Nitric Oxide and Oxidative Cluster Conversion

Chemistry of Model [4Fe-4S] Clusters

By Ryan L. Lehane

B.S. U.Mass. Dartmouth 2013

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

  

© Copyright 2018 Ryan L. Lehane

iii  

This dissertation by Ryan L. Lehane 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: ____________ ________________________

Date: ____________ ________________________

Date: ____________ ________________________

Approved by the Graduate Council

Date: ____________ ________________________

Eunsuk Kim, Ph.D. - Advisor

Paul Williard, Ph.D. - Reader

Shouheng Sun, Ph.D. - Reader

Andrew G. Campbell, Ph.D

Dean of the Graduate School

iv  

Curriculum Vitae Ryan L. Lehane

Education and Academic History Brown University, Providence, RI Ph.D. Chemistry, May 2018 U. Mass. Dartmouth, North Dartmouth, MA B.S., Biochemistry, May 2013 Marianapolis Preparatory School, Thompson, CT Graduated May 2009 Research Experience Graduate Research Assistant, Brown University January 2015-Present Advisor: Dr. Eunsuk Kim, Ph.D. Bioinorganic Synthesis/Analysis Project Title: Nitric Oxide and Oxidative Reactivity of Model [4Fe4S] Clusters Project Description:

Synthesized and characterized model [4Fe4S] clusters. Studied reactivity of clusters with nitric oxide and oxidative species

Graduate Research Assistant, Brown University September 2013-December 2014 Advisor: Dr. Wesley Bernskoetter, Ph.D. Organometallic Synthesis/Analysis Project Title: Novel Co1- Complexes for CO2 Coupling to Small Molecules Project Description:

Synthesize and characterized novel Co1- complexes. Studied the ability of the complexes to couple CO2 to ethylene to make

acrylate. Undergraduate Research Assistant, U.Mass. Dartmouth June 2011-June 2013 Advisor: Dr. David Manke, Ph.D. Organic/Inorganic Synthesis Project Title: CO2 Capture Using Lewis Base Derivatized Metal-Organic Frameworks Project Description:

Synthesized novel MOF linkers with Lewis Basic sites Studied the aptitude for MOFs with the linkers to selectively capture CO2 at

atmospheric concentrations Awards and Citations Dissertation Research Fellow, Brown University Fall 2017 Summer Research Fellow, Brown University Summer 2013 Commonwealth Scholor Honoree, U.Mass. Dartmouth Spring 2013 AIC Student Awardee, NEIC Spring 2013 Jean Boissevein Lectureship Research Fellow, Dreyfus Foundation Summer 2012 Honors Summer Research Grant, U.Mass. Dartmouth Summer 2012 Office of Undergraduate Research Awardee, U.Mass. Dartmouth Spring 2012 ACS Division of Inorganic Chemistry Award, U.Mass Dartmouth Spring 2012

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Leadership Experience Head Teaching Assistant, Brown University

CHEM0500 – Inorganic Chemistry Spring 2015 Teaching Assistant, Brown University

CHEM0500 – Inorganic Chemistry Spring 2014 and Spring 2015 CHEM0330 – General Chemistry Fall 2013, Fall 2014, Spring 2016

Teaching Assistant, U.Mass. Dartmouth

CHM153 – General Chemistry Fall 2011 – Spring 2013 Senior Resident Assistant Fall 2012 – Spring 2013

Cedar Dell West Community Resident Assistant, U.Mass. Dartmouth

Cedar Dell South Community Fall 2010 – Spring 2012 Publications Cao, R.; Elrod, L.T.; Lehane, R.L; Kim, E.; Karlin, K.D. “A Peroxynitrite Complex: Formation via Cu-NO and Cu-O2 Intermediates and Reactivity via O-O Cleavage Chemistry” J. Am. Chem. Soc. 2016, 138, 16148-16158.

Lehane, R.L.; Golen, J.A.; Rheingold, A.L.; Manke, D.R. “Crystal structure of 9H-carbazole-2,7-dicarboxylic acid dimethyl ester” Acta Cryst. 2015, E71, o784-o785.

Lehane, R.L.; Golen, J.A.; Rheingold, A.L.; Manke, D.R. “Dimethyl 2,2’-dinitrobiphenyl-4,4’-dicarboxylate” Acta Cryst. 2014, E70, o305.

Lehane, R.L.; Golen, J.A.; Rheingold, A.L.; Manke, D.R. “Dimethyl 2-aminobiphenyl-4,4’-dicarboxylate” Acta Cryst. 2013, E69, o797.

vi  

Preface and Acknowledgements

The last five years have not at all gone as I planned for them to and have certainly

not been what I would call a normal graduate student experience. Thinking back on it I

realize that it is not something I could have possibly done on my own, and I have had the

fortune to share it with some amazing people along the way.

First, I would like to thank my Advisor Dr. Eunsuk Kim. Her patience, guidance,

and compassion have been limitless, and I consider myself incredibly lucky to have worked

so closely with her for so long. I cannot thank her enough for the encouragement and

support she has lent me to help get me through some very hard times and over some very

hard obstacles.

Thank you to my committee members, past and present: Dr. Paul Williard, Dr.

Shouheng Sun, and Dr. Wesley Bernskoetter for all your help and the challenges you have

given me over the years. Particularly to Dr. Bernskoetter, I am very grateful for all the help

you gave me throughout my first year as a graduate researcher. I learned a lot about what

it is to be a chemist and more specifically the kind of chemist I want to be from you, and

those are things I will never forget.

I would also like to thank my group mates from both the Kim and Bernskoetter

groups. From Hongwei, Dong, YuanYuan, Alex, and Liz in my first research family under

Dr. Bernskoetter to Jessica, Kevin, and Taylor under Dr. Kim, all of you have played an

important role in my development as a person and a scientist. I would also like to thank

the rest of the Chemistry Department at Brown University for the help and support, great

and small, they have provided to me over the past five years.

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The list of family and friends to whom I owe a debt of thanks for love and support

is both staggering and humbling. My entire extended family has been incredible in keeping

me balanced and smiling, and I owe a particularly large amount of my remaining sanity to

the many nights of fun and laughter with my DeSormier cousins. Additionally, I owe a

great deal of thanks to all the incredible friends I have been lucky enough to make and keep

over the past thirteen years from undergrad and high school. Your companionship has made

it easier to keep going when things got difficult.

Finally, my family. My parents, Michelle and Kevin, my brother Michael, and my

wife-to-be Brittany. Without the four of you I could never have gotten this far. There are

neither enough words, nor time to thank you for all that you have each done for me. There

has never been a time when I needed you that you weren’t there. Late nights, early

mornings, successes, and failures: you have been there for it all, and I owe a great deal of

who I am and what I have been able to accomplish to the four of you. I will never be able

to repay you for what you have meant to me, and I know you would never let me. I thank

you all from the bottom of my heart for all the emails, texts, talks, hugs, and kisses that

have gotten me through the last five years. Without you none of this would ever have been

possible.

Thank you.

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Table of Contents Signature Page iii

Curriculum Vitae iv

Acknowledgements vi

Table of Contents viii

List of Figures xi

List of Tables xvi

List of Schemes xvii

List of Equations xix

Chapter 1: Introduction 1

1.1.Iron-sulfur proteins 2

1.2.Nitric Oxide 8

1.3.Hydrogen Sulfide 17

1.4.Reactivity of Iron-Sulfur Clusters with NO 19

1.5.Oxygen and Reactive Oxygen Species 24

Chapter 2: Thiol-Dependent NO Reactivity of Model [4Fe-4S] Clusters Producing H2S

and Roussin’s Red Esters 41

2.1 Abstract 42

2.2 Introduction 43

2.3 Results and Discussion 49

2.4 Conclusions 66

2.5 Experimental Methods 67

ix  

2.6 References 72

Chapter 3: Preliminary Studies of Thiol-Dependent NO Reactivity with a Water-Stable

and Soluble Synthetic Model [4Fe-4S] Cluster 75

3.1 Abstract 76

3.2 Introduction 77

3.3 Results and Discussion 85

3.4 Conclusions 94

3.5 Experimental Methods 95

3.6 References 99

Chapter 4: Model [4Fe-4S]2+ to [2Fe-2S]2+ Cluster Facilitate by O2, Thiolate, and

Disulfide 103

4.1 Abstract 104

4.2 Introduction 105

4.3 Results and Discussion 111

4.4 Conclusions 121

4.5 Experimental Methods 121

4.6 References 125

Chapter 5: Model Cluster Conversion from [4Fe-4S]2+ to [2Fe-2S]2+ Facilitated by

Superoxide 128

5.1 Abstract 129

5.2 Introduction 130

5.3 Results and Discussion 135

x  

5.4 Conclusions 142

5.5 Experimental Methods 142

5.6 References 145

xi  

List of Figures

Figure 1.1: Schematic representation of known iron-sulfur clusters 4

Figure 1.2: Schematic representation of synthetic model iron-sulfur clusters 7

Figure 1.3: Molecular orbital diagram for nitric oxide 9

Figure 1.4: Schematic representation of linear and bent NO coordination 11

Figure 1.5: Schematic representation of trans-[Co(en)2(NO)Cl]+ 12

Figure 1.6: Schematic representations of Roussin’s Red Ester, Red Salt, Black Anion 14

Figure 1.7: General form of mononuclear dinitrosyl iron complexes 16

Figure 1.8: Simplified representation of known cluster conversions 25

Figure 1.9: Illustrative representation of biological H2O2 mediation 28

Figure 2.1: Schematic representation of protein bound and synthetic [4Fe-4S] clusters 44

Figure 2.2: Common products of [Fe-S] cluster nitrosylation 45

Figure 2.3: FTIR, UV-Vis, and EPR spectral comparison for thiol dependent nitrosylation

of [Et4N]2[Fe4S4(SPh)4] (1) 50

Figure 2.4: 31P NMR analysis for elemental sulfur production from thiol dependent

nitrosylation of [Et4N]2[Fe4S4(SPh)4] (1) 52

Figure 2.5: EPR spectrum for the reaction product of [Et4N]2[Fe4S4(SPh)4] nitrosylation in

the presence of HSPh 53

xii  

Figure 2.6: Crude 1H NMR spectral comparison of the product of [Et4N][Fe4S4(SPh)4] in

the presence of HSPh with authentic Fe2(μ-SPh)2(NO)4 54

Figure 2.7: FTIR and UV-Vis analysis for ether soluble product of [Et4N]2[Fe4S4(StBu)4]

nitrosylation in the presence of HStBu 55

Figure 2.8: FTIR, UV-Vis, and EPR analysis for the THF soluble product of

[Et4N][Fe4S4(StBu)4] nitrosylation in the presence of HStBu 56

Figure 2.9: GC-MS calibration curve for triphenylphosphine sulfide 57

Figure 2.10: Illustrative pictures of positive and negative responses of lead acetate paper

to hydrogen sulfide 59

Figure 2.11: Lead acetate paper results of thiol dependent nitrosylation of

[Et4N]2[Fe4S4(SPh)4] 60

Figure 2.12: Experimental setup for headspace transfer and analysis with C7Az 61

Figure 2.13: Fluorescence spectrum of C7Az solution after exposure to the headspace of

[Et4N]2[Fe4S4(SPh)4] nitrosylation in the absence and presence of HSPh 62

Figure 2.14: Calibration curve for C7Az fluorescence response as a function of hydrogen

sulfide concentration 63

Figure 3.1: Schematic representation of protein-bound and synthetic [4Fe-4S] clusters 78

Figure 3.2: Common products for iron-sulfur cluster nitrosylation 80

Figure 3.3: Schematic representation of [Fe4S4(SEtOH)4]2- 85

xiii  

Figure 3.4: FTIR and UV-Vis spectral analysis for reaction products of

[Et4N]2[Fe4S4(SEtOH)4] in the absence of HSEtOH 86

Figure 3.5: FTIR analysis for ether soluble product of [Et4N]2[Fe4S4(SEtOH)4]

nitrosylation in the presence of HSEtOH 87

Figure 3.6: FTIR and UV-Vis analysis for THF soluble product of [Et4N]2[Fe4S4(SEtOH)4]

nitrosylation in the presence of HSEtOH 88

Figure 3.7: FTIR and UV-Vis analysis of MeCN soluble product of

[Et4N]2[Fe4S4(SEtOH)4] nitrosylation in the presence of HSEtOH 89

Figure 3.8: Overlay of solution IR monitoring of [Et4N]2[Fe4S4(SEtOH)4] nitrosylation in

the presence of HSEtOH 90

Figure 3.9: Illustrative pictures of negative and positive lead acetate paper responses to

hydrogen sulfide 91

Figure 3.10: Lead acetate paper results for thiol dependent nitrosylation of

[Et4N]2[Fe4S4(SEtOH)4] 92

Figure 3.11: Experimental setup for headspace transfer and hydrogen sulfide analysis by

C7Az 93

Figure 3.12: Fluorescence spectrum of C7Az solution after exposure to reaction headspace

of [Et4N]2[Fe4S4(SEtOH)4] nitrosylation in the absence and presence of HSEtOH 94

Figure 4.1: Simplified representation of known cluster conversions 106

Figure 4.2: Schematic representation of protein-bound and synthetic [4Fe-4S] clusters 108

xiv  

Figure 4.3: UV-Vis spectral comparison of [Et4N]2[Fe4S4(SPh)4] (1) and the reaction

product of 1 with 10 equivalents each diphenyldisulfide and tetraethylammonium

phenylthiolate 112

Figure 4.4: Crude 1H NMR spectrum for the reaction product of [Et4N]2[Fe4S4(SPh)4] with

10 equivalents each diphenyldisulfide and tetraethylammonium phenylthiolate 113

Figure 4.5: UV-Vis comparison of authentic [Et4N]2[Fe2S2(SPh)4] with the reaction

product of [Et4N]2[Fe4S4(SPh)4] with 1 equivalent of diphenyldisulfide and 2 equivalents

of tetraethylammonium phenylthiolate 115

Figure 4.6: Crude 1H NMR spectral analysis for the reaction product of

[Et4N]2[Fe4S4(SPh)4] with 2 equivalents of tetraethylammonium phenylthiolate and 1

equivalent of diphenyldisulfide 116

Figure 4.7: Crude 1H NMR spectral analysis of the reaction of [Et4N]2[Fe4S4(SPh)4] with

10 equivalents of tetraethylammonium phenylthiolate and 1 hour of air exposure 118

Figure 5.1: Schematic representation of protein-bound and synthetic [4Fe-4S] clusters 133

Figure 5.2: Crude 1H NMR spectral analysis for the reaction product of

[Et4N]2[Fe4S4(SPh)4] with 4 equivalents (15-Crown-5)KO2 136

Figure 5.3: Crude 1H NMR spectral analysis for the reaction product of

[Et4N]2[Fe4S4(SPh)4] with 6 equivalents (15-Crown-5)KO2 138

Figure 5.4: Crude 1H NMR spectral analysis for the reaction product of

[Et4N]2[Fe2S2(SPh)4] with 1 equivalents (15-Crown-5)KO2 139

xv  

Figure 5.5: Illustrative overlay of UV-Vis monitored reactivity of [Et4N]2[Fe4S4(SPh)4]

with 4 equivalents of (15-Crown-5)KO2 141

xvi  

List of Tables

Table 1.1: Functions of biological iron-sulfur proteins 6

Table 1.2: Summary of NO active iron-sulfur clusters and their functions 13

xvii  

List of Schemes

Scheme 1.1: Thiol-dependent nitrosylation reactivity of synthetic [2Fe-2S] clusters 22

Scheme 1.2: Thiolate-dependent nitrosylation reactivity of synthetic [4Fe-4S] clusters 23

Scheme 2.1: Thiol dependent nitrosylation reactivity of synthetic [2Fe-2S] clusters 46

Scheme 2.2: Nitrosylation products of the [4Fe-4S] cluster in FNR 47

Scheme 2.3: Thiolate dependent nitrosylation reactivity of synthetic [4Fe-4S] clusters 48

Scheme 2.4: Thiol dependent nitrosylation reactivity of synthetic [4Fe-4S] clusters 64

Scheme 2.5: Working mechanism for synthetic [4Fe-4S] cluster nitrosylation in the

presence of thiol 65

Scheme 3.1: Thiol dependent nitrosylation reactivity of synthetic [2Fe-2S] clusters 79

Scheme 3.2: Thiolate dependent nitrosylation reactivity of synthetic [4Fe4S] clusters 82

Scheme 3.3: Thiol dependent nitrosylation reactivity of synthetic [4Fe4S] clusters 83

Scheme 3.4: Workup summary for nitrosylation products of [Et4N]2[Fe4S4(SEtOH)4] in the

presence of HSEtOH 90

Scheme 4.1: Graphic representation of O2-mediated [4Fe-4S] – [2Fe-2S] cluster

conversion in FNR 107

Scheme 4.2: Pyridine facilitated conversion of a [4Fe-4S]4+ cluster to two [2Fe-2S]2+

clusters 109

Scheme 4.3: Proposed [4Fe-4S]2+ to [2Fe-2S]2+ cluster conversion facilitated by disulfide

and thiolate 110

xviii  

Scheme 4.4: Products of the reaction of [Et4N]2[Fe4S4(SPh)4] with 10 equivalents each of

diphenyldisulfide and tetraethylammonium phenylthiolate 115

Scheme 4.5: Products of the reaction of [Et4N]2[Fe4S4(SPh)4] with 1 equivalent of

diphenyldisulfide and 2 equivalents of tetraethylammonium phenylthiolate 118

Scheme 4.6: Stoichiometric dependent cluster conversion reactivity of

[Et4N]2[Fe4S4(SPh)4] with disulfide and thiolate 121

Scheme 5.1: Graphic representation of O2-mediated [4Fe-4S] – [2Fe-2S] cluster

conversion in FNR 131

Scheme 5.2: Pyridine facilitated conversion of a [4Fe-4S]4+ cluster to two [2Fe-2S]2+

clusters 134

Scheme 5.3: Reaction summary of [Et4N]2[Fe4S4(SPh)4] (1) with superoxide to produce

[Et4N]2[Fe2S2(SPh)4] (2) 137

xix  

List of Equations

Equation 1.1: General synthesis of [Fe4S4(SR)4]2- 8

Equation 1.2: Ligand replacement reaction of [Fe4S4(StBu)4]2- 8

Equation 1.3: Disulfide mediated [4Fe-4S] – [2Fe-2S] cluster conversion 27

Equation 2.1: Triphenylphosphine sulfide production through reaction of

triphenylphosphine with elemental sulfur 57

Equation 2.2: Relevant reaction for lead acetate paper sensing of hydrogen sulfide 59

Equation 3.1: Relevant reaction for lead acetate paper sensing of hydrogen sulfide 91

1  

Chapter 1: Introduction

2  

1.1 Iron-Sulfur Proteins

1.1.1 Introduction to Iron-Sulfur Proteins. Iron-sulfur proteins are ubiquitous

in biology1 and common to almost all forms of life, from the most ancient to the most

modern2,3. There are few organisms that can survive in the absence of iron and therefore

iron-sulfur proteins. These species include Borrelia burgdorferi (Lyme disease), and

certain members of the Lactobassilus family, which play a role in human digestion by

helping convert lactic acid into sugars for respiration4,5. The iron-sulfur family of proteins

is identified by inclusion of a core within the protein containing iron and inorganic sulfur

atoms in one of many conformations6. The most common of these arrangement is the [4Fe-

4S] cubane core, where the iron and sulfur atoms alternate around the corners of a cube.

Also common in biology are the [2Fe-2S] rhomboid core, with the iron and sulfur

alternating corners in a diamond. While these two clusters make up the majority of iron-

sulfur protein instances, there are many more less common cores that include up to 8 iron

atoms1,7,8. The different kinds of cluster will be further discussed in a subsequent section.

The variance in the protein cores also indicates the wide variety of functions these proteins

can have.

Even though they are common to all kinds of life, iron sulfur proteins were not

recognized in biology until a relatively short time ago. In the 1962 researchers discovered

the first example of an iron sulfur protein when it was isolated from a bacterium9. Shortly

thereafter, separate researchers isolated another iron sulfur cluster from spinach-the first

plant-based iron sulfur cluster10. In these cases, the researchers found that the proteins

played a role in electron transfer and categorized these new proteins under the name

Ferridoxin, which would soon become known in other bacteria, plants, and animals11. This

category grew quickly and now, approximately 50 years later, the crystallographic database

3  

contains more than 1300 proteins that contain iron-sulfur clusters in their structure. While

there is no definitive core-to-function translation in iron-sulfur proteins, the many roles

these proteins perform are vital to everyday life—these functions will be covered in a future

section.

1.1.2 Iron-Sulfur Cluster Structures. As was previously mentioned, there is a

large variability in the iron-sulfur cores contained in iron-sulfur proteins. In fact, the iron-

sulfur family of proteins is second in prevalence and variety only to iron-oxo protein

species, which includes hemoglobin and other oxygen carrier proteins1. The two most

common iron-sulfur cores are the cuboidal [4Fe-4S] and rhomboidal [2Fe-2S] clusters. Of

those two, the [4Fe-4S] cluster is the more common. Schematic representations of these

and other clusters can be found in Figure 1.1. Most often, these clusters are ligated by the

thiolate region of reduced cysteine residues which can themselves bind in a number of

ways. There are clusters in which there is one thiolate ligand per iron center, as well as two

per iron, and in some rare cases there are thiolate ligands that bridge two iron centers. In

more rare cases, the clusters can be ligated by N-bound histidine residues, which is the case

for the Reiske12 and mitoNEET clusters13. Additionally, the iron-sulfur clusters can vary

in structure and number of iron atoms from one ligated by four thiolate ligands all the way

to eight iron atoms with seven or eight inorganic bridging sulfides seen in nitrogenase and

up to sixteen iron atoms in rarer cases8,14-21. An iron sulfur protein is defined as having at

least one of these clusters present in its structure, but in many cases, there are more than

one cluster per protein. Clusters can fill many roles in a protein, from being a participating

member of the active site22,23, to providing a route for electron transfer to the active site8,

or even simply stabilizing the overall structure of the protein itself without playing a

4  

meaningful part in the reactivity24. This dissertation focuses on trying to understand how

clusters react with their environment.

