catalytic OAT chemistry reported by Duhme-Klair9

and co-workers was investigated. Replacing the one equivalent of Mo(O)2(LBr)(MeOH)

(1) ([cat] = 5mM) with 0.5 equivalents of Mo2O3(LBr)2(THF)2·2THF (2) ([cat]=2.5mM)

under the catalytic conditions reported for the OAT reaction between triphenylphosphine

and DMSO yielded a similar pseudo-first order rate constant (kobs) of 18s- that is similar

to the reported kobs of 21s-. Dissolution of 0.5 equivalents of 2 in DMSO results in the

formation of 1 equivalent of a dioxo species that then follows the previously reported

catalytic cycle. These findings suggest that under the catalytic conditions reported that

the dinuclear 2 directly participates in the OAT reaction (Scheme 3.2), or an equilibrium

with a MoIVO and MoVIO2 species and 2 exists allowing for reactivity with the MoIV

mono-oxo species (Scheme 3.1). Regardless of the route, 2 has been shown to participate

in the catalytic cycle, and further demonstrates MoV2O3 ability to take part in useful OAT

reactions.

91

The OAT substrate scope for Mo2O3(LBr)2(THF)2·2THF (2) was further expanded

to include biologically and environmentally relevant nitrate (NO3−). Treatment of

Mo2O3(LBr)2(THF)2·2THF (2) with [Bu4N][NO3] in THF was accompanied by the

gradual dark brown to orange color change reminiscent of the reaction of 2 with DMSO.

The UV-Vis of the of the reaction mixture (Figure 3.14) features the loss of the 450 nm

absorption band and contains the absorption bands at 320 and 414 nm found in authentic

Mo(O)2(LBr)(MeOH) (1) and Mo(O)2(LBr)(THF) (3). 1H NMR of the reaction product in

acetone-d6 (Figure 3.15b) further confirms the formation of 3 from nitrate reduction with

2. KBr IR of the reaction mixture (Figure 3.16) contain two stretches at 938 and 900 cm−

attributed molybdenum-oxo stretches (νMo=O (1) = 906, 940 cm− ), however the spectrum

is dominated by tetrabutylammonium features (~1500 cm−). Attempts to remove

tetrabutylammonium products from the nitrate reduction reaction mixture were

unsuccessful due to similar solubility of the di-oxo molybdenum product.


92

Figure 3.15. Overlay of a) Mo2O3(LBr)2(THF)2·2THF (2) with [Bu4N][NO3] (0.5 equiv.) b) Mo2O3(LBr)2(THF)2·2THF (2) with [Bu4N][NO3] (1 equiv.) c) Mo2O3(LBr)2(THF)2·2THF (2) with [Bu4N][NO2] (1 equiv.) d) Mo(O)2(LBr)(MeOH) (1) in acetone-d6 (400 MHz).

Figure 3.16. KBr IR of reaction mixture of Mo2O3(LBr)2(THF)2·2THF (2) with [Bu4N][NO3] (1 equivalent).

a

b

c

d

93

Monitoring the reaction of Mo2O3(LBr)2(THF)2·2THF (2) with [Bu4N][NO3] in

THF by UV-Vis demonstrates the concentration dependence of the reaction. At UV-Vis

concentrations (0.15 mM) the loss of the 454 nm absorption band is accompanied with

the growth of the 320 nm absorption band, analogous to the reaction of 2 and DMSO, and

the emergence of new absorption features at 365 and 400 nm (Figure 3.17). Monitoring

the reaction of 2 and [TBA][NO3] at the synthetic scale concentration (1.6 mM) (Figure

3.18) by UV-Vis over the same time results in the loss of the 454 nm absorption band

accompanied with the growth of the 320 and 414 nm absorption bands observed in 1 and

3.


94


The fate of the nitrite (NO2−) generated from the OAT from nitrate (NO3−) by

Mo2O3(LBr)2(THF)2·2THF (2) was examined. After extracting the dried reaction mixture

with ultrapure water, Griess reagent test,28 the most commonly used method of detection

of nitrite, was then used to detect the nitrite in the reaction products. Treatment of the

water-soluble material with the Griess reagent resulted in the formation of the azo dye

with an absorbance band at 548 nm (Figure 3.19) indicative of the presence of nitrite.

However, only a trace amount (<1% yield) of nitrite was obtained based off the prepared

nitrite calibration curve.

95

Figure 3.19. Room temperature UV-vis in H2O for positive Griess reagent test for nitrite generated from Mo2O3(LBr)2(THF)2·2THF (2) (1 equiv.) [Bu4N][NO3] (1 equiv.) Prepared nitrite calibration curve (inset).