 

Figure 1.1: Simplified representations of crystallographically confirmed protein iron-

sulfur cluster sites containing 1, 2, 3, and 4 iron atoms, including symmetric and

asymmetric ligation. Adapted from published review by Rao et al8.

1.1.3 Iron Sulfur Cluster Reactivity. Iron sulfur proteins perform a large number

of functions in biology and the clusters therein complete different tasks depending on what

is required of them. Originally it was believed that the sole function for iron-sulfur proteins

was electron transport for biological reactions. Iron-sulfur clusters are uniquely suited to

this kind of reactivity because of their ability to delocalize electron density throughout their

core. For example, the iron ions in the most common cubane [4Fe-4S] cluster are generally

assigned as two ferrous (Fe2+) and two ferric (Fe3+) iron ions. While this does account for

5  

the overall core charge of +2 with the addition of the four bridging sulfide ions with a

charge of -2 on each, it is not a true representation of the electronic structure of the species.

In fact, the entire +10 charge is spread out over the entirety of the core, meaning each iron

is better approximated as a +2.5 oxidation state. All of the iron centers are

antiferrmagnetically coupled, resulting in an overall spin of S = 0, just as in the [2Fe-2S]

case25. Clusters known to be involved in electron transfer include [2Fe-2S], [3Fe-4S], [4Fe-

4S], or [8Fe-7S] cores8. The most common outer ligand for any iron sulfur cluster is

cysteinate, providing a pseudo-tetrahedral environment around the iron atoms. Less

common ligands include aspartate26, histidine12, serine27, or amide groups from the

backbone of an amino acid residue. These clusters largely act as one electron carriers, but

the [8Fe-7S] cluster has been shown to be able to carry two electrons15.

While electron transport is still one of the most common jobs for iron sulfur proteins

to do, there have been many iron sulfur clusters that have been discovered to perform a

wide variety of different roles. Some of these more recently discovered roles include iron

or cluster storage, substrate binding or activation, gene regulation, oxygen sensing,

disulfide reduction, and sulfur donation28. A summary of clusters and their functions can

be found in Table 1.1.

6  

Table 1.1: Functions of biological [Fe-S] clusters adapted from Johnson et al28.

Function Example Cluster Type

Electron Transfer Ferredoxins [2Fe2S]

PCET Nitrogenase [8Fe7S]

Substrate Binding/Activation Hydratases [4Fe4S]

Iron/Cluster Storage Polyferredoxins [4Fe4S]

Gene Regulation SoxR [2Fe2S]

Oxygen Sensing FNR [4Fe4S]-[2Fe2S]

Disulfide Reduction Heterodisulfide Reductase [4Fe4S]

Sulfur Donation Biotin synthase [2Fe2S]

In addition to all of the functions listed here, iron-sulfur clusters have been found

to be a target for very small but significant biological molecules—nitric oxide29,30,

molecular oxygen31, and reactive oxygen species32,33. The reactivity of iron sulfur clusters

with nitric oxide and oxidative species will be explored more fully later, but this reactivity,

along with the inherent difficulties that come with working with proteins led researchers to

try to find an easier way to study the reactivity of iron sulfur clusters. One method

researchers used to accomplish that was using synthetic models.

1.1.4. Synthetic Model Clusters. While iron sulfur clusters were discovered in

biological systems in the 1960’s, it was not until the 1970’s that the first synthetic model

clusters started to appear. Utilizing a number of methods, researchers were able to produce

species containing the desired iron-sulfur cores ligated by simple molecules—most often

thiolates. Synthetic models have been produced for a wide variety of clusters including,

but not limited to, [2Fe-2S], [3Fe-4S], and [4Fe-4S] clusters8, 34-36.

7  

Figure 1.2: Schematic representations of synthetic iron sulfur clusters containing one, two,

three, and four iron atoms. R-groups represent a wide range of alkyl and aryl groups.

Adapted from published review by Rao et al8.

In addition to more simplified chemical reactivity compared to protein bound clusters, the

ability to vary the external ligands (generally thiolates) allows for greater probing into the

nature of cluster reactivity under different electronic conditions. For example

phenylthiolate is a commonly used ligand for model clusters. The aryl group on the thiolate

allows for easy modification, and through that modification the electronic structure of the

cluster core can be tuned for deeper mechanistic and reactivity investigation. More recently

researchers in the Meyer group have been able to effectively synthesize structural models

for two of the less common iron sulfur cluster types: the Rieske center37,38, a [2Fe-2S]

cluster ligated by two cysteinates on one iron and two histidines on the other, and the

mitoNEET center39, with three cysteinates and one histidine residue bound to a [2Fe-2S]

cluster. These will not be further explored in this dissertation, but their synthesis is worth

note. The main focus of this dissertation is the reactivity of the cubane [4Fe-4S] cluster

type, although the rhombic [2Fe-2S] cluster is a product in a number of the reactions and

used for control reactions itself.

8  

The syntheses for the [4Fe-4S] clusters used in this work have been known since

the early 1970’s40 and involve the self-assembly of the [4Fe-4S]2+ core with four thiolate

ligands. The reaction formula can be found in Equation 1.1.

(1.1) FeCl3 + 4 NaOMe + 3 HSR + 3 NaSH → Na2[Fe4S4(SR)4] + other products

Also included in the products for the reaction are twelve equivalents of NaCl and 4 residual

equivalents of MeOH—the reaction solvent. Utilizing different thiols during this reaction

allows for the production of [4Fe-4S] clusters bound by different thiolates. One of the most

useful starting clusters is the tBu thiolate derivative: as the most weakly bound of the

thiolate groups, the tBu cluster can be transformed into any of the other clusters simply by

adding the appropriate thiol41, as demonstrated in Equation 1.2.

(1.2) [Fe4S4(StBu)4]2- + HSR → [Fe4S4(SR)4]2- + HStBu

Once the synthetic clusters have been synthesized, they can be used to approximate the

reactivity of protein bound clusters with a variety of substrates and species.

1.2 Nitric Oxide

1.2.1 Introduction to Nitric Oxide. Nitric oxide is a small, diatomic molecule

consisting of one nitrogen atom and one oxygen atom connected by a triple bond. In

addition to the three bonds, there is one additional electron left in the 2p energy level from

the oxygen atom that contributes greatly to the chemistry of nitric oxide—it makes the

molecule a radial in its neutral state. The molecular orbital diagram for nitric oxide is shown

in Figure 1.3.

9  

Figure 1.3: Molecular orbital diagram for NOꞏ. The 2s sigma bonding and antibonding

orbitals are full, as well as the sigma and pi orbitals in the 2p energy level. There is one

electron in the pi star orbital, imparting radical character onto the molecule.

In addition to its radical nature, nitric oxide being a neutral molecule allows it to dissolve

in water and pass across membranes. Originally nitric oxide was thought to simply be a

toxic gas42—in high enough concentrations it leads to cell death. In the last few decades it

has been discovered that nitric oxide also plays roles vital to survival43. In fact, in human

tissues it has been found to play roles in the central nervous44, circulatory45, and immune

response46. It has also been found that it performs these tasks largely by interacting with

10  

metalloproteins in the cellular matrix. In some cases, it nitrosylates the amino acid residues,

and in others it binds directly to the metal47.

1.2.2 Metal Binding of Nitric Oxide. The coordination chemistry of nitric oxide

has been widely studied48-50. When binding to a metal center, nitric oxide acts as what is

known as a, “non-innocent,” ligand51. In most cases, when a ligand interacts with a metal,

the orbitals from the ligand are much lower in energy than the metal centered orbitals.

Therefore, there is poor overlap between the two and very little room for the sharing of

electrons. As mentioned previously, however, nitric oxide has one electron in a pi start,

anti-bonding orbital. Due to the inherent high level of energy these orbitals have, they fall

much closer in energy to metal centered d-orbitals. This allows for more complicated

electron sharing to take place.

Nitric oxide has three possible binding modes to a metal center—as NO+, NOꞏ, or

NO-. One way to get a better idea of how the nitric oxide molecule is binding to a metal

center is the geometry of the bond. In the first extreme case, NO+, NO acts as a two-electron

donor to the metal center and the metal-nitrogen-oxygen angle will take on a linear, almost

180º orientation. In the opposite extreme, NO-, NO acts as a three-electron donor to the

metal center and the metal-nitrogen-oxygen angle is far more bent, coming in closer to

120º52. Schematic representations of these binding modes can be found in Figure 1.4.

11  

Figure 1.4: Extreme binding modes of nitric oxide to a metal center. Left: bent,

characteristic of NO-. Right: Linear, characteristic of NO+. Figure adapted from Jayarathne

et al. 53

In addition to the change in geometry of the bond depending on how the ligand

binds, there is also a concomitant shift in the infrared spectral shift of the N-O stretching

frequency. In the bent, NO-, case the IR frequency is approximately in the 1650cm-1 range,

close to where a C=O stretch can be found. In the NO+, linear, case the IR frequency for

the bond shifts to approximately 1840cm-1. This would seem to indicate that the

determination of the nature of the metal-NO bond can be simply made through the use of

infrared spectroscopy or x-ray crystallography. Unfortunately, this is not the case: there are

examples of metal-nitrogen-oxygen bond angles throughout the entire window of 125-

180º, and N-O stretching frequencies similarly throughout the entire window of 1650-

1840cm-1 (54). Since there is no definitive way to know how the NO is binding to the metal,

there is also no way to be sure how many electrons are being donated to the metal d-orbitals,

which has a direct impact on the oxidation state of the metal.

12  

1.2.3 Enermark-Feltham Notation. To make some sense of such systems, in 1974

Enermak and Feltham devised a notation that avoided attributing any electrons as distinctly

metal centered or NO centered55. In this system, a metal center with x nitrosyl ligands

would be described as {M(NO)x}n, where n denotes the total number of NO π* electrons

and metal d-orbital electrons. A proof of concept is the complex [Co(en)2(NO)Cl]+, shown

in Figure 1.5.

Figure 1.5: Coordination compound trans-[Co(en)2(NO)Cl]+ with two ethylenediamine

ligands and one each NO and Cl ligands trans to each other. The crystal structure for the

compound indicates a bent cobalt-nitrogen-oxygen bond angle.

One way to view this complex would be both NO and Cl being x-type, anionic ligands. In

that case, the cobalt center would formally be viewed as a CoIII center, explaining the

overall +1 charge on the complex. In this case, the cobalt center would contribute six d-

orbital electrons and the NO ligand, as previously discussed, would have two π* electrons.

As such, using the Enemark-Feltham notation, this complex would be described as

{Co(NO)}8. While this fits the NO- binding mode, it also perfectly describes CoII-NOꞏ and

13  

CoI-NO+ (56). In doing so, the Enemark-Feltham notation removes the need to try to

determine exactly how the NO is binding, and therefor formally assign a metal oxidation

state.

1.2.4 Nitrosylation of Iron-Sulfur Proteins. As previously mentioned nitric oxide

is a toxic gas in high enough concentrations with the capability of killing pathogens and

cancerous cells46. One of the ways it accomplishes this task is through targeting iron-sulfur

proteins57. Iron-sulfur clusters, due to their inherent redox reactivity, are susceptible to

reactive oxygen species (ROSs), which will be covered later, and reactive nitrogen species

(RNSs), including nitric oxide. In many cases, interactions with these species result in

cluster transformations or conversions, or complete cluster loss. This reactivity makes iron-

sulfur clusters ideal for sensing ROS and RNS, and the stresses they cause. Additionally,

at low enough (nM) concentrations, NO acts as a signaling molecule that interacts with a

variety of iron-sulfur proteins. A summary of such proteins and their functions can be found

in Table 1.2.

Table 1.2: Summary of NO Active Iron-Sulfur Proteins and their Functions.

Protein Cluster Type Function

SoxR [2Fe2S] Redox/O2-/NO stress sensor.

NsrR [2Fe2S]/[4Fe4S] Global regulator of NO stress response.

FNR [4Fe4S] Global regulator of O2 sensor. Secondary NO sensor.

IRP1 [4Fe4S] Iron regulation.

Wbl [4Fe4S] NO sensor. Control cell developmental processes.

14  

In order to study some of these reactivities more closely, researchers have employed the

previously discussed synthetic model clusters to determine possible products and reactivity

patterns.

1.2.5 Nitrosylated Iron-Sulfur Products. Over the course of the past few decades

researchers have discovered a number of nitrosylated iron products, both in biological and

synthetic cases. In some cases, the reactivity of synthetic clusters matches perfectly with

the reactivity of protein bound clusters. Some of the most common non-heme nitrosylated

iron products are mono- and dinitrosyliron complexes (MNICs and DNICs), and Roussin’s

Red Salt (RRS), Red Ester (RRE) and Black Anion (RBA). The latter three were

discovered in 1858 by French chemist Roussin, and RRS is recognized as the first synthetic

iron sulfur cluster58. The structures of RRE, RRS, and RBA are shown in Figure 1.6: their

structures have been known for decades59-61.

Figure 1.6: Schematic representations of Roussin’s Red Salt, Red Ester, and Black Anion.

As shown in the schematic, Roussin’s Black Anion has a formula of [Fe4(NO)7(μ3-S)3]1-

containing four iron centers, seven total nitric oxide ligands, and three sulfide ions bridging

15  

three iron centers each62. Despite its complex structure, the synthesis of RBS is

straightforward—combining iron(II) sulfate with sodium nitrite and ammonium sulfide

results in the self-assembly of RBA, stable up to 120º 63,64. Of all known iron-nitrosyl

complexes, RBA is the most stable. Its NO ligands, however, are photolabile which allows

for the delivery of NO65,66 –making RBA a functional antimicrobial agent against the

growth of gram-positive and gram-negative bacteria64.

Roussin’s Red Salt has a similar activity to RBA, but is not as good of a

antimicrobial agent due to its instability67-69. RRS has only two iron centers, each ligated

by two NO molecules and connected via two μ2-sulfide ions to give a bi-tetrahedral

structure with the overall formula [Fe2S2(NO)4]2- 62,65,66. One synthetic route to RRS utilizes

RBA as a starting material—adding sodium hydroxide to RBA results in the production of

RRS. Of a similar structure is Roussin’s Red Ester, which contains the same bi-tetrahedral

structure as RRS, but instead of two μ2-sulfide ions, it has two μ2-thiolate groups. There

are a number of synthetic routes to RREs, which are EPR silent and diamagnetic due to

antiferromagnetic coupling between the two iron centers70,71. RREs have been investigated

for their ability to deliver NO for therapeutic applications, along with another common

class of nitrosylated iron species—dinitrosyl iron complexes (DNICs).

1.2.6 Dinitrosyl Iron Complexes (DNICs). Dinitrosyl iron complexes are one of

the most common NO-derived cellular adducts72. Of particular abundance are mononuclear

dinitrosyl iron complexes, with a single iron center, two nitric oxide ligands and two ligated

thiolate groups, generally cysteinate or glutathionate residues in biological systems63. The

complexes were first discovered in the 1960’s by EPR spectroscopy, in which they have a

distinct signal at g = 2.03, originally found in yeast and animal cells73-75. Due to their

16  

prevalence in biology, a wide range of synthetic DNICs have also been produced to study

their reactivity. The most common general structure (with to NO ligands and two thiolate

ligands) is shown in Figure 1.7.

Figure 1.7: General form of a DNIC with two NO ligands and two thiolate ligands.

While thiolate bound DNICs are the most common in the literature and in biology, there

have been many others found as well. These include N-bound, O-bound, and P-bound

derivatives. Although their exact functions are unknown, they are being investigated as NO

or iron storage, transport, and delivery vehicles to facilitate their biological activity76-78.

There is also the possibility that their presence is an indicator of NO related

cytotoxicity79,80, or an active switch for gene regulation81-83. These and other biological

roles can be found in reviews by Vanin84, Richardson85, and Lewandowska86. To date there

are more than 80 synthetic model DNICs in the literature, and each of them help to provide

insight into what their biological roles might be.

17  

1.3 Hydrogen Sulfide

1.3.1. Introduction to Hydrogen Sulfide. In cells H2S is produced from the sulfur

containing amino acid residue L-cysteine by four different synthetic paths: (a)

cystathionine β synthase (CBS) acts on L-cysteine to produce H2S and L-serine; (b)

cystathionine γ synthase (CSE) forms thiocysteine from cystine, which can rearrange to

form H2S; (c) cysteine aminotransferase (CAT) catalyzes the reaction of L-cysteine with

keto acids to for 3-mercaptopyruvate, which is then desulfurated by 3-mercaptopyruvate

sulfurtransferase (3-MST) to for H2S; and (d) cysteine lyase (CL) converts L-cysteine and

sulfite to L-cysteate and H2S57. Through these avenues, cells produce small, micromolar

concentrations of H2S that can be utilized and consumed by various tissues. These tissues

use the H2S and produce various other sulfur-containing species, including thiosulfate,

sulfite, and sulfate.

Hydrogen sulfide is lipophilic, which allows it to easily cross through cellular

membranes without the use of any specific transfer agents. Similar to nitric oxide, hydrogen

sulfide (H2S) was originally thought to simply be a poisonous gas molecule87. More

recently though, it became the third recognized gasotransmitter molecule; along with

carbon monoxide and nitric oxide87,88. All three, in high concentrations, are toxic and can

lead to death, but at much lower concentrations they facilitate chemical messages being

sent throughout biological systems87-89. Specifically, H2S is active in the central nervous

system contributing to brain health, memory, and learning, the cardiovascular system

affecting vasodilation, and the immune system by helping produce the inflammation

response. The inflammation response is specifically interesting in that it varies with

concentration: at low concentrations H2S causes anti-inflammatory effects, whereas at

18  

higher concentrations it can actually induce pro-inflammatory effects. Additionally, H2S

can affect cellular energetics by influencing cytochrome c oxidase and it can also bind to

hemoglobin—bloods oxygen carrier, another interaction it shares similarity with NO.

1.3.2 Hydrogen sulfide and nitric oxide crosstalk. As has been mentioned in two

previous sections nitric oxide and hydrogen sulfide carry out a multitude of functions at

the cellular level. Along with carbon monoxide, H2S and NO allow various parts of the cell

and organisms to communicate with each other and carry out functions and reactions

required for survival. One of the interesting things about H2S and NO specifically though

is the extent of the overlap their functions have with one another: both operate in the

nervous, immune, and circulatory systems90-93. Furthermore, in many cases they act much

more specifically in the same areas of those systems. For example, both molecules act in

the central nervous system to promote brain activity and learning. Based on these overlaps,

researchers have investigated the two molecules and searched for ways the two of them

could communicate with each other to be able to work so closely.

Over the years, studies have unearthed biological mechanisms that can help explain

the overlapping functions between NO and H2S. In the case of vasorelaxtion, H2S and NO

are mutually dependent on one another and their reactivity converges at cGMP94-96. NO

helps to generate cGMP by activating soluble guanylyl cyclase. Once it has been produced,

H2S helps maintain cGMP levels by inhibiting its degradation. It does this through its

ability to deactivate phosphodiesterase-5 which breaks cGMP down. Without NO cGMP

could not be easily produced, and without H2S it would be broken down before it was able

to accomplish its task. The exact mechanism for crosstalk between H2S and NO has

remained mostly elusive, even though efforts have been made to understand it through

19  

reactivity of H2S with nitroprusside94, S-nitrosothiols97, and peroxynitrite (ONOO-)98. In

order to probe this prospect of crosstalk a little deeper, our group has recently made

advances using the hypothesis that iron-sulfur clusters play a central role in the crosstalk

between NO and H2S.

1.4 Reactivity Studies with NO and Iron Sulfur Clusters

1.4.1 Reactivity of NO with Protein-bound Iron Sulfur Clusters. Since the

previously discussed discovery of iron-sulfur proteins as a target for NO reactivity,

researchers have looked into exactly what those interactions result in28. Studying reactivity

of protein-bound iron sulfur clusters has proven difficult, but researchers have still been

able to determine some nitrosylated iron products, as were previously mentioned in other

sections of this introduction. Interestingly, the products for the [2Fe-2S] cluster case is

more or less uniform—they always form DNICs. In 2000, Ding and Demple were able to

show the production of DNICs upon the reaction of NO with a known protein-bound [2Fe-

2S] cluster in the SoxR protein29. After exposure to NO, the researchers were able to get

an electron paramagnetic resonance (EPR) spectrum of the entire cell medium. The

characteristic EPR signal at g=2.03 provided evidence that DNICs had been formed during

the reaction99. These results agree with synthetic model reactivity studies which will be

covered in more detail shortly. What the protein studies did not make clear, however, was

the fate of the bridging sulfide ligands during the clusters reaction with NO which will be

discussed more in a subsequent section. While the [2Fe-2S] cluster reactivity is universal,

the [4Fe-4S] reactivity studies have led to a number of different of nitrosylation products.

Work in the LeBrun group with protein-bound [4Fe-4S] clusters in the FNR

enzyme have shown that different products are possible100-102. The researchers used NRVS

20  

to study the products of reactions after exposing the FNR protein to NO and they found

that the products fell into two categories—there were clusters that converted to a Roussin’s

Red Ester type product, and clusters that converted to a product similar, but not identical,

to Roussin’s Black Anion. In order to reconcile the latter results, the researchers proposed

a complex known as Roussin’s Black Ester, in which at least one of the bridging sulfide

ligands present in RBA are exchanged for a bridging thiolate group. The researchers were

also able to determine that under their reactivity conditions bridging sulfides were released

as three parts sulfane (Sx) and one part sulfide (S2-)102. Although the researchers were able

to provide a great deal of information about the products from cluster nitrosylation, they

also admitted that there were still questions to be answered. They called upon groups to

use synthetic models to help understand the reactivity being seen, and many groups,

including our own, have answered that call.

1.4.2. Reactivity of Synthetic Iron-Sulfur Clusters with NO. To more closely

study the reactivity of iron sulfur clusters with NO, research groups have utilized synthetic

clusters that allow for more convenient reaction monitoring and more simplistic product

isolation. Our group has been among those that has looked at the reactivity pattern of [2Fe-

2S] clusters using synthetic models and those studies have helped formulate our hypothesis

that iron-sulfur clusters act as a significant point of crosstalk between hydrogen sulfide and

nitric oxide.

This hypothesis began due to the knowledge that NO specifically targets iron sulfur

clusters in many cases. Additionally, there is a source of sulfide (the bridging sulfides)

readily available for donation to produce hydrogen sulfide. The work done by Dr. Camly

21  

Tran in our group studied the effects of the reaction environment on the ability of clusters

to produce H2S upon reaction with NO.

The earliest studies looked at the presence of acid during the degradation process

that transformed a [2Fe-2S] cluster into a nitrosylated iron product. Specifically, Dr. Tran

found that in the presence of an external acid source (HCl) and NO, a [2Fe-2S] cluster

[Fe2S2Cl4]2- would decompose into a mononitrosyl iron complex (MNIC) [Fe(NO)Cl3]1-,

and one of the bridging sulfides would also be lost as H2S103. Emboldened by these results,

Dr. Tran then moved on to study the effects of more relevant biological species on more

relevant model clusters.

Starting with thiolate bound model cluster [Fe2S2(SPh)4]2-, Dr. Tran found that the

presence or absence of an external thiol in the reaction mixture had drastic impact on the

fate of the bridging sulfide ligands. In the absence of an external thiol source, the [2Fe2S]

cluster broke down into two equivalents of the corresponding DNIC [Fe(NO)2(SPh)2]2-,

releasing the bridging sulfides as sulfane. In the presence of thiophenol (HSPh) in the

reactive mixture, however, she found that the fate of the bridging sulfides was very

different. With the thiol in solution, the DNIC was still formed, but the bridging sulfides

were released as hydrogen sulfide instead of sulfane104. This reaction is detailed in Scheme

1.1.

22  

Scheme 1.1: Differential reactivity of model [2Fe-2S] clusters in the absence and presence

of an external thiol source. The addition of thiol produces hydrogen sulfide from the

bridging sulfide ligands instead of sulfane, which is seen in the reaction with no external

thiol. Scheme adapted from Tran et al104.

Since thiols, including cysteine residues and glutathione tripeptides, are found in

the cellular matrix in millimolar concentrations105, the addition of an external thiol to the

reaction medium is one that certainly carries biological relevance. Continued reactivity

studies with phenol and different substituted phenylthiolate derivatives also showed that

the mechanism involved the thiol as a Hꞏ donor, and indicated that more electron donating

thiols and thiolate bound clusters favored more hydrogen sulfide production during the

reactivity with NO. In all cases, the majority of the bridging sulfide ligands were released

as hydrogen sulfide, with only a minority being released as sulfane. Based on these

significant results were formulated the hypothesis that [2Fe-2S] clusters acted as a point of

crosstalk between H2S and NO. We then set out to see whether or not that role could be

extrapolated to more than just the [2Fe-2S] cluster motif.

23  

While the [2Fe-2S] rhombic core is very common in biological systems, the [4Fe-

4S] cubane core, as has been previously mentioned, is the most common iron-sulfur cluster

motif. Work has been done by other groups to determine the reactivity of these clusters

with NO using synthetic models, and the results have been interesting. Work in the Lippard

group106,107 has shown that, much like the [2Fe-2S] case, the reaction environment is very

important to the products observed from the nitrosylation reactions. The reactions can be

seen in Scheme 1.2.

Scheme 1.2: Reactions of model [4Fe4S] cluster in the absence and presence of external

thiolate. The different conditions produce different iron containing products, but bridging

sulfides are always released as sulfane. Scheme adapted from Lippard et al.

The Lippard group researchers found that they would get different iron containing products

out of the reactions depending on whether or not they included an external source of

24  

thiolate in the reaction mixture. When there was no thiolate present, addition of excess NO

to a synthetic [4Fe-4S] cluster resulted in the production of RBA. In the presence of

thiolate, however, the researchers found that they obtained four equivalents of the

corresponding DNIC instead. In both cases, however, the bridging sulfides were always

lost as sulfane. The lack of H2S from such a system is not surprising however, since there

is no proton source. The first half of this dissertation will describe our group’s effort to

determine whether the reactivity of synthetic [4Fe-4S] clusters follows the same pattern as

the synthetic [2Fe-2S] clusters discussed in this section—does the inclusion of an external

thiol provide the Hꞏ source required to produce H2S instead of sulfane from the bridging

sulfide ligands.

1.5 Redox Reactivity and Cluster Conversion

1.5.1 Introduction to Cluster Conversions. As mentioned in Section 1.2, nitric

oxide is not the only species that iron-sulfur clusters interact with. Due to the inherent redox

reactivity of the iron-sulfur core, they interact with a wide variety of redox active species31.

In many cases these reactions lead to what are known as cluster conversions108. For the

purposes of this dissertation, the phrase cluster conversion will refer to any time a reaction

causes an iron-sulfur cluster to transform from one motif to another. Examples of these

kinds of conversions can be found in Figure 1.8.

25  

Figure 1.8: Schematic representation of some common cluster conversions observed for

biological and synthetic iron-sulfur cluster systems. Other ligands and charges excluded

for clarity. Adapted from published review by Holm et al108.

One of the most common examples of this is the conversion between the [2Fe-2S] rhombic

cluster and the [4Fe-4S] cubane cluster.

One example of this reaction that will be more fully examined in a subsequent

section is the O2 sensing ability of the fumarate and nitrate reductase (FNR) protein family,

which is particularly well studied in E. coli32, 109-112. Under anaerobic conditions, FNR

exists as a dimer that binds to DNA and inhibits the transcription of certain genes that are

required for aerobic respiration. When exposed to high concentrations of O2, the [4Fe-4S]

cubane structures undergo a cluster conversion reaction and transform into [2Fe-2S]

rhombic centers. This transformation triggers a structural change in the protein, prohibiting

the binding of DNA and allowing for the aerobic respiration genes to be transcribed. Using

this mechanism, many bacteria thrive in both aerobic and anaerobic conditions. While the

26  

conversions themselves are well documented in biological systems, their mechanisms and

other chemical insight are lacking due to a scarcity of model systems able to reliably

undergo similar reactivity.

1.5.2 Synthetic Cluster Conversion Reactions. There are only two known

examples to undergo the [4Fe-4S] to [2Fe-2S] cluster conversion. The first example, from

the Holm group113 in the 1970’s, involves using the chloride bound [Fe4S4Cl4]2- cluster. In

the presence of an oxidizing agent, such as ferricenium, and extra chloride ligand, the

cluster will undergo the transformation to [Fe2S2Cl4]2-. This is the first known example of

this conversion by chemical means. More recently, in 2016, Tatsumi and coworkers114 were

able to affect the [4Fe-4S] to [2Fe-2S] cluster conversion on synthetic clusters bearing

amide ligands. Again, however, the researchers found that to make the [4Fe-4S] cluster

split, it first had to be oxidized to its all ferric (FeIII) form. Just as Holm had, the researchers

used ferricenium as an outer-sphere oxidant to provide the oxidizing power to achieve such

a species. Excess pyridine in the reaction solution then acted as the stabilizing ligands to

produce a [2Fe-2S] core ligated by two of the original amide ligands and two new pyridine

ligands. While both of these systems do provide the sought-after cluster conversion

reactivity, neither of them is able to undergo the process with biologically relevant

oxidants. Ferricenium, while a good outer sphere oxidant, is not a species likely to be found

in a biological system to get these clusters to undergo the transformations we see in bacteria

and other organisms.

1.5.3 Biological oxidizing agents. When considering oxidizing agents that would

be relevant and useful for this transformation we first analyzed what we needed in the

reaction mixture to effect the desired change. The most glaring need to go from

27  

[Fe4S4(SR)4]2- to two equivalents of [Fe2S2(SR)4]2- were the four additional thiolate bound

ligands in the latter compared to the former. Furthermore, there is a two-electron oxidation

required to go from the [4Fe-4S]2+ cubane core to two equivalents of the [2Fe-2S]2+

rhombic core. The simplest way to provide these required species is the addition of one

equivalent of the corresponding disulfide and two equivalents of the corresponding

thiolate. This transformation is shown in Equation 1.3.

(1.3) [Fe4S4(SPh)4]2+ + PhSSPh + 2 -SPh → 2 [Fe2S2(SPh)4]2-

Looking at biological systems, disulfide is a very relevant oxidant to oxidation

chemistry115.

As was mentioned in a previous chapter, biological thiols exist in concentrations

up to the millimolar range105. In fact, these thiols exist in an equilibrium with their oxidized

forms, including disulfide116,117. When a cell enters oxidative stress, this equilibrium is

disturbed and favors the formation of disulfide species far more than under normal cellular

conditions. This increase in disulfide concentration provides us with a biologically relevant

oxidant that could affect the cubane to rhombic core conversion. In addition to the indirect

products of oxidative stress, we were also interested in studying the effects of the direct

causes of oxidative stress on iron sulfur clusters as well—Reactive Oxygen Species

(ROSs).

1.5.4 Reactive Oxygen Species and Iron Sulfur Clusters. Reactive Oxygen

Species have been studied for many years for the effects that they have on biological

systems. Among this category are reduced forms of molecular oxygen: superoxide, the

singly reduced form, and peroxide, the doubly reduced form. Both of these species have

28  

been known to induce deleterious effects to organisms, and many organisms have therefore

evolved to include a defense mechanism against their production.

The production of reactive oxygen species is unavoidable. During respiration and

other processes in the mitochondria, reactive oxygen species are produced as side products

or the vital reactions taking place. Unfortunately, ROSs have been shown to damage all

important species in the cell: DNA, proteins, and lipids. Therefore, cells must have a

mechanism in place to deal with the production of these species. Taking hydrogen peroxide

as an example, when there is an increased concentration, a number of enzymes step in to

get rid of it. First, glutathione (GSH) is oxidized into its disulfide form (GSSG) by

glutathione peroxidase(GPx). This can then undergo reduction back to glutathione with the

help of glutathione reductase (GR) and NADPH, with its concomitant transformation to

NADP+. The NADP+ can then be converted back to NADPH separately by the Penthoses

Phosphate Pathway (PPP)118. This mechanism is shown in Scheme 1.5.

Figure 1.9: H2O2 mediation through glutathione oxidation and subsequent reduction by

Glutathione Peroxidase (GPx) and Glutathione Reductase (GR) respectively. Scheme

adapted from Lamas et al118.

29  

While these defenses are in place, sometimes the ROSs are able to avoid them, and

they start to cause problems in the cell. One area that has been found to be a target for

ROSs is iron sulfur clusters. Superoxide in particular has been shown to interact with the

clusters119, and to disastrous effect33. Looking at a system we have already discussed, work

by Kiley and coworkers in 2003 showed that the [2Fe-2S] cluster in the oxidized,

monomeric form of FNR is susceptible to interactions with superoxide. The addition of

superoxide to the purified protein resulted in complete loss of the cluster, and

decomposition of the protein. While this shows the importance of the previously mentioned

defense mechanism against reactive oxygen species, it also provides insight that iron sulfur

clusters can be targets for the species themselves, not just their indirect products. Once

again, even though these effects have been studied in biological systems, there are no

working synthetic models to date that provide the functionality required to better

understand this reactivity.

1.6 Concluding remarks and statement of research objectives

Iron sulfur clusters have long been known to provide vital reactivity and

functionality to organisms in all classifications of life. In order to study these complexes

more closely, researchers have utilized synthetic models to simplify reactivity and gain

greater chemical understanding. Previous work in the Lippard group has shown that

synthetic model clusters can react with NO to produce RBA and DNICs depending on the

reaction conditions, always releasing the bridging sulfide ligands as elemental sulfur. This

is in argument with the results seen by the LeBrun group however, where nether RBA nor

DNICs are detected as iron containing products, and not all bridging sulfide ligands are

lost as sulfane. The first portion of this dissertation seeks to uncover what other possible

30  

iron containing products there could be under different reaction conditions, and whether or

not there are other possible fates for the bridging sulfides of [4Fe4S] clusters during

nitrosylation.

The second portion of this dissertation examines the reactivity of model [4Fe-4S]

clusters with oxidative species, namely disulfide and superoxide, and the effects those

reagents have on the cluster. Specifically, the work aims to show that these species can

undergo redox reactivity with the clusters, triggering the oxidative degradation of the [4Fe-

4S] cubane cluster into the [2Fe-2S] rhombic cluster and unknown side products. This

provides the first evidence of model clusters able to undergo these transformations with

biologically relevant oxidants, opening the door for more full characterization and analysis

of this reactivity to gain greater insight into the biological processes they mimic.

31  

1.7 References

(1) Beinert, H.; Holm, R.H.; Munk, E. Science, 1997, 277, 653.

(2) Lovenberg, W. Iron-sulfur Proteins: Molecular Properties; Academic Press: New

York, 1973.

(3) Huber, C. Science 1997, 276, 245.

(4) Archibald, F. FEMS Microbiol. Lett. 1983, 19, 29.

(5) Posey, J. E. Science 2000, 288, 1651.

(6) Rees, D. C. Science 2003, 300, 929.

(7) Meyer, J. J. Biol. Inorg. Chem. 2008, 13, 157.

(8) Rao, P.V.; Holm, R.H. Chem. Rev. 2003, 104, 527.

(9) Mortenson, L. E.; Valentine, R. C.; Carnahan, J. E. Biochem. Biophys. Res.

Commun. 1962, 7, 448.

(10) Tagawa, K.; Arnon, D. I. Nature 1962, 195, 537.

(11) Valentine, R. C. Bacteriol. Rev. 1964, 28, 497.

(12) Link, T.A. In Advances in Inorganic Chemistry; Sykes, A.G., Ed.; Academic Press:

1999; Vol. 47, p 83.

(13) Bak, D.W.; Elliot S.J. Curr. Opin. Chem. Biol. 2014, 19, 50.

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Chapter 2: Thiol-Dependent NO Reactivity of Model

[4Fe-4S] Clusters Producing H2S and Roussin’s Red Ester

42  

2.1 Abstract

New products have been identified in the nitric oxide (NO) mediated degradation

of synthetic model [4Fe-4S] clusters through the inclusion of an external thiol source in the

reaction environment. In the absence of thiol, [Et4N]2[Fe4S4(SR)4] (1a R=Ph, 1b R=tBu)

reacts with NO to produce [Et4N][Fe4S3(NO)7] (2) , Roussin’s Black Salt, and one

equivalent of elemental sulfur. Upon the inclusion of thiol in the mixture the reaction

produces two equivalents of the corresponding dinitrosyl iron complex (DNIC)

[Et4N][Fe(SR)2(NO)2] (3a R = Ph, 3b R = tBu), and one equivalent of the corresponding

Roussin’s Red Ester (RRE) [Fe2(μ-SR)2(NO)4] (4a R = Ph, 4b R = tBu). Concomitant with

the production of these iron-containing products the reaction also releases hydrogen sulfide

(H2S), a vital signaling molecule whose functions share a great deal of overlap with nitric

oxide. These results provide evidence that [4Fe-4S] clusters could serve as a point of

crosstalk between NO and H2S.

43  

2.2 Introduction

Nitric oxide (NO), originally thought to simply be a toxic gas1, has more recently

been investigated for its role in cellular signaling2. Along with carbon monoxide (CO) and,

more recently, hydrogen sulfide (H2S), NO has been labeled as one of the three important

gasotrasmitters in biological systems. Specifically, NO is known to be most active in the

central nervous system3 where it effects brain development, memory, and learning, the

immune response4, where it plays a part in the inflammatory response, and the circulatory

system5 where it acts as a vasorelaxer and vasodilater to aid circulation. Interestingly, many

of the functions performed by NO overlap with the functions of H2S. Due to this overlap,

researchers have been looking for a point of crosstalk for the two for years6.

One example of this crosstalk is there action on the secondary messenger cGMP.

In this case, NO facilitates the production of cGMP by activating soluble guanylyl cyclase.

Once it has been produced, H2S acts to delay cGMP’s degradation by inhibiting

phosphodiesterase-57. While examples of indirect crosstalk such as this exist, any evidence

of direct crosstalk between the two species has remained elusive until more recently.

Research into the possibility of this kind of crosstalk has been focused on the reactivity of

H2S with nitroprusside8, S-nitrosothiols9, and Peroxynitrite10. Our research group has

focused on a different possible target for NO-H2S crosstalk—iron sulfur clusters.

Iron sulfur clusters are ubiquitous in biological systems11. Common to the most

ancient and modern forms of life, they carry out many functions required for survival—

electron transport, substrate activation and catalysis, and cellular sensing and signaling12.

Among the functions of iron sulfur clusters in biological systems, they have been found to

44  

be a major target for reactivity of nitric oxide, and that reactivity has been studied and well

characterized in many systems13. To study the reactivity of iron sulfur clusters with nitric

oxide, researchers have broadly taken two different routes—protein bound clusters to study

reactivity in their native environment, and synthetic model clusters, which allow for the

chemical reactivity and mechanisms to be more easily studied.

Figure 2.1: Schematic representations of (a) protein-bound and (b) synthetic model iron-

sulfur clusters. The biological cluster is shown in its most common form, ligated by four

cysteine residues. The synthetic model is shown ligated by four thiolate residues where R

can represent various arryl and alkyl groups.

While the synthetic models are rarely stable or soluble in biologically relevant aqueous or

buffer systems, they have been shown to provide reactivity patterns comparable with those

of protein-bound systems.

As researchers studied the NO reactivity of iron-sulfur clusters, both protein-bound

and synthetic, they started to see patterns emerge in the kinds of iron-containing species

45  

these reactions produced. Indeed, some of the products were found to be relevant to both

synthetic and protein-bound systems, further proving that synthetic clusters could provide

useful insight into protein-bound cluster behavior.

Figure 2.2: Commonly occurring nitrosylated iron species found in the reactivity of both

protein-bound and synthetic model iron sulfur clusters with nitric oxide.

One of the most promising results for synthetic models providing useful information is the

reactivity of the protein-bound [2Fe-2S] cluster in the SoxR protein14, and various synthetic

[2Fe-2S] clusters15,16. In both cases, when the clusters are exposed to NO the only iron

containing species produced are the corresponding dinitrosyl iron complexes (DNICs).

46  

Scheme 2.1: Differential reactivity patterns of synthetic [2Fe-2S] clusters in the absence

(top curve) and presence (bottom curve) of an external thiol source. In the presence of thiol

the reaction produced hydrogen sulfide instead of elemental sulfur.

Work in our research group by Dr. Camly Tran17,18, summarized in Scheme 2.3, using

synthetic [2Fe-2S] clusters indicated that the reaction environment can play a major role in

the species produced from their nitrosylation. Specifically, Dr. Tran looked at the effect of

adding an external thiol source to the reaction mixture. It was found that regardless of thiol,

the DNIC was still the only iron containing species produced: the major difference

observed was in the fate of the bridging sulfide ligands. In the absence of thiol, these were

released into the reaction solution as elemental sulfur. In the presence of thiol, though, the

bridging sulfides were released as varying amounts of hydrogen sulfide, with the electronic

character of the cluster and thiol playing a role in the amount of hydrogen sulfide produced.

These results emboldened our group’s hypothesis that iron sulfur clusters acted as a point

of direct crosstalk between NO and H2S. At this point we only had evidence pertaining to

47  

the [2Fe-2S] cluster system though, and we wanted to investigate whether that motif could

be expanded to other clusters including the cubane-type [4Fe-4S] cluster—the most

common cluster form.

There has been work carried out on [4Fe-4S] cluster systems with nitric oxide

before, using both protein-bound and synthetic model systems. Unlike the [2Fe-2S]

systems, however, there is no consistency in the iron containing species produced from the

reactivity, nor is there agreement in the fate of the bridging sulfides. The LeBrun group has

utilized protein-bound clusters found in the fumarate and nitrate reductase (FNR) family

of proteins to study their reactivity toward nitric oxide19-21. During these reactivity studies

the researchers determined that the iron-containing products included the neutral Roussin’s

Red Ester (RRE), and a species that was similar to, but not the same as, Roussin’s Black

Anion (RBA). They also determined that the bridging sulfides were released in two

different ways: three of them were released as elemental sulfur, but the fourth was released

as a sulfide ion. These reactivity findings are shown summarized in Scheme 2.2.

Scheme 2.2: Schematic representation of FNR-bound [4Fe4S] clusters reactivity with NO,

where R = cysteine residue.

48  

While the researchers were able to discover a good deal of information about the reactivity

patterns of protein-bound [4Fe-4S] clusters with nitric oxide, they also admitted that more

information could be found through the use of synthetic model clusters20.