15N NMR studies were carried out to further investigate the fate of nitrite. The

consumption of nitrate with generation of nitrite can be readily identified by the 15N

NMR chemical shifts for [Bu4N][15NO3] at 6.46 ppm and [Bu4N][15NO2] at 243.67 ppm

in acetone-d6. Addition of [Bu4N][15NO3] to 2 in acetone-d6 results in the complete loss

of the 6.46 ppm signal in 15NMR without the formation of the 243.67 ppm signal for

nitrite (Figure 3.20). In previous work by Holm11, 21, clean nitrate reduction via MoV2O3

complexes required nitrite scavenging sulfamic acid to prevent further reactivity of the

generated nitrite with the di-oxo molybdenum products. The loss of the nitrite signal in

the 15N NMR and low concentrations of nitrite as determined by the Griess reagent test

suggests that Mo(O)2(LBr)(THF) (3) generated through OAT with

Mo2O3(LBr)2(THF)2·2THF (2) and [Bu4N][NO3] can further react with nitrite. This was

confirmed by reacting two equivalents of Mo(O)2(LBr)(MeOH) (1) with [Bu4N][15NO2] in

96

acetone-d6. The rapid reaction was accompanied by precipitation of yellow solid with the

loss of the 243.67 ppm signal in 15N NMR (Figure 3.21) and the formation of a broad

shoulder in at 360 nm in the UV-Vis spectrum (Figure 3.22) and new peaks in the 1H

NMR (Figure 3.23).

Figure 3.20. Room temperature 15N NMR (in acetone-d6) spectra of a) [Bu4N][15NO3] showing nitrate signal at 6.46 ppm, b) reaction of Mo2O3(LBr)2(THF)2·2THF (2) and [Bu4N][15NO3].

97

Figure 3.21. Room temperature 15N NMR (in acetone-d6) spectra of a) Bu4N[15NO2] showing nitrate signal at 243.67 ppm, b) reaction of Mo(O)2(LBr)(MeOH) (1) and [Bu4N][15NO2 ] (2 equivalents).

Figure 3.22. Room temperature UV-vis of Mo(O)2(LBr)(MeOH) (1) (1 equivalent) with [Bu4N][NO2] (2 equivalent) in THF.

243.67 a

b

98

Figure 3.23. Overlay of a) Mo(O)2(LBr)(MeOH) (1) b) Mo(O)2(LBr)(MeOH) (1) (1 equivalent) with [Bu4N][NO2] (2 equivalents) in acetone-d6 (400 MHz).

When the headspace of the reaction mixture of Mo(O)2(LBr)(MeOH) (1) with two

equivalents of [Bu4N][NO2] in THF was analyzed by IR spectroscopy, the characteristic

signals29 reported for nitrous oxide (N2O) were observed at 2236 and 2212 cm–1 (Figure

3.24).

a

b

99

Figure 3.24. IR spectra (gas cell) of headspace from the Mo(O)2(LBr)(MeOH) (1) (1 equivalent) with [Bu4N][NO2] (2 equivalents) (orange solid) and that of authentic N2O (blue dotted).

Transferring the headspace of the reaction to an alkaline solution of pyrogallol30, a

commonly used oxygen scavenger, resulted in darkening of the solution, and the

appearance of an absorbance band at 409 nm in the UV-Vis spectrum (Figure 3.25)

indicating that O2 is generated in addition to N2O. The absorption band at 365 nm is

attributed to decomposition of Mo(O)2(LBr)(THF) (3) with nitrite generated from the

reaction of Mo2O3(LBr)2(THF)2·2THF (2) with [Bu4N][NO3] (Figure 3.17). Previous

work by our group has demonstrated the ability of Sc(OTf)3 to act as a nitrite scavenger,

resulting in the formation of nitrous oxide and O2 (Section 2.4). Monitoring the reaction

of Mo2O3(LBr)2(THF)2·2THF (2) (0.15 mM) with [Bu4N][NO3] (10 equivalents) and

Sc(OTf) (2 equivalents) in THF by UV-Vis demonstrates the ability cleanly convert 2 to

3 when the NO2− is scavenged by the Lewis acid additive (Figure 3.26).

100

Figure 3.25. UV-vis of pyrogallol in 50% KOH (aq) before (blue trace) and after head space transfer (orange trace) from the reaction of Mo(O)2(LBr)(MeOH) (1) (1 equivalent) with [Bu4N][NO2] (2 equivalents).

Figure 3.26. Mo2O3(LBr)2(THF)2·2THF (2) (0.15 mM), Sc(OTf)3 (0.15 mM), and [Bu4N][NO3] (1.5 mM) followed by UV-Vis spectroscopy at room temperature in THF for 2 hours. The spectral changes correspond to the conversion of Mo2O3(LBr)2(THF)2·2THF (2) to Mo(O)2(LBr)(THF) (3).

101

The ability to generate Mo(O)2(LBr)(THF) (3) from OAT from nitrite to

Mo2O3(LBr)2(THF)2·2THF (2) was also examined. Addition of [Bu4N][NO2] to 2

generated an orange solution in shorter reaction times (~ 5 mins) than the reaction of 2

and [Bu4N][NO3] under analogous reaction conditions. The UV-Vis spectrum of the

reaction mixture in THF (Figure 3.27) is remarkably similar to the UV-Vis spectrum of

Mo(O)2(LBr)(MeOH) (1) with two equivalents of [Bu4N][NO2] (Figure 3.22), with both

exhibiting the loss of the 454 nm absorption band of 2 with the appearance of a broad

shoulder around 360-370 nm. The 1H NMR of the reaction of 2 with [Bu4N][NO2] does

not show clean formation of 3 (Figure 3.15), but is reminiscent of the reaction of 1 with

[Bu4N][NO2] (Figure 3.28). Generation of 3 from NO2− followed by decomposition

seems plausible, due to the observed decomposition of 1 with nitrite.


102

Figure 3.28. Overlay of a) Mo2O3(LBr)2(THF)2·2THF (2) with [Bu4N][NO2] (1 equiv.) and b) Mo(O)2(LBr)(MeOH) (1) (1 equivalent) with [Bu4N][NO2] (2 equivalents) in acetone-d6 (400 MHz).

To further investigate nitrite reactivity, 2 was reacted with 0.5 equivalents of

[Bu4N][NO3]. The formation of Mo(O)2(LBr)(THF) (3) is shown in the UV-Vis spectrum

of the reaction product in THF (Figure 3.29) and matches the UV-Vis of authentic

Mo(O)2(LBr)(MeOH) (1), and Mo(O)2(LBr)(THF) (3) generated by OAT from DMSO or

trimethylamine n-oxide and 2 (see above). 1H NMR of 2 and 0.5 equivalents of

[Bu4N][NO3] (Figures 3.15 and 3.30) further indicates the consumptions of 2 with the

formation of 4 equivalents of dioxo species. The formation of the dioxo species 3 from 2

and 0.5 equivalents of [Bu4N][NO3], suggests that 2 reacts with nitrate forming two

equivalents of 3 and the nitrite formed is then consumed with the remaining 2 in solution

forming an additional two equivalents 3 and a nitroxyl (NO−) product (Scheme 3.3). It is

possible that nitroxyl (NO−) generated from the nitrite reduction could further react to

form N2O through formation of HNO with trace water or H+ followed by rapid

103

dehydrative dimerization31-32. Formation of the proposed nitroxyl product from the

reaction of 2 with 0.5 equivalents of [Bu4N][NO3] is supported by the appearance of N2O

in the headspace of the reaction mixture as identified by IR spectroscopy (Figure 3.31).

Figure 3.29. Room temperature UV-vis of Mo2O3(LBr)2(THF)2·2THF (2) (1 equivalent) with [Bu4N][NO3] (0.5 equivalent) in THF.

Figure 3.30. Mo2O3(LBr)2(THF)2·2THF (2) with [Bu4N][NO3] (0.5 equivalents) in acetone-d6 (400 MHz) demonstrating the ~4:1 ratio of Mo(O)2(LBr)(sol) (3) to tetrabutylammonium. −CH3 of 3 at 1.22 ppm. −CH3 of tetrabutylammonium at 0.99 ppm and −CH2−CH3 of tetrabutylammonium at 1.44 ppm.

104

Figure 3.31. IR spectra (gas cell) of headspace from the Mo2O3(LBr)2(THF)2·2THF (2) (1 equivalent) with [Bu4N][NO3] (0.5 equivalents) (orange solid) and that of authentic N2O (blue dotted).

Scheme 3.3. Proposed Mo2O3(LBr)2(THF)2·2THF (2) reactivity with 1.0 and 0.5 equivalents NO3−.