As was previously mentioned, prior reactivity studies between synthetic [4Fe-4S]

clusters and nitric oxide have not agreed with the findings utilizing protein-bound clusters.

Research in the Lippard group22,23 has indicated a number of different products from such

reactions, but they do not match those seen in the biological systems by the LeBrun group.

The Lippard group’s findings are summarized in Scheme 2.3.

Scheme 2.3:  Effects of an external thiolate source on the reactivity of

[Et4N]2[Fe4S4(SPh)4]. Scheme adapted from Lippard et al23.

49  

In the Lippard group’s investigation, they studied the effects of adding an external thiolate

source to the reaction mixture. They found that in the absence of an external thiolate source

the only iron containing species produced from the reaction was Roussin’s Black Anion.

While the LeBrun group found a product similar to RBA in their biological studies, they

were adamant in their declaration that RBA itself was not the product. Upon the addition

of an external thiolate source, the Lippard group found that the only iron containing product

was the corresponding DNIC. This product is not found in the LeBrun group’s

investigation. Additionally, in both reaction conditions studied by Lippard and coworkers,

the only product derived from the bridging sulfides was elemental sulfur. This, again,

disagrees with LeBrun’s findings, where one of the bridging sulfides was released as a

sulfide ion. Looking at these results, in conjunction with our own group’s work with the

[2Fe-2S] cluster system and our hypothesis about iron-sulfur clusters as a point of crosstalk

for NO and H2S, we decided to investigate the effect of adding an external thiol source to

our reaction mixtures. Herein we report the findings of such reactivity studies.

2.3 Results and Discussion

2.3.1 Effects of Thiol Inclusion on Reaction Products. The first question we

sought to answer was whether or not adding an external thiol source to the reaction mixture

would provide different iron containing species as products. To make that determination,

nitric oxide was added to synthetic [4Fe4S] clusters [Et4N]2[Fe4S4(SPh)4] (1a) and

[Et4N]2[Fe4S4(StBu)4] (1b) using two different methods: as a purified gas, and through the

50  

addition of S-trityl thionitrite (TTN). In both cases, we also investigated the effects of

adding an external thiol source, in the form of HSPh or HStBu respectively. The reaction

products for the thiol containing reactions were then compared to those from a reaction

with no external thiol added—from the previous Lippard reactivity studies, RBA was

expected to be the product of these control reactions. Comparative product analysis is

shown in Figure 2.3.

Figure 2.3: UV-Vis (a), FTIR (b), and EPR (c) spectral comparisons for the reactivity of

[Et4N]2[Fe4S4(SPh)4] with NO gas in the absence (black line) and presence (red line) of

HSPh. Note: RBA is EPR-silent.

Utilizing UV-Vis, FTIR, and EPR spectroscopy it was clear that the reaction containing

the external thiol source produced drastically different products from the reactions with no

51  

external thiol present. The reactions without the external thiol source present in the reaction

produced products that by UV-Vis and FTIR matched exactly with literature values for

RBA24. In the presence of thiol, however, the spectra did not match those of RBA at all.

An EPR spectrum provided a signal at g = 2.03, indicating that a DNIC was present as an

iron containing species25. Qualitative measures were also taken to try to ascertain the fate

of the bridging sulfides as well. After the reactions were complete, the solution was filtered

to remove any insoluble material, which was then combined with triphenylphosphine

(TPP) to determine whether or not there was an elemental sulfur produced from the

reaction. After a one-hour reaction time the solution was reduced to dryness and

phosphorus NMR was used to determine whether any triphenylphosphine sulfide (TPPS)

was produced. TPPS is a known product of TPP and elemental sulfur22,26,27. The results for

such NMR studies can be seen in Figure 2.4.

52  

Figure 2.4: 31P NMR analysis of authentic TPPS (blue), reaction of (1) with NO in the

absence of an external thiol source (red), and reaction of (1) with NO in the presence of

HSPh (green).

Compared to a control of authentic TPPS, the reaction with no thiol was found to produce

TPPS and therefore elemental sulfur. The reaction with an external thiol source, however,

was found to produce no TPPS, meaning no elemental sulfur was produced during the

reaction. Determining that the addition of external thiol did indeed have a significant

impact on the reactivity pattern of the synthetic [4Fe-4S] cluster, the next steps were to

more definitively determine the species produced.

2.3.2 Determination of Iron-Containing Species. The first determination to be

made was the iron-containing species being produced by the reactions. We knew the

53  

reactions were not producing RBA but were not sure what they were. The EPR analysis,

shown in Figure 2.5, indicated that there was at least some DNIC present in the product.

Figure 2.5: Room temperature EPR spectrum of the reaction product of (1a) with NO in

the presence of HSPh. Sample prepared in MeCN.

However, crude NMR analysis, shown in Figure 2.6 also showed there was some kind of

diamagnetic species present as well—the DNIC is paramagnetic and NMR silent.

54  

Figure 2.6: Crude 1H NMR analysis of authentic [Fe2(μ-SPh)2(NO)4] (4a) (red) and the

crude reaction product of [Et4N]2[Fe4S4(SPh)4] (1a) and NO in the presence of HSPh

(blue).

The NMR spectrum closely matched that of the phenylthiolate derived RRE28. During

reactivity studies with (1a) it was difficult to separate the paramagnetic DNIC from the

diamagnetic RRE present in the NMR. To make a more careful and specific determination,

another synthetic [4Fe-4S] cluster [Et4N]2[Fe4S4(StBu)4] (1b) was utilized due to increased

solubility of the corresponding RRE, which should make it much easier to separate the

RRE and DNIC species.

Upon the reaction of (1b) with purified TTN or NO gas, the solution was observed

to change color from a green-black to a dark brown. In agreement with the results of the

reactivity studies of (1a), the product of such a reaction was determined to be RBA by UV-

55  

Vis and FTIR analysis. Upon the inclusion of tertbutylthiol in the reaction mixture, the

products once again produced vastly different spectral traces. During reactions with TTN

and purified NO gas, the reaction solution was observed to change from black-green to

brown-red. The crude reaction product was isolated as a dark brown solid. Through further

workup of the product, including a wash with diethyl ether and THF, the crude reaction

product was found to be two separate species. FTIR and UV-Vis analysis of the ether

soluble material, shown in Figure 2.7, indicated the presence of tertbutyl RRE29 (4b).

Figure 2.7: FTIR (left, KBr) and UV-Vis (right, MeCN) spectra for a the ether soluble

reaction product of [Et4N]2[Fe4S4(StBu)4] (1b) with NO in the presence of HStBu.

Similar analysis of the THF soluble material using UV-Vis, FTIR, and EPR spectroscopies

indicated the presence of the tertbutyl DNIC30 (3b). This analysis is shown in Figure 2.8.

56  

Figure 2.8: Room temperature FTIR (top left, KBr), UV-Vis (top right, MeCN), and EPR

(bottom, MeCN) spectra of the THF soluble product of [Et4N]2[Fe4S4(StBu)4] (1) and NO

in the presence of HStBu. Marked features match literature values for

[Et4N][Fe(StBu)2(NO)2] (3b).

These results confirmed the original hypothesis: the addition of an external thiol source to

the reaction environment drastically changed the reaction products. Instead of producing

RBA, the reaction produced RRE and DNIC in a 1:2 ratio as confirmed by isolated yields.

This provides the first evidence of RRE production from the reactivity of synthetic model

[4Fe-4S] clusters with nitric oxide and more closely matched the reactivity seen using

protein-bound clusters. Now the identities of the iron containing products were known, but

unlike the reaction in the absence of thiol, there are no products that contain bridging

57  

sulfides, and there is no evidence of elemental sulfur production. This led to the next course

of action, which was determining the fate of those sulfides and whether they were released

as hydrogen sulfide or some other sulfur product.

2.3.3 Determining the Fate of the Bridging Sulfides. As has previously been

mentioned, a very common method of confirming the presence of elemental sulfur is the

addition of triphenylphosphine (TTP) to produce triphenylphosphine sulfide (TTPS) by

equation 2.1.

(2.1) P(Ph)3 + Sx → S=P(Ph)3

TPPS can then be easily found via 31P NMR and quantified utilizing a calibration curve

produced via GC-MS, shown in Figure 2.9.

Figure 2.9: Calibration curve for triphenylphosphinesulfide (S=P(Ph)3).

The first step was to examine the control reaction with no external thiol, which has already

been shown to produce RBA and one equivalent of elemental sulfur. The addition of excess

58  

TTN or NO to a solution of 1a or 1b in acetonitrile resulted in the production of a small

amount of pale yellow solid. This solid was isolated by filtration and a solution of TTP was

added. After an overnight reaction time, the solution was reduced to dryness and two

samples were prepared: one NMR sample in CD3CN and one 100-fold dilute GC-MS

sample in MeCN with 200mM diphenyldisulfide for calibration purposes. In both cases,

the spectra indicated the presence of TPPS in the final reaction product meaning elemental

sulfur had been present in the original reaction product mixture.

Running the same analysis for the reactions containing external thiol provided very

different results. In reactions of both 1a and 1b in the presence of HSPh and HStBu,

respectively, the presence of elemental sulfur was greatly reduced. As mentioned in the

previous section, none of the iron containing products of the reactions contained any

bridging sulfides, meaning that there are four sulfur atoms unaccounted for. The TTPS

analysis did not provide satisfactory results—there were nowhere near four equivalents of

elemental sulfur produced after the reaction had run its course. In fact, elemental sulfur

accounted for less than 25% of the bridging sulfide equivalents in an average sample. Since

the majority of the bridging sulfides still had not been found, we turned to methods used

previously in our and other labs to determine whether or not the thiol containing reactions

were producing H2S as we had hypothesized.

To start, we decided to attempt to determine qualitatively whether or not any

hydrogen sulfide could be detected from the reaction products before attempting any

further quantitative measurements. The first such method we employed was the use of lead

acetate paper31—hydrogen sulfide is detected via its interaction with lead acetate on the

paper, producing lead sulfide via the equation shown in Equation 2.2.

59  

(2.2) H2S + Pb(OOAc)2 → PbS + 2 HOOAc

Lead sulfide is a dark brown-black solid, so when the paper is exposed to hydrogen sulfide

in even trace concentrations (as low as 5ppm32), the paper turns from bright white to dark

brown. An example of this color change is shown in Figure 2.10.

Figure 2.10: Representative pictures of lead acetate paper in a reaction flask before (left)

and after (right) the production of H2S in the flask. This control reaction was carried out

using NaSH and HCl to produce the required H2S.

Having a good qualitative handle for H2S detection, lead acetate paper was hung in the

neck of a reaction flask (as shown above) while reactions in the absence and presence of

thiol were run. The lead acetate paper results are summarized in Figure 2.11.

60  

Figure 2.11: Lead acetate paper results for (a) no reaction, and 3 hour reaction times of

(b) 1a with 10 equivalents of NO, (c) 1a with 10 equivalents of HSPh, and (d) 1a with 10

equivalents of HSPh and 10 equivalents of NO. The negative control with HSPh gives a

slight false positive but is easily distinguished from the NO and HSPh reaction.

As the results above show, there is no evidence of H2S formation in the reaction of 1a in

the absence of an external thiol source, which was the expected result. In the presence of

excess HSPh, however, there is a clear reaction with the lead acetate paper, indicating that

hydrogen sulfide was produced during the nitrosylation of 1a in the presence of HSPh.

Emboldened by these results, we decided to attempt to quantify the amount of H2S

produced through these reactions.

Previous work in our group has utilized fluorescence sensors to determine the

presence of hydrogen sulfide in the headspace of a reaction flask. The setup for headspace

capture can be seen in Figure 2.12.

61  

Figure 2.12: Setup for headspace capture and H2S detection using C7Az fluorescence

sensor.

While previous work used sulfidefluor-1 (SF1)33 this work utilizes a newer sensor called

7-azido-4-methylcoumarin (C7AZ)34 due to its commercial availability and ease of use.

Both sensors have a linear response to increasing concentration of H2S, which means they

can be used to quantify the amount of H2S produced during the course of a reaction. Since

this was our first time using this sensor, we decided to run control experiments to ensure

that our methods of H2S collection and detection would be sufficient. The results of such

tests are shown in Figure X.X.

62  

Figure 2.13: Fluorescence spectra of headspace from reactions of 1a in the absence (black

line) and presence (red line) of HSPh. The reaction in the presence of thiol shows a 30-fold

increase in fluorescence response compared to the reaction in the absence of thiol.

The control studies showed that the sensor would work under the conditions we were

planning to use. The next step was to create a calibration curve to allow for quantitative

analysis. Utilizing the calibration curve, shown in Figure 2.14, we will be able to determine

the amount of bridging sulfides being released as H2S. Studies still ongoing.

63  

Figure 2.14: Calibration curve for hydrogen sulfide concentration-dependent fluorescence

intensity of C7Az.

This is the first evidence of H2S as a product of nitrosylation using synthetic model [4Fe4S]

clusters. It also indicates that our hypothesis of iron sulfur clusters as a point of crosstalk

between NO and H2S may be able to be expanded from just the [2Fe-2S] clusters to include

the [4Fe-4S] cluster motif as well—the most common of all cluster types in biology.

2.3.4 Discussion and Working Reaction Model. Based on the results of the work

described above, we are able to provide a new scheme for the reactivity of synthetic model

[4Fe-4S] clusters in the absence and presence of an external thiol source. This new

reactivity is summarized in Scheme 2.4.

64  

Scheme 2.4: Differential reactivity of synthetic model [4Fe-4S] clusters in the absence

(path a) and presence (path b) of an external thiol source. Both the iron containing products

and fate of the bridging sulfides are different in each reaction.

Using this reaction scheme and what we already know about the reactivity of these

synthetic clusters, i.e. they are stable in the presence of excess thiol alone, we started to

formulate an idea for what the reaction mechanism could be to lead us to these products.

Our proposed working model for such a mechanism is shown in Scheme 2.5.

65  

Scheme 2.5: Working model for the mechanism of a reaction between synthetic model

[4Fe4S] clusters (shown as 1a) and NO in the presence of an external thiol source. The

conversion of (4) to (3) is a known reaction.

Since we know that there is no reaction between the thiol and the cluster, the first step of

the reaction must include the addition of NO ligands. This could be followed by a one

electron oxidation of two bridging sulfide ligands, which has been seen previously18,

producing a radical type character on those atoms. This is where the reactivity between the

absence and presence of thiol could diverge. Depending on the reaction conditions, this

transient species could be oxidized, be reduced, or disproportionate. In the absence of thiol,

it would stand to reason that one of the sulfides could be oxidized an additional time to

produce elemental sulfur en route to Roussin’s Black Anion. In the presence of thiol,

66  

however, another possible pathway is open. The radical character on the sulfide ligands

could each attack one equivalent of thiol, abstracting a formal Hꞏ and leaving a total of two

equivalents of ꞏSPh behind which could then combine to form one equivalent of PhSSPh.

With the hydrogen addition to one of the bridging sulfides, the cubane cluster would break

into two modified rhombic pieces. Further reaction of this species with four additional

equivalents of NO could then promote bond formation between the two bridging sulfide

ligands, producing a transient HSS- species and (4a). The HSS- species would then provide

the path to the other final iron containing product: upon reaction with a total of six more

equivalents of thiol, the two equivalents of HSS- could produce two equivalents of -SPh

and two equivalents of PhSSPh. In a reaction known to the literature35, the two equivalents

of thiolate could then interact with one equivalent of RRE to produce two equivalents of

the corresponding DNIC (3a). Through this mechanism we are able to produce the

biologically relevant iron containing product RRE, and the vital gasotransmittor H2S from

the nitrosylation of a model [4Fe4S] cluster in the presence of an external thiol source, a

biologically relevant agent36, in the reaction media.

2.4 Conclusion

The reactivity of synthetic model [4Fe-4S] clusters with nitric oxide is very clearly

dependent on the reaction conditions. As has been shown by the Lippard group in the past,

the addition of thiolate changes the iron products from RBA to DNIC, with the bridging

sulfides released as elemental sulfur in both cases. Herein we report the first production of

Roussin’s Red Ester, a biologically relevant nitrosylation product, and hydrogen sulfide, a

vital gasotransmittor, through the reaction of model [4Fe-4S] clusters. These products more

closely resemble the products of protein-bound [4Fe-4S] clusters and extend the possibility

67  

of iron sulfur clusters as a point of crosstalk between NO and H2S to include the most

common form of iron sulfur cluster in biology, the [4Fe-4S] cubane motif. More reactivity

studies are currently underway to determine the effect of electronic changes to the cluster

using substituted aryl thiolate ligands, but no data is available at the present time.

2.5 Experimental methods

2.5.1 General Considerations. All reactivity studies and manipulations, unless

otherwise specified, were carried out under inert atmospheric conditions: either in an

MBraun glovebox under dewar supplied nitrogen (>0.1ppm O2, >0.1ppm H2O), or via

conventional Schlenk techniques under argon (UHP 5.0). All solvents were passed through

an alumina column and dried over molecular sieves (3Å) under a glovebox atmosphere.

Thiols were purchased from Sigma Aldrich and degassed by freeze-pump-thaw and stored

under an argon atmosphere. All other chemicals were ordered from Sigma Aldrich and

used as received. Nitric oxide (Corp Bros.) was purified following a literature method,

where the gas is passed through an Ascarite column and distilled at -80ºC.

2.5.2 Physical Measurements. Unless otherwise specified, all samples were

prepared under a nitrogen glovebox atmosphere. Infrared spectra were recorded on a

Bruker Tensor 27 FT-IR—samples were analyzed as KBr pellets or solution samples using

a fiber optic immersion probe. UV-Visible spectra were recorded on a Varian Cary 50 Bio

spectrometer, NMR were recorded on a Bruker Avance 400MHz spectrometer, and GC-

MS data were recorded on a Hewlett-Packard (Agilent) GCD 1800 GC-MS spectrometer.

68  

2.5.3 Synthesis. TTN22, [Et4N]2[Fe4S4(SPh)4] (1a)37, [Et4N]2[Fe4S4(StBu)4] (1b)38,

[Et4N][Fe(NO)2(SPh)2] (3a)28, [Et4N][Fe(NO)2(StBu)2] (3b)29, [Fe2(NO)4(μ-SPh)2] (4a)28,

and [Fe2(NO)4(μ-StBu)2] (4b)29 were synthesized according the literature procedures.

2.5.4 General Reaction of [Et4N]2[Fe4S4(SR)4] (1) with NO(g) in the Absence of

HSR (1a R=Ph, 1b R=tBu). A 10mL Schlenk flask was charged with a solution of (1a)

(15mg, 0.014mmol) in 5mL MeCN, a dark red-black solution. To this stirring solution,

10mL of purified NO gas was added via gas-tight syringe. The solution was allowed to stir

at room temperature for three hours. After that time all volatiles were removed in vacuo

and the residue was moved to the glovebox and redissolved in minimal MeCN. The

solution was layered with ether and place in a freezer at -35ºC overnight and

microcrystalline material precipitated out of solution. The precipitate was filtered and

washed with Et2O to yield RBA (7.1mg, 75%), confirmed by FTIR (KBr, cm-1): 1732,

1711, 1689 (νNO) and UV-Vis (MeCN, λmax, nm): 207, 259, 357, 428, 580.

An analogous reaction utilizing (1b) resulted in a similar formation of Roussin’s

Black Anion in a 72% yield. UV-Vis and FTIR analysis confirmed the production of RBA.

2.5.5 General Reaction of [Et4N]2[Fe4S4(SR)4] (1) with NO(g) in the Presence

of HSR (1a R=SPh, 1b R=tBu). A 10mL Schlenk flask was charged with solution of 1a

(15mg, 0.014mmol) in 5mL MeCN. To this reaction was added 0.014mL (0.14mmol, 10

equivalents) of HSPh under an argon atmosphere. After 5 minuets stirring at room

temperature, the argon flow was closed and 10mL of purified NO gas was added to the

flask via a gas-tight syringe. The solution was stirred at room temperature for an additional

three hours. There was no definitive change in color for the reaction. All the volatiles were

removed in vacuo and moved to the glovebox for further workup. The reaction product was

69  

washed with pentane and ether and redissolved in. IR, EPR, and NMR studies indicated

the presence of 3a and 4a in a 2:1 ratio. 3a: FTIR (KBr, cm-1): 1743, 1707, 1682 (νNO).

EPR (MeCN) g = 2.03. 3b: FTIR (KBr, cm-1): 1739, 1695 (νNO). NMR (CD3CN, 1H): 7.275

(bs), 7.35 (bs), 7.53 (bs).

Analogous reactions were carried out using 1b in the presence of HStBu, arriving

at the same reaction products, 3b and 4b in a 2:1 ratio as confirmed by IR, EPR, and UV-

Vis analysis. 3b: FTIR (KBr, cm-1): 1722, 1678 (νNO). UV-Vis (MeCN, λmax, nm): 313,

381, 435. EPR (MeCN) g = 2.03. 4b: FTIR (KBr, cm-1): 1778, 1730 (νNO). UV-Vis (MeCN,

λmax, nm): 314, 358, 423. EPR-silent.

2.5.6 General Reaction of [Et4N]2[Fe4S4(SR)4] (1) with TTN in the Absence of

HSR (1a R=Ph, 1b R=tBu). A 10mL Schlenk flask was charged with 15mg (0.014mmol)

of 1a. The resulting solution was put on the Schlenk line and stirred at room temperature.

43mg of TTN was dissolved in 3mL MeCN and injected via syringe to the reaction mixture.