3.5. Conclusion

Nature utilizes high valent MoIV/VI metal centers in mononuclear oxotransferase

enzymes that are capable of a wide variety of oxygen atom transfer reactions. While

105

molybdenum(V) oxo formation is precluded in biological systems, it is pervasive in

oxotransferase model complexes, and is widely viewed as undesirable when attempting to

model the enzyme active sites. Strategies to prevent dimer formation have been

developed and are widely utilized. While MoV2O3 formation can complicate reactivity,

it’s formation does not necessarily prevent stoichiometric or catalytic OAT product

formation. We have demonstrated that Mo2O3(LBr)2(THF)2·2THF (2) can reduce nitrate,

a pervasive ground water contaminant, to gaseous products, in addition to being involved

in the catalytic OAT activity previously reported by Duhme-Klair9.

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μ-Peroxo Iron/Copper Complexes. Inorg. Chem. 2005, 44 (20), 7014-7029.

110

31. Hughes, M. N., Relationships between nitric oxide, nitroxyl ion, nitrosonium

cation and peroxynitrite. Biochimica et Biophysica Acta (BBA) - Bioenergetics 1999,

1411 (2), 263-272.

32. Shafirovich, V.; Lymar, S. V., Nitroxyl and its anion in aqueous solutions: Spin

states, protic equilibria, and reactivities toward oxygen and nitric oxide. Proceedings of

the National Academy of Sciences 2002, 99 (11), 7340-7345.

111

Chapter 4: Lewis Acid Assisted Perchlorate Reduction

with Dinuclear Molybdenum(V)(µ-Oxo) Complex

112

4.1. Abstract

Perchlorate is a pervasive groundwater contaminant. The long term environmental

impact and health effects from perchlorate contamination is still largely unknown.

Remediation is challenging due to the high solubility of perchlorate salts in water and

their relative kinetic inertness to reduction. Nature utilizes the high valent molybdenum

oxotransferase enzyme perchlorate reductase (PcrAB) to reduce perchlorate (ClO4− ) and

chlorate (ClO3−) to chlorite (ClO2−). While structural perchlorate reductase active site

models exist, they are not capable of the difficult reduction of perchlorate. Synthetic

systems capable of perchlorate reduction to chloride have been reported but are rare.

Presented is the reduction of perchlorate by a MoV2O3 thiosemicarbazone complex.

Addition of the Lewis acid Sc(OTf)3 dramatically enhances the rate of perchlorate

reduction to the proposed chloride product.

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4.2. Introduction.

Perchlorate (ClO4−) salts are used in a wide variety of industrial and agricultural

applications including manufacture of industrial lubes, dyes, rubbers, fertilizers, paints, in

electroplating processes, and battery manufacturing. Over 90% of all perchlorates salts

are manufactured as the strong oxidant ammonium perchlorate. Ammonium perchlorate

is widely used in solid rocket fuels and in the production of pyrotechnics and munitions

owing to its high oxygen content.1-2 Perchlorate salts are a pervasive ground water

contaminant due to their high solubility in water and organic solvents. Even though

perchlorates are strong oxidants and can be highly reactive solids, when dissolved

perchlorate salts become nonreactive and stable due to high kinetic barriers.1, 3 The extent

of perchlorate contamination of groundwater was not realized prior to 1997. Only after

the development of better techniques for perchlorate detection in groundwater has the

extent of perchlorate contamination been established.3 While long-term health effects of

consumption of perchlorate contaminated water are still largely unknown, it is known

that perchlorate competitively inhibits the uptake of iodide by the thyroid gland, altering

the production of hormones in the body, and is a potential source of hypothyroidism.4-5

Chlorate and chlorite formed from the reduction of perchlorate can lead to hemolytic

anemia in mammals, and have been shown to be taken up by plants grown in

contaminated soils.4-5 Remediation of perchlorate is needed and remains a challenge.

Typical water treatments, such as carbon adsorption, is complicated by the low tendency

of perchlorate to adsorb to surfaces and its kinetic inertness to reduction.2-3

Like nitrate (NO3−), perchlorate is utilized by some bacteria for metabolic

processes. Perchlorate reductase (PcrAB) is a member of the dimethylsulfoxide reductase

114

(DMSOR) family of enzymes and uses perchlorate and chlorate as the terminal electron

acceptors during anaerobic respiration.6-7 The active site of perchlorate reductase from A.

suilum is closely related to the active site of the membrane-bound respiratory nitrate

reductase NarG found in E. coli, with both featuring two pyranopterin cofactors and

aspartate (Asp) residue bound to a MoIV/VI metal center (Scheme 4.1). Perchlorate

reductase (PcrAB) reduces perchlorate (ClO4− ) and chlorate (ClO3−) to chlorite

(ClO2−) through oxygen atom transfer (OAT) to the MoIV center to form a MoVI-oxo

species that releases water on the addition of two external electrons and protons.6, 8

Perchlorate reducing bacteria, such as A. oryzae, often contain chlorite dismutase (Cld), a

Fe heme containing enzyme, that further converts chlorite (ClO2−) to molecular oxygen

and chloride (Cl−) for the overall reduction of chlorine shown in Scheme 4.2.9-10

Scheme 4.1. Perchlorate reduction by A. suilum PcrAB (adapted from Ref. 6).