The flask was covered in foil and the solution was stirred for 3 hours. The volatiles were

removed in vacuo and the flask was moved back to the glovebox for workup. The solid

was washed with ether and redissolved in MeCN, layered with ether and put in the freezer

to yield RBA (8mg, 87% yield), confirmed by FTIR (KBr, cm-1): 1732, 1711, 1689 (νNO)

and UV-Vis (MeCN, λmax, nm): 207, 259, 357, 428, 580.

An analogous reaction utilizing (1b) resulted in a similar formation of Roussin’s

Black Anion in a 78% yield. UV-Vis and FTIR analysis confirmed the production of RBA.

2.5.7 General Reaction of [Et4N]2[Fe4S4(SR)4] (1) with TTN in the Presence of

HSR (1a R=Ph, 1b R=tBu). A 10mL Schlenk flask was charged with solution of 1a (15mg,

70  

0.014mmol) in 5mL MeCN. To this reaction was added 0.014mL (0.14mmol, 10

equivalents) of HSPh under an argon atmosphere. Approximately 43 mg of TTN was

dissolved in 3mL of MeCN and added to the reaction mixture via syringe. The solution

was stirred at room temperature for an additional three hours. There was no definitive

change in color for the reaction. All the volatiles were removed in vacuo and moved to the

glovebox for further workup. The reaction product was washed with pentane and ether and

redissolved in. IR, EPR, and NMR studies indicated the presence of 3a and 4a in a 2:1

ratio. 3a: FTIR (KBr, cm-1): 1743, 1707, 1682 (νNO). EPR (MeCN) g = 2.03. 3b: FTIR

(KBr, cm-1): 1739, 1695 (νNO). NMR (CD3CN, 1H): 7.275 (bs), 7.35 (bs), 7.53 (bs).

Analogous reactions were carried out using 1b in the presence of HStBu, arriving

at the same reaction products, 3b and 4b in a 2:1 ratio as confirmed by IR, EPR, UV-Vis,

and NMR analysis. 3b: FTIR (KBr, cm-1): 1722, 1678 (νNO). UV-Vis (MeCN, λmax, nm):

313, 381, 435. EPR (MeCN) g = 2.03. 4b: FTIR (KBr, cm-1): 1778, 1730 (νNO). UV-Vis

(MeCN, λmax, nm): 314, 358, 423. EPR-silent.

2.5.8 General Method for Sx Detection and Quantification. After the completion

of a reaction, a canula filter was used to remove all soluble products from any insoluble

material. While the soluble material went through the continued workup process described

above, the insoluble material was kept separate in the original Schlenk flask. To this flask

was added 16.8mg of triphenylphosphine in 3mL of MeCN. The resulting reaction mixture

was stirred for three hours. The volatiles were removed in vacuo and the solid product was

used for NMR and GC-MS samples.

Quantification was carried out using GC-MS with the preparation of a calibration

curve of TPPS with concentrations between 0.1 and 0.5mM. The results from the

71  

experimental reactions could then be compared to the curve, and Sx production could be

quantified.

2.5.9 General Method for H2S Detection and Quantification. After the

completion of a reaction, the vacuum arm of the reaction flask was connected to a 3-way

stopcock attached to a round bottomed flask. The RBF was charged with 1mL of 1mM

C7Az solution and cooled in liquid nitrogen for 10 minutes. The Schlenk flask vacuum

arm and RBF stopcock were opened allowing the headspace to move from the reaction

flask to the RBF. The headspace was transferred for a total of 2 minutes. The stopcock was

closed and the RBF was warmed to room temperature and the sensor solution was stirred

for one hour before being used for fluorescence analysis.

The results could be quantified utilizing a calibration curve made through the same

procedure using only NaSH and HCl was reagents in the reaction flask to produce the H2S

for analysis. This procedure was carried out using varying amounts of the reagents to

produce concentrations of H2S between 0.2 and 1mM. The results of experimental

reactions can then be compared to the curve to determine the amount of H2S produced.

72  

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Halliwell, B.; Moore, P.K. J. Neurochem. 2004, 90, 765.

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(12) Johnson, D.C.; Dean, D.R.; Smith, A.D.; Johnson, M.K. Annual Review of

Biochemistry 2005, 74, 247.

(13) Crack, J.C.; Green, J.; Thomson, A.J.; Le Brun, N.E. Current Opinion in Chemical

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(14) Ding, H.; Demple, B. Proc. Nat. Acad. Sci. U.S.A. 2000, 97, 5146.

(15) Hyduke, D.R.; Jarboe, L.R.; Tran, L.M.; Chou, K.J.; Liao, J.C. Proc. Nat. Acad. Sci.

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(16) Landry, A.P.; Duan, X.; Huang, H.; Ding, H. Free Radical Biol. Med. 2011, 50, 1582.

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(18) Tran, C.T.; Kim, E. Inorg. Chem. 2012, 51, 10086.

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Kamali, S. Yoda, Y.; Zhao, J.; Hu, M.Y.; Alp, E.E.; Oganesyan, O.; Le Brun, N.E.; Cramer,

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(23) Victor, E.; Lippard, S.J. Inorg. Chem. 2014, 53, 5311.

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(25) Vanin, A.F.; Serezhenkov, V.A.; Mikoyan, V.D.; Genkin, M.V. Nitric Oxide 1998, 2,

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(26) Donahue, J.P.; Chem. Rev. 2006, 106, 4747.

(27) Lu, T.T.; Huang, H.W.; Liaw, W.F. Inorg. Chem. 2009, 48, 9027.

(28) Harrop, T.; Song, D.; Lippard, S.J. J. Inorg. Biochem. 2007, 101, 1730.

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75  

Chapter 3: Thiol-Dependent NO Reactivity with a Water-

Stable and Soluble Synthetic Model [4Fe-4S] Cluster

76  

3.1 Abstract

Herein we report the reactivity of nitric oxide (NO) with a new model cluster

system—[Fe4S4(SEtOH)4]2- (1), a cluster that is both soluble and stable in water and

aqueous buffer media. This synthetic model undergoes thiol-dependent degradation to

iron-nitrosyl compounds so far unknown to the scientific literature. In the absence of thiol,

1 reacts with NO to produce Roussin’s Black Anion: [Fe4S3(NO)7]1- (RBA) and elemental

sulfur. In the presence of excess HSEtOH, 1 degrades into three separate species. The THF

soluble product exhibits FTIR features (νNO = 1761, 1736) similar to those for the ethyl

thiolate derived Roussin’s Red Ester: [Fe2(μ-SEt)2(NO)4]. The ether soluble product

exhibits weak FTIR features νNO = 1749, 1774, that do not match with any known iron-

nitrosyl product. The ether and THF insoluble product produces a single strong, broad

FTIR feature νNO = 1649, that also is not a match for any known iron-nitrosyl species.

Definitive identification for these products is still ongoing, but reactions in the presence of

thiol did not produce elemental sulfur, instead releasing the bridging sulfide ions as

hydrogen sulfide. This lends further evidence to iron-sulfur clusters as a point of crosstalk

between H2S and NO, and sets the foundation for probing that reactivity in aqueous buffer

media.

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3.2 Introduction

Nitric oxide (NO) is one of the three important gasotransmittors found in biological

systems1, even though it was originally thought to just be a toxic gas2. Along with carbon

dioxide and, most recently, hydrogen sulfide (H2S)3,4, NO acts as a messenger within the

cell to transmit chemical messages vital to the survival of the organism. In human beings

alone NO has been found to act in a number of ways: the immune system where it takes

part in the inflammation response5, the cardiovascular system where it acts as a vasodilator

and vasorelaxer6, and the central nervous system where it has been found to act in the

development of the brain as well as memory and learning capabilities7. Interestingly, there

is a great deal of overlap in the functions performed by NO and H2S8, which acts in the

same systems in the same fashions mentioned above. This overlap of responsibility

between two of the vital gaseous signaling molecules led researchers to search for a point

of crosstalk between the two, which has met with varying degrees of success9-11.

One example of indirect crosstalk between NO and H2S is their convergence on the

secondary messenger cGMP. First, NO helps facilitate the generation of cGMP by

activating an enzyme known as soluble guanylyl cyclase. Then H2S maintains cGMP levels

by delaying its degradation through its inhibition of phosphodiesterase-512-14. This,

however, is only an indirect example of crosstalk between the two. A direct point of

crosstalk has been elusive until recently with some help from our lab.

One known target for NO reactivity in biological systems is iron-sulfur clusters.

These clusters are common to the most ancient and modern forms of life15 and make up the

second most diverse category of enzymatic motifs—following only iron-oxo species found

in proteins such as hemoglobin16. Most often ligated by S-bound cysteine residues within

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proteins, the iron sulfur cores are comprised of iron and bridging inorganic sulfide ligands.

These bridging sulfides are what first brought our group’s attention to these clusters as a

possible point of crosstalk for NO and H2S. Our first investigations were carried out by Dr.

Camly Tran17,18 and utilized the simplest form of the iron-sulfur cluster—the rhombic [2Fe-

2S]2+ core. A great deal of work has been carried out on these clusters, using both protein-

bound19 clusters and more simplified synthetic models20,21. By Using synthetic models,

researchers have been able to gain greater chemical insight into the reactivity of

biologically relevant complexes, including iron-sulfur clusters. In the place of the cysteine

residues that usually ligate these clusters in biological systems, researchers use much more

simplified thiolate ligands to provide the stabilizing scaffold for the iron-sulfur core.

Schematic representations of these clusters and the synthetic models are shown in Figure

3.1, visualizing the most common cluster core—the [4Fe-4S] cubane motif.

Figure 3.1: Schematic representations of (a) biological [4Fe-4S] clusteres with four

cysteine residues as ligands for the cluster and (b) synthetic model clusters where the

cysteines are replaced with simple thiolate ligands. For this work the cluster is

[Et4N]2[Fe4S4(SCH2CH2OH)4] 1.

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Utilizing synthetic model clusters, Dr. Tran was able to show that under the right

conditions, namely the inclusion of an external thiol source during nitrosylation, the

synthetic clusters would release their bridging sulfides as hydrogen sulfide rather than

elemental sulfur, which had been found in previous studies. These reaction patterns are

summarized in Scheme 3.1.

Scheme 3.1: Differential reactivity of model [2Fe-2S]2+ clusters in the absence (top path)

and presence (bottom path) of an external thiol source. Scheme adapted from Tran et al18.

In this case, the addition of nitric oxide to a synthetic [2Fe-2S] cluster resulted in the

production of a dinitrosyl iron complex (DNIC). This was a significant result because

previous results by Ding and Demple had shown that DNICs were the most likely product

of nitrosyation of the [2Fe-2S] cluster containing SoxR protein19. They were able to

determine this using a full-cell EPR spectrum that showed a signal at g = 2.03, a

characteristic signal for DNIC species22. The model reactions also produced hydrogen

sulfide, which provided evidence that iron-sulfur clusters could indeed be a point of direct

crosstalk between nitric oxide and hydrogen sulfide. The agreement between the iron

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containing products of both the synthetic and protein-bound reactions led us to believe that

the production of hydrogen sulfide was also a biologically relevant result. We then turned

our attention to whether this reactivity, and role in H2S—NO crosstalk, could be expanded

to include other iron-sulfur clusters, starting with the most common—[4Fe-4S] clusters.

Unlike the [2Fe-2S] cluster case, previous work with protein-bound and synthetic

[4Fe-4S] clusters had led to a mix of results and disagreement in the products. Using protein

bound [4Fe-4S] clusters in the fumarate and nitrate reductase (FNR) family of proteins23-

25, the LeBrun group had determined that the iron containing products of nitrosylation were

Roussin’s Red Ester (RRE) and a complex similar to, but not the same as, Roussin’s Black

Anion. Common nitrosylated products of iron sulfur clusters are shown in Figure 3.2.

Figure 3.2: Nitrosylated iron species generated from the reaction of iron-sulfur clusters

with nitric oxide in biological and model reactions.

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Additionally, the bridging sulfides were found to be released as two different products

during the reaction: three of them were released as elemental sulfur, and one of them was

released as sulfide anion.

Synthetic model reactivity, however, has yet to match these findings. Work done in

the Lippard26,27 group has indicated that the reaction environment can have a drastic impact

on the products produced by synthetic model clusters. They found that when excess nitric

oxide was added to a synthetic cluster alone the major product was Roussin’s Black Anion,

(2) in Figure 3.2. If they added an external thiolate source to the reaction mixture, though,

they found that they would instead produce four equivalents of the corresponding DNIC

(4) was produced. The reaction scheme is shown in Scheme 3.2.

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Scheme 3.2: Differential reactivity for the nitrosylation of synthetic [4Fe4S] clusters in the

absence (path a) and presence (path b) of an external thiolate source. Adapted from Lippard

et al.27

Furthermore, in both cases, elemental sulfur is the only product derived from the bridging

sulfides. Therefore, there is very little agreement between the synthetic and protein bound

cluster systems—both have elemental sulfur as one of their products, and the biological

systems indicate a product similar to RBA, but it is not an exact match and elemental sulfur

is not the only product from the bridging sulfides. Another set of work from our lab set out

to reconcile the two systems.

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Scheme 3.3: Differential reactivity of synthetic model [4Fe4S] cluster in the absence (path

a) and presence (path b) of an external thiol source. This work is discussed in the previous

chapter where R = Ph or tBu.

Using synthetic model [4Fe-4S] clusters with phenyl- and tert-butylthiolate supporting

ligands, we investigated the effect of adding an external thiol source—similar reactivity to

that studied by Tran and coworkers discussed previously. While the results still did not

match those found by the LeBrun group perfectly, they did provide some new products.

Specifically, we found that the addition of thiol to the reaction mixture resulted in the

production of biologically relevant species—Roussin’s Red Ester and hydrogen sulfide.

While this reaction still includes the DNIC, which is implicated in some [4Fe-4S] cluster

reactivity but not others28,29, it also provided the first evidence of RRE produced via

nitrosylation of a synthetic model [4Fe-4S] cluster as well as the first evidence of hydrogen

sulfide production form a similar reaction. Emboldened by these results, we decided to start

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to investigate the effects of one of the major points of contention for synthetic model

complexes—the reaction medium.

Synthetic model clusters offer researchers much more freedom to utilize a wide

range of chemical and analytical techniques to gain more insight into the reactivity of

biologically relevant species, but it often comes with a major drawback—insolubility and

instability in water. For example, the clusters used in the previously mentioned study to

investigate the addition of an external thiol source, [Fe4S4(SPh)4]2- and [Fe4S4(StBu)4]2-,

are exceedingly stable compounds at room temperature both in solid form and in an

acetonitrile solution, but they are equally unstable in the presence of water. Therefore,

while these clusters have been shown to provide invaluable chemical insight into

biologically relevant reactivity, the inability for them to be studied in more biologically

relevant solvent environments can still lead to questions about the validity of the results.

Toward this end, our group decided to utilize one of the few water-soluble and water-stable

clusters known to the literature30,31 to see if the reactivity of such a cluster in aqueous media

would match that seen for the synthetic model clusters in organic media. Herein we report

preliminary reactivity studies of a water-soluble and water-stable synthetic model [4Fe4S]

cluster [Fe4S4(SEtOH)4]2- (1), shown in Figure 3.3.

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Figure 3.3: Schematic representation of [Fe4S4(SEtOH)4]2- (1). For the purposes of this

work, the cluster was synthesized and used with a tetraethylammonium counterion.

This cluster, first synthesized in the 1970’s by the Holm group32, has been shown to be

stable and soluble in dimethylsulfoxide-water solutions with ratios ranging from 9:1 to 2:8

DMSO:water. It is also soluble and stable in pure acetonitrile, which was used for the

purposes of these preliminary reactivity studies.

3.3 Results and Discussion

3.3.1 Effects of the New Cluster System on Reaction Products. The first question

to answer is whether the new cluster system followed the same reactivity pattern as the

clusters used in the previous chapter. To match, we would need to have the same reaction

product for a reaction in the absence of an external thiol source, RBA and elemental sulfur,

and the same change in reactivity pattern based on the inclusion of that thiol source. The

first set of reactions, in the absence of a thiol source provided the same data that bad been

seen for the previous two cluster systems—the nitrosylation of [Et4N]2[Fe4S4(SEtOH)4] (1)

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in the absence of an external thiol source produced Roussin’s Black Anion in large yields,

confirmed by UV-Vis and FTIR33 analysis shown in Figure 3.4, and produced

approximately 1 equivalent of elemental sulfur.

Figure 3.4: FTIR (left) and UV-Vis (right) spectra for the reaction product of (1) with NO

in the absence of an external thiol source. Both spectra confirmed the production of

Roussin’s Black Anion (2).

Though the confirmation of RBA production through reaction in the absence of thiol is

important to establishing a reaction pattern, it is more important that the reaction products

be different upon the inclusion of a thiol source.

Upon the addition of a nitric oxide source to (1) in the presence of HSEtOH, the

solution was observed to become red-brown in color. Further workup of the crude reaction

product discovered three separate products, one soluble in ether, THF, and acetonitrile.

Since there were only two products observed for the previous studies of the phenyl and

tertbutyl thiolate-based clusters, work immediately began trying to determine the identities

of these new products.

87  

3.3.2 Ongoing Identification of Iron-containing Products. Prior to

experimentation, the hypothesis was that the products of this set of reactions would match

that of the previously utilized clusters. As soon as three products were discovered rather

than two, that was no longer a valid assumption. As mentioned in Section 3.3.1, washing

the crude reaction product with diethyl ether, THF, and acetonitrile resulted in the

separation of three products. The solutions in ether and THF were pale and bright red,

respectively, and the MeCN extraction was found to be dark brown in color. The ether

extraction was found to have the least, only trace amounts of solid product upon removal

of volatile species in vacuo. An FTIR spectrum, shown in Figure 3.5, shows only slight

peaks in the NO-stretching region.

Figure 3.5: FTIR spectrum (KBr pellet) of the ether soluble product of the reaction

between (1) and NO in the presence of HSEtOH. Marked peaks are in the typical NO

stretching frequency range.

88  

Since the ether extraction made up such a small proportion of the product, it was left alone

for the time being. The bright red, THF-soluble product provided much more interesting

information. The UV-Vis and FTIR spectra, shown in Figure 3.6, provide evidence to

suggest a couple of different products.

Figure 3.6: The FTIR (left, KBr pellet) and UV-Vis (right, MeCN) spectra for the THF

soluble reaction product of (1) with NO in the presence of HSEtOH (black solid line). The

red dotted line in the FTIR spectrum is authentic [Fe2(μ-SEt)2(NO)4] for comparison.

The UV-Vis spectrum matches very well to the RBA UV-Vis trace, but the FTIR spectrum

does not match RBA. Instead, it shares a significant overlap with the peaks observed in the

FTIR spectrum (Figure 3.6, red dotted line) of the authentic ethylthiolate-based Roussin’s

Red Ester34 complex [Fe2(μ-SEt)2(NO)4]. Not pictured in Figure 3.6 is a significant

increase in signal at 3500cm-1, where the -OH stretching frequency is found. We took these

results to indicate that the 2-mercaptoethanol-based Roussin’s Red Ester, [Fe2(μ-

SEtOH)2(NO)4], had been produced as a result of the reaction in the presence of thiol. An

x-ray crystal structure of the complex remains elusive. Based on the results of the THF

soluble material, our next thought was that the acetonitrile soluble product could be the 2-

89  

mercaptoethanol-based DNIC, [Et4N][Fe(SEtOH)2(NO)2] and we presumed that the IR

spectrum should once again match that of the ethylthiolate-based DNIC,

[Et4N][Fe(SEt)2(NO)2] . The results of our FTIR and UV-Vis analysis, shown in Figure

3.7, did not indicate that result.

Figure 3.7: The FTIR (left, KBr pellet) and UV-Vis (right, MeCN) spectra for the MeCN

soluble reaction product of (1) with NO in the presence of HSEtOH (black line). The red

dotted line in the FTIR spectrum is of authentic [Et4N][Fe(SEt)2(NO)2] for comparison.

Clearly, the MeCN product does not match the ethyl-based DNIC35 as well as the THF

soluble product matched the RRE. Further characterization is under way for this product

as well, though a crystal structure remains elusive. While no definitive comparison can be

made at this time, the FTIR spectrum of the MeCN soluble product are reminiscent of the

known species [Fe4S4(NO)4]1-, which displays a similar broad single peak at 1725cm-1 in a

DCM solution27. The characterization of this product is still ongoing. The results of this

experiment and the products it produces are summarized in Scheme 3.4.

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Scheme 3.4: Summary of the separation of the three species produced from the

nitrosylation of 1 in the presence of excess HSEtOH. There was no product found in a

previous pentane wash, and there was no solid remaining after the final precipitate was

dissolved in MeCN.

The results of these experiments indicate that the reaction pattern for this cluster

system may be entirely new. To gain greater understanding of the reaction taking place,

solution IR was carried out using a submersion probe, and the spectrum was collected every

few minutes for a total of 3 hours. A demonstrative spectrum is shown in Figure 3.8.

91  

Figure 3.8: Solution IR spectrum of the reaction between [Et4N][Fe4S4(SEtOH)4] (1) and

NO in the presence of HSEtOH before the NO addition (black line), 5 minutes after

addition (blue dotted line) and 3 hours after addition (red line).

The solution IR provided two pieces of insight: first, the acetronitrile-soluble product was

the major product, and second, the ether extraction product seems as though it could be an

intermediate species of some kind. During the reaction the peaks assigned to that species,

at approximately 1770 and 1750cm-1, initially grew into the spectrum over the course of

the first few minutes and then receded back to the baseline to give the final spectrum, the

red line in the above figure. This also explains why there was so little of the product.