115

Scheme 4.2. Reduction of perchlorate (ClO4−) to chloride (Cl−) and molecular oxygen by perchlorate reductase (PcrAB) and chlorite dismutase (Cld).

New remediation technologies have taken advantage of the enzymes utilized by

nature. Perchlorate reduction to chloride and molecular oxygen utilizing free and lipid

encapsulated perchlorate reductase and chlorite dismutase isolated from A. oryzae has

been achieved, and highlights the potential of biocatalysts in perchlorate remediation.9, 11

In addition to biocatalysis, the reduction of perchlorate to chloride through hypochlorite

(ClO−) has been achieved using transition metal complexes. Catalytic reduction of

perchlorate to chloride under mild conditions utilizing organic sulfides with the air and

water stable ReV(O)(hoz)2Cl or [ReV(O)(hoz)2(OH2)]OTf (hoz = [2-(2’-hydroxyphenyl)-

2-oxazoline], OTf= trifluoromethanesulfonate) was reported by Abu-Omar in 200012 .

Recently Mosch-Zanetti reported similar reduction of perchlorate to chloride with Rev-

oxo complexes with tetradentate iminophenolate ligands (Scheme 4.3).13 In addition to

nitrate reduction, catalytic perchlorate reduction to chloride and oxide products with a

FeII azafulvene-amine complex was recently reported by Fout and co-workers.14

Interestingly, a molybdenum bis(dithiolene) structural and functional DMSOR model

complex reported by Holm15 that was capable of nitrate reduction was not capable of

perchlorate reduction.

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Scheme 4.3. Catalytic reduction of perchlorate (ClO4−) to chloride (Cl−) by ReV-oxo complexes (adapted from Ref. 13).

Inspired by Fout and co-workers’ ability to reduce perchlorate with a complex

that also demonstrated nitrate reduction capabilities, we investigated the ability to reduce

perchlorate using biomimetic Mo2O3(LBr)2(THF)2·2THF (2). The effect of the Lewis acid

additive Sc(OTf)3 on the reactivity was also investigated. Preliminary results indicate the

successful reduction of perchlorate to chloride with 2. The addition of Sc(OTf)3 results in

a dramatic decrease in the reaction time needed to convert 2 to the proposed di-oxo

product Mo(O)2(LBr)(sol) (3).




gloveblox or under argon using standard Schlenk line techniques. 3Å molecular sieves

were dried under vacuum for 24 hours at 250 °C prior to use. THF was degassed then

dried using an MBraun solvent purification systems under an Ar atmosphere, and stored

over activated 3 Å molecular sieves. Tetrabutylammonium perchlorate was purchased

from Sigma-Aldrich and used as received. Scandium triflate was purchased from Strem

and used as received. Acetone-d6 was purchased from Cambridge Isotopes, degassed via

freeze-pump-thaw cycle (5x), stored over 3 Å molecular sieves for 6 hours, then filtered

117

from sieves before use. Mo(O)2(LBr)(MeOH) (1) was prepared following the published

procedure.16


nitrogen glovebox unless otherwise noted. Room temperature UV-Vis spectra were

recorded on a Varian Cary 50 Bio spectrometer with using screw cap UV-Vis cuvettes,

Schlenk cuvette, or a 2 mm Hellma All-Quartz Immersion probe fitted in a 24/40 Schlenk

tube with a 14/20 female joint sealed with a rubber septum. 1H and 35Cl NMR were

recorded with a Bruker 400MHz Avance III ultrashield spectrometer. 1H NMR was

referenced to acetone-d6 residual solvent signal (δ 2.05).

CATUION: Perchlorate salts pose a significant risk when used. The risk of explosion

was reduced by performing the reactions on the smallest scale possible. A PTFE coated

spatula was used to weigh out [Bu4N][ClO4] and care was taken to not crush or grind

the material.

Synthesis of Mo2O3(LBr)2(THF)2·2THF (2). Polymer supported triphenylphosphine

(125 mg, ~0.375 mmol) was added to a stirring solution of Mo(O)2(LBr)(MeOH) (1)

(115.0 mg, 0.250 mmol) in 10.0 mL THF. The resulting dark brown solution was stirred

for 6 h then filtered through a pad of celite and concentrated to approximately 5 mL.

Recrystallization by vapor diffusion of pentane into the THF solution yielded 115.9 mg

(82.2 %) of 2 as blue-black crystals. UV-Vis (THF): λmax (εM) 236 nm (22200), 252 nm

(19700), 316 nm (13300), 454 nm (12200). IR (KBr): 970 cm-. 1H NMR (Acetone-d6, 400

MHz): δ 9.00 (s, 1 H), 7.93 (dd, 1 H, J = 5.9 Hz, 2.5 Hz) 7.83 (d, 1H, J= 2.5 Hz) 7.64

(dd, 1 H, J = 8.9 Hz, 2.4 Hz), 7.09 (br, 2H), 3.64 (m, 8H), 3.53 (m, 2H), 1.80 (m, 8H),

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1.27 (q, 3H). Anal. Calcd for C36H52Br2Mo2N6O9S2: C, 38.10; H, 4.64; N, 7.45. Found:

C, 38.21; H, 4.65; N, 7.50.

Reaction of Mo2O3(LBr)2(THF)2·2THF (2) and [Bu4N][ClO4] (1:10). [Bu4N][ClO4]

(15.1 mg, 44.3 μmol) was added to a stirring solution of Mo2O3(LBr)2(THF)2·2THF (2)

(5.0 mg, 4.43 μmol) The dark brown solution turned orange/brown after 72 hours of

stirring. Following an additional day of stirring, the reaction was dried to an orange

residue, dissolved in acetone-d6 and 1H and 35Cl NMR were recorded.

Reaction of Mo2O3(LBr)2(THF)2·2THF (2), Sc(OTf)3 and [Bu4N][ClO4] (1:1:1). Stock

solutions of Sc(OTf)3 and [Bu4N][ClO4] were prepared in THF. Sc(OTf)3 (4.43 μmol) and

[Bu4N][ClO4] (4.43 μmol) were added to a stirring solution of 2 (5.0 mg, 4.43 μmol) in 5

mL THF. The solution turned orange with 5 minutes of stirring. After an additional 30

mins of stirring the UV-Vis in THF was recorded and the reaction was dried to an orange

residue. The residue was dissolved in acetone-d6 and 1H and 31Cl NMR were recorded.

Reaction of Mo2O3(LBr)2(THF)2·2THF (2), Sc(OTf)3 and [Bu4N][ClO4] (1:1:0.25).

Reaction run analogously to above reaction with of 2 (5.0 mg ,4.43 μmol), Sc(OTf)3 (4.43

μmol), and [Bu4N][ClO4] (1.11 μmol).

UV-Vis monitoring of the reaction of Mo2O3(LBr)2(THF)2·2THF (2), Sc(OTf)3 and

[Bu4N][ClO4] (1:1:10). After blanking spectrometer with 5.0 mL THF, 1.0 mL of a stock

solution of 2 (0.86 μmol) was injected and the spectrum of 2 was recorded. 1.5 mL of a

stock solution of [Bu4N][ClO4] (8.6 μmol) was diluted mixed with a 0.5 mL of a stock

solution of Sc(OTf)3 (0.86 μmol) in THF was injected into the solution of 2, after which

a series of spectra were measured for 5 minutes.

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4.4. Results and Discussion

Addition of excess [Bu4N][ClO4] to Mo2O3(LBr)2(THF)2·2THF (2) in THF

resulted in the dark brown solution gradually turning orange brown over the course of

three days. UV-Vis of reaction aliquots at 12, 24 and 36 hours (data not shown) showed

little change from the starting spectrum of 2 in THF. After three days of stirring at

ambient temperature the solution was noticeably orange. UV-Vis of the reaction mixture

(Figure 4.1) was reminiscent of Mo(O)2(LBr)(sol) (3) generated by the reaction of 2 with

[Bu4N][NO3]. The absorption band at 454 nm was significantly diminished and shifted to

444 nm, suggesting that oxygen atom transfer (OAT) from perchlorate to 2 was

successful but likely not complete. After an additional day of stirring the reaction was

dried to an orange brown residue. 1H NMR of the residue in acetone-d6 (Figure 4.2b) is

similar to 1H NMR of authentic Mo(O)2(LBr)(MeOH) (1) and Mo(O)2(LBr)(sol) (3)

generated through OAT with NO3 and 2, further suggesting that perchlorate reduction

was successful. Attempts to detect Cl containing products through 35Cl NMR were

unsuccessful (Figure 4.3a). The only chlorine product detected in the reaction mixture by

35Cl NMR was the excess with only the excess [Bu4N][ClO4] located at 1000 ppm.