Further efforts are ongoing to determine the identity of all three chemical species.

3.3.3 The Fate of the Bridging Sulfides. The final part of the question pertaining

to the reaction pattern of this new cluster is whether or not the bridging sulfides are still

released as hydrogen sulfide in the presence of an external thiol source. To determine if

92  

that is the case, lead acetate paper36 and the turn-on fluorescence sensor 7-azido-4-

methylcoumarin (C7Az)37 were employed.

Lead acetate paper is a common technique for qualitative determination of

hydrogen sulfide production, detecting concentrations as low as 5ppm38. The paper is

coated in lead acetate (Pb(OOAc)2), which reacts with hydrogen sulfide to produce lead

sulfide and acetic acid by Equation 3.1.

(3.1) Pb(OOAc) + H2S → PbS + 2 HOOAc

Lead sulfide is a dark brown-black color, so upon interaction with H2S the paper

dramatically changes color. Examples of pre- and post-reaction lead acetate paper can be

seen in Figure 3.9.

Figure 3.9: Lead acetate paper suspended in the neck of a reaction flask before (left) and

after (right) interaction with a headspace containing H2S.

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To determine if H2S was produced during the reaction of 1 in the absence and presence of

HSEtOH, lead acetate paper was hung in the neck of a reaction flask for the duration of the

reaction time. The results of such reactions are summarized in Figure 3.10.

Figure 3.10: Lead acetate paper (a.) before a reaction, (b.) after a reaction between

[Et4N]2[Fe4S4(SEtOH)4] (1) and NO, (c.) after a reaction between 1 and HSEtOH, (d.) after

a reaction between 1, NO, and HSEtOH.

The results clearly indicate that the H2S portion of the reactivity match that of the

previously tested clusters. In the absence of any thiol, there is no indication of a reaction

with the lead acetate paper. As was seen in the case of HSPh, there is a slight false positive

when stirring the cluster in the presence of HSEtOH with no NO. This result is far

outstripped by the true positive result, shown in part d of the above figure. This result shows

that H2S is only produced during nitrosylation in the presence of HSEtOH, with none

produced in its absence.

To get a greater understanding of the fate of the bridging sulfides, we utilized the

turn-on fluorescence H2S sensor 7-azido-4-methylcoumarin (C7Az). In the presence of

H2S the azide group is reduced to an amine, and 7-amino-4-methylcoumarin is fluorescent

94  

with λmax=365nm and λemm=434nm. By transferring the headspace of the reaction via the

method illustrated in Figure 3.11, it is possible to determine whether H2S is produced

during the reaction or not.

Figure 3.11: Setup for headspace transfer and analysis by C7Az.

For reactions in the absence of an external thiol source, there is no response from

the fluorescence sensor. When an external thiol source is added to the reaction mixture,

however, a strong fluorescence response is produced. These results are summarized in

Figure 3.12.

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Figure 3.12: Fluorescence response for the reactions of [Et4N]2[Fe4S4(SEtOH)4] (1) with

NO in the absence (black) and presence (red) of HSEtOH.

These results provide evidence that the presence of an external thiol source changes the

reaction of this [4Fe-4S] cluster, allowing for the production of H2S. This result allows for

the continued hypothesis that [4Fe-4S] clusters are a center for the crosstalk of H2S and

NO. Efforts to quantify H2S production are ongoing.

3.4 Conclusion

The present study shows that the inclusion of an external thiol source (HSEtOH) in

the reaction environment for the nitrosylation of [Et4N]2[Fe4S4(SEtOH)4] (1) greatly

influences the reaction products. In the absence of such a thiol source the iron-containing

product is RBA [Fe4S3(NO)7]1- , with one bridging sulfide lost as elemental sulfur. In the

presence of the thiol, however, the bridging sulfides are instead lost as H2S and the iron-

96  

containing products change drastically. One of the products, soluble in ether, appears to be

residual amounts of an intermediate species with FTIR features at νNO = 1749 and 1774.

The second, ether insoluble and THF soluble product appears to be a complex similar to

independently synthesized [Fe2(μ-SEt)2(NO)4], the ethyl derived Roussin’s Red Ester, with

FTIR features at νNO = 1736 and 1761. Identification of the final, THF insoluble and MeCN

soluble product remains elusive but displays a consistent strong, broad FTIR feature at νNO

= 1649, and we await a diffraction-quality crystal to provide definitive characterization.

3.5 Experimental Methods

3.5.1 General Considerations. All reactivity studies and manipulations, unless

otherwise specified, were carried out under inert atmospheric conditions: either in an

MBraun glovebox under dewar supplied nitrogen (>0.1ppm O2, >0.1ppm H2O), or via

conventional Schlenk techniques under argon (UHP 5.0). All solvents were passed through

an alumina column and dried over molecular sieves (3Å) under a glovebox atmosphere.

Thiols were purchased from Sigma Aldrich and degassed by freeze-pump-thaw and stored

under an argon atmosphere. All other chemicals were ordered from Sigma Aldrich and

used as received. Nitric oxide (Corp Bros.) was purified following a literature method39,

where the gas is passed through an Ascarite column and distilled at -80ºC.

3.5.2 Physical Measurements. Unless otherwise specified, all samples were

prepared under a nitrogen glovebox atmosphere. Infrared spectra were recorded on a

Bruker Tensor 27 FT-IR—samples were analyzed as KBr pellets or solution samples using

a fiber optic immersion probe. UV-Visible spectra were recorded on a Varian Cary 50 Bio

spectrometer, NMR were recorded on a Bruker Avance 400MHz spectrometer, and GC-

MS data were recorded on a Hewlett-Packard (Agilent) GCD 1800 GC-MS spectrometer.

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3.5.3 Synthesis. [Et4N]2[Fe4S4(SEtOH)4 (1)32 and TTN28 were synthesized

according to literature precedent. 1 was synthesized from [Et4N]2[Fe4S4(StBu)4] which was

also synthesized according to literature precedent40.

3.5.4 General Reaction of [Et4N]2[Fe4S4(SEtOH)4] (1) with NO in the Absence

of Thiol. A 10mL Schlenk flask was charged with 15mg (0.016mmol) of 1 in 3mL MeCN.

The solution was put under argon on the Schlenk line and stirred at room temperature.

10mL of headspace were removed from the flask and replaced with purified NO gas. The

flask was wrapped in foil and the solution was stirred at room temperature for a total of 3

hours. All volatiles were removed from the flask in vacuo and the flask was returned to the

glovebox for workup. The solid was redissolved in MeCN and layered with ether in the

freezer to yield RBA (10mg, 85% yield), confirmed by FTIR (KBr, cm-1): 1732, 1711,

1689 (νNO) and UV-Vis (MeCN, λmax, nm): 207, 259, 357, 428, 580.

3.5.5 General Reaction of [Et4N]2[Fe4S4(SEtOH)4] (1) with NO in the Presence

of Thiol. A 10 mLSchlenk flask was charged with 15mg (0.016mmol) of 1 in 3mL of

MeCN and put on the Schlenk line under argon. 1.25mg (0.16mmol) of HSEtOH was added

to the solution via stock solution in MeCN. The resulting mixture was stirred at room

temperature for 5 minutes. 10mL of static argon headspace was removed from the flask

and replaced with purified NO gas. The flask was covered in foil and the solution was

stirred at room temperature for 3 hours. All volatiles were removed in vacuo and the flask

was moved to the glovebox for workup. The products were extracted by ether, THF, and

MeCN washes. The ether extraction was analyzed by FTIR (KBr, cm-1): 1774, 1749 (νNO).

The THF extraction was analyzed by FTIR (KBr, cm-1): 1761, 1736 (νNO) and UV-Vis

98  

(MeCN, λmax, nm): 207, 259, 357, 428, 580. The MeCN extraction was analyzed by FTIR

(KBr, cm-1): 1649 (νNO) and UV-Vis (MeCN, λmax, nm): 219, 295, 555.

3.5.6 General Reaction of [Et4N]2[Fe4S4(SEtOH)4] (1) with TTN in the Absence

of Thiol. A 10mL Schlenk flask was charged with 15mg (0.016mmol) of 1 in 3mL MeCN.

The flask was removed from the glovebox and stirred at room temperature under argon on

the Schlenk line. 48mg (0.16mmol) of TTN was dissolved in 3mL MeCN and added to the

stirring solution at room temperature. The solution was stirred for 3 hours with the flask

wrapped in foil. The volatiles were removed in vacuo and the flask was returned to the

glovebox for workup. The solid was washed with either and redissolved in MeCN and

layered with ether in the freezer to produce RBA (8.4mg, 80% yield), confirmed by FTIR

(KBr, cm-1): 1732, 1711, 1689 (νNO) and UV-Vis (MeCN, λmax, nm): 207, 259, 357, 428,

580.

3.5.7 General Reaction of [Et4N]2[Fe4S4(SEtOH)4] (1) with TTN in the

Presence of Thiol. A 10mL Schlenk flask was charged with 15mg (0.016mmol) of 1 in

3mL MeCN. The flask was moved out of the glovebox and put under argon on the Schlenk

line. 1.25mg (0.16mmol) of HSEtOH was added to the solution via syringe in MeCN. 48mg

(0.16mmol) of TTN was added via syringe in 3mL of MeCN. The flask was wrapped in

foil and the solution was stirred at room temperature for 3 hours. All volatiles were

removed in vacuo and the flask was moved to the glovebox for workup. The products were

extracted by ether, THF, and MeCN washes. The ether extraction was analyzed by FTIR

(KBr, cm-1): 1774, 1749 (νNO). The THF extraction was analyzed by FTIR (KBr, cm-1):

1761, 1736 (νNO) and UV-Vis (MeCN, λmax, nm): 207, 259, 357, 428, 580. The MeCN

99  

extraction was analyzed by FTIR (KBr, cm-1): 1649 (νNO) and UV-Vis (MeCN, λmax, nm):

219, 295, 555.

3.5.8 General Procedure for Sx Analysis. After the completion of a reaction, a

canula filter was used to remove all soluble products from any insoluble material. While

the soluble material went through the continued workup process described above, the

insoluble material was kept separate in the original Schlenk flask. To this flask was added

16.8mg of triphenylphosphine in 3mL of MeCN. The resulting reaction mixture was stirred

for three hours. The volatiles were removed in vacuo and the solid product was used for

NMR and GC-MS samples.

3.5.9 General Procedure for H2S Analysis. After the completion of a reaction, the

vacuum arm of the reaction flask was connected to a 3-way stopcock attached to a round

bottomed flask. The RBF was charged with 1mL of 1mM C7Az solution and cooled in

liquid nitrogen for 10 minutes. The Schlenk flask vacuum arm and RBF stopcock were

opened allowing the headspace to move from the reaction flask to the RBF. The headspace

was transferred for a total of 2 minutes. The stopcock was closed and the RBF was warmed

to room temperature and the sensor solution was stirred for one hour before being used for

fluorescence analysis.

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(6) Ignarro, L.J. Annu. Rev. Immunol. Toxicol. 1990, 30, 535.

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(13) Quiroga, S.L.; Almaraz, A.E.; Amborebieta, V.T.; Perissinotti, L.L.; Olabe, J.A.

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(14) Yong, Q.C.; Cheong, J.L.; Hua, F.; Deng, L.W.; Khoo, Y.M.; Lee, H.S.; Perry, A.;

Wood, M.; Whiteman, M.; Bian, J.S. Antioxid. Redox Signal 2011, 14, 2081.

(15) Lovenberg, W. Iron-sulfur Proteins: Molecular Properties; Academic Press: New

York, 1973.

(16) Beinert, H.; Holm, R.H.; Munk, E. Science, 1997, 277, 653.

(17) Tran, C.T.; Kim, E. Inorg. Chem. 2012, 51, 10086.

(18) Tran, C.T; Williard, P.G.; Kim, E. J. Am. Chem. Soc. 2014, 136, 11874.

(19) Ding, H.; Demple, B. Proc. Nat. Acad. Sci. U.S.A 2000, 97, 5146.

(20) Hyduke, D.R.; Jarboe, L.R.; Tran, L.M.; Chou, K.J.; Liao, J.C. Proc. Nat. Acad. Sci.

U.S.A. 2007, 104, 8484.

(21) Landry, A.P.; Duan, X.; Huang, H.; Ding, H.; Free Radical Biol. Med. 2011, 50, 1582.

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Chapter 4: Model [4Fe-4S]2+ to [2Fe-2S]2+ Cluster

Conversion Facilitated by O2, Thiolate, and Disulfide

105  

4.1 Abstract

Iron sulfur clusters are ubiquitous in biology and carry out many functions vital to

survival. One interesting reactivity pattern of these clusters is the ability to undergo

transformations from one cluster core to another, most commonly converting between the

[4Fe-4S] cubane and the [2Fe-2S] rhombic cores. This specific interaction has been shown

to effect gene regulation in response to molecular oxygen through the [4Fe-4S] cluster in

fumarate and nitrate reductase (FNR) found in many bacteria. Previous attempts at

affecting this transformation in synthetic model clusters have been successful, but only

through the use of harsh, biologically irrelevant oxidants and supporting ligands. In this

work we report the conversion of an all thiolate bound [4Fe-4S]2+ cubane cluster

[Et4N]2[Fe4S4(SPh)4] (1) directly to the corresponding [2Fe-2S]2+ rhombic cluster

[Et4N]2[Fe2S2(SPh)4] (2) through the use of biologically relevant reagents including

thiolate, disulfide, and molecular oxygen. This work provides the first evidence of such a

transformation under mild conditions in synthetic model systems.

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Introduction

Iron sulfur clusters are ubiquitous in biology1 and common to the most ancient and

modern forms of life2. Utilized in many ways, iron sulfur proteins facilitate the survival of

organisms through electron transport3, structural support4,5, and substrate binding and

activation during catalysis6. Additionally, these clusters have been shown to play a role in

cellular signaling and small molecule sensing7-9. One function that has been extensively

studied is its interaction with nitric oxide, one of the three vital gasotransmittors. This

reactivity has been shown to play a role in iron and gene regulation, both of which are vital

to organisms’ survival. Another reaction of iron-sulfur clusters that has been shown to

affect the same cellular processes is O2 sensing10. This function is carried out by way of a

reaction mechanism that has been well documented11 but is not entirely understood—

cluster conversion.

For the purposes of this work, cluster conversion refers to any reaction by which an

iron sulfur clusters changes from one nuclearity to another. For example, a [4Fe-4S] cluster

can undergo an oxidation that converts it to a [2Fe-2S] cluster. Figure 4.1 shows a

schematic representation of cluster conversions that have been observed in biological and

synthetic systems.

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Figure 4.1: Schematic representations of cluster conversions observed in biological and

synthetic systems. Terminal ligands removed for clarity. Figure adapted from a published

review by Holm et al11.

This work will focus solely on the conversion of a [4Fe-4S] cluster to a [2Fe-2S] cluster, a

reaction that has been shown to be important for biologically relevant processes. One

system that is particularly well studied is the oxygen sensing activity of fumarate and

nitrate reductase (FNR) proteins in E. coli12-16.

Some bacteria can survive in both the absence and presence of oxygen. To do this,

they must be able to sense the availability of O2 in their surroundings and adjust their

respiration processes to match those conditions. In E. coli this function is carried out by

the FNR enzyme13. Under anaerobic conditions, FNR has two [4Fe-4S] clusters and exists

as a dimer that binds to DNA and inhibits the transcription of certain genes that are required

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for aerobic respiration15. When the concentration of oxygen gets high enough, the clusters

undergo an oxidation and convert into [2Fe-2S] clusters along with a release of the other

two iron atoms to an unknown product. Additionally, the Le Brun group determined that

the two extra bridging sulfide ligands were released and captured by two of the cysteine

residues ligating the iron-sulfur core, forming cysteine persulfide ligands. This product

could then be cycled back to the original [4Fe-4S] cluster through the addition of an iron(II)

source, cysteine, and cysteine desulfurase enzyme16. A representation of this cluster

conversion is shown in Scheme 4.1.

Scheme 4.1: Graphical representation of the reversible cluster conversion reaction

undergone by the [4Fe-4S] clusters in FNR. Evidence suggests the inclusion of two

bridging sulfides to produce cysteine persulfide ligands16.

This cluster conversion leads directly to a conformational change in the protein itself,

breaking the dimer into two monomers and interrupting the binding to DNA13. Once that

binding is broken, those genes are open to transcription and the cellular machinery can

produce the species needed for aerobic respiration. Though this reaction is well

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documented in biological systems, it is not very well understood chemically—in part due

to a lack of synthetic model complexes that undergo the same reaction pattern.

There have been many reactivity patterns studied for iron sulfur clusters, and a great

deal of insight in those patterns has been found through using synthetic analogous clusters.

While protein bound clusters provide a more direct look at the reactivity found in the

biological systems, they are very difficult to use. First, they need to be isolated and purified

in a large enough quantity to be useful. Second, due to their large size relative to molecular

species, it is much more difficult to use a wide range of analytical techniques for product

and mechanistic determinations. To get past these obstacles, researchers have taken to

using synthetic model clusters17-22—these clusters contain the same iron-sulfur cores, but

instead of being ligated by cysteine residues in a protein, they are instead ligated by simple

thiolate molecules. Figure 4.2 shows the difference between these kinds of clusters.

Figure 4.2: Schematic representations of (a) protein bound iron sulfur clusters ligated by

cysteine residues and (b) synthetic model clusters ligated by thiolate molecules, where R

represents a wide range of alkyl and aryl groups.

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By using these smaller species, more analytical techniques can be employed to ascertain

information about the reactions taking place that is not as easily found using protein bound

clusters. Additionally, there have been cases where synthetic clusters have been shown to

demonstrate the same reactivity as their protein-bound counterparts with a high degree of

fidelity23. For the [4Fe-4S] to [2Fe-2S] cluster conversion reaction, however, there are very

few examples of synthetic models successfully undergoing the transformation.

In the 1970’s Holm and his coworkers24 were able to demonstrate the ability of a

synthetic [4Fe-4S] cluster, [Fe4S4Cl4]2- to undergo a cluster conversion to two equivalents

of [Fe2S2Cl4]2- in the presence of ferricenium as an outer-sphere oxidant with excess

chloride ion in solution to stabilize the resulting rhombic cluster. Much more recently,

Tatsumi and coworkers25 we able to show the interconversion of a [4Fe-4S] cluster bearing

amide ligands to its [2Fe-2S] counterpart utilizing pyridine as the additional ligand source.

Once again, this reaction relied on ferricenium to provide a highly oxidized [Fe4S4]4+

cluster core, which then degrades to the two equivalents of [Fe2S2]2+, with the additional

terminal ligand positions filled by pyridine from the reaction solvent. This reaction is

summarized in Scheme 4.2.

Scheme 4.2: Reversible cluster conversion from a [4Fe-4S]4+ core to two equivalents of

[2Fe-2S]2+ core upon the addition or removal of pyridine. Reaction scheme adapted from Tatsumi

et al25.

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Although this work does showcase the desired reactivity, it still relies on a harsh and

biologically irrelevant oxidant to produce a cluster oxidized to the point that it will readily

degrade into two smaller clusters. This work looks to induce the same reactivity using much

more neutral and, more importantly, biologically relevant conditions. Toward that end, we

started to determine which sources of oxidation we should consider for experimentation.

First, we decided that we wanted to utilize a model cluster that was representative

of the most common conditions found in biological systems. We settled on [Fe4S4(SPh)4]2-

as our candidate, as it provided a [4Fe-4S] cluster core ligated by four thiolate molecules,

reminiscent of the cysteine residues found in most biological cluster systems. We then

delved into what species we would need to affect the transformation we were pursuing and

settled on the reaction shown in Scheme 4.3 for our first attempt.

Scheme 4.3: Proposed reaction scheme for the conversion of a [4Fe4S]2+ cluster to two

equivalents of [2Fe2S]2+ core facilitated by one equivalent of disulfide and two additional

equivalents of the corresponding thiolate.

112  

Examining the reaction, we found that to move forward a two-electron oxidation and four

total thiolate ligands were required. The easiest way to deliver those requirements was

through one equivalent of disulfide to provide the oxidation and two of the thiolate ligands,

and then an additional two free thiolate ligands to stabilize the final products. Disulfide, it

turns out, is an oxidant that is very relevant to biological systems26,27.

During homeostasis, the cell has a very high, millimolar, concentration of thiols28.

These concentrations have many components, including amino acid residues such as

cysteine, small polypeptides such as glutathione, and open cysteine residues in other

proteins. These thiols exist in a state of equilibrium with various oxidized derivatives. One

of the most notable derivatives is disulfides, playing many roles including structural

determination and support for proteins. Most disulfide bonds are between residues helping

form and fold proteins into their tertiary structures, though some disulfides are free in the

cellular matrix under the previously mentioned equilibrium. That equilibrium, however,

can be upset29. When a cell undergoes oxidative stress, there are many targets effected by

the reactive oxygen species, one of which is cellular thiols. Under oxidative stress the

concentration of disulfides rises well above equilibrium, and they become a new source for

oxidizing power. This work sets out to investigate the usage of disulfide to affect the cluster

conversion reaction of a synthetic model [4Fe-4S] cluster its corresponding [2Fe-2S]

analog. Additionally, the cluster conversion reaction by molecular oxygen and supporting

thiolate ligands is explored.

4.3 Results and Discussion

4.3.1 Reactivity of [Et4N]2[Fe4S4(SPh)4] (1) with Excess Diphenyldisulfide and

Et4N(SPh). The first reaction condition explored was the addition of excess disulfide and

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thiolate corresponding to the cluster being used. In this case (1) was the cluster in question,

so the phenyl derivatives were used. The cluster was heated to reflux in the presence of 10

equivalents of each species and stirred under argon for 3 hours. During the course of the

reaction, the solution was observed to change from dark red-black to dark purple.

Comparing the UV-Vis spectrum of the final product to the starting material indicated that

a reaction had taken place.

Figure 4.3: UV-Vis trace in MeCN of (1) (black line) and the reaction product of (1) with

10 equivalents each diphenyldisulfide and tetraethylammonium phenylthiolate heated to

reflux for 3 hours (red line). Insert focuses on visible region.

Close inspection of the product UV-Vis trace, the red line in Figure 4.3, indicated the [2Fe-

2S] cluster [Et4N]2[Fe4S4(SPh)4] (2) could be the major reaction product. An NMR sample

was prepared and analyzed in CD3CN, and the diamagnetic spectrum peaks indicated the

[2Fe-2S] cluster was indeed a major product of the reaction. Integration of the peaks

114  

indicated that the cluster was only there in an approximate 50% yield (1 equivalent per (1)

used). Inspection of the paramagnetic regions of the NMR spectrum (shown in Figure 4.4)

identified the other product—two equivalents of [Et4N]2[Fe(SPh)4], (3).

Figure 4.4: Crude 1H NMR (CD3CN) spectrum of the reaction product of

[Et4N]2[Fe4S4(SPh)4] with 10 equivalents each diphenyl disulfide and tetraethylammonium

phenylthiolate showing residual thiolate (blue), [Et4N]2[Fe2S2(SPh)4] (2) (red), and

[Et4N]2[Fe(SPh)4] (3) (black).

This result hinted toward two different possibilities: the simpler explanation was the three

hours at reflux (approximately 80ºC in acetonitrile) was causing the cluster to decompose

into two Fe3+ ions, which formed the rhombic cluster, and two Fe2+ ions, which were

stabilized as tetrathiolate complex anions. The more interesting, if less likely, possibility is

115  

that the cluster was being oxidized to all Fe3+ ions, with two forming the rhombic core and

two being expelled into the reactions mixture. It is known from the literature that

[Fe(SPh)4]1- will auto reduce to [Fe(SPh)4]2- in the presence of excess thiolate, during

which disulfide is formed as the oxidized product. This could be further investigated

through stoichiometric control, but this path is not investigated any further in this

dissertation.

To avoid thermal decomposition of (1), the reaction was run under all the same

conditions except for time and temperature. In this case the reaction was carried out at room

temperature and run for 18 hours instead of only three. In the presence of excess thiolate

and disulfide at room temperature, the reaction still followed the same reaction path: one

equivalent of (2) was produced along with two additional equivalents of (3). This reactivity

is summarized in Scheme 4.4.

Scheme 4.4: Results of the reaction between 1 and excess amounts of diphenyldisulfide

and tetraethylammonium phenylthiolate, producing one equivalent of [Et4N]2[Fe2S2(SPh)4]

(2) and two equivalents of [Et4N]2[Fe(SPh)4] (3).

Since changing the temperature did not have any immediate effect, the reaction

stoichiometry was the next factor to consider.

116  

4.3.2 Reactivity of [Et4N]2[Fe4S4(SPh)4] (1) in the Presence of Stoichiometric

Amounts of Thiolate and Disulfide. To avoid the previously mentioned thermal

decomposition, these reactions were similarly run at room temperature. Instead of using

excess thiolate and disulfide, the exact amount needed for the proposed reaction were

combined—one equivalent of disulfide and two equivalents of thiolate per (1). Again, the

reaction was stirred at room temperature for a total of 18 hours, during which the color was

still observed to change from dark red-black to dark purple. The UV-Vis trace of the

reaction product was again found to match the spectrum for authentic, independently

synthesized (2). The spectral comparison can be seen in Figure 4.5.

Figure 4.5: UV-Vis spectra for (2) (black line) and the reaction product of (1) with 1

equivalent diphenyldisulfide and 2 equivalents of tetraethylammonium phenlthiolate (red

line). Insert focuses on visible region.

117  

NMR analysis (Figure 4.6) of the reaction product found that (2) was the overwhelmingly

major product.

Figure 4.6: Crude 1H NMR (CD3CN) of the reaction product of [Et4N]2[Fe4S4(SPh)4] (1)

with 2 equivalents of tetraethylammonium phenylthiolate and 1 equivalent of

diphenyldisulfide showing residual 1 (blue), [Et4N]2[Fe2S2(SPh)4] (2) (red) and

[Et4N]2[Fe(SPh)4] (3) (black).

There was still a small amount of (3) formed, but it made up less than 5% of the total

product on average over several runs. To our knowledge, this is the first evidence of the

direct conversion of a [4Fe-4S]2+ cluster to a [2Fe-2S]2+ cluster using a mild, biologically

relevant oxidizing agent.

These reaction conditions were also used in reactions at reflux temperatures, run

for a total of three hours. In these cases, the amount of tetrathiolate was increased slightly,

118  

but still significantly lower than the 2:1 ratio of tetrathiolate to [2Fe2S] cluster found in the

excess reactions run at reflux. These findings are summarized in Scheme 4.5.

Scheme 4.5: Reaction results of a stoichiometric reaction of 1 with one equivalent of

diphenyldisulfide and two equivalents of tetraethylammonium phenylthiolate, producing

two equivalents of [Et4N]2[Fe2S2(SPh)4] (2).

This seems to indicate that the production of tetrathiolate is more dependent on having

excess thiolate in solution than it is about the temperature at which the reaction is run.

There are still questions to be answered about this differential reactivity that may provide

some insight into a reaction mechanism for the transformation, but no more work is done

toward that end in this dissertation.

4.3.3 Reactivity of [Et4N]2[Fe4S4(SPh)4] (1) with Molecular Oxygen. In order to

determine whether or not this reactivity could be expanded to include molecular oxygen,

the subject cluster (1) was exposed to molecular oxygen. The solution was stirred for a

total of 24 hours before the reaction was stopped, with NMR experiments taken over the

course of the reaction. Over time the starting material was observed to decay, but no

significant peaks were observed to increase. After the course of 24 hours the solution was

observed to be pale yellow in color with a large amount of orange solid precipitate in the

119  

bottom of the reaction vessel. This solid was insoluble in any conventional organic solvent

system and was presumed to be iron oxide. Therefore, it was determined that the cluster

had moderate stability under molecular oxygen, but over time it decomposed. An NMR of

the final solution showed only thiolate as a diamagnetic species.

4.3.4 Reactivity of [Et4N]2[Fe4S4(SPh)4] (1) with Molecular Oxygen in the

Presence Thiolate. To determine whether the reactivity pattern could be changed by

increased ligand stabilization for the desired product (2), the starting cluster (1) was

combined with four equivalents of phenylthiolate and stirred at ambient temperature in the

glovebox for a total of 18 hours with no visible change to the solution and no significant

spectral changes. After 18 hours the solution was removed from the glovebox and exposed

to air during transfer to a reaction flask. The solution was stirred for a total of 1 hour after

air exposure, during which time the solution was observed to change from dark red-black

to dark purple. The product was isolated from solution under vacuum and UV-Vis and

NMR analysis were performed.

Based on NMR analysis (Figure 4.7), it was determined that approximately one half

of an equivalent of [2Fe-2S] cluster was produced from the reaction.

120  

Figure 4.7: Crude 1H NMR (CD3CN) spectrum for the reaction product of

[Et4N]2[Fe4S4(SPh)4] (1) with 10 equivalents of tetraethylammonium phenylthiolate and 1

hour of air exposure. Spectrum shows [Et4N]2[Fe2S2(SPh)4] (2) (red) and a mixture of

residual tetraethylammonium thiolate and diphenyldisulfide produced from thiolate

oxidation (blue).

Subsequent reactions with different reaction times after air exposure produced a wide

variety of yield in [2Fe2S] cluster. The tetrathiolate product was not detected in any of the

reaction products. Additional reactivity studies with authentic [2Fe-2S] cluster found that

upon exposure to air the cluster would decompose to mostly insoluble black material, most

probably due to the formation of various iron oxides and sulfides. This provides insight

into why the yields of [2Fe-2S] vary so widely—as the cluster is transformed from the

cubane core to the rhombic core, the product is then reacting with the oxygen itself to

provide the decomposition products, so as more [2Fe-2S] cluster is produced, more of it is

121  

also decomposing, but not at the same rate. Therefore, depending on how long the reaction

is stirred after the oxygen exposure, there will be different amounts of product cluster.

Further optimization of this reaction to maximize product cluster is currently under way

but will not be covered in this dissertation.

To our knowledge this is the first example of a synthetic model [4Fe-4S] cluster

undergoing a conversion reaction to yield its [2Fe-2S] derivative in the presence of only

additional supporting ligand and molecular oxygen as the oxidant.

4.3.4 Discussion of Reactivity Results. After carrying out the previously discussed

reactivity studies, we found, in the disulfide oxidation case, we had two different reaction

pathways, and which path a reaction followed was dependent upon the reaction conditions.

This reactivity is summarized in Scheme 4.4.

Scheme 4.6: Differential reactivity observed for a model [4Fe-4S] cluster in the presence

of excess (path a) and stoichiometric (path b) amounts of disulfide and thiolate. All anionic

species have tetraethylammonium as a cation.

122  

Additionally, we know that the reaction of molecular oxygen with the same model cluster

is dependent on the reaction environment. In the presence of just molecular oxygen, the

cluster exhibits moderate stability, slowly degrading into iron oxide products. In the

presence of an external thiolate source, however, the cluster instead undergoes a core

conversion from the [4Fe-4S] cubane cluster to its [2Fe-2S] rhombic counterpart.

Unfortunately, given the instability of the rhombic cluster in the presence of O2, any

reaction optimization has been elusive. Still, to our knowledge this is the first example of

a synthetic model [4Fe-4S] cluster undergoing this cluster conversion under mild oxidative

conditions.

123  

4.6 Conclusion

Herein we report the first evidence of a synthetic model [4Fe-4S] cluster with the

ability to undergo a biologically relevant core conversion to its [2Fe-2S] rhombic

counterpart. First, this reaction was facilitated by disulfide, a relevant oxidant due to its

role in the cell during oxidative stress. Second, the reaction was carried out using molecular

oxygen, the species responsible for the conversion in biological systems. It is our hope that

by further exploring this system we can gain deeper understanding of the underlying

chemistry at play in iron sulfur proteins.

4.5 Experimental Methods

4.5.1 General Considerations. All reactivity, unless otherwise specified, was

carried out under an inert nitrogen atmosphere in a MBraun glovebox (O2 < 0.1ppm, H2O

< 0.1ppm), or under an inert argon atmosphere using standard Schlenk techniques. All

solvents were passed through an alumina column and dried over molecular sieves (3Å)

under a glovebox atmosphere. Thiols were purchased from Sigma Aldrich and degassed

by freeze-pump-thaw and stored under an argon atmosphere. All other chemicals were

ordered from Sigma Aldrich and used as received. Oxygen gas was ordered from Corp

Bros. and used as received.

4.5.2 Physical Measurements. Unless otherwise specified, all samples were

prepared under a nitrogen glovebox atmosphere. Infrared spectra were recorded on a

Bruker Tensor 27 FT-IR—samples were analyzed as KBr pellets or solution samples using

a fiber optic immersion probe. UV-Visible spectra were recorded on a Varian Cary 50 Bio

124  

spectrometer, NMR were recorded on a Bruker Avance 400MHz spectrometer, and GC-

MS data were recorded on a Hewlett-Packard (Agilent) GCD 1800 GC-MS spectrometer.

4.5.3 Synthesis. (1)30, (2)31, and (3)31 were prepared according to literature

precedent. Phenylthiolate was prepared using acid-base chemistry to produce the sodium

salt, followed by cation exchange with Et4NCl in acetonitrile to yield the

tetraethylammonium salt.

4.5.4 General Reaction of (1) with Excess Diphenyldisulfide and Et4N(SPh) at

Reflux. A 10mL Schlenk flask was charged with 15mg (0.014mmol) (1), 31mg

(0.14mmol) diphenyldisulfide, and 34mg (0.14mmol) tetraethylammonium phenylthiolate

in 5mL acetonitrile. The solution was removed from the glovebox and attached to a water-

cooled reflux condenser under a positive argon flow. The solution was heated to reflux

(approximately 80ºC) and stirred for 3 hours. After three hours all volatiles were removed

in vacuo and the solid product was moved to the glovebox. Crude NMR and UV-Vis

samples were prepared as necessary and the product was washed with ether and THF, and

redissolved in MeCN and layered with ether to afford a black powder. NMR analysis

indicated (2) and (3) in a 1:2 ratio. (CD3CN, ppm: 2: 9.30 (bs), 4.90 (bs), 3.22 (bs), 3.14

(bs), 1.19 (bs). 3: 22.77 (bs), -16.63 (bs), -24.39 (bs). UV-Vis, λmax, nm: 2: 480)

4.5.5 General Reaction of (1) with Excess Diphenyldisulfide and Et4N(SPh) at

Room Temperature. A 20mL scintillation vial was charged with 15mg (0.014mmol) (1),

31mg (0.14mmol) diphenyldisulfide, and 34mg (0.14mmol) tetraethylammonium

phenylthiolate in 5mL MeCN. The solution was stirred at room temperature for a total of

18 hours in the glovebox. Volatiles were removed in vacuo and crude NMR and UV-Vis

samples were prepared as needed. The solid product was washed with ether and THF,

125  

redissolved in MeCN and layered with ether to yield a black powder. NMR analysis

indicated (2) and (3) in a 1:2 ratio. (NMR, CD3CN, ppm: 2: 9.30 (bs), 4.90 (bs), 3.22 (bs),

3.14 (bs), 1.19 (bs). 3: 22.77 (bs), -16.63 (bs), -24.39 (bs). UV-Vis, λmax, nm: 2: 480)

4.5.6 General Reaction of [Et4N]2[Fe4S4(SPh)4] (1) with Stoichiometric

Amounts of Diphenyldisulfide and Et4N(SPh) at Room Temperature. A 20mL

scintillation vial was charged with 15mg (0.014mmol) 1, 3mg diphenyldisulfide

(0.014mmol), and 6.7mg of tetraethylammonium phenylthiolate (0.028mmol) in 5mL of

acetonitrile. The reaction mixture was stirred at room temperature for a total of 18 hours,

during which time the solution changed color from dark red-black to dark purple. The

volatile species were removed from the mixture in vacuo and crude UV-Vis and NMR

samples were prepared as needed. The solid product was purified through consecutive

washings with diethyl ether and THF, followed by redissolving in acetonitrile and layering

with ether in the freezer to afford (2). (NMR, CD3CN, ppm: 2: 9.30 (bs), 4.90 (bs), 3.22

(bs), 3.14 (bs), 1.19 (bs). UV-Vis, λmax, nm: 480)

4.5.7 General Reaction of [Et4N]2[Fe4S4(SPh)4] (1) with O2 in the Absence of

Et4N(SPh). A 20mL scintillation vial was charged with 15mg (0.014mmol) 1 in 3mL

MeCN and stirred at room temperature for 10 minutes in the glovebox. The vial was

removed from the glovebox and the solution was opened to air and stirred at room

temperature for a total of between 3 and 18 hours. Over the course of the reaction very little

spectral change was observed until the solution became pale yellow with a large amount of

orange solid. NMR analysis of the solution showed only Et4N(SPh), indicating

decomposition of the starting cluster.

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4.5.8 General Reaction of [Et4N]2[Fe4S4(SPh)4] (1) with O2 in the presence of

Et4N(SPh). A 20mL scintillation vial was charged with 15mg (0.014mmol) 1 and 13.4mg

(0.056mmol) of Et4N(SPh) in 3mL MeCN and stirred at room temperature for 10 minutes

in the glovebox. No changes in color or spectra were observed. The solution was removed

from the glovebox, exposed to air, and stirred for an additional hour. During the hour the

solution was observed to dramatically change color from dark red-black to a dark purple.

All volatile species were removed in vacuo and the solid was moved back into the glovebox

for workup. Crude NMR samples were prepared in CD3CN. The solid was washed with

THF and redissolved in MeCN and layered with ether in the freezer to yield 2. (NMR,

CD3CN, ppm: 2: 9.30 (bs), 4.90 (bs), 3.22 (bs), 3.14 (bs), 1.19 (bs). UV-Vis, λmax, nm: 480) 

127  

4.6 References

(1) Beinert, H.; Holm, R.H.; Munk, E. Science, 1997, 277, 653.

(2) Lovenberg, W. Iron-sulfur Proteins: Molecular Properties; Academic Press: New

York, 1973.

(3) Rao, P. V.; Holm, R. H. Chem. Rev. 2004, 104, 527.

(4) Kuo, C.; McRee, D.; Fisher, C.; O'Handley, S.; Cunningham, R.; Tainer, J. Science

1992, 258, 434.

(5) Guan, Y.; Manuel, R. C.; Arvai, A. S.; Parikh, S. S.; Mol, C. D.; Miller, J. H.; Lloyd,

R. S.; Tainer, J. A. Nat. Struct. Biol. 1998, 5, 1058.

(6) Flint, D. H.; Allen, R. M. Chem. Rev. 1996, 96, 2315.

(7) Crack, J.C.; Green, J.; Thomson, A.J.; Le Brun, N.E. Acc. Chem. Res. 2014, 47, 3196.

(8) Fleischhacker, A.S.; Kiley, P.J. Curr. Opin. Chem. Biol. 2011, 15, 335.

(9) Beinert, H.; Kiley, P.J. Curr. Opin. Chem. Biol. 1999, 3, 152.

(10) Green, J.; Crack, J.C.; Thomson, A.J.; Le Brun, N.E. Curr. Opin. Microbiol. 2009, 12,

145.

(11) Holm, R.H.; Lo, W. Chem. Rev. 2016, 116, 13685.

(12) Crack, J.C.; Green, J.; Cheesman, M.R.; Le Brun, N.E.; Thomson, A.J. Proc. Nat.

Acad. Sci. U.S.A. 2007, 104, 2092.

128  

(13) Lazazzera, B.A.; Beinert, H.; Khoroshilova, N.; Kennedy, M.C.; Kiley, P.J. J. Biol.

Chem. 1996, 271, 2762

(14) Crack, J.C.; Gaskell, A.A.; Green, J.; Cheesman, M.R.; Le Brun, N.E.; Thomson, A.J.

J. Am. Chem. Soc. 2008, 130, 1749.

(15) Khoroshilova, N.; Popescu, C.; Munk, E.; Breinert, H.; Kiley, P.J. Proc. Nat. Acad.

Sci. U.S.A. 1997, 94, 6087.

(16) Zhang, B.; Crack, J.C.; Subramanian, S.; Green, J.; Thomson, A.J.; Le Brun, N.E.;

Johnson, M.K. Proc. Nat. Acad. Sci. U.S.A. 2012, 109, 15734.

(17) Tran, C.T.; Kim, E. Inorg. Chem. 2012, 51, 10086.

(18) Tran, C.T; Williard, P.G.; Kim, E. J. Am. Chem. Soc. 2014, 136, 11874.

(19) Hyduke, D.R.; Jarboe, L.R.; Tran, L.M.; Chou, K.J.; Liao, J.C. Proc. Nat. Acad. Sci.

U.S.A. 2007, 104, 8484.

(20) Landry, A.P.; Duan, X.; Huang, H.; Ding, H.; Free Radical Biol. Med. 2011, 50, 1582.

(21) Harrop, T.C.; Tonzetich, Z.J.; Reisner, E.; Lippard, S.J. J. Am. Chem. Soc. 2008, 130,

15602.

(22) Victor, E.; Lippard, S.J. Inorg. Chem. 2014, 53, 5311.

(23) Ding, H.; Demple, B. Proc. Nat. Acad. Sci. U.S.A 2000, 97, 5146.

(24) Wong, G.B.; Bobrik, M.A; Holm, R.H. Inorg. Chem. 1978, 17, 578.

(25) Tanifuji, K.; Tajima, S.; Ohki, Y.; Tatsumi, K. Inorg. Chem. 2016, 55, 4512.

129  

(26) Gilbert, H. J. Biol. Chem. 1982, 257, 12086.

(27) Ondarza, R.N. Bioscience Reports 1989, 9, 594.

(28) Winther, J.R.; Thorpe, C. Biochim. Biophys. Acta. 2014, 2, 1840.

(29) Kemp, M.; Go, Y.M.; Jone, D.P. Free Radical Biol. Med. 2008, 44, 921.

(30) Averill, B.A.; Herskovitz, T.; Holm, R.H.; Ibers, J.A. J. Am. Chem. Soc. 1973, 95,

3523.

(31) Hagen, K.S.; Reynolds, J.G.; Holm, R.H. J. Am. Chem. Soc. 1981, 103, 4054.

130  

Chapter 5: Model Cluster Conversion from [4Fe-4S]2+ to

[2Fe-2S]2+ Facilitated by Superoxide

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5.1 Abstract

Iron sulfur clusters are ubiquitous in biology and common to the most ancient and

modern forms of life. One pattern of reactivity that has been of interest recently is the

ability for a cluster to convert from one core to another. The best studied of these cases is

the conversion of a [4Fe-4S] cluster to a [2Fe-2S] cluster in the fumarate and nitrate

reductase (FNR) family of proteins in response to O2, regulating transcription of genes

required for aerobic respiration. This reactivity has been found to be amplified in the

presence of reactive oxygen species such as superoxide. Previous efforts to replicate this

reactivity with synthetic models have resorted to using harsh and biologically irrelevant

oxidants and supporting ligands. Previous work in our group (described in Chapter 4),

discovered the transformation could be performed by [Et4N]2[Fe4S4(SPh)4] (1) in the

presence of disulfide or molecular oxygen and extra thiolate ligand to form

[Et4N]2[Fe2S2(SPh)4] (2). Herein is described the first example of this same transformation

of a synthetic model [4Fe-4S] cluster to its corresponding [2Fe-2S] cluster mediated only

by superoxide ion.