120

Figure 4.1. Room temperature UV-vis of Mo2O3(LBr)2(THF)2·2THF (2) (1 equivalent) and [Bu4N][ClO4] (10 equivalents) in THF.

Figure 4.2. 1H NMR overlay of a) Mo2O3(LBr)2(THF)2·2THF (2) (1 equivalent), Sc(OTf)3 (1 equivalent) and [Bu4N][ClO4] (0.25 equivalents) b) Mo2O3(LBr)2(THF)2·2THF (2) (1 equivalent) and [Bu4N][ClO4] (10 equivalents) c) Mo(O)2(LBr)(MeOH) (1) in acetone-d6.

c

b

a

121

Figure 4.3. 35Cl NMR overlay of a) Mo2O3(LBr)2(THF)2·2THF (2) (1 equivalent) and [Bu4N][ClO4] (10 equivalents) b) Mo2O3(LBr)2(THF)2·2THF (2) (1 equivalent), Sc(OTf)3 (1 equivalent) and [Bu4N][ClO4] (0.25 equivalents) in acetone-d6.

Encouraged by the preliminary perchlorate reduction results, and previous success

inducing OAT from nitrate with Lewis acid additives, the effect of Sc(OTf)3 on the

perchlorate reactivity was investigated. Addition of Sc(OTf)3 (1 equivalent) to a stirring

solution of 2 (1 equivalent) and [Bu4N][ClO4] (1 equivalent) resulted in the formation of

an orange solution in under 5 minutes. UV-Vis of the reaction mixture (Figure 4.4) shows

the loss of the absorption band at 454 nm and formation of the 414 nm absorption band

found in the UV-Vis spectrum of 1 and 3, suggesting that oxygen atom transfer (OAT)

from perchlorate to 2 to generate a dioxo species was successful. Monitoring the reaction

of 2 (1 equivalent) and [Bu4N][ClO4] (10 equivalent) with Sc(OTf)3 (1 equivalent) by

UV-vis (Figure 4.5) indicates that the UV-Vis spectrum in Figure 4.1, featuring the 444

nm absorption band, was from the incomplete reaction of perchlorate and 2. Sc(OTf)3

greatly reduces the reaction time for 2 and [Bu4N][ClO4] from days to minutes.

a

b

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Figure 4.4. Room temperature UV-vis of Mo2O3(LBr)2(THF)2·2THF (2) (1 equivalent), Sc(OTf)3 (1 equivalent), and [Bu4N][ClO4] (1 equivalent) in THF.

Figure 4.5. Mo2O3(LBr)2(THF)2·2THF (2) (0.13 mM), Sc(OTf)3 (0.13 mM), and [Bu4N][ClO4] (1.3mM) followed by UV-Vis spectroscopy at room temperature in THF for 5 minutes. The spectral changes correspond to the conversion of Mo2O3(LBr)2(THF)2·2THF(2) to Mo(O)2(LBr)(sol) (3).

The ability to reduce [Bu4N][ClO4] to chloride was investigated through the

reaction of 2 (1 equivalent) and [Bu4N][ClO4] (0.25 equivalent). Without the addition of

123

Sc(OTf)3 no change in the UV-vis spectrum of the reaction was observed, even with

reaction times of up to 1.5 weeks. However, addition of Sc(OTf)3 (1 equivalent) to 2 (1

equivalent) and [Bu4N][ClO4] (0.25 equivalent) in THF results in the formation of an

orange solution in under 5 minutes. The UV-Vis of the reaction mixture (Figure 4.5)

matches the spectrum of the reaction of 2 (1 equivalent) with [Bu4N][ClO4] (1

equivalent) and Sc(OTf)3 (1 equivalent) (Figure 4.4). The 1H NMR of the dried reaction

mixture dissolved in acetone-d6 (Figure 4.2c) further indicates that 2 has been converted

to the di-oxo species 3. The complete loss of the perchlorate signal (1000 ppm) in the

35Cl NMR (Figure 4.3b) and the apparent consumption of Mo2O3(LBr)2(THF)2·2THF (2)

by UV-vis and 1H NMR, suggests that the reduced Cl products chlorate (ClO3−), chlorite