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5.2 Introduction

Common to the most ancient and modern forms of life1, iron sulfur clusters and the

proteins that house them are ubiquitous in biological systems2. They make up one of the

most common and most diverse metalloprotein motifs—second only to the iron-oxo motif

found in proteins such as hemoglobin. Also diverse is the number of functions these

clusters can have in their various proteins. They have been shown to be involved

extensively in electron transport3, structural support4,5, and the binding and activation of

substrates during catalysis6. They have also been implicated in cellular signaling and

molecular sensing7-9. The most well studied reaction in this set is the reactivity of iron-

sulfur clusters with nitric oxide.

Iron sulfur clusters have been shown to be a major target for the gasotransmittor

nitric oxide (NO). There are numerous examples of NO-iron sulfur reactivity in biological

proteins including the [2Fe-2S] cluster in SoxR10 and the [4Fe-4S] cluster in the fumarate

and nitrate reductase (FNR)11 family of proteins—both have which have been studied using

synthetic model clusters12-15. These reactions give a wide variety of nitrosylated iron

products including DNICs, Roussin’s Red Esters, and Roussin’s Black Anion. While the

reactivity of iron sulfur clusters has been studied for many years and is fairly well

understood, the FNR family of proteins also illustrates another, less studied function of

iron-sulfur clusters—O2 sensing16.

Many bacteria, including E. coli, are able to survive and even thrive in both aerobic

and anaerobic conditions, and that is partly due to the activity of the FNR family of

proteins17. These proteins help the bacteria transition from aerobic and anaerobic

respiration by regulating the transcription of genes required for aerobic respiration. In

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anaerobic conditions, FNR exists as a dimer with two relevant domains—the iron sulfur

cluster domain, and the DNA binding domain. When the protein is bound to the DNA, the

transcription of those genes is inhibited by the inaccessibility of the genes to the

transcription machinery of the cell. In the presence of higher concentrations of O2, the iron

sulfur cluster domain undergoes a transformation characteristic of some clusters—cluster

conversion17-21.

Under anaerobic conditions, the iron sulfur clusters in the cluster domain exist as

two [4Fe-4S] cubane cores. Under increased O2 concentrations, those clusters transform

into [2Fe-2S] rhombic clusters. Additionally, the Le Brun group determined that the two

extra bridging sulfide ligands are released and then captured by two of the cysteine residues

ligating the iron-sulfur core. This reaction can be reversed through the addition of an

iron(II) source, cysteine, and cysteine desulfurase enzyme21. This reactivity is summarized

in Scheme 5.1.

Scheme 5.1: Graphical representation of the [4Fe4S] to [2Fe2S] cluster conversion

observed in the FNR family of proteins, showing the formation of cysteine persulfide

ligands upon addition of O2 and reaction reversal upon addition of Fe2+, cysteine, and

IscS21.

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This conversion triggers a structural change in the protein itself and inhibits the DNA

binding capability of the protein. When the bonds are broken, the genes are no longer

blocked, and the cellular machinery can take over and begin to transcribe DNA into RNA,

which can then be translated into the proteins required for aerobic respiration to take over.

While this transformation is well documented for proteins in bacteria, there is not a great

deal of chemical insight into how the change takes place. This is mainly due to a lack of

synthetic model species that have been able to exhibit the same reactivity pattern.

There has been a great deal of work done on iron-sulfur proteins using purified

protein and various reagents. While these experiments do give insight into the reactions

taking place in biological systems, it can often be difficult to gain any insight into the

chemical reactions taking place beyond the starting material and the finished product. This

is mainly due to the immense size, relatively speaking, of protein molecules and the

difficulty that is associated with many analytical techniques when using species that size.

To work around that particular obstacle, researchers have turned to using synthetic model

clusters with simple terminal ligands, most commonly thiolate groups to mimic the S-

bound cysteine residues generally found in iron-sulfur proteins. This transition is illustrated

in Figure 5.1.

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Figure 5.1: Schematic representations of (a) a biological [4Fe4S] cluster ligated by four

cysteine residues, and (b) a synthetic model [4Fe4S] cluster ligated by thiolate molecules,

where R represents a wide range of alkyl and aryl groups.

Using these smaller species allows for much easier use of analytical techniques such as IR,

NMR, and UV-Vis. Furthermore, there have been a number of synthetic systems13 that

have been shown to undergo the same chemical transformations as their protein bound

counterparts10 when exposed to the same reagents, proving the use of such studies. In the

case of the [4Fe-4S] to [2Fe-2S] cluster conversion reaction, there have only been two

synthetic systems shown to mimic the reactivity. In the 1970’s, Holm and coworkers22 were

able to affect the desired cluster conversion reaction in an all chloro-bound cluster

[Fe4S4Cl4]2-. In the presence of ferricenium as an oxidant and excess chloride ion to

stabilize the final product, the researchers were able to convert the cubane cluster into two

equivalents of the rhombic [Fe2S2Cl4]2- cluster. While this reactivity does match the cluster

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conversion seen in biology, it relies on a strong oxidant and utilizes a cluster with ligands

not seen in proteins.

Much more recently, Tatsumi and coworkers23 were able to affect the same core

conversion utilizing a N(TMS)2-bound cluster. Once again, ferricenium was utilized to

provide the necessary oxidizing power, and pyridine as both a solvent and the stabilizing

ligand for the product cluster. This work showed that the cluster needed to be oxidized to

the all ferric [Fe4S4]4+ form before the cluster would fracture into two pieces, and the L-

type pyridine ligand could stabilize the new rhombic product. This reactivity is summarized

in Scheme 5.2.

Scheme 5.2: Reactivity observed by Tatsumi and coworkers using a neutral, all ferric

[Fe4S4]4+ core to produce two equivalents of a neutral [Fe2S2]2+ species. Scheme adapted

from Tatsumi et al23.

While this did help provide insight into the reactivity being observed, it still required a

harsh oxidant and still did not have the S-bound terminal ligands found in the

overwhelming majority of protein bound clusters.

Previous work in our group, described in the previous chapter, utilized an all S-

bound synthetic model cluster and mild, biologically relevant oxidants—disulfide and

137  

molecular oxygen—to effect a [Fe4S4]2+ to [Fe2S2]2+ core conversion. In this chapter we

explore the ability for another biologically important species, superoxide anion, to affect

the same change.

In protein bound systems, superoxide has been found to play a role in the cluster

conversion reaction discussed. In fact, it takes part in the reaction in two ways. First, it has

been shown to amplify the oxygen-induced conversion through its own dismutation to

hydrogen peroxide and oxygen24. Additionally, it has been shown to have deleterious

effects on the [2Fe-2S] cluster present in the monomeric, post-conversion form of the

protein25. Superoxide could also affect cluster oxidation in another, indirect way. When

superoxide concentrations rise in the cell, it enters what is known as oxidative stress. While

in this state, a number of equilibria in the cell get compromised. One of these is the thiol-

disulfide equilibrium26,27. The cellular concentration of thiol is very high28, when the cell

enters oxidative stress that equilibrium is disrupted, and the cell becomes a more oxidizing

environmen29. For the purposes of this chapter, the focus is on the direct interaction of

superoxide anion with the iron-sulfur cluster.

5.3 Results and Discussion

5.3.1 Reactivity of [Et4N]2[Fe4S4(SPh)4] (1) with 1-4 Equivalents of Superoxide

Anion. Under and inert glovebox atmosphere, synthetic cluster (1) was combined with

various amounts of postassium superoxide (KO2), solubilized by the inclusion of 15-

crown-5, in acetonitrile. Upon the addition of one equivalent, the majority of the (1) starting

material was found to still be present in the crude reaction product. Also found in the

product, however, was a small amount of [2Fe-2S] cluster (2). Additionally, an unknown

product with peaks that matched sparingly with [Et4N][SPh] was also produced. To our

138  

knowledge, this is the first example of a synthetic model cluster undergoing a [4Fe4S]2+ to

[2Fe2S]2+ core conversion in the presence of superoxide. Emboldened by this result,

additional equivalents of superoxide were added to the starting cluster. For molar

equivalencies from 1-4, the starting [4Fe-4S] cluster was still found in the crude reaction

product, although the amount decreased with the addition of more superoxide. In addition

to the [2Fe-2S] cluster produced, there was a small amount of [Fe(SPh)4]2-, presumably

from some side reaction, and a small amount of [Fe3S4(SPh)4]3-, which could be an

intermediate in the conversion from the [4Fe-4S] core to the [2Fe-2S] core. The best yield

of [2Fe-2S] cluster came with the reaction of the starting cluster (1) with 4 equivalents of

superoxide, the NMR spectrum for which is shown in Figure 5.2.

Figure 5.2: Crude 1H NMR spectrum of the reaction product of (1) with 4 equivalents of

[15-Crown-5]KO2 prepared in CD3CN showing residual (1) (blue stars) and the desired

product (2) (red stars). Integration referenced of 4 equivalents of 15-Crown-5 (80 protons,

3.53ppm).

139  

In this case the yield of product was approximately 50% compared to the amount of reacted

cluster by difference with remaining 1 in the reaction product. Reactivity is summarized in

Scheme 5.3.

Scheme 5.3: Reaction summary of interaction of 1 with superoxide to produce 2 and

decomposition products.

5.3.2 Reactivity of [Et4N]2[Fe4S4(SPh)4] (1) with >4 Equivalents of Superoxide

Anion. Once the 1-4 equivalent reactions had been run and determined to retain the starting

material (1) in the reaction product, greater numbers of equivalents, including 6 and 8

equivalents of superoxide per mole of (1), were attempted. The 8-equivalent reaction

resulted in a major product of pale orange solid insoluble in any conventional solvent

system produced almost instantaneously upon mixing. This product was presumed to be

iron oxide from the addition of more superoxide than the cluster was stable in. Therefore,

the equivalents were reduced to 6, and the reaction once again ran for 18 hours without

producing such immediate and significant decomposition. Upon further analysis of the

products by NMR, shown in Figure 5.3, it was found that while there was significantly less

140  

starting material (1) present in the reaction product, there was also a lower yield of (2) at

the end of the reaction.

Figure 5.3: Crude 1H NMR spectrum of the reaction of (1) with 6 equivalents of [15-

Crown-5]KO2 prepared in CD3CN showing residual (1) (blue stars) and desired product

(2) (red stars). Integration referenced to 6 equivalents of 15-Crown-5 (120 protons,

3.58ppm).

The yield of this reaction, comparing the amount of (2) produced by the reaction with the

amount of (1) consumed was only 15%, much lower than the almost 50% yield per (1)

consumed seen in the reaction with 4 equivalents of superoxide This result indicated to us

that the desired product (2) may not be stable under the reaction conditions, and encouraged

us to carry out control experiments.

5.3.3 Reactivity of [Et4N]2[Fe2S2(SPh)4] (2) with 1 Equivalent of Superoxide

Anion. The first suspicion for why the yield of [2Fe2S] cluster was reduced came from the

141  

reactivity discussed in the previous chapter between the model [4Fe4S] cluster an

molecular oxygen. In that case, the product (2) was not stable in the presence of O2 and

would decompose after a short amount of time exposed to it. Therefore, as we consumed

more starting material (1), we also started to degrade our reaction product, resulting in

lower overall yields. To test this hypothesis, independently synthesized (2) was combined

with [15-Crown-5]KO2 in acetonitrile in a 1:1 ratio and stirred at room temperature for 18

hours. Upon the analysis of the reaction product by NMR, shown in Figure 5.4, there was

still approximately 50% of the starting [2Fe-2S] cluster present.

Figure 5.4: Crude 1H NMR of the reaction of 2 with 1 equivalent of [15-Crown-5]KO2

prepared in CD3CN. The spectrum shows residual 2 (red stars) and the production of an

unknown product (black stars).

Additionally, there was a black insoluble material produced by the reaction and a

significant amount of the unknown product produced as well. This provided us with two

142  

insights: first, the desired reaction product (2) is not stable in the presence of O2-, and

second, the unknown product could be produced from the degradation of (2) under those

conditions rather than as a side product of the original reaction. With that knowledge

reaction condition optimization has been attempted, but due to the inherent instability of

(2) it was nearly impossible to make significant improvement on the 4-equivalent reaction,

which remains the most successful reaction to date.

5.3.4 UV-Vis Monitoring of [Et4N]2[Fe4S4(SPh)4] + 4[15-Crown-5]KO2. To get

a better understanding of the reaction taking place, a UV-Vis monitoring reaction was set

up. 3mL of a 58.2μM solution of 1 was prepared in a Schlenk cuvette in acetonitrile. 4

equivalents of [15-Crown-5]KO2 via 1mM solution in a gas tight syringe under argon flow.

This stoichiometry was decided on because it had provided the best results for the 1 to 2

conversion reaction. The reaction was monitored by UV-Vis spectra taken every 15

minutes at room temperature for a total of 18.5 hours. The results are summarized in Figure

5.5.

143  

Figure 5.5: UV-Vis spectral monitoring for the reaction of [Et2N]2[Fe4S4(SPh)4] 1 with 4

equivalents of [15-Crown-5]KO2 at room temperature. Spectra were taken every 15

minutes for a total of 18.5 hours.

During the course of the overall reaction the spectra were observed to lose the features of

1, particularly the bands at 258 and 453nm. Additionally, features were observed to grow

into the spectrum at 242 and 382nm. The characteristic band of 2 at approximately 480nm

is not visible in the final spectrum, which we attribute to the relatively low yield of the

reaction, producing a concentration of 2 that is too small to be seen in the UV-Vis spectrum.

Immediately upon the addition of the superoxide to the solution, a new feature at 300nm

grew into the spectrum and over the course of the following 18 hours it reduced in sized

down to almost non-existence, indicating there could be an intermediate species produced

during the reaction. There is also what appears to be an isosbestic point between the 242

144  

and 258nm features at 250nm. In addition to these results, NMR and x-ray crystallography

determined that some amount of the [3Fe-4S] cluster [Fe3S4(SPh)4]3- with counter cations

of tetraethylammonium and potassium was also produced, but only in the reactions with

lower concentrations of superoxide. When greater than 4 equivalents of superoxide were

added to the solution, there was no [3Fe-4S] cluster observed. Other variables are currently

being explored, including the addition of extra thiolate and variable temperature to try to

ascertain more information, but no results are available at this time.

5.5 Conclusions

The present studies demonstrate the conversion of [Et4N]2[Fe4S4(SPh)4] to

[Et4N]2[Fe2S2(SPh)2] through reaction with superoxide, an important oxidizing agent and

a biologically relevant agent of oxidative stress. To our knowledge this is the first example

of a synthetic model [4Fe-4S] cluster to undergo such a conversion with superoxide. These

results open a pathway to greater understanding of iron sulfur clusters undergoing oxidative

stress and may be the foundation for mechanistic insights into that interaction. At the

present time the subsequent reactivity of the product [2Fe-2S] cluster with superoxide has

made it difficult to determine the reaction products and mechanistic information.

5.5 Experimental Methods

5.5.1 General Considerations. Unless otherwise specified, all reactions and

manipulations were carried out under and inert nitrogen atmosphere in an MBraun

glovebox (O2 >0.1ppm, H2O >0.1ppm) or under and inert argon atmosphere using standard

Schlenk techniques (Corp Bros., UHP 5.0). All solvents were purified by passage over an

145  

alumina column under an argon atmosphere and stored over activated molecular sieves

(3Å). Potassium superoxide (98%) was ordered from Strem and used as received.

5.5.2 Physical Measurements. Unless otherwise specified, all sample preparation

was carried out under an inert glovebox atmosphere. All UV-Vis characterization was

carried out using a Varian Cary 50 Bio spectrometer. All NMR characterization was carried

out using a Bruker Avance 400MHz spectrometer.

5.5.3 Synthesis. Compounds [Et4N]2[Fe4S4(SPh)4] (1)30 and [Et4N]2[Fe2S2(SPh)4]

(2)31 were synthesized according to literature methods.

5.5.4 General Reaction of [Et4N]2[Fe4S4(SPh)4] (1) with [15-Crown-5]KO2.

15mg (0.014mmol) of (1) were dissolved in 5mL of acetonitrile, producing a dark red-

black solution. The solution was stirred at room temperature for 5 minutes before the

addition of varying amounts (0.2 – 1.6mL) of [15-Crown-5]KO2 (0.07M) solution prepared

in acetonitrile depending on the number of equivalents (1-8) required for the reaction in

question. After the addition the solution was stirred at ambient temperature for a total of

18 hours. For lower ( >4) equivalent reactions, no significant color change was observed

during the reaction. For higher (4 – 6) equivalent reactions the reaction mixture changed

from dark red-black in color to dark purple. For 8 equivalents the solution became pale

yellow with a large amount of orange solid almost immediately upon the superoxide

addition. The volatile species were removed from the mixture in vacuo and crude UV-Vis

and NMR samples were prepared as necessary. The desired product (2) was isolated and

purified by consecutive washing with THF and acetone, and physical characterization

methods (UV-Vis and NMR) were in good agreement with literature values for

146  

[Et4N]2[Fe2S2(SPh)4] (2). (NMR, CD3CN, ppm: 2: 9.30 (bs), 4.90 (bs), 3.22 (bs), 3.14 (bs),

1.19 (bs). UV-Vis, λmax, nm: 480)

5.5.5 General Reaction of [Et4N]2[Fe2S2(SPh)4] (2) with [15-Crown-5]KO2.

15mg (0.017mmol) of (2) were dissolved in 5mL of acetonitrile. The solution was stirred

at room temperature for approximately 5 minutes before 0.25mL of 0.07M [15-Crown-

5]KO2 (0.017mmol) was added to the solution. The solution was stirred at room

temperature for a total of 18 hours. All volatile species were removed in vacuo and crude

UV-Vis and NMR samples were prepared as needed. The crude product was washed with

ether and THF, redissolved in MeCN and layered with ether in the freezer to afford a black

powder. The solid was isolated by filtration to give 16mg of total product.

5.5.6 UV-Vis Monitoring Reaction of [Et4N]2[Fe4S4(SPh)4] (1) with 4

Equivalents of [15-Crown-5]KO2. A 58.2μM solution of 1 was prepared using 62mg of

1 dissolved in 10mL of MeCN. The solution was then diluted by a factor of 100 to produce

the reaction solution. A 3mL Schlenk cuvette was charged with 3mL of the reaction

solution and the first spectrum was taken. 0.2mL of a 1mM solution of [15-Crown-5]KO2

(4 equivalents) was added to the reaction solution in the Schlenk cuvette by syringe under

argon. The cuvette was returned to the instrument and spectra were collected every 15

minutes for a total of 18.5 hours. Data was collected for all runs and a representative sample

of spectra were selected for inclusion.

147  

5.6 References

(1) Lovenberg, W. Iron-sulfur Proteins: Molecular Properties; Academic Press: New

York, 1973.

(2) Beinert, H.; Holm, R.H.; Munk, E. Science, 1997, 277, 653.

(3) Rao, P. V.; Holm, R. H. Chem. Rev. 2004, 104, 527.

(4) Kuo, C.; McRee, D.; Fisher, C.; O'Handley, S.; Cunningham, R.; Tainer, J. Science

1992, 258, 434.

(5) Guan, Y.; Manuel, R. C.; Arvai, A. S.; Parikh, S. S.; Mol, C. D.; Miller, J. H.; Lloyd,

R. S.; Tainer, J. A. Nat. Struct. Biol. 1998, 5, 1058.

(6) Flint, D. H.; Allen, R. M. Chem. Rev. 1996, 96, 2315.

(7) Fleischhacker, A.S.; Kiley, P.J. Curr. Opin. Chem. Biol. 2011, 15, 335.

(8) Beinert, H.; Kiley, P.J. Curr. Opin. Chem. Biol. 1999, 3, 152.

(9) Crack, J.C.; Green, J.; Thomson, A.J.; Le Brun, N.E. Acc. Chem. Res. 2014, 47, 3196.

(10) Ding, H.; Demple, B. Proc. Nat. Acad. Sci. U.S.A 2000, 97, 5146.

(11) Serrano, P.N.; Wang, H.; Crack, J.C.; Prior, C.; Hutchings, M.I.; Thomson, A.J.;

Kamali, S.; Yoda, Y.; Zhao, J.; Hu, M.Y.; Alp, E.E.; Oganesyan, V.S.; Le Brun, N.E.;

Cramer, S.P. Angew. Chem. Int. Ed. 2016, 55, 14575.

(12) Tran, C.T.; Kim, E. Inorg. Chem. 2012, 51, 10086.

(13) Tran, C.T; Williard, P.G.; Kim, E. J. Am. Chem. Soc. 2014, 136, 11874.

148  

(14) Harrop, T.C.; Tonzetich, Z.J.; Reisner, E.; Lippard, S.J. J. Am. Chem. Soc. 2008, 130,

15602.

(15) Victor, E.; Lippard, S.J. Inorg. Chem. 2014, 53, 5311.

(16) Green, J.; Crack, J.C.; Thomson, A.J.; Le Brun, N.E. Curr. Opin. Microbiol. 2009, 12,

145.

(17) Lazazzera, B.A.; Beinert, H.; Khoroshilova, N.; Kennedy, M.C.; Kiley, P.J. J. Biol.

Chem. 1996, 271, 2762.

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