(ClO2−) and hypochlorite (ClO−) are further utilized for OAT to 2 generating the

proposed dioxo product Mo(O)2(LBr)(sol) (3). 35Cl NMR of authentic [Bu4N][Cl] has a

single broad feature located at ~ 2.0 ppm in acetone-d6 (data not shown). To date

detection of chloride by 35Cl NMR has been unsuccessful. In previous work by Abu-

Omar the chloride (Cl−) generated through perchlorate reduction was precipitated as

AgCl and isolated upon the addition of AgBF4 at the end of the reaction.12 Identification

and isolation of the proposed chloride (Cl−) product is ongoing, but future plans include

the addition of AgBF4 at the end of the reaction of 2 (1 equivalent), Sc(OTf)3 (1

equivalent) and [Bu4N][ClO4] (0.25 equivalent) in attempts to isolate AgCl.

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Figure 4.5. Room temperature UV-vis of Mo2O3(LBr)2(THF)2·2THF (2) (1 equivalent), Sc(OTf)3 (1 equivalent), and [Bu4N][ClO4] (1/4 equivalent) in THF.

4.5. Conclusions.

Perchlorate is a pervasive contaminant in groundwater due to the high solubility

and stability in water. New remediation technologies are required to combat

contamination and lessen the potential health risks associated with the perchlorate

consumption. Taking inspiration from nature, where perchlorate reductase enzymes

utilize high valent molybdenum active sites to achieve the difficult reduction of

perchlorate, we have investigated the ability of Mo2O3(LBr)2(THF)2·2THF (2) to reduce

perchlorate. While perchlorate reduction with 2 is successful it is very slow. Addition of

the Lewis acid additive greatly accelerates the reduction of perchlorate. Conversion of 2

to the proposed di-oxo product Mo(O)2(LBr)(sol) (3) was achieved using 0.25 equivalents

of [Bu4N][ClO4] suggesting that perchlorate is reduced to chloride. This works

demonstrates the ability of Lewis acid additives to enhance reactivity associated with

difficult chemical transformations, such as perchlorate reduction.

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4.6. References

1. Motzer, W. E., Perchlorate: Problems, Detection, and Solutions. Environmental

Forensics 2001, 2 (4), 301-311.

2. Brown, G. M.; Gu, B., The Chemistry of Perchlorate in the Environment. In

Perchlorate: Environmental Occurrence, Interactions and Treatment, Gu, B.; Coates, J.

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3. Abu-Omar, M. M., Effective and Catalytic Reduction of Perchlorate by Atom

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4. Coates, J. D.; Achenbach, L. A., Microbial perchlorate reduction: rocket-fuelled

metabolism. Nat Rev Micro 2004, 2 (7), 569-580.

5. PerBardiya, N.; Bae, J.-H., Dissimilatory perchlorate reduction: A review.

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6. Youngblut, M. D.; Tsai, C.-L.; Clark, I. C.; Carlson, H. K.; Maglaqui, A. P.; Gau-

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7. Youngblut, M. D.; Wang, O.; Barnum, T. P.; Coates, J. D., (Per)chlorate in

Biology on Earth and Beyond. Annual Review of Microbiology 2016, 70 (1), 435-457.

8. Jormakka, M.; Richardson, D.; Byrne, B.; Iwata, S., Architecture of NarGH

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9. Hutchison, J. M.; Poust, S. K.; Kumar, M.; Cropek, D. M.; MacAllister, I. E.;

Arnett, C. M.; Zilles, J. L., Perchlorate Reduction Using Free and Encapsulated Azospira

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10. Schaffner, I.; Mlynek, G.; Flego, N.; Pühringer, D.; Libiseller-Egger, J.; Coates,

L.; Hofbauer, S.; Bellei, M.; Furtmüller, P. G.; Battistuzzi, G.; Smulevich, G.; Djinović-

Carugo, K.; Obinger, C., Molecular Mechanism of Enzymatic Chlorite Detoxification:

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11. Hutchison, J. M.; Zilles, J. L., Biocatalytic perchlorate reduction: kinetics and

effects of groundwater characteristics. Environmental Science: Water Research &

Technology 2015, 1 (6), 913-921.

12. Abu-Omar, M. M.; McPherson, L. D.; Arias, J.; Béreau, V. M., Clean and

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13. Zwettler, N.; Schachner, J. A.; Belaj, F.; Mösch-Zanetti, N. C., Oxidorhenium(V)

